[TRANS] LECTURE UNIT 01: INTRODUCTION TO MYCOLOGY

MYCOLOGY • General rule: All fungi are nonmotile

• The term “mycology” is derived from Greek word o Bacteria: can be motile or nonmotile
“mykes” meaning mushroom.
o Mycology = study of mushrooms
• Raymond Sabouraud (1910),
o Published his book Les Teignes,
which was a comprehensive
study of dermatophytic fungi
o Regarded as father of medical
mycology
• Sabouraud Dextrose Agar (SDA)
o Culture media used for the
isolation of saprophytic fungi

General Properties of Fungi

Differences of Fungi and Plant Cells
• They are eukaryotic
• Fungal cells can be also differentiated from plant cells
o Cells contain membrane bound cell organelles
including nuclei, mitochondria, Golgi apparatus, o Fungi are chemoheterotrophs
endoplasmic reticulum, lysosomes etc. § Require organic compounds for both carbon and
§ Fungi and animal cells have almost the same energy sources
cellular components. o Fungi lack chlorophyll and are therefore not
• Exhibit mitosis autotrophic.
• They obtain nutrients as saprophytes (live off of § Plant cells have chlorophyll
decaying matter) or as parasites (live off of living
matter).
o Fungi are saprophytic which means they are
ubiquitous in nature.
§ If the environment provides a good nutrient for
them to grow, they can easily cultivate.
o Just like bacteria and viruses, fungi are parasites
which can infect humans and cause infection
• All are obligate aerobes
o All fungi require water and oxygen and there are no
obligate anaerobes
• All are non-motile
o Due to the rigid characteristic of their cell wall
component which is the chitin. CLASSIFICATION BASED ON SEXUAL
REPRODUCTION
Differences of Fungi and Animal Cells
(1) Zygomycetes
• Fungal cells can be differentiated to animal cells within
two components:
o (1) Fungi have ergosterols in their membranes and • Produce through production of
possesses 80s ribosomes zygospores.
o (2) All fungi possess cell wall made of chitin.
§ Have a rigid cell wall and are therefore non- Zygospores
motile

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 LECTURE UNIT 01: INTRODUCTION TO MYCOLOGY

(2) Ascomycetes • Sporogenesis is the endogenous process of spore

formation within a sac
• Produce endogenous spores called ascospores in cells
called asci Conidia
o Asci – plural form of • Arise either by budding off conidiogenous hyphae or by
ascus differentiation of preformed hyphae.
• Ascus is a sac-form • Develop following mitosis of a parent nucleus and are
formed in any manner except involving cytoplasmic
o Hold and keep the cleavage.
ascospores intact • Conidia production may be blastic or thallic.
o Ascospores are located
inside the ascus Blastic Development

(3) Basidiomycetes • The conidium begins to enlarge and a septum is

• Produces exogenous spores called basidiospores in formed.
cells called basidia. • Conidium originates from part of parent.
• Budding form – forming a bud from the parent cell.
o Basidia – plural form
of basidium Thallic Mode of Development
o Basidia are often
referred to as parent • The conidium is differentiated by a septum before its
cells as it reproduces differentiation.
sexually to produce
o One parent will develop and form a septum
basidiospores
(daughter • Conidium results from the conversion of entire parent
spores/cells). cells into the conidium.
• 3 classifications:
• Sterigma – connecting filament that connects the
basidiospores to the basidium. o Thallic-solitary
o Thallic-arthric
(4) Deuteromycetes (Fungi imperfecti) o Alternate-arthric
• Classified as fungi imperfecti since they do not belong
to the other classifications.
• These fungi are not known to produce any sexual
spores (ascospores or basidiospores).
• This is a heterogeneous group of fungi where no sexual
reproduction has yet been demonstrated.
Asexual Reproduction
Thallic-solitary Thallic-arthric Alternate-arthric
• Asexual spores are produced following mitosis,
whereas sexual spores are produced following meiosis.
Conidiogenous Cell
• Asexual reproduction is NOT part of the classification of
fungi.
• The cell that gives rise
Sporogenesis to a conidium
• Conidiophores are
• (1) The asexual spores of zygomycetes, which are specialized hyphae that
known as sporangiospores, form within the sac-like bear conidia or
structure known as sporangia. conidiogenous cells
o Sporangia – plural form of sporangium. o Phialides holds the
conidia
• (2) The sporangiospores result from the mitotic o Conidiophores hold the phialides
cleavage of cytoplasm in the sporangium.
• In many cases conidiogenous cells are referred as
o Sporangiospores – formed after mitotic division
phialides
and located inside the sporangium
• (3) The sporangia are
borne on special hyphae
called sporangiophore.
o Sporangiophore –
holds the sporangium
o Sporangium – holds
the sporangiospores.

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 LECTURE UNIT 01: INTRODUCTION TO MYCOLOGY

CLASSIFICATION BASED ON MORPHOLOGY • Septate Hyphae

• Classified into yeast form and mold form o Show frequent crow-walls occurring perpendicularly
to the pouter walls of the hyphae
Molds § Cross-walls are in a good interval
• Most molds have a fuzzy or woolly appearance because o Pattern of the septation is constant and the septum
of the formation of mycelia distance is almost the same
o Two forms of mycelia • Sparsely Septate Hyphae
§ Aerial mycelia o Have few cross-walls at irregular intervals
§ Vegetative mycelia
§ It has a septum but sparsely
• Aerial mycelia extend
Hyaline Versus Phaeoid
above the surface of the colony and are responsible for
the fuzzy appearance
o Aerial mycelia also support the reproductive
structures that produce conidia
§ Conidia, in many
cases, are used to
identify different
fungal genera
§ Example: different
Aspergillus Hyaline hyphae Phaeoid hyphae
species have • Another characteristic useful in identification is
different patterns pigmentation
of conidia • Hyaline (moniliaceous) hyphae
NOTE: Conidia alone cannot be solely used to identify special o They are nonpigmented or lightly pigmented
level of fungi, same goes with bacteria o No melanin
Microscopic Appearance • Phaeoid (dematiaceous) hyphae
o They are darkly pigmented because of the presence
• The microscopic appearance often aids in the
identification of molds. of melanin in the cell wall

o Aid – microscopic appearance only helps to identify Dimorphism and Polymorphism

the genus level of fungi.
Dimorphism
• In some species, antler, racquet, rhizoids, or spiral
hyphae are formed • Ability of some fungi to exist in two forms, dependent on
growth conditions
Rhizoids
• Fungi is able to transform into two forms based on a
different thermal requirement.
Thermal Requirements
• Very common
• Root-like appearance • (1) Mold Phase
o seen when the organism is
grown at room temperature
(22° to 25°C) in ambient air
Septate Hyphae & Sparsely Septate Hyphae conditions
o Outside temperature
o Fuzzy appearance
• (2) Yeast or Tissue state
o seen in vivo or when the
organism is grown at 37°C
with increased CO2
o Body temperature
• Majority of the pathogenic fungi are dimorphic
Septate hypae Sparsely septate hyphae o They can live in the environment and can be
accidentally ingested by humans
• Both are septate o Dimorphic fungi can survive at normal human
• Their difference lies on the manner of the cross-walls temperature of 37°C
or septum
• Not all fungi are dimorphic

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 LECTURE UNIT 01: INTRODUCTION TO MYCOLOGY

o Majority of the saprophytic fungi only have one o Capsules can be

phase, which is the mold phase demonstrated by
negative staining
§ Visible in the naked eye methods using India Capsule

• Does this organism cause infection? ink or Nigrosin.

o Are fungi dimorphic or not? § Staining with
polysaccharide
§ Dimorphic – causes infection (most common)
§ Not dimorphic and enters at 37°C – cannot
survive and cannot cause infection o Capsule itself can be stained by Meyer Mucicarmine
stain.
• Thermally dimorphic fungal species associated with
human disease include Blastomyces dermatitidis, • (2) Some yeasts are pigmented.
Coccidioides immitis, Histoplasma capsulatum var.
o Rhodotorula sps. produces
capsulatum, Paracoccidioides brasiliensis, Sporothrix
pink colonies due to
schenckii, and Penicillium marneffei
carotenoid pigments.
o Phaeoannellomyces weneckii
and Piedraia hortae.
§ Are dematiaceous,
producing brown to
olivaceous colonies in
culture media.

• Several other fungi also possess this ability but have PATHOGENESIS OF FUNGAL DISEASES
not been described as agents of human mycoses (MYCOSES)
o Mycoses – infections caused by fungi • Most fungi are saprophytic or parasitic to plants and are
§ Ex: superficial mycoses, cutaneous mycoses, adapted to their natural environment.
systemic mycoses o Saprophytic – ubiquitous in nature

Polymorphism § Commonly seen in the environment where they

are free floating
• Polymorphic fungi have both yeast and mold forms in § In Bacteriology, the presence of a fungal
the same culture. element is a good indicator of a contamination.
o Majority of the polymorphic fungi are not clinically w Determine whether the fungi isolated is a true
significant pathogen or a contaminant

Yeast • Fungal infection in humans rarely occurs, especially to

immunocompetent individuals.
• Yeast are single vegetative cells that typically form a o Occurs only when conditions are favourable such as
smooth, creamy, bacterial-like colony without aerial low immune system, taking immunosuppressive
hyphae. drugs, or having diseases such as HIV, AIDS.
o Looks like bacteria in a
• Except for few fungi such as dimorphic fungi that
culture media and
cause systemic mycoses and dermatophytes, which are
resembles large cocci
primary pathogens, the rest are only opportunistic
under the microscope
pathogens.
o Usually misidentified as
gram positive cocci • Human body is a hostile environment and offers great
resistance to fungal invasion.
§ Cocci – small
o Some fungi such as Candida and Malasezzia furfur
§ Yeast – spherical but
have adapted to the human environment and exist
large
as commensals
• Yeasts reproduce by budding or fission
§ When the normal flora is disturbed, commensals
o Budding involves maturation of the bud to an such as Candida will over reproduce, causing
independent blastoconidium (daughter cell) infection
§ Parent cell form a bud formation and try to • The complex interplay between fungal virulence
release daughter cell factors and host defense factors will determine if a
fungal infection will cause a disease.
• (1) Some yeast such as Cryptococcus and the yeast
• Infection depends on the inoculum size and the general
form of Blastomyces dermatatidis produce
immunity of the host.
polysaccharide capsule.
o Inoculating large amounts of organisms can cause
infection, as well as with the interplay with the host’s
immunity.

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 LECTURE UNIT 01: INTRODUCTION TO MYCOLOGY

REVIEW
Triad of Disease

Agent

• Includes the virulence factors (similar to bacteria)

• Examples:
o Ability to adhere to host cells by way of cell wall
glycoproteins
o Production of capsules allowing them to resist
phagocytosis
o Ability to damage host by secreting enzymes
(keratinase, elastase, collagenase)
o Capability to secrete mycotoxins
• These are virulence factors produced by fungi that can
attack the host cell.

Host

• The host has different mechanisms to counteract the

virulence factors possessed by the infectious agent.
• Examples:
o Physical barriers (skin and mucus membranes)
o Fatty acid content of skin
o pH of the skin, mucosal surfaces and body fluids
o other components of innate immunity
• If the host defense factors are impeded, the fungi can
still cause infection to the human.

Environment

• Plays a very important role in the interplay of the agent

and host infection
• Examples:
o Prolonged antibiotic therapy
o Underlying diseases
o Age
o Immunosuppressive drugs
• If the environment or host defense factors will fail to
protect the host, the infectious agent can cause disease
to the host

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 [TRANS] LECTURE UNIT 02: LABORATORY DIAGNOSIS OF FUNGAL DISEASES

SPECIMEN COLLECTION • One of the most common clinical samples seen in the
Microbiology Laboratory for fungal identification
• Diagnosis of fungal infection depends entirely on
selection, collection, transport, and processing of Processing consideration
clinical specimen
• Specimens may be stored at room temperature if
Selection processing is completed within 2 hours.

• In terms of (selecting the) appropriate clinical sample o E.g., You have 5 samples of sputum, and the
samples can be processed within 2 hours. When
• Fungi are classified based on their site of infection
processing one sample, the other 4 can be stored at
o E.g., Superficial Mycosis room temperature.
▪ Superficial infection • If processing is delayed, specimens should be
▪ Fungi that cause skin infection refrigerated at 4oC
o E.g., Systemic Mycosis o If the sample is processed beyond 2 hours, the
integrity of the sample will be compromised.
▪ Systemic infection
▪ Include samples like CFS and blood ▪ The sample might contain bacteria and it will try
to multiply.
Ringworm & Athlete’s Foot ▪ These contaminants can hinder the replication of
fungi
 Cause a negative result in culture
▪ Evaluate what you can accomplish within two
hours.
• The most common specimens collected for culture
NOTE: Respiratory tract secretions at the upper respiratory
• Clinical manifestation caused by superficial mycosis tract contains normal flora
o Its clinical manifestation is skin infection • Normal flora: bacteria that normally reside in the upper
▪ The appropriate collection is to do a swab or respiratory tract that will try to contaminate the sample.
scrape o Fungi: contaminant in bacteriology
▪ In this case, skin scraping is better o Bacteria: contaminant in mycology
Collection • Antibacterial antibiotics in the battery of media to be
used
• In this section, collection is hard because we are not
trained on how to collect the sample in Mycology o Use antibiotics to control the normal flora in culture
• Majority of the sample is collected by the physician media
• We only have common knowledge about collecting
• Problem: Fungi are too concentrated in respiratory
sample like skin scraping, swab, or collection of
tract secretions
• If CSF, we are not allowed to collect
o Overgrowth of molds causes problem in
o Call the assistance of a physician identification
CLINICAL SAMPLES • Cycloheximide (0.5 mL) in at least one of the culture
media
Respiratory Tract Secretion
o Used to prevent overgrowth of molds
• Sputum, Induced sputum,
BW (Bronchial • Why do we add antibiotics in culture media?
Wash/Bronchial Washing), o Usual samples used in laboratory are not sterile,
BAL (Bronchial Alveolar especially in the respiratory tract.
Lavage), Tracheal aspiration o Lower respiratory tract (lungs) is sterile
▪ However, the way of collection of sample
(sputum) makes it not sterile since it passes

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 LECTURE UNIT XX: TITLE

through the normal flora in the upper respiratory ▪ Reporting should be done after 21 days (Mahon
tract. and Bailey & Scott)
▪ Temp. should be 30°C because H. capsulatum is
o Bacteria (e.g. normal flora) can hinder with the
a dimorphic fungus
identification
o Addition of antibiotics helps in the isolation of fungi  can transform to yeast form at 30-37°C

• Proper collection of sputum and respiratory tract • The optimal temperature for fungal blood cultures is
secretions must be collected in a screw cap, wide- 30°C, and the suggested incubation time is 21 days.
mouth bottle
Urine
o Can be either glass or plastic
o Properly labelled • Processed immediately after collection
• 24-hour sample – unacceptable
Cerebrospinal Fluid
o Always double check if the sample submitted is
• Filtered through a 0.4 um appropriate for the test
membrane filter attached to a ▪ E.g., If a physician requests a fungi culture on a
sterile syringe 24 hour sample, the MT should correct and
o Commonly, it is used with a educate him/her
centrifuge. However other • Centrifuge and sediment cultured using loop
countries utilize filter.
o Fungi will be trapped in the o Sediments from a centrifuged urine is used for
pores of the syringe filter culture
o Once the filter is removed, the trapped organisms
▪ Fungi are heavy and large. Hence, they do not
will be placed in the culture media
float
o According to Bailey & Scott, if filter is utilized, it
▪ It is more likely that fungi are part of the
should be placed in different areas of the culture
sediment
media and should be examined daily
o Supernatant is not used
• If less than 1 ml submitted – centrifuge and 1 drop
aliquots placed on several areas of the culture media Hair, Skin, and Nail Scrapings
o Should be processed promptly – if not, stored at • Obtained by scraping the skin with scalpel/microscope
room temperature or at 30°C slide
o Should not contain antibacterial or antifungal agents
o Microscope slide is usually used
▪ Since CSF is considered sterile, if bacteria grow
in culture media means that other than fungal ▪ Should be autoclaved first
infection, it is possible that meningitis is caused o Scalpel is preferred
by a bacteria
▪ Consider bacteria other than fungal Hairs
▪ The concern in the upper respiratory tract is
fungi • Plucking with forceps
• Temp. 30°C for 21 days before reporting negative
• If the samples are considered sterile, no need to add • Mycosel agar with chloramphenicol and
antibacterial cycloheximide
o It can have a double identification o Choice of culture for hair, skin, and nail scraping
o Sterile areas such as blood and CSF must have no samples
organisms in it
Eye (Corneal Scrapings or Vitreous Humor)
NOTE: CSF specimens should never be refrigerated
• Corneal Scrapings
Blood
o Collected by a physician
• Blood culture system o Should be placed directly onto microscopic slides
and inoculated onto non-inhibitory media
o E.g., BACTEC
• Vitreous Humor
• Lysis centrifugation system for
heavy incidence of dimorphic o Collected by a
fungi physician
o effective in isolation of H. ▪ Method of
capsulatum collection is
o H. capsulatum optimum inserting a
recovery: 10-14 days needle via
syringe into
▪ Blood culture should be extended until 14 days the globe of
o Temp. 30°C for 21 days before reporting negative the eye (red dot in the picture)

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 [TRANS] LECTURE UNIT 03: LABORATORY DIAGNOSIS OF FUNGAL DISEASES
(MICROSCOPIC METHODS & CULTURE MEDIA)

INTRODUCTION
 Some clinical samples require direct microscopic Negative Staining
observation
 India Ink or Nigrosin
o Example: Urine samples - perform gram staining
before proceeding to culture o Identify capsule of the yeast Cryptococcus
neoformans in CSF (India Ink stain)
 Goal: to aid physicians rule out infectious agents
 10% KOH + India Ink = India Ink stain
(bacteria or fungi)
 10% KOH + Nigrosin = Nigrosin stain
MICROSCOPIC METHODS  Other references use deionized water
instead of 10% KOH
Wet Preparations

 Saline or 10% KOH – most common microscopic  Quick and easy

methods used both direct & after culture visualization of C.
 Saline mount – direct observation of fungal elements neoformans under the
microscope
o Disadvantage: does not dissolve keratin in clinical
 Zone of Clearance / Halo
samples (e.g., skin & nails)
o Particles of the ink
 10% KOH – dissolves keratin in skin, hair, or nails pigment do not enter the capsule that surrounds the
o Recommended by laboratory handbook of medical spherical yeast cell.
mycology for direct detection of fungal elements in o Difficult to interpret
clinical samples
o When positive, provides valuable clues on etiologic Lactophenol Cotton Blue (LPCB)
agent of infection  Imparts a blue color to cell wall of fungal element
o Principle: disrupts cellular sheets or clumps of
 Used in tease mount (wet preparation) or slide culture
protein tissues, debris that may be present, and
 Performed usually in aerial mycelia infection
clears the specimen for easier fungal detection
o A piece of the aerial
 Calcofluor White Stain (brightening agent) mycelia is taken and
o Enhances visibility of fungal elements stained with LPCB.
o Hair is examined for endothrix or ectothrix o The picture depicts typical
infections Aspergillus spp.

 Ultraviolet Wood’s Lamp  Has many species.

 Microscopic appearance
o Used in the absence of Calcofluor White Stain cannot identify species
o Endothrix infection will not fluoresce level of Aspergillus
o Some exothrix infection may fluoresce bright green
or yellow green Giemsa Or Wright’s Stain
o Positive result: fluorescent bright green or yellow
green  Detection of intracellular Histoplasma capsulatum in
blood, lymph nodes, lung, liver, or BM (bone marrow)
Endothrix vs Ectothrix o If the physician requests for
H. capsulatum detection,
 Endothrix
Giemsa stain is not
o endo = inside performed in microbiology
o Dermatophyte infection of laboratory.
the hair that invade the hair o Rather, the sample is
shaft and internalize into forwarded to the
the hair cell histopathology section.
 Ectothrix
o exo/ecto = outside
o Dermatophyte infection is
confined on the hair

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 LECTURE UNIT 03: LABORATORY DIAGNOSIS OF FUNGAL DISEASES

Periodic Acid Shiff PROCEDURE

 Internal details STEP 1.

 If a culture is suspected to be C.
 Useful in staining fungal elements albicans, prepare a serum.
from deeply seated tissues STEP 2.
o Subcutaneous mycoses –  Get a colony from the culture.
fungal agents that can cause STEP 3.
 Emulsify the colony in the serum.
infection in deeply seated
tissue STEP 4.
 Incubate at 37°C for 1-3 hours.
Gomori’s Methanamine Silver Stain STEP 5.
 Observe for budding.

 Serum used can be either from rabbit or human

 Stains viable and non-viable
fungi o Rabbit serum – preferred but expensive
o Human serum – usually used in the laboratory

 If mother cells produce pseudohyphae, it is C. albicans

NOTE: PAS and Gomori’s methanamine silver stain
o Can be used to identify the etiologic or fungal agent
o Both can also stain viable and non-viable fungi CULTURE MEDIA
 Types of Culture Media:

Mayer’s Mucicarmine Stain o Primary Recovery Media

o Differential Test Media
 Demonstration of the mucoid
 Similar with the principle in Bacteriology
capsule of C. neoformans
o Characterize the colony characteristic of a fungal
agent
o Pointed by the arrow: capsule
of [Link] How to Read?

Surface Texture
Hematoxylin and Eosin (H & E)
 Cottony or Wooly  Powdery
 Determine hyaline and  Granular  Silky
Dematiaceous fungi  Chalky  Glabrous (Smooth, Creamy)
o Determined by the  Velvety  Waxy Glass
pathologist
Pigmentation
 but you can screen as a
microbiologist  Observation in the Surface and Reverse Plate

Fontana-Masson Stain o Surface – reading of colony on the surface of the

culture media
o Reverse – flip the culture media and observe the
 Demonstration of melanin or pigmentation
melanin-like substances in the
 Each specie in Mycosporum has different
lightly pigmented agents of
reverse plate characteristic
phaeohyphomycosis

Germ Tube

 For presumptive identification of C. albicans

 Uses serum for at 37°C, 1-3 hours

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 LECTURE UNIT 03: LABORATORY DIAGNOSIS OF FUNGAL DISEASES

Table [Link] and Microscopic Characteristics of  To evaluate microscopic characteristics of keratinized

Medically Important Fungi specimen, 10% QH is used
o 10% QH will dissolve the keratin
Culture
Microscopy  Can replace SDA-CC for recovery of dermatophytes
Characteristic
Sterile hyphae, o CC stands for
Downy white to terminal cycloheximide
Microsporumaudouinii salmon-pink colony chlamydoconida,
FAVIC  CC suppress other
Disease: Tinea capitis Reverse: tan to saprophytic fungi
salmon pink CHANDELIERS, and
pectinate bodies including normal flora
Membranous with
feathery periphery;  Has antibacterial and
Microsporumcanis center of colony is antifungal property
Thick walled, spindle
white to buff over shaped, multiseptated Mycosel/Mycobiotic
Disease: Tinea capitis orange yellow.
associated with rough walled
macroconidia
alopecia Reverse: Lemon-  Similar to DTM
yellow or yellow
orange apron o Has the same purpose with DTM, to isolate
Cinnamon-colored, Thick walled, rough, dermatophytes
Microsporumgypseum
powdery colony elliptical multiseptated  Not the same with DTM in terms of the content
Disease: Tinea capitis macroconidia
Reverse: Light tan

Saboraud Dextrose Agar (SDA)

Culture Media
 Saprobic and pathogenic fungi
 Generally must contain nitrogen and carbon sources,
o Raymond Saboraud –
and vitamins
father of mycology
o Sugars: glucose, fructose and mannose, sucrose o SDA is equivalent to nutrient
(table sugar) agar in bacteriology
o Peptone, yeast extract, malt extract, amino acids,
 SDA allows growth with
ammonium and NO2 compounds
all fungi either saprobic
o Salts (Fe, Zn, Mn) – for defined media
or pathogenic fungi
o Thiamine (B1) and biotin (B12)
 Cycloheximide is added to isolate pathogenic
 Culture media used in mycology is somewhat the same fungi only
in bacteriology. SDA – (Cycloheximide)
Primary Isolation Media
 Pathogenic fungi
Brain-heart infusion agar (BHI)  Bacteria saprobe fungi – inhibited

 Bacteriology: BHI is used to isolate fastidious organisms o Majority of the saprobe fungi will be inhibited
together with the normal flora
o Fastidious: maaarte na organism
 Add cyclohexemide to isolate only pathogenic
 Mycology: BHI is used to isolate saprophytic and fungi since it has antibacterial and antifungal
pathogenic fungi from sterile sites property
BHI w/ Antibiotics  it will also inhibit the normal flora
SDA with oil
 Some samples may be contaminated with normal flora
 Pathogenic fungi, specimens contaminated with  For isolation of Malassezia furfur
bacteria or saprobes fungi
o Olive oil – most commonly used oil
o Bacteria: add antibiotics
 Overlayed with SDA
o Saprobes fungi: add Cycloheximide (0.5mL)
Potato Dextrose Agar (PDA)
 Cycloheximide (0.5mL) in at least one of the
culture media prevents overgrowth of molds,
especially saprobes  Grows a wide range of fungi
o An alternative for SDA and
same with nutrient agar
Dermatophyte Test Medium (DTM)

 Dermatophyte test medium is used in keratinized

specimen subjected to culture

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 LECTURE UNIT 03: LABORATORY DIAGNOSIS OF FUNGAL DISEASES

Differential Media
PREPARATION FOR SLIDE CULTURE
Birdseed (niger agar) STEP 1. Prepare the SDA

 Isolates C. neoformans producing phenol oxidase STEP 2.

Prepare the sterile
plate while preparing
o Resulting to production of melanin (brown-black the SDA
color) Place two glassrods
STEP 3. parallel to each other in
 Colonies that did not
produce melanin are the sterile plate
not C. neoformans STEP 4. Place a glass slide
 Differentiates C. over the glass rods
neoformans from Add distilled water
other pathogenic STEP 5. inside (1-2mL) to
fungi provide moisture
during incubation
Cornmeal agar with tween 80

 Isolation of Chlamydospore,
production of Candida species

Cut the SDA

horizontally and
Czapek agar and MALT agar STEP 6. vertically using a
scalpel until small
blocks are formed

 Isolation Asperigillus species

Malt Agar (MA)

 Useful in isolation of
Ascomycota

STEP 7. Place the block of SDA

on top of the slide

Slide Culture Using a wire needle,

collect the organism
STEP 8. from the culture and
 Optimal examination method for preservation of fungal
inoculate it on top of
morphology
the block
o It requires a culture media - usually SDA STEP 9. Add cover slip
Advantages
STEP 10. Close using the upper
lid of the plate
 No need to remove a portion of the fungus from a
Incubate
culture plate and transfer it to the slide.
 This reduces the chance of damage to fragile STEP 11. (- 24°C for Mycelia
reproductive structures or spore-bearing structures of - 37°C for Yeast/Fungi)
fungi.

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 LECTURE UNIT 03: LABORATORY DIAGNOSIS OF FUNGAL DISEASES

Adhesive Tape Preparation

 Similar to parasitology’s cellophane scotch tape method

PROCEDURE

STEP 1. Prepare a scotch tape or

adhesive tape and the
applicator stick

STEP 2. Loop the adhesive tape

to the applicator stick

STEP 3. Applicator stick is inserted into

the butt of the tube to get the
mycelial part of the fungi

STEP 4. After obtaining the mycelial part

of the fungi, it is placed in a
slide

 slide should
contain
Lactophenol
Cotton Blue
(LPCB) prior to
placement of tape

STEP 5. Put the scotch tape to the

LPCB-containing slide

STEP 6. Perform microscopic

characterization

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 [TRANS] LECTURE UNIT 04: SUPERFICIAL MYCOSES

INTRODUCTION DERMATOPHYTES
• Mycoses – refers to a fungal infection • Break down and utilize keratin – source of Nitrogen
o The word before “mycoses” indicates the location. • Incapable of penetrating in subcutaneous tissue
• Classified into 3 genera, with different sites of infection:
▪ Superficial mycoses – fungal infection that can
o Genus Trichophyton – infects skin, hair and nails
be seen superficially (skin, nails, & hair)
o Genus Microsporum – infects skin and hair (not
▪ Subcutaneous mycoses – fungal infection that
nails)
can cause subcutaneous infection, including the
o Genus Epidermophyton – infects skin and nails
tissue
(not hair)
 Fungi are facultative anaerobes, hence they
can survive even without the presence of • These organism cause TINEA aka “Ringworm”
oxygen. • Onchomycosis – infection in the nails
• For superficial mycoses, the fungal agents have a Tinea
specific microscopic description.
• Latin word for worm or ringworm
o E.g., Typical • Characterized by another Latin word to designate the
characteristic of Microconidia - meatballs
area of the body involved
Malassezia furfur • Two Latin words:
▪ Described as o Tinea – ringworm
“spaghetti and o Location – e.g. barbae
meatballs”
▪ Spaghetti – Table 1. Various Forms of Dermatophytoses and the
hyphae Respective Affective Sites
Hyphae - spaghetti
▪ Meatballs –
microconidia Type of Body part
Ringworm affected

SUPERFICIAL MYCOSES
• Non-invasive, involves top layer of the skin, hair, or
nails Tinea barbae Beard
• Caused by dermatophytes
• Classified as:
• DERMATOPHYTES
o Group of fungi that are able to damage and utilize
keratin found in the skin, hair, and nails
o 3 genera:
Tinea capitis Scalp/Head
▪ Trichophyton
▪ Epidermophyton
▪ Microsporum
o The 3 genera of dermatophytes are the most
common fungal agents of superficial mycoses
• NON-DERMATOPHYTES
o Fungal agents that can cause superficial mycoses Body (glabrous
Tinea corporis
that does not belong to dermatophytes skin)
o Examples:
▪ Malassezia
▪ Trichosporon
▪ Piedra
▪ Exophiala
Tinea cruris Groin (Jock Itch)

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 LECTURE UNIT 04: SUPERFICIAL MYCOSES

• (1) Urease

• (2) Hair baiting test

Feet (Athlete’s o Also called hair
Tinea pedis
foot) perforation test

• (3) Red pigmentation on

SDA
o Deep red
o Red pigmentation
should be seen on the
Nails reverse or bottom of
Tinea unguium
(Onchomycosis)
the culture
▪ Surface – white
▪ Reverse – deep red
o T. mentagrophytes has a red pigmentation on
reverse, but not deep red.
NOTE:
• Tinea barbae is common on men uses the same Trichophyton mentagrophytes
shave/doesn’t change shave
o The fungi can survive on the surface of the shave
• Microconidia – Grapelike
and causes infection
• Superficial mycoses can cause infection only if the
individual is immunocompromised.
o Stress can also be a predisposing factor
o Asthmatic individuals can be potentially affected
• Macroconidia – Cigar shaped
Trichophyton spp.

• Trichophyton spp. infect the hair, skin, and nails

COLONY CHARACTERISTICS:
Table 2. Trichophyton spp.
• Colonies are generally flat,
white to cream in colour,
Specie General Characteristics with a powdery to granular
surface.
Trichophyton verrucosum Rat tail microconidia • Reverse: yellow-brown
to reddish-brown color
Trichophyton violaceum Violet colony on SDA with oil
o Hard to differentiate
Favic chandelier, causes with T. rubrum
Trichophyton schoenleinii because both produce
alopecia
a reddish-brown color
Trichophyton “Balloon microconidia”, “black o T. mentagrophytes produce a reddish-brown color in
tonsurans dot ringworm old cultures
• Schoenleinii – German word for beautiful Trichophyton rubrum

Tests to Differentiate T. tonsurans and T. rubrum

Table 3. Differentiation of T. tonsirans and T. rubrum

T. mentagrophytes T. rubrum

Urease + - Microconidia Macroconidia

Hair baiting Test + - • Microconidia: clavate or peg-shaped (teardrop-

shaped)
Red pigmentation
on SDA
- + • Macroconidia: Produce three to eight-celled cylindrical

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 LECTURE UNIT 04: SUPERFICIAL MYCOSES

COLONY CHARACTERISTICS: Trichophyton verrucosum

• Surface: white downy to pink granular • Hyphae

• Reverse: yellow when the colony is young, red color
o The tips of some
when old culture
hyphae are broad
and club-shaped,
and occasionally
divided, giving the
so-called “antler”
effect.
• Chlamydospores – Often in chains
o Looks by pair in pictures or seldom in chains
Surface Reverse • Macroconidia
o Only rarely produced, but have a characteristic tail
NOTE: They have the same characteristic with Trichophyton or string bean shape when present
mentagrophytes.
• Additional Biochemical tests must be done COLONY CHARACTERISTICS:
o e.g., Hair baiting Test • Slow growing, small, button or disk-shaped, white to
cream-colored,
• Molecular diagnostic test • Suede-like to velvety surface, raised center, and flat
o This can be utilized in order to be sure of the periphery with some submerged growth
organism that has been isolated. • Reverse
o Pigment may vary from non-pigmented to yellow
Trichophyton tonsurans

• Hyphae – relatively broad,

irregular, much-branched with
numerous septa
• Microconidia
o Varying in size and shape from
long clavate to broad pyriform
o Borne at right angles to the
hyphae – looks like a leaf that
Trichophyton schoenleinii
branches
• Macroconidia • schoenleinii – German word for beautiful
• No macroconidia and microconidia are seen in
o Very occasional smooth, thin- routine cultures
walled, irregular, clavate • Hyphae
o Characteristic antler “nail head”
COLONY CHARACTERISTICS: hyphae or “favic chandeliers”
• Cultures
• Colonies show considerate o Difficult to maintain in their
variation in texture and color typical convoluted form, and
rapidly become flat and downy
• Cultures are difficult to maintain in
their typical convoluted form, and
rapidly become flat and downy.
• Reverse: color varies from yellow • No reverse pigmentation is
to reddish-brown to deep present
mahogany
o Reverse – color is white
Tinea favosa/favus

• Infection of the hair follicles

NOTE: Culture Characteristic together with Microscopic o Hair will stop growing
Characteristic are not definitive because the roots are
• Need to associate laboratory diagnosis to the clinical damaged.
manifestation of the patient

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 LECTURE UNIT 04: SUPERFICIAL MYCOSES

• Can progress to formation of crusty, cup shaped lesion o If compared to bacteria, it looks like dienes
(scutula) which is made up of dead epithelial cells and phenomenon in Proteus mirabilis and Proteus
fungal mycelia. vulgaris
o Foul odor due to dead epithelial cells.
• Can lead to hair loss and scar tissue formation
• Caused by: Trichophyton schoenleinii

Microsporum spp.

• Microsporum spp. may infect the hair and skin but rarely
the nails.
o Other reference states that it can cause infection to
Surface Reverse
nails but generally, microsporum species are body
location restricted.
o The 3 genera of
dermatophytes are
NON-DERMATOPHYTES
classified based on
location of infection: Malassezia furfur
▪ Trichophyton - hair,
• Associated with poor personal hygiene
nails, skin
▪ Microsporum - hair o Those who only
and skin take a bath once a
week are at risk
o In the clinical setting, fungi are much more diverse
o It is not advisable
▪ Possibly but rarely infect other body parts, to take a bath 3-4
including the nails. times a day
because the
• Characterized by presence of large, spindle-shaped, normal flora will be
thick-walled, multiseptate macroconidia washed out.
▪ Street children have a stronger immune system
Table 4. Microsporum spp.
compared to people who take a bath 3-4 times a
day because normal flora is under innate
Specie Characteristics immunity.
• Fluoresce under UV wood’s lamp
M. canis Grows on rice grains • Grows on Sabouraud Dextrose Agar (SDA) with Olive
Oil
M. audouinii Apple green fluorescence under UV light • Microscopy/ Microscopic Appearance: Spaghetti and
Meatballs
Geophilic – can be transferred in the form of soil
M. gypseum o Hyphae – Spaghetti
(termed geophilic dermatophytes)
o Microconidia – Meatballs
• Causative agent of Tinea Versicolor or Pityriasis
Epidermophyton spp. Versicolor
o “ap-ap” in bisaya
Epidermophyton floccosum

• Only member Tinea versicolor / Pityriasis versicolor

• Most common cause of tinea
cruris and tinea pedis.
• Club shaped, smooth walled • Superficial brownish or scaly areas on light skinned
macroconidia with two to four individuals
cells described as ‘beaver-tail’ • Irregular patches or non-
• Does not produce microconidia. pigmented or untanned
• In their macroconidia, there is skin on dark skinned
2-4 cells described as “beaver- individual
tail” o More visible to dark
skinned individuals
COLONY CHARACTERISTICS:
• Khaki green surface; powdery green appearance
• Reverse: yellowish brown with folds

ALESNA. AMOLORIA. ANIBAN. ASOY. DE LEON. IBONES. OSTIQUE. PADILLO. RAMOS. TEVES. TOLO. MONARES, BSMLS 3 4
MONTEROSO.
 LECTURE UNIT 04: SUPERFICIAL MYCOSES

Exophiala werneckii NOTE:

• In the Philippines, it is seldom to encounter superficial
• Causative agent of Tinea mycoses because majority of the Filipinos don’t go to
Nigra Palmaris hospitals when infected nor it develops severely.
o Location: Palm area o E.g. Skin infection with skin discoloration.
o Discoloration on the
center of the palm • Thus, it is rare to receive mycology samples due to low
to no numbers of patients going to hospital for diagnosis
• Palms of the hand (black to and treatment.
scaly patches)

Piedraia hortae

• Causative agent of Black

Piedra
o It usually infects on the
surface of the hair
shaft.
• Hard, brown black in
crusts on the outside of
the hair shaft
Trichosporon beigelii

• Causative agent of White

Piedra
o Light brown, soft nodules
on the beard or mustache
that are less firmly attached
than those of the black
piedra

SCHEMATIC OF HAIR INFECTIONS

Ectothrix

• Fungal agents that can cause

infection only to the surface of
the hair sac.

Endocervix

• Fungal agents that can cause

infection inside the hair sac.

Favic

• Problem is within the roots of the

hair
o probability of having hair loss
is high.

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 MV [LEC] – LU5. Subcutaneous Mycoses

SUBCUTANEOUS MYCOSES Chromoblastomycosis

• Fungal diseases that affect subcutaneous tissue
• Also known as verrucous dermatitidis and
o Superficial and Cutaneous mycoses: Fungal chromomycosis
agents that affects epidermis and dermis • Chromoblastomycosis occurs worldwide but is most
common in tropical and subtropical regions of the
▪ Affect the upper part of the skin
Americas and Africa
o Subcutaneous tissue (fat): the area where
o Even in the PH there are little recorded cases.
subcutaneous
o Majority of the cases are found in America and
mycoses fungal
Africa
agent will infect
causing Chromoblastomycosis Infectious Agents
infection
▪ Affect the • Fonsecaea compacta
lower part of • Fonsecaea pedrosoi
the skin • Phialophora verrucosa

• The causative o Most common fungal agent that can cause

agents responsible are organisms commonly found in chromoblastomycosis
soil or on decaying vegetation • Cladophialophora carrionii
o Geophilic and are usually occupationally related • Rhinocladiella aquaspersa
▪ E.g., farmers, gardeners, or other occupations Clinical Manifestations
that deal directly with soil (high risk)
▪ As medical technologist, less contact with the • Chromoblastomycosis is a chronic mycosis of the skin
soil, therefore, lesser risk to be infected with and subcutaneous tissue that develops over a period of
subcutaneous mycoses months or, more commonly, years
 However, if immunocompromised or has a o Chronic = long term
traumatic puncture in the skin, then infection
can be acquired • mostly asymptomatic in the absence of secondary
complications, such as bacterial infections,
• Organisms causing subcutaneous mycoses belong to a carcinomatous degeneration, and elephantiasis
variety of genera in the form class under
Hyphomycetes o Patient showing no symptoms
• Although some are moniliaceous (hyaline or light- o Presence of secondary complications =
colored), many are phaeoid, producing darkly symptomatic
pigmented colonies and containing melanin in their cell Clinical Manifestations
walls
o There are 2 groups of diseases (Mahon/B&S): • Chromoblastomycosis is a chronic mycosis of the skin
and subcutaneous tissue that develops over a period of
▪ Phaeohyphomycosis – mainly phaeoid months or, more commonly, years
▪ Chromoblastomycosis – either phaeoid or
moniliaceous o Chronic = long term

• Subcutaneous fungal infections may be grouped • It is mostly asymptomatic in the absence of secondary
together by the disease processes they cause or by the complications, such as bacterial infections,
causative agents involved carcinomatous degeneration, and elephantiasis
o Grouped according to their infection: o Presence of secondary complications = symptoms
manifest
▪ Chromoblastomycosis
▪ Sporotrichosis • Lesions are usually confined
▪ Mycetoma (eumycotic) to the extremities, often the
▪ Phaeohyphomycosis feet and lower legs, and are
a result of trauma to these
NOTE: Subcutaneous mycoses are either moniliaceous areas
(hyaline or light-colored) or phaeoid (darkly pigmented)
o E.g. farmers

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 LECTURE UNIT XX: TITLE

o Hallmark characteristic of chromoblastomycosis is • Mycetomas may be caused by fungi or bacteria. Those

cauliflower-like surface of the lesions caused by bacteria are referred to as actinomycotic
mycetomas, and those caused by fungal agents are
Laboratory Diagnosis referred to as eumycotic mycetomas
• Brown, round sclerotic bodies, which are nonbudding Table 2. Description of Granules Seen in Eumycotic
structures occurring singly or in clusters, are seen in Mycetomas
tissues
Fungus Color Size Texture
• These sclerotic bodies
reproduce by dividing in Pseudallescheria
White 0.5-1.0 Soft
various planes, resulting boydii
in multicellular forms Acremonium
White 0.2-0.5 Soft
o Sclerotic bodies are falciforme
also called Medlar Madurella
Black 0.5-5.0 Hard
bodies mycetomatis

• The presence of sclerotic bodies is diagnostic for Madurella grisea Black 0.3-0.6 Soft
this disease
Exophiala spp. Black 0.2-0.3 Soft
o Sclerotic bodies/Medlar bodies in tissue are a
• Exophiala spp.
diagnostic tool to confirm that the infection is
chromoblastomycosis o In other reference books, these are not under the
Eumycotic Mycetomas
• Culture: growth is moderate to slow, and colonies are
velvety to woolly and gray-brown to olivaceous black Pseudallescheria boydii
• Species are not differentiated by colony morphologies
because they all produce similar characteristics • The anamorphic form of P. boydii is the septate
filamentous fungus Scedosporium boydii.
Table 1. Microscopic Morphology of Fungi Causing
o Anamorphic form -
Chromoblastomycosis
Asexual, mitosis
Organism Microscopic Morphology ▪ Scedosporium boydii
Conidiogenous cells, phaeoid, flask-shaped
Phialophora
phialides, with collarettes o Polymorphic or
verrucosa Teleomorph form - Sexual, meiosis
Conidia oval, one celled, occur in balls at tips
▪ Pseudallescheria boydii
of phialides
Primary one-celled conidia formed on ▪ Hallmark characteristic
Fonsecaea is the formation of
sympodial conidiophores
pedrosoi cleistothecia
Primary conidia function as conidiogenous  globose, darkly
cells to form secondary one-celled conidia
pigmented structure
Some conidia are similar to those seen in  contains ascospores
Cladosporium sp., some are similar to those
in Rhinocladiella sp., and some are similar to • It produces oval conidia singly at the tips of
those in Phialophora sp. conidiogenous cells (cells that make conidia) known as
Fonsecaea
Similar to F. pedrosoi but with more compact annellides
conidial heads
compactum
Acremonium falciforme
Conidia are subglobose rather than ovoid
Erect conidiophores bearing branched Culture Characteristic
Cladophialophora
chains of one-celled, brown blastoconidia
carrionii
Conidium close to tip of conidiophore, • Colonies grow slowly and
termed shield cell are grayish brown,
becoming grayish violet
Fragile chains
Conidiophores erect, dark, bearing conidia o Grayish violet – old
Rhinocladiella culture
only on upper portion near the tip
aquaspersa
Conidia elliptic, one celled, produced
sympodially

Eumycotic Mycetomas

• Mycetoma is an infection of the subcutaneous tissues

that arises at the site of inoculation
• The disease is characterized by swelling, with
characteristic exudate draining to the skin surface
through sinus tracts

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 LECTURE UNIT XX: TITLE

Exophiala dermatidis
o Grayish brown –
young culture • Forms conidia at the tip of
the phialides
o Phialides – specialized
conidiophore cells,
producing conidia in
basipetal succession
without increasing the
Microscopic Characteristic length
• This group produces olivaceous to black colonies that
• Produces mucoid clusters of are initially yeastlike but become velvety at maturity
single or two-celled, slightly
curved conidia borne form Sporothrix schenckii
phialides at the tips of long,
unbranched, multiseptated Clinical Manifestations
conidiophores.
• The most commonly seen presentation of S. schenckii
Madurella spp. is lymphocutaneous sporotrichosis

• Madurella spp. o E.g., A rose’s thorn you plucked happens to have S.

are phaeoid, schenckii and pricked you. The spot heals, but re-
septate fungi. appears after a week on another area and with
• Approximately increasing infection
50% of the o Lymphocutaneous – the infection progresses
isolates of M. proximally along the lymph channels, causing
mycetomatis continuous infections
produce • Gardener’s disease
conidia from • This chronic infection
the tips of phialides. is characterized by
• This species grows very slowly but is initially white, and nodular and
becomes yellow, olivaceous, or brown, with a ulcerative lesions
characteristic diffusible brown pigment with age. along the lymph
• It grows best at 37°C, with slower growth at 40°C channels that drain
Madurella grisea the primary site of
inoculation
• Only sterile hyphae are Laboratory Diagnosis
observed
• Direct examination of tissue might reveal S. schenckii
o Sterile hyphae – fungal
as small, cigar-shaped yeast
cells that do not
produce sexual or • Microscopic examination from culture reveals thin,
asexual productive delicate hyphae bearing conidia developing in a rosette
structures pattern at the ends of delicate conidiophores
o Rosette pattern resembles the rouleaux formation of
▪ Do not bear spores
RBCs
• This isolate grows slowly, produces olive brown to black
colonies, and may produce a reddish-brown pigment.
• Optimal growth temperature is 30°C

Exophiala spp.

• Conidia are borne from annellides, with conidia

aggregating in masses at the tips of the conidiophore • Since S. schenckii is dimorphic, cultures are examined
o Annellides – specialized chonidiogenous cell, at:
producing conidia in basipetal succession by a o 22°C for the mold form
series of short, percurrent proliferation (annellations) o 37°C for the yeast form
▪ The tip of an
annellide increases
in length and
becomes narrower
as each subsequent
conidium is formed

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 [TRANS] LECTURE UNIT 6: SYSTEMIC MYCOSES

SYSTEMIC MYCOSES Coccidiodes immitis

 The systemic mycoses fungal agents are
geographically limited. Laboratory Diagnosis
 Dangerous because the entry of the fungi is through the
 Microscopic examination:
bloodstream and it infects the internal organs
o Fertile hyphae arising at
o It can infiltrate the brain tissues, such as the
right angles to the
Cryptococcus neoformans. (classified as systemic
vegetative hyphae,
mycoses before)
producing alternating
 Cryptococcus neoformans is part of opportunistic (separated by a disjunctor
mycoses cell) hyaline arthroconidia.
 Signs are not easily seen unlike cutaneous, so it will  Hallmark characteristic:
take a longer time to know if one is infected or not Hyphae together with
 Systemic fungal agents can adjust with the body’s alternating hyaline
temperature, and they can penetrate the organs and arthroconidia.
circulatory system o Only the [Link]
produces this hyaline
Blastomyces dermatitidis arthroconidia.

 Other names:  Should be processed in

Biosafety Level 3 (BSL-3).
o Gilchrist disease
o North American blastomycosis,
o Chicago disease  Initial growth, which occurs within 3 to 4 days, is white
 Memorize other names too since “Blastomyces to gray, moist, and glabrous.
dermatitidis” isn’t usually used o Colonies rapidly develop abundant aerial
mycelium.
Laboratory Diagnosis
 Mature colonies usually
 Sample: become tan to brown to
o Tissue or purulent material in cutaneous skin lesions lavender.
o Blood
 NOTE: Whenever there is a
 Systemic Mycoses – Dimorphic fungi cottony-like or powdery-like
culture, do not try to open the
o The organism can have a hyphal phase at 24o C or
plate or flask.
22o C
o Will have 2 different characteristic o This is because the spores
o Can survive at 2 different temperature of C. immitis is very
infectious that can cause
 Culture: disease.
o 22o C: The organism can produce a variety of
 There is no reported C. Immitis
colony morphologies. Colonies can be white, tan, or
cases in the Philippines.
brown and may be fluffy to glabrous
o 37o C: Broad based, budding yeast cells  Geographically limited/restricted – specific
geographic location, species cannot exist in other types
 Photo (from left-right) of geography.
o Left - Front (of the plate)
o Right - Reverse Plate (Butt of the plate) Histoplasma capsulatum

 Other names:
o Reticuloendothelial cytomycosis
o Cave disease
o Spelunker’s disease
o Darling disease

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 LECTURE UNIT 06: SYSTEMIC MYCOSES

Laboratory diagnosis  Systemic mycoses can survive outside the environment

that can be acquired by humans through transmission
 Direct Smear and can survive in our body
Preparations:
o Small yeast cells
measuring 2 to 3 μm
x 4 to 5 μm.
 Giemsa or Wright stain
o Yeast cells are
commonly seen
within monocytes and macrophages occurring in
significant numbers.
Paracoccidioides brasiliensis
Disease

 Paracoccidioidomycosis
o South American blastomycosis
o Brazilian blastomycosis
o Lutz-Splendore-Almeida disease
o Paracoccidioidal granuloma
 A chronic, progressive fungal disease endemic to
Central and South America

Hallmark Characteristic

 Multipolar budding at the

periphery
 Resemble a mariner’s
wheel

QUESTION: Mycoses are classified as superficial, cutaneous,

subcutaneous, and systemic. Which of the 4 is the most
serious and can cause serious diseases? Why?

 Systemic mycoses
o It spreads and affects the internal organs
o It can infiltrate into the deep tissues.

Summary

 Systemic are most dangerous because of their

characteristics
o Dimorphic
 Major virulence factor of systemic mycoses
 These fungi are the only organisms that can
cause a systemic infection since they can
survive at body temperature
 Although subcutaneous can slightly survive but
they require a lighter temperature
 Even if one will ingest molds but it does not have the
capacity to transform or survive, they cannot survive

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[TRANS] LECTURE UNIT 07: OPPORTUNISTIC MYCOSES

OPPORTUNISTIC MYCOSES
• They are commonly found in cultures of respiratory
• The tissue-invasive opportunistic mycoses are a group
secretions, skin scrapings, and other specimens.
of fungal infections that occur almost exclusively in
immunocompromised patients • A. flavus, A.
fumigatus and A.
o Immunocompromised patients have underlying niger are the three
diseases such as lymphoma, leukemia, diabetes most commonly
mellitus, or another defect of the immune system. encountered fungi in
the laboratory.
• Many patients who undergo transplantation, are placed
• A. terreus is a re-
on treatment with corticosteroids, cytotoxic drugs, or
emerging Aspergillus
other immunosuppressive agents to control rejection of
fungal infection.
the transplanted organ.
o There are many
o May accommodate fungal agents that can cause
recorded cases of
opportunistic mycoses.
fungal infections
• Many fungi previously believed to be nonpathogenic are involving A. terreus
now recognized as etiologic agents of opportunistic
fungal infections. Pathogenesis and Spectrum of Disease

o If the patient is immunocompromised, then the • Aspergillus spp. usually manifest the same
fungal agents can cause disease. characteristic of infection
• Aspergillus spp. are capable of causing disseminated
• Aspergillus fumigatus
infection, as is seen in immunocompromised patients
o Aspergillus is ubiquitous in nature • It also causes a wide variety of other types of infections,
o Inhalation of spores (conidia) can actually cause including:
infection
o Pulmonary or sinus fungus ball
▪ Mode of transmission of Aspergillus spp. is
inhalation. Therefore, the primary infection
occurs in the lungs.
o Allergic bronchopulmonary aspergillosis
o External otomycosis
▪ A fungus ball of the external auditory canal.
o Other diseases include:
▪ Mycotic keratitis
▪ Oncomycosis
▪ Sinusitis
▪ Endocarditis
▪ Infections involving the central nervous system
(CNS)
Aspergillus spp.

• Several Aspergillus spp. are among the most commonly General Laboratory Diagnosis
encountered fungi in the laboratory.
• Direct Detection Methods
o They are the most commonly encountered fungi in • Cultivation
the lab because of its epidemiology
o Most Aspergillus spp. are susceptible to
▪ Aspergillus spp. are widespread in the cycloheximide
environment
▪ Therefore, culture media should not be
▪ They colonize everywhere, hence they are
supplemented with cycloheximide
ubiquitous in nature
o The conidia of the Aspergillus spp. are easily
dispersed in the environment and humans become
infected by inhaling them.

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Table 1. Species of Aspergillus Recovered from Clinical Specimens During a 10-year Period at the Mayo Clinic

Clinical Specimen Source

Skin,
Respiratory Blood, Bone, CNS,
Organisms Gastrointestinal Genitourinary Subcutaneous
Secretions* Other
Tissue
Aspergillus clavatus 97/93 1/1 - 1/1 -
Aspergillus flavus** 1298/740 10/10 11/11 177/131 2/2
Aspergillus
3247/2656 11/9 14/14 175/137 8/8
fumigatus**
Aspergillus glaucus 503/307 1/1 - 8/8 1/1

Aspergillus nidulans 52/48 - - 5/3 -

Aspergillus niger** 1484/1376 18/18 17/17 151/124 11/11

Aspergillus terreus** 164/146 - - 23/21 3/3

Aspergillus versicolor 1237/1202 6/6 24/22 226/224 16/16

Other Aspergillus
3463/3418 18/14 32/32 319/314 16/16
species

CNS, Central nervous system.

*Numerator, Number of cultures; denominator, number of patients.
**Aspergillus spp. that are of focus

▪ Phialides – carry the conidia

Aspergillus fumigatus ▪ Conidia → phialides → metulae (if biseriate) →
vesicle → hyphae → stem
• Most commonly recovered species from
immunocompromised patients
• Species most often seen in the clinical laboratory
Culture of A. fumigatus

• Rapidly-growing mold (2-6

days) that produces a fluffy to
granular, white to blue-green
colony.
o White in the periphery
o Blue to green on the inside
• Mature sporulating colonies
most often have a blue-green,
powdery appearance. Mature mold form of A. Microscopic Characteristics of A. fumigatus
fumigatus
• Thermotolerant
o Able to withstand temperatures up to 45 °C • (1) Hyphae: short or
long conidiophores
General Structure of A. fumigatus with a characteristic
“foot cell” at their base
• Divided into two: o Long
o Uniseriate conidiophore –
most commonly
▪ Phialides arising directly from a vesicle as in seen in labs if A.
Aspergillus fumigatus
o Biseriate o Since they are
uniseriate, they
▪ Phialides arising from metulae as in the genus have no metulae
Aspergillus
▪ Metula – a sterile cell below the phialides of ▪ Phialides carry
some Aspergillus and Penicillum species the conidia
 Structure in between the vesicle and phialides • (2) Long chains of small (2-3 mm in diameter),
spherical, roughwalled, green conidia form a columnar
 Metulae – plural form mass on the vesicle

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• Conidiophore: coarsely roughened near the vesicle

o They are rough and spiny
o Difficult to identify under the microscope

Aspergillus niger

• Commonly seen in the clinical laboratory, but its

association with clinical disease is somewhat limited.
• This organism is a cause of fungus ball and otitis
externa.
Culture of A. niger

• Mature colonies within 2-6 days.

• Growth begins initially as a yellow colony that soon
develops a black, dotted surface as conidia are
Aspergillus flavus
produced.
• Sometimes, it is recovered from immunocompromised o With age, the colony becomes jet black and
patients are represents a common isolate in the clinical powdery
microbiology laboratory
• Reverse: buff or cream-colored
Culture of A. flavus

• Rapidly growing species (1-5 days) that produced a

yellow-green colony
o Yellow → young colony
o Green → matured colony

Microscopic Characteristic of A. niger

• Hyphae: long conidiophores

supporting spherical vesicles
Microscopic Characteristic of A. flavus giving rise to large metulae and
smaller phialides (biseriate),
from which long chains of brown
• Vesicles are globose, to black, rough-walled conidia
and phialides are are produced.
produced directly from • The entire surface of the versicle
the vesicle surface is involved in sporulation
(uniseriate) or from a
primary row of cells o Other Aspergillus spp. –
called metulae sporulation is only with
(biseriate) conidia
o Aspergillus flavus can
either be uniseriate or
biseriate
o Biseriate A. flavus:
▪ Vesicle → Phialide Metulae
Metulae →
Phialides (carries Vesicle
the conidia)
o Also has a foot cell
• The Phialides give rise to
short chains of yellow-
orange elliptical or
spherical conidia that
become roughened on
Conidiospore
the surface with age

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Aspergillus terreus Microscopy

• A significant cause of infection in immunocompromised • Hyphae: Give rise to phialides

patients producing either single-celled
• Innately resistant to amphotericin B. microconidia, usually borne in
gelatinous heads or large, multicelled
o If Aspergillus spp. is recovered, it should be
macroconidia that are sickle- or boat-
determined whether it is terreus, niger, fumigatus, or
shaped and contain numerous
flavus.
septations
o Amphotericin B will be given which is an antifungal
• Some cultures of Fusarium spp.
agent
Commonly produce numerous
▪ If not effective, it is Aspergillus terreus chlamydoconidia
Culture of A. terreus Other Hyaline Septate Opportunistic Molds

• Tan colonies that resemble cinnamon Geotrichum candidum

• Culture alone cannot identify Aspergillus organisms
• Is an uncommon cause of infection but has shown to
cause wound infections and oral thrush; it is an
opportunistic pathogen in immunocompromised hosts.
Culture

• White to cream colored, yeast-like colony; some

isolates may appear as white, powdery molds.
Microscopy

• Hyphae: septate and produce numerous rectangular to

Microscopic Characteristic of A. terreus cylindrical to barrel-shaped arthroconidia.
• Arthroconidia do not alternate but are continuous, in
• Vesicles are hemispherical, and phialides cover the contrast to Coccidioides spp.
entire surface and are produced from a primary row
o Coccidioides – alternating with a gap and
metulae (biseriate)
arthrospore
• Phialides produce globose to elliptical conidia arranged
o Both Geotrichum candidum and Coccidiodes spp.
in chains
have arthroconidia but Geotrichum candidum has no
• This species produces lager cells, aleurioconidia, which gap.
are found on submerged hyphae o Illustration labelled as A presents with a gap with the
o Aleurioconidia is larger than conidia picture labelled as B presents without a gap.

o Blastoconidia: not produced

Acremonium spp.

• Associated with disseminated infection, fungemia (fungi

in the blood), subcutaneous lesions, and esophagitis.
Fusarium spp. Culture
• Fusarium solani species complex – the most
commonly isolated organisms within this group o Rapid-growing
• Fusarium oxysporum species complex – the second and also may
most common group responsible for human disease appear yeast-
like when initial
Culture Characteristics growth is
observed.
• Grow rapidly, within 2 to 5 days, and are fluffy to cottony o Mature colonies
and may be pink, purple, yellow, green, or other colors, become white
depending on the species. to gray to rose
or reddish
orange.

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 LECTURE UNIT 07: OPPORTUNISTIC MYCOSES

Microscopy Paecilomyces variotii

o Hyphae: produce single, unbranched, tube-like • Has also been shown to be an important pathogen,
phialides are observed. causing endocarditis, fungemia, and invasive disease.
o Phialides give rise to cluster to elliptical, single- Culture
celled conidia contained in a gelatinous cluster at
the tip of the phialide.
o Velvety, tan to olive brown,
Penicillium spp. and somewhat powdery.

• Are among the most common organisms recovered by

the clinical laboratory.
Culture

• Green or blue-green, but pink, Microscopy:

white or other colors may be
seen. o Phialides of
• The surface of the colonies Paecilomyces
may be velvety to powdery spp. are long,
because of the presence of delicate, and
conidia. tapering

Microscopy
LABORATORY DIAGNOSIS
• Hyphae: Brushlike
conidiophores Direct Detection Methods Antigen – Protein
• Conidiophores: Produce
metulae from which flask- Galactomannan Assay
shaped phialides producing
chains of conidia arise.
o Has a flashlight like
appearance

Purpureocillium lilacinum

• Previously known as
Paecilomyces lilacinus
• Appears to be the most
pathogenic species and
has been associated with
endophthalmitis • Targets antigens of Aspergillus spp., the most common
(inflammation of the interior source of invasive fungal infections caused by the
cavity of the eyes), hyaline septate molds.
cutaneous infections, and • Disadvantage:
arthritis
o However, the assay may yield false-positive results
because of cross-reactivity with other non-
Aspergillus molds, including Talaromyces
Culture marneffei, Histoplasma capsulatum, Fusarium
oxysporum, Paecilomyces spp., and Alternaria spp.
o Exhibits colonies that are considered lilac in color • Primary antibody (Monoclonal Antibody) – first
exhibiting shades of lavender to pink. antibody that is embedded with the
Microscopy o If there is a sample that contains Aspergillus,
therefore, it has Galactomannan protein.
o Chlamydospores: absent o Galactomannan is an antigen that will bind to the
antibody.
o If it will bind to the antibody, it cannot be observed
since both antibodies and antigen are colorless.
Therefore, a secondary antibody should be added.
• Secondary Antibody – carries the peroxidase enzyme
on the Fc region of the secondary antibody.

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 LECTURE UNIT 07: OPPORTUNISTIC MYCOSES

o Purpose of peroxidase: Differentiating C. albicans and C. glabrata

▪ The antigen to antibody sandwich interaction
• Temperature requirement
produces a colorless reaction, therefore, it is
needed so that if chromogen (which will serve as o Both can survive at 37, 42 and 45 degrees celsiu.
the substrate) is added, it will be consumed by
peroxidase. • Cornmeal agar
▪ If chromogen will be consumed by peroxidase, o Pseudo and true Hyphae
the chromogen will produce a color reaction. o Can be checked by the use of germ tube method
▪ The intensity of the color reaction is directly
proportional to the concentration of ▪ (+) Candida albicans
Galactomannan. ▪ (-) Candida glabrata
• Cyclohexamide
Beta-D-glucan assay
o Resistant: Candida albicans
o Susceptible: Candida glabrata
• Urea and nitrite
o Cannot be used as a differential tool because both
of the organisms are negative

Pneumocystis spp.

• Inhabit the lungs of many mammals.

• P. carinii was originally classified with the protozoa, but
nucleic acid sequencing showed conclusively that the
• Designed to detect antigens common to all clinically organism is a fungus
important fungi. • It is apparent from nucleic acid studies that P. carinii is
• Beta-D-glucan can be detected in the serum of patients not a single species.
infected with systemic aspergillosis.
o P. carinii is the species most commonly found in rats
o Serves as screening; if tested positive, the organism
is a fungal source. • P. jirovecii is the species most often recovered from
o Beta glucans are not present in bacteria. humans
o HIV is under Baltimore’s classification, group VI
• Disadvantage:
(group 6)
o The predictive value is not specific to infectious o Baltimore classification group VII (group 7) includes
with Aspergillus spp. hepadnavirus.
▪ Beta glucans can also be found with other fungi.

Direct Detection Methods – Molecular Methods

• The most reliable gold standard identification of the

fungal agent is the molecular methods.
Nucleic Acid Amplification Assays

• Are not commonly performed to detect or identify these

fungi
MALDI-TOF MS

• For the identification of fungal isolates has the potential

to provide quick and accurate species identification.
Candida

• Can be both systemic and opportunistic

• Candida spp. Are commonly present as normal biota
of the mucosa, skin, and digestive tract, and they are
also the most notorious agents of yeast infection.
• Clinical diseases ranges from superficial skin infections
to disseminated diseases.
• C. albicans currently reigns as the premier cause of
yeast infection in the world.

ALESNA. AMOLORIA. ANIBAN. ASOY. DE LEON. IBONES. OSTIQUE. PADILLO. RAMOS. TEVES. TOLO. MONARES. BSMLS 3 6
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Table 2. Differentiating Characteristics of Yeasts

Temperature Growth (°C) at Cornmeal Agar

Pseudo
37° 42° 45° True Hyphae Arthroconidia Cyclohexamide Urea Nitrate
Hyphae

CANDIDA

C. albicans + + + + + - R - -
C. dubliniensis + - - + + - R - -
C. glabrata + + + - - - S - -
C. guilliermondii + + - + - - R - -
C. krusei + + - + - - S V -
C. lusitaniae + + + + - - V - -
C. parapsilosis + - - + - - S - -
C. stellatoidea + + + + + - S - -
[Link] + + + + - - V - -
CRYPTOCOCCUS

C. albidus - - - - - - S + +
C. neoformans + - - - - - S + -
Trichosporon spp. + V - + + + R + -
+, Positive; -, negative; R, resistant; S, sensitive; V, variable.

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 [TRANS] LECTURE UNIT XX: BASIC CONCEPTS IN VIROLOGY

VIROLOGY
• Study of viruses and virus-like agents including but not
limited to their taxonomy, disease producing properties,
cultivation and genetics
• Often considered as part of microbiology or pathology
Viruses

• Smallest infectious agent that can pass through filters

o 0.45nm
▪ Most commonly used filter
▪ Filters are used to clinical samples to eliminate
bacteria & bacteria and isolate only viruses
• Most frequent cause of human infectious diseases
o 30%-40% of diseases are caused by viruses General Characteristics
o E.g., SARS-CoV-2, SARS-CoV-1, MERS-CoV,
Influenza, Polio, Hepatitis A, B, C, D, and E, HIV, • Obligate intracellular parasite
Ebola virus
o Requires human host cell to replicate
• Infects humans, lower animals, insects, plants, bacteria
and fungi ▪ Viruses don’t replicate outside the human host
cell
o Bacteriophage: Viruses that infects bacteria
o Considered as non-living things (inanimate) when
▪ Specific only to its bacteria outside the host cell
▪ If bacteriophage is used as a therapy or use as a
treatment to a specific bacterial infection → the • Cannot multiply by binary fission
bacteriophage will only target the bacteria and • Only contain either RNA or DNA
not to the whole cells o Cannot be both RNA or DNA
• Divided into genera and species o RNA: Coronavirus, HIV, Hepatitis C, Rabies
o DNA: Hepatitis B
o Genus: HIV o One best example: Chlamydia
▪ Species: HIV1, HIV2 ▪ Was classified before as virus
o Genus: Herpes simplex virus (HSV) ▪ They found out that chlamydia has DNA and
RNA, therefore it cannot be called as a virus
▪ Species: HSV-1 ▪ It was taken out and put into the last part of
• Viruses are much smaller compared to bacteria bacteriology (it was kicked out from the virus
family)
o Smallpox
• Lack ribosomes, ATP
▪ Largest virus o They don’t generate their own ATP
▪ 200 nm x 300 nm o They only carry their genome so therefore they need
▪ Under the poxvirus (DNA virus) the human host’s ribosomes to produce their own
▪ Can pass through filters proteins
o Poliovirus • Size: 20-250/300 nm
• 22 families associated with human infections (updated)
▪ Smallest virus
▪ 30 nm o 14 – RNA viruses
▪ Under the picornavirus (RNA virus) o 8 – DNA viruses
▪ Because there are two non-human viruses
before that cause zoonotic infections now, and
they are classified under medical virology

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 LECTURE UNIT XX: TITLE

Viral Structure • The protein

shell, or coat,
that encloses
the nucleic acid
genome which
is either helical
or icosahedral
in symmetry
o Functions:
▪ To protect the viral genome from destructive
agents in the external environment
▪ To introduce the viral genome into the host cell
Virion

• The complete set of your virus components (infectious (4) Nucleocapsid

viral particle)
• Viruses aside from its classification based on their • The combination of
genome (either RNA or DNA), they are also classified capsid together
based on the presence of envelope with the enclosed
o If the virus has envelope, they are called nucleic acid
“enveloped virus” • The combination of
o If the virus does not have envelope, they are called capsomeres (basic
“naked virus (non-enveloped virus)” unit of capsid)

• The envelope is termed “peplos” which is a Latin term o The nucleic

for “cape” acid is present
inside the capsid
o If the virus doesn’t have peplos, they are called
“naked” (5) Peplos/Viral envelope
• Remember: In virion, if we say complete infectious viral • Lipoprotein envelope that covers the capsid
particle, if it is an enveloped virus, it must have an • Function is for the viral protein/spikes on the surface of
o Envelope the envelope which will allow the attachment to the
o Capsid – many capsomeres whole cells
o Capsomere – basic unit of capsid • Some
o Nucleic acid (either DNA or RNA) – inside of capsid viruses
o Nucleocapsid – combination of capsid and nucleic have
acid envelopes
o If you have the presence of all of these, this is called from the
virion, pertaining to its complete infectious viral host cell
particle that includes all parts of the viruses membrane

• For naked, it should not have an envelope, but the rest o If the
of the parts should be present virus
goes
1. Nucleic Acid Core inside the cell, it will leave its viral envelope on the
surface of the cell or the virus can also go inside the
cell without leaving its viral protein/spikes on the
• Constitutes the genetic surface of the cell
material or viral genome o Once inside the cell, the virus will be naked. Leading
which can be single or to the release of its genome
double stranded DNA or o The genome will head towards the ribosome to
RNA create proteins for spike and envelope protein.
o These proteins will then go back and attach to the
o Either a single or
surface
double stranded DNA
o Once the virus will go outside the host cell, it will
or RNA
acquire an
2. Capsomere envelope with
protein spikes
• The nucleic acid is protected by capsomere • Some viruses have
• Complex morphologic units of the capsid which consist envelopes from the
of several identical or different protein molecules nuclear membrane
o The purpose of capsomere is to protect the nucleic (some has
acid from external environment phospholipid
envelope) they
3. Capsid

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 LECTURE UNIT XX: TITLE

acquire from the host cell. • The virus can be identified in picture based on the
o The virus (double-stranded either DNA or RNA) that characteristics of their spikes
is enveloped and icosahedral (due to the orientation Classification of Viruses According to:
of the capsid) will go inside the host cell and leaves
its envelope on the surface of the host cell • Morphology – type of capsid (Icosahedral, helical)
▪ The capsid will dissolve, releasing dsDNA • Type of genome – viruses are classified based on the
genome they carry
o The DNA virus will go inside the nucleus
o E.g., DNA or RNA (single or double stranded)
▪ Majority of the DNA viruses goes inside the o Positive-sense or Negative-sense
nucleus
▪ Majority of the RNA viruses replicate on the • Means of replication
cytoplasm o E.g., Enteroviruses have single-stranded RNA that
o Once inside the nucleus, the DNA virus will produce synthesize strands of RNA directly
proteins and a new virion and then its new o E.g., Retroviruses make RNA in two steps
synthesized DNA will go inside it (Synthesizing DNA first followed by RNA)
o If it already created a complete nucleocapsid, it is
Baltimore Classification
now ready to go out
o During the replication cycle, proteins will also be • Developed by David Baltimore
secreted and goes outside the nuclear membrane
o Once the nucleocapsid goes out the nucleus, it will o Working as a professor in Caltech
now acquire an envelope from the cytoplasm o Made a new classification of viruses
o He was able to identify and noticed that
• Some viruses have no envelopes and termed naked hepadnaviruses and retroviruses have reverse
o Naked viruses don’t have the same replication cycle transcriptase
compared with enveloped viruses. • A virus classification system that groups viruses into
families, depending on their type of genome (DNA,
(6) Spikes
RNA, single-stranded(ss), double-stranded (ds) and
• Glycoprotein molecules that bind to host cell during their method of replication.
attachment, readily visible under the electron o There are 7 groups of Baltimore’s classification
microscope
• One of the most important parts in terms of Classification of Viruses
transmission
o It facilitates the virus to enter the host cells
• Example 1:
o Glycoproteins present:
▪ HA1 (hemagglutinin)
▪ NA (neuraminidase)
 By the spike
alone, we can
identify that the
virus is influenza. • Viruses are classified into two:
 Among all the o RNA viruses
viruses, the only virus with both HA1 and NA
spikes is the influenza. ▪ Icosahedral
 The genome is just an additional because the  Naked
genome of the influenza is segmented  Enveloped
• Example 2: ▪ Helical
o Glycoproteins present:  Enveloped
 gp120 o DNA viruses
 gp141 ▪ Icosahedral
 Through these  Naked
glycoproteins, we  Enveloped
can identify that the
virus is HIV.  Naked/Env. (cytoplasmic)
 It also has reverse transcriptase, p24, and ▪ Helical
cone-like shape capsid  Enveloped

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 LECTURE UNIT XX: TITLE

▪ NOTE: exemption of poxviruses which is • Reovirus

complex
o Common cause of upper respiratory tract infections
Family Name of Viruses
DNA VIRUSES
• Poxvirus
• Baculovirus
o Zoonotic infection that is not clinically significant in
for now, but re-emerging
• Iridovirus
o Zoonotic infection that is not clinically significant for
now, but re-emerging
• Herpes virus (1 & 2)
o Part of the sexually-transmitted viruses
• Hepadnavirus
o Hepatitis B
• Adenovirus (Adenoviridae)
o Vector used for the vaccine Group I: Double-stranded DNA
• Papovavirus
• Classified into two:
o Combination of two viruses
o (1) Non-enveloped
▪ Papillloma + Polyoma
▪ Based on genome orientation
• Parvovirus (Parvoviridae) ▪ Circular
o Parvovirus B19  Papillomaviridae
▪ common cause of death in dogs  Polyomaviridae – formerly grouped together
as the Papovaviridae
• Arenavirus (Arenaviridae)
▪ Linear
• Paramyxovirus
 Adenoviridae - vector used for vaccines
o Respiratory syncytial viruses
o (2) Enveloped
• Orthomyxovirus
▪ Herpesviridae
o Influenza viruses ▪ Hepadnaviridae
• Bunyavirus (Bunyaviridae)  Not part of group I, but group VII
• Rhabdovirus  Contains reverse transcriptase, an enzyme
o Rabies that converts RNA to DNA
• Filovirus  A DNA type of virus

o Marburgvirus, Ebolavirus, Reston virus  Cause hepatitis; infect hepatocytes

o Complex Enveloped
• Coronavirus
▪ Poxviridae
o SARS CoV 1, SARS CoV 2, MERS-CoV
• Retrovirus Group II: Single-stranded DNA

o HIV • Non-enveloped
• Togavirus o Parvoviridae – Parvovirus B19; commonly infect
dogs
o Chikungunya virus
• Flavivirus RNA VIRUS

o Dengue 1 and 2
• Picornavirus
o Polio, Enteroviruses, Hepatitis C
• Caliciviruses
• Birnavirus
o Not clinically significant

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 LECTURE UNIT XX: TITLE

 Picornaviruses are the smallest viruses in all

families
▪ Caliciviridae

Group 5: Single-stranded Negative Sense

• Enveloped
o Helical
▪ Orthomyxoviridae
 Influenza
▪ Paramyxoviridae
 Respiratory Syncytial Virus (causative agent
for common respiratory infections to children)
• Birnavirus and Indovirus are clinically insignificant
▪ Rhabdoviridae
Group III: Double-stranded RNA  Rabies
• Non-enveloped ▪ Filoviridae

o Icosahedral  Ebola virus

 Marburg virus
▪ Reoviridae
 Reston virus – an Ebola variant from the
Group IV: Single-stranded Positive Sense Philippines
▪ Bunyaviridae
• The positive and negative sense pertains to the
▪ Arenaviridae
readability of the mRNA
o Positive sense is equivalent to mRNA to human; Summary of Baltimore’s Classification
therefore, they can be easily translated to the
ribosome to proteins
• Enveloped
o Icosahedral
▪ Flaviviridae
 Dengue virus I, II, III & IV
▪ Togaviridae
 Chikungunya virus
 Chikungunya is common in the Philippines
 Has the same symptoms with dengue and has
similar treatment but differ with their causative
agent
• Group I (Double stranded)
▪ Retroviridae
 HIV o DNA

 Removed from group IV because it contains ▪ Positive Sense

reverse transcriptase ▪ Negative Sense
 reclassiffied to Group VI • Group II (Single stranded)
o Helical o DNA
▪ Coronaviridae ▪ Positive Sense
 SARS-CoV, MERS-CoV • Group III (Double stranded)
• Non-enveloped o RNA
o Icosahedral ▪ Positive Sense
▪ Negative Sense
▪ Picornaviridae
 Poliovirus, Enterovirus • Group IV (Single stranded)
 RNA type of virus o RNA
 Has a pico (small) characteristic ▪ Positive Sense
• Group V (Single stranded)

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o RNA o Single-stranded RNA with reverse transcriptase

▪ Negative Sense ▪ It will make DNA
▪ Then, from DNA, mRNA will be created
• Group VI (Single stranded)
• GROUP VII
o RNA
o Double-stranded DNA with reverse transcriptase
▪ Positive Sense
▪ Used to be part of Group IV. However, was ▪ Same mechanism with group VI
excluded due to the presence of the reverse
o The purpose of producing a lot of mRNAs is to
transcriptase
produce a lot of protein
▪ Re-classified to Group VI
▪ More mRNA, more protein will be made
• Group VII (Double stranded)
▪ More proteins, more virions will be made
o DNA
Viral Replication Cycle (Infectious Cycle)
▪ Positive Sense
▪ Negative Sense • Hepatitis C Virus (HCV)
▪ Considered to be part of Group I. However, was o A small, enveloped, single-stranded, positive sense
excluded due to the presence of the reverse RNA virus
transcriptase o Under Baltimore’s classification group IV
Central Dogma
▪ Group I - DNA, positive, double stranded
▪ Group II - single stranded
• mRNA ▪ Group III - RNA, double stranded
o A specific requirement to be translated into protein ▪ Group IV - single stranded, positive sense
o Requires a specific enzyme from each genetic
(1) Attachment
molecule
• Group I (Double stranded DNA) o Recognize and binds to suitable host cells
o Glycoprotein spikes binds to host cell CHO
o Has DNA-dependent RNA polymerase that will receptors
produce mRNA o Phenomenon called Virus Tropism
• Group II (Single stranded DNA) ▪ Specific attachment of the viral glycoprotein
o Needs to be in a double stranded state first, before spikes to human protein receptors
DNA-dependent RNA polymerase produces mRNA ▪ Not all virus can actually infect cells
 Example: Hepatitis C - their cell of tropism is
• Group III (Double stranded RNA) hepatocytes
o Has RNA-dependent RNA polymerase because its o In the presence of glycoproteins receptors, viruses
baseline is RNA can attach to host cell
• Group IV (Single stranded RNA)
(2) Penetration
o Has to create a template of negative sense so that
the genome would not be consumed upon producing o Virus enters host cell
mRNA o Fusion of viral envelope
▪ If the genome is used as positive sense to ▪ Often leads to syncytia formation
produce mRNA, the genome will be consumed, ▪ During penetration, as it enters, the
then nothing will infiltrate the capsid glycoproteins of the virus sometimes remain in
the surface of the cell and then bind to the other
 There would be no genome in the
cells
nucleocapsid
 Syncytia Formation = making one large cell
o From RNA positive, it will make a template
o From RNA negative, it will go through RNA  Not all viruses are like this
polymerase and produce complementary  Example: Respiratory Syncytial Virus
▪ Complementary = positive o Phagocytosis
▪ RNA negative will become a template to produce
many RNA positive sense as mRNA ▪ Activation of immune cells
▪ Since the presence of glycoproteins in the cell
o The negative sense will then generate plenty which is protein (highest form of antigen), the
amounts of positive sense antibodies will opsonize.
• Group V (Single stranded RNA) ▪ In the presence of the antibody, exposing the Fc
portion, the neutrophil and other immune cells
o Utilizes RNA-dependent RNA polymerase enzymes that have a Fcc III receptor will recognize the Fc
to produce mRNA portion of the antibodies, activating phagocytosis
• GROUP VI (3) Uncoating

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o Virus loses its capsid and exposes its viral genome • Majority of the transmission are through person to
(DNA or RNA) person via:
o RNA viruses
o Fecal-oral
▪ Releases genome in the cytoplasm
▪ Hepatitis A and E
▪ Exception: Influenza virus; even though they
▪ Poliovirus
are RNA virus, genome will be release in the
nucleus o Sexual contact
o DNA viruses ▪ HIV
▪ Herpes
▪ Releases genome in the nucleus
▪ Exception: Poxvirus, genome will be released in o Trauma
the nucleus
▪ Bite (Rabies)
(4) Macromolecular Synthesis ▪ Vector base (Dengue)
o Injection of contaminated objects
o Production of nucleic acid and protein polymers
o Tissue transplants (BT)
o Synthesis of messenger RNA (mRNA) which
encodes early and late viral CHONs ▪ Blood transfusion (Hepatitis B and HIV)
o Early CHONs
o Arthropod or animal bites
▪ Non-structural (enzymes)
▪ Chikungunya
▪ NS2, NS4A, NS5A, NS4B, NS5B
▪ Dengue 1, 2, 3, and 4
o Late CHONs
o Transplacental
▪ Structural components
▪ Placental HIV
o Macromolecule synthesis of Hepatitis C, the
NOTE:
ribosome will produce a large macromolecule
protein then the enzymes produced earlier will try to
• Inside host
cut each part of the large macromolecule. Each part
will serve as the capsid, spike proteins, o Virus infects susceptible cells, frequently in the
nucleocapsid, and etc. upper RT

(5) Assembly • Local infections lead to viremia

o Then to other tissues (systemic)
o Structural CHONs, genome, and enzymes assemble
o All of the systemic viral infections have a MOT via
into virions
with respiratory tract infection, eventually
▪ Virions – already a complete virus disseminating to other systems
▪ Acquisition of envelope – final step
• Oncogenic virus
(6) Release o Virus that has the ability to simulate uncontrolled
growth of host cells (Papilloma virus)
o Also termed as “Egress”
o Intact virions are released during: ▪ Under Baltimore’s group classification I
▪ Cell lysis Antiviral Agents
 The virus destroys the cell as it exit • Approximately 40 antiviral drugs used
▪ Budding by cytoplasmic membrane
o Majority comes from HIV
 Some viruses just bud by cytoplasmic
membrane • Mostly used in HIV, and other viral infections
• Categorized according to their mode of action
 Other term: Lysogenic
NOTE: SUPPLEMENTARY VIDEO: THE CENTRAL DOGMA
• Each infected cell = 100, 000 virions • From information encoded in genomic DNA, proteins
• As few as 1% of these may be infectious or viable are made.
• Non-infectious virions result from errors in replication or o Chromosomes are located inside the nucleus
mutation o Genomic DNA binds to histones; it is folded many
times over
VIRAL PATHOGENESIS • When chromosomes are unfolded, DNA waiting
transcription can be seen
Mode of Transmission
o Information for protein synthesis is transcribed in
Person-person: this area
Preparing Transcription

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• Preparation for transcription from DNA to RNA begins o Slides along mRNA to find the initiation site for
when various transcription factors gather around DNA translation
• One of the transcription factors finds a transcription
• Transfer RNAs (tRNAs) arrive
initiation site on DNA and docks
• Each transcription factor has its role o There are many types of tRNAs that each carry
• The leading player in transcription, RNA polymerase II, different amino acids
conducts the most important process for transcribing o Each recognize a certain codon on mRNA and
genomic DNA to RNA leaves the amino acid specified by the codon
• The long, extended area shaped like a tail is called CTD ▪ In this manner, amino acids are linked in the
o RNA transcribed from DNA is processed here correct order
• Using aggregated transcription factors as a scaffold, • A protein is thus formed in accordance with the
RNA polymerase II docks to DNA synthesis information written in mRNA
• While being formed, the protein is folded sterically
o Transcription factor
• Once the end of translation is recognized, the ribosome
▪ It has the role of undoing the double-helical breaks down
structure of DNA o The resulting protein is carried to the appropriate
▪ The transcription factor helps RNA polymerase II site
complete its work
• Translation – occurs concurrently to produce multiple
• Preparations for transcription are complete
copies of the same protein from one mRNA
Transcription of Genetic Information o Sometimes, incorrect tRNAs get in, so they are
expelled as a result of codon mismatches
• DNA opens up
• Unnecessary transcription factors leave DNA • After a certain number of proteins are made, the mRNA
• RNA polymerase II – changes shape to begin ring is broken
transcription
o The mRNA is broken down when it has completed
o It begins to read DNA information its role
• Nucleotides are taken in • Central dogma – series of processes where genomic
o Nucleotides - materials for RNA synthesis information is used to make the necessary proteins for
biological activities
• Protein synthesis information is serially transcribed from
DNA to RNA SUPPLEMENTARY VIDEO: INTRODUCTION TO
• After transcription progresses to a certain degree, RNA VIRUSES
is subjected to various processes
• 5’cap – attached to the head • A separate kingdom of the living world
• Splicing – takes place to eliminate from the RNA the • Very small objects that can duplicate themselves only
information not used for protein synthesis by penetrating living cells and diverting the cell
▪ Intron – unused area macromolecular synthesis towards making more virus
▪ Exons – necessary areas • They can have RNA or DNA as their genetic material
and can grow in all kinds of cells; (e.g., animal, plant
• Each intron is bent, cut, removed, and immediately and even bacterial (bacteriophages)
broken down, and only the exons are linked
Central Dogma & Updated Dogma (1970)
o In this manner, only the information needed for
protein synthesis remains in RNA
Central Dogma
• RNA is then carried outside the nucleus
• Once the necessary information is transcribed, RNA
polymerase II leaves DNA
• At one end of the separated RNA, a poly-A tail is
formed
• Now, the completed RNA has the necessary information
for protein synthesis
• As a functioning messenger RNA (mRNA), it is
transported outside the nucleus
• Elucidated by Francis Crick
Translation of Genomic Information • (1) Replication
• The protein synthesis information encoded in mRNA o DNA can duplicate itself
consists of sets of three nucleotides or codon o Can go from cell to cell in an exact copy
o Codon – a set of three nucleotides • (2) Transcription
• mRNA forms a ring to be translated o DNA can be transcribed into RNA
• Ribosome – is responsible for translating mRNA
information and protein synthesis • (3) RNA

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o RNA encodes proteins o Duplicates a DNA strand to make a double strand

that can be transcribed into mRNA
• (4) Translation
• (5) Protein ▪ More complicated

Updated Dogma (1970) RNA viruses

• Type III
o Double-stranded RNA viruses
o Has RNA Polymerase
▪ Can copy double-stranded RNA into a single-
stranded mRNA
• Type IV
o Single-stranded RNA viruses
• (1) Replication
• (2) Transcription/Reverse transcription ▪ Only has the plus strand/sense strand
o Reverse transcription – is the ability to reverse the o Plus strand is copied into a minus strand which
ordinary flow of information so that RNA gives rise to becomes a template for mRNA
DNA
▪ Ex. Common cold virus
▪ This ability is particular to viruses
• Type V
• (3) RNA
o Single-stranded RNA viruses
• (4) Translation
• (5) Protein ▪ Only has the minus/negative strand
Classifying Viruses by How they relate to mRNA  Copied into mRNA directly but must be
duplicated so they must copy back the plus
strand into the minus strand
• Type VI
o Retrovirus-kind
o Single-stranded RNA viruses
▪ Only has the plus strand/sense strand
o Copies single-stranded RNA (plus strand) into a
single-stranded DNA (negative strand), then to a
double-stranded DNA
▪ Finally, double-stranded DNA is transcribed into
mRNA
SUPPLEMENTARY VIDEO: HEPA C LIFE CYCLE
• (1) HCV is usually transmitted through blood-to-blood
contact
• (2) HCV replicates in the hepatocytes of the liver and
• The mRNAs that encode proteins are the most circulates throughout the body
important endpoint of a molecular system that allows for o Entry into hepatocytes occur with the interaction of
controlling cell behavior the viral envelope with receptors on the surface of
• Viruses are classified on how they make their mRNA the host cell

DNA viruses ▪ Enters the hepatocyte through interaction with:

 CD81
• Class I
 SR-B1
o Double-stranded DNA viruses
o The standard kind of DNA found in the nucleus of  CDLN-1
organisms  OCLN-1
o To make mRNAs, it uses DNA-dependent RNA
polymerase that copies DNA into RNA • (3) The virus undergoes a fusion and uncoating step,
releasing positive-strand viral RNA
▪ Viruses either hijack the host cells’ DNA- • (4) HCV utilizes host cell’s proteins and molecules to
dependent RNA polymerase or they bring in their replicate
own • (5) Translation of viral RNA genome
• Class II into a polyprotein
• (6) Polyprotein is processed by both host cell and viral
o Single-stranded DNA viruses proteases into structural and non-structural protein

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• (9) The interaction of the host cells’ protein Cyclophilin

A with the viral proteins NS5A and NS5B
o This enables the functional replication complex
• (10) Replication Complex is now formed
o Generates new viral RNA
• (11) Negative strand RNA intermediate is formed
o Used as a template for the synthesis of positive-
strand viral RNA
• (12) Positive-strand viral RNA is packaged to create
new HCV virion
• (13) Virion matures in the Golgi apparatus
• (14) It is released form the host cell via exocytosis

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 [TRANS] LECTURE UNIT 08: SPECIMEN COLLECTION & LABORATORY DIAGNOSIS FOR VIRUSES

VIROLOGY: SPECIMEN COLLECTION  C – Synthetic phase

AND PROCESSING o new virus particles are assembled (macromolecular,
micro molecule or protein synthesis)
Considerations o starts 12 hours post-infection
Collection depends on specific disease syndromes, viral  D – Latent period
etiologies suspected, and time of year
o no extracellular virus can be detected
 (1) Based on disease  all viruses are inside the cell already
o Complicated since some viruses enter certain areas o max plateau
yet infects distant tissues/organs
 Difference with bacterial growth curve:
 Ex: enter respiratory tract → become systemic virus plateau &haveno decline phase
 (2) Etiologic virus
Viral Shedding
o Complicated since many similar clinical syndromes
may be caused by different viruses  Usually greatest in the early stage of infection
 Sensitivity of viral culture may decrease rapidly 3 days
 Ex: respiratory disease viruses – produce the after onset of symptoms
same clinical symptoms but are caused by
different viruses/ causative agents o In the lab, upon receiving sample, ask the infectious
disease collector/physician/specialist for the onset of
 Influenza, syncytial virus – different viruses
fever
but produces the same clinical manifestations
 If they collected sample 4 days since onset of
 (3) Seasonal appearance of virus
fever → sensitivity of culture is less
o Influenza  If negative result → inform physician that this
could probably due to time of collection
 more increased during winter
 decreased during summer (less likely a o The longer the time passed before collection, the
causative agent) more the sensitivity of the cell culture decreases
o Dengue Sample Collection
 considered seasonal before but not anymore
since cases are found in the Philippines  Aseptic collection – IS A MUST!
throughout the year o Golden rule
Specimen must be collected as early as possible following  Aspirated specimens – most preferable sample
onset of symptomatic disease  Swabs (Dacron or Rayon) – can also be used
 Calcium Alginate Swabs(not recommended)
 Ex: fever – should go to hospital and have sample
collected o Inhibits replication of some viruses and inhibits
nucleic acid amplification tests.
o Difficult in the PH since Filipinos don’t go to the o A common swab used in bacteriology which may be
hospital for consultation unless they’re severe mistakenly used.
o May cause a false-negative result for viral infection.
 Virus titers (concentration of virus) – usually highest
in the early part of the illness  Tissue samples – must be kept moist by using:
Viral One Step Growth Curve o Viral Transport Medium (VTM) – commonly used in
hospitals and virology laboratories.
 A – Adsorption o Saline
B o Trypticase Soy Broth
B o initial phase
D
o attachment
Areas of Collection for Common Specimens
 B – Eclipse phase
o uncoating step – releases NOTE:The area of sample collection depends on the clinical
genome manifestation of the patient.
o lasts for 10-12 hours o Highest concentration of virus is where the
A C symptoms manifest.

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Samples that can be collected without the use of VTM:

 Throat,
Nasopharyngeal swabs  Samples in liquid form:
or aspirate
 Bronchial and o Blood
Bronchoalveolar o Bone Marrow
washings o CSF
 Rectal swabs and stool specimens o Amniotic Fluid
o Urine
 Urine
o Pericardial fluid
 Skin and Mucous membrane lesions
o Pleural fluid
 Blood
o Plasma for Viral Load
o Serum for Antibody testing Viral Transport Media (VTM)

 Sterile body fluids other than blood  Purpose: make sample moist
o CSF, synovial fluid, etc.  Moisture will protect the virus
 Dry environment will make the virus dry up and easily
 Bone Marrow destroyed
o Specifically in patients suspected with Parvo B19 Examples of Virus Transport Media
o Parvovirus B19 – usually goes to the bone marrow
and attacks the RBC progeny cells.  Amies Medium
 Tissue  Stuart’s
o Recommended
o Stuart’s transport media used in the bacteriology
Virus Transport Media (VTM)
laboratory can be used to collect virus
Components:  Leibovits – Emory Medium
 Hanks Balanced Salt Solution (HBSS)
 Buffered Isotonic Solution  Eagles Tissue Culture Media
 Albumin, gelatin, or serum
 Commercial Kit – follow manufacturer’s specification
proteins
o To protect less stable Virus Transport Processing
viruses
Types of Blood Specimen for Viral Diagnosis
o Less stable virusesare
enveloped viruses  Whole Blood
 Envelope - composed of lipids that are easily o Heparinized – ideal for virus isolation
destroyed by substances such as alcohol. o Virus isolation: culture media + saline
 Once envelope is removed, the virus
 Saline will not grow without culture media
becomes non-viable. Thus, the whole virion
must be protected.  Plasma
 Envelope also contains glycoproteins that binds o EDTA – ideal for viral load/ molecular tests (e.g.
to the specific cell (tropism). PCR)
o Can also be used for serological tests
 Added with antibacterial and antifungal agents  Serum
o Penicillin – common antibacterial agent o Collected in plain tube, normal clotting
o Streptomycin – common antifungal agent o Collected with clot activator – hastens clotting
o Collected with clot activator and serum separator
Samples that CAN be collected using VTM: o Allow to clot before centrifuge
o Common sample used in serology
 Samples in solid form: Viral Culture Samples
o Respiratory Swabs
o Tissue Samples  Must be processed immediately
 Placed in ice during transport
NOTE: VTM keeps the sample moist in order to protect the  Processed within 12 – 24 hours upon receiving
virus. A dry sample will make the virus dry up and easily
destroyed. o Some viruses such as respiratory syncytial virus
(RSV) may be difficult to recover even a few hours
of delay
o RSV is common in children
 If delay is expected – samples should be stored at 4°C

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 Frozen at -70°C not at -20°C – if delay will take up to

more than 4 days
o Sample can be thawed once only
o Thawing twice can destroy the virus
 Specimen for viral culture – process immediately
 Must be accompanied with a request form
o Patient I.D. and demographics (age and sex)  Single formation where two form or two cells are
o Source of specimen combined
 Must be noted since clinical samples other than
blood samples have the same appearance  Right Photo: CMV Infected Monocytes
 Clinical history or virus suspected o Papilloma Viruses are DNA viruses so inclusions
o Identification of suspected virus is important should be observed on the nucleus
o Must know the identifying characteristics of the  Nucleus are highly dense, hence, the part where
suspected virus the inclusions can be observed.
 Date and time of collection  Inclusion: Dense Color Blue or Purple color

o If unavailable – call the physician/person carrying o Inclusion usually appears where the virus is
the samples released
o Virus is very sensitive and are easily destroyed
o If the sample is too old, it will be destroyed
 Occurrence of a false-negative result
 Should occur in Biosafety cabinet
o Depends on the level of the agent
o Viruses are usually on Biosafety Level 2
o BSL 3 (e.g., influenza, HIV, COVID)
 Could be processed using a protective plexiglass on
tabletops
 Pipetting should be performed behind a shield
 Discard all materials used in an appropriate disinfectant  Left Photo: Papilloma Virus Inclusion stained with H
 Wear PPE at all times and E

LABORATORY DIAGNOSIS FOR VIRUSES  Since Papilloma Viruses are DNA viruses,
hence, are found on the nucleus
Virology: Laboratory Methods
 Right Photo: Cervical PAP smear showing the Human
 Direct detection of the virus in clinical specimens papilloma virus infected squamous cell
 Nucleic Acid-based detection
o Characteristic:
 Isolation of viruses in cell cultures
 Serologic assays  Folding of squamous cells

Microscopy Microscopy

 Cytology and Histology  Cytology

o Use bright-field microscopy for presence of o Frequently used to detect VZV and HSV
characteristic viral inclusions (p. 749)  VZV - varicella-zoster virus
 Inclusions formed by CMV, adenovirus,  Varicella – causative agent of chicken pox in
papilloma virus stained with Hand. E children
 Zoster – causative agent of shingles in adults,
Cytopathic Effects (CPE) reoccurring chicken pox
 HSV (herpes simplex virus) Type 1/ HSV-1 -
 CPE are Indicated by the changes in whole cell the lesion is observed on the upper extremity
morphology which are caused by target infecting virus  HSV (herpes simplex virus) Type 2/ HSV-2 -
 The common visual observations of the host cells are the lesion is observed on the lower extremity
swelling or sinkage, rounding, lysis, plaque, clumping, (example: genital herpes)
syncytia formation, and inclusion  Tzanck Smear
 Left Photo: CMV Inclusion
o Smear of cells from the base of skin vesicle
o Using the H and E stain o Simple and cheap test that relies on viewing and
o Characteristic is described as an owl-eye inclusion interpretation of a single cell (cytology)

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o ArnaultTzanck described the technique in 1947 to  In medical virology, the electron microscope is used in
distinguish various blistering conditions viruses that do not readily grow on cell cultures.
 Used to detect viruses that do not grow readily in cell
 Usually caused by VZV and HSV (specifically
culture
type 1)
 Negative staining – specimen is placed in a grid
o Stained with potassium phosphotungstate/uranyl
acetate

 Inclusion for rabies virus

o Detected by examining tissues
o Rabies – Negri bodies (pointed by the arrow)
 Negri bodies are the infectious virion of rabies
virus

Viruses that can be viewed using EM

 Noroviruses
 Norwalk virus
 Coronaviruses
o Can be cultivated in culture media because there
are now good culture media for the isolation of
Direct Fluorescence Assays (DFA) and Indirect
coronaviruses
Fluorescence Assay (IFA)
o The structure of the coronavirus cannot be observed
 Can also be a flexible tool to detect viral agents in the in cell cultures. Only the cytopathic effect can be
clinical specimen observed.
 Presence of antigen means there is the presence of a  Astroviruses
virus. Thus, it will result in a positive test for DFA  HSV, measles, and JC polyomavirus

Electron Microscopy Problems with Electron Microscopy

 Microscope that uses a beam of accelerated electrons  Expensive equipment

as a source of illumination.  Expensive maintenance
o used to investigate the ultrastructure of a wide  Require experienced observer
range of biological and inorganic specimens  Sensitivity often low
including microorganisms, cells, large molecules, o Microscope should be
biopsy samples, metals, and crystals. paired with other

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 LECTURE UNIT 08: SPECIMEN COLLECTION & LABORATORY DIAGNOSIS FOR VIRUSES

molecular tests to really confirm the presence of  Inclusion bodies are not 100% directed to
virus. specific organism or virus. Therefore, we can
o Ex. PCR Test use molecular test for confirmation. We can
identify and report the virus if it is amplified.
 (3) May be a problem with mixed cultures – would have
to assay for all organisms causing the infection.

Advantages of Using Molecular Test o If there is mixed cultures, there is a need to

separately identify each viruses first and to confirm it
 (1) High sensitivity individually using molecular test with specific primer.
o Can theoretically detect the presence of a single Virology: Laboratory Methods
organism Molecular Techniques: DNA Review
 PCR or molecular test can amplify the certain
 In double stranded linear DNA, 1 end of each strand
organism as long as presented with correct
has a free ‘5 carbon and phosphate and 1 end has a 3’
primers.
OH group
 (2) High specificity o The two strands are in the opposite orientation with
o Can detect specific genotypes respect to each other (antiparallel).
o Can determine drug resistance
 Adenines always base pair with thymines (2 hydrogen
o Can predict virulence
bonds) and guanines always base pair with cytosines
 (3) Speed (3 hydrogen bonds)
o Quicker than traditional culturing for certain
organisms Target Amplification
o Most important advantage for molecular test
 Target amplification requires that the DNA to be tested
 RNA extraction may take 30 min to 1 hour for be amplified
depending on the number of samples
 PCR test may take up to 1 hour o Basic Requirement:DNA
 The minimum number of days in culture that the o The number of copies of the DNA is increased
sample can be observed is 7 days. The highest  E.g. One organism isolated contains at least 1
number to observe the culture to confirm if it is DNA, which can be amplified or increased
positive is 1 month.
 Review the activity of the enzyme, DNA polymerase,
 It is better for medical technologists to perform
that is used to amplify the DNA
diagnostic tool that is fast that has high
 DNA polymerase cannot initiate synthesis on its own.
sensitivity and specificity. Molecular tests
such as PCR can provide that. o It needs a primer to prime or start the reaction
o The primer is a single stranded piece of DNA that is
Disadvantages of Using Molecular Test complementary to a unique region of the sequence
to be amplified
 (1) Expensive
o RNA extraction or DNA extraction costs around o DNA polymerase
50,000-80,000. PCR reagent costs 80,000-100,000 sticks to the primer,
creating the
 (2) It will miss new organisms unless sequencing is complementary
done as it will be done in the lab for our molecular strand.
unknowns (not practical in a clinical setting) o Adenine paired with
o The advantage of having high specificity pulls a Thymine; Guanine
great disadvantage because it will not detect new paired with Cytosine
organisms unless sequencing is done which is not  AATTGCG  TTAACGC (double stranded)
practical in the laboratory. In the laboratory we will
receive unknown sample, therefore, we do not have
any idea of the possible organisms present in the  In molecular test, machines are used instead of human.
sample. Therefore, machines have different thermal requirement
o In bacteriology, once we receive the sample we can compared to humans.
actually plate that one to primary culture media. o DNA polymerase requires 37°C optimally
(MacConkey Agar and Chocolate Agar) o PCR machines/ Molecular Machines/ Diagnostic
 Growth in BAP Agar and Chocolate Agar; no tools require high temperature.
growth in MacConkey Agar = gram positive  PCR - 95°C
 MacConkey has an inhibitor which is Crystal  DNA polymerase in human is not applicable
Violet that inhibits gram positive organisms,
 Taq polymerase (“Taq pol”) is a thermostable
only gram negative organisms can grow.
polymerase isolated from Thermus aquaticus, a
 In virology, cell culture is used first

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 LECTURE UNIT 08: SPECIMEN COLLECTION & LABORATORY DIAGNOSIS FOR VIRUSES

bacterium that lives in hot springs and hydrothermal o To do this, DNA polymerase extends the primers
vents. that annealed in the annealing step of the reaction.
 “Taq polymerase” is an abbreviation of Thermus o The temperature used is 72°C.
Aquaticus Polymerase.
 This is the optimum reaction temperature for
 It is often used in polymerase chain reaction, since it is
the thermostable polymerase that is used in
reasonably cheap and it can survive PCR conditions.
PCR.
 dNTPs include:
Molecular Techniques: Polymerase Chain Reaction (PCR)
o Cytosine
 Polymerase Chain Reaction – used to amplify o Thymine
something found in such small amounts that without o Adenine
PCR it would be undetectable o Guanine
o Addresses the low sensitivity of the previous  The extension is where the Taq polymerase will start its
methods mentioned to identify viruses. activity by providing the complementary dNTPs of the
o Uses two primers, one that binds to one strand of a sequence.
double-stranded DNA molecule, and the other which
binds to the other strand of the DNA molecule  PROCESS:
o A thermostable DNA polymerase Taq
Polymerase o (1) The primer is already attached (A,A,T,C,G).
o (2) There is free-floating A, T, G, and C which are
PCR: Three (3) Basic Steps included as part of the reagent.
o (3) If there will be DNA polymerase that will attach to
1) Denaturation the primers (A,A,T,C,G), the Taq polymerase will
check what base-pair is needed.
 Denature – when DNA is denatured, it is separated into
single strands  AT
 AT
o DNA needs to be denatured to have primers.  TA
Primers should bind to a specific part of DNA  CG
sequence  GC
 Primers cannot attach to a specific DNA o (4) dsDNA will now be produced.
sequence if its double stranded. Thus, o (5) The process will start over again with
denaturation is needed for it to become single denaturation phase.
stranded DNA.
 The combination of denaturation, annealing, and
o In the PCR reaction, this is accompanied by heating extension constitue 1 cycle in a PCR reaction.
at 95°C for 15 seconds to 1 minute
o In 1 cyle, the three phases must be complete.
 Thermostable DNA polymerase (Taq
polymerase) should be used.  Most PCR reaction use 25 to 30 of these cycles to
amplify the target DNA up to a million times the starting
o The single stranded DNA generated will serve as
concentration.
template for DNA synthesis
o From 1 DNA, 25 to 30 cycles of PCR can produce
million times of DNA.
2) Annealing

 Anneal – to anneal is to come together through PCR Types

complementary base-pairing (hybridization)
o During this stage in the PCR reaction, the primers Conventional PCR
base-pair with their complementary sequences on
the SS template DNA generated in the denaturation  Uses Agarose gel for detection of PCR amplification at
step of the reaction. the final phase or end-point of the PCR reaction.
 The results are seen by separating the PCR products
 Annealing: If the primer attach to the specific sequence by agarose/ethidium bromide electrophoresis.
part of the DNA (red box).
o If the sequence of ssDNA is already open, the
primer can attach to the specific sequence.

 PROCESS:
3) Extension
o (1) The sample is placed in a thermal cycler.

 Extension – during this stage of the PCR reaction, the  The thermal cycler measures the temperature of
DNA polymerase will use dNTPs to synthesize DNA the sample during the three phases
complementary to the template DNA. (Denaturation, Annealing, and Extension).

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 LECTURE UNIT 08: SPECIMEN COLLECTION & LABORATORY DIAGNOSIS FOR VIRUSES

o (2) The sample is then placed in an agarose gel.  Duck eggs, that are usually around 9-12 days, are
o (3) The sample is then placed in an electrophoresis. prepared
 Brown: betadine to make it sterile
 The control sample has a band = Positive
 If sample 1 do not have a band just like the
control = Negative
 If sample 2 has a band just like the control =
Positive
 REPORTING:Qualitative
o Positive (+)  Inoculate the specimen by inserting the needle into the
o Negative (-) hole in the shell, and using a short stabbing motion,
pierce the chorioalantoic membrane and inoculate
Real-Time PCR 200μL, seal and incubate the eggs
o To identify the hollow part of the duck egg, put it
 Real-time detection of amplification by use of probes,
under the light. Then, use a scalpel (or anything
detection of fluorescence emission.
sterile) to make a hole
 Eliminates Post-PCR processing
o After finding the chorioalantoic membrane, the virus
o Do not utilize agarose gel. can now be introduced, and allow the virus to infect
the embryonated egg
 PROCESS:
o (1) The sample is placed in the PCR machine which
is connected to a laptop.
o (2) The laptop will produce a graph whether there is
amplification or not.
 Allows quantification

Air sac
Virus Isolation

Gold Standard  Harvesting virus form inoculated eggs

o With sterile forceps, gently break the shell over the
 Animal inoculation test
air sac. Push aside the allantoic membrane with the
o Costly forceps. Using a 10mL pipette, aspirate the allantoic
o Used ONLY in reference lab fluid, avoiding the veins and place it in the labelled
centrifuge tube
 Embryonated eggs o Store the viral isolate at 4°C. Perform
o Rarely used hemagglutination test
o E.g., Vaccine for Influenza
Hemagglutination Assay (Indirect)
 Egg nucleation test is the best method to
produce a high number copies of influenza.
 Cell culture
o Either you get it from primary cell culture line or
continuous cell line

Embryonated Egg Inoculation

 It is used as seed viruses for the production of the  Post test used for egg embryonated inoculation test
majority of influenza vaccines or identification  Used to identify if the virus amplified
o In virology, seed is term used, whereas in  Visible clumping of RBC
bacteriology, the term used is inoculation
o The virus was able to amplify
 Seed = Inoculation
 Influenza A and B
o Hemadsorb and hemagglutinate guinea pig RBC
betadine
 Parainfluenza and mumps
o Hemadsorption only

Cell Culture

 The most preferred method

o Easy to perform and easy to maintain

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 LECTURE UNIT 08: SPECIMEN COLLECTION & LABORATORY DIAGNOSIS FOR VIRUSES

 Conventional cell culture  Continuous cell line

o Routinely incubated in a
roller drum tilted at 5- HOW TO OBTAIN PRIMARY CULTURE
7°, revolve at ½ to 1
RPM at 37°C  (1) Acquisition of sample
o Test tube for cell
culture must be o For ex. Rat, rabbit, horse, etc.
regularly rotated  (2) Isolation of tissue
 Can also be incubated in a stationary rack
o Metabolism of growing cells – produced CO2 and
acidifies the medium
 Minimum requirement of CO2 is 5% for cells to
survive
o Bicarbonate buffering system – keep cells at
physiologic pH
o Phenol red indicator –
monitor adverse pH o Rat: remove skin and kidneys → transfer to petri
changes plate
 Acidic: yellow  (3) Dissection and or disaggregation (Mechanical
disaggregation)
 It can be toxic to cells and possibly cause the
death of the cells o Collecting cells that spill out when tissue is sliced =
 E.g., Presence of contaminant (bacteria or Scrapping/spillage
fungi). The cell culture media contains o Pressing the dissected tissue through series of
glucose, so once glucose is consumed, it will sieves – Sieving
release by-products, which can change the o Forcing the tissue fragments through syringe
pH condition of the cell culture media, making o Pipetting repeatedly – less destruction of cell
it acidic  (4) Culture after seeding into culture vessel
 Alkaline: red o Culture vessel = petri plate
 Indicates a good environment o Then incubate at 5°C
o Influenza (optimum temperature) = 33°C while for
Cell Culture with Specimen other viruses = 37°C
 Incubated 1-4 weeks (depending on the virus Checking the Culture Flask Contamination Confluency
suspected)
o Incubation requirement is at least 1 week - 1 month o Most common contamination is bacteria, fungi
depending on the virus o Confluency – union term it means that the cell is
confluent
 For examplein lab, influenza = maximum of 7
days; majority can produce CPE at 3rd day but if Suspension
at 7th day + 1 = reseed again to another culture
media to confirm positive or negative result  cells are suspended in liquid as single cells or as free-
floating clumps of a few cells
o Coronavirus cell culture: at least 7 days but majority
produce CPE or cell destruction in cell line is at 1-3 o To passage cell cultures, a proportion of the cells in
days culture are diluted into a larger volume of medium
o Passage in virology: while in bacteriology =
 Periodically inspected microscopically for the presence subculture
of virus indicated by areas of dead or dying cells known
called as Cytopathic Effect (CPE)

Confluency
Cell Culture

 The maintenance of cells outside of the living animal for  How “covered” the
easier experimental manipulation and regulation of growing surface
controls. appears
o Problem:
evaluation is
Categories of Cell Culture subjective
o Must wait for it
 Primary cell line to be confluent
 Low passage cell line before

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 LECTURE UNIT 08: SPECIMEN COLLECTION & LABORATORY DIAGNOSIS FOR VIRUSES

performing passage o Incubated at 35o – 37o

o Respiratory viruses – optimal at 33o

 Optimal confluency for moving cells to a new dish is o Incubated for two weeks (14 days)
70-80%
 Recovery is variable depending on the virus type
o Cannot perform passage if confluency did not reach
70-80% Cultures

 Examined for CPE using phase contrast

Passage number microscopy
o Need Control cell
 The number of times the cells
 For reference of normal cell appearance
have been removed (or “split”)
from the plate and re-plated. o Examples:
 Always write this on your plate or
 Cells rounding, clumping, vacoulation,
flask as P#
granulation
 Giant multinucleated cells, cell fusion or syncytial
formation
 There is a need to consider cell culture lines that are
 Cell destruction or lysis
required for passage because there are cultures that:

 Passes only once or twice since harvesting  Typical cytopathic effect (CPE) caused by HSV-1
o If will undergo passage for more than twice, it will
change the morphology of the cells
o Cells coming from animals
o Examples:
 Human embryonic kidney cells (HEK), rabbit
kidney (RK)
 Primary monkey kidney (PMK),
 Cynomolgus Monkey Kidney (CMK)
 African green monkey kidney (AGMK)
 Diploid cell lines/Low passage cell lines – must have
at least 75% of cells with same karyotype as the normal  Syncytia – Multinuclear Cell, Response to the
cells which they are derived. (20 – 50 passages). expression of measles virus genes

o More than 50 passage will change the karyotype of

the cell
o Cells coming from origin of human cells
o Examples:
 Human Neonatal Lung (HNL)
 Human Diploid Fibroblasts (HDFs)
o Derived from human kidney or lung
fibroblasts.
 Heteroploid or immortal cell lines (Continuous cell
lines)
o More than 25% of cells have abnormal karyotype  Viral Cytopathic effect in Cell Culture – Measles
compared to normal cells Virus
o Frequently derived from malignant tissues or
other transformed cells
o Undergo continuous/unlimited subcultures/passage
o Examples:
 HeLa (derived from human cervical carcinoma)
 Hep-2 (derived from carcinoma of the human
larynx)
 KB (derived from nasopharyngeal carcinoma)
 A – 549 (derived from human lung carcinoma) –
usually used
 Vero (derived from AGMK)
Table No.1 Quantification of cell culture CPE
 Inoculated cell cultures:

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 LECTURE UNIT 08: SPECIMEN COLLECTION & LABORATORY DIAGNOSIS FOR VIRUSES

o Viral gene products or antigens are detected not

Quantification Interpretation CPE
 SVCE
Negative Unifected monolayer
Atypical alteration of monolayer o Originally developed to detect CMV
Equivocal (+/_)
involving few cells  CMV is the causative agent of kissing disease
1+ 1%-25% of monolayer exhibits CPE o Commercially available for HSV, VZV, Adenovirus
o Influenza A and B
2+ 25%-50% of monolayer exhibits CPE  not commonly used
 but used for speeding up the isolation of
3+ 50%-75% of monolayer exhibits CPE influenza viruses
76%-100% of monolayer exhibits
4+
CPE

 The quantification of your cell culture CPE actually

directly proportional to the number of your virus VIRAL SEROLOGY
o The higher the quantification reporting of your CPE,  Detect immune status and make diagnosis of infections
the higher the number of virus present in the cell
in situations in which the virus cannot be cultivated in
culture
cell culture
o The problem with virology is that either the virus is
difficult to culture and or the virus is culturable, it
Shell Vial Centrifugation – Enhanced (SVCE) Virus requires time
Detection o When it comes to the laboratory, it would be difficult
for the physician to manage the patient if the
 Centrifugation of the specimen into viruses sensitive cell causative agent is not known
grown on cover slips at the bottom of shell vials o Therefore, viral serology is used
 We use antibody to identify if the patient
produces antibodies against specific causative
agent
 Can be used to diagnose primary or reinfection
 Specimen: Serum or Plasma
 Criteria for Diagnosing Primary Infection
o Presence of IgM antibody
o Seroconversion
 There will be less IgM and increased IgG
concentration

o (1) Using a pipette, get the cell culture and transfer

to the vial and centrifuge
o (2) When it spins, a part of the cell culture will attach
to the coverslip
o (3) Use stains and observe on the microscope
Typical Serological Profile After Acute Infection
 Produce more rapid result (incubation decreased from
1-5 days depending on the virus)
 Low speed centrifugation – enhances viral infectivity
 Somehow similar to cytospin principle in histopathology

After centrifugation

 Cells are fixed

 Fluorescein labelled
monoclonal/polyclonal
antibody is added
o Monoclonal – only
one antibody  Criteria for diagnosing Re-infection:
o Polyclonal – many
antibodies o Fourfold or more increase in titre of IgG or total
antibody between acute and convalescent sera
 Detection of antigen – antibody binding o Absence or slight decrease in IgM

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 LECTURE UNIT 08: SPECIMEN COLLECTION & LABORATORY DIAGNOSIS FOR VIRUSES

 Reinfection o Minimum of 3mL whole blood in plastic violet top or

EDTA tube
o The patient actually had an infection before
o Minimum of 3mL whole blood in plastic red top or
therefore the body is able to respond faster by
plain tube
producing IgG
o Minimum of 3mL whole blood in plastic yellow top or
 Class switching is the term used for the shift of IgM to serum separator tube
IgG  Requires prior centrifugation to separate the
Examples of Viral Serology Tests serum from the clot
 It is important to secure the tube cover with
 Complement fixation (CF) test parafilm during centrifugation to prevent
 Enzyme Linked Immunosorbent Assay (ELISA) accidental spillage
 Indirect immunofluorescence  Do not uncover the tube once the centrifugation
is done
 Western blotting
 Make sure that all specimens are labeled:
S
SUPPLEMENTAL VIDEO  Age and sex
 Date and Time of Collection
COLLECTION, PACKAGING, AND TRANSPORT OF
SPECIMESN FOR EBOLA VIRUS DISEASE TESTING  Failure to do so can invalidate the sample
testing
 Laboratory confirmation is needed:
 Do not attempt to open the collection tube or
o to classify a patient as to confirm the case of Ebola aliquot specimens prior to sending to RITM
Virus Disease and
o to initiate appropriate clinical management and
epidemiologic investigation Packaging and Shipments of Specimens

General Guidelines  Sending laboratory is responsible in packaging the

specimens and ensuring that the specimens reaches
 Before the collection of specimens contact the RITM the RITM in good condition
Surveillance Unit (02) 994-1887  Specimen for shipment should package following the
 Obtain only the specimens essential for diagnosis basic triple packaging system:
o Specimen should be obtained by trained staff with Procedure
experience in specimen collection
 Do not use glass specimen collection devices or Grab the sample
containers container with
STEP 1. absorbent
Timing of Specimen material

 Onset of symptoms
o The Ebola Virus can be detected in blood after on
set of symptoms most notably fever requires Place the
o Specimens ideally should be taken when specimen in a
symptomatic patient reports to a healthcare facility STEP 2. resealable plastic
and is suspected of having Ebola Virus Disease bag
exposure
o It takes up to 3 days after onset of symptoms for
reach the detectable levels
o If the sample is taken less than 3 days after onset of When shipping
multiple
symptoms, a follow-up specimen is required to
specimens, grab
completely rule out Ebola Virus Disease each specimen
with absorbent
Appropriate PPE for Specimen Collection STEP 3. material
Each specimen
 Double layer gloves must be place in
 N95 Respirator a separate
 Full face shield or Goggles resealable plastic
 Impermeable long-sleeved laboratory lab gown or bag
cover-all
Place the
Appropriate Specimens for Ebola Virus Testing specimen in a
STEP 4. water tight leak
 For patients under investigation for suspected Ebola proof container
Virus Disease collect the following specimens: (secondary
container)

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 LECTURE UNIT 08: SPECIMEN COLLECTION & LABORATORY DIAGNOSIS FOR VIRUSES

After filling out the

Be sure to forms, place them
remove the outer in the sealed
STEP 5.
gloves at this STEP 12. plastic bag and
point tape it in the
shipment
container

Disinfect the
secondary Address the
STEP 6. container before shipment to
placing it in the
shipment box Dr. Celia C.
Carlos Chief
Laboratory
Prepare at least 6 Research Division
frozen ice packs STEP 13. Research Institute
to maintain the for Tropical
STEP 7. prescribe Medicine
temperature of 5 Filinvest
to 8 degrees Corporate City,
Celsius Alabang,
Muntinlupa City

Place the ice

packs in the
STEP 8. bottom and side
of the shipment
box  RITM currently performs:
o Detection method recommended by the World
Health Organization
Place the o Antigen detection ELISA, IgM ELISA, and PCR for
secondary the detection of the acute phase of the disease
container of the o IgG ELISA for the detection of the antibodies during
sample tubes
inside the
the convalescent phase of the disease
STEP 9. shipment box  TURNAROUND TIME: 2 days
(secondary
container is o for all tests from receiving samples from RITM to
surrounded by the release of results
ice packs)
 CUT-OFF TIME: 10:00 AM
o For any questions on collection, transport, and
shipments of specimens, contact the RITM
Cover and seal Surveillance Unit at (02) 994 – 1887 or through
STEP 10. the shipment box email: ritmsu@[Link]

Prepare the Ebola

Virus Disease
Case
Investigation
Form and the
RITM official
STEP 11. laboratory request
form for special
diagnostic test
The laboratory
request form can
be downloaded at
the RITM website

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 [TRANS] LECTURE UNIT 09: DNA VIRUSES (GROUP I AND II)

BALTIMORE CLASSIFICATION GROUP I uncontrolled, causing Epidermodysplasia

verruciformis or “tree man”.
• Divided into two:
o (3) Sexually transmitted venereal warts (HPV-6 &
o Circular HPV-11)
▪ Describes the orientation of ▪ Female genital warts
the genome
 Somewhat dry; not painful since no active
o Linear inflammation occurs

Baltimore Classification Group I – Circular ▪ Male genital warts

 Warts usually infect the epithelial cells,
Papovaviruses therefore only the lining will be infected and
not the head.
• Group of DNA viruses that shares a common
▪ Anal warts
characteristic
• Double stranded DNA  Very common when people perform anal sex
• Icosahedral  Distinguishable from hemorrhoids
• Naked
• 45-55 nm – somewhat small
• Members:
o Papillomavirus (PA) – common warts
o Polyomavirus (PO)
o Simian vacuolating virus (VA)
▪ Thus, the name PAPOVA
Human papillomavirus (HPV)

NOTE: As an infectious disease specialist, you need to have a

• Under the papillomavirus of papovaviruses
good skill in ruling out other diseases.
• More than 60 genetic types exist
• Associated with a variety of cutaneous lesions and • For example, differentiating anal warts from
benign growths including: hemorrhoids, and male genital warts from syphilis.
o (1) Plantar warts (HPV-1) o Syphilis tends to infect the head part and other
o (2) Common warts (HPV-2 bacteria infect the shaft or the body.
& HPV-4)
Transmission
▪ The virus causing such
warts does not cause • Direct contact, sexual contact
harmful effects in terms
of fever, etc. o Genital infections: associated with neoplastic lesions
▪ HPV contains integrase including cervical carcinoma (oncogenic) (HPV-16 &
HPV-18)
 Integrase – enzyme
capable of integrating Laboratory Diagnosis
the viral DNA
genome to the host • Histopathologic/cytologic examination of cutaneous
genome biopsy or cells
▪ e.g., a human DNA and virus circular DNA • DNA probe assays – gold standard
 Virus circular DNA will
release integrase during Treatment
replication, which will
then try to integrate the • Surgical or chemical removal
viral DNA genome to
the host genome Prevention
 If left untreated, there
• Avoid contact with infected tissues
would be a possibility
that HPVs will be

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 LECTURE UNIT 09: DNA VIRUSES (GROUP I AND II)

NOTE: If sexually active, check the partner for any visible Human adenovirus
lesions or warts.
Other Strains • Divided in to 6 groups (A-F)
• Replicated and produce disease in the epithelial cells of
• Polyoma strains JC and BK respiratory tract, gastrointestinal tract, urinary tract,
liver, and in the eye.
o produce mild asymptomatic infection. • Usually does not spread beyond the regional lymph
o Mode of transmission: Spread through respiratory node.
secretions. • Restricted to adenoids.
• Group C
Baltimore Classification Group 1 – Linear
o Persist as latent infections for years in adenoids and
Adenoviridae tonsils
o Shed in feces for many months after initial
• Name derives from infections.
their initial isolation
from human • Adenoid is located in the upper pallet of the tongue.
adenoids in 1953
• Belongs to the Disease Associated of Human Adenoviruses
genus Respiratory diseases
Mastadenoviridae
• Naked, icosahedral,
dsDNA, with the size • Cough, nasal congestion, fever, and
of 70-90nm sore throat
• Has 49 serotypes, o commonly manifested in infants
which implies this and children and usually involves
can cause infection Group C
49 times
• Adenovirus Type 3, 7, 21
o Humans have immunity against some strains of the
virus; however, the body cannot produce a good o linked for more than 10-20%
long-term immunity to some viruses with the same pneumonias in childhood
strains, thus the occurrence of reinfections. • Adenovirus Type 4 and 7, occasionally type 3
• Serotypes 1-8, 11, 21, 35, 47, and 40 o cause of acute respiratory distress syndrome
o Commonly isolated in infections (ARDS) among military recruits

• Sore eyes, or conjunctivitis is an infection caused by

adenoviruses. Table 1. X-ray fields of the lungs

o Sore eyes – general term

Characteristic Description Picture
o Conjunctivitis – medical terms
Virus capsomere
An x-ray field of a normal
• Penton base Normal lung lung presents no shadows

o Carries a toxin-like activity

that cause rapid
appearance of cytopathic
effect (CPE) and Has the causative agent
Lobar Streptococcus pneumoniae,
detachment of cells from
pneumonia which are facultative
their surface. anaerobe
• Hexon fiber
o Contains type specific antigens that are important in Has a shadow on the apical
serotyping part of the lung therefore,
o Associated with hemagglutinating activity Mycobacterium the organism is a facultative
tuberculosis or obligate aerobic bacteria
Classification into 2 genera such as Mycobacterium
tuberculosis.

• (1) Aviadenovirus Dispersed shadow spots

due a virus that does not
o Infects birds require oxygen to grow and
Adenoviruses would likely be caused by
• (2) Mastadenovirus adenovirus and other
viruses that can cause
o Infects mammals
respiratory diseases.

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 LECTURE UNIT 09: DNA VIRUSES (GROUP I AND II)

Eye Infections: Laboratory Diagnosis of Adenoviruses

• Mild ocular involvement may be • Virus isolation culture that requires human cells:
part of the respiratory- o Primary human embryonic kidney cells
pharyngeal syndromes
• Pharyngoconjunctival fever ▪ most susceptible/usually not available
o Occurs in outbreaks such o Hep-2, HeLa, KB
as at children's summer
▪ Sensitive/difficult to maintain
camps – "swimming pool
conjunctivitis" • Characteristic CPE
o Associated with types 3
o rounding and clustering of swollen cells
and 7
• Immunofluorescence test
• Conjunctivitis or sore eyes is a
general condition that could be o Using an anti-hexon antibody
possibly caused by other
▪ It will use an antibody that will detect the hexon
external factors such as dust,
part, and will produce a coloration
possibly also bacteria, fungi
(not common), and viruses • Shell vial technique
o Sore eyes cannot be solely linked to adenoviruses o for rapid detection
• Epidemic • Serology using ELISA or latex agglutination,
keratoconjunctivitis Complement Fixation/CF test
o Most serious disease Epidemiology
o Occurs mainly in adults
and highly contagious • widespread
o Caused by types 8, 19,
and 37 Treatment
o Characterized by acute conjunctivitis (reddening of
the eyes), followed by keratitis that usually resolves • no specific treatment
in 2 weeks but may leave subepithelial opacities in
the cornea for up to 2 years Prevention and Control

• Adenovirus can remain viable for several weeks on • Careful handwashing - easiest way
sinks and hand towels (as sources of transmission) • Environmental surface - disinfected with sodium
▪ e.g. public baths hypochlorite/alcohol
o Can be shed to feces but not all types • The best way to contro; adenovirus infection

Gastrointestinal Disease NOTE: Patients infected with conjunctivitis/ sore eyes, are
restricted to touch inanimate objects because Adenovirus can
• Types 40 & 41(most common) survive outside the host cell.

o Associated with infantile gastroenteritis (5 -

15% of viral gastroenteritis in young children) Baltimore Classification Group I – Envelope

Other Diseases: • Most commonly herpesviruses

Classification (Human Pathogens)

• Types 11 & 21
o Acute hemorrhagic cystitis in children • (1) Alphaherpesvirinae
o Herpes simplex virus type 1 HSV-1
• Children receiving liver transplants o Herpes simplex virus type 2 HSV-2
o May develop adenovirus hepatitis in the o Varicella-Zoster Virus VZV
allograft possibly but rarely ▪ Causative agent of smallpox (varicella) and
shingles (zoster)
• Children with heart transplants
• (2) Betaherpesvirinae
o May develop myocardial adenovirus
infections and are at risk of graft loss o Cytomegalovirus CMV
o Human herpesvirus Type 6 HHV-6
• Patients with AIDS o Human herpesvirus Type 7 HHV-7
o May suffer adenovirus infections • (3) Gammaherpesvirinae
• Children are more vulnerable because of their immune o Epstein-Barr virus EBV
system and lifestyle
▪ Causative agent of kissing disease

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 LECTURE UNIT 09: DNA VIRUSES (GROUP I AND II)

Herpesvirus • Neonatal HSV-2

o Acquired during delivery
• Spherical o Attacks infant’s CNS (death may possibly occur)
• 150-200 nm in diameter
• Icosahedral ▪ This happens when the mother doesn’t go to
• Double-stranded DNA check-ups
▪ The virus enters after delivery and attacks the
o Envelope with viral infant’s CNS.
glycoproteins
o Produces latent viral Laboratory diagnosis
infection in WBCs and
peripheral nerves • Specimen – lesions or conjunctival lesion
• Direct examination of virus:
• Reactivation
▪ Electron Microscopy, Pap’s smear, DFA, indirect
o Associated with immunosuppressed patients, with fluorescence, immunoperoxidase, cell cultures,
underlying conditions and those undergoing shell via culture
chemotherapy or corticosteroid therapy
• Characteristic CPE:
o e.g. Varicella-Zoster Virus - causative agent of
smallpox o Round, clumping, syncytial giant cells
▪ If you have acquired and recovered from Other Infections caused Herpes simplex virus
smallpox, it is possible that the Varicella-Zoster
Virus have latency on your body Herpetic Keratitis, Herpetic withlow
 Latency – dormant, resting or not actively
dividing or replicating but the virus is still • Infects fingers (occupational hazard)
inside the body • Seen in AIDS
▪ If stress occurs, the latent Varicella-Zoster virus
will cause shingles (second chickenpox)

Herpes simplex virus- 1 (HSV-1)

• “Oral strain”
• Mild infection and less resistant to treatment
• Transmission:
o Through active ulceration of the mucous
membranes Varicella-Zoster Virus (VZV)

• Cause infections above the waist • Agent of varicella/ chicken pox,

• Associated with: herpes zoster, or shingles (a
o Gingivostomatitis reactivation form of latent
o Ulcerative mouth varicella)
lesions o If the chickenpox has
o Fever blisters become latent (varicella
zoster virus) during
• May spread to the lips
childhood, and the person is
and cheek
stressed, then they can
acquire shingles.
• Chicken pox – a childhood
Herpes simplex virus- 2 (HSV-2) disease; generalized skin rash
with raised, fluid filled lesion
• “Genital strain”
• Cause infections below the waist o It will start with papule, becomes a blister, then it will
• 80 - 90% of genital herpes, a common STD become ulcer once it burst
• Signs:
• Latency is in the spinal cord
o Fever
o Virus remains latent in the dorsal root ganglia or
o Malaise
peripheral cranial nerves.
o Inguinal
lymphadenopathy • Shingles – a disease of the elderly and
immunosuppressed patients.
• Primary lesion appears
in: o Also to those who previously had chickenpox
o vagina, cervix, glans, or penile shaft, recurrent • Complications: Neuralgia, keratitis ophthalmia, hearing
lesions may occur loss, facial paralysis, aseptic meningitis.

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 LECTURE UNIT 09: DNA VIRUSES (GROUP I AND II)

o Aseptic meningitis – aseptic means clean (no • Isolated in saliva and mononuclear cells
bacteria seen) • Transmission – respiratory route
• Agent of exanthema subitem or roseola
• Question: Is it possible to acquire shingles even
though you haven’t had chicken pox yet? o Known as the “sixth disease”
o Benign childhood disease (6 months – 3 years)
o NO. A person should acquire chicken pox first.
o Shingles is a reactivation of the varicella zoster Epstein-Barr Virus (EBV/HHV-4)
virus.
• Agent of Infectious Mononucleosis
Pathways of a Herpes Infection: • Member of subfamily Gammaherpesvirinae
• (1) Herpesvirus enters • EBV – shed in saliva and transmitted through oral
the body contact
o Enters through skin o Kissing’s disease
or mucous o Asymptomatic
membranes
• Virus replicates first in the epithelial cells of the pharynx,
• (2) Herpesvirus lies which causes sore throat or pharyngitis
dormant in the nerves o A very typical symptom (seen in bacterial and other
o After viral infection, herpesvirus settles in nerves viral agents)
near the spine
• Latency and target cells:
• (3) Herpesvirus is reactivated, causing another o B cells that are invaded via their CD-21 receptor
outbreak. (shingles)
• Cell immune response involve cytotoxic CD8 positive B
o Herpesvirus travels along the nerves, back to the
cells against the infected B cells, resulting in enlarged
skin to form new blisters
atypical lymphocytes.
Cytomegalovirus (CMV/HHV-5) o Downey cells (Atypical
lymphocytes)
• Formerly known as “Salivary Gland Virus”
• Member of the family Betaherpesvirinae ▪ Reactive lymphocytes
• Opportunistic infection; transmitted through direct associated with EBV.
contact with saliva, blood transfusion, organ transplants ▪ Termed by Hal Downey
in 1923
• Produce asymptomatic or mild infections.
• Latent in: WBCs, endothelial cells and other organs. • Disseminates in the reticuloendothelial system (RES)
• Congenital CMV such as the liver, spleen, and lymph nodes
• Disease characteristics/signs:
o Occurs as primary infection
during pregnancy if the o Fever
mother lacks antibody to o Sore throat
the virus. o Enlarged lymph node and
o Future mothers should tonsils
have a complete screening o Splenomegaly,
test, especially those who hepatomegaly and
are sexually active. elevated liver enzymes
• Specimen used: urine, saliva, ▪ Observed late in the
tears, milk, semen, and vaginal disease course
secretion ▪ Usually suspected as
• Direct cytology: “Owl’s eye” a bacterial infection
instead of EBV
o Hallmark characteristic
o Large cells with basophilic  If antibiotic is given to the patient, EBV can
staining inclusion within the cause allergic rash.
nucleus o Peak incidence of the illness occurs during
o Two fused cells adolescence and early adult life
o Isolated in cell culture and shell vial. o In children, the initial symptoms of mono occur
• CPE: rounding of cells or owl’s eye appearance about 10 days after exposure

Human Herpesvirus Type 6 (HHV-6) • EBV does not require long-term treatment because it is
a self-limiting disease
• Member of the subfamily • Complications:
Betaherpesvirinae o Splenic rupture
• First known as Human Lymphotropic o Hemolytic anemia
Virus o Encephalitis
o Chronic EBV infections.
o Defected infecting B-cells

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 LECTURE UNIT 09: DNA VIRUSES (GROUP I AND II)

• Question: Why is there a chronic complication o Encodes epidermal growth factor and
when EBV is self-limiting? transforming growth factor-α
o Herpes viruses have latency and, in this case, EBV ▪ accounts for proliferative diseases
has latency to B cells
o Poxvirus encoded host defense modifiers
▪ Latency – capable of being dormant
▪ Proteins which inhibit host defense mechanism
 They will not replicate so the virus will not be such as Tumor necrosis factor (TNF) receptor
detected. ▪ IL-1 receptors, and Complement binding protein
Review of the Herpesviridae ▪ Under innate immune system
Small pox (variola virus) Infection
• Alpha viruses – establishes latency in neurons
• Beta herpes – establishes latency where virus lies • Ancient disease that killed
dormant until reactivated in leukocytes millions
• Gamma Herpesviridae – establishes latency in cells of • Transmission
the immune system such as B cells
o Direct respiratory contact and
multiply in lymph nodes
Baltimore Classification Group I – Complex
• Smallpox was declared
• Ali Maow Maalin, the last case of smallpox in Somalia, eliminated in 1980.
1977 • Variolation and Vaccination
o The last naturally occurring case of smallpox o Helps eradicate smallpox.
(Variola minor) was diagnosed on October 26,1977
• In 1980 the WHO declared that smallpox is completely Laboratory Diagnosis
eradicated through vaccination • Skin Lesion
• The nature and structure of smallpox does not belong to o Specimen of choice
envelopes and naked. o Poxvirus are stable and may remain viable in
• Double stranded DNA specimens without refrigeration.
Poxviruses • Virus Isolation

• Largest and most complex viruses, oval or brick shaped o Most reliable laboratory tests but not usually
or ellipsoid (400 nm x 230 nm) performed
• External surface shows ridges, contains core and lateral o Inoculation of vesicular fluid onto the chorioallantoic
bodies (dumbbell shape) membrane of chick embryo
• Double stranded DNA, enveloped with multiple • PCR (Polymerase Chain Reaction)
membranes
• Family members: o Available
o Fast
o Variola – small pox virus;
o Vaccinia virus – agents of cowpox and monkey pox. • Serology

▪ The first vaccine is derived from cowpox o Enzyme Linked Immunosorbent Assay (ELISA)
▪ Somewhat similar to variola in terms of structure o Radioimmunoassay (RIA)
o Immunofluorescence tests
NOTE: Research about poxvirus/smallpox virus is still active in
Russia and US in cases of bioterrorism.

Treatment
• Vaccinia immune globulin
• Methisazone (USAN) or Metisazone (INN)
o Chemotherapeutic agent
o An antiviral drug that works by inhibiting mRNA and
protein synthesis, especially in pox viruses.
• Reproductive cycle of poxviruses ▪ Directly inhibits the virus genome function.
o Two distinct infectious virus particle exists o It has been used in the past to treat smallpox.
▪ (1) Intracellular mature virus (IMV=MV) Monkeypox Infection
▪ (2) Extracellular enveloped virus (EEV=EV)
• Virus encoded proteins help evade host immune • Caused by a species of Orthopoxvirus
defense system • 1958 – First recognized in captive monkeys
• 1970 – Human infections with the virus
o Very resistant to inactivation and restricts the
immune system action o Discovered in West Africa and Central Africa

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 LECTURE UNIT 09: DNA VIRUSES (GROUP I AND II)

• Acquired by direct contact Human Tanapox Infection

with wild animals
o Physical or occupational • Tanapox and Yaba Monkey Tumor Poxvirus
contact (animal as a Infections
source of food) o Tana river Kenya, Zoonosis, B type inclusion, Massif
• Tanapox
Cowpox Infection
o Common skin infection in
parts of Africa, mainly in
• Caused by a species of
Kenya and Democratic
Orthopoxvirus
Republic of Congo
• Milder form of pox diseases
occurs in cattle • Natural host is Monkey
• Human Infections: • Classified as the
o Occur by direct milking Yatapoxvirus genus
o Lesion of milkers is o Morphologically similar to Orthopoxvirus genus
usually confined in the
hands • Human infection via direct contact (occupational
exposure)
• In immunology, Edward Jenner observed that cow
o One of the major sources of food is hunting wild
handlers or milk milkers that have cowpox will not
animals such as monkeys
develop a severe infection if exposed to smallpox (only
manifests fever) BALTIMORE CLASSIFICATION GROUP II
o There is a cross-reactivity between cowpox and
smallpox. Parvovirus B19
o To vaccinate against smallpox, people are exposed
to the milder cowpox
Buffalopox Infection

A derivative of
vaccinia virus that
has persisted in India
in water buffalo
• Can be transmitted to
humans through
direct contact.
o Culture or Occupational exposure
Orf virus Infection
• (1) Parvovirus B19 infects the proerythroblast
(pronormoblast) lineage of red cell maturation.
• Caused by the virus of Orf
o Species of Parapoxvirus • (2) The proerythroblast will become giant
proerythroblast.
• Causes disease in sheep o If the giant proerythroblast cannot contain the virus
and goats anymore, it will undergo apoptosis
o Worldwide prevalence o Thus, causes the decrease of red cell production
• Transmitted to humans via direct contact Parvovirus
o Culture or Occupational exposure
• Single-stranded DNA
Molluscum Contagiosum o Group I are double stranded, while group II are
single stranded
• Benign epidermal tumor
that occurs only in • Icosahedral symmetry (18-26 nm in diameter)
humans • Extremely resistant to inactivation
• Causative agent: o Immediately look at the characteristic structure of
o Molluscipoxvirus the naked virus
genus o Naked

• Not transmitted to ▪ Majority of naked viruses are resistant to

animals inactivation compared to enveloped viruses.
• Sexually transmitted and is seen in AIDS ▪ Envelope proteins are lipid proteins; proteins can
be easily inactivated or control.
o Common in people with AIDS

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 LECTURE UNIT 09: DNA VIRUSES (GROUP I AND II)

• Stable at pH 3 and 9, withstand heating at 56 oC for 60

minutes.
• Inactivated by formalin, β-propiolactone and oxidizing
agents Hydrops fetalis Fetus
Fatal anemia and
abortion of fetus
o Can cause death to the organism or virus when
used
• Known to infect mice, hamsters, cats, and dogs
(common)
2 sub-families Erythema infectiosum (fifth disease)

• (1) Parvovirinae – infect vertebrates (Genus Parvovirus • A childhood illness (measles, rubella, VZV, roseola), a
and Erythrovirus) common childhood exanthem
o Able to replicate autonomously in rapidly dividing o Exanthem – “rash”
cells
• Mode of transmission:
• (2) Densovirinae – infects insects (defective members)
o Respiratory route
o Depend on a helper virus for replication (adenovirus o Parenterally (blood transfusion)
or herpesvirus) o Vertically (Mother-fetus)
• Parvovirus B-19 • Clinical features:
o Known to infect humans (only member of genus o Fever
Erythrovirus) o Unique “Slapped-Cheek” rash for Parvo B19.
• Canine Parvovirus – Infects dogs ▪ Has affinity to RBC precursors which may lead to
anemia
Table 2. Some of the viruses in the family Parvoviridae o For older patients – butterfly rash associated with
SLE
Subfamily Genus Species
• Incubation period: 1-2 weeks
Dependovirus Adeno-associated virus 2
Transient Aplastic anemia
Minute virus of mice
Parvovirus
Parvovirinae Feline panleukopenia virus
• May complicate chronic hemolytic anemia
Erythrovirus B19 virus
o Sickle cell disease, Thalassemias, and acquired
Bocavirus Human bocavirus
hemolytic anemia in adults
Densovirinae Iteravirus Bombyx mori densovirus
• Occurs after BM transplantation
• Dependovirus – it depends on adenovirus and retrovirus • Symptoms:
• Erythrovirus – infects humans o Abrupt cessation of RBC synthesis in BM, reflected
in the absence of erythroid precursors
o Discovered in 1975 by an Australian virologist
Yvonne Cossart o Accompanied by rapid worsening anemia
o It gained its name because it was discovered or Pure Red cell Aplasia
tested positive in well B19 of large series of
microtiter plates • B19 may persist and cause chronic suppression of BM
• “Parvo” – Latin for small and chronic anemia in immunocompromised patients
• Severe anemia patients depend on blood transfusion
• Observed in:
Disease Association
o Patients with AIDS
Table 3. Human Diseases Associated with B19 Parvovirus o Malignancies
o Organ transplants
Host or o Congenital deficiencies
Syndrome Clinical Features
Condition
Erythema Infection during pregnancy
Cutaneous
infectiosum (Fifth Children/Adults
rash/Arthralgia-arthritis
disease) • Hydrops fetalis and fetal death due to severe anemia
Severe acute anemia
Transient Underlying o Pose serious risk to fetus
• Targets one step
aplastic crisis hemolysis in erythrocyte • Fetal death occurs during pregnancy
lineage
Pure Red Cell Immuno- o Also common with Neisseria gonorrhoea, N.
Chronic anemia meningitidis and Listeria monocytogenes (bacteria)
aplasia deficiencies

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 LECTURE UNIT 09: DNA VIRUSES (GROUP I AND II)

Laboratory Diagnosis

• PCR (most sensitive)

• Serology:
o Detection of B19 IgM antibodies – for recent
infections
o B19 - IgG – persist through years
• Virus – difficult to grow
o Culture is not typically done

Epidemiology

• Widespread

Treatment and Prevention

• Treatment
o Commercial Immunoglobulin (Ig) preparation contain
neutralizing antibody
o No available vaccine
• Prevention/Control
o Good hygienic practices (hand washing, not sharing
of drinks)

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 [TRANS] LECTURE UNIT XX: RNA VIRUSES (BCIII AND IV)

BALTIMORE CLASSIFICATION GROUP III • (1) The virus enters the body through the mouth, often
via a contaminated thumb. The viral particles then pass
Non-enveloped segmented dsRNA Viruses: Reoviruses
through the stomach and into the small intestine
Rotavirus • (2) VP4 proteins attach the virus to the epithelial cell
lining the gut
• Naked o Viral protein 4 (VP4)
o Does not have an envelope ▪ Proteins attach the virus to the epithelial lining in
• 75 nm in diameter with two the gut
protein layer surrounding the  Viral tropism: the VP4 is able to attach to the
capsid epithelial cell linings
o The protein surrounding the • (3) Then the VP4 spikes and the outer shell are shed,
capsid is not an envelope and the rest of the particle (and the subparticle) enters
o The envelope characteristics should be in lipid form the cytoplasm
• (4) There the viral gene direct production of thousand
• Most common cause of viral gastroenteritis in infants
new viral particles and of toxin molecules that can
and children
poison even uninfected cells and instigate fluid release
• Has 11 double-stranded RNA segments
from the intestinal tissue
Transmission • (5) Waves of new virus stream out of the infected cells
to invade healthy ones, and the cycle repeats itself
• (6) Dead epithelial cells and fluids from the stomach
and tissues fill the gut and exit the body as profuse
diarrhea

Diagnostic Tools

• Enzyme-linked Immunosorbent Assay (ELISA)

• Spread by the oral-fecal route o Unknown – viral antigen

• Incubation period of 1 to 4 days o Virus are proteins thus, there are antigens

o Incubation period - the period between the • Latex Agglutination Test (viral antigens in fecal
infection of an individual by a pathogen and material)
manifestation of the illness or disease o Unknown – viral antigen
• Sudden onset of symptoms includes: o The particle used should be embedded with
antibody
o Vomiting
o Diarrhea ▪ Antibody will bind to the viral antigen causing
o Fever agglutination
o In some cases, abdominal pain & respiratory • Electron Microscopy Examination (not sensitive)
symptoms
Human Bovine Virus Vaccine (2006)
How Rotavirus Attacks
• Since the virus is very common to infant and children,
• The virus is shed in large quantities in the stool and can vaccine is already provided
cause nosocomial outbreaks in the absence of good • Developed countries use rotavirus vaccine but for third
hygiene world countries, there are very rare number of vaccines
for the rotavirus

BALTIMORE CLASSIFICATION GROUP IV (NON-

ENVELOPED)
• Polio
o One of the common viruses under this classification
o Already eliminated all over the world
• Dr. Jonas Salk
o Invented the Salk vaccine for Polio

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 LECTURE UNIT XX: TITLE

o During his time, people who doesn’t believe in • Mode of transmission: Viruses are spread via
vaccines accused that Salk vaccine is not safe, not aerosol, the fecal-oral route, and fomites
effective, etc. • Portal entry: the alimentary canal via the mouth
▪ His response was, “Risks, I like to say, always • The viruses replicate initially in the lymphoid tissue of
the pharynx and gut
pay off. You learn what to do or what not to do
o The polio vaccine is a not a Salk vaccine because it
Non-enveloped ssRNA Viruses: Picornaviridae is given orally (Sabin vaccine)

• Picornaviridae ▪ Salk vaccines (1954) are injected

intravascularly compared with Sabin vaccines
o “Pico” is the Latin which are given orally
word for small
 Created in 1954
o Chain of characteristic
is “RNA”  Inactivated vaccine
o “Viridae” – family  Mode of introduction is injection
o Family of RNA viruses
that are small ▪ Sabin vaccines are given orally because they
are attenuated vaccines.
• Is one of the largest families of viruses, with more than  Produced after Salk
230 members
 Attenuated - weakened version of the virus
• It contains many important human and animal
pathogens  Given orally
o They are very small with only 27 nm in size  Therefore, the mode of initiation should be
• Naked orally because the mode of transmission of
polio viruses initially replicates in the pharynx
o No peplos
and gut
• Reflected on the picture:
o Capsid  The Sabin vaccine will copy the course of
infection of polio
o Capsomeres – a set of capsids
o ssRNA (single-stranded RNA) – 7478 bases mean
it’s quite long
o VPg
• Four genera with clinical significance belong to the
family: Enterovirus, Rhinovirus, Hepatovirus, and
Parechovirus
• The enteroviruses found in the genus include:
o Poliovirus (1 to 3)
o Coxsackieviruses (A1 to A23)
o Coxsackieviruses (B1 to B6)
o Enterovirus (68 to 72)
o Echovirus (1 to 32)
o Parechovirus (1 to 4)
• (1) If the enterovirus may enter via aerosol or ingestion,
▪ These are the viruses that are under the four
it will replicate in the oropharynx and tonsils including
genera that have human clinical significance the lymphoid tissues
• (2) After that, there is a possibility of primary viremia.
Enteroviruses Once it circulates, it will proceed to secondary viremia.

• Small, naked o Viremia - virus in the blood

viruses’ case • (3) In the secondary viremia, the virus goes to its
various conditions specific organs
including fever of
unknown origin, o Poliovirus and Coxsackievirus - Brain
aseptic meningitis, ▪ Causes encephalitis and paralysis
paralysis,
cardiomyopathy, exanthemas, pharyngitis, and o Echovirus, Poliovirus, and Coxsackievirus -
conjunctivitis Meninges
o Aseptic Meningitis - when cultured, there is no ▪ Meningitis
growth (not bacteria or fungi) o Hepatitis A virus - Liver
• Infections are more prevalent in areas with poverty, ▪ Hepatitis A
overcrowding, poor hygiene, and poor sanitation.
o Echo and Cox A- Skin
o The mode of transmission is too close contact, you
can easily transmit it to other people ▪ Hand foot mouth disease and Rash herpangina

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 LECTURE UNIT XX: TITLE

o Echo, Cox A and B - Muscle • They can be isolated from the feces or as long as 6
weeks afterwards.
▪ Myocarditis, Pericarditis, Pleurodynia
o Stool/feces – best sample and most common
• Enteroviruses have the same portal of entry, via
aerosol or ingestion • Specimen from the throat. feces, rectum, and CSF, and
• Viremia can result in the virus spreading to the spinal conjunctiva are recommended
cord, heart, and skin • Enteroviruses have no group antigen, so they must be
identified individually by a serum neutralization test
Poliovirus
Serum Neutralization Test
• The polioviruses tend to infect the nervous system and
can cause paralysis in a small percentage of infected • Test to neutralize the virus
patients • Virus are proteins, therefore, they are antigen
• The viruses destroy their host cells. In the intestines, • To neutralize antigen, it will need antibodies
damage is temporary because the cells lining the gut • Poliovirus can be classified as 1, 2 and 3
are rapidly replaced
o If antigens and antibodies interact with each other,
neutralization happens
o Once neutralized, cells are added (in this case,
enterocytes)
▪ Enterocytes – target cell of poliovirus
o Does the virus infect the cell (enterocytes)?
▪ NO
▪ If CPE is negative → positive for poliovirus 1

Hand, Foot, and Mouth Disease (HFMD)

• Caused primarily by Coxsackievirus type A5, 10 and

16 and occasionally enterovirus 71
• It is spread by fomites or the oral-fecal route
• Occurs in young children
• A mild-prodromal phase:
malaise, headache, and
• (1) Oral ingestion of poliovirus abdominal pain. Small painful
• (2) PV will try to infect the alimentary tract sores suddenly appear on the
• (3) It will eventually go to the blood circulation tongue, buccal mucosa, and
• (4) It will then cross the Blood Brain Barrier soft palate
o Viruses can cross the BBB o Prodromal phase – symptoms before the main
symptoms
• (5) Once it crosses the BBB, it will then go to the
Central Nervous System (CNS) • Simultaneously, a
• (6) Replication in the CNS which can cause paralysis maculopapular rash
• 2 pathways of the poliovirus: appears on the hands, feet,
and buttocks, followed by
o Skeletal pathway
bullae on the soles of the foot
o Neural pathway
and palms of the hands
• In contrast, neurons are not replaced, so neuron death o Bulla/Bullae – a fluid-filled sac or lesion that
can result in permanent paralysis appears when fluid is trapped under a thin layer of
• No vaccines are available for enteroviruses other than the skin
poliovirus
▪ A type of a blister
o Good personal and nosocomial hygiene and good ▪ To be classified as a bulla, the blister must be
sanitation can reduce the incidence of the infection larger than 0.5 cm in diameter
• Excellent poliovirus vaccines of either attenuated or o Smaller blisters are called vesicles
inactivated viruses are available
• The virus can be isolated from specimens from swabs
o Two types of polio vaccines of the mouth and bullae
▪ Attenuated or Sabin vaccine • CoxsackievirusA16 grows in PMK and human diploid
▪ Inactivated or Salk vaccine fibroblast cells and can be identified by serum
neutralization tests
• Since 1988 the polio vaccine has been crucial to the
WHO's effort to eradicate polio worldwide Hepatitis A virus
• Enteroviruses can be cultured from the pharynx
immediately before the onset of symptoms and 1 to 2 • Aka Enterovirus 72
weeks afterward • Small, icosahedral, naked single-stranded RNA virus

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 LECTURE UNIT XX: TITLE

• The sole member of the Non-enveloped ssRNA Viruses: Caliciviridae

genus Hepatovirus in the
family Picornaviridae Norovirus
• HAV is almost always
transmitted by the fecal-oral • Small, ssRNA, round virus of 27 to 30 nm in diameter
route • These viruses are currently placed in the genus
• Risk factors include sexual Norovirus, family Caliciviridae
or household contact with an infected person, day-care • The most common
contacts, food-borne or water-borne outbreaks IV drug cause of infectious
use and international travel. gastroenteritis in the
United States
o There are some areas where hepatitis A is highly
accounting for as
prevalent, including the Philippines
many as 23 million
cases annually
• Transmission is most
commonly food-
borne, although
water-borne and
person-to-person transmission can be significant
o Most common source of Norovirus (food-borne):
Shellfish
• The incubation period (between exposure and first
symptom) is 24 to 48 hours, and the onset of severe
nausea, vomiting, diarrhea, and low-grade fever
• The infection rate can be high as 50%
• Immunity may be short-lived, leading to the potential for
multiple infections throughout life

• The virus is shed in large amounts in the feces during o The antibodies or the Immune system that regulates
the incubation period (approximately 1 month) the Norovirus in the body, would only have a short-
• The incubation period is 0-1 week after exposure life span
• ALT will increase as a response for the incubation BALTIMORE’S CLASSIFICATION GROUP IV
period, because the virus enters the hepatocytes
(ENVELOPED)
o Response of the liver is to increase ALT
o Together with the IgM titer • One of the clinically significant viruses is the Dengue
o After 2 months, IgG will be produced Virus

• Hepatitis A can be neutralized with antibodies, o On 2015, Dengvaxia vaccine was made available to
specifically IgG the public
o However, with the administration of the vaccine, a
o As time progresses, the production of the antibodies lot of children/people died
increases
• Flaviviridae
• After individuals are infected, they experience a
transient viremia, after which the virus reaches the o Dengue – Type I to
liver and replicates in hepatocytes Type IV

o ALT is the liver marker for Hepatitis A

• Self-limiting, with convalescence possibly lasting weeks
• Low mortality and no persistence, and it does not cause
chronic liver damage, compared to Hepatitis B • Togaviridae
• The best method for laboratory diagnosis of HAV is to
o Chikungunya
demonstrate IgM to HAV
o Even as early as the first month ▪ Similar to flavivirus in
terms of clinical
o Viremia – Blood
o Hepatitis A – Stool manifestation

• Coronaviridae
o Corona viruses

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Enveloped ssRNA Viruses: Flaviviridae Dengue Virus

General Replication Cycle of Flaviviruses • Can cause two

distinct diseases:
o (1) Classic
Dengue Fever
(DF)
o (2) Dengue
Hemorrhagic
Fever (DHF)
• Has four
serotypes:
o Dengue Virus 1
o Dengue Virus 2
o Dengue Virus 3
• They share the same replication cycle of Picornaviruses o Dengue Virus 4
• Flaviviruses – single stranded RNA virus
• The virus is transmitted by Aedes mosquitoes, including
o Positive sense – They will try to inject their genome A. aegypti and A. albopictus
to the infected cell • Patient with
▪ Since their genome is a positive sense, they Dengue Fever
cannot use it as a polypeptide precursor (DF) develop
▪ If they will use immediately the genomic RNA fever, headache,
positive sense, it will be depleted that is why myalgia, and
they use complementary genomic RNA bone pain
(resulting in the
o Negative sense – Complementary genomic RNA nickname “break-
▪ If they already have negative sense, they will try bone fever”)
to either use their own RNA dependent RNA o Hallmark characteristic of dengue fever
polymerase or they will use that of a human o Some patients also develop a rash
 Depends on the virus
• Dengue Hemmorrhagic Fever (DHF)
▪ RNA Dependent RNA Polymerase
o Although DF is a mild
 It will pass through negative sense disease, DHF is not
 It will produce a lot of mini copies of positive o Patients develop DHF
single stranded RNA or also called as mRNA after they have
already been
• Structural and Non-structural Proteins exposed to one of the
o It includes for the assembly other three serotypes
o It will go back for genomic RNA (Positive sense) ▪ Requirement: You need to be exposed first from
• Progeny Virus – It will make new progeny virus Dengue virus.
Serotypes of Dengue Virus
Flaviviridae

• The family • Exposure to two different serotypes of Dengue virus

Flaviviridae contains appears necessary for developing DHF
a number of important • Patients DHF develop the symptoms of classic DF, long
human pathogens, with thrombocytopenia, hemorrhage and shock, and
many of which are sometimes death
zoonotic arboviruses
including:
o Japanese Encephalitis virus
o Dengue virus
▪ Common in Philippines
o Yellow fever
o St. Louise Encephalitis virus • (1) Monocyte
o West Nile virus o It has a Fc gamma receptor for the Fc portion of IgG
• (2) Antibodies will bind to DENV
o IgG will bind to the virus

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• (3) After it binds, monocyte will bind to the Fc portion of and cause encephalitis.
the antibody and engulf it. • Chikungunya
o The function of monocyte is to engulf. o Produces the same clinical symptoms with dengue
o Family: Togaviridae
• (4) After it engulfs, the virus can actually survive inside o Genus: Alphavirus
o Virus will produce or increase the viral load
• Dengue
Antibody-dependent Enhancement (ADE) o Family: Flaviviridae
o Genus: Flavivirus

Eastern Equine Encephalitis (EEE) Virus

• Occurs primarily in the eastern half of the United States.

• Infections have a significant mortality rate of about 36%.
• Family: Togaviridae
• Cases of EEE Virus in the Philippines is very low to
none
o Geographically restricted
▪ It is only highly
prevalent in
certain areas
• As many as half of
those who survive the
infection suffer
permanent CNS
damage
• Birds are the natural
• Dengue Hemorrhagic Fever reservoir of the virus,
and it is spread to
o Needs to two exposures to the strains because of humans and horses
ADE via bite of
mosquitoes.
• Initial Infection (Type I Dengue virus)
• Vector: Mosquitoes
o (1) Dengue virus will enter the cell • Amplifying host: Bird
o (2) The virus will replicate • Dead-end hosts: Human and/or Horses
o (3) B cells will produce antibodies to neutralize the
virus West Equine Encephalitis (WEE) Virus
o (4) After neutralization, the macrophage will engulf
the virus, and destroy it • Causes milder diseases than EEE virus
• 30% of infected children and infants will suffer
• Second Infection (Type 2 Dengue virus) permanent CNS damage.
o (1) Memory B-cells will produce antibodies against • Mortality is about 3%.
Dengue virus type 1 • Since 1964 there have been 639 cases in the United
o Dengue type 1 and type are almost identical States.
o (2) Antibodies against Dengue virus type 1 is
weaker
o (3) Macrophage will engulf the virus
o (4) Since the antibodies are weaker, the Dengue
virus type 2 can survive inside the macrophage
• In the second infection, the antibody enhanced the entry
of the virus inside the macrophage
• Development of dengue haemorrhagic fever is faster
than dengue fever
Enveloped ssRNA Viruses: Togaviridae
• Vector: Culex tarsalis and Aedes melanimon
• The family Togaviridae
contains the genera Rubella Virus
Alphavirus,
Rubivirus, and • It causes the disease rubella
Arterivirus or German measles, a mild
febrile illness accompanied by:
• Many of the viruses in
the genus Alphavirus o Erythematous,
are mosquito-borne Maculopapular, discrete

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rash with postauricular (ear) and suboccipital (eyes) • Small (32-34nm) classified in the genus Hepevirus,
lymphadenopathy. family Hepeviridae
• Rubella virus is o Classified before as Caliciviridae but were removed
transmitted by eventually
droplets
• Along with HCV, HEV is the other historic non-A,non-B
o Large (NANB) hepatitis virus
infectious • HEV causes an acute, self-limiting disease with clinical
droplets symptoms that are similar to those of HAV
▪ 1-3 feet • The incubation period is 2 to 9 weeks
• Problem: Not
o Small infectious droplets screened in blood
▪ 3-5 feet bags but may
eventually cause
o Infectious droplets nuclei infection once
transfused to a
▪ 5-160+ feet
person, like pregnant
▪ Airborne; very small and can spread far
women, eventually
• The virus is present in nasopharyngeal specimens or leading to the death
any secretion or tissue of infected infants of the infant
• A rash starts on the face and spread to the trunk and • Incubation period: 2-9 weeks
limbs. No rash appears on the palm and soles • Viral shedding in the feces has been shown to persist
for several weeks
o Hallmark characteristic of Rubella virus
• The mortality rate is 1%-3% overall, with a higher
▪ Rash will always start on the face likelihood of death in pregnant women (15-25%)
o NOTE: Immediately rule out Rubella virus if rashes • Recommendation: Include Hepatitis E screening in
are present on the palm and soles blood bags
• ELISA test – developed to detect IgG and IgM
• As many as 50% of individuals with rubella are antibodies to HEV
asymptomatic. • HEV testing – not currently performed in diagnostic
o An individual may spread the virus despite not laboratories in the US and Philippines
having rashes Enveloped ssRNA Viruses: Coronaviridae
o Hence, vaccination is important
• Can cross the placenta of pregnant women and Coronavirus
disseminate to fetus tissues; a condition referred to as
• Have distinctive club-shaped
Congenital Rubella Syndrome
projections on their surfaces
o It is important that pregnant women must avoid • Responsible for 15% of cold-like
people manifesting symptoms of German measles infections in adults, but higher
seroconversion rates are seen in
• The impact on the children
embryo is worse when
• Corona – not corona for ‘crown’, but for ‘sun’
the infection develops
in the earliest stage o During an eclipse, when the sun is blocked, the
of pregnancy, crown of light formation on the outer parts = corona
because the virus
• Extremely fragile – they are enveloped
halts or slows the
growth of the cells in • Difficult to culture, but it is possible to test specimens
infants directly by IF (immunofluorescence) and EIA (enzyme
• An effective attenuated vaccine is available and should immunoassay) methods
be administered to all children and young women before o Culture - used in the laboratory to identify
they will become sexually active. coronavirus
o Exposure to German measles prior to pregnancy • A novel coronavirus was the causative agent of a
leads to decreased risk of reacquiring the virus pandemic of respiratory disease that emerged from
Hong Kong in late 2002.
• Direct examination of specimens by IF or EIA is
recommended o In a 6-month period, the infection spread rapidly to
• Serologic procedures are effective because any Rubella 26 countries in Asia, Europe, South America, and
antibody is presumed to be protective North America
• The most sensitive serologic assays are the solid- o Lasted for 7 months
phase and passive hemagglutination test
• Mortality rate: Approximately 10%
Enveloped ssRNA Viruses: Hepeviridae • The disease was characterized by:
o High fever – sudden onset
Hepatitis E Virus
o Pneumonia

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o Acute respiratory distress syndrome (for some • MERS-CoV, 2012

patients)
o Saudi Arabia
o 27 countries
o 2,500 cases
o 850 deaths
• SARS-CoV-2, 2019
o Wuhan, China
• The disease was named SEVERE ACUTE o 143 countries
RESPIRATORY SYNDROME (SARS) o 153,517 cases
o 5,735 deaths
o Causative agent: SARS-associated coronavirus
(SARS-CoV) Intermediate host: Pangolin
o No vaccine or antiviral agent was able to fight the
pandemic, which ultimately ended with intense • The discovery of multiple lineages of pangolin
public health intervention including: coronavirus and their similarity to SARS-CoV-2
suggests that pangolins should be considered as
▪ Massive screening programs possible hosts in the emergence of new coronaviruses
▪ Voluntary quarantine
and should be removed from wet markets to prevent
▪ Travel restrictions zoonotic transmission
• Jumped from its normal animal host (possibly a civet • Used as possible treatment for cancer by the Chinese
cat), into a human
Coronoviridae Family
o Bat → civet cat → human

• Order
o Nidovirales
• Family
• SARS-CoV-1 (2003) o Coronaviridae
o Bat – natural host • Genus
o Civet cat – intermediate host
o Human – disease outbreaks o alphacoronavirus
o betacoronavirus
• MERS-CoV (2012) o gammacoronavirus
o Bat – natural host o deltacoronavirus
o Camel – intermediate host
o Human – disease outbreaks • Lineage
o A
o B
▪ SARS-CoV
▪ MERS-CoV
▪ SARS-CoV-2
o C
o D
• The SARS-CoV-2 virus is a betacoronavirus, like
MERS-CoV and SARS-CoV. All three of these viruses
have their origins in bats

• SARS-CoV, 2003
o Guangdong, China
o 30 countries
o 8,096 cases
o 774 deaths

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SARS-CoV-2: Structure and Genomic Organization Table 1. SARS-CoV-2 vaccines available in the Philippines (1)

Oxford
Pfizer BioNTech
AstraZeneca
(RNA)
(Viral vector)
Dose and 2 doses, 21 days 2 doses, 4-12
Frequency apart [a] weeks apart [a]
Storage
-80 to -60°C [a] 2 to 8 °C [a]
Requirements
-70.4% against
Vaccine Efficacy symptomatic
95% against
based on phase COVID-19 [a]
• Spike protein III Clinical Trial
symptomatic
-100% against
COVID-19 [b]
o Used to attach itself to the host cell (CT) severe COVID-19
o Does this via Angiotensin converting enzyme 2 [b]
(ACE2) Sinovac Gamelya Sputnik
CoronaVac V
SARS-CoV-2 Life cycle (Inactivated) (Viral vector)
Dose and 2 doses, 28 days 2 doses, 21 days
Frequency apart [a] apart [c]
Storage -18°C and below
2 to 8 °C [a]
Requirements (liquid form) [e]
-91.6% against
Vaccine Efficacy 65-91% (based on symptomatic
based on phase Brazil, Indonesia COVID-19 [b]
III Clinical Trial and Turkey trials) -100% against
(CT) [a] moderate or
severe cases [b]
Table 2. SARS-CoV-2 vaccines available in the Philippines (2)

Bharat Bio Tech Moderna

• The SARS-CoV-2 is a novel strain, meaning it is new. (Inactivated) (RNA)
As a result, humans have note developed immunity to Dose and 2 doses,14 days 2 doses, 28 weeks
COVID-19, and therefore can be infected. Acquired Frequency apart [c] apart [b]
immunity is when humans have been exposed or Storage -25 to -15 °C; 2 to
vaccinated 2 to 8 °C [e]
Requirements 8 °C (30 day)
• Life Cycle -94.1% against
o (1) SARS-CoV-2 is inhaled into the lungs 80.6% against symptomatic
Vaccine Efficacy
o (2) Goes into the lungs via Spike protein & ACE2 PCR-confirmed COVID-19 [b]
based on phase III
symptomatic -100% against
▪ Triggers an immune response Clinical Trial (CT)
COVID-19 [e] severe COVID-19
o (3) Immune response will try to neutralize the virus [b]
however it can survive and causes disease Novavax Janssen
progression (Subunit) (Viral vector)
SARS-CoV-2 Detection Dose and 2 doses, 21 days
1 dose [f]
Frequency apart [c]
• Detection method include electron microscopy, -20°C (2 yrs.)
Storage
ELISA, and RT-PCR 2 to 8°C [h] 2 to 8 °C (3
Requirements
months) [d]
o There is still no ELISA for Coronavirus-2 66.1-66.9%
Vaccine Efficacy Awaiting Official against confirmed
• Antibody can be detected by Western Blot
based on phase III Phase III Interim moderate to
o Can be detected together with MERS-CoV, SARS- Clinical Trial (CT) Journal Publication severe/critical
CoV &SARS-CoV-2 COVID-19 [I]
SARS-CoV-2: Vaccine

• No SARS vaccine, trials with an intranasal

immunization and a UV/formalin, inactivated vaccine
have shown potential.
• For COVID-19, however, already has a vaccine

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 [TRANS] LECTURE UNIT 11: RNA VIRUSES (BC V)

BALTIMORE’S CLASSIFICATION GROUP V o Genome is segmented

(NEGATIVE SENSE SINGLE STRANDED) ▪ Like reoviruses & rotaviruses, which have 11
Negative-stranded RNA Viruses segmented double-stranded RNAs
(-RNA) Influenza A
↓ ←RDRP
• Influenza A virus remains one of the most crucial health
+RNA (mRNA)→Proteins (replicative) problems worldwide.
↓ ↲ o During 2018-2019, many samples are still being
received by laboratories.
-RNA→Proteins (structural) o Prior to COVID-19 outbreak, as many as 30-40
↓ ↲ samples per day for culture.
o It is cultured for 7 days for:
Progeny Virus
▪ (1) Identifying the sample’s CPE (cytopathic
• Genome: negative stranded RNA viruses (-RNA) effect) characteristic such as how fast it is
▪ (2) Typing the culture whether it is H1N1, H2N2,
o Negative sense RNA H3N4, etc.
• RNA-Dependent RNAPolymerase (RDRP) ▪ (3) Surveillance purposes for vaccine
preparation.
o Creates an anti-sense (+ RNA = equivalent to an
mRNA)  Reports are prepared and submitted to the
o mRNA is ready for translation into proteins government for preparation of next year’s
vaccine.
• mRNA is translated to proteins
o production of structural component of viruses (e.g.,
capsomeres, capsid)
• From + RNA template → create – RNA using RDRP
• Once structural proteins are formed, genome (-RNA)
can enter → forming Progeny Virus

EnvelopedssRNA Negative Sense Viruses:

Orthomyxoviridae

• Influenza Virus Type A, B and C

o Originate as zoonoticinfections, being carried by a
number of different species of birds and mammals
Trend of Influenza A Viruses throughout the Years
▪ H7N3 virus – for domestic ducks
▪ H7N9 virus – for wild birds
▪ Multiple H9N2 viruses – for domestic poultry • In the pandemic of 1918 and 1919, the virus is
estimated to have killed between 20 and 50 million
people.
o H / HA – hemagglutinin
▪ Spike of influenza
▪ Function: introduction
o N / NA– neuraminidase
▪ Function: removes/cleaves the cell surface from
inside in order to allow the virus to exit the cell

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 LECTURE UNIT 11: RNA VIRUSES – BALTIMORE’S CLASSIFICATION GROUP V

Basic Structure of influenza Antigenic Drift

• Each year antigenic drift occurs, caused by RNA

replication error of the virus
o RNA viruses don’t have a correcting mechanism
leading to high chances of having replication error
• A drift is a minor change in antigenic structure as
mutations accumulate

Antigenic Shift

• Sometimes the surface antigens can change drastically,

• Hemagglutinin Spikes (H) causing an antigenic shift, resulting in a new H or N
o Involved in cell attachment antigen
o 16 separate H types in influenza A • Can lead to pandemics or epidemics
Two Mechanisms:
• Neuraminidase (N)
o Enzyme involved in release form infected cell • (1) Genetic reassortment of the 8 ssrNA of 2 separate
▪ Degrades the inner layer of the cell to allow for influenza strains
virus release.
o 9 separate N types in influenza A
• RNA genome
o Single-stranded RNA is highly mutable
o Due to 8 separate segments that permit genetic
“reassortment” between viruses during double
infection
▪ Orthomyxoviridae – 8 separate segments;
single stranded RNA
▪ Reovirus and Rotavirus – 11 segments; double-
stranded RNA
• COMBINATIONS:
o EXAMPLES: H6N2 and H1N4

Influenza Virus type A, B and C

o Genome characteristics (segmented) lead to antigen
• There are 16 H shifts
antigens, o Needs 2 parent viruses
although human
infections usually ▪ E.g. H2N2 and H1N1
only occur with ▪ Reassortment leads to the creation of a new
H1, H2, and H3. type of virus
There are total of o Pigs have receptors of both avian and human
9 N; human influenza viruses, as well as swine influenza viruses,
infections usually and be co-infected with all three types of viruses
occur with N1 and N2.
• EXAMPLE: A/Fujian/411/2002 (H3N2) ▪ Pigs are “recyclers” because they are capable
of recycling all different types of influenza viruses
o A – Virus type → creating a new type of virus → causing
o Fujian – Geographic origin epidemic strains
o 411 – Strain number
o 2002 – Year of isolation • (2) Adaptive Mutation
o (H3N2) – Virus Subtype o A novel virus slowly adjusts and becomes
Mutations transmissible from a mammalian (including human)
host
o Common factor involved is the environment
• TWO TYPES: o Example
o (1) Antigenic drift
▪ A virus will try to survive and try to replicate.
o (2) Antigenic shift
Mutation is needed by viruses to enter hosts e.g.
• The key to persistence of the humans
influenza virus is its antigenic
variation

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o External factor forces the virus to mutate to adapt to • (3) If the virus cannot enter the nucleus, it cannot create
the environment a virion.
Influenza Virus Type A, B, C
ENVELOPED ssRNA NEGATIVE SENSE VIRUSES
• Spread by aerosol
Paramyxoviridae
• The viruses attack the ciliated epithelial cells lining the
respiratory tract, causing necrosis, and sloughing of the • Several genera belong to the family Paramyxoviridae,
cells including:
• Incubation period is 1 to 4 days
• Can be identified in respiratory secretions by Direct o Paramyxovirus
Fluorescence Assay (DFA), EnzymeImmunoAssay o Rubulavirus
(EIA), and optical immunoassays o Morbillivirus
• Culture lines: PMK and MDCK cells o Pneumovirus

Laboratory Diagnosis • The genome

characteristics of
Paramyxoviridae:
• Nasopharyngeal swabs, washes, or aspirates collected
early in the course of the disease are the best o Helical
specimens
▪ Unlike the Orthomyxoviridae that is segmented.
• Flocked swabs are reported to collect significantly
more epithelial cells than rayon swabs from the o Hemagglutinin-neuraminidase (HN)
nasopharynx
▪ The HN is combined already in one antigen.
o Flocked swabs are bigger and are capable for high
yield collections o Fusion protein (F)

Rapid Kits Parainfluenza Virus (PIV)

• Four types of PIV:

• (1) Detect and distinguish between influenza A and B
• (2) Detect both but not distinguishable between them o Human PIV 1 and 3
• (3) Detect only influenza A ▪ genus Paramyxovirus
Treatment o Human PIV 2 and 4

• Amantadine and Rimantadine ▪ genus Rubulavirus

o Prevent infection or reduce the severity of symptoms • PIV are enveloped helical
if administered within 48 hours. RNA viruses with two
surface antigens:
• Neuraminidase inhibitors
o Hemagglutinin-
o New antiviral drug neuraminidase (HN)
antigen
NOTE:All DNA viruses will replicate in the nucleus except o Fusion (F) antigen
poxvirus. On the contrary, majority of the RNA viruses will
replicate in the cytoplasm except influenza virus. • PIV can enter the nucleus
because it uses large RNA
• Influenza replicate or release their nuclei inside the polymerase protein.
nucleus.
Mode of Action of the Amantadine Drug
• PIVs are a major
cause of respiratory
disease in young
children.
o They cause
croup.
▪ This is an inflammation on the airway specifically
the trachea.
• PIVs 1 and 2 cause the most serious illness in children
between 2 and 4 years of age.
• CROUP: a tough disease where there’s inflammation or
• (1) The amantadine does not allow the uncoating of the swollen tissue of the trachea
influenza virus. • PIV-1 is the primary cause of croup
• (2) Thus, the genome of the virus cannot be released (laryngotracheobronchitis) in children
and enter the nucleus. • The best specimens for viral culture are aspirated
secretions and nasopharyngeal washes.

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• Specimen for viral isolation: PMK cells o Virus isolation is preferable, although physician
• Serologic assays are more valuable for epidemiology rarely have trouble recognizing mumps clinically.
studies than for diagnostic purposes.
• NO VACCINES are available Measles virus
• Termed as “parainfluenza virus” because they have
• Classified in the genus Morbillivirus
almost the same clinical manifestation of influenza
• Highly contagious and spreads by aerosol
o “para”: almost same/ almost alike
o Initial replication takes place in the mucosal cells of
o Same clinical manifestation with influenza but
the RT then in the local lymph nodes, circulates in
different causative agent
the T and B cells and monocytes and spread
Mumps Virus systemically.
o After spreading it will
• Related to the PIVs and are classified in the genus be seen as skin rashes
Rubulavirus
• Incubation period is to 7 to
• With HN and F antigens 10 days
• Spread by droplets of infected saliva and has a • Prodromal symptoms:
worldwide distribution fever, sneezing, cough,
red eyes and Koplik’s spot
(enanthem)
▪ Enanthem– rash inside the body (Ex: Koplik’s
spot)
▪ Exanthem – rush outside the body
o About 2-3 days later, a
maculopapular rash
(main symptom)
appears on the head
and trunk
o Easily diagnosed
clinically
• Related to the PIVs and are classified in the genus o The virus is fragile and
Rubulavirus must be handled
• With HN and F antigens carefully
• Spread by droplets of infected saliva and has a o
worldwide distribution
▪ Note: all envelope
• It causes acute illness producing unilateral or bilateral
viruses are fragile
swelling of the parotid glands although other glands
compared to the
such as testes, ovaries, and pancreas can be infected
naked viruses
• The primary infection of the ductal epithelium cells in
▪ Treat all virus as
the glands results in the cell death and inflammation.
fragile
o For boys infected with mumps, there is a high
o Specimen of choice:
chance for infertility to occur since the virus can
Nasopharynx and urine
reach the testes
o Culture: use primary
• The virus infects primarily children and adolescents monkey culture cells,
• Confers long-lasting immunity kidney cells and
observed the cytopathic
o There is a very small chance for reinfection but it effect
always depends on your immune system o CPE on PMK: formation of distinctive spindle-shape
o Multiple infections are still possible only to people or multinucleated cells
with poor immunity o multinucleated cells are due to f protein
• The mumps virus can be isolated from infected saliva ▪ can actually spindle or bind to other cells =
and swabs rubbed over the Stensen’s duct. fusion and forming large nucleated cells
• The virus can also be isolated: Urine and CSF
• Relatively fragile: Specimens may be examined directly • Methods: Serum neutralization test (poliovirus 1,2, and 3),
by IF and EIA methods. enzyme immunoassays (EIA), or immunofluorescence (IF)
• Virus isolation is preferable, although physicians rarely tests
have trouble recognizing mumps clinically • Acute phase of the disease: Serology (IgM measles-
specific)
o Inflammation on the parotid area is only associated
with mumps virus Respiratory Syncytial virus (RSV)
o If the patient is an infant, clinical manifestations
similar to mumps virus can be associated with • Member of the Pneumovirus
bacterial infection (Corynebacterium diphtheriae) • Most common virus isolated from infants with lower tract
respiratory infection or LRTI

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• Causes croup, • The structure of Rhabdoviridae is bullet formed

bronchitis,
o Presence of matrix protein, lipid bilayer, helical
bronchiolitis or
single-stranded RNA virus
interstitial
pneumonia • The sample rabies under the electron microscope is a
• Infection does not brain
confer immunity;
multiple infections
can occur
throughout life
o Problem: produced antibodies and immune
response are very poor causing multiple infections
• Can be severe to elderly, immune-compromised and
with lung problems.
o For example: asthmatic
o In Japan, high cases of RSV in children and elderly
• RSV can be identified in specimens from
• Humans usually acquire the rabies virus when they are
nasopharyngeal swabs and washes by direct
bitten or scratched by rabid animals such as dogs, cats,
fluorescence assay and/or enzyme immunoassays
bats, etc.
• Virus is
extremelyfragile. o (1) The virus enters tissue from saliva of the biting
• Grows readily in animal
continuous o (2) The virus replicates in the muscle near the bite
epithelial cell lines, o (3) The virus moves up the peripheral nervous
such as HEp2, system to the CNS in the spinal cord
forming syncytia. o (4) The virus ascends to the spinal cord
o (5) Virus reaches brain and causes fatal encephalitis
o still a o (6) Virus enters salivary glands and other organs of
multinucleated victim
cell like measle
• Prodromal period: pain in the exposure site and have
• Antiviral compound Ribavirin vague flulike symptoms
is approved as a treatment.
o The closer to the brain,
o approved for the treatment of the faster the
respiratory single-cell virus manifestation of infection
• Mental status changes, such
as anxiety, irritability, and
ENVELOPED ssRNA NEGATIVE SENSE VIRUSES depression, may also
become evident
Rhabdoviridae
• After prodromal period:
hallucinations, paralysis, excessive salivation,
Rabies Virus
hydrophobia, and seizures
• Rabies is caused by several o This is because the infected part of the brain is in
strains of viruses belonging to the cerebellum and hippocampus
the genus Lyssavirus
• 40 years ago, most rabies • Rabies virus is one of the best examples of the
infections occurred in dogs, ascending virus
with some infections also o Even though the victim is bitten at one’s foot, it will
occurring in cats, foxes, and ascend and go to the brain
skunks
• Laboratory diagnosis involves determining whether an
o Pets should be vaccinated for rabies animal that has bitten a human has rabies
• Animal is killed → head is removed → sent to reference
laboratory
o In the Philippines, the common practice is to monitor
the animal (not necessarily remove the head
immediately) – and immediate treatment for the
bitten person
• The fastest and most sensitive method of identifying
rabies viruses in a specimen is by using direct
Structure of the Rhabdoviridae and Rabies virus under EM
immunofluorescent techniques.

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 LECTURE UNIT 11: RNA VIRUSES – BALTIMORE’S CLASSIFICATION GROUP V

• Impression smears of the brain: Hippocampus, pons, • Is the more virulent species
cerebella, and medulla oblongata • Mortality rate is 88%
• Rabies cannot be successfully treated once
o Only 12% chance of survival
symptoms appear.
o However, post exposure prophylaxis is 100% • Ebola virus is somehow the same with COVID in terms
effective in preventing the disease if the patient is of origin
treated sufficiently early. o Its origin are bats and are transferred to
intermediate hosts and to human hosts
▪ Prophylaxis → either antibodies that will
neutralize the rabies virus so that it will not Ebola-Sudan (EBO-S)
replicate and therefore will not reach the brain
• Mortality rate is 53%
Ebola Virus Disease

• Post exposure prophylaxis

o (1) Vigorously cleaning the wound site
o (2) Providing human rabies immune globulin
▪ If bitten within 24 hrs., it should be administered
immediately
o (3) Administering three injection series of rabies
vaccine • Ebola, which first appeared in outbreaks in Sudan and
Dr Congo in 1976, is a severe and often fatal disease
with no known specific treatment or vaccine. It has
ENVELOPED ssRNA NEGATIVE SENSE VIRUSES since killed more than 1,500 people in parts of Africa.
• Source
FILOVIRIDAE
o In Africa, particular species of fruit bats are
Ebola Virus considered possible natural hosts for Ebola

• Named after the Ebola river in the Democratic Republic • Transmission

of Congo, 1976 o Infected bats are thought to transmit the disease to
• In Zaire, a patient treated at a village hospital for a humans, or indirectly through other animals which
bloody nose probably introduced the virus into the are hunted for their meat.
hospital → nosocomial → nuns routinely reused syringe
o Reusing of syringe has been a problem in 1976 due
to lack of materials
o Possible routes
▪ Close contact with the blood, secretions, organs
or other bodily fluids of infected or dead animals
▪ Consumption of infected bushmeat
▪ Touching objects that have come in contact with
the virus.
• Damage
o Incubation period is from 2 to 21 days.
o Death from the disease is often caused by multiple
organ failure and tissue death.
o Targets in the body:
• Diagnosis of the infection can be made using PCR, IF ▪ Hepatocytes, functional cells of the liver
(immunofluorescence), or viral culture methods ▪ Endothelial cells, which form the linings of the
blood vessels
o PCR is the most reliable ▪ Phagocytes, blood cells that absorb foreign
Ebola-Zaire (EBO-Z) particles

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 LECTURE UNIT 11: RNA VIRUSES – BALTIMORE’S CLASSIFICATION GROUP V

o Symptoms
▪ Fever
▪ Sore throat
▪ Severe headache
▪ Muscle pain
▪ Intense weakness
▪ Vomiting
▪ Diarrhea
▪ Impaired liver and kidney function
▪ Internal and external bleeding
Ebola-Reston (EBO-R)

• Third type of Ebola virus isolated from the Philippines

o Philippines is actively exporting monkey cells for the
laboratory diagnosis in the United States. In the
U.S., they tested the monkeys and it tested positive
for EBO-R
• Weakest Ebola specie and can cause asymptomatic
infection
• It was named after Reston, Virginia, United States
where the virus was first discovered
• In the Philippines, four workers in the animal facility
developed antibodies to EBO-R but did not develop
disease.

ASUMBRA. BABAO. CATAPANG. CHIN. COCHING. JUNIO. MAHINAY. MARAVILLA. PIENCENAVES. RELATOR. ROSALINDA. BSMLS 3 7
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 [TRANS] LECTURE UNIT 12: RNA VIRUSES GROUP VI AND VII

RNA VIRUSES GROUP VI: HUMAN o A published report showing the demographic of the
IMMUNODEFICIENCY VIRUS-I HIV in the Philippines per 3 months
o WHO – gets the demographic and numbers of male
• Originally, Human Immunodeficiency Virus-I (HIV-I) is
and females who are infected with HIV in the
under Baltimore’s Classification Group IV Philippines
o However, HIV-I contains reverse transcriptase
(RT), thus transferring it into group VI Transmission

Background Transmission via:

• Epidemiology (according to WHO)

o 33.2 million living with HIV infection
o 2.5 million newly infected per year
o 2.1 million died of AIDS
• HIV is a virus that causes the HIV infection, while
Acquired Immunodeficiency Syndrome (AIDS) is a
condition.
o Contracting HIV can lead to the development of
• Unprotected sex
AIDS
o However, having an HIV infection does not o Practice safe sex
guarantee the development of AIDS because there
are antiretroviral drugs available • Pregnancy, childbirth, and breastfeeding
• Injecting drugs
§ Antiretroviral drugs – taken by HIV-infected
patient to control the viral load o HIV can survive outside the body

w If the viral load will not be controlled, HIV will • Working in healthcare
develop to AIDS o Health care workers have a higher risk of getting
• AIDS develops when HIV causes serious damage to the HIV
immune system § Does not necessarily mean that if you are
o The tropism of HIV-I and HIV-II is lymphocytes (key working in a hospital or health care facility you
players of the immune system) can get HIV
§ The target is the lymphocytes and the mode of • Blood transfusions and organ/tissue transplant
replication is lysogenic or destroying the cell à o Before blood transfusion all blood should be tested
leads to a decreased number of lymphocytes. with HIV
w There will be fewer lymphocytes that will kill o Organ/tissue transplant will be tested before the
opportunistic organisms such as bacteria and transplant
fungi.
HIV is not transmitted by:
• Former names:
o Human T-cell lymphotropic virus-type III (HTLV-III)
o Lymphadenopathy-associated virus (LAV)
o AIDS-associated retrovirus (ARV)
• HIV-1 – most common worldwide
• HIV-2 – commonly found in Africa
o Less pathogenic and has a lower rate of
transmission • Insect bites
• Toilet seats
• In the Philippines, health professionals also detect HIV-
2 in screening tests, since it is included along with HIV- • Kissing
1. o There are some cases that the virus is contained in
• The Department iof Health (DOH) in the Philippines has the saliva but according to some journals, 2 liters of
HIV/AIDS & ART (antiretroviral therapy) Registry saliva is needed in order to transmit the virus
• Sharing cutlery

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 LECTURE UNIT 12: RNA VIRUSES GROUP VI AND VII

o Lesser chance to transmit HIV as compared to other components (e.g., transmembrane,

Hepatitis A and E which has an oral-fecal route receptors and envelope proteins)
(higher chance to get the virus)
• (3) Reverse transcriptase
• Touching
Structural Genes
o Needs penetration and high concentration of body
fluids to get HIV • Matrix – p6, p9, p17, p24
• Attachment – gp160, gp120, gp41
Characteristics of HIV
• Replication – p66, p51, p31, p10
• Enveloped • Regulation – p14, p15, p16, p19, p23, p27
• Capsid – cone appearance
Replication
• Composition:
o Family: Retroviridae
Spike Protein
§ It has a Reverse
Transcriptase –
meaning, it is
capable of going
back
Lymphocyte or
o Genus: Lentivirinae
macrophage
§ Lenti (latin word) – slow
§ HIV is a slow progressing virus, but there are
some strains that are high progressive
o A retrovirus
o Single stranded RNA (ssRNA), with Reverse
transcriptase (RT) à Group VI • The virus uses the co-receptors:
§ No RT and a (+) sense à Group IV o CXCR4 for lymphocytes
o Spherical, 100-120nm (small) o CCR5 for macrophages
• (1) Once the virus enters the cell, it will inject its
Western Blot Pattern
genome into the cytoplasm.
• gp 160 and gp120 – envelope • (2) With the function of reverse transcriptase, it will
protein create a complementary single-stranded DNA (ssDNA)
• p66 – reverse transcriptase from the virus’ RNA.
• gp41 – transmembrane • (3) The ssDNA will then enter the nucleus, where
protein replication occurs.
• gp 31 – integrase • (4) With the use of integrase, the genome of the host
will be cut and provirus will be integrated into it
• p24 – core protein
• (5) During replication, mRNA will be produced, which
o Usual marker used for will now produce proteins
quantifying the viral load • (6) Since the cell regularly replicates, the virus would
also be replicated, resulting to a higher production of
the viral load.
Three Enzymes of HIV
Summary of replication
• (1) Integrase
o An enzyme that will • (1) Always starts with uncoating because the virus is
integrate the enveloped
genome of the • (2) Once the virus is uncoated, it will go into the
virus to human cytoplasm since HIV is an RNA virus
genome, which is the o RNA viruses with RNA genome must be in the
reason why HIV have cytoplasm
a high tendency of latency
• (3) Reverse transcriptase makes a complementary
• (2) Protease DNA
o An enzyme that will degrade the peptide • (4) Integration – will enter the nucleus, cut the human
o Problem of HIV – during their replication cycle, genome and insert provirus inside the nucleus
specifically during translation, they produce a large, o Must take place in the nucleus since reverse
long peptides. transcriptase turns RNA into DNA and DNA is
§ Will not become a viral component placed inside the nucleus
§ Proteases will cut the long peptides of HIV to • (5) Transcription – produce mRNA.
produce short peptides which is either part of
• (6) Translation – to produce proteins.

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 LECTURE UNIT 12: RNA VIRUSES GROUP VI AND VII

o Since the protein production of HIV is large, it needs o (2) CXCR4 Inhibitor
to be cut by protease
• Integrase inhibitors
• (7) Assembly • Highly Active Antiretroviral Therapy (HAART)
• (8) Acquisition of envelope
o Combination of two
• (9) Egress or exit o Can be nucleoside analogue inhibitor + one
Immunologic Manifestations protease inhibitor
o Combination of two drugs will depend on the
• Increased p24 antigen – initial viral replication physician and the guidelines given by WHO
o Usually, test kits have p24 antigen such as ELISA to Laboratory Testing of HIV
identify and determine the p24 concentration.
• anti-p24 – first antibodies formed CD4 T- cell Enumeration

o Followed by envelope, polymerase, then regulatory • One of the criteria to classify if the HIV infection already
antibodies progressed to AIDS is by CD4 count
o Window period - takes approximately 6 months to • Flow cytometry
1 year (gold standard)
§ Within 6 months, antibodies are not produced o Forward scatter
yet or other immunological parameters are still – measures cell
negative size
o Side scatter –
• Envelope – most immunogenic measures
o Comprised of proteins and lipids granularity
• Cytotoxic lymphocytes can suppress replication and
spreading the virus.
Detection of HIV antibody
Evasion Mechanisms
ELISA (Enzyme-linked immunoassay)
• Mutation
o HIV is an RNA virus which has a high mutation rate • Antibodies are used for screening in the laboratory
• (1) ELISA Kit Immunochromatography
• Down-regulation of MCH class I on cell surface
o Used for screening
• Silent proviral state – due to HIV integrase. o ex: screening HIV in blood bags
Acquired Immunodeficiency Syndrome (AIDS) • (2) ELISA
• Cd4 count – less than o Done if the ELISA kit immunochromatography is
200/ul positive
• Profound o Submitted to the reference laboratory
immunosuppression Western blot
• Resurgence of viremia
o Results to a high • Confirmatory test
concentration of p24 • Detects the antigen or viral proteins
antigen. • Positive predictive
o Using PCR, there is value:
high viral load.
o >99% for low
• Life-threatening infections and high risk
o Patients does not typically die due to AIDS, but • Positive result
rather due to secondary infections, specifically
bacteria and fungi. o At least two of
o The virus kills the lymphocytes, which weakens the the following:
immune system § p24
• Malignancies § gp41
§ gp120/gp160
Treatment of HIV
• If intermediate
• Nucleoside analogue RT inhibitors o ex: a line on p55 & p24
• Nonnucleoside RT inhibitors o Only p24 is considered positive
• Protease inhibitors o Do not report negative result and rerun
• Fusion inhibitors
• Coreceptor antagonists § If the results are still the same, follow the
guidelines for WHO
o (1) CCR5 Inhibitor

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 LECTURE UNIT 12: RNA VIRUSES GROUP VI AND VII

w Repeat testing after 1 month, 3 months, or 6 o Branched-Chain DNA assay (bDNA)

months (depends on the country or in the
laboratory) § Hybridization
o Nucleic Acid Sequence-Based Amplification
• Negative
(NASBA)
o No band formed
BALTIMORE CLASSIFICATION: GROUP VII
Densitometry
Hepatitis B Virus
• Measures intensity of bands to quantitate levels of
antibody • Originally under group I Baltimore’s Classification
because it is a double-stranded DNA incomplete but
NOTE: transferred to group VII because it contains reverse
transcriptase.
• Densitometry – quantitate antibody
• Western blot – detect different proteins of HIV and o Due to this, David Baltimore created an additional
serve as a gold standard group that possesses the same mechanism as
Group VI – Human Immunodeficiency Virus
o Detects the antigen of the viruses
§ HIV was originally under Group IV but
Other Confirmatory Tests transferred to Group VI
§ Hepatitis B virus was originally Group I but
• IFA (Immunofluorescence Assay) transferred to Group VII.
• RIPA (Radioimmunoprecipitation Assay)
• Line immunoassays Structure and Characteristics
• Rapid confirmatory tests

Detection of HIV Antigen

P24 Antigen Testing

• Very common – In research, this test is usually used.

• Enveloped
o Contains
• dsDNA incomplete
• Genus: Hepadnavirus
o Hepa – Liver
o DNA – DNA
• (1) Anti-p24 monoclonal antibody is embedded on § A DNA virus that infects the liver.
the wells • Family: Hepadnaviridae
• (2) The sample is added, which will attach to the • Mode of Transmission:
monoclonal antibody
• (3) Add biotinylated anti-p24 polyclonal antibody o Parenteral
o Sexual
o p24 antigen is sandwiched between the biotinylated o Perinatal
anti-p24 polyclonal antibody and anti-p24 o Oral-Fecal Transmission
monoclonal antibody
o biotinylated anti-p24 polyclonal antibody has • Hepatitis B Proteins:
peroxidase
o Envelope CHON – HBsAg
• (4) Add labelled streptavidin § HBsAg - Hepatitis B Surface Antigen
o Once this is attached to the peroxidase, it will o Structural nucleocapsid core CHON – HBcAg
release HNO3 and will produce color reactions
§ HBcAg – Hepatitis B Core Antigen
Nucleic Acid Testing
o Soluble nucleocapsid CHON – HbeAg
• Viral load assays § Hepatitis B E antigen
o PCR § B-related antigen

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 LECTURE UNIT 12: RNA VIRUSES GROUP VI AND VII

o Majority thinks that the E is for the envelope but it’s

not
• One incomplete strand
• Viral DNA is incorporated to host’s DNA
o Q: If Hepatitis B has the capability to incorporate
their viral DNA to host DNA, does it mean it they
have integrase?
§ Ans: Hepatitis B doesn’t have integrase enzyme.
However, they are able to integrate parts of their
genome to the host cells.
o “Surface” - can be seen in the envelope part
Signs and Symptoms • HIV is not transmitted by touching, penetration is
needed in order for HIV to be transmitted
• Long incubation hepatitis – • There should also be higher concentration of body fluid
45-90 (ex. Semen)
o 45-90 days before the
appearance of first Characteristics of HIV
symptoms either
jaundice • Has spike protein envelope
• Capsid has a cone appearance
• Vasculitis,
• Contains 3 enzymes:
glomerulonephritis, arthritis
and dermatitis may o Integrase, protease and reverse transcriptase
manifest
§ Protease : degrade peptide
o This is due to the
w HIV during translation produce a large long
production of antibodies that will try to neutralize the
peptides, so the virus bring its own protease
antigen and will have the antigen-antibody reaction,
to cut the long peptides of HIV to produce a
then will be filtered to different parts of the body
short peptides which is part of other
including the glomerulus (which is why we acquire
components (transmembrane proteins,
glomerulonephritis)
receptors or envelope proteins).
• ~1% fulminant liver disease – high fatality Composition
o More or less 1% fulminant liver disease
o High fatality if this is developed • Family: Retroviridae
• Chronic infection = viral load correlates with the risk for o It has reverse transcriptase so it is capable to go
cirrhosis and hepatocellular carcinoma back.
o Goal is to at least neutralize the virus in order for the • Genus: Lentivirinae
viral load to decrease
o Lenti is a latin word for slow
• Most frequently asymptomatic/ subclinical o Virus is actually a slow progressing virus.
• A retrovirus
Diagnostic Evaluation • ssRNA
o RT- GROUP VI
• Hepatitis B Surface antigen (HBsAg)
o First detectable marker • Spherical, 100-120 nm
o Acute: peaks, then declines upon recovery Western Blot Pattern
o Chronic: remains elevated for ≥ 6 months
o Active infection
§ Positive for HBsAg

• Envelope protein: gp160 and 120

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 LECTURE UNIT 12: RNA VIRUSES GROUP VI AND VII

• P66 reverse transcriptase • Immunohistochemistry (liver biopsy tissues)

• Gp31 integrase
• P24 core protein
o Marker for quantifying the viral load
Acute Hepatitis

• Incubation period: 90 days (other references)

Prevention and Treatment

• Vaccination (Hepatitis B vaccine)

• Proper donor screening
• Liver transplant

• Incubation: average 8-13 weeks

o The virus is still observing
• Acute infection: 2 weeks – 3 months
o Manifestation of HBsAg, HBeAg, symptoms
• Early recovery: 3-6 months
o Anti-HBc total and anti-HBc IgM will be produced
§ Total = IgG and IgM
• Recovery: 6-12 months
o Anti-HBs and anti-HBe
Chronic Hepatitis
• Has persistent HBsAg and total anti-HBc
o Stationary HBsAg
• IgM anti-HBc lowers in the long run

Diagnostic Evaluation

• Detection of Hbs antigen by chemiluminescent

microparticle immunoassay
• Qualitative chemiluminescent immunoassay
• Qualitative EIA (for viral antigens and viral antigen
antibodies)
• Real-time PCR (quantitative)

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