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 Porcine Viruses
From Pathogenesis to Strategies for Control

Edited by
Hovakim Zakaryan

Caister Academic Press

Porcine Viruses
From Pathogenesis to Strategies for Control

https://doi.org/10.21775/9781910190913

Edited by

Hovakim Zakaryan

Group of Antiviral Defense Mechanisms

Institute of Molecular Biology of the
National Academy of Sciences
Yerevan
Armenia

Caister Academic Press

Copyright © 2019

Caister Academic Press

Norfolk, UK

www.caister.com

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library

ISBN: 978-1-910190-91-3 (paperback)

ISBN: 978-1-910190-92-0 (ebook)

Description or mention of instrumentation, software, or other products in

this book does not imply endorsement by the author or publisher. The author
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their use.

All rights reserved. No part of this publication may be reproduced, stored in

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 Contents

Prefacev

1 African Swine Fever Virus 1

Erik Arabyan, Armen Kotsinyan, Astghik Hakobyan and
Hovakim Zakaryan

2 Classical Swine Fever Virus 21

Sandra Blome

3 Foot-and-Mouth Disease Virus 43

Francisco Sobrino, Flavia Caridi, Rodrigo Cañas-Arranz and
Miguel Rodríguez-Pulido

4 Porcine Circoviruses 81
Sheela Ramamoorthy and Pablo Piñeyro

5 Porcine Epidemic Diarrhoea Virus 107

Changhee Lee

6 Porcine Parvovirus 135

André Felipe Streck and Uwe Truyen

7 Porcine Reproductive and Respiratory Syndrome Virus 149

Alexander N. Zakhartchouk, Sujit K. Pujhari and
John C.S. Harding

8 Swine Vesicular Disease Virus 181

Estela Escribano-Romero, Miguel A. Martín-Acebes,
Angela Vázquez-Calvo, Emiliana Brocchi, Giulia Pezzoni,
Francisco Sobrino and Belén Borrego

Index199
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www.caister.com
 Preface

For humans, pork meat is one of the most complete dietary sources of protein. According to
the Food and Agriculture Organization of the United Nations, animal protein production
will grow three times by 2050, and meat production, including of pork, will double. An
increase in intensification is necessary, because arable land cannot be increased in propor-
tion. However, viral diseases cause significant animal losses and represent a major threat to
global pig farming industry. Therefore, the understanding of molecular biology, pathogen-
esis, host–virus interaction and epidemiology of these viruses is essential for reducing the
burden of viral outbreaks.
The overall aim of this book is to review the most important and dangerous porcine
viruses that have emerged in the global swine population. It covers different DNA and RNA
viruses about which we have learnt much during the last decades. In Chapter 1, my col-
leagues and I discuss African swine fever virus (ASFV), the causative agent of highly lethal
haemorrhagic fever of domestic pigs and wild boar. It is a large, enveloped, double-stranded
DNA virus that is the only known DNA arbovirus since it is transmitted by soft ticks of the
genus Ornithodoros. Most recently, ASFV was introduced into Georgia and then spread to
the Russian Federation, Belarus, Ukraine, and some EU member states. In the absence of
effective vaccines and antiviral drugs, this virus continues to pose a global risk for pig indus-
try. Chapter 2 is about classical swine fever virus (CSFV), a small enveloped RNA virus of
the genus Pestivirus in the Flaviviridae virus family. Although live attenuated vaccines are
currently available, it remains a major threat to profitable pig production worldwide because
CSFV infection is still associated with high mortality rates. Sandra Blome summarizes
CSFV properties, pathogenesis, clinical picture and control options. Chapter 3 focuses on
foot-and-mouth disease virus (FMDV), which is the prototypic member of the Picorna-
viridae family. It causes an acute systemic vesicular disease affecting livestock worldwide.
Francisco Sobrino and colleagues discuss different aspects of FMDV infection, including
new strategies for viral control by vaccination and other antiviral strategies. This chapter also
covers the current approaches for virus diagnosis. In Chapter 4, Sheela Ramamoorthy and
Pablo Piñeyro provide an overview of porcine circoviruses, focusing particularly on porcine
circovirus strain 2 (PCV2), which was first isolated in 1997. PCV2 is a single-stranded DNA
virus belonging to the Circoviridae family. PCV2 infection by itself causes only mild disease
but co-factors such as other infections are involved in the development of severe diseases.
Molecular biology, pathogenesis, immune response, diagnosis and control strategies are
discussed in detail. Chapter 5 is about porcine epidemic diarrhoea virus (PEDV), the aetio-
logical agent of severe diarrhoea and dehydration. Although this virus was first reported in
vi | Preface

Europe, it has become problematic in Asian countries such as China, Japan, Thailand and
the Philippines. Owing to high morbidity and mortality in piglets, PEDV has a substantial
economic burden in affected countries. Changhee Lee discusses molecular and cellular biol-
ogy of the virus, as well as diagnostic procedures, epidemiology and control strategies. In
Chapter 6, André Felipe Streck and Uwe Truyen describe the biology, pathogenic potential
and strain variation of porcine parvovirus (PPV). This virus is considered the main cause
of reproductive disorders in pigs with no maternal clinical signs. PPV is a small, non-envel-
oped, single-stranded DNA virus belonging to the Parvoviridae family. Although losses are
low in vaccinated herds, PPV can cause devastating abortion storms in unvaccinated herds,
or in those herds, where new antigenic types are circulating. Chapter 7 is devoted to por-
cine reproductive and respiratory syndrome virus (PRRSV), a small enveloped RNA virus
belonging to the Arteriviridae family. PRRSV causes reproductive failure in herds and res-
piratory tract illness in young pigs. In this chapter, Alexander Zakhartchouk and colleagues
summarize the current understanding of PRRSV, including the virus molecular biology,
virus–host cell interactions, pathogenesis, diagnostic procedures and epidemiology. They
also provide an overview of currently available vaccines and a novel vaccine development.
The last chapter (Chapter 8) is about swine vesicular disease virus (SVDV), which belongs
to the Enterovirus genus within the Picornaviridae family. SVDV is genetically highly related
to the human coxsackie virus B5. It causes a vesicular disease with clinical signs similar to
those of foot-and-mouth disease. Francisco Sobrino, Belén Borrego and colleagues discuss
different aspects essential for understanding the infectious cycle of SVDV. They also provide
an overview of current strategies for SVDV control by vaccination and other antiviral strate-
gies.
It took about a year to complete this book, during which time the chapters were writ-
ten, edited and re-edited in order to improve and present high-quality reading material. My
primary acknowledgement must go to all our contributing authors for their significant work,
enthusiasm and cooperation. In addition, I thank Annette Griffin from Caister Academic
Press for her great assistance and patience during all this time. Finally, but most importantly,
I wish to extend appreciation to my family members, particularly to my beautiful wife,
Kamila, for her invaluable support throughout the writing and editing process.

Dr Hovakim Zakaryan
African Swine Fever Virus
Erik Arabyan, Armen Kotsinyan, Astghik Hakobyan and
Hovakim Zakaryan*
1
Group of Antiviral Defense Mechanisms, Institute of Molecular Biology of the National Academy of
Sciences, Yerevan, Armenia.
*Correspondence: [email protected]
https://doi.org/10.21775/9781910190913.01

Abstract
African swine fever virus (ASFV) is a large DNA virus belonging to the family Asfarviri-
dae. It is the causative agent of an acute haemorrhagic fever in domestic pigs and wild boar
with high fatality rates, up to 100%, in the acute forms. The disease is currently present in
Africa and Europe, where it causes a high socio-economic impact. In this chapter we have
addressed different aspects of ASFV including the biology of the virus which is relevant for
understanding the viral disease. We have also discussed the pathological changes caused by
high and low virulence ASFV strains. Finally, this chapter addresses the current approaches
for ASFV diagnosis, as well as presents an overview of research efforts towards the develop-
ment of effective vaccines during the past decades. As an alternative to vaccine development,
the current state in antiviral research is also presented.

History
The disease caused by African swine fever virus (ASFV) was first observed in settlers’ pigs in
Kenya in 1909 and was reported by Montgomery (1921) as an acute haemorrhagic disease
distinct from classical swine fever. After the first report, the disease was also observed in
South Africa and Angola (Gago da Câmara, 1932; Steyn, 1932). Although Gago da Câmara
determined that the disease observed in pigs was not erysipelas, the viral identity was con-
firmed in 1943 (Mendes, 1994). Interestingly, pigs belonging to the indigenous population
of Angola demonstrated increased resistance to African swine fever (ASF) suggesting that
they served as a source of infection for the pigs farmed by European settlers (Mendes, 1994).
During the same period, the link between warthogs and ASF was shown (Montgomery,
1921). However, the possible involvement of an argasid tick vector in the epidemiology of
ASF was confirmed much later, in 1960s, when Sánchez Botija (1963) reported about the
argasid tick Ornithodoros erraticus in the epidemiology of ASF in Spain. Studies undertaken
in southern and eastern Africa revealed ASFV in O. moubata complex ticks inhabiting both
pig shelters and warthog burrows (Penrith et al., 2004).
2 | Arabyan et al.

The first cases of ASF outside of Africa was reported in Portugal in 1957 and again in
1960 with its further establishment in the Iberian Peninsula for many decades (Wilkinson,
1984). ASF has also caused sporadic outbreaks in other European countries, such as France
(1964, 1967 and 1974), Italy (1967, 1969 and 1993), Andorra (1975), Belgium (1985),
Malta (1978), and The Netherlands (1986). However, in 1995, the disease was eradicated
from the continental Europe, except Sardinia, where it has been endemic since 1978. It is the
only territory beyond Africa that is considered endemic for ASF at the present time. In 2007,
ASF re-emerged in Europe for a second time, following a single introduction into Georgia in
the Caucasus, from which it spread rapidly to the Russian Federation, Armenia, Azerbaijan
and a number of countries in Eastern Europe, including some EU countries (Estonia, Lithu-
ania, Latvia, Poland, the Czech Republic and Romania). Owing to the lack of vaccines or
antiviral drugs, as well as in a context of increased trade activity, there is widespread concern
that ASF may spread beyond affected countries.

Classification
ASFV was initially classified as an iridovirus, based mainly on the similarity of virion mor-
phologies. However, increasing knowledge of ASFV biology, particularly genome analysis,
led to its reclassification as a new DNA virus family, Asfarviridae (Asfar, African swine fever
and related viruses) (Penrith et al., 2004). Until recently, ASFV was considered to be the
only known virus of this family. In 2009, the complete sequence of the type B DNA poly-
merase (PolB) gene of Heterocapsa circularisquama DNA virus revealed a close link with
Asfarviridae, suggesting a co-occurrence of marine and terrestrial viruses in the same family
(Ogata et al., 2009). In 2015, La Scola and colleagues isolated eight strains of a new giant
virus named faustovirus from Vermamoeba vermiformis (Reteno et al., 2015). The genomes of
faustoviruses showed that they are related to ASFV, although the faustovirus gene repertoire
is three times larger than that of ASFV. Two other viruses, kaumoebavirus and pacmanvirus,
were branched within the cluster encompassing ASFV and faustovirus (Bajrai et al., 2016;
Andreani et al., 2017) (Fig. 1.1). Thus, these findings and future viruses may deeply alter the
current knowledge of Asfarviridae family in many ways.
The comparison of ASFVs based on the partial sequence of B646L gene has allowed the
differentiation of ASFV isolates collected worldwide. Using results from partial sequencing,
phylogenetic analysis has divided ASFV isolated into 23 genotypes (Bastos et al., 2003). The
newest genotype was reported in Ethiopia (Achenbach et al., 2017).
ASFV is also classified as the only known arbovirus (arthropod-borne virus) with a DNA
genome, since soft ticks are involved in the sylvatic transmission cycle of ASFV in Africa (O.
moubata) and in Europe (O. erraticus).

Molecular biology of the virus

Structure of ASFV particles

The ASFV particle has an icosahedral shape with an average size of 200 nm. Two-dimensional
analysis of ASFV purified by Percoll gradient centrifugation has identified 54 structural pro-
teins with different molecular weights (Esteves et al., 1986). These proteins are localized in
several concentric domains: an internal core containing viral DNA and nucleoid coated by a
 African Swine Fever Virus | 3

Methanohalophilus mahii, Euryarchaeota

Faustovirus
100/1/100
Faustovirus ST1
99.9/1/100

Pacmanvirus A23
47.5/0.655/57
Heterocapsa ciruclarisquama
DNA virus 01
98/1/86

99.9/1/100 Kaumoebavirus

Georgia 2007/1

99.8/1/100

ASFV
Benin 97/1
85.6/0.955/74
Ken06.Bus

Klebsiella pneumoniae UHKPC27, Gammaproteobacteria

0.4

Figure 1.1 Phylogenetic tree based on the amino acid sequence alignment of DNA polymerase
beta of viruses from Asfarviridae family.

thick protein layer (core shell); an inner lipid envelope; and the capsid (Andrés et al., 1997).
The extracellular ASFV particles possess an external envelope acquired by budding from the
plasma membrane. Although the external envelope is similar to the plasma membrane, viral
proteins, p12, p24 and pE402R, have been reported to localize at the outer envelope (Sanz
et al., 1985; Carrascosa et al., 1993; Rodríguez et al., 1993).
The capsid is composed of about 2000 capsomers arranged in a hexagonal lattice. Com-
puter-filtered electron micrographs have revealed capsomers with a hexagonal outline and a
hole in the centre. The intercapsomer distance varies from 7.4 to 8.1 nm (Carrascosa et al.,
1984). Protein p72, encoded by viral B646L gene, is the main component of capsomers,
accounting for about one third of the protein mass of ASFV (Carrascosa et al., 1986). It has
been shown that the correct folding of p72 requires the expression of another viral protein,
pB602L, which acts as a molecular chaperone (Epifano et al., 2006a). Another component
of the capsid is pE120R whose incorporation into the virus particle is dependent on p72
expression. This protein is involved in the microtubule-mediated transport of ASFV par-
ticles from the viral factories to the plasma membrane (Andrés et al., 2001). The lack of
structural protein pB438L leads to the formation of viral particles with a tubular structure,
suggesting that it is likely to be involved in the stabilization of the icosahedral vertices of
ASFV particles (Epifano et al., 2006b).
In addition, the virion contains proteins for the transcriptional machinery, capping,
polyadenylation, nucleoside triphosphate phosphohydrolases and nucleoproteins such as
the DNA-binding protein p10 and pA104R, which is similar to the histone-like proteins of
bacteria (Salas, 1999; Andrés et al., 2002).
4 | Arabyan et al.

Viral genome structure

The viral genome is a linear double-stranded DNA molecule that varies in length between
different isolates from 170 to 193 kbp (Chapman et al., 2008). The differences in genome
length are due to gain or loss of open reading frames (ORF) from the multigene fami-
lies (MGF) encoded by virus. ASFV contains five MGFs (MGF 100, 110, 300, 360 and
505/530,) named to reflect the average number of codons present in each gene. Compari-
son of the genome sequences of ASFV isolates revealed multiple gene deletions in MGF 110
and 360 (Chapman et al., 2008). Moreover, repeated passages of field isolates through cell
cultures result in the loss of genes of the MGF 110 family, suggesting that these genes may
determine the virulence of ASFV in different hosts (Pires et al., 1997). The ASFV genome
contains a conserved, centrally located genomic core (125 kbp) in which major changes are
rare and high variability sequences confined to the left (38 to 47 kbp) and right (13 to 16
kbp) terminal regions (Fig. 1.2). Both strands of DNA encode genes that are closely spaced
and do not contain introns. Promoter sequences are short and A + T rich. The genome
termini are covalently cross-linked hairpin loops presented in two forms, inverted and com-
plementary to each other (González et al., 1986).

Cellular biology of the virus

Viral entry
The viral cycle starts with the infection of monocytes and macrophages, the natural target
cells of ASFV in pig. The restricted cell tropism of ASFV indicates that a monocyte/
macrophage-specific receptor is required for infection. CD163 is the high affinity scaven-
ger receptor whose expression is specific for the cells of monocyte/macrophage lineage. It
has been shown to be important for ASFV entry, since incubation of macrophages with a
specific anti-CD163 antibody decreases the level of infection in a dose-dependent manner
(Sánchez-Torres et al., 2003). However, more recent studies have reported that pigs pos-
sessing a complete knockout of CD163 by CRISPR/Cas9 are not resistant to infection
with ASFV Georgia2007/1 strain (Popescu et al., 2017), thereby suggesting that other
macrophage surface proteins are also involved in the infection process. On the other hand,
viral proteins responsible for ASFV binding to host cells have been described. Neutralizing
antibodies against viral proteins p12, p32, p54 and p72 inhibited both ASFV attachment
to and internalization into Vero cells and macrophages, indicating that these proteins are
involved in the early stages of ASFV infection (Carrascosa et al., 1991; Gomez-Puertas et

ASFV BA71V Genome

Left variable region Genomic core Right variable region

Genes: MGF300; MGF360; MGF505/530 Genes: MGF100;

MGF360; MGF505/530

Figure 1.2 Genomic regions of ASFV BA71V strain. Left and right variable regions contain
multigene families (MGFs).
 African Swine Fever Virus | 5

al., 1996). Interestingly, baculovirus-expressed p32, p54 and p72 proteins were not able to
protect pigs against the virulent virus (Neilan et al., 2004). This result suggests that other
viral proteins should be involved in ASFV entry, and therefore further studies may define
both viral and cellular proteins responsible for ASFV entry.
After attachment, ASFV enters the cell via two endocytic pathways: clathrin-mediated
endocytosis and micropinocytosis. The fact that ASFV utilizes clathrin-mediated endo-
cytosis to infect host cells is unexpected. This pathway is usually used by small- and
intermediate-sized viruses, whose diameter does not proceed 100 nm (Schelhaas, 2010).
Nonetheless, detection of ASFV particles within enlarged clathrin-coated vesicles indicates
that they can be strained to adapt to larger viruses like ASFV (Hernáez et al., 2016). Other
studies showed that ASFV uptake by clathrin-mediated endocytosis requires dynamin
GTPase activity, as well as the presence of clathrin-coated pit component Eps15 and cho-
lesterol in cellular membranes (Hernaez and Alonso, 2010; Galindo et al., 2015). Sánchez
et al. (2012) used a combination of optical and electron microscopy to show that ASFV
utilizes a macropinocytosis-like pathway as the primary means of entry into Vero cells and
porcine macrophages. They also showed that ASFV entry was significantly decreased, when
cells were treated with inhibitors of the key regulators of macropinocytosis such as Na+/
H+ channels, Pak-1, PI3K, actin cytoskeleton, EGFR, Rac-1 and tyrosine kinases. In addi-
tion to monocytes and macrophages, ASFV infects other cells like vascular endothelial cells
and lymphocytes. Therefore, it is possible that the usage of macropinocytosis and clathrin-
mediated endocytosis may increase the ability of ASFV to enter different target cells during
the infection process.
Once internalized, ASFV particles move throughout the entire endolysosomal system.
The inhibitors of endosomal maturation like wortmannin and nocodazole significantly
affect ASFV infection, suggesting that virus trafficking throughout endocytic vesicles are
essential for a productive infection in target cells (Hernáez et al., 2016). Immediately after
infection (15–30 minutes post infection), nearly intact ASFV particles are detectable in
early endosomes (Hernáez et al., 2016). At later times (30–90 minutes post infection),
viral decapsidation occurs in late endosomes and lysosomes with acidic pH. Interestingly,
exposure of purified virus to low pH mimics the observed disassembly of ASFV, indicat-
ing that the acidic pH of late endosomes is a signal that triggers ASFV disassembly. Once
disassembled, the inner envelope of viral particles fuses with the limiting membrane of the
endosomes and naked cores are released into cytoplasm and delivered in perinuclear cyto-
plasmic viral assembly sites in order to start DNA replication.

ASFV factory, gene expression and DNA replication

Microtubules provide the major long-range intracellular transport mechanism within the
cytoplasm. They play an important role in ASFV transport to the perinuclear area, where
viral factories are formed. Early studies showed that incoming ASFV particles were closely
associated with microtubules in cytoskeletons obtained Triton X-100 extraction of taxol
treated cells (de Matos and Carvalho, 1993). Further studies revealed that the viral struc-
tural protein p54 can directly interact with the dynein motor protein and such interaction
may constitute a molecular mechanism for microtubule-mediated ASFV transport (Alonso
et al., 2001). The viral factory can be characterized as a single and large area localized close to
the nucleus, where viral proteins and DNA are accumulated and new virions are assembled.
Besides electron microscopy, fluorescent dyes (DAPI or Hoechst) staining cellular and viral
6 | Arabyan et al.

DNA allow the identification of viral factories, where DNA replication occurs. The recom-
binant ASFV expressing p54 protein fused to the enhanced green fluorescent protein is also
used for visualization of ASFV factories (Hernaez et al., 2006). Alternatively, Karalyan et al.
(2018) described a simple method of visualizing the viral factory and measuring the number
of viral genomes using Feulgen-Naphthol Yellow staining technique and image cytometry.
The scheme of viral gene expression revealed that four groups of virus-specific polypep-
tides, designated as immediate early, early, intermediate and late, are synthesized during
ASFV infection in a coordinated manner in which the synthesis of viral DNA presages a
major switch in the programme of ASFV gene expression. Immediate early and early genes
are expressed before the onset of DNA replication. These genes encode for enzymes that are
involved in the nucleotide metabolism and DNA replication, as well as expression of tran-
scription factors that are important for late gene expression (Rodríguez and Salas, 2013).
The expression of intermediate and late genes are not detected, when cells are infected in
the presence of DNA replication inhibitors, suggesting that their expression depends on
the accomplishment of viral DNA replication. These genes encode for structural proteins,
polymerases and transcription factors that are packed into the new virions. However, little
is known about the mechanisms that control ASFV gene expression during different stages
of infection. Recently, in collaboration with Dr Walter Doerfler (Institute for Virology,
Friedrich-Alexander-University Erlangen-Nürnberg, Germany), we showed that ASFV
DNA does not become de novo methylated in the course of infection, and therefore DNA
methylation does not interfere with ASFV gene transcription (Weber et al., 2018).
In situ hybridization and radioactive labelling assays demonstrated that ASFV DNA
replication has two distinct stages, an early stage in the cell nucleus and a later stage within
perinuclear cytoplasmic viral assembly sites (ASFV factory). The genome fragments syn-
thesized in the nucleus are relatively short and not transformed into higher genome length
fragments, indicating that the main part of viral DNA replication occurs in the cytoplasm
(Rojo et al., 1999). These small DNA fragments exit from the nucleus by disassembling of
the lamina network (Ballester et al., 2011). Although the role of nuclear stage in viral DNA
replication remains unclear, it has been shown that ASFV growth is inhibited in enucleated
cells, suggesting that the nucleus may provide important factors required for viral DNA
replication (Ortin and Vińuela, 1977). The precise molecular mechanism by which ASFV
DNA replication occurs also remains unclear. However, the presence of head–head and tail–
tail genomic intermediates in infected macrophages at late times post infection suggests that
ASFV may share a similar replication model to vaccinia virus since the same intermediates
have been observed during vaccinia virus DNA replication (Baroudy et al., 1982; Rojo et al.,
1999). Data from electron microscopy and in situ hybridization indicate that viral DNA con-
denses into a pronucleoid structure that is inserted into icosahedral ‘empty’ particles during
virion maturation (Brooks et al., 1998). Viral polyproteins pp220 and pp62 are important
for the correct assembly and maturation of the core of ASFV particle. Their suppression
results in formation and egress of empty viral particles (Suárez et al., 2010).

Control of apoptosis and autophagy

Infected cells can undergo apoptosis as an antiviral response to viral infection, thereby limit-
ing viral infection. ASFV infection can activate endoplasmic reticulum stress and unfolded
protein response. These processes are reflected by activation of caspase-12, leading to the
subsequent activation of mitochondrial caspase-9 and effector caspase-13 (Galindo et al.,
 African Swine Fever Virus | 7

2012). As ASFV requires a viable cell for replication, the death of the cell limits cellular func-
tions that are required for the virus. Therefore, ASFV encodes for proteins that inhibit cell
death, thereby allowing viral infection to continue. Early studies identified two viral genes,
A179L and A224L, with similarity to cellular Bcl-2 and IAP family members, respectively
(Neilan et al., 1993; Chacón et al., 1995). The A179L protein is conserved among different
ASFV isolates and contains domains similar to all BH domains. This protein is expressed
at early and late times post infection and localizes at the mitochondria or endoplasmic
reticulum (Hernaez et al., 2013). It has been shown that A179L protein can bind to several
BH-3 only proteins, including the activated truncated forms of the Bid protein. The bind-
ing of A179L to pro-apoptotic Bak and Bax has been also observed (Galindo et al., 2008).
Another ASFV protein, A224L, inhibits caspase-3 activation and activates NF-κB (Nogal
et al., 2001; Rodríguez et al., 2002). The expression of this protein is detected late during
infection and it is incorporated into virus particles. Increased caspase-3 activity has been
observed in cells infected with an EP153R gene-deletion mutant, indicating that EP153R
protein is a cell-death inhibitor encoded by ASFV. Further studies revealed that EP153R is
able to reduce the transactivating activity of the cellular protein p53 following induction of
apoptosis (Granja et al., 2004; Hurtado et al., 2004).
Autophagy is an important vacuolar process of the cell, leading to lysosomal degrada-
tion and recycling of intracellular content, including proteins and organelles. This cellular
process can also take over bacterial or viral proteins inside autophagosomes and degrade
them directly in autolysosomes. Some virus have developed strategies to escape this deg-
radation, by expression of specific proteins. It has been shown that A179L protein interacts
not only with Bcl2 family proteins but also inhibits autophagy by targeting Beclin1 through
its BH3 homology domain (Hernaez et al., 2013). The same authors showed that induction
of autophagy prior or at the time of infection reduced the number of infected cells, and
therefore ASFV inhibited autophagosome formation in cells for productive infection.

Pathogenesis
The entry of ASFV into pigs occurs via oral or nasal routs, although other routes such as
tick bites or scarification are also detected. Depending on circulating viral strain, clinical
signs may vary from the highly lethal form with 100% mortality to subclinical and almost
unapparent disease. The incubation period of ASF may last for a few days or longer in case
of the subacute forms of the disease. However, the primary viraemia occurs until 24 h post
infection and virus is detected in almost all organs after 30 h post infection. During viraemia,
ASFV is found associated with erythrocytes, neutrophils and lymphocytes (Plowright et al.,
1994). It is widely accepted that the destruction of macrophages plays an essential role in
the development of pathogenesis, particularly in the impaired haemostasis.

Pathological changes in blood cells

Early studies showed that ASFV infection is accompanied by leucopenia and thrombocyto-
penia, but their nature may vary depending on the strain virulence and host factors. During
acute infection, pigs demonstrate marked neutropenia and lymphopenia from 2 to 3 days
post infection (Karalyan et al., 2012). Interestingly, band-to-segmented neutrophils ratio
became ten times higher in infected pigs than in the control group. The large number of
other immature cells such as metamyelocytes is also observed during the course of infection
8 | Arabyan et al.

indicating that acute ASFV infection is accompanied by the emergence of immature cells in
the host blood (Karalyan et al., 2012). Karalyan and colleagues have also found that more
than 10% of blood cells in the final phase of acute infection are represented by atypical lym-
phocytes (Karalyan et al., 2012a,b). Furthermore, they can be observed in the lymphoid
organs, particularly in the spleen and lymph nodes (Zakaryan et al., 2015). These cells have
high metabolic activity and altered nuclear shape with hyperdiploid DNA content (Karalyan
et al., 2016). However, acute ASFV infection leads to a 2-fold decrease in the number of
lymphocytes by the last day of infection (Zakaryan et al., 2015). Affected lymphocyte sub-
sets are macrophages, B-lymphocytes, CD4+ T-helper cells (Ramiro-Ibanez et al., 1997).
While previous studies have shown that thrombocytopenia develops at the final stage of
acute infection (Anderson, 1986; Anderson et al., 1987), we observed a significant decrease
in the number of platelets from the day 3 post infection (Zakaryan et al., 2014), suggesting
that the level and extent of pathological changes may vary between different virulent strains.
Animals infected with a moderately virulent ASFV strain demonstrate minor changes
in the number of circulating blood cells, especially a slight increase in neutrophils and
decrease in lymphocytes (Wardley, 1982). In chronically infected pigs, macrophages and
B-lymphocytes increase in number by 100% within the first week of infection (Ramiro-
Ibanez et al., 1997). However, these values are back to normal after the second week of
infection, whereas the number of CD4+ and CD8+ T-lymphocytes increases during the
same period of time. The expression of SLA I and SLA II on peripheral mononuclear cells
is also elevated, thereby indicating for stimulation of the immune system. At the third week
post infection, the expression of SLA I and SLA II is significantly decreased, which may lead
to impaired antigen presentation. The recovery of SLA expression during ASFV infection
can be associated with an effective immune response against the virus, and thereby with
survival of infected pigs.

Gross pathological changes

Upon infection with a virulent strain, pigs display high fever, severe depression, anorexia,
vomiting, watery to bloody diarrhoea, conjunctivitis, accelerated respiratory and pulse rate,
abortion in pregnant sows, reddened skin, cyanosis, and incoordination. Acute lethal forms
are also accompanied by vascular lesions, which include bleeding in gastrohepatic and renal
lymph nodes, petechial haemorrhage, hyperaemic splenomegaly, pulmonary oedema and
disseminated intravascular coagulation (Gómez-Villamandos et al., 2003).
ASF is classified as a viral haemorrhagic disease due to haemorrhagic lesions consistently
reported in organs that do not contain a fixed vascular macrophage population, particularly
gastrohepatic and renal lymph nodes. However, haemorrhages also occur in other organs
including lung, intestine and heart. Although vascular changes in acute forms of the disease
are less intense than in subacute forms of ASF, the final stages of acute infection are charac-
terized by extensive bleeding and changes in coagulation system (Neser and Kotzé, 1987).
Initially, it was supposed that endothelial damage was the main cause of bleeding (Colgrove
et al., 1969). However, ultrastructure experiments have revealed that when bleeding is
observed in the lymph nodes and kidneys, there is no virus replication in the endothelial
cells of these organs (Carrasco et al., 1997a). Thus, this hypothesis has been ruled out as the
initial cause of haemorrhages, although endothelial damages can be implicated in bleeding in
the final stage of infection, when ASFV replication is observed in the capillary endothelium.
Phagocytic activation of capillary endothelial cells as an alternative hypothesis has been
 African Swine Fever Virus | 9

proposed. Upon ASFV infection, activated endothelial cells coincide with extensive infec-
tion and destruction of neighbouring monocytes and macrophages. Therefore, phagocytic
activation of endothelial cells is likely due to cytokine release from infected macrophages
(Salguero et al., 2005). Pro-inflammatory cytokines such as IL-1 and TNF-α are known to
trigger a procoagulant state of the endothelium and activate the coagulation cascade in viral
haemorrhagic fevers (Akıncı et al., 2013; Basler, 2017).
After the lymph nodes, spleen is the major target organ of ASFV. In acute infection,
the spleen becomes six times larger than in healthy pigs, crossing the entire abdominal
cavity from one side to the other. Affected spleen has rounded edges and purplish-black
colour (Carrasco et al., 1995). Microscopic lesions tend to be more intense in those areas,
where macrophages are abundant. Particularly, ASFV replication and cytopathic effect are
observed in splenic red pulp macrophages, leading to their massive destruction and activa-
tion of the coagulation system, which in turn leads to the accumulation of erythrocytes in
splenic cords (Carrasco et al., 1997b). Thus, the main function of the spleen, blood clear-
ance, is impaired during acute ASF. Pigs infected with moderately virulent or low-virulent
strains demonstrate mild hyperaemic splenomegaly, which is progressively reversed at the
end of infection.
Another pathological change is pulmonary oedema, which is observed in pigs infected
with highly virulent ASFV strains. The population of macrophages in porcine lungs consists
of interstitial macrophages, free rounded alveolar macrophages and pulmonary intravascular
macrophages located in pulmonary capillaries adjacent to the endothelium. The latter cells
appear to be the main target of ASFV in the lung. The secretory activation of macrophages
before the viral entry into the lung indicates that it is induced by circulating chemical media-
tors produced by primary replication organs (Carrasco et al., 1996, 2002). At the same time,
cell debris triggers phagocytic activation of pulmonary intravascular macrophages. This
leads to increased intravascular pressure, prompting a number of vascular changes, including
interstitial and alveolar oedema.

Diagnosis
Recognition of the clinical signs of ASF is the first alarm that the virus is circulating among
wild or domestic pigs. Early detection allows authorities to rapidly implement control meas-
ures in affected areas. However, the diagnosis of ASF is complicated by the fact that other pig
diseases, particularly classical swine fever, porcine dermatitis and nephropathy syndrome,
swine erysipelas and salmonella have similar clinical signs. Therefore, the rapid, sensitive
and specific detection of ASFV is important not only for control measures but also for the
differential diagnosis of other pig diseases. According to the World Organization for Animal
Health recommendations, ASFV diagnosis includes virus isolation, fluorescent antibody
tests, real-time and conventional PCR assays (Oura et al., 2013). For large-scale testing of
samples, enzyme-linked immunosorbent assay (ELISA) is also used, although it has lower
analytical sensitivity than that of PCR assays (Oura et al., 2013) (Fig. 1.3).
Several conventional PCR assays have been described (Steiger et al., 1992; Wilkinson,
2000; Bastos et al., 2003). However, real-time PCR has distinct advantages over conven-
tional PCR, and therefore the latter one is now superseded by real-time PCR assays. King et
al. (2003) developed the first real-time TaqMan PCR assay. It targets the viral B646L gene
and includes an artificial mimic, thereby validating negative results. This assay was validated
10 | Arabyan et al.

Figure 1.3 Diagnostic tests available for detection of ASFV in field and laboratory samples.

against 25 different ASFV isolates and it did not cross react with other pig viruses. Zsak
et al. (2005) developed an alternative real-time TaqMan PCR assay, which was performed
in a single tube containing PCR reagents. This assay also targets the viral B646L gene but
it has higher analytical sensitivity than the assay described by King et al. (2003). Another
real-time PCR assay was reported by McKillen et al. (2010). This assay was designed against
the 9GL gene of the ASFV genome, and was found to be sensitive and specific. It was
validated against 15 different ASFV isolates. However, the most sensitive PCR assay was
developed based on the Linear-After-The-Exponential-PCR (LATE-PCR) principle. The
assay was designed to amplify the B646L gene and was validated using 19 ASFV DNA cell
culture virus strains and three tissue samples from infected pigs. The analytical sensitivity is
between one and ten copies and it was designed to be used in both laboratory settings and
portable PCR machine (Ronish et al., 2011). A single-tube multiplexed real-time PCR assay
detecting six different transboundary viruses, including ASFV, was described by Wernike
et al. (2013). Other multiplex PCR assays for simultaneous detection and differentiation
of porcine viruses were also developed (Haines et al., 2013; Grau et al., 2015; Hu et al.,
2016). However, all these assays have one or two log lower analytical sensitivity than other
real-time PCR assays.
In low-income countries, where authorities cannot afford to purchase expensive PCR
systems, the antigen ELISA assay is an attractive alternative for virus detection. Commer-
cially produced antigen ELISA kits (ELISA INGEZIM K3, Ingenasa, Spain; IDvet, France;
SVANOVIR, Sweden) are currently available, although very few data on the sensitivity of
these assays are known.

Vaccines
Although different strategies have been employed in order to develop an effective vaccine
for this disease, no commercially available vaccine exists to prevent ASF. Approaches used
in vaccine development have included live-attenuated ASFV (traditional and engineered),
inactivated virus and ASFV subunit vaccines (Zakaryan and Revilla, 2016).
During the first outbreaks of ASF in Spain and Portugal, ASFV isolates were collected
and used for traditional attenuation by cell passages. Pigs infected with attenuated ASFV
develop long-term potent resistance against challenge with homologous but rarely with
heterologous strains (Leitão et al., 2001; King et al., 2011; Mulumba-Mfumu et al., 2016).
Interestingly, the boundaries of homologous cross-protection are not clear, since closely
related ASFV isolates fail to produce cross-protection but diverse ASFV may induce some
 African Swine Fever Virus | 11

immunity (King et al., 2011; Lacasta et al., 2015). In addition to this uncertainty, live-
attenuated ASFV strains cause several side-effects and post-vaccination reactions including
necrotic foci, abortion and pneumonia. Chronic ASF was detected in pigs vaccinated with
ASFV isolate attenuated by serial passages in bone marrow cells (Manso-Ribeiro et al., 1963).
Hypergammaglobulinaemia and systematic immune activation with increased numbers of
B lymphocytes, CD8+ T lymphocytes and macrophages were also observed in vaccinated
pigs (Leitão et al., 2001). Thus, the application of live-attenuated ASFV is thwarted by safety
issues and poor cross-protection against heterologous strains.
Hypothetically, recombinant ASFV strains containing specific deletions of genes are
safety alternatives of naturally attenuated ASFV vaccines. In the last two decades compara-
tive genomic research has identified ASFV genes associated with virulence. The deletion
mutant ASFV BA71ΔCD2 conferred protection not only against lethal challenge with
ASFV BA71 strain but also against the heterologous ASFV E75 (Monteagudo et al., 2017).
However, the effect of virulence-related gene deletion on viral attenuation and immuno-
genicity seems to be strain and genotype dependent. For instance, deletion of the thymidine
kinase gene from ASFV strains Malawi and Georgia attenuates these strains but only the
TK-deleted Malawi is able to induce a protective immunity in inoculated pigs (Sanford
et al., 2016). The deletion of the NL gene from virulent strains attenuates the European
E70 strain but has no effect on two other strains (Afonso et al., 1998; Neilan et al., 2002;
Reis et al., 2016). Multiple mutations in ASFV strains may produce more attenuated and
safer ASFV vaccines but recent studies showed that when two virulence-related genes are
deleted from ASFV, the protection of immunized pigs is reduced. For example, it has been
shown that the B119L and MGF 360/505 gene cluster-deleted ASFV Georgia strain was
incapable of inducing protective immune responses in inoculated pigs (O’Donnell et al.,
2016). Interestingly, the same strain containing single deletions in either B119L or the MGF
360/505 gene cluster genes protected animals from challenge with virulent ASFV Georgia
strain (O’Donnell et al., 2015a,b).
All attempts to induce effective immunity by using inactivated virus have failed. These
attempts include purified and inactivated virions, infected glutaraldehyde-fixed mac-
rophages, and detergent-treated, infected alveolar macrophage cell cultures (Forman et al.,
1982; Kihm et al., 1987; Mebus et al., 1988). Inactivated ASFV failed to induce protective
immunity in pigs even in the presence of modern adjuvants (Blome et al., 2014). The com-
plexity of the virus which contains more than 50 structural proteins may explain this failure.
Subunit vaccines containing specific viral antigens and optimized delivery system have
lower chances to cause adverse reactions. However, identifying which antigens best stimu-
late the immune system is a tricky and time-consuming process. It has been shown that pigs
co-immunized with p30 and p54 by baculovirus vector conferred protection against lethal
challenge with ASFV E75 isolate (Gómez-Puertas et al., 1998), but the combination of p30,
p54 and p72 proteins did not protect against lethal challenge with highly virulent ASFV
Malawi isolate (Neilan et al., 2004). Similarly, when pigs were immunized with a fusion pro-
tein containing CD2v, p30 and p54 coupled to ubiquitin, they were not protected against
ASFV E75 isolate (Argilaguet et al., 2013). Recently, Jankovich et al. (2018) delivered 47
antigens to pigs by DNA prime and recombinant vaccinia virus boost and then pigs were
challenged with a lethal dose of ASFV Georgia 2007/1 isolate. All pigs developed clinical
signs consistent with acute ASFV. However, further studies are required in order to iden-
tify effective viral antigens for induction of robust protective immunity in pigs. Improved
12 | Arabyan et al.

immunization strategies including delivery of the putative ASFV antigens, doses and their
proper presentation to the host also need further studies.

Antiviral drugs
Although it is always better to prevent disease rather than to treat, a possible application of
antivirals developed at a reasonable cost may have very beneficial effects. Antiviral therapy
can prolong the host survival allowing the infected pigs to generate a productive immune
response against ASFV. Therefore, such therapies can be applied in affected farms in order
to isolate the epidemic area and prevent further spread.
Nucleoside analogues are the major group of antiviral agents that inhibit viral replica-
tion by incorporation into viral nucleic acids or interfering with their essential enzymes
such as polymerases. Various nucleoside analogues were tested for their antiviral activity
against ASFV infection in vitro (Gil-Fernández and De Clercq, 1987; Gil-Fernández et al.,
1987; Arzuza et al., 1988). (S)-9-(3-hydroxy-2 phosphonylmethoxypropyl)adenine and
carbocyclic 3-deazaadenosine were found to exhibit a much higher anti-ASFV activity than
other nucleoside analogues. Recently, we found that two nucleoside analogues, 5-(Perylen-
3-ylethynyl)-arabino-uridine (aUY11) and 5-(Perylen-3-ylethynyl)uracil-1-acetic acid
(cm1UY11), possess a potent, dose-dependent inhibitory effect on ASFV infection in Vero
cells and porcine macrophages (Hakobyan et al., 2018). The major antiviral effect (3.5 log
reduction) was observed when we added aUY11 and cm1UY11 at the internalization stage
of ASFV infection. These compounds belong to a novel family of nucleoside analogues,
called RAFI (rigid amphipathic fusion inhibitors) (Fig. 1.4). It has been previously shown
that RAFIs inhibit the infectivity of unrelated enveloped viruses by targeting the envelope

Chemical Formula: C31H22N2O8 Chemical Formula: C28H16N2O4

Molecular Weight: 518,5250 Molecular Weight: 444,4460

Figure 1.4 Rigid amphipathic fusion inhibitors. (A) 5-(Perylen-3-ylethynyl)-arabino-uridine

(aUY11) and 5-(Perylen-3-ylethynyl)uracil-1-acetic acid (cm1UY11) have antiviral activity
against ASFV. (B) Other RAFIs with unknown biological activity.
 African Swine Fever Virus | 13

lipids to prevent the curvature changes required for the fusion of viral and cellular mem-
branes during viral entry (St Vincent et al., 2010; Colpitts et al., 2013). Thus, it is possible
that aUY11 and cm1UY11 can serve as antiviral agents for interference with ASFV infection
because their antiviral effect likely relies on biophysical mechanisms.
Because of low side effects and high availabilities, natural compounds have been the
centre of attention among researchers working in antiviral drug discovery (Zakaryan et
al., 2017). For example, sulfated polysaccharides were shown to affect ASFV attachment
and subsequent replication because of negatively charged sulfate groups interacting with
positively charged amino acids in the viral envelope (García-Villalón and Gil-Fernández,
1991). The aqueous extracts from different plants and marine microalgae demonstrated
anti-ASFV activity in a dose-dependent manner (Fabregas et al., 1999; Fasina et al., 2013).
Polyphenolic phytoalexins, such as resveratrol and oxyresveratrol, significantly reduced
ASFV production by inhibiting viral DNA replication, late viral protein synthesis and viral
factory formation (Galindo et al., 2011). Similar results were observed when we treated
ASFV-infected Vero cells with 4′,5,7-trihydroxyflavone, also known as apigenin (Hakobyan
et al., 2016). This flavonoid was highly effective at the early stages of infection, reducing the
viral yield by more than 99.99%. ASFV-infected cells continuously treated with apigenin did
not display a cytopathic effect, and ASFV was not detected, neither by viral antigen ELISA
nor by conventional titration. Recently, we also reported that genistein, another flavonoid,
affected ASFV infection in Vero cells and porcine macrophages (Arabyan et al., Antiviral
Research, submitted). The effect was maximal when it was added to cells at middle phase of
infection (8 hpi), disrupting viral DNA replication. We revealed the presence of fragmented
ASFV genomes in cells exposed to genistein, suggesting that this molecule may act as an
ASFV-topoisomerase II poison. Molecular docking studies showed that genistein may inter-
act with four residues of the ATP-binding site of ASFV-topoisomerase II (Asn-144, Val-146,
Gly-147 and Leu-148), showing more binding affinity than ATP4–, emphasizing the idea
that this viral enzyme can be a good target for drug development against ASFV. Indeed,
siRNA knockdown experiments showed that ASFV-topoisomerase II plays an essential
role in viral DNA replication (Freitas et al., 2016). Particularly, a significant decrease in the
number of both infected cells and viral factories per cell and in virus yields (up to 2.5 log)
was found only in cells transfected with siRNA targeting ASFV-topoisomerase II.
In conclusion, ASFV represents a significant pig welfare problem in need of immediate
redress. Although so far all efforts to generate effective vaccines have failed, further studies
to riddle the details of the interaction of ASFV with host cells will provide new opportuni-
ties for vaccine development and antiviral drug discovery. We believe that vaccines alone or
in combination with antiviral treatments will reduce or completely eliminate the occurrence
of disease.

References
Achenbach, J.E., Gallardo, C., Nieto-Pelegrín, E., Rivera-Arroyo, B., Degefa-Negi, T., Arias, M., Jenberie,
S., Mulisa, D.D., Gizaw, D., Gelaye, E., et al. (2017). Identification of a new genotype of African swine
fever virus in domestic pigs from Ethiopia. Transbound. Emerg. Dis. 64, 1393–1404. https://doi.
org/10.1111/tbed.12511
Afonso, C.L., Zsak, L., Carrillo, C., Borca, M.V., and Rock, D.L. (1998). African swine fever virus NL gene
is not required for virus virulence. J. Gen. Virol. 79, 2543–2547.
Akıncı, E., Bodur, H., and Leblebicioglu, H. (2013). Pathogenesis of Crimean-Congo hemorrhagic fever.
Vector Borne Zoonotic Dis. 13, 429–437. https://doi.org/10.1089/vbz.2012.1061
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