Arterivirus Molecular Biology and Pathogenesis 2013

Journal of General Virology (2013), 94, 21412163
DOI 10.1099/vir.0.056341-0
Arterivirus molecular biology and pathogenesis
Review
Eric J. Snijder,1 Marjolein Kikkert1 and Ying Fang2,3

1
Correspondence
Molecular Virology Department, Department of Medical Microbiology, Leiden University Medical

Center, Leiden, The Netherlands
Eric J. Snijder
E.J.Snijder@lumc.nl
Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings,
South Dakota, USA
Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine,

Kansas State University, Manhattan, Kansas, USA
Received 25 June 2013

Accepted 7 August 2013
Arteriviruses are positive-stranded RNA viruses that infect mammals. They can cause persistent or
asymptomatic infections, but also acute disease associated with a respiratory syndrome, abortion or
lethal haemorrhagic fever. During the past two decades, porcine reproductive and respiratory
syndrome virus (PRRSV) and, to a lesser extent, equine arteritis virus (EAV) have attracted attention as
veterinary pathogens with significant economic impact. Particularly noteworthy were the porcine high
fever disease outbreaks in South-East Asia and the emergence of new virulent PRRSV strains in the
USA. Recently, the family was expanded with several previously unknown arteriviruses isolated from
different African monkey species. At the molecular level, arteriviruses share an intriguing but distant
evolutionary relationship with coronaviruses and other members of the order Nidovirales. Nevertheless,
several of their characteristics are unique, including virion composition and structure, and the
conservation of only a subset of the replicase domains encountered in nidoviruses with larger
genomes. During the past 15 years, the advent of reverse genetics systems for EAV and PRRSV has
changed and accelerated the structurefunction analysis of arterivirus RNA and protein sequences.
These systems now also facilitate studies into host immune responses and arterivirus immune evasion
and pathogenesis. In this review, we have summarized recent advances in the areas of arterivirus
genome expression, RNA and protein functions, virion architecture, virushost interactions, immunity,
and pathogenesis. We have also briefly reviewed the impact of these advances on disease
management, the engineering of novel candidate live vaccines and the diagnosis of arterivirus infection.
Introduction
About two decades ago, genome sequencing established
that arteriviruses qualified as a separate family of positivestranded RNA (+RNA) viruses (reviewed by Cavanagh,
1997; Snijder & Meulenberg, 1998). A family with, on the
one hand, an intriguing but distant evolutionary relationship to coronaviruses and other members of the order
Nidovirales but, on the other hand, characteristics unique
among currently known +RNA viruses. These include
virion composition and structure, and the conservation of
only a subset of the replicase domains encountered in
nidoviruses with larger genomes. Shortly after the family
Arteriviridae was created, equine arteritis virus (EAV) and
porcine reproductive and respiratory syndrome virus
(PRRSV) were the first nidoviruses for which reverse
genetics systems became available (Meulenberg et al., 1998;
van Dinten et al., 1997). This breakthrough not only
revolutionized the structurefunction analysis of RNA and
protein sequences, but also facilitated studies into pathogenesis and immune responses, and the engineering of novel
candidate live vaccines.
056341 G 2013 SGM
Printed in Great Britain
The consequences of arterivirus infection can range from

persistent asymptomatic infection to acute disease, associated with abortion, fatal age-dependent poliomyelitis or
lethal haemorrhagic fever. Three of the currently recognized
arterivirus species [EAV, lactate dehydrogenase-elevating
virus (LDV) of mice and simian haemorrhagic fever virus
(SHFV)] were first isolated about 50 years ago (reviewed by
Snijder & Kikkert, 2013; Snijder & Meulenberg, 1998).
However, during the past two decades, it was particularly the
fourth arterivirus, PRRSV, that moved into the spotlight by
causing tremendous economic losses to the swine industry
worldwide. In the late 1980s, highly diverged variants of this
virus emerged almost simultaneously in Western Europe
(genotype 1) and North America (genotype 2), causing
numerous acute respiratory disease outbreaks and abortion
storms. Subsequently, the porcine high fever disease
outbreaks in South-East Asia (Tian et al., 2007) and the
emergence of the highly virulent MN184 strain in the USA
(Han et al., 2006) have attracted special attention.
Disease prevention and control will clearly benefit from
the dissection of the molecular and cellular biology of
2141
E. J. Snijder, M. Kikkert and Y. Fang
arterivirus infection. In this review, we will summarize what

has been learned, in particular since the advent of reverse
genetics, with respect to arterivirus genome expression, RNA
and protein functions, virion architecture, host responses,
and pathogenesis. We will also briefly review the impact of
these advances on disease management, vaccine development and the diagnosis of arterivirus infection.
2142
100
10
0
10
0
PRRSV
VR2332
SHFV
krtg1 SHFV
krtg2
0
The arterivirus genome is a polycistronic +RNA (Figs 2

and 3), with 59 and 39 NTRs of 156224 and 59117 nt,
respectively, that flank an array of 1015 known ORFs. The
large replicase ORFs 1a and 1b occupy the 59-proximal
three-quarters of the genome, with the size of ORF1a being
much more variable than that of ORF1b. ORF1a translation
0100
0
100 1
10
Genome structure and expression

Genome properties and organization
The arterivirus genome is 1216 kb long, 39-polyadenylated
and presumably 59-capped. Full-length genome sequences
(Table 1) have been obtained for European and North
American EAV isolates, a large number of genotype 1 and 2
PRRSVs, two LDV strains, and five distantly related monkey
viruses, tentatively grouped under the name SHFV.
SHFV
LVR
LDV-C
PRRSV
Lelystad
As nidovirus discovery continued, in a remarkably wide

variety of hosts, the order was further expanded with
nidoviruses infecting shrimp (family Roniviridae; Cowley
et al., 2000), fish (genus Bafinivirus; subfamily Torovirinae
in the family Coronaviridae; Schutze et al., 2006) and most
recently also insects (proposed family Mesoniviridae; Nga
et al., 2011; Zirkel et al., 2011). The recently identified
wobbly possum disease virus (WPDV) appears most closely
related to arteriviruses (Dunowska et al., 2012). Furthermore, several additional monkey arteriviruses, only distantly
related to SHFV, were discovered recently (Lauck et al., 2013).
Together with the large evolutionary distances between current
arterivirus species (Fig. 1), this may well prompt a future
revision of the internal taxonomic structure of the family.
SHFV
krc1
10
About 15 years ago, the family Arteriviridae was united

with the family Coronaviridae in a new order Nidovirales
(Cavanagh, 1997; Snijder & Meulenberg, 1998). In spite of
striking differences in genome size and virion structure, the
genome organization and expression of arteri- and
coronaviruses are strikingly similar, and key replicase
domains were postulated to share a common ancestry (den
Boon et al., 1991). A prominent feature of nidovirus
genome expression is the nested set of subgenomic (sg)
mRNAs that was the basis for the order name Nidovirales
(Latin nidus5nest). The complex evolutionary relationship
between arteriviruses and other nidoviruses has been reviewed
extensively elsewhere (Gorbalenya et al., 2006; Nga et al., 2011;
Snijder & Meulenberg, 1998). Essentially, related replicase
genes have become associated with seemingly unrelated sets of
structural protein genes, with RNA recombination likely
playing an important role in nidovirus evolution.
SHFV
krc2
60
The expanding order Nidovirales
LDV-P
EAV
EAV
Bucyrus
s3685
0.1
WPDV
Fig. 1. Phylogeny of representatives of currently recognized

arterivirus species (EAV, PRRSV, LDV and SHFV), four recently
discovered monkey arteriviruses distantly related to SHFV (Lauck
et al., 2013), and WPDV (Dunowska et al., 2012). This neighbourjoining phylogenetic tree is based on amino acid sequences
translated from a codon-based ORF1b nucleotide sequence
alignment. The analysis highlights the distances between current
and tentative members of the family Arteriviridae. Bootstrap values
(based on 1000 replicates of the data) are shown on branches;
scale bar, amino acid substitutions per site. Courtesy of Dr Tony L.
Goldberg, University of Wisconsin, Madison, WI, USA.
yields replicase polyprotein (pp) 1a (17272502 aa), whereas

ORF1b is expressed by 21 programmed ribosomal frameshifting (PRF) (den Boon et al., 1991), which C-terminally
extends pp1a into pp1ab (31753959 aa). Recently, a short
transframe (TF) ORF was found to overlap the nsp2-coding
region of ORF1a in the +1 frame and to be expressed by 22
PRF (Fang et al., 2012). Remarkably, the TF ORF is
conserved in the genomes of PRRSV, LDV and SHFV, but
this part of the EAV genome is considerably truncated.
The 39-proximal genome part has a compact organization
and contains eight to 12 relatively small genes, most of
which overlap with neighbouring genes (Fig. 2). These
ORFs encode structural proteins and are expressed from a
39-co-terminal nested set of sg mRNAs (Fig. 3). The
organization of these ORFs is conserved, but downstream
of ORF1b, SHFV and all recently identified SHFV-like
viruses contain three or four additional ORFs (~1.6 kb)
that may be derived from an ancient duplication of ORFs
24 (Godeny et al., 1998; Lauck et al., 2013). Together with
the size variation in ORF1a, this presumed duplication
explains the genome size differences among arteriviruses.
Journal of General Virology 94
Table 1. Arterivirus genomes and database accession numbers

Virus (isolate)
EAV (Bucyrus)
LDV (P)
PRRSV genotype 1 (Lelystad)
PRRSV genotype 2 (16244B)
SHFV (LVR 42-0)
SHFV-like (krc1)
SHFV-like (krc2)
SHFV-like (krtg1)
SHFV-like (krtg2)
Host
Genome size (kb)
GenBank accession no.
Horse, donkey
Mouse
Swine
Swine
Macaques
Red colobus
Red colobus
Red-tailed guenon
Red-tailed guenon
12.7
14.1
15.1
15.4
15.7
15.5
15.2
15.2
15.3
NC_002532
NC_001639
M96262
NC_001961
NC_003092
HQ845737
HQ845738
JX473847
JX473849
RNA structures in replication, transcription and translation
the coding sequences (Molenkamp et al., 2000a; Tijms et al.,

2001). Detailed RNA secondary structure models were
developed for the EAV 59 and 39 NTRs (Fig. 4). The 59
NTR is involved in translation, replication and transcription (van den Born et al., 2004), and contains the so-called
Several RNA signals involved in arterivirus replication have been identified. In EAV, a minimum of
~300 nt from both genome termini is required for efficient
replication, meaning that replication signals extend into
Expressed from genomic RNA

EAV
P P2
TM
TM
S TM
replicase ORF1b
replicase ORF1a
LDV
P
1
P P2
TM
replicase ORF1a 2 TM
TF
PRRSV
P
P2
TM
2 TM
replicase ORF1a
TM
TM
S TM
TF
SHFV
P P
P2
TM
replicase ORF1a 2 TM
TF
0
TM
S TM
10
11
12
N
7
(A)n
4 5a
6
GP 5
M
a
GP
GP
N
5
7
3
13
(A)n
4 5a
GP 5
a
E GP
GP
2a 3
5
2b
GP
replicase ORF1b
6
M
GP
5
N
2a
GP
3
2b 3
1
P
2a
GP
E
2b
replicase ORF1b
4 5a
GP 5
a
2b
GP
E
2a
replicase ORF1b
1
TM
Expressed from sg mRNAs

4 5a
6
2b
M
GP GP 5
a
(A)n
E
GP
GP
N
2a 3
5
7
14
15
6
M
N
7
16
(A)n
kb
Fig. 2. Arterivirus genome organization. The family prototype EAV is shown at the top. The replicase ORFs 1a and 1b [the latter
expressed by 1 programmed ribosomal frameshifting (PRF)] are followed by the genes encoding the minor and major envelope
proteins and the N protein. GP, glycoprotein; M, membrane; E, envelope; N, nucleocapsid. The 39-proximal region of the SHFV
genome carries a large insertion (pink) containing four ORFs that may encode additional virion proteins. With the exception of
EAV, arterivirus genomes contain an alternative transframe (TF) ORF in the non-structural protein 2 (nsp2)-coding region, which
is expressed by 2 PRF (Fang et al., 2012). The positions corresponding to (known or predicted) polyprotein cleavage sites are
depicted above the replicase ORFs; red arrowheads, sites cleaved by the nsp4 SP (S); blue arrowheads, sites cleaved by PLP
domains (P) in the nsp1/nsp2 region. The processing scheme of the SHFV nsp1 region remains to be elucidated. Three
ORF1a-encoded (putative) transmembrane domains (TM) and four highly conserved ORF1b-encoded domains are depicted:
RNA-dependent RNA polymerase (R), (putative) multinuclear zinc-binding domain (Z), RNA helicase (H), and NendoU
endoribonuclease domain (N). Modified from Snijder & Kikkert (2013).
http://vir.sgmjournals.org
2143
Binding
Release
Fusion
Endocytosis
Genome
AAA
Translation
pp Cleavage
RTC
assembly
Exocytosis
()Genome
sg ()RNAs
Genome
Golgi
AAA
AAA
AAA
AAA
AAA
AAA
AAA
RTC
membrane
association
Budding
AAA
Genome
encapsidation
sg mRNAs
N protein
DMV
Envelope
protein
maturation
Envelope proteins
AAA
AAA
Endoplasmic
Reticulum
leader TRS hairpin (LTH) that is crucial for sg mRNA

production. The relevance of other EAV 59 NTR structures
remains to be investigated as few of these are conserved in
other arteriviruses (Lu et al., 2011; van den Born et al., 2004).
A 39-proximal EAV RNA hairpin is required for RNA
synthesis and its loop was implicated in an essential
pseudoknot interaction with an upstream hairpin within
the N protein gene (Beerens & Snijder, 2007). This conformation appears conserved in all arteriviruses and may
constitute a molecular switch that, for example, regulates a
step in negative-strand RNA synthesis. In addition, a 39terminal CC motif upstream of the poly(A) tail was implicated in a critical step in replication, possibly the initiation
of negative-strand RNA synthesis (Beerens et al., 2007). In
PRRSV, a kissing interaction between the loops of RNA
hairpins in the 39 NTR and N protein gene was found to be
crucial for replication (Verheije et al., 2002a).
Arterivirus genome translation presumably initiates through
conventional ribosomal scanning of the 59 NTR (van den
Born et al., 2005), but also entails one or two ribosomal
frameshifting events. As in all other nidoviruses, a 21 PRF
in the short ORF1a/1b overlap region is used to express
ORF1b. The estimated frameshift efficiency is 1520 % (in a
reporter system), which derives from the concerted action of
a slippery sequence and a downstream RNA pseudoknot
structure (den Boon et al., 1991; Firth & Brierley, 2012). In
addition, all arteriviruses except EAV employ 22 PRF to
express a conserved TF ORF that overlaps the nsp2-coding
region (Fig. 2) (Fang et al., 2012). This frameshift site
2144
AAA
AAA
Nucleus
Fig. 3. Overview of the arterivirus infection

cycle. Following entry by receptor-mediated
endocytosis and disassembly, genome translation yields replicase polyproteins pp1a and
pp1ab. These polyproteins are cleaved by
internal proteinases and the viral non-structural
proteins assemble into a replication and
transcription complex (RTC) that first engages
in minus-strand RNA synthesis. Both fulllength and subgenome-length minus strands
are produced, the latter serving as templates
for the synthesis of sg mRNAs required to
express the structural protein genes, which
reside in the 39-proximal quarter of the
genome. Novel genomes are packaged into
nucleocapsids that become enveloped by
budding from smooth intracellular membranes,
after which the new virions leave the cell using
the exocytic pathway. For further details, see
text. Modified from Snijder & Kikkert (2013).
dictates both efficient 22 and 21 PRF (estimated

efficiencies in PRRSV-infected cells of 1620 % and 7 %,
respectively) and consequently three N-terminally collinear
products are produced: nsp2, nsp2TF and nsp2N. A
downstream RNA sequence (CCCANCUCC) is a critical
determinant of this first documented case of 22 PRF in
eukaryotic cells, but is not predicted to be part of a particular
frameshift-directing RNA structure. The two PRF mechanisms create a complex series of non-structural protein
expression ratios. Of the ribosomes that translate the PRRSV
nsp1 region, y20 % and y7 % synthesize nsp2TF and
nsp2N, respectively, with y73 % translating the remainder
of ORF1a (nsp38) and probably only y15 % subsequently
translating ORF1b (nsp912).
Proteinases and replicase polyprotein processing
As in all nidoviruses (Ziebuhr et al., 2000), the posttranslational processing of arterivirus replicase polyproteins involves a complex proteolytic cascade that is directed
by three (EAV) or four (PRRSV/LDV) ORF1a-encoded
proteinase domains (Figs 2 and 5). Cleavage of pp1a and
pp1ab generates 13 (EAV) or 14 (PRRSV/LDV) processing
end products, named nsp112, including nsp7a/7b (PRRSV/
LDV/EAV) and nsp1a/1b (PRRSV/LDV). In PRRSV, LDV
and SHFV, the 22 PRF event described above yields the
truncated nsp2 variants nsp2TF and nsp2N (Fang et al.,
2012). The nsp38 region is subject to two alternative
processing cascades, with cleaved nsp2 acting as a co-factor
to promote the nsp4/5 cleavage in the major processing
(a)
Leader
TRS
12520
LTH
SL3
5
1
12560
J 313
(b)
SL2
ot
kn
o
ud
Ps
12440
SL5
12690
SL1
12600
SL4
12650
(A)n 3
Fig. 4. RNA structures at the EAV genome termini. (a) Secondary structure model of the 59-proximal 313 nt of the EAV genome
(van den Born et al., 2004). The leader TRS hairpin (LTH; hairpin G), leader transcription-regulatory sequence (TRS) region and
replicase ORF1a initiation codon are highlighted. (b) RNA secondary structure model of the 39-proximal 300 nt, with stemloop
(SL) structures 15 indicated. An RNA pseudoknot (conserved in all arteriviruses) that can be formed between the SL4 and
SL5 loops was found to be critical for EAV replication. Modified from Beerens & Snijder (2007) with permission.
pathway (Wassenaar et al., 1997). EAV cleavage-site

mutagenesis underlined the critical role of replicase polyprotein processing in virus replication (van Aken et al.,
2006b; van Dinten et al., 1999).
Two papain-like proteinases (PLPs) and one chymotrypsinlike serine proteinase (SP) are functional in all arteriviruses,
and reside in nsp1/1b (PLP1b), nsp2 (PLP2) and nsp4 (SP)
(for reviews, see Fang & Snijder, 2010; Snijder &
Meulenberg, 1998; Ziebuhr et al., 2000). In PRRSV and
LDV, a fourth non-structural proteinase (PLP1a) mediates internal nsp1 cleavage into nsp1a and nsp1b (den
Boon et al., 1995). The presence of two PLP domains may
reflect an ancient duplication event, but PLP1a has
become inactivated in EAV. The sequence analysis of
the SHFV nsp1 region revealed a potentially even more
complex organization, with an array of three (potential) PLP
domains in the 480 aa region upstream of the (predicted)
nsp1/2 junction.
During the past decade, crystal structures have been reported
for all four arterivirus proteinase domains (Fig. 5). Consistent
with earlier bioinformatics, expression and mutagenesis
studies (den Boon et al., 1995), PLP1a (Sun et al., 2009) and
PLP1b (Xue et al., 2010) are very compact PLP domains
employing a CysHis tandem as active-site residues. They
appear to exclusively cleave their own C-terminus, which is
retained in the substrate-binding pocket, thus precluding
further proteolytic activity.
In contrast, the PLP2 domain in nsp2 exhibits both cis and

trans cleavage activities (Han et al., 2009; Snijder et al.,
1995). In addition to performing the critical nsp2/3 cleavage,
PLP2 is capable of removing ubiquitin (Ub) and Ub-like
modifiers like ISG15 (an IFN-stimulated gene) from host
cell substrates (Frias-Staheli et al., 2007; Sun et al., 2010,
2012b; van Kasteren et al., 2012). Structurally, PLP2 also
differs from PLP1a/b and was proposed to be distantly
related to so-called ovarian tumour domain-containing
(OTU) deubiquitinases (DUBs) (Makarova et al., 2000).
However, the compact EAV PLP2 structure (Fig. 5d; van
Kasteren et al., 2013) is distinct from other OTU superfamily
members and incorporates a structurally important zinc
finger (ZF) domain. Given these features, PLP2 represents a
unique subclass of zinc-dependent OTU DUBs.
Crystal structures were solved for both EAV and PRRSV nsp4
(Fig. 5e), which includes the SP that is the arteriviral main
proteinase and cleaves all sites downstream of nsp3 (BarretteNg et al., 2002; Tian et al., 2009). The protein contains two
domains that form the typical chymotrypsin-like two-b-barrel
fold and a C-terminal domain that is dispensable for
proteolytic activity, but may be involved in fine-tuning of
replicase polyprotein processing (van Aken et al., 2006a).
Non-structural protein functions
During or following pp1a and pp1ab cleavage, arterivirus

non-structural proteins assemble into a membrane-associated
2145
(a)
PRF
PRF
PRRSV
nsp1
HVR
P P2
TM
2
P P P2
EAV
TM
TM
TM
TM
5 67 7 8
TM
H
10
N
11 12
PRF
(c) PRRSV nsp1
(b) PRRSV nsp1
(d) EAV PLP2
(e) PRRSV nsp4
NTD
ZF domain
ZF
Zn
Zn
Asp
Cys
His
His
Linker
Ser
Linker
Zn
His
Cys
HisCys
Asn
CTE
CTE
CTD
Fig. 5. Arterivirus proteinases and replicase polyprotein processing. (a) Domain organization and proteolytic processing of EAV
and PRRSV pp1ab. For domain abbreviations, see Fig. 2; HVR, hypervariable region. Demonstrated and predicted zinc-binding
domains are indicated in green. Adapted from Snijder & Kikkert (2013). (be) Crystal structures of arterivirus-encoded
proteases made using PYMOL (http://www.pymol.org/) and Protein Data Bank (PDB) entries (b) 3IFU, (c) 3MTV, (d) 4IUM and
(e) 3FAN, respectively. Proteinase active-site residues are highlighted in red. Courtesy of Puck van Kasteren (Leiden University
Medical Center). (b) PRRSV nsp1a (Sun et al., 2009), including the N-terminal zinc finger (ZF) domain (blue, zinc-binding
residues highlighted in yellow) and C-terminal PLP1a domain (red/pink). After cleavage, the C-terminal extension (CTE)
domain (green) remains in the substrate-binding pocket. (c) PRRSV nsp1b (Xue et al., 2010), including N-terminal domain
(NTD; blue), linker sequence (grey), PLP1b domain (red/pink) and CTE (green). (d) EAV PLP2 proteinase domain (van Kasteren
et al., 2013), including its ZF with zinc-binding residues highlighted (yellow). (e) PRRSV nsp4 (Tian et al., 2009), including the
typical chymotrypsin-like two-b-barrel fold of the SP domain (red/pink) that is the arterivirus main proteinase. The pink
bidirectional arrow represents a loop devoid of visible electron density in the crystal structure. The nsp4 C-terminal domain
(CTD; blue) is dispensable for proteolytic activity in EAV.
enzyme complex that directs viral replication and transcription. In addition to encoding the proteinases discussed above,
ORF1a encodes three (putative) transmembrane proteins
(nsp2, nsp3 and nsp5) that are thought to anchor the viral
RNA-synthesizing machinery to modified intracellular membranes. These membrane structures as well as the role of nsp1
proteins in transcriptional control and innate immune
evasion will be discussed separately below.
The functions of nsp68 and a conserved cysteine-rich
domain in the C-terminal part of nsp2 have remained
enigmatic thus far. Interestingly, the latter domain is lacking
from the truncated, 22 PRF-derived nsp2TF and nsp2N
proteins. nsp2N lacks a hydrophobic domain and is predicted
to be cytosolic, whereas nsp2TF is fitted with an alternative
transmembrane region that appears to target the protein to
the exocytic pathway (Fang et al., 2012). Full-length nsp2,
however, is a key component of the viral replication
structures. Additional nsp2- and nsp2TF-related proteins,
likely resulting from further post-translational modification
and/or cleavage, were observed in PRRSV-infected cells
2146
(Fang et al., 2012; Han et al., 2010), but their biological

significance needs to be studied in more detail. It is
interesting to note that nsp2N and nsp2TF both contain
the N-terminal PLP2 domain, and may thus target this viral
DUB to alternative locations in the cell.
In addition to PLP2 and the 22 PRF mechanism, arterivirus
nsp2 stands out for its hypervariable region in which
deletions and insertions have occurred in both EAV and
PRRSV (Balasuriya et al., 2004b; reviewed by Fang & Snijder,
2010). Although its interactions with the immune system
remain to be elucidated, a cluster of immunodominant B
cell epitopes and potential T-cell epitopes is associated with
the hypervariable region. Variation in this nsp2 region was
initially linked to viral virulence, in particular for the highly
pathogenic Asian PRRSVs, but this claim continues to be
debated (reviewed by Fang & Snijder, 2010). Nevertheless,
certain nsp2 regions that are non-essential for replication
may play an important role in PRRSV pathogenesis in vivo.
Arterivirus ORF1b encodes the two core enzymes for viral
RNA synthesis: the RNA-dependent RNA polymerase
(RdRp) in nsp9 and the helicase domain in nsp10.

Phylogenetic analyses provided strong evidence for a
common ancestry of these arterivirus enzymes and their
coronavirus equivalents (den Boon et al., 1991; Gorbalenya
et al., 2006). Nsp10, which has been studied quite
extensively, also contains a nidovirus-wide conserved Nterminal zinc-binding domain, which was implicated in sg
mRNA synthesis (van Dinten et al., 1997). The predicted
NTP-binding and superfamily 1 helicase activities of nsp10
were corroborated by in vitro assays, which also revealed the
59A39 polarity of the unwinding reaction (Seybert et al.,
2000).
A fourth conserved arterivirus ORF1b domain encodes a
puzzling endoribonuclease function (originally named
NendoU, for nidovirus endonuclease specific for uridylate;
Ivanov et al., 2004; Snijder et al., 2003a). Its importance for
arterivirus replication was established by site-directed
mutagenesis (Posthuma et al., 2006), but the exact function
of NendoU and, in particular, the identity of its natural
substrate(s) in infected cells remains enigmatic. Recombinant EAV and PRRSV nsp11 display broad substrate
specificity in vitro, cleaving ssRNA and dsRNA substrates 39
of pyrimidines (Nedialkova et al., 2009). The individual
expression of nsp11 is extremely toxic and a similar broad
activity towards viral RNA substrates in infected cells
would clearly make NendoU expression potentially suicidal. This suggests that its access to RNA substrates is
strictly controlled in the context of infection, possibly by
compartmentalization of nsp11 in replication structures.
Two ORF1b-encoded replicase domains that remain to be
characterized are the conserved N-terminal third of nsp9
and the small C-terminal nsp12 cleavage product. It is also of
note that the enzymes and signals ensuring the 59 capping
and 39 polyadenylation of arterivirus mRNAs remain to be
identified.
Synthesis and translation of sg mRNAs
A hallmark of arteriviruses and other nidoviruses is the

synthesis of a 39-co-terminal nested set of sg mRNAs, from
which the structural proteins are translated (Fig. 6). Each
arterivirus sg mRNA contains a common 59 end leader
sequence (156211 nt) that is identical to the 59-proximal
part of the genome (de Vries et al., 1990). This property is
shared with coronaviruses, but not with several other groups
of nidoviruses. The fusion of the 59 leader to the coding
part (or body) of the sg mRNA is thought to rely on
discontinuous RNA synthesis a mechanism that resembles
copy-choice RNA recombination and that has been reviewed
extensively elsewhere (Pasternak et al., 2006; Sawicki et al.,
2007; Sola et al., 2011). Briefly, discontinuous negativestrand RNA synthesis is thought to generate a nested set of
subgenome-length, negative-stranded RNAs, which subsequently serve as templates for sg mRNA synthesis. Base
pairing of short conserved transcription regulatory sequences
(TRSs) plays a key role, as was first verified for EAV using
reverse genetics (Pasternak et al., 2001; van Marle et al., 1999).
At the 39 end of the nascent subgenome-length negative

strand, the body TRS complement can base pair with the TRS
that is present at the 39 end of the leader sequence (leader
TRS) in the genomic template.
Arterivirus sg mRNAs are produced in non-equimolar, but
relatively constant amounts, thus providing a mechanism
to regulate structural protein expression. Transcription
depends on TRS duplex formation and, in general, the
relative amounts of sg mRNA correlate with the calculated
stability of this duplex (Pasternak et al., 2003). Structural
studies (van den Born et al., 2004) placed the leader TRS in
a single-stranded loop referred to as the LTH (Fig. 4a) a
critical signal in EAV discontinuous RNA synthesis (van
den Born et al., 2005).
Several replicase subunits have been implicated in transcriptional regulation, with specific mutations in both EAV
nsp1 and nsp10 causing the (near-) complete inactivation
of sg mRNA synthesis (Tijms et al., 2001; van Dinten et al.,
1997). EAV nsp1 is thought to control the accumulation of
genome and sg mRNAs by determining the levels at which
their negative-stranded templates are produced (Nedialkova
et al., 2010). An N-terminal ZF domain was implicated in
this function, but other nsp1 domains are also important
(Tijms et al., 2001). In PRRSV, where nsp1 is internally
cleaved into nsp1a and nsp1b, the ZF-containing nsp1a
subunit appears to fulfil a similar role in transcriptional
regulation (Fig. 5b; Kroese et al., 2008; Sun et al., 2009).
With the exception of the smallest transcript, arterivirus sg
mRNAs are structurally polycistronic but are presumed to
be functionally monocistronic. Notable exceptions are the
functionally bicistronic mRNAs from which the partially
overlapping gene sets E/GP2 and ORF5a/GP5 are expressed
(Firth et al., 2011; Godeny et al., 1998; Johnson et al., 2011;
Snijder et al., 1999).
Virion, structural proteins and assembly
Virion and nucleocapsid properties
The arterivirus genome is encapsulated by a single N
protein of 110128 aa, forming a core that is wrapped in a
lipid envelope containing various surface GPs and other
membrane proteins (Fig. 7). The roughly spherical or ovalshaped virions have a diameter of 5060 nm and reported
buoyant densities of 1.131.17 g cm23 in sucrose (Snijder &
Meulenberg, 1998; Spilman et al., 2009). They possess a
relatively smooth envelope (Fig. 7), which is likely explained
by the small ectodomains of the two major envelope
proteins, GP5 and M protein.
The arterivirus nucleocapsid has long been assumed to be
isometric, but recent cryo-electron tomography studies
of PRRSV particles rather suggest a pleomorphic core
structure (mean diameter 39 nm), possibly resembling that
proposed for coronaviruses in terms of being a helical coil
or an even more loosely organized filamentous structure
(Dokland, 2010; Spilman et al., 2009). The N protein is
phosphorylated, which may modulate nucleic acid binding
2147

5
Genome
Leader
Progeny genomes
Leader
Anti-lea
Anti-genome
Leader
Genome
REPLICATION
RdRp complex
TRANSCRIPTION
5
+B
Genome
Leader
LTH
+L
B
3
+B
3 B
+B
3
5
nsp1
Nascent
subgenome-length minus strand
Anti-leader
Anti-leader
Anti-leader
5
Nested set of subgenome-length minus strands
5
5
Leader
Leader
Leader
Nested set of sg mRNAs
Fig. 6. Arterivirus RNA synthesis. Model for replication and transcription using a hypothetical genome encoding three sg
mRNAs. The top half of the scheme depicts the replication of the genome from a full-length minus-strand intermediate (antigenome). The bottom half illustrates how minus-strand RNA synthesis can be interrupted at a body TRS (+B), after which the
nascent minus strand, having a body TRS complement (B) at its 39 end, is redirected to the leader TRS (+L) near the 59 end of
the genome. Guided by a base-pairing interaction between the B and +L sequences, RNA synthesis is resumed to add the
anti-leader sequence to each nascent subgenome-length minus strand. Subsequently, the latter serves as template to produce
a sg mRNA. The RdRp complexes engaged in replication and transcription may differ, as transcription-specific regulatory
protein factors, like EAV nsp1, have been described (Nedialkova et al., 2010). For further details, see text. Adapted from Snijder
& Kikkert (2013).
or proteinprotein interactions (de Vries et al., 1992;

Wootton et al., 2002). Crystal structures were obtained for
the capsid-forming C-terminal domain of the PRRSV and
EAV N proteins (Fig. 7f). The proteins N-terminal domain
contains many positively charged residues and presumably
binds RNA (reviewed by Dokland, 2010). Based on cryoEM studies of frozen hydrated PRRSV particles, the core
was proposed to consist of two intertwined layers of N
protein dimers that sandwich the genome (Fig. 7gi;
Spilman et al., 2009). Specific RNA encapsidation signals
that interact with the N protein have not been identified
thus far.
The arterivirus envelope is now presumed to contain two
major and five minor envelope proteins (Fig. 7e). With the
exception of the recently discovered ORF5a protein (Firth
et al., 2011), they are all critical requirements to produce
infectious progeny (Molenkamp et al., 2000b; Wissink et al.,
2005). In reverse genetics studies, EAV mutants lacking one
of the minor GP genes (see below) produced non-infectious subviral particles that consisted of RNA, and the N,
M and GP5 proteins, suggesting that these form the basic
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virion scaffold. The defect could be complemented in trans

by expression of the deleted gene, thus generating a system
to produce disabled infectious single-cycle viruses with
potential vaccine applications (Welch et al., 2004; Wieringa
et al., 2004; Zevenhoven-Dobbe et al., 2004).
Major envelope proteins
The two major envelope proteins are the major GP (GP5

in EAV, PRRSV and LDV) and the non-glycosylated M
protein, which form a disulphide-linked heterodimer (de
Vries et al., 1995; Faaberg & Plagemann, 1995; Snijder et al.,
2003b). The EAV GP5/M heterodimer is essential for virus
assembly, possibly by inducing the membrane curvature
required for virus budding. The N-terminal half of the
conserved M protein presumably traverses the membrane
three times, exposing only a short ectodomain (1018 aa)
on the virion surface (de Vries et al., 1992; Faaberg &
Plagemann, 1995). The major GP is the most variable
structural protein, although important structural features
are conserved between arteriviruses. The proteins consist of
(b)
(a)
(d)
(c)
TM domains
Spike
complex
52 nm
6.5 nm
42 nm
(f)
(e)
3 nm
+RNA genome
NUCLEOCAPSID
f~39 nm
N protein
E
C
N
5a
Small hydrophobic proteins

(g)
Minor GP trimer
ENVELOPE
f~55 nm
GP3
Major envelope
protein dimer
Lipid bilayer
N-linked glycans
(h)
(i)
GP4
GP5
GP2
Fig. 7. Arterivirus structure. Transmission electron microscopy (EM) images of (a) extracellular PRRSV particles and (b) a
budding EAV particle. Adapted from Snijder & Meulenberg (1998). (c) Negatively stained, purified PRRSV particles. All bars,
25 nm. Adapted from Spilman et al. (2009). (d) Cryo-EM of a representative PRRSV particle in vitreous ice. A (putative) spike
protein complex and striations possibly corresponding to the transmembrane domains of envelope proteins are indicated.
Adapted from Spilman et al. (2009). (e) Schematic representation of the structure of an arterivirus particle. The stoichiometry of
virion components was chosen (more or less) arbitrarily. For abbreviations, see main text. Adapted from Snijder & Kikkert
(2013). (f) Ribbon diagram of the crystal structure of the dimer of the C-terminal dimerization domain of the PRRSV N protein
(PDB entry 1P65). Adapted from Snijder & Kikkert (2013). (gi) Cryo-EM-based tomographic reconstruction of a PRRSV
particle, suggesting that the virion core is not solid but consists of a two-layered shell that surrounds a hollow central cavity.
Adapted from Spilman et al. (2009). (g) Cutaway view of the internal core, which appears to be disorganized and to consist of
density strands bundled together into a ball. The core is shown as an isosurface, coloured by the radius from the centre of the
particle (from red to blue). (h) The core has been cut open to show its internal structure, including the central density (red)
typically seen in the tomograms. (i) A 6.3 nm thick slab through the centre of one PRRSV particle tomogram, with several copies
of the crystal structure of the dimeric C-terminal domain of N [see (f)], rendered at a comparable resolution and superimposed
on the oblong densities in the core. (c, d, fi) Courtesy of Dr Terje Dokland, University of Alabama at Birmingham, Birmingham,
AL, USA.
an ectodomain, a central hydrophobic region predicted to

span the membrane three times and a sizeable cytoplasmic
C-terminal domain of 5075 aa. An N-terminal signal
sequence is predicted to be cleaved from the ectodomain
(Dokland, 2010), which is post-translationally modified
with one to three N-linked glycans (de Vries et al., 1992;
Faaberg & Plagemann, 1995; Zhang et al., 2010) that can
become very heterogeneous due to the attachment of polyN-acetyllactosamine structures of different size during GP5
maturation in the Golgi complex. The GP5 ectodomain is
an important target for neutralizing antibodies and for
LDV (Li et al., 1998), for example, its variable N-glycosylation
was linked to important differences in neutralization and
pathogenesis (see below).
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Minor envelope proteins
The structural properties of arterivirus minor envelope

proteins have been reviewed recently by Dokland (2010).
The minor GPs (GP2, GP3 and GP4) are present in virions
as disulphide-linked GP2GP3GP4 heterotrimers and
GP2GP4 heterodimers (Das et al., 2010; de Lima et al.,
2009; Wieringa et al., 2003a, b; Wissink et al., 2005). GP2 is
a conventional class I integral membrane protein containing an N-terminal signal peptide, an ectodomain with one to
four potential N-glycosylation sites, and a C-terminal
transmembrane segment and short cytoplasmic tail.
Cysteine residues in the GP2 ectodomain are critical for
the formation of intramolecular and intermolecular disulphide bridges, and the interaction with the GP4 ectodomain
(Wieringa et al., 2003a). Arterivirus GP3 is a heavily
glycosylated integral membrane protein containing a noncleaved N-terminal signal sequence and a hydrophobic Cterminal domain (de Lima et al., 2009; Wieringa et al.,
2002). The protein may be anchored in the membrane with
both termini (Wieringa et al., 2002). Like GP2, GP4 is a class
I membrane protein with an N-glycosylated ectodomain
from which a signal peptide is removed (Meulenberg et al.,
1997; Wieringa et al., 2002). A recent PRRSV study suggests
that, in addition to being part of the GP2GP3GP4
heterotrimer, PRRSV GP4 interacts with the major GP, GP5
(Das et al., 2010).
The two remaining arterivirus envelope proteins are small
non-glycosylated poly peptides that are predicted to be
largely embedded in the membrane. In EAV (Snijder et al.,
1999) and PRRSV (Wu et al., 2001), the small E protein
was identified as a minor structural protein, which
associates with the minor GP heterotrimer (Wieringa
et al., 2004). The E protein is essential for virus infectivity
and, although its membrane topology is unknown, was
proposed to oligomerize and form an ion channel that
could play a role during viral entry and/or fusion (Lee &
Yoo, 2006). The E protein contains a conserved myristoylation signal (Thaa et al., 2009) that is important but not
absolutely required for virion infectivity.
Recently, a second small hydrophobic protein, encoded by
the previously undiscovered ORF5a, was identified for EAV
and PRRSV (Firth et al., 2011; Johnson et al., 2011). The
ORF5a protein was detected in purified PRRSV. Although
EAV reverse genetics demonstrated that it is not essential
for infectivity, knockout mutants produce significantly
reduced progeny titres.
Assembly and release
Since the N protein is found in association with viral

replication structures, arterivirus genome encapsidation
may be initiated at the site of viral RNA synthesis (Tijms
et al., 2002). Recent electron tomography studies of EAVinfected cells revealed a network of N protein-containing
sheets and tubules of unknown function, which appear
intertwined with the membranous replication structures
(see below). Arterivirus budding involves the wrapping of a
2150
preformed nucleocapsid by membranes of the smooth

endoplasmic reticulum (ER) or Golgi complex (Magnusson
et al., 1970; Wood et al., 1970), in which the viral envelope
proteins appear to be retained. The formation of the GP5/M
heterodimer is a primary determinant of virus assembly and
triggers the transport of both proteins to the Golgi complex
a step correlated with the production of infectious progeny
(Snijder et al., 2003a; Verheije et al., 2002b; Wieringa et al.,
2004; Wissink et al., 2005). Once released into the lumen of
secretory pathway compartments, virions are transported to
the plasma membrane and released, while undergoing
extensive glycan processing in the Golgi complex.
Arterivirushost interactions
Tropism, entry and receptors
With the exception of EAV, arterivirus tropism is very
restricted (Plagemann & Moennig, 1992; Snijder &
Meulenberg, 1998). EAV replicates efficiently in primary
horse macrophages and kidney cells, and in a remarkable
variety of cell lines. LDV grows in primary mouse
macrophages, but not in macrophage or other cell lines. In
addition to replicating in primary macrophages from their
respective hosts, SHFV and PRRSV replicate in certain
African green monkey kidney cell lines (MA-104) and
derivatives thereof, such as MARC-145 (Duan et al., 1998).
Arterivirus entry occurs through standard clathrin-mediated
endocytosis, and the viral nucleocapsid is released into the
cytosol following endosome acidification and membrane
fusion (Kreutz & Ackermann, 1996; Nauwynck et al., 1999).
However, fusion activity has not been convincingly assigned
to any of the viral envelope proteins. Likewise, the debate on
which protein(s) determine tropism and receptor binding
has not been settled. In cell culture, a chimeric PRRSV
equipped with the EAV minor GPs and E protein gained the
broad tropism that is typical of EAV (Tian et al., 2012),
whereas EAV/PRRSV chimeras with swapped GP5 and M
ectodomains did not display such a changed tropism
(Dobbe et al., 2001; Verheije et al., 2002b). Thus, the GP5/
M heterodimer may be the key player in envelope formation
and budding, while the minor GPs are the prime determinants of host cell binding and possibly also fusion and
entry.
The host factors that mediate entry have only been studied in
detail for PRRSV (for recent reviews, see Van Breedam et al.,
2010a; Welch & Calvert, 2010). Heparin-like molecules on
the cell surface (Delputte et al., 2002) and sialic acids on the
virion surface (Delputte & Nauwynck, 2004) were implicated in PRRSV binding to porcine alveolar macrophages.
Subsequently, internalization would be mediated by sialoadhesin (CD169) a macrophage-restricted member of the
siglec family of immunoglobulin-like lectins that carries
sialic acids with which the ectodomains of the GP5/M
heterodimer can interact (Van Breedam et al., 2010b).
Expression of porcine CD169 in non-susceptible cell lines
can mediate PRRSV internalization, but not disassembly and
productive infection. However, a CD169 homologue is not
expressed by the MARC-145 cell line that fully supports

PRRSV infection (Duan et al., 1998), suggesting the existence
of alternative receptors. In particular, CD163, a member of
the scavenger receptor cysteine-rich family, was identified as
its expression rendered a variety of non-permissive cell lines
susceptible to PRRSV infection (Welch & Calvert, 2010). It
was postulated that CD169 and CD163 work together in
porcine macrophages, with the former serving as a receptor for
internalization and the latter playing a key role in uncoating
and genome release (Van Gorp et al., 2008). Clearly, several
questions regarding arterivirus entry remain to be addressed,
despite the considerable recent progress made for PRRSV.
Replication structures
As in all +RNA viruses, arterivirus replicative proteins

assemble into an enzyme complex for viral RNA synthesis
that is associated with virus-induced membrane structures.
In the case of arteriviruses and other nidoviruses, these are
not simple invaginations, but more complex networks of
modified intracellular membranes in the perinuclear region
of the infected cell (Fig. 8ac; Li et al., 2012; van der Meer
et al., 1998). All arterivirus replicase subunits studied to
date co-localize in this area, except for the nsp1 and
nsp2TF proteins (Chen et al., 2010; Fang et al., 2012; Tijms
et al., 2002). Infection triggers the formation of large
numbers of double membrane vesicles (DMVs) (Fig. 8d),
with which the viral replication and transcription complex
(RTC) is thought to be associated (Pedersen et al., 1999; van
Hemert et al., 2008). The formation of this membranous
scaffold for the RTC has been attributed to the ORF1aencoded (putative) membrane-spanning proteins nsp2, nsp3
and nsp5, and paired membranes and DMVs can be induced
by the expression of nsp2 and nsp3 alone (Snijder et al., 2001).
A recent electron tomography study revealed that, as in
coronaviruses, DMVs can be interconnected by their outer
membranes (Fig. 8eh), thus forming a network of modified
ER in EAV-infected cells (Knoops et al., 2012). A striking
additional similarity with coronaviruses is the presence of
dsRNAs inside the DMVs. Although these may represent
double-stranded intermediates of replication and transcription, their enclosure by a double membrane is puzzling and
their functionality in RNA synthesis remains to be demonstrated.
Little is known about host proteins that may interact with
arterivirus RNA or transmembrane non-structural proteins, or that may be recruited to replication structures.
Common sets of (as-yet unidentified) host proteins bind in
vitro to the 3 end of the genome or anti-genome of SHFV,
EAV and PRRSV, and were implicated in the initiation of
arterivirus RNA synthesis (Hwang & Brinton, 1998; Maines
et al., 2005). The RNA-synthesizing activity of EAV RTCs
associated with crude membrane fractions from infected
cells does depend on membrane integrity. Moreover, an asyet unidentified host protein factor or complex from the
cytosolic fraction of the cells is required for in vitro RTC
function (van Hemert et al., 2008). Recent EAV and
PRRSV inhibitor studies with the drug cyclosporin A

suggest the involvement of members of the cyclophilin
family, most likely cyclophilin A, in arterivirus RNA
synthesis (de Wilde et al., 2013). Furthermore, it has been
suggested that the autophagy marker microtubule-associated protein 1 light chain 3 (LC3) and the ER degradationenhancing a-mannosidase-like1 (EDEM1) associate with
EAV DMVs (Monastyrska et al., 2013), but the functional
relevance of this observation for viral RNA synthesis remains
unclear since the analysis was only performed late in
infection. In any case, in this study EAV replication was
unchanged in autophagy-deficient cells, although it was
affected by the depletion of LC3, specifically in its nonlipidated form. In contrast, several recent PRRSV studies
(for example Sun et al., 2012; Liu et al., 2012) suggested that
autophagy promotes arterivirus replication, as judged from
the effect of inhibiting the autophagy pathway using
pharmacological and siRNA approaches. Clearly, additional
research is needed to establish the precise role of autophagy
or individual factors from this pathway in the arterivirus
replicative cycle.
Arterivirus proteins in the nucleus
Despite the cytoplasmic replication cycle, some arterivirus

proteins are directed (in part) to the nucleus of infected
cells, specifically the nsp1 proteins and the N protein. Their
possible involvement in modulating host cell responses is
discussed in the next section, together with other viral
proteins implicated in similar functions.
Whereas the signals or interactions that target arterivirus
nsp1 proteins to the nucleus remain to be defined, residues
4147 of the PRRSV N protein were identified as a nuclear
localization signal that interacts with the nuclear transporters importin a and b (Rowland et al., 2003). Like its EAV
counterpart (Tijms et al., 2002), the PRRSV N protein
accumulates in the nucleoli of infected cells. Remarkably, a
leptomycin B-induced block of CRM1-mediated nuclear
export resulted in the nuclear accumulation of the EAV N
protein, indicating that the protein must shuttle between
the cytoplasm and nucleus before playing its role in cytoplasmic virus assembly. Various nuclear host proteins
interact with the PRRSV N protein (reviewed by Sun et al.,
2012a), including fibrillarin, nucleolin and poly(A)-binding
protein, but the functional implications of these findings
remain to be studied. Using reverse genetics, a knockout
mutant for the nuclear localization signal in the PRRSV N
protein was engineered, which was viable, but seriously
attenuated. Pigs infected with this mutant developed
reduced viraemia and significantly higher neutralizing
antibody titres (Lee et al., 2006).
Immune responses
Innate immune responses
Arterivirus infections generally elicit poor innate immune
responses, probably explaining the weak adaptive immune
2151
(b)
(a)
(c)
dsRNA
nsp3
(d)
nsp2
nsp2TF
nsp9
(e)
(f)
ER
(g)
(h)
Fig. 8. Arterivirus replication structures. (ac) Subcellular localization of EAV (a, b) and PRRSV (c) non-structural proteins in
infected Vero-E6 (a, b) or MARC-145 (c) cells as observed by immunofluorescence microscopy. (a) Double labelling for EAV
nsp3 (red) and dsRNA (green), presumably highlighting structures involved in viral RNA synthesis. (b) Staining for EAV nsp9,
which contains the RdRp domain. (c) Double labelling showing that PRRSV nsp2TF (red) is not targeted to replication
structures (labelled for nsp2; green). (dh) Electron micrographs and tomograms of EAV-infected cells. (d) Membrane changes
in infected BHK-21 cells with the rectangle indicating a region containing many DMVs. Arrows, virions budding from smooth
intracellular membranes. Bar, 250 nm. (e, f). Tomogram slices showing close-ups of DMVs in EAV-infected Vero-E6 cells
preserved by high-pressure freezing. Arrows indicate connections of DMV outer membranes with ER (e) or other DMVs (f).
Bars, 50 nm. (g, h) Three-dimensional surface-rendered model of DMVs from a tomogram of EAV-infected Vero-E6 cells (7 h
post-infection). DMV membranes, brown; DMV cores, blue; ER, beige; mitochondria, red; N protein tubules, green. Bar,
250 nm. (h) Close-up showing neck-like connections of DMVs to ER (arrows). (a, b) Courtesy of Dr Yvonne van der Meer
(Leiden University Medical Center); adapted from Snijder & Kikkert (2013). (c, d, eh) Adapted from Fang et al. (2012), Fang &
Snijder (2010) and Knoops et al. (2012), respectively, with permission.
responses and viral persistence. For a detailed overview of

the immune response to arterivirus infection, the reader is
referred to other reviews (Darwich et al., 2010; Kimman et al.,
2009; Sun et al., 2012a) and references therein. Viral protein
functions that may help arteriviruses to modulate or evade host
immune responses will be discussed in the next section.
2152
It has not been firmly established which pattern recognition receptors of the innate immune system are involved
in the sensing of arterivirus infection. The involvement of
Toll-like receptor 3 (TLR3) during PRRSV infection in
macrophages has been suggested (Darwich et al., 2010 and
references therein), but also other TLRs were implicated in
triggering innate immune response. Experimental LDV

infection of knockout mice established the importance of
TLR7 in the induction of the type I IFN response (Ammann
et al., 2009). An EAV study in knockout mouse embryonic
fibroblasts suggested that the retinoic acid inducible I
(RIG-I)-like receptors, including melanoma differentiationassociated protein 5 (MDA5) and RIG-I, both have a role in
countering arterivirus infection (van Kasteren et al., 2012).
The extent to which arterivirus infection suppresses the
innate immune response varies among genetically diversified isolates. It was demonstrated that different PRRSV
field isolates varied in their ability to induce IFN-a
expression in porcine alveolar macrophages an observation that may account for differences in virulence and
pathogenicity (Lee et al., 2004). Multiple studies reported
that PRRSV infection induces IL-8 expression, while the
induction of IL-6 and IL-10 is still debated (Darwich et al.,
2010), most likely due to variation between PRRSV isolates
tested. In EAV, virulent and non-virulent strains differed in
their ability to induce the expression of TNF-a, IL-1b, IL-6
and IL-8, and the magnitude of the cytokine response in
cultured macrophages reflects virus virulence in the field
(Balasuriya et al., 2004a; Moore et al., 2003).
The role of innate cytokines in LDV infection is rather
unclear. LDV normally induces early and transient expression of pro-inflammatory cytokines, including IL-6 and IL12, but their effect on virus replication and pathogenesis
remains to be elucidated (Coutelier et al., 1995; MarkineGoriaynoff et al., 2001). LDV-induced IFN-a expression
was also reported, but the virus is insensitive to a systemic
IFN-a response in mice (Ammann et al., 2009). LDV infection activates NK cells and consequently a large increase in
IFN-c production was observed, but these responses were
unable to control LDV replication in vivo (MarkineGoriaynoff et al., 2002).
Innate immune evasion
Arteriviruses likely employ a combination of functions to

modulate the hosts innate immune response and several
proteins have been implicated: PRRSV nsp1a, nsp1b, nsp2,
nsp11 and N protein, and EAV nsp2 and N protein.
However, many of these studies employed expression of
individual viral proteins rather than infected cells, which
may influence parameters like expression kinetics and
subcellular localization of viral proteins. Furthermore, thus
far only a few studies addressed the (postulated) immunemodulating activities of PRRSV non-structural proteins
during replication and pathogenesis in animals. Thus,
corroborating these (proposed) innate immune evasion
functions in infected cells and animal models remains a
critical step. An interesting example is the proposed
modulation of IFN-b production by the PRRSV nsp11
NendoU endoribonuclease (Sun et al., 2012a), which is
known to be highly cytotoxic upon its individual expression
in a variety of systems. The cytosolic version of the enzyme
may target the overall RNA population of the cell, thus
inducing a translational shut-off (see above; Nedialkova et

al., 2009), whereas NendoU activity in infected cells is likely
to be restricted by, for example, association with membranous replication structures.
PRRSV nsp1a and nsp1b were proposed to be major
modulators of the type I IFN response (reviewed by Fang &
Snijder, 2010; Sun et al., 2012a). Their expression suppresses
IFN-b activation following the induction of innate immune
responses by Sendai virus infection or dsRNA transfection
(Beura et al., 2010; Chen et al., 2010; Song et al., 2010). The
nsp1a is able to suppress the activation of NFkB, while
nsp1b has the ability to inhibit IFN regulatory factor 3
(IRF3)-mediated type I IFN production. Both proteins
localize to the nucleus of infected cells and a recent study
suggests that nsp1a interferes with IFN-b production
through degradation of the transcription factor CREB
(cAMP response element-binding protein)-binding protein
(CBP) in the nucleus (Han et al., 2013). In addition, nsp1b is
able to inhibit downstream IFN-induced signalling pathways
by interrupting STAT1 phosphorylation and nuclear
translocation of IFN-stimulated gene factor 3 (ISGF3) in
the JAK (Janus kinase)STAT (signal transducer and
activator of transcription) pathway (Chen et al., 2010;
Patel et al., 2010). The nsp1a and nsp1b domains critical for
their function in IFN suppression have been identified (e.g.
by alanine-scanning mutagenesis) (Beura et al., 2012). Most
recently, a highly conserved nsp1b motif was implicated in
inhibiting the IFN response (Li et al., 2013), as its
mutagenesis yielded viable recombinant viruses inducing
increased expression of IFN-a, IFN-b and ISG15.
The arterivirus PLP2 (Fig. 5) is a dual-specificity proteinase
that not only cleaves the nsp2/3 junction in the replicase
polyproteins (see above), but is also able to disrupt innate
immune signalling by removing Ub and Ub-like modifiers
from host cell substrates. EAV and PRRSV PLP2 were
found to exhibit general DUB activity towards cellular Ub
conjugates and to also remove the IFN-induced Ub
homologue ISG15, which is thought to have antiviral
activity (Frias-Staheli et al., 2007; Sun et al., 2012b). The
biological significance of this activity was supported by the
fact that PLP2-DUB could inhibit type I IFN production by
interfering with NFkB activation, which is regulated by
polyubiquitination (Sun et al., 2010). PLP2 overexpression
in cell culture can suppress IFN-b induction by RIG-I-like
receptors. The proteinase is able to remove K63-linked
polyubiquitin from RIG-I in order to inhibit downstream
signalling (van Kasteren et al., 2012). Recently, based on
the structure of the EAV PLP2Ub complex, targeted
mutagenesis was used to inhibit DUB activity without
affecting nsp2/3 cleavage (van Kasteren et al., 2013). The
resulting virus mutants displayed WT replication properties and their strongly reduced PLP2-DUB activity translated into strikingly enhanced innate immune signalling in
infected equine cells. The mRNA levels for IFN-b increased
by nearly an order of magnitude, suggesting that this
approach could be applied to next-generation vaccine
development (see below).
2153
As described above, arterivirus N proteins are transported

to the nucleus and interactions with various host cell proteins
have been described (reviewed by Sun et al., 2012a).
However, no firm conclusions on the functional significance
of these interactions have been drawn. The PRRSV N protein
may be responsible for the early upregulation of IL-10 during
infection, suggesting that it modulates the production of
regulatory T cells to suppress the innate immune response
(Wongyanin et al., 2012), but this hypothesis requires further
investigation.
Humoral immune responses
In general, arterivirus infection induces the early production of high antibody levels. Antibodies in EAV-infected
horses mainly recognize the structural proteins of GP2,
GP5, M and N (Chirnside et al., 1995; MacLachlan et al.,
1998), and non-strutural proteins of nsp2, nsp4, nsp5 and
nsp12 (Go et al., 2011b). In PRRSV-infected animals,
antibody responses against all GPs, M and N proteins were
described, with the anti-N response being the first and
strongest (Darwich et al., 2010; Lopez & Osorio, 2004).
PRRSV nsp1a, nsp1b, nsp2 and nsp7 also induced a
humoral immune response (Brown et al., 2009; Johnson
et al., 2007), with antibodies specific for these proteins
being detected as early as 14 days post-infection (d.p.i.)
and lasting to at least 202 d.p.i.
Neutralizing antibodies in EAV-infected horses can develop
as early as 714 d.p.i. In contrast, neutralizing antibodies in
PRRSV- or LDV-infected animals are generated late and
their titres remain low (reviewed by Balasuriya et al., 2004a;
Lopez & Osorio, 2004). In arterivirus-infected animals,
neutralizing antibodies are predominantly directed to the
major GP, GP5. Various neutralizing epitopes were
mapped to the GP5 ectodomain in EAV, PRRSV and LDV
(Balasuriya et al., 1997; Plagemann et al., 2002; Plagemann,
2004), and also to the PRRSV GP4 ectodomain (Meulenberg
et al., 1997). Synthetic peptides representing neutralizing
epitopes from LDV and PRRSV GP5 did not induce virusneutralizing antibodies, suggesting that additional residues
or structural features are critical for antigenicity (Lopez &
Osorio, 2004; Plagemann, 2001). The four major neutralizing epitopes in EAV GP5 are conformation dependent and
GP5/M heterodimerization is thought to be critical for the
induction of neutralizing responses in both PRRSV and EAV
(Balasuriya et al., 2004a; Lopez & Osorio, 2004).
The delayed or weak induction of neutralizing antibodies
upon arterivirus infection has frequently been linked to
GP5 glycosylation, particularly for PRRSV and LDV. It
was postulated that non-neuropathogenic LDV strains
can establish persistent infections due to the fact that
neutralizing antibodies bind less efficiently to their
highly glycosylated GP5 ectodomain (Chen et al., 1997;
Li et al., 1998). In PRRSV, expression of a hypoglycosylated GP5 variant induced significantly higher neutralizing antibody levels in infected piglets (Vu et al., 2011).
An immuno-dominant decoy epitope was identified in
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PRRSV GP5, which is thought to subvert the immune

systems ability to focus on an adjacent neutralizing
epitope (Ostrowski et al., 2002). It has been proposed
that LDV- and PRRSV-specific antibodies may contribute to antibody-dependent enhancement of infection
(Cafruny & Plagemann, 1982; Yoon et al., 1996), in
particular through virus opsonization by antibodies
recognizing certain non-neutralizing N and GP5 epitopes, leading to enhanced virus internalization by
macrophages (Cancel-Tirado et al., 2004). Finally,
PRRSV infection was proposed to manipulate and delay
neutralizing antibody production by preventing the
development of a normal B cell repertoire in piglets
(Butler et al., 2008).
The humoral immune response against SHFV infection
varies between monkey species and virus isolates tested
(Gravell et al., 1986). Virulent SHFV strains, which cause
acute disease in patas monkeys, induced neutralizing
antibodies by 7 d.p.i., leading to complete virus clearance
by 21 d.p.i. In contrast, SHFV strains that cause persistent
infections in these monkeys induce very low antibody
titres.
Cell-mediated immune responses
The cell-mediated immune (CMI) response to arterivirus

infection has not been characterized in great detail. Cellmediated cytotoxic responses against EAV were studied in
experimentally infected ponies, in which CD8+ T-cellmediated cytotoxicity was virus strain-specific and genetically restricted (Castillo-Olivares et al., 2003). The EAVspecific CTL precursors persisted for at least 1 year after
infection.
CMI responses, including CD4+, CD8+ and CD4+/CD8+
double-positive T cells, have been detected in PRRSVinfected animals, and they appear transiently between 2
and 8 weeks post-infection (reviewed by Darwich et al.,
2010; Murtaugh et al., 2002). The abundance of PRRSVspecific T cells and IFN-c-producing cells in both acute and
persistently infected swine appears to be highly variable,
and there is no apparent correlation with the viral load in
lymphoid tissues. It was reported that PRRSV has the
ability to downregulate the expression of MHC class I and
II molecules on the surface of antigen-presenting cells.
Several PRRSV structural proteins (GP4, GP5, M and N)
were found to be strong CMI inducers and a set of T-cell
epitopes was identified (reviewed by Darwich et al., 2010).
In addition, potential T-cell epitopes were identified in
nsp9 and nsp10 (Parida et al., 2012).
LDV-specific CD4+ and CD8+ T-cell immune responses
in infected mice did not suffice to achieve virus clearance
(Even et al., 1995; van den Broek et al., 1997). Additional
studies are required to determine whether there is a
correlation between T-cell responses in vitro and protection
in vivo, and overall it seems likely that arteriviruses manipulate the CMI response, directly or indirectly.
Arterivirus disease, diagnostics and prevention

Epidemiology and disease
The natural host range of arteriviruses is restricted to
equids (EAV), swine (PRRSV), mice (LDV), and several
genera of African and Asian monkeys (SHFV).
Macrophages appear to be the primary target cell for all
arteriviruses (Plagemann & Moennig, 1992). Receptors for
arterivirus entry have only been studied for PRRSV and have
been discussed above.
Equine viral arteritis is a respiratory and reproductive
disease that is encountered in equine populations worldwide. In the field, EAV infections are frequently subclinical
or persistent, but the virus is also capable of inducing a
variety of symptoms, including characteristic lesions of the
small muscular arteries (arteritis), acute anorexia and fever,
oedema, abortion in pregnant mares, and interstitial
pneumonia in neonates (for reviews, see Balasuriya &
MacLachlan, 2004; Holyoak et al., 2008; Timoney &
McCollum, 1993). EAV persistence in the ampulla of the
reproductive tract of infected stallions can result in a
carrier state and the risk of virus transmission through
infected semen. The prevalence of infection varies between
horse breeds and recent genome-wide association studies
pinpointed genetic differences associated with susceptibility
of CD3+ T lymphocytes to EAV infection in vitro (Go et al.,
2011a), and possibly also with the risk of persistent infection
in stallions (Go et al., 2012).
Following its emergence ~25 years ago, PRRSV has rapidly
become one of the most important viral diseases of swine
(Zimmerman et al., 2012). The virus continues to evolve
(Stadejek et al., 2013), causing numerous acute disease
outbreaks, resulting in swine mortality and abortion
storms, or in atypical porcine reproductive and respiratory
syndrome. The highly pathogenic PRRSV variants that
emerged in China in 2006 caused mortality rates between
20 % and 100 %, depending on the age and health of the
infected animals (Fig. 9). Reported primary target cells for
PRRSV replication are fully differentiated porcine lung
alveolar macrophages and other cells of the monocyte/
macrophage lineage (Duan et al., 1997; Thanawongnuwech
et al., 2000). Clinical manifestations include occasional
discolouring of the skin, most often on the ears (blue ear
disease) and vulva. Further symptoms are fever, anorexia,
breathing difficulties, lymphadenopathy, lung lesions and
abortion. After spreading through the circulation, PRRSV
can replicate persistently in the tonsils, lungs and lymphoid
organs. The pathogenesis and epidemiology of PRRSV has
been discussed in many excellent recent reviews (Shi et al.,
2010; Zimmerman et al., 2012).
LDV infection causes increased serum levels of lactate
dehydrogenase. The virus was initially isolated from laboratory mice, but subsequently also found in wild mice (Li
et al., 2000). LDV infection is life-long but asymptomatic
and is maintained by continuous rounds of cytocidal virus
replication in a renewable subpopulation of macrophages.
The virus has been used extensively as an in vivo research
model due to its ability to escape immune surveillance

(Cafruny, 1989; Plagemann & Moennig, 1992).
SHFV was originally discovered following an outbreak of
fatal haemorrhagic fever in captive rhesus macaques
(Tauraso et al., 1968), which likely contracted the virus
from asymptomatically infected African monkeys in the
same animal facility. Clinical signs in macaques included
early fever, oedema, dehydration and various haemorrhagic
manifestations, usually culminating in death within 2 weeks
with mortality rates approaching 100 %. Although SHFVlike viruses have never been isolated from human specimens,
the resemblance of the clinical symptoms in macaques with
those caused by, for example, Ebola and Marburg viruses in
humans has attracted considerable attention. SHFV and its
recently identified distant relatives appear to be endemic
among several species of African monkeys, including primates
(Lauck et al., 2013; London, 1977). They apparently cause
asymptomatic acute or persistent infections and the
zoonotic potential of this arterivirus reservoir is a highly
intriguing but as-yet unexplored matter.
Current practices and new diagnostic assays
For laboratory diagnosis of EAV, reverse transcriptase (RT)PCR assays can be used on nasopharyngeal swabs or washings,
conjunctival swabs, and blood samples. In addition, immunohistochemistry using specific mAbs to individual EAV
proteins is a reliable method for EAV diagnosis in tissues. A
virus neutralization assay remains the gold standard for
detection of serum antibodies against EAV (Holyoak et al.,
2008).
PRRSV can be detected using immunohistochemistry, virus
isolation and (indirect) fluorescent antibody (IFA or FA)
tests in serum or tissue samples. In serology, the IDEXX
ELISA is the most widely used test to detect antibodies
against PRRSV, but IFA and neutralization assays can also be
used. Furthermore, (real-time) RT-PCR assays are available
to specifically detect viral RNA in serum, semen and tissue
samples. Sequence analysis of the ORF5 region has routinely
been used for strain identification and differentiation, and a
related sequence database has been established (Zimmerman
et al., 2012). Although these traditional assays provide good
sensitivity and specificity in controlled laboratory settings,
experience from the field suggests that substantial improvement is needed in most of these areas.
Recently, assays using swine oral fluids have been evaluated
as an alternative to serology (Langenhorst et al., 2012; Prickett
& Zimmerman, 2010). Oral fluid is a complex mixture of
saliva and gingival crevicular fluid, which more closely
resembles serum than salivary gland secretions. Oral fluid
sampling methods are an inexpensive, safe and non-invasive
alternative to blood sampling to assess both acute infection
and immune prevalence in a swine population. Oral fluidbased diagnostic assays, including ELISA and (real-time)
RT-PCR, are being further developed and implemented in
major animal disease diagnostic laboratories. In addition,
2155
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 9. Clinical symptoms typical of infection with highly pathogenic PRRSV. (a) Infected piglet shivering with high fever. (b)
Characteristic purple/blue colouring of the ears. (c) Adult sow that has succumbed to porcine high-fever disease. In most
cases, infection in pregnant sows leads to abortion, but occasionally also to death of the sow. (d) Aborted fetuses as a result of
infection. (e) Kidney from an infected pig showing numerous blood spots (red arrows), found in 2030 % of the cases. (f)
Severe lesions and haemorrhage (red arrows) of lungs unique to highly pathogenic PRRSV infection. (a, e, f) Courtesy of Dr K.
Tian and Professor G. F. Gao, Chinese Academy of Sciences, Beijing, PR China; reprinted from Tian et al. (2007) under the
Creative Commons license. (c, d) Courtesy of the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of
Agriculture Sciences, Fuzhou, PR China.
the fluorescent microsphere immunoassay (Luminex technology) has been adapted to the use of oral fluid and serum
in order to improve the sensitivity of the assay. It allows the
uniform, simultaneous detection of multiple antigens or
antibodies within a small volume of a single sample
(Langenhorst et al., 2012). In traditional antibody detection
assays, PRRSV N protein was used as antigen, but recently
also certain non-structural proteins (nsp1a, nsp1b, nsp2 and
nsp7) have been explored as more accurate indicators of
infection (Brown et al., 2009; Langenhorst et al., 2012).
Arterivirus vaccines
Both modified live virus (MLV) and inactivated vaccines

are commercially available for the arteriviruses of veterinary
importance. For EAV, Arvac (MLV; Ford Dodge
Laboratories) and Artervac (inactivated; Ford Dodge
Laboratories) have been used in the field. Since the first
PRRSV MLV (RespPRRS/Ingelvac PRRS MLV; Boerhringer
Ingelheim Animal Health) became available in 1994, various
other MLV and inactivated vaccines have been released in
the USA and Europe (Mengeling, 2005). Together with
management strategies, these vaccines suffice to control
disease outbreaks. However, they are derived from single
virus strains, and are not always efficacious in preventing
reinfection and transmission. Furthermore, there are MLV
safety concerns, in particular regarding reversion to
virulence and the possibility of recombination with field
2156
isolates (Btner et al., 1997; Li et al., 2009; Storgaard et al.,

1999). The efficacy of MLV vaccines in protecting against a
broad spectrum of heterologous field isolates has also been
questioned (Kimman et al., 2009). In general, killed vaccines
are safer but less efficacious in inducing protection, which
makes the development of a safe and broadly protective
PRRSV vaccine a major challenge.
For EAV, several genetically engineered candidate vaccines
have been tested in vivo. Vaccination of ponies with a
subunit vaccine based on the GP5 ectodomain conferred
certain levels of protection (Castillo-Olivares et al., 2001).
More promising results were obtained with GP5/Mexpressing Venezuelan equine encephalitis virus replicon
particles. Horses vaccinated with this candidate vaccine
produced neutralizing antibodies, shed little or no virus
and developed only mild symptoms after a challenge
(Balasuriya et al., 2002). Using reverse genetics, a candidate
EAV live marker vaccine was constructed by deletion of the
major neutralization domain of GP5 (Castillo-Olivares et al.,
2003). This recombinant virus produced an asymptomatic
infection in ponies, which were subsequently protected
against a challenge with virulent EAV. A GP5 peptide-based
ELISA was developed to distinguish vaccinated animals
from those infected with WT virus.
Several experimental subunit/vectored vaccines based on
plasmid DNA and viral vectors that carry PRRSV structural
proteins (GP3, M and/or GP5) have been developed (reviewed
by Huang & Meng, 2010; Kimman et al., 2009). However, it

remains to be determined whether such vaccines could
generate better protection than the existing MLV and killed
whole-viral vaccines. In terms of efficacy in the field, PRRSV
MLV vaccines still are the most promising approach (Rock,
2007), and attempts to improve their safety and efficacy are
ongoing. A recent development is the application of
knowledge regarding determinants of virulence and immune
antagonist functions. Using reverse genetics, recombinant
chimeras of virulent field strains and attenuated vaccine
strains were generated, which were attenuated to variable
degrees in animal models (Kwon et al., 2008; Wang et al.,
2008). Furthermore, recombinant viruses carrying specific
mutations or deletions in viral immune evasion proteins
have been engineered (Beura et al., 2012; Li et al., 2013; Sun
et al., 2010, 2012b; van Kasteren et al., 2013).
Another drawback of current PRRSV vaccines is that
vaccinated animals cannot be distinguished from pigs that
have recovered from a natural infection. Several laboratories
have explored the possibility of constructing genetically
engineered marker vaccines (reviewed by Fang & Snijder,
2010). Using type 1 or 2 PRRSV infectious clones, an nsp2 B
cell epitope was deleted to create a negative marker, whereas
an antigenic protein or peptide tag was inserted into the
nsp2-coding sequence of the same construct to add a positive marker. To monitor the vaccination status of animals,
companion diagnostic assays were designed (Fang et al.,
2008). These genetically modified live (marker) viruses may
well provide the most rational approach to future PRRSV
vaccine development, although the use of recombinant
viruses in the field continues to be a matter of debate
between vaccine developers, swine practitioners and animal
health authorities.
Outlook
The arterivirus genome expression strategy is among the
most complex of currently known +RNA viruses and
includes an intriguing variety of regulatory mechanisms that
operate at the co- and post-translational level. Consequently,
EAV and increasingly also PRRSV are being used extensively
as research models to study basic aspects of +RNA virus
replication at large, and nidovirus molecular biology in
particular. Thus far most of the ~25 arterivirus proteins have
only been defined in basic terms and their functional characterization is one of the major challenges for future research.
This will enhance our understanding of, for example, the
intricacies of arterivirus RNA synthesis, replicase function,
replication structures and virushost interactions. The
recently discovered non-canonical translation mechanism
that produces nsp2TF/nsp2N adds another layer of complexity to arterivirus genome expression. This highly
efficient 22 PRF event is unprecedented in eukaryotic
systems and may have implications beyond the field of RNA
virology. Furthermore, the in-depth characterization of
nsp2TF/nsp2N function will likely reveal novel arterivirus
host interactions.
Following the discovery of the ORF5a protein as the

eighth arterivirus structural protein, the dissection of the
functional interactions between structural proteins during
particle assembly and disassembly has become an even
more complex issue. Recent studies revealed several
unique features of the arterivirus nucleocapsid and
envelope proteins, which in part link to unanswered
questions regarding attachment and entry. Although
progress has been made towards understanding host
factors involved in PRRSV entry, issues like the specific
functions of these host proteins and the role of
carbohydrate moieties on the virion and cell surface
remain to be addressed. The arterivirushost molecular
interplay may ultimately provide an explanation for the
highly specific tropism of this group of viruses. This
property, in turn, connects to the intriguing question
whether arteriviruses are able to cross species barriers and
emerge in other hosts. Considering the unapparent and
persistent infections caused by currently known arteriviruses, it may be difficult to detect arterivirus-induced
disease in other species. However, modern virus detection
techniques may compensate for this and are increasingly
likely to identify additional family members, as evidenced
by the recent discovery of multiple additional SHFV-like
viruses in African monkeys.
The development of improved vaccines and disease control
strategies has been a major driving force behind studies
into arterivirus molecular biology. EAV and PRRSV are
significant veterinary pathogens, and the latter is one of
the economically most important swine pathogens. The
recent Asian PRRSV outbreaks continue to cause enormous economic losses. The complete control of PRRSV and
EAV infection is hampered by natural characteristics
common to all RNA viruses, in particular rapid evolution
yielding genetically and antigenically diverse virus populations. Our poor understanding of arterivirushost interactions and immunology further complicates the control of
infection (e.g. due to major deficits in our knowledge of the
events initiating the immune response upon infection, the
key targets for B and T-cell-based protection, and the
mechanisms regulating the maturation of the immune
response). Persistent infection is another significant factor
impeding arterivirus control, as current diagnostic methods are unable to reliably identify persistently infected
animals. Recent advances in arterivirus non-structural
protein research has resulted in the development of
improved diagnostic assays and the identification of
various potential innate immune antagonists, with implications for the rational design of novel vaccines. Reverse
genetics has become a key tool to manipulate immune
antagonist functions in the context of vaccine development. The initial steps toward genetically engineered
MLV vaccines have been taken and illustrate how
molecular virological knowledge of arterivirus infection,
supported by structural biology and immunology, may
provide a rational basis for future disease prevention and
control.
2157
Acknowledgements
Btner, A., Strandbygaard, B., Srensen, K. J., Have, P., Madsen,

K. G., Madsen, E. S. & Alexandersen, S. (1997). Appearance of acute
We would like to acknowledge the contributions, over many years, of

a large number of colleagues who worked with us in the arterivirus
field. Our research was and is supported by the Netherlands Organization of Scientific Research (NWO grants 700.57.301, 700.10.352 and
700.59.008), the European Commission (grants 260644 and 264286),
the US Department of Agriculture (grants 2005-35204, 2007-01745,
2008-55620 and 2011-02925) and the US National Pork Board (grants
05-155, 06-173, 09-234, 11-037, 11-143 and 12-127).
PRRS-like symptoms in sow herds after vaccination with a modified

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Journal of General Virology (2013), 94, 21412163

Arterivirus molecular biology and pathogenesis

Eric J. Snijder,1 Marjolein Kikkert1 and Ying Fang2,3

Molecular Virology Department, Department of Medical Microbiology, Leiden University Medical

Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine,

Received 25 June 2013

Printed in Great Britain

The consequences of arterivirus infection can range from

E. J. Snijder, M. Kikkert and Y. Fang

arterivirus infection. In this review, we will summarize what

The arterivirus genome is a polycistronic +RNA (Figs 2

Genome structure and expression

As nidovirus discovery continued, in a remarkably wide

About 15 years ago, the family Arteriviridae was united

The expanding order Nidovirales

Fig. 1. Phylogeny of representatives of currently recognized

yields replicase polyprotein (pp) 1a (17272502 aa), whereas

Arterivirus molecular biology and pathogenesis

Table 1. Arterivirus genomes and database accession numbers

Genome size (kb)

GenBank accession no.

RNA structures in replication, transcription and translation

the coding sequences (Molenkamp et al., 2000a; Tijms et al.,

Expressed from genomic RNA

Expressed from sg mRNAs

E. J. Snijder, M. Kikkert and Y. Fang

leader TRS hairpin (LTH) that is crucial for sg mRNA

Fig. 3. Overview of the arterivirus infection

dictates both efficient 22 and 21 PRF (estimated

Arterivirus molecular biology and pathogenesis

pathway (Wassenaar et al., 1997). EAV cleavage-site

In contrast, the PLP2 domain in nsp2 exhibits both cis and

During or following pp1a and pp1ab cleavage, arterivirus

E. J. Snijder, M. Kikkert and Y. Fang

(b) PRRSV nsp1

(d) EAV PLP2

(e) PRRSV nsp4

(Fang et al., 2012; Han et al., 2010), but their biological

Arterivirus molecular biology and pathogenesis

(RdRp) in nsp9 and the helicase domain in nsp10.

A hallmark of arteriviruses and other nidoviruses is the

At the 39 end of the nascent subgenome-length negative

E. J. Snijder, M. Kikkert and Y. Fang

Nested set of sg mRNAs

or proteinprotein interactions (de Vries et al., 1992;

virion scaffold. The defect could be complemented in trans

The two major envelope proteins are the major GP (GP5

Arterivirus molecular biology and pathogenesis

Small hydrophobic proteins

an ectodomain, a central hydrophobic region predicted to

E. J. Snijder, M. Kikkert and Y. Fang

Minor envelope proteins

The structural properties of arterivirus minor envelope

Since the N protein is found in association with viral

preformed nucleocapsid by membranes of the smooth

Arterivirus molecular biology and pathogenesis

expressed by the MARC-145 cell line that fully supports

As in all +RNA viruses, arterivirus replicative proteins

PRRSV inhibitor studies with the drug cyclosporin A

Despite the cytoplasmic replication cycle, some arterivirus

E. J. Snijder, M. Kikkert and Y. Fang

responses and viral persistence. For a detailed overview of

Arterivirus molecular biology and pathogenesis

triggering innate immune response. Experimental LDV