Veterinary Microbiology 167 (2013) 181–204

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Equine infectious anemia and equine infectious anemia virus in 2013: A review R.F. Cook a, C. Leroux b, C.J. Issel a,* a b

Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546, USA INRA Universite´ Lyon 1 UMR754, Retrovirus and Comparative Pathology, Universite´ de Lyon, France

A R T I C L E I N F O

A B S T R A C T

Article history: Received 5 February 2013 Received in revised form 16 September 2013 Accepted 21 September 2013

A detailed description of equine infectious anemia virus and host responses to it are presented. Current control and eradication of the infection are discussed with suggestions for improvements to increase their effectiveness. ß 2013 Elsevier B.V. All rights reserved.

Keywords: Lentivirus Equidae Equine infectious anemia Pathogenesis Transmission Diagnosis and control

1. Historical perspective Equine infectious anemia virus (EIAV) is the etiological agent for equine infectious anemia (EIA), a disease with an almost worldwide distribution. EIA is of considerable importance to the equine industry being one of the only eleven notifiable equine specific diseases listed by OIE, the world organization for animal health. The first report of clinical signs associated with EIA occurred in 1843 (Lignee, 1843) with a more complete description of the different stages of the disease (acute, subacute and chronic) along with the fact that it is caused by a ‘‘filterable agent’’ published as early as 1904 Carre´ and Valle´e (Vallee and Carre, 1904). In addition, these French authors described experimental reproduction of the disease by the transfer of small amounts of blood, transmission from a horse to a donkey and the fact that the virus persists after the resolution of the clinical signs. Consequently, EIA is the first retrovirus induced animal disease to be assigned a

* Corresponding author. Tel.: +1 859 218 1096. E-mail address: [email protected] (C.J. Issel). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.09.031

viral etiology, preceding by several years the major discovery of filterable causes for avian leukemia (Ellerman and Bang, 1908) and avian sarcoma (Rous, 1910, 1911). The discovery of an endogenous ‘‘Foamy-like’’ endogenous retroviral element in the coelacanth (Latimeria chalumnae) genome suggest the Retroviridae family are ancient viruses that have been coevolving with their hosts for at least 400 million years (Han and Worobey, 2012a) and may explain why the host range of these viruses includes all known vertebrates. In contrast, the lentiviruses are generally considered to be very recent arrivals although the discovery of rabbit endogenous lentivirus K (RELIK) in rabbits and hares, two leporid species that diverged some 12 million years ago (Katzourakis et al., 2007; Keckesova et al., 2009; van der Loo et al., 2009) demonstrate the genus is older than the 1 million years, suggested from estimates based on nucleotide substitution rates (Wertheim and Worobey, 2009) However a comparatively recent origin could account for the fact the host range of extant lentiviruses is restricted to equids, bovids, small ruminants, members of the Felidae and some primates. Furthermore, lentiviruses are not distributed throughout a mammalian phylogenetic order, in that for

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example, the Equidae are the only members of the Perissodactlya, known to be infected with one of these viruses, suggesting that infection of equids occurred after they had diverged from the rhinoceros and tapir some 55– 40 million years ago. Alternatively, the discovery of endogenous lentiviruses in lagomorphs (Katzourakis et al., 2007), prosimian primates (Gifford et al., 2008; Gilbert et al., 2009) and the Mustelidae (Cui and Holmes, 2012; Han and Worobey, 2012b) representing another order within the Carnivora suggests that at one time lentiviruses may have had a wider host-range distribution. However, the fact remains the Equidae are currently among a very limited number of mammalian species that act as natural hosts for these viruses. This review ranges from the molecular biology of EIAV to ongoing control efforts and highlights some important differences with the genetically more complex lentiviruses. Indeed EIAV has simplest genome organization

of any extant lentivirus and so while it has been labeled the ‘‘country cousin’’ of HIV (Leroux et al., 2004) it might in fact be a rural grandfather. 2. Virus properties Analysis of EIAV by electron microscopy reveals a mixture of oval or circular particles, approximately 115 nm in diameter, in which the typical lentiviral conical core is encased in a proteinaceous matrix and bounded by a lipid membrane containing numerous 6–8 nm projections (Matheka et al., 1976; Weiland et al., 1977) (Fig. 1A). Within the core are two copies of the single stranded RNA genome. After infection, this RNA undergoes reverse transcription by virally encoded reverse transcriptase (RT) and RNase H producing a double stranded DNA molecule designated the provirus or proviral DNA. Reverse transcription along with integration of the proviral DNA

Fig. 1. (A) Model of an EIAV particle showing probable locations of the major structural antigens. (B) EIAV proviral genome organization. The structural proteins of EIAV are produced from polyprotein precursor molecules that are cleaved either by a virally encoded Protease (Gag/Pol) or by cellular endoproteinases (SU/TM). LTR, long terminal repeat; PR, protease; RT-RH, reverse transcriptase-RNase H; DU, dUTPase; IN, integrase; tat, transcriptional trans-activator; rev, regulator of viral mRNA splicing and transport; SU, surface unit glycoprotein; TM, transmembrane glycoprotein. (C) Diagram showing the location of variable regions within SU (gp90) along with potential N-linked glycosylation sites and neutralizing determinants (including the principal neutralizing determinant PND) in a fibroblast-cell adapted variant (EIAVPV) of EIAVWY.

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Fig. 2. Phylogenetic analysis of EIAV genomic sequences. A phylogenetic tree of aligned (ClustalW) complete proviral sequences was constructed by the neighbor joining method with bootstrap values determined over 1000 iterations. Branch lengths are proportional to the distance existing between the sequences. GenBank accession numbers are JX480631–JX480634 for the four EIAVIRE sequences (IRE 1–4), AF327877 for EIAVLIA (LIA) plus AF327878 for an attenuated EIAVLIA strain LIA-Vac, AF016316 for EIAVWY along with two derivatives of this strain: AF016316 (EIAVUK), AF247394 (EIAVWSU5) and JX003263 for EIAVMIY.

into the host-cell genome via virally encoded integrase are two major hallmarks of the Retroviridae. The proviral DNA of all EIAV strains sequenced to date comprises approximately 8.2 kbp (Cook et al., 1998; Dong et al., 2012a; Perry et al., 1992; Quinlivan et al., 2013; Tu et al., 2007), and possess a prototypical retroviral genomic organization (Fig. 1b) with the major genes gag, pol and env bounded at both ends by long terminal repeats (LTR). In common with all members of the lentivirus genus, EIAV possesses additional open reading frames encoding the regulatory proteins Tat and Rev (Fig. 1b). Although several full length EIAV sequences have been described they were until recently all derived from just two field strains originating in North America (EIAVWYOMING or EIAVWY) and China (EIAVLIAONING or EIAVLIA). This includes EIAV isolates designated V70 and V26 that while described as being of Japanese origin (Zheng et al., 2000) were almost certainly isolated from horses inoculated with in vivo passaged variants of EIAVWY imported from the United States (Kobayashi and Kono, 1967; Kono et al., 1970), a conclusion supported by recent nucleotide sequence analysis (Dong et al., 2012a). However, two additional full length EIAV proviral sequences have now been published, one derived from a strain (EIAVMIYAZAKI2011-A or EIAVMIY) isolated from Misaki horses in southern Japan (Dong et al., 2012a) while the other (EIAVIRELAND or EIAVIRE) was obtained from horses infected

during the 2006 EIA outbreak in Ireland (Quinlivan et al., 2013). These strains share 80% nucleotide sequence identity, form separate monophyletic groups upon phylogenetic analysis (Fig. 2) and do not appear to be derived from each other by recombination events (Dong et al., 2012a; Quinlivan et al., 2013). Therefore, it appears that each of these strains has evolved independently since diverging from a common ancestor.

3. Viral proteins Both gag and pol gene products are produced from a genomic full-length viral mRNA while those encoded by env are translated from a singly spliced viral mRNA species and the accessory proteins (Tat, Rev, S2) are generated from mRNAs that have undergone multiple splicing events (Fig. 3). As the gag and pol gene coding sequences are on different reading frames, a minus 1 ribosomal frameshifting event is required to enable translation of the Pol protein products. The essential ribosomal frame shifting components, namely an AAAAAAC slippery sequence, downstream 5-base GC-paired segment and a pseudoknot structure (Chen and Montelaro, 2003), are conserved in all EIAV strains sequenced to date. Translation of the gag and pol genes results in a polyprotein that is subsequently cleaved by a virally encoded protease.

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Fig. 3. Major transcripts produced from the EIAV genome. An mRNA encoding a truncated transmembrane protein (Ttm) is produced but it has not been well characterized.

3.1. Gag proteins Cleavage of the Gag-precursor (PR55gag) produces four major structural EIAV proteins: p15 matrix (MA), p26 capsid antigen (CA), p11 or the genomic RNA-binding nucleocapsid (NC) and a ‘‘late domain’’ protein (p9). Crystallographic structures have been determined for p15, Tat, Protease and dUTPase. In the case of p15 this structural analysis has demonstrated the molecule comprises five long a-helices along with a 310 helical loop. Therefore despite a very different primary amino acid sequence it possesses a comparable structure to MA of HIV-1 and SIV with the exception that the N and C termini are in closer proximity (Hatanaka et al., 2002). In addition, EIAV p15 contains a putative leucine-rich (11LKKLEKVTV19 in an EIAVWY derived strain) nuclear export signal (NES) and while the sequence of the NES in HIV-1 MA is different both are located in the N-terminal domain. However, unlike HIV-1 or SIV MA, EIAV MA does not contain a nuclear localization signal or N-terminal myristoylation sites (Hatanaka et al., 2002). The MA of HIV plays a critical role in virus particle assembly by binding to phosphatidylinositol 4,5-diphosphate (PIP2) located on the inner surface of the host cell plasma membrane, an association that is mediated by the N-terminal myristate. Despite the absence of this N-terminal modification EIAV p15 also attaches to the inner surface of membranes although studies conducted in vitro suggest that it may bind with higher affinity to phosphatidylinositol 3-phoshate (PI3P) than PIP2 (Chen et al., 2008; Fernandes et al., 2011). While binding of MA to membrane associated phosphoinositides initiates lentiviral particle assembly, the CA acts to control packing and when released by the viral protease it polymerizes as a capsid shell around the genomic RNA and NC proteins to generate the core of the mature virion. The CA protein contains the major homology region (MHR), a twenty amino acid motif, that plays a significant role in both the immature (prior to complete polyprotein cleavage) and mature stages of virus assembly (Grund et al., 1994; Purdy et al., 2008; Strambiode-Castillia and Hunter, 1992). Within the MHR the polar amino acids glutamine (Q3), glutamic acid (E7) and arginine (R15) are extensively conserved among retroviruses including EIAV. Packaging of genomic RNA into progeny lentivirus particles is mediated by NC. This is accomplished by the

fact all lentiviral NC proteins contain two CX2CX4HX4C zinc binding motifs (CCHC) connected by a variable length linker having a preponderance of basic amino acids. The EIAV p11 is unusual in possessing a short five amino acid linker along with a proline (P)–glycine (G) sequence preceding the histidine (H) residue of the CCHC motif (Amodeo et al., 2006). The EIAV p9 contains the late (L) domain that in EIAVWY strains has been shown to consist of a four amino acid motif (tyrosine Y, proline P, aspartic acid D and leucine L) (Chen et al., 2001; Jin et al., 2005a; Li et al., 2002; Puffer et al., 1997). This viral protein associates with cellular ALG-2-interacting protein-X (ALIX) and the m2 subunit of the AP-2 adaptor protein complex (MartinSerrano, 2007; Martin-Serrano et al., 2003; Puffer et al., 1997; Strack et al., 2003). Therefore as ALIX binds to charged multivesicular body protein 4B (CHMP4B), a member of the endosomal sorting complexes required for transport-III (ESCRT-III), p9 recruits this pathway to mediate release of progeny virus particles (Puffer et al., 1997; Strack et al., 2003; Zhai et al., 2008). Analysis of EIAVWY p9 using circular dichroism spectroscopy and nuclear magnetic resonance suggests the C-terminus of the molecule adopts a helical conformation while the Nterminal half will assume an unstructured state in hydrophobic environments analogous to those likely to exist in close proximity to lipid membranes (Sharma et al., 2009). Although p9 is clearly very important in viral budding, recent studies have demonstrated viruses containing mutations in the L domain can be rescued by overexpression of the Bro 1 domain of ALIX (Bello et al., 2012). However, this Bro-1 mediated restoration of viral budding was dependent on p11, suggesting NC also plays a role in pathways involved in the release of virions from the cell (Bello et al., 2012). Almost all of the structural and functional studies on EIAV Gag proteins have been conducted with EIAVWYderived viruses. However, several complete EIAV gag gene sequences have been published from virus strains isolated in North America, South America, Europe and Asia (Capomaccio et al., 2012; Cappelli et al., 2011; Cook et al., 1998; Dong et al., 2012a; Nagarajan and Simard, 2007; Perry et al., 1992; Quinlivan et al., 2007, 2013; Tu et al., 2007). Comparison of these sequences demonstrates significant variation although it is not distributed evenly throughout the gag gene. Nucleotide sequence identity between strains examined to date is highest in sequences

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Fig. 4. Alignment of predicted amino acid sequences of Gag p9 in different EIAV strains. Viral isolates obtained from the 2006 EIA outbreaks in Ireland (IRE 1– 4) and Italy (Ita 1–2) thought to originate from the same source are boxed. For comparison sequences are shown from an Italian isolate (Ita 5) that is unrelated to the 2006 outbreak along with isolates from Romania (Rom 1), China (EIAVLIA), Pennsylvania (PA), North Carolina (NC), Florida (FL), Canada (Can 1, 3, 7, 10) and Brazil (95, 99). GenBank Accession Numbers not provided elsewhere EF418582, EF418583, EF418584, EF418585, AB008196, EU24073, EU375543, EU375544, EU741609, GQ265785, GQ229581.

encoding p26 at approximately 89%, followed by p11 and p15 with identities of 78% and 75% respectively. Interestingly, nucleotide sequence identity between different EIAV strains in p9 is 42% which is low figure considering 25% is the level expected for randomly chosen DNA sequences having similar lengths and base composition (Cappelli et al., 2011; Quinlivan et al., 2007). Although the mechanism responsible for such extensive inter-strain variation in p9 is not known it is tempting to speculate the unstructured state adopted by the N-terminal half of the molecule (Sharma et al., 2009) might permit a wider range of amino acid substitutions than in proteins possessing more rigid secondary and tertiary structures. The fact that most of the variation occurs within the N-terminal half of p9 tends to support this hypothesis (Fig. 4). Fortunately, a lack of sequence identity in p9 between strains may prove to be extremely useful in tracing the origin and progression of EIA outbreaks. For example, the 2006 outbreaks in Italy and Ireland are believed to have originated from the same source, namely a contaminated horse plasma product administered to foals (Cappelli et al., 2011; Quinlivan et al., 2007, 2013). This conclusion is strongly supported by the fact that viruses isolated from both outbreaks possess identical p9 sequences (Fig. 4). Although there is significant variation in predicted amino acid sequence throughout the gag gene between geographically distinct EIAV isolates all studies conducted to date demonstrate extensive conservation of important structural and/or functional motifs. This is seen for example, in the form of highly conservative amino acid substitutions in the putative NES of p15, preservation of the CCHC zinc binding motifs in p11 and despite the considerable differences in overall sequence between strains, in absolute conservation of the L-domain (YPDLFig. 4) within p9 (Capomaccio et al., 2012; Dong et al., 2012a; Quinlivan et al., 2013). 3.2. Pol proteins Cleavage of the EIAV Gag-Pol precursor (PR 180gag/pol) generates an aspartic proteinase (Pr) (Powell et al., 1994,

1996), RT that can contain RNase H activity, DU and an integrase (INT). Unlike gag, comparative pol gene sequence information between different EIAV strains is currently limited to EIAVWY, EIAVLIA, EIAVIRE and EIAVMIY. Nucleotide sequence identity in Pr, RT, DU and INT between these strains varies between 80.4–84.6%, 79.8–83.2%, 80.0–90% and 85.3–85.7% respectively. Furthermore, when differences occur, they are single residue substitutions rather than deletions or insertions and generally involve conservative amino acid changes. A situation that might be expected since each Pol protein performs highly specific enzymatic functions. Furthermore, these molecules possess similar structures although not primary amino acid sequences to Pol proteins encoded by other retroviruses. Xray crystallographic analysis demonstrates the only major difference between EIAVWY and other retroviral proteinases is the presence of a unique second a-helix within the C-terminus of the molecule (Gustchina et al., 1996). Similarly, EIAVWY encodes a typical RT generally comprising of a heterodimer containing 66 kDa (p66) and 51 kDa (p51) subunits in which the p51 lacks RNase H activity (Coggins, 1981; Rausch et al., 1996; Souquet et al., 1998; Wohrl et al., 1994). In common with other retroviruses, the RT of EIAV is highly error prone with a tendency to catalyze A:C mismatches. Furthermore, these transcription errors are not corrected because initial viral replication occurs in the cell cytoplasm where cell-proofreading activities are absent and RT does not possess 30 to 50 exonuclease activity (Bakhanashvili and Hizi, 1993, as reviewed in Smyth et al., 2012). As a consequence of the high error rate, each round of reverse transcription is predicted to result in the introduction of at least one mutation in the viral genome producing significant increases in genetic diversity. The resultant populations of genetically related viruses competing within a highly mutagenic environment have been termed ‘‘viral swarm’’ or ‘‘quasispecies.’’ Lentiviruses are unusual among the Retroviridae in that they can replicate in non-proliferating cells, a fact that has made them, including EIAV, attractive as potential gene therapy vectors (Mitrophanous et al., 1999; Olsen, 1998). As non-proliferative cells do not undergo extensive DNA

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synthesis they generally have low intracellular deoxynucleotide (dNTP) pools with the exception of deoxyuridine 50 -triphosphate (dUTP) that accumulates due to limited expression of deoxyuridine 50 -triphosphate nucleotide hydrolase (dUTPase). Although incorporation of dUTP instead of dTTP is not considered a mutagenic event per se, uracil (U) is more prone than thymine (T) to mis-pairing with guanine (G). Furthermore, the presence of U rather than T can alter promoter function (Verri et al., 1990). Therefore, the combination of low intracellular dNTP concentrations plus high dUTP levels in nonproliferating cell types constitutes a hostile environment for viral replication. Therefore the dUTPase encoded by several lentiviruses including EIAV may prevent U incorporation into the virus genome. The fact this enzyme is important for EIAV replication in non-proliferative host-cell types has been confirmed by site directed mutagenesis experiments on laboratory cell-adapted variants of EIAVWY (Malmquist et al., 1973). These variants possess the ability to propagate in fibroblastic cells in addition to equine cells of the macrophage/monocyte lineage. Although deletion of DU does not affect progeny virus production in dividing cells it reduces replication by at least 100-fold in equine macrophage cultures and 10–100-fold in ponies compared with viruses containing wild-type DU sequences (Lichtenstein et al., 1995; Threadgill et al., 1993). The amino acid sequence of EIAVWY DU contains five domains that show extensive conservation not only with other retroviruses but also with DU from humans and Escherichia coli (Dauter et al., 1999). Sequences encoding DU share approximately 85% identity between EIAVWY, EIAVLIA, EIAVIRE and EIAVMIY and in each case the overall domain structure appears to be conserved. In the IN protein, the HHCC (Histidine [H]12, H16, Cytosine [C]40, C43) zinc binding motif and key residues in the hydrophobic core domain (isoleucine [I]5, alanine [A]8 leucine [L]22 A33 and I36) are conserved between the primate lentiviruses and all EIAV sequences examined to date (Dong et al., 2012a; Eijkelenboom et al., 1997; Quinlivan et al., 2013). 3.3. Env proteins The organization of the EIAV env gene is typical of that described for other retroviruses in that it encodes both surface (SU or gp90) and transmembrane (TM or gp45) envelope glycoproteins. These are translated as a polyprotein that is subsequently cleaved by cellular endoproteinases. It is not known if the SU/TM heterodimer associates to form a multimeric structure on the surface of the mature EIAV particle similar to the SU/TM trimers that occur in HIV-1 (Liu et al., 2008). However, gp90 like gp120 of HIV-1, is extensively glycosylated and pathogenic EIAV strains contain at least 17 potential N-linked glycosylation sites in their SU coding sequences (Leroux et al., 1997; Cook et al., 1998; Dong et al., 2012a; Payne et al., 1998; Quinlivan et al., 2013). Indeed approximately half the apparent molecular weight of EIAV gp90 is composed of glycosylated residues (Fig. 1C) although as shown by longitudinal studies in ponies infected with a pathogenic fibroblast adapted variant (EIAVPV) of EIAVWY

the positions of many potential N-glycosylation sites in the SU coding region are subject to change during the course of infection (Leroux et al., 2001). To date only one structural model for SU has been published suggesting that at least in the case of the fibroblast cell adapted variant of EIAVWY (Malmquist et al., 1973) the protein contains a loop structure analogous to the V3 domain in HIV gp120 (Ball et al., 1992). As this region contains two of the three neutralizing epitopes recognized by mouse monoclonal antibodies it has been termed the principal neutralizing domain or PND (Ball et al., 1992; Hussain et al., 1987, 1988). However, during the course of infection sequences associated with the PND may be deleted from SU (SUDPND) conferring a neutralizationresistant phenotype on the resultant viruses (Leroux et al., 1997; Craigo et al., 2002). Interestingly, immunosuppression of EIAVSUDPND infected horses to permit increased viral replication results in the production of strain-specific, neutralizing antibodies demonstrating the PND is not an essential viral neutralization determinant or indispensable for immune-mediated control of EIAV (Craigo et al., 2002). The SU of EIAV, in common with other retroviruses, is responsible for attachment to the host cell receptor. This host cell protein has been identified as a member of the tumor necrosis factor family of receptor proteins and designated as equine lentivirus receptor-1 (ELR-1) (Zhang et al., 2005). In vitro experiments have established that EIAV-receptor entry occurs via a low-pH-dependent endocytic pathway (Brindley and Maury, 2005, 2008; Brindley et al., 2008; Jin et al., 2005b) and revealed that the ELR1-binding domains are located in the C-terminal two-thirds of EIAV gp90, as a complex of discontinuous determinants (Sun et al., 2008). A probable membrane-fusion domain has been identified located within the N-terminal domain of TM (Chong et al., 1991). To date SU or gp90 is the only EIAV protein shown to contain epitopes recognized by neutralizing antibodies (Hussain et al., 1987, 1988). However, the tertiary or quaternary structure adopted by SU may confer resistance to neutralization unless the effector antibodies are present in high titers. This is suggested by the fact that nonconservative amino acid substitutions induced by serial passage of a cell-adapted variant of EIAVWY in fetal donkey dermal (FDD) cells result in increases in the sensitivity to neutralizing antibodies by 1000–10,000 fold (Cook et al., 1995). One of the major factors contributing to the establishment of persistent EIAV infections is the ability of gp90 to undergo substantial changes in sequence without significant loss of function. Furthermore, these changes are not limited to simple nucleotide transitions or transversions but as indicated above in the case of the PND may involve relatively large deletions or insertions (Leroux et al., 1997, 2001; Zheng et al., 1997). It appears the predominant selective force driving gp90 evolution in vivo is the hostimmune response. EIAV was one of the first viruses to be associated with ‘‘antigenic drift’’ as each sequential febrile episode in an EIAV infected equid is caused by an antigenic variant that differs in gp90 coding sequence from its predecessors (Kono, 1988; Kono et al., 1973; Leroux et al.,

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Fig. 5. Alignment of predicted SU amino acid sequences. SU sequences for EIAVWY (WY), EIAVMIY (MIY), EIAVLIA (LIA), EIAVIRE (IRE) and EIAVPA (PA) are shown with hypervariable regions indicated by boxes. Conserved cysteine residues are highlighted.

1997, 2001; Rwambo et al., 1990a; Zheng et al., 1997). The fact that antibodies can induce changes in nucleotide sequence in the gp90 encoding region is supported by experiments where antigenic variants of EIAV have been produced by growth in the presence of neutralizing antiserum (Rwambo et al., 1990b). In addition to evading neutralizing antibodies, substitutions also result in escape from specific cell mediated immune responses (Mealey et al., 2003). The net result of continual selective immune pressure is that sequences encoding gp90 become increasingly divergent from those present in the original infecting strain. For example in one experimentally infected pony, predicted amino acid sequences in gp90 differed from the parental virus by 6% at 260 dpi (days post infection) and by 13% at 1219 dpi (Craigo et al., 2007b). Interestingly, variation in gp90 within individual infected animals is not randomly distributed throughout the molecule but primarily restricted to eight hypervariable regions (Leroux et al., 1997, 2001; Zheng et al., 1997) (Figs. 1C and 5). Considering the potential for change inherent in viral sequences encoding gp90 it is not surprising there is significant variation between different EIAV strains. For example EIAVWY has predicted amino acid identities of 65.3%, 63.1%, 56.9% and 63.4% with equivalent sequences in EIAVLIA, EIAVIRE, EIAVMIY and EIAVPA, a field isolate from Pennsylvania (Craigo et al., 2009). Analysis of gp90 sequences from horses infected during the 2006 EIA outbreak in Ireland resemble results obtained from longitudinal studies in experimentally infected horses in that most variation occurs within the eight hypervariable regions (Quinlivan et al., 2013). Interestingly, it is possible to accurately predict in silico variable and constant regions in SU glycoproteins of EIAV, HIV-1, small ruminant lentivirus (SRLV) simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV) and feline immunodeficiency virus (FIV) using algorithms based on hidden Markov models (Boissin-Quillon et al., 2007). Although variation in gp90 within a single strain appears to be restricted to certain locations, alignments between different EIAV strains demonstrates that amino acid changes can occur throughout the molecule (Fig. 5) with the exception of apparently conserved cysteine residues suggesting

disulphide bonds are essential for maintaining the molecular architecture of this glycoprotein. 3.4. Accessory proteins In the absence of Tat (Transactivator of Transcription protein) only low levels of basal transcription are observed from the LTR of EIAV (Dorn et al., 1990; Dorn and Derse, 1988; Sherman et al., 1988). The protein acts by enhancing the elongation efficiency of host RNA polymerase II (Pol II), a process requiring interaction with the nascent viral RNA transcript after it forms a specific stem-loop structure termed the trans-activation response (TAR) element (Carvalho and Derse, 1991; Hoffman et al., 1993; Hoffman and White, 1995). The interactions between Tat and TAR lead to recruitment of cellular factors including positive transcription elongation factor b (P-TEFb), a protein complex comprising of the CDK9 (cyclin-dependent kinase 9) and cyclin T1 that is thought to increase processivity by phosphorylating the carboxyl domain of the largest subunit of RNA Pol II (Sune et al., 2000). EIAV Tat contributes to host-species restriction because its interactions are limited to equine and canine orthologs of cyclin T. Interactions can only occur with human cyclin T1 if valine, as found in HIV-1 Tat, is substituted for leucine at position 29 in EIAV Tat (Taube et al., 2000). The crystallographic structure of EIAVWY Tat reveals flexible N- and C-terminal domains, a core domain and a basic region (Willbold et al., 1994). Therefore it possesses a structure similar to HIV-1 Tat with the exception of a cysteine-rich domain. The core domain provides a scaffold for the flexible regions and is relatively conserved between HIV-1 and EIAVWY. In EIAVWY Tat the core domain comprises 15 amino acids delineated by two tyrosine residues at amino acid positions 35 and 49 (Willbold et al., 1994). The predicted amino acid sequence of EIAVWY Tat shares around 75% identity with the equivalent amino acid sequences in EIAVIRE, EIAVLIA and EIAVMIY. However, a majority of the amino acid substitutions appear to be conservative (although experimental studies are required to confirm this) and in all four strains the overall domain organization is preserved.

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The S2 protein is unique to EIAV and mutagenesis studies have demonstrated that while it is not essential for replication in vitro it is an important virulence determinant in vivo (Fagerness et al., 2006; Li et al., 1998, 2000). In pony infection experiments with EIAV strains genetically engineered to prevent S2 expression (DS2) plasmaassociated viral loads are several orders of magnitude lower than those observed in animals inoculated with wild-type viruses. This attenuated phenotype has resulted in EIAVDS2 variants being evaluated as modified live vaccines (Fagerness et al., 2006; Li et al., 2000, 2003). The molecular mechanism of action for S2 has yet to be fully elucidated although pro-inflammatory cytokine expression is increased in equine macrophages infected with a wild-type EIAVWY derived strain compared to its DS2 variant (Covaleda et al., 2010a). Furthermore, in a yeast two-hybrid system, S2 interacts with equine homologs of proteasome 26S ATPase subunit 3 (PSMC3) and osteosarcoma amplified 9 (OS-9) (Covaleda et al., 2010b). However, while S2 variation over time in individual infected equids appears limited (Li et al., 2000) the protein is not extensively conserved between different virus strains with EIAVWY sharing only 46.8%, 54.4% and 39.7% amino acid identity with EIAVIRE, EIAVLIA and EIAVMIY respectively. Furthermore, there is inter-strain variation in sequences putatively identified in an EIAVWY derived virus as Nucleoporin, SH3 domain binding and nuclear localization signal motifs (Li et al., 2000). This being said the amino acid substitutions in the Nucleoporin motif appear to be conservative while the C-terminus in S2 of all strains contains a preponderance of positively charged amino acids (Fig. 6). The EIAV Rev protein facilitates nuclear export of incompletely spliced viral RNA and when it is absent only the bicistronic tat/rev mRNA is present in the cytoplasm (Belshan et al., 2000; Gontarek and Derse, 1996; Martarano et al., 1994; Stephens et al., 1990). This protein is translated from exons 3 and 4 of the bicistronic tat/rev mRNA via leaky scanning of the Tat CUG codon (Carroll and Derse, 1993). All Rev coding sequences are contained within the env gene with exon 3 being in the same reading frame as SU while exon 4 is in a different reading frame overlapping the

region encoding the cytoplasmic tail of TM. Rev binds to viral RNA via the specific Rev-response element (RRE) and then undergoes multimerization with passage across the nuclear membrane dependent upon a nuclear export signal (Hope, 1997). EIAV and FIV Rev utilize an atypical NES that differs from other Rev proteins by the lack of a core tetramer and the organization of hydrophobic residues (Fridell et al., 1993; Mancuso et al., 1994). However, despite these differences, nuclear export of incompletely spliced viral RNA in EIAV infected cells is inhibited by leptomycin B suggesting that as in other lentiviral systems it is mediated by a host-cell CRM1 (required for chromosomal region maintenance) or exportin dependent pathway (Nishi et al., 1994; Fornerod et al., 1997; Otero et al., 1998). While the RRE of HIV-1 is predicted to form a highly structured stem loop located near the SU/TM junction there are two independent cis-acting elements, RRE1 and RRE2 in EIAV. RRE1 consists of 555 nucleotides encompassing the first Rev exon (exon 3 in the tat/rev bicistronic mRNA) while RRE2 occurs immediately downstream and is 1698 nucleotides long (Belshan et al., 1998; Martarano et al., 1994). In reporter gene assays RRE1 and RRE2 have 52% and 17% respectively of the activity observed with the intact RRE sequence. Furthermore, RRE1 contains the 57-nucleotide purine-rich exon splicing enhancer (ESE) that can bind EIAV Rev and to a limited extent mediate the nuclear export of incompletely spliced RNA (Belshan et al., 2000). Interestingly, by binding at or near the ESE, Rev down-regulates its own synthesis by preventing exon 3 splicing (Belshan et al., 2000). In ponies experimentally infected with EIAV significant variation occurs in sequences encoding Rev over time (Belshan et al., 2001; Leroux et al., 1997) and while most non-synonomous nucleotide changes are found in regions outside known functional domains, the resultant polymorphisms have significant effects on the biological activity of this accessory protein (Belshan et al., 1998, 2001). Comparison of predicted Rev amino acid sequences reveals identities of 68% to 73% between EIAVWY, EIAVIRE, EIAVLIA and EIAVMIY and while sequences within the Nterminal polar effector domain (Mancuso et al., 1994) vary between these strains, there is complete conservation of

Fig. 6. Alignment of S2 predicted amino acid sequences. Putative functional motifs (boxed) identified in EIAVWY (WY) are shown in comparison with EIAVMIY (MIY), EIAVLIA (LIA) and EIAVIRE (IRE).

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the hydrophobic residues (L36, W41 L45, L49) shown by sitedirected mutagenesis to be required for the nuclear export function of Rev (Belshan et al., 1998; Harris et al., 1998). However, there are differences between strains in the Cterminal amino acid residues implicated in RNA binding (Harris et al., 1998; Lee et al., 2006) suggesting that viruses associated with the separate monophyletic groups may find different ‘‘solutions’’ in terms of primary amino acid sequence to the same biological problems associated with replication and persistence in their equid hosts. However it is not known if these individual ‘‘solutions’’ are equivalent as regards to the biological fitness of each viral population although EIAVWY, EIAVLIA plus EIAVIRE (Cullinane et al., 2007; Kemeny et al., 1971; Quinlivan et al., 2007; Tu et al., 2007) are certainly capable of inducing disease and EIAVMIY has proven sufficiently viable to have persisted within the Misaki horse population for many years (Dong et al., 2012a, 2012b). 3.5. Long terminal repeats (LTR) The long terminal repeat (LTR) contained within the proviral DNA of all retroviruses share the same basic organization comprising a unique 30 segment (U3) a short repeated region (R) and a unique 50 -end (U5). The 50-nt long U5 region of EIAV is shorter than observed in many retroviruses. Comparative alignment studies demonstrate the length of the LTR varies between EIAV strains with EIAVWY, EIAVLIA, EIAVIRE and EIAVMIY having nucleotide lengths of 323 bp, 316 bp, 313 bp and 306 bp respectively. In addition to size differences, there is less than 80% nucleotide sequence identity in the LTRs between the four different strains although the majority of this variation occurs in the U3 region upstream from known transcription factor

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binding motifs. The enhancer region within U3 appears to be extensively conserved between the different strains in that they all contain methylated DNA-binding protein site (MDBP), a TATA Pol II recognition sequence and three PU.1 motifs (Fig. 7). PU.1 is a member of the ets (E-twenty six) transcription-factor family and the presence of its binding motifs in the EIAV LTR is critical for its expression in equine macrophages (Hines et al., 2004; Maury, 1994). In contrast EIAV19-2, an infectious molecular clone derived from a laboratory-adapted variant of EIAVWY (Payne et al., 1994), lacks two PU.1 sites but contains a PEA-2 binding motif (Fig. 7) consistent with its ability to replicate in nonmonocytic cell-types (Maury et al., 2000, 2005). The only enhancer motifs that are not conserved between the four strains are the putative CAAT boxes present in EIAVWY (Fig. 7), although their influence on viral transcription in macrophages is unknown. The EIAV LTR also contains a poly-A addition site and the TAR stem-loop motif, that are both conserved among all isolates characterized to date. Additional information on the regulation of EIAV RNA expression can be found in a previously published review by Maury (1998). 3.6. EIAV and host-specified retroviral restriction factors Insertional mutagenesis caused by integration of proviral DNA into host cell chromatin can produce significant longterm, highly detrimental effects. This may explain why mammalian species have evolved several molecular defense mechanisms against retroviruses, in addition to innate and adaptive immune responses. These host-specified retroviral restriction factors include the apolipoprotein b editing complex 3 (APObEC3) cytidine deaminase family members that introduce lethal mutations by converting C to U in the

Fig. 7. LTR enhancer region. The enhancer regions within the LTR of EIAVWY (WY), EIAVMIY (MIY), EIAVLIA (LIA), EIAVIRE (IRE) and a molecularly cloned fibroblast adapted strain of EIAVWY (19-2) are shown with transcription factor binding motifs indicated by boxes.

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viral genome (Blanco-Melo et al., 2012; Sheehy et al., 2002), tripartite motif containing protein 5a (TRIM5a) that disrupts viral uncoating (Stremlau et al., 2004) and tetherin that ‘‘tethers’’ progeny virions to the host cell surface thereby preventing their release (Neil et al., 2008; Van Damme et al., 2008). In response to these host-specified defenses, some retroviruses and particularly lentiviruses have developed countermeasures the form of additional ORFs such as vif, vpu and nef that encode proteins capable of effectively inhibiting the action of the different restriction factors. For example, the virus infectivity factor (vif) protein binds to APObEC3 and promotes its proteasomal degradation thereby removing it from the site of viral budding and so preventing it from becoming incorporated into progeny virions (Marin et al., 2003; Mehle et al., 2004; Yu et al., 2003). Interestingly an ortholog of the vif gene is encoded by all known lentiviruses with the exception of EIAV. Therefore an important question is how does EIAV survive without any of the additional ORFs encoded by other lentiviruses to defend against host retroviral restriction factors? It is not that horses lack APObEC3 coding sequences, in fact the genome of Equus caballus encode six members of this gene family giving the species one of the highest APObEC3 copy number observed to date. (Bogerd et al., 2008; LaRue et al., 2008; Munk et al., 2008; OhAinle et al., 2006). A potential solution to this dilemma is reported by Bogerd et al. (2008) who found that EIAV is inherently resistant to APObEC3 cytidine deaminase activity. However a subsequent study has questioned this finding instead suggesting the equine APObEC3 protein family members with the highest inhibitory activity against EIAV, A3Z3 and A3Z2c-Z2d, are poorly expressed in macrophages, the predominant cellular host for this virus (Zielonka et al., 2009). Lentiviruses appear to develop resistance to Trim 5a produced by their natural hosts as suggested by the fact that primate orthologs of this protein only partially restrict their cognate lentiviruses (Nisole et al., 2004). It is not known if EIAV is inherently resistant to equine Trim 5a. However, the bovine equivalent of this protein is not effective with bovine immunodeficiency virus (BIV) or EIAV despite possessing significant inhibitory activity against HIV-1 and FIV replication (Si et al., 2006). Alternatively, Equidae may resemble the Canidae and simply not possess a fully functional Trim 5a gene (Sawyer et al., 2007). Two distinct mechanisms have been developed by Lentiviruses to reduce the effectiveness of host-specified Tetherin. In HIV-1 the Vpu ancillary protein downregulates this host-cell protein, removing it from viral assembly points in the plasma membrane and possibly targeting it for degradation (Douglas et al., 2009; Gupta et al., 2009; McNatt et al., 2009; Perez-Caballero et al., 2009; Rong et al., 2009) whereas in some HIV-2 strains, the Env proteins act as effective tetherin antagonists by inducing sequestration within the trans-Golgi network (Abada et al., 2005; Hauser et al., 2010; Le Tortorec and Neil, 2009; Noble et al., 2006). This Env mediated antagonist activity is dependent on a YXX-hydrophobic amino acid motif (YXXu) located just downstream of the membrane spanning domain in the transmembrane (TM) and a second less clearly defined motif present in the

ectodomain of the envelope glycoprotein (Abada et al., 2005; Le Tortorec and Neil, 2009). While EIAV clearly does not encode a Vpu-like molecule several YXXu motifs are distributed throughout EIAV Env proteins although none are located immediately downstream of the membrane spanning domain in TM as in HIV-2. However, if EIAV is resistant to the actions of equine Tetherin, assuming the protein is expressed in horses, this resistance does not extend to Tetherins produced by all species as virus particle release in expression experiments with a fibroblast adapted variant of EIAVWY in human HeLa cells is enhanced significantly by co-expression of both HIV-1 Vpu and HIV-2 Env (Abada et al., 2005). 4. Pathogenesis of disease Clinical signs of EIA are triggered when viral burdens attain a critical threshold value that in an experimental horse/pony challenge model correlates with plasmaassociated EIAV RNA loads of 5  107 to 1  108 copies/ ml (Cook et al., 2003). However reaching or exceeding this threshold amount is dependent on the virus strain, the degree of individual innate resistance to disease and the equid species. In a comparative study using horse virulent EIAV strains, horses or ponies (E. caballus) experienced severe or even fatal disease while similarly infected donkeys (Equus asinus) remained clinically normal with peak of EIAV titers at least a 1000-fold lower than in E. caballus (Cook et al., 2001). At present it is not known if donkeys are inherently resistant to induction of disease by EIAV or if once this virus has been adapted to one equid species it cannot replicate optimally in another. However, horse derived EIAV strains have equivalent replication rates in both horse and donkey monocyte-derived macrophage cells (Cook et al., 2001) suggesting the protective mechanism in donkeys is not dependent on simple host cell restriction. The clinical signs associated with acute EIA are mediated by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFa), interleukin 6 (IL-6) and transforming growth factor b (TGFb) that are released when tissue associated viral burdens reach threshold levels (Costa et al., 1997; Sellon et al., 1999; Tornquist and Crawford, 1997; Tornquist et al., 1997). It is not known if the major source of this cytokine production is from bystander cells or infected macrophages although it has been shown that infection with EIAV disrupts regulation of host-cell gene expression increasing the production of TNFa, IL-1a and IL-1b, and IL-6 (Esser et al., 1996; Lechner et al., 1997; Lim et al., 2005; Swardson et al., 1997; Yoo et al., 1996). Once released, IL-6 and TNFa can induce febrile responses by activating the arachidonic pathway to increase production of prostaglandin E2 (PGE2) whereas thrombocytopenia can be instigated by TNFa/TGFb mediated suppression of equine megakaryocyte colony growth (Tornquist and Crawford, 1997) and anemia in part produced by downregulation of erythropoiesis by TNFa (Dufour et al., 2003; Felli et al., 2005; Zamai et al., 2000; Moldawer et al., 1989). In mice TNFa induces a profound thrombocytopenia by stimulating cells expressing the widely distributed 55 kDa tumor necrosis factor receptor 1

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(TNFR1) to release platelet agonists such as thrombin, plasmin and serotonin (Tacchini-Cottier et al., 1998). Although clinical signs in acute EIA are for the most part triggered by pro-inflammatory cytokines, adaptive immune responses, when present, also play a role in the pathogenesis of EIA. In EIAV infected horses platelets become destined for immune mediated destruction because of bound IgG or IgM. In addition to exacerbating thrombocytopenia this process results in splenomegaly and hepatomegaly (Banks et al., 1972; Clabough et al., 1991). Furthermore, complement C3-coated erythrocytes may undergo phagocytosis, an event that not only contributes to anemia but results in the presence of hemosiderin granules in macrophages of the liver, spleen and lymph nodes (Perryman et al., 1971; Sentsui and Kono, 1987). In addition, chronically EIAV infected equids often have thickened glomerular tufts within the kidney associated with deposition of immunoglobulin and complement C3 on the basement membranes and mesangial areas (Henson and McGuire, 1971). Additional more detailed information concerning EIA pathogenesis can be found in earlier reviews (Cheevers and McGuire, 1985; Clabough, 1990). Oxidative stress may play an important role in lentiviral infections facilitating viral replication, and inflammatory responses while simultaneously decreasing immune cell proliferation. Based on sample collection from a large cohort of working horses, a recent study has shown that EIAV infection can modify the oxidant/antioxidant equilibrium, by altering glutathione peroxidase and uric acid levels. These effects were more pronounced in recently infected horses (