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Background: Equine infectious anemia (EIA) is a viral disease, caused by the Equine Infectious Anemia virus (EIAV) belonging to the Retroviridae family, genus Lentivirus. Horses (or equids) infected with EIAV are lifelong carriers and they remain contagious for other horses even in the absence of clinical signs. So far, EIAV infection has been reported among horses in North and South America, France, Germany, Italy, Hungary and Romania, with no publication regarding the presence of EIAV in horses in Serbia. To determine the circulation of EIAV among, approximately, the 5000 horses of the Vojvodina region, northern part of Serbia, 316 serum undergone serological testing for EIA. Then, identification and full genome sequencing using next generation sequencing was performed from one EIA positive horse., Results: the 316 sera were tested with 3 different commercial agar gel immunodiffusion (AGID) tests and two different commercial enzyme-linked immunosorbent assay (ELISA). With the three AGID kits, 311 (98.4%) among the 316 tested sera were negative and only five (1.6%) sera were positive for EIA. Some discrepancies were seen for the two ELISA kits tested since one exhibited the same results as AGID test and the second gave 295 sera with negative results, five with a positive result and 16 with doubtful outcome. Phylogenetic analysis performed using the full genome sequence showed that EIAV characterized from a horse in Serbia is different from those identify so fare around the world and form a distinct and separate group together with another EIAV strain., Conclusions: This study demonstrate for the first time that EIAV is circulating at a low level in the horse population from the Northern part of Serbia. Interestingly, phylogenetic data indicates that this EIAV from the western Balkan region of Europe belongs to a new cluster.

Equine infectious anemia (EIA), a disease caused by equine infectious anemia virus (EIAV), is considered an obstacle to the development of the horse industry. There is no treatment or vaccine available for EIA, and its pathogenesis, as well as the immune response against the virus, is not fully understood. Therefore, an immunohistochemistry assay was developed for the detection of viral antigens in tissues of equids naturally infected with EIAV. Sections of organs of six equids from Apodi-RN, Brazil, that tested positive for EIA by serological tests (ELISA and AGID) were fixed in 10% formalin solution and embedded in paraffin. Immunohistochemistry was performed using a polyclonal anti-EIAV antibody. EIAV antigens were observed in red spleen pulp cells and hepatic sinusoids, as well as bronchiolar and alveolar epithelial cells of the lungs and proximal and distal tubules of the kidneys. The presence of EIAV in the spleen and liver was expected due to viral tropism by macrophages, which are abundantly present in these organs. However, EIAV was also found in lung and kidney epithelial cells, indicating that the virus infects cell types other than macrophages. In conclusion, the immunohistochemical assay standardized in this study was able to detect EIAV antigens in spleen, liver, kidney and lung cells from naturally infected EIAV equids. Immunostaining observed in the spleen confirms viral tropism by mononuclear phagocytes; however, the presence of EIAV in lung and kidney epithelial cells indicates that virus may be eliminated in urine and/or oronasal secretions, suggesting new routes for viral excretion.

Equine infectious anemia virus (EIAV) is a persistent lentivirus that causes equine infectious anemia (EIA). In Brazil, EIAV is endemic in the Pantanal region, and euthanasia is not mandatory in this area. All of the complete genomic sequences from field viruses are from North America, Asia, and Europe, and only proviral genomic sequences are available. Sequences from Brazilian EIAV are currently available only for gag and LTR regions. Thus, the present study aimed for the first time to sequence the entire EIAV genomic RNA in naturally infected horses from an endemic area in Brazil. RNA in plasma from naturally infected horses was used for next-generation sequencing (NGS), and gaps were filled using Sanger sequencing methodology. Complete viral genomes of EIAV from two horses were obtained and annotated (Access Number: MN560970 and MN560971). Putative genes were analyzed and compared with previously described genes, showing conservation in gag and pol genes and high variations in LTR and env sequences. Amino acid changes were identified in the p26 protein, one of the most common targets used for diagnosis, and p26 molecular modelling showed surface amino acid alterations in some epitopes. Brazilian genome sequences presented 88.6% nucleotide identity with one another and 75.8 to 77.3% with main field strains, such as EIAV Liaoning, Wyoming, Ireland, and Italy isolates. Furthermore, phylogenetic analysis suggested that this Brazilian strain comprises a separate monophyletic group. These results may help to better characterize EIAV and to overcome the challenges of diagnosing and controlling EIA in endemic regions.

Equine infectious anemia virus (EIAV) is responsible of acute disease episodes characterized by fever, anemia, thrombocytopenia and anorexia in equids. The high mutation rate in EIAV genome limited the number of full genome sequences availability. In the present study, we used the SureSelect target enrichment system with Illumina Next Generation Sequencing to characterize the proviral DNA of Equine Infectious Anemia Virus (EIAV) from asymptomatic horses. This approach allows a direct sequencing of the EIAV whole genome without cloning or amplification steps and we could obtain for the first time the complete genomic DNA sequences of French EIAV strains. We analyzed their phylogenetic relationship and genetic variability by comparison with 17 whole EIAV genome sequences from different parts of the world. The results obtained provide new insights into the molecular detection of EIAV and genetic diversity of European viral strains., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Equine infectious anemia (EIA) has a worldwide distribution, and is widespread in Brazil. The Brazilian Pantanal presents with high prevalence comprising equine performance and indirectly the livestock industry, since the horses are used for cattle management. Although EIA is routinely diagnosed by the agar gel immunodiffusion test (AGID), this serological assay has some limitations, so PCR-based detection methods have the potential to overcome these limitations and act as complementary tests to those currently used. Considering the limited number of equine infectious anemia virus (EIAV) sequences which are available in public databases and the great genome variability, studies of EIAV detection and characterization molecular remain important. In this study we detected EIAV proviral DNA from 23 peripheral blood mononuclear cell (PBMCs) samples of naturally infected horses from Brazilian Pantanal using a semi-nested-PCR (sn-PCR). The serological profile of the animals was also evaluated by AGID and ELISA for gp90 and p26. Furthermore, the EIAV PCR amplified DNA was sequenced and phylogenetically analyzed. Here we describe the first EIAV sequences of the 5' LTR of the tat gene in naturally infected horses from Brazil, which presented with 91% similarity to EIAV reference sequences. The Brazilian EIAV sequences also presented variable nucleotide similarities among themselves, ranging from 93,5% to 100%. Phylogenetic analysis showed that Brazilian EIAV sequences grouped in a separate clade relative to other reference sequences. Thus this molecular detection and characterization may provide information about EIAV circulation in Brazilian territories and improve phylogenetic inferences.

Equine infectious anemia is an important infectious disease that affects equids worldwide. Control of the disease is currently based on detection of anti-p26 EIAV by Agar Gel Immunodiffusion (AGID). In this work, 62 animals were examined by AGID and nested-PCR using primers for the gag gene. Fifty-three samples (85.5%) were positive by nested-PCR, whereas only 33 samples (53%) were positive for AGID. Fifteen amplicons obtained by nested-PCR were sequenced and the aligned results subjected to phylogenetic analysis. The analysis suggests that the Brazilian EIAV form a cluster with WSU5, EIAVUK and Wyoming strains from United States.

Molecular and serological techniques for Equine Infectious Anemia Virus (EIAV) diagnosis were compared using samples from 59 clinically normal horses stabled on five farms in the Santa Fe Province of Argentina. Of these 26 (44.1%) were positive in official AGID tests and/or gp45/gp90-based ELISA. Surprisingly 18 of the 33 seronegative horses were positive in a PCR against viral sequences encoding gp45 (PCR-positive/AGID-negative) with all but one remaining EIAV-antibody negative throughout a two year observation period. The gp45 PCR results are supported by fact that 7/18 of these horses were positive in the Office International des Epizooties (OIE) recommended EIAV gag gene specific PCR plus 2 of this 7 also reacted in a PCR directed predominantly against the 5' untranslated region of the viral genome. Furthermore sufficient quantities of serum were available from 8 of these horses to verify their seronegative status in sensitive Western Blot tests and demonstrate by ELISA the absence of EIAV-specific antibodies was not attributable to abnormalities in total IgG concentration. Studies involving 7 of the PCR-positive/AGID-negative horses to measure lymphocyte proliferation in the presence of PHA showed no significant differences between this group and control animals. In addition, lymphocytes from 2 of these 7 horses responded to peptides derived from gp90 and gp45. Together these results demonstrate that apparently clinically normal horses with no gross signs of immunodeficiency in terms of total IgG concentration or T helper-cell function can remain seronegative for at least 24 months while harboring EIAV specific nucleic acid sequences., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Equine infectious anemia virus (EIAV) causes lifelong infections ranging from acutely fatal, to chronic, to asymptomatic. Within infected animals, EIAV is found as a quasispecies. Many experimental studies on EIAV, carried out in the U.S. over the past 70 years, have used either the highly virulent Wyoming (EIAVWYO) field strain or various derivatives of that strain. These infections have provided insights into the variety of genetic changes that accumulate in the env gene and LTR in experimentally infected horses. In the current study, we obtained EIAV sequences from blood samples collected from naturally infected Texas horses between 2000 and 2002. We found surface (SU) and long terminal repeat (LTR) sequences clearly related to EIAVWYO and its cell culture-adapted derivatives. Some blood samples yielded SU or LTR sequences belonging to 2 discrete clusters. In these cases, SU and LTR variation between animals was no greater than sequence variation within animals. In contrast, a portion of integrase (IN) was more homogeneous within animals than between animals. These results suggest that specific selective pressures are applied to SU and LTR sequences, potentially driving generation of two distinct sequence clusters within a horse. We speculate that viruses in one cluster may be more highly expressed and easily transmitted while those in the second cluster support long-term inapparent infection. The presence of homogeneous IN sequences within a horse supports the hypothesis that SU and LTR sequences diverged after the initial infection.

Although equine infectious anemia virus (EIAV) poses a major threat to the equine industry worldwide, the molecular epidemiology of this virus is poorly understood. Recently, an EIAV strain (EIAVMiyazaki2011-A) representing a new monophyletic group was discovered in feral horses in southern Japan. In the present study, the EIAVMiyazaki2011-A proviral genome is compared with evolutionarily divergent EIAV isolates to investigate conservation of functional elements or motifs within the long terminal repeats (LTRs) and structural genes. This analysis represents a significant step forward in increasing understanding of the molecular conservation and variation between geographically distinct strains of this equine lentivirus.

In January 2010, the United Kingdom notified cases of equine infectious anaemia (EIA) in two horses introduced from Belgium. The animals came from one assembly centre in Romania and had transited through Belgium with 16 other horses. Nine of them, bought by a Belgian horse breeder, were investigated in Belgium and revealed one additional EIA-positive animal. Afterwards, the Belgian Federal Agency for the Safety of the Food Chain (FASFC) organized a serological EIA survey of the horses introduced into Belgium from Romania between 2007 and 2009. Among the 95 horses identified, six additional serological positive cases were found that had been introduced into Belgium in 2008 (n = 4) and in 2009 (n = 2). The survey was extended to the horses in contact with the positive cases, but all contact animals were negative, indicating the absence of transmission. Virological examination performed on tissue samples collected from two seropositive animals demonstrated the presence of viral DNA of EIA virus. Phylogenetic analysis based on the sequences of EIA virus gag gene clustered the Belgian isolates with Romanian strains isolated in 2009. The presumption of a common Belgian origin could be rejected., (© 2012 Blackwell Verlag GmbH.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Although equine infectious anemia (EIA) was described more than 150 years ago, complete genomic sequences have only been obtained from two field strains of EIA virus (EIAV), EIAV(Wyoming) and EIAV(Liaoning). In 2011, EIA was detected within the distinctive feral Misaki horse population that inhabits the Toi-Cape area of southern Japan. Complete proviral sequences comprising a novel field strain were amplified directly from peripheral blood of one of these EIAV-infected horses and characterized by nucleotide sequencing. The complete provirus of Miyazaki2011-A strain is 8208 bp in length with an overall genomic organization typical of EIAV. However, this field isolate possesses just 77.2 and 78.7 % nucleotide sequence identity with the EIAV(Wyoming) and EIAV(Liaoning) strains, respectively, while similarity plot analysis suggested all three strains arose independently. Furthermore, phylogenetic studies using sequences obtained from all EIAV-infected Misaki horses against known viral strains strongly suggests these Japanese isolates comprise a separate monophyletic group.

The genetically distinct wild horse herds inhabiting Shackleford Banks, North Carolina are probably the direct descendents of Spanish stock abandoned after failed attempts to settle mid-Atlantic coastal regions of North America in the Sixteenth Century. In a 1996 island survey, 41% of the gathered horses were discovered seropositive for Equine Infectious Anemia Virus (EIAV) with additional cases identified in 1997 and 1998. As a result of their unique genetic heritage, EIAV seropositive individuals identified in the two latter surveys were transferred to a quarantine facility on the mainland. In September 2008 two of the horses SB1 and SB2 after 10 and 11 years in quarantine respectively, developed clinical signs of EIA. In the case of SB2 these were so severe that the only humane option was euthanasia. Although SB1, survived it experienced a second clinical episode one month later. In May 2009, a third horse in quarantine, SB3, developed extremely severe clinical EIA and was euthanized. This demonstrates naturally infected long-term inapparent carriers can experience recrudescence of very severe disease many years after initial exposure to EIAV. Phylogenetic analysis of complete EIAV gag gene sequences obtained from each of three Shackleford horses demonstrated they were infected with very closely related viruses. Although these were distinguishable from all other strains examined, they belong to a monophyletic group comprising almost exclusively of New World isolates that is distinct from a number of recently characterized Central European EIAV strains., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Equine infectious anaemia virus (EIAV) is classified within the Retroviridae and, like other lentivirus, has the propensity for considerable antigenic variation. An extensive phylogenetic analysis in Bayesian fashion, with significant amounts of new EIAV gag sequence information, revealed a strong geographic compartmentalization clearly related to the phylogeographic history of modern horses, pointing out that New World EIAV strains form a distinct group with a potentially common origin. This evidence suggests that a single founder event may have occurred during the reintroduction of horses to the Americas by European colonists in the 15th century, a possibility that raises many interesting scenarios with implications for all evolutionary and ecological studies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The attenuated Chinese equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. In this pilot study, to determine whether this attenuated vaccine can induce broadly neutralizing antibodies, we immunized four horses with the attenuated Chinese vaccine strain EIAVFDDV and then observed the evolution of neutralizing antibodies against different EIAV strains. During the vaccination phase, all vaccinees rapidly developed high levels of neutralizing antibodies against the homologous vaccine strain (pLGFD3V), and 3 out of 4 horses showed a gradual increase in serum neutralizing activity against two relatively heterologous virulent variants of the challenge strain (pLGFD3Mu12V and DLV34). After challenge, the three horses that had developed high levels of neutralizing antibodies against pLGFD3Mu12V and DLV34 did not show signs of infection, which was demonstrated by immune suppression, while the one horse producing serum that could only neutralize pLGFD3V developed a febrile episode during the 8-month observation period. To assess whether the broadly neutralizing activity is associated with immune protection, sera drawn on the day of challenge from these four vaccinees and an additional four EIAVFDDV-vaccinated horses were analyzed for neutralizing antibodies against pLGFD3V, pLGFD3Mu12V and DLV34. Although there was no significant correlation between protection from infection and serum neutralizing activity against any of these three viral strains, protection from infection was observed to correlate better with serum neutralizing activity against the two heterologous virulent strains than against the homologous vaccine strain. These data indicate that EIAVFDDV induced broadly neutralizing antibodies, which might confer enhanced protection of vaccinees from infection by the challenge virus.

The application of molecular diagnostic techniques along with nucleotide sequence determination to permit contemporary phylogenetic analysis of European field isolates of equine infectious anemia virus (EIAV) has not been widely reported. As a result, of extensive testing instigated following the 2006 outbreak of equine infectious anemia in Italy, 24 farms with a history of exposure to this disease were included in this study. New PCR-based methods were developed, which, especially in the case of DNA preparations from peripheral blood cells, showed excellent correlation with OIE-approved agar gel immunodiffusion (AGID) tests for identifying EIAV-infected animals. In contrast, the OIE-recommended oligonucleotide primers for EIAV failed to react with any of the Italian isolates. Similar results were also obtained with samples from four Romanian farms. In addition, for the first time complete characterization of gag genes from five Italian isolates and one Romanian isolate has been achieved, along with acquisition of extensive sequence information (86% of the total gag gene) from four additional EIAV isolates (one Italian and three Romanian). Furthermore, in another 23 cases we accomplished partial characterization of gag gene sequences in the region encoding the viral matrix protein. Analysis of this information suggested that most Italian isolates were geographically restricted, somewhat reminiscent of the "clades" described for human immunodeficiency virus type 1 (HIV-1). Collectively this represents the most comprehensive genetic study of European EIAV isolates conducted to date.

Background: Equine infectious anemia virus (EIAV), a lentivirus that infects horses, has been utilized as an animal model for the study of HIV. Furthermore, the disease associated with the equine lentivirus poses a significant challenge to veterinary medicine around the world. As with all lentiviruses, EIAV has been shown to have a high propensity for genomic sequence and antigenic variation, especially in its envelope (Env) proteins. Recent studies have demonstrated Env variation to be a major determinant of vaccine efficacy, emphasizing the importance of defining natural variation among field isolates of EIAV. To date, however, published EIAV sequences have been reported only for cell-adapted strains of virus, predominantly derived from a single primary virus isolate, EIAVWyoming (EIAVWY)., Results: We present here the first characterization of the Env protein of a natural primary isolate from Pennsylvania (EIAVPA) since the widely utilized and referenced EIAVWY strain. The data demonstrated that the level of EIAVPA Env amino acid sequence variation, approximately 40% as compared to EIAVWY, is much greater than current perceptions or published reports of natural EIAV variation between field isolates. This variation did not appear to give rise to changes in the predicted secondary structure of the proteins. While the EIAVPA Env was serologically cross reactive with the Env proteins of the cell-adapted reference strain, EIAVPV (derivative of EIAVWY), the two variant Envs were shown to lack any cross neutralization by immune serum from horses infected with the respective virus strains., Conclusion: Taking into account the significance of serum neutralization to universal vaccine efficacy, these findings are crucial considerations towards successful EIAV vaccine development and the potential inclusion of field isolate Envs in vaccine candidates.

Molecular analysis of the regulatory and structurally important genetic segments of equine infectious anemia virus (EIAV) in mules is presented. We have previously reported clinicopathological and laboratory findings in mules infected with EIAV, both naturally and after experimental inoculation. In this study the fragment coding for integrase, gp90, tat and the fusion domain of gp45 of the proviral genome from these animals was sequenced and compared with one another and with that of EIAV strains already published in the literature. Significant variations were observed mainly in the sequences of the gp90 surface protein. In the two wild type sequences, there were substitutions in the V5 hypervariable domain of this protein. In the sequences of the experimentally inoculated animals and the donor strain, variations were due to insertions/duplications in the V3 principal neutralizing domain (PND) and substitutions in the V5 hypervariable domain. Finally, when compared with the already published strains, the wild type sequences had single amino acid substitutions across the whole protein and multiple substitutions in the V4-V6 variable domains. In general, the two Greek wild type sequences were closer to two of the American strains (WSU5 and Massachusetts), than to the two Japanese (V26 and V70) or the third American strain (Wyoming_wi) used in this study.

Cytotoxic T lymphocytes (CTL) are important for controlling equine infectious anemia virus (EIAV). Because Gag matrix (MA) and capsid (CA) are the most frequently recognized proteins, the hypothesis that CTL from EIAV-infected horses with diverse MHC class I alleles recognize epitope clusters (EC) in these proteins was tested. Four EC were identified by CTL from 15 horses and 8 of these horses had diverse MHC class I alleles. Two of the eight had CTL to EC1, six to EC2, five to EC3, and four to EC4. Because EC2-4 were recognized by CTL from >50% of horses with diverse alleles, the hypothesis was accepted. EC1 and EC3 were the most conserved EC and these more conserved broadly recognized EC may be most useful for CTL induction, helping overcome MHC class I polymorphism and antigenic variation.

Lentiviruses exist in vivo as a population of related, nonidentical genotypes, commonly referred to as quasispecies. The quasispecies structure is characteristic of complex adaptive systems and contributes to the high rate of evolution in lentiviruses that confounds efforts to develop effective vaccines and antiviral therapies. Here, we describe analyses of genetic data from longitudinal studies of genetic variation in a lentivirus regulatory protein, Rev, over the course of disease in ponies experimentally infected with equine infectious anemia virus. As observed with other lentivirus data, the Rev variants exhibited a quasispecies character. Phylogenetic and partition analyses suggested that the Rev quasispecies comprised two distinct subpopulations that coexisted during infection. One subpopulation appeared to accumulate changes in a linear, time-dependent manner, while the other evolved radially from a common variant. Over time, the two subpopulations cycled in predominance coincident with changes in the disease state, suggesting that the two groups differed in selective advantage. Transient expression assays indicated the two populations differed significantly in Rev nuclear export activity. Chimeric proviral clones containing Rev genotypes representative of each population differed in rate and overall level of virus replication in vitro. The coexistence of genetically distinct viral subpopulations that differ in phenotype provides great adaptability to environmental changes within the infected host. A quasispecies model with multiple subpopulations may provide additional insight into the nature of lentivirus reservoirs and the evolution of antigenic and drug-resistant variants.

A primary mechanism of lentivirus persistence is the ability of these viruses to evolve in response to biological and immunological selective pressures with a remarkable array of genetic and antigenic variations that constitute a perpetual natural experiment in genetic engineering. A widely accepted paradigm of lentivirus evolution is that the rate of genetic variation is correlated directly with the levels of virus replication: the greater the viral replication, the more opportunities that exist for genetic modifications and selection of viral variants. To test this hypothesis directly, we examined the patterns of equine infectious anemia virus (EIAV) envelope variation during a 2.5-year period in experimentally infected ponies that differed markedly in clinical progression and in steady-state levels of viral replication as indicated by plasma virus genomic RNA assays. The results of these comprehensive studies revealed for the first time similar extents of envelope gp90 variation in persistently infected ponies regardless of the number of disease cycles (one to six) and viremia during chronic disease. The extent of envelope variation was also independent of the apparent steady-state levels of virus replication during long-term asymptomatic infection, varying from undetectable to 10(5) genomic RNA copies per ml of plasma. In addition, the data confirmed the evolution of distinct virus populations (genomic quasispecies) associated with sequential febrile episodes during acute and chronic EIA and demonstrated for the first time ongoing envelope variation during long-term asymptomatic infections. Finally, comparison of the rates of evolution of the previously defined EIAV gp90 variable domains demonstrated distinct differences in the rates of nucleotide and amino acid sequence variation, presumably reflecting differences in the ability of different envelope domains to respond to immune or other biological selection pressures. Thus, these data suggest that EIAV variation can be associated predominantly with ongoing low levels of virus replication and selection in target tissues, even in the absence of substantial levels of plasma viremia, and that envelope variation continues during all stages of persistent infection as the virus successfully avoids clearance by host defense mechanisms.

Most in vivo studies with equine infectious anemia virus (EIAV) have been performed in horses and ponies (Equus caballus) with little published information available detailing the clinical responses of donkeys (Equus asinus) to infection with this virus. Consequently, donkeys were inoculated with two strains of EIAV (EIAV(PV) and EIAV(WY)) which have been documented to produce disease in E. caballus. Four ponies, 561, 562, 564 and 567 and two donkeys, 3 and 5 were infected with EIAV(PV) and one horse (94-10) and one donkey (4) were infected with EIAV(WY). Although the horse and ponies all experienced clinical signs of disease, which in some cases were severe, the donkeys remained asymptomatic throughout a 365-day observation period, except for mild transient reductions in platelet counts. The results from serological assays, virus isolation from plasma and detection of plasma-associated viral RNA by RT-PCR, indicated that initial replication of EIAV(PV) and EIAV(WY) was lower in donkeys than in horses and ponies. This conclusion was confirmed using competitive RT-PCR, in which viral RNA levels in the plasma of EIAV(PV)-infected ponies was up to 100,000-fold higher than in infected donkeys during the first 20 days post-infection (dpi). Similar results were obtained in the EIAV(WY)-infected animals, in which viral RNA burdens in the donkey at 20 dpi were 1000-fold less than in the horse. However, infection of donkey and horse monocyte-derived macrophage cultures with EIAV(PV) demonstrated that these cells in vitro were equally susceptible to virus-induced cytopathic effects and yielded similar levels of progeny virus. This result suggests that factors other than host cell permissiveness mediate the clinical differences observed between horses and donkeys infected with EIAV(PV) or EIAV(WY).

Genetic and biological variation in the regulatory protein Rev of equine infectious anemia virus (EIAV) were examined throughout a clinically dynamic disease course of an experimentally infected pony. Following infection with the virulent EIAV(Wyo), the pony underwent a variable disease course, including an acute fever episode at 12 days postinfection (DPI), multiple recurrent fever episodes until 135 DPI, a prolonged subclinical period, and two late fever episodes. Viral RNA was isolated from the inoculum and sequential sera samples, and the rev exon 2/gp45 overlapping ORFs were amplified, cloned, and sequenced. Novel variants were found throughout infection, and genetic analyses indicated that both the Rev and gp45 ORFs were under selective pressure. The Rev variant predominant in the inoculum, R1, remained predominant during the early periods following infection (until 35 DPI); however, R1 was replaced by new predominant variants during the recurrent fever period (67-135 DPI). R1 reemerged as the predominant variant during the afebrile period, but a new predominant variant, R93, was associated with the late fever episodes. Rev variants predominant during recurrent febrile and late-febrile periods had significantly higher Rev-mediated nuclear export activity than the variants predominant during the acute and afebrile periods. Statistical correlation was found between Rev activity and different stages of clinical disease. Together, these results suggest that genetic and biological variation in rev may be a contributing factor in EIAV disease progression., (Copyright 2001 Academic Press.)

The Wyoming strain of equine infectious anaemia virus (EIAV) is a highly virulent field strain that replicates to high titre in vitro only in primary equine monocyte-derived macrophages. In contrast, Wyoming-derived fibroblast-adapted EIAV strains (Malmquist virus) replicate in primary foetal equine kidney and equine dermis cells as well as in the cell lines FEA and Cf2Th. Wyoming and Malmquist viruses differ extensively both in long terminal repeat (LTR) and envelope region sequences. We have compared the promoter activities of the Wyoming LTR with those of LTRs derived from fibroblast-adapted viruses by examining their abilities to drive a luciferase reporter gene as well as by construction of infectious molecular clones differing only in LTR sequence. Our results indicate that LTR sequences are a major restriction for growth of the Wyoming strain of EIAV in fibroblasts.

Equine infectious anemia virus (EIAV) is a lentivirus that infects and persists in the monocyte/macrophage populations of blood and tissues. We employed polymerase chain reaction to investigate the distribution and the level of genome variability of EIAV DNA in different tissues of a horse infected with a highly virulent variant of the Wyoming strain of the virus. Long terminal repeat, gag, and pol primer pairs were used to direct the amplification of EIAV DNA from the peripheral blood mononuclear cells and from cells, presumably the macrophage subtypes, of the kidney, spleen, liver, lymph node, and cerebellum and periventricular regions of the brain. Polymerase chain reaction analysis of four domains within the envelope showed that viral subtypes with particular envelope domains segregated to different tissues as defined by the presence or absence of a given type of domain. Collectively, these results show that virus variants in the env gene home to different tissues, presumably because of selection for tissue-specific envelope determinants.

The reported serological relatedness between the major glycoproteins of human immunodeficiency virus (HIV gp120) and equine infectious anaemia virus (EIAV gp90) was examined using purified antigens in radioimmunoprecipitation (RIP), radioimmunoassay (RIA) and immunoblot assays with reference serum from acquired immunodeficiency syndrome (AIDS) patients, an anti-gp120 goat serum and EIAV-infected horse serum. To assess the contributions of glycoprotein oligosaccharide and peptide components to any observed reactivities, antigens treated with endoglycosidase F to remove carbohydrate were assayed in parallel with the intact glycoprotein. The results of the experiments indicated that the reactivity observed for each antigen was dependent on the immunoassay employed. The RIP and RIA analyses demonstrated that HIV gp120 is equally reactive with the AIDS patient serum, the goat anti-gp120 serum and the EIAV-infected horse serum, whereas the EIAV gp90 reacted only with the horse serum. In immunoblot assays, the HIV gp120 reacted with AIDS patient serum, but not with the EIAV-infected horse serum. Deglycosylation of the HIV gp120 evidently increased its reactivity with the AIDS patient serum, had no significant effect on its reactivity with the goat antiserum, and essentially abolished its reactivity with the EIAV reference serum. Thus, it appears that the serological cross-reactivity observed between HIV gp120 and sera from EIAV-infected horses can be attributed to the oligosaccharide rather than the peptide components of the viral glycoprotein. These studies also emphasize the necessity of employing several assay procedures in assessing lentivirus antigenicity.

Equine infectious anemia virus (EIAV) has a density of 1.154 g/cm3 in sucrose a high-molecular-weight RNA similar in size to Rauscher murine leukemia virus, and an internal virion reverse transcriptase that utilizes the synthetic RNA template poly(rA) but not the synthetic DNA template poly(dA), both with (dT)12 as primer. Although capable of utilizing manganese at low concentrations (approximately 0.1 mM), EIAV reverse transcriptase showed highest activity in the presence of 9 mM magnesium. The major protein of EIAV has a slightly lower molecular weight than the comparable protein of type C viruses and co-electrophoresed with 125I-labeled p25 of Mason-Pfizer monkey virus. A reference horse serum with antibodies to the major EIAV protein reacted only with EIAV and not with other type C or non-type C retraviruses. Reciprocally, a broadly reactive serum to type C virus p30s and specific sera to a variety of non-type C retraviruses did not react with EIAV. We recommend the inclusion of EIAV in the family Retraviridae.