Identification of a Novel Post-transcriptional Transactivator from the Equine Infectious Anemia Virus.

All lentiviruses encode a post-transcriptional transactivator, Rev, which mediates the export of viral mRNA from the nucleus to the cytoplasm and which is required for viral gene expression and viral replication. In the current study, we demonstrate that equine infectious anemia virus (EIAV), an equine lentivirus, encodes a second post-transcriptional transactivator that we designate Grev. Grev is encoded by a novel transcript with a single splicing event that was identified using reverse transcription-PCR (RT-PCR) and RNA-seq in EIAV-infected horse tissues and cells. Grev is about 18 kDa in size, comprises the first 18 amino acids (aa) of Gag protein together with the last 82 aa of Rev, and was detected in EIAV-infected cells. Similar to Rev, Grev is localized to the nucleus, and both are able to mediate the expression of Mat (a recently identified viral protein of unknown function from EIAV), but Rev can mediate the expression of EIAV Gag/Pol, while Grev cannot. We also demonstrate that Grev, similar to Rev, specifically binds to rev-responsive element 2 (RRE-2, located in the first exon of mat mRNAs) to promote nuclear export of mat mRNA via the chromosome region maintenance 1 (CRM1) pathway. However, unlike Rev, whose function depends on its multimerization, we could not detect multimerization of Grev using coimmunoprecipitation (co-IP) or bimolecular fluorescence complementation (BiFC) assays. Together, these data suggest that EIAV encodes two post-transcriptional transactivators, Rev and Grev, with similar, but not identical, functions. IMPORTANCE Nuclear export of viral transcripts is a crucial step for viral gene expression and viral replication in lentiviruses, and this export is regulated by a post-transcriptional transactivator, Rev, that is shared by all lentiviruses. Here, we report that the equine infectious anemia virus (EIAV) encodes a novel viral protein, Grev, and demonstrated that Grev, like Rev, mediates the expression of the viral protein Mat by binding to the first exon of mat mRNAs via the chromosome region maintenance 1 (CRM1) pathway. Grev is encoded by a single-spliced transcript containing two exons, whereas Rev is encoded by a multiple-spliced transcript containing four exons. Moreover, Rev is able to mediate EIAV Gag/Pol expression by binding to rev-responsive element (RRE) located within the Env-coding region, while Grev cannot. Therefore, the present study demonstrates that EIAV encodes two post-transcriptional regulators, Grev and Rev, suggesting that post-transcriptional regulation patterns in lentivirus are diverse and complex.

Modelling Mutation in Equine Infectious Anemia Virus Infection Suggests a Path to Viral Clearance with Repeated Vaccination.

Equine infectious anemia virus (EIAV) is a lentivirus similar to HIV that infects horses. Clinical and experimental studies demonstrating immune control of EIAV infection hold promise for efforts to produce an HIV vaccine. Antibody infusions have been shown to block both wild-type and mutant virus infection, but the mutant sometimes escapes. Using these data, we develop a mathematical model that describes the interactions between antibodies and both wild-type and mutant virus populations, in the context of continual virus mutation. The aim of this work is to determine whether repeated vaccinations through antibody infusions can reduce both the wild-type and mutant strains of the virus below one viral particle, and if so, to examine the vaccination period and number of infusions that ensure eradication. The antibody infusions are modelled using impulsive differential equations, a technique that offers insight into repeated vaccination by approximating the time-to-peak by an instantaneous change. We use impulsive theory to determine the maximal vaccination intervals that would be required to reduce the wild-type and mutant virus levels below one particle per horse. We show that seven boosts of the antibody vaccine are sufficient to eradicate both the wild-type and the mutant strains. In the case of a mutant virus infection that is given infusions of antibodies targeting wild-type virus (i.e., simulation of a heterologous infection), seven infusions were likewise sufficient to eradicate infection, based upon the data set. However, if the period between infusions was sufficiently increased, both the wild-type and mutant virus would eventually persist in the form of a periodic orbit. These results suggest a route forward to design antibody-based vaccine strategies to control viruses subject to mutant escape.

Truncation of the Cytoplasmic Tail of Equine Infectious Anemia Virus Increases Virion Production by Improving Env Cleavage and Plasma Membrane Localization.

Envelope glycoproteins (Envs) of lentiviruses harbor unusually long cytoplasmic tails (CTs). Natural CT truncations always occur in vitro and are accompanied by attenuated virulence, but their effects on viral replication have not been fully elucidated. The Env in equine infectious anemia virus (EIAV) harbors the longest CT in the lentiviral family, and a truncated CT was observed in a live attenuated vaccine. This study demonstrates that CT truncation significantly increased EIAV production, as determined by comparing the virion yields from EIAV infectious clones in the presence and absence of the CT. A significant increase in a cleaved product from the CT-truncated Env precursor, but not the full-length Env, was observed. We further confirmed that the presence of the CT inhibited the cleavage of the Env precursor and found that a functional domain located at the C terminus was responsible for this function. Moreover, CT-truncated Env was mainly localized at the plasma membrane (PM), while full-length Env was mainly localized in the cytoplasm. The CT truncation caused a dramatic reduction in the endocytosis of Env. These results suggest that the CT can modulate the processing and trafficking of EIAV Env and thus regulate EIAV replication. IMPORTANCE The mature lentivirus envelope glycoprotein (Env) is composed of a surface unit (SU) and a transmembrane unit (TM), which are cleaved products of the Env precursor. After mature Env is heterodimerically formed from the cleavage of the Env precursor, it is trafficked to the plasma membrane (PM) for incorporation and virion assembly. Env harbors a long cytoplasmic tail (CT), which has been increasingly found to play multiple roles in the Env biological cycle. Here, we revealed for the first time that the CT of equine infectious anemia virus (EIAV) Env inhibits cleavage of the Env precursor. Simultaneously, the CT promoted Env endocytosis, resulting in weakened Env localization at the PM. We also validated that the CT could significantly decrease EIAV production. These findings suggest that the CT regulates the processing and trafficking of EIAV Env to balance virion production.

Identification and genetic characterization of equine infectious anemia virus in Western Balkans.

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.

Molecular detection of equine infectious anemia virus in clinically normal, seronegative horses in an endemic area of Mexico.

Equine infectious anemia (EIA) is a highly infectious disease in members of the Equidae family, caused by equine infectious anemia virus (EIAV). The disease severity ranges from subclinical to acute or chronic, and causes significant economic losses in the equine industry worldwide. Serologic tests for detection of EIAV infection have some concerns given the prolonged seroconversion time. Therefore, molecular methods are needed to improve surveillance programs for this disease. We attempted detection of EIAV in 6 clinical and 42 non-clinical horses in Nuevo Leon State, Mexico, using the agar gel immunodiffusion (AGID) test for antibody detection, and nested and hemi-nested PCR for detection of proviral DNA. We found that 6 of 6, 5 of 6, and 6 of 6 clinical horses were positive by AGID, nested PCR, and hemi-nested PCR, respectively, whereas 0 of 42, 1 of 42, and 9 of 42 non-clinical horses were positive by these tests, respectively. BLAST analysis of the 203-bp 5'-LTR/tat segment of PCR product revealed 83-93% identity with EIAV isolates in GenBank and reference strains from other countries. By phylogenetic analysis, our Mexican samples were grouped in a different clade than other sequences reported worldwide, indicating that the LRT/tat region represents an important target for the detection of non-clinical horses.

Comparative analysis of CpG islands in equine infectious anemia virus strains.

Increasing evidence suggests that DNA methylation has key roles in the replication of retroviruses, including lentiviruses, and pathogenesis of diseases. However, the precise characteristics of CpG islands are not known for many retroviruses. In this study, we compared the distribution of CpG islands among strains of equine infectious anemia virus (EIAV), a lentivirus in the family Retroviridae and a model for HIV research. We identified CpG islands in 32 full-length EIAV genomic sequences obtained from the GenBank database using MethPrimer. Only one CpG island, from 100 to 120 bp, was identified in the genomes of EIAV strains DV10, DLV3-A, and DLV5-10 from China, V26 and V70 from Japan, and IRE H3, IRE F2, IRE F3, and IRE F4 from Ireland. Importantly, the CpG island was located within the Rev gene, which is required for the expression of viral cis-acting elements and the production of new virions. These results suggest that the distribution, length, and genetic properties of CpG islands differ among EIAV strains. Future research should focus on the biological significance of this CpG island within rev to improve our understanding of the precise roles of CpG islands in epigenetic regulation in the species.

Molecular detection, histopathological analysis, and immunohistochemical characterization of equine infectious anemia virus in naturally infected equids.

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.

High Genomic Variability in Equine Infectious Anemia Virus Obtained from Naturally Infected Horses in Pantanal, Brazil: An Endemic Region Case.

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.

Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly.

Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.

Molecular characterization of Equine Infectious Anemia Viruses using targeted sequence enrichment and next generation sequencing.

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.

Attenuation of Equine Lentivirus Alters Mitochondrial Protein Expression Profile from Inflammation to Apoptosis.

Equine infectious anemia virus (EIAV) is an equine lentivirus similar to HIV-1, targets host immune cells, and causes a life-long infection in horses. The Chinese live EIAV vaccine is attenuated from long-term passaging of a highly virulent strain in vitro The parent pathogenic strain (EIAVDLV34) induces a host inflammatory storm to cause severe pathological injury of animals. However, the vaccine strain (EIAVDLV121) induces a high level of apoptosis to eliminate infected cells. To investigate how these processes are regulated, we performed a comparative proteomics analysis and functional study in equine monocyte-derived macrophages (eMDMs) and found that the divergent mitochondrial protein expression profiles caused by EIAV strains with different virulence led to disparate mitochondrial function, morphology, and metabolism. This in turn promoted the distinct transformation of macrophage inflammatory polarization and intrinsic apoptosis. In EIAVDLV34-infected cells, a high level of glycolysis and increased mitochondrial fragmentation were induced, resulting in the M1-polarized proinflammatory-type transformation of macrophages and the subsequent production of a strong inflammatory response. Following infection with EIAVDLV121, the infected cells were transformed into M2-polarized anti-inflammatory macrophages by inhibition of glycolysis. In this case, a decrease in the mitochondrial membrane potential and impairment of the electron transport chain led to increased levels of apoptosis and reactive oxygen species. These results correlated with viral pathogenicity loss and may help provide an understanding of the key mechanism of lentiviral attenuation.IMPORTANCE Following viral infection, the working pattern and function of the cell can be transformed through the impact on mitochondria. It still unknown how the mitochondrial response changes in cells infected with viruses in the process of virulence attenuation. EIAVDLV121 is the only effective lentiviral vaccine for large-scale use in the world. EIAVDLV34 is the parent pathogenic strain. Unlike EIAVDLV34-induced inflammation storms, EIAVDLV121 can induce high levels of apoptosis. For the first time, we found that, after the mitochondrial protein expression profile is altered, EIAVDLV34-infected cells are transformed into M1-polarized-type macrophages and cause inflammatory injury and that the intrinsic apoptosis pathway is activated in EIAVDLV121-infected cells. These studies shed light on how the mitochondrial protein expression profile changes between cells infected by pathogenic lentivirus strains and cells infected by attenuated lentivirus strains to drive different cellular responses, especially from inflammation to apoptosis.

Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT-PCR assay to aid diagnosis under field conditions.

BACKGROUND: Control of equine infectious anaemia (EIA) currently depends on serological diagnosis of infected equids. However, recently infected equids may not produce detectable anti-EIAV antibodies up to 157 days post infection and so present a high transmission risk. Therefore, direct nucleic acid detection methods are urgently needed to improve EIAV surveillance and management programs in counties where the disease is endemic. OBJECTIVES: To evaluate a field-deployable, reverse transcription-insulated isothermal PCR (RT-iiPCR) assay targeting the conserved 5' untranslated region (5' UTR)/exon 1 of the tat gene of EIAV. STUDY DESIGN: The analytical and clinical performance of the newly developed EIAV RT-iiPCR was evaluated by comparison with a EIAV real-time RT-PCR (RT-qPCR) along with the AGID test. METHODS: Analytical sensitivity was determined using in vitro transcribed RNA containing the target area of the 5' UTR/tat gene and samples from two EIAV-positive horses. Specificity was verified using nine common equine viruses. Clinical performance was evaluated by comparison with EIAV RT-qPCR and AGID using samples derived from 196 inapparent EIAV carrier horses. RESULTS: EIAV RT-iiPCR did not react with other commonly encountered equine viruses and had equivalent sensitivity (95% detection limit of eight genome equivalents), with a concordance of 95.41% to conventional EIAV RT-qPCR. However, the RT-qPCR and RT-iiPCR had sensitivities of 43.75 and 50.00%, respectively, when compared to the AGID test. MAIN LIMITATIONS: Low viral loads commonly encountered in inapparent EIAV carriers may limit the diagnostic sensitivity of RT-PCR-based tests. CONCLUSIONS: Although EIAV RT-iiPCR is not sufficiently sensitive to replace the current AGID test, it can augment control efforts by identifying recently exposed or "serologically silent" equids, particularly as the latter often represent a significant transmission risk because of high viral loads. Furthermore, the relatively low cost and field-deployable design enable utilisation of EIAV RT-iiPCR even in remote regions.

Enzyme-linked immunosorbent assay and agar gel immunodiffusion assay for diagnosis of equine infectious anemia employing p26 protein fused to the maltose-binding protein.

A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented excellent values. The AGIDrec showed 100% sensitivity and specificity, and the ELISArec showed 100% sensitivity and 99.64% specificity. In addition, MBP-p26rec was stabile after three years of storage at 4 °C, maintaining its immunoreactivity.

Equine infectious anemia virus in naturally infected horses from the Brazilian Pantanal.

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.

Molecular characterization of equine infectious anaemia virus from a major outbreak in southeastern France.

In 2009, a major outbreak of equine infectious anaemia (EIA) was reported in the south-east of France. This outbreak affected three premises located in the Var region where the index case, a 10-year-old mare that exhibited clinical signs consistent with EIA, occurred at a riding school. Overall, more than 250 horses were tested for EIAV (equine infectious anaemia virus) antibodies, using agar gel immunodiffusion test, and 16 horses were positive in three different holdings. Epidemiological survey confirmed that the three premises were related through the purchase/sale of horses and the use of shared or nearby pastures. Molecular characterization of viruses was performed by sequencing the full gag gene sequence (1,400 bp) of the proviral DNAs retrieved from the spleen of infected animals collected post-mortem. Phylogenetic analysis confirmed epidemiological data from the field, as viruses isolated from the three premises were clustering together suggesting a common origin whereas some premises were 50 km apart. Moreover, viruses characterized during this outbreak are different from European strains described so far, underlying the high genetic diversity of EIAV in Europe.

Dynamics of lentiviral infection in vivo in the absence of adaptive immune responses.

Understanding the dynamics of acute viral infection is crucial for developing strategies to prevent and control infection. In this study, lentiviral dynamics in a host without adaptive immunity were examined in order to determine kinetic parameters of infection and quantify the effect of neutralizing antibodies in preventing infection, using mathematical modeling of data from equine infectious anemia virus (EIAV) infection of horses with severe combined immunodeficiency (SCID). Estimated parameters were used to calculate the basic reproductive number and virus doubling time and found that the rate that antibodies neutralized virus was ~18 times greater than the virus clearance rate. These results establish EIAV replication kinetics in SCID horses and the minimal efficacy of antibodies that blocked infection. Furthermore, they indicate that EIAV is at most mildly cytopathic. This study advances our understanding of EIAV infection and may have important implications for the control of other viral infections, including HIV.

Detection and molecular characterization of equine infectious anemia virus in Mongolian horses.

The genetic characterization and actual prevalence of EIAV in Mongolian horse in the disease endemic region is currently unknown. Here, 11 of 776 horse serum samples from four Mongolian provinces tested positive on agar gel immunodiffusion test. Genomic DNA extracted from all seropositive samples was subjected to nested PCR assay. Among these, three samples tested positive with nested PCR assay and were identified by sequencing analysis based on long termination repeat and tat gene of the virus. Two of the three sequences were identical, with 94.0% identity with the third. These two independent Mongolian EIAV sequences were retained functional motifs, with no dramatic changes but some variability in the U5 region; they were clustered with genotypes from European countries but not with those from China, U.S.A., or Japan.

Nonsynonymous changes of equine lentivirus receptor-1 (ELR1) gene in amino acids involved in the interaction with equine infectious anemia virus (EIAV).

Equine lentivirus receptor-1 (ELR1) has been characterized as the specific functional receptor that mediates equine infectious anemia virus (EIAV) entrance to horse macrophages. This receptor is tumor necrosis factor receptor superfamily member 14 (TNFRSF14). The aim of this study was to investigate the occurrence of allelic variants in the coding sequence of equine TNFRSF14 gene by screening for single-nucleotide polymorphisms (SNPs) in different equine populations. Forty seven horse samples were randomly selected from a reservoir of EIAV-seropositive and seronegative samples collected from different outbreaks and regions of Argentina. DNA samples were scanned via PCR and direct sequencing of exon 3 and exon 5 of TNFRSF14 gene. A total of 21 SNPs were identified, of which 11 were located in coding sequences. Within exon 5, four SNPs caused nonsynonymous substitutions, while two other SNPs caused synonymous substitutions in crucial residues (Ser112 and Thr114) implicated in the interaction with EIAV. Despite some of exon 5 variants occurred exclusively in EIAV-positive or EIAV-negative horses, critical residues for the function of the mature protein were conserved, accounting for selective pressures in favor of preserving the specific function of TNFRSF members and the host immune response. To our knowledge, this is the first report of the existence of allelic variations involving some crucial amino acid residues in horse ELR1. Further, it could be an initial step to test the possible functional relevance and relationship of these variants with EIAV infection and disease progression as well as to develop preventive strategies.

Evaluation of six serological ELISA kits available in Italy as screening tests for equine infectious anaemia surveillance.

BACKGROUND: ELISAs are known to have a higher diagnostic sensitivity than the agar gel immunodiffusion (AGID) when employed for serological diagnosis of equine infectious anaemia (EIA). For this purpose, an "in-house" and five commercial ELISAs available in Italy were assessed by the National Reference Centre for EIA for their analytic specificity (Sp); precocity, defined as capability of detecting first antibodies produced during a new infection; precision based on repeatability and reproducibility, estimated from the coefficient of variation (CV); accuracy, estimated from multiple K and relative Sp and sensitivity (Se). Two serum panels, positive for non-equine retroviruses and the most frequent equine viruses, were employed to measure analytic Sp. ELISA precocity was also compared to that of one "in-house" and three commercial AGID kits, employing a panel of sera, collected weekly from horses infected with modified EIA viruses. Precision and accuracy were defined using results of a panel containing positive and negative sera examined in an inter-laboratory trial with the participation of the ten Official Laboratories. Furthermore, a questionnaire was used to assess the appropriateness of each kit for routine use. RESULTS: Analytic Sp was 100%, while the 75th percentile of CVs for positive sera varied from 0.4% to 12.73% for repeatability and from 1.6% to 44.87% for reproducibility. Although CV of the negative serum was constantly high, its outcome was unaltered. Relative Se ranged from 98.2% to 100%, relative Sp was constantly 100% and multiple K ranged from 0.95 to 1. Precocity differed among the assays: three kits detected 4.8% and 42.9% positive samples on 21 days post infection (dpi), all assays detected positive samples on 28 dpi, between 47.6% and 95.2%. Precocity of ELISAs was superior to that of the AGIDs except for two assays. In view of the feedback obtained from the questionnaires, all kits were considered appropriate for routine use. CONCLUSION: All ELISAs having high Se and precocity are preferable as a screening test in EIA surveillance programmes to the AGID tests examined. These two tests can be incorporated in a serial diagnostic pathway to improve the efficacy of a surveillance plan.