Intrahost Selection Pressure Drives Equine Arteritis Virus Evolution during Persistent Infection in the Stallion Reproductive Tract.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a reproductive and respiratory disease of horses. Following natural infection, 10 to 70% of infected stallions can become carriers of EAV and continue to shed virus in the semen. In this study, sequential viruses isolated from nasal secretions, buffy coat cells, and semen of seven experimentally infected and two naturally infected EAV carrier stallions were deep sequenced to elucidate the intrahost microevolutionary process after a single transmission event. Analysis of variants from nasal secretions and buffy coat cells lacked extensive positive selection; however, characteristics of the mutant spectra were different in the two sample types. In contrast, the initial semen virus populations during acute infection have undergone a selective bottleneck, as reflected by the reduction in population size and diversifying selection at multiple sites in the viral genome. Furthermore, during persistent infection, extensive genome-wide purifying selection shaped variant diversity in the stallion reproductive tract. Overall, the nonstochastic nature of EAV evolution during persistent infection was driven by active intrahost selection pressure. Among the open reading frames within the viral genome, ORF3, ORF5, and the nsp2-coding region of ORF1a accumulated the majority of nucleotide substitutions during persistence, with ORF3 and ORF5 having the highest intrahost evolutionary rates. The findings presented here provide a novel insight into the evolutionary mechanisms of EAV and identified critical regions of the viral genome likely associated with the establishment and maintenance of persistent infection in the stallion reproductive tract.IMPORTANCE EAV can persist in the reproductive tract of infected stallions, and consequently, long-term carrier stallions constitute its sole natural reservoir. Previous studies demonstrated that the ampullae of the vas deferens are the primary site of viral persistence in the stallion reproductive tract and the persistence is associated with a significant inflammatory response that is unable to clear the infection. This is the first study that describes EAV full-length genomic evolution during acute and long-term persistent infection in the stallion reproductive tract using next-generation sequencing and contemporary sequence analysis techniques. The data provide novel insight into the intrahost evolution of EAV during acute and persistent infection and demonstrate that persistent infection is characterized by extensive genome-wide purifying selection and a nonstochastic evolutionary pattern mediated by intrahost selective pressure, with important nucleotide substitutions occurring in ORF1a (region encoding nsp2), ORF3, and ORF5.

Downregulation of MicroRNA eca-mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus.

Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. In this study, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of microRNAs (miRNAs). Most importantly, we demonstrated that long-term EAV persistence is associated with the downregulation of an SE-associated miRNA (eca-mir-128) and with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. The findings presented here suggest that SE eca-mir-128 is implicated in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. This is a novel finding and warrants further investigation to identify its specific mechanism in modulating the CXCL16/CXCR6 axis in the reproductive tract of the EAV long-term carrier stallion.IMPORTANCE Equine arteritis virus (EAV) has the ability to establish long-term persistent infection in the stallion reproductive tract and to be shed in semen, which jeopardizes its worldwide control. Currently, the molecular mechanisms of viral persistence are being unraveled, and these are essential for the development of effective therapeutics to eliminate persistent infection. Recently, it has been determined that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and is maintained despite induction of local inflammatory, humoral, and mucosal antibody responses. This study demonstrated that long-term persistence is associated with the downregulation of seminal exosome miRNA eca-mir-128 and enhanced expression of its putative target, CXCL16, in the reproductive tract. For the first time, this study suggests complex interactions between eca-mir-128 and cellular elements at the site of EAV persistence and implicates this miRNA in the regulation of the CXCL16/CXCR6 axis in the reproductive tract during long-term persistence.

Allelic Variation in CXCL16 Determines CD3+ T Lymphocyte Susceptibility to Equine Arteritis Virus Infection and Establishment of Long-Term Carrier State in the Stallion.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of horses and other equid species. Following natural infection, 10-70% of the infected stallions can become persistently infected and continue to shed EAV in their semen for periods ranging from several months to life. Recently, we reported that some stallions possess a subpopulation(s) of CD3+ T lymphocytes that are susceptible to in vitro EAV infection and that this phenotypic trait is associated with long-term carrier status following exposure to the virus. In contrast, stallions not possessing the CD3+ T lymphocyte susceptible phenotype are at less risk of becoming long-term virus carriers. A genome wide association study (GWAS) using the Illumina Equine SNP50 chip revealed that the ability of EAV to infect CD3+ T lymphocytes and establish long-term carrier status in stallions correlated with a region within equine chromosome 11. Here we identified the gene and mutations responsible for these phenotypes. Specifically, the work implicated three allelic variants of the equine orthologue of CXCL16 (EqCXCL16) that differ by four non-synonymous nucleotide substitutions (XM_00154756; c.715 A → T, c.801 G → C, c.804 T → A/G, c.810 G → A) within exon 1. This resulted in four amino acid changes with EqCXCL16S (XP_001504806.1) having Phe, His, Ile and Lys as compared to EqCXL16R having Tyr, Asp, Phe, and Glu at 40, 49, 50, and 52, respectively. Two alleles (EqCXCL16Sa, EqCXCL16Sb) encoded identical protein products that correlated strongly with long-term EAV persistence in stallions (P<0.000001) and are required for in vitro CD3+ T lymphocyte susceptibility to EAV infection. The third (EqCXCL16R) was associated with in vitro CD3+ T lymphocyte resistance to EAV infection and a significantly lower probability for establishment of the long-term carrier state (viral persistence) in the male reproductive tract. EqCXCL16Sa and EqCXCL16Sb exert a dominant mode of inheritance. Most importantly, the protein isoform EqCXCL16S but not EqCXCL16R can function as an EAV cellular receptor. Although both molecules have equal chemoattractant potential, EqCXCL16S has significantly higher scavenger receptor and adhesion properties compared to EqCXCL16R.

Detection of equine arteritis virus by two chromogenic RNA in situ hybridization assays (conventional and RNAscope(®)) and assessment of their performance in tissues from aborted equine fetuses.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, a respiratory and reproductive disease of equids. EAV infection can induce abortion in pregnant mares, fulminant bronchointerstitial pneumonia in foals, and persistent infection in stallions. Here, we developed two RNA in situ hybridization (ISH) assays (conventional and RNAscope(®) ISH) for the detection of viral RNA in formalin-fixed paraffin-embedded (FFPE) tissues and evaluated and compared their performance with nucleocapsid-specific immunohistochemistry (IHC) and virus isolation (VI; gold standard) techniques. The distribution and cellular localization of EAV RNA and antigen were similar in tissues from aborted equine fetuses. Evaluation of 80 FFPE tissues collected from 16 aborted fetuses showed that the conventional RNA ISH assay had a significantly lower sensitivity than the RNAscope(®) and IHC assays, whereas there was no difference between the latter two assays. The use of oligonucleotide probes along with a signal amplification system (RNAscope(®)) can enhance detection of EAV RNA in FFPE tissues, with sensitivity comparable to that of IHC. Most importantly, these assays provide important tools with which to investigate the mechanisms of EAV pathogenesis.

Development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay for detection of equine arteritis virus in equine semen and tissue samples using the POCKIT™ system.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses. Most importantly, EAV induces abortion in pregnant mares and can establish persistent infection in up to 10-70% of the infected stallions, which will continue to shed the virus in their semen. The objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the detection of EAV in semen and tissue samples. The newly developed assay had a limit of detection of 10 RNA copies and a 10-fold higher sensitivity than a previously described real-time RT-PCR (RT-qPCR). Evaluation of 125 semen samples revealed a sensitivity and specificity of 98.46% and 100.00%, respectively for the RT-qPCR assay, and 100.00% and 98.33%, respectively for the RT-iiPCR assay. Both assays had the same accuracy (99.2%, k=0.98) compared to virus isolation. Corresponding values derived from testing various tissue samples (n=122) collected from aborted fetuses, foals, and EAV carrier stallions are as follows: relative sensitivity, specificity, and accuracy of 88.14%, 96.83%, and 92.62% (k=0.85), respectively for the RT-qPCR assay, and 98.31%, 92.06%, and 95.08% (k=0.90), respectively for the RT-iiPCR assay. These results indicate that RT-iiPCR is a sensitive, specific, and a robust test enabling detection of EAV in semen and tissue samples with very considerable accuracy. Even though the RT-qPCR assay showed a sensitivity and specificity equal to virus isolation for semen samples, its diagnostic performance was somewhat limited for tissue samples. Thus, this new RT-iiPCR could be considered as an alternative tool in the implementation of EAV control and prevention strategies.

Complete Genome Sequence of Noncytopathic Bovine Viral Diarrhea Virus 1 Contaminating a High-Passage RK-13 Cell Line.

A high-passage rabbit kidney RK-13 cell line (HP-RK-13[KY], originally derived from the ATCC CCL-37 cell line) used in certain laboratories worldwide is contaminated with noncytopathic bovine viral diarrhea virus (ncpBVDV). On complete genome sequence analysis, the virus strain was found to belong to BVDV group 1b.

Validation of an improved competitive enzyme-linked immunosorbent assay to detect Equine arteritis virus antibody.

The objective of the present study was to validate a previously described competitive enzyme-linked immunosorbent assay (cELISA) to detect antibody to Equine arteritis virus (EAV) based on GP5-specific nonneutralizing monoclonal antibody (mAb) 17B7(9) using the World Organization for Animal Health (OIE)-recommended protocol, which includes the following 5 in-house analyses. 1) The assay was calibrated with the OIE-designated reference serum panel for EAV; 2) repeatability was evaluated within and between assay runs; 3) analytical specificity was evaluated using sera specific to related viruses; 4) analytical sensitivity was evaluated with sera from horses vaccinated with an EAV modified live virus (MLV) vaccine; and 5) the duration of cELISA antibody detection following EAV vaccination was determined. The positive cELISA cutoff of ≥35% inhibition (%I) was confirmed by receiver operating characteristic plot analysis. Analytical sensitivity of the cELISA was comparable to the serum neutralization (SN) assay in that it detected EAV-specific antibody as early as 8 days postvaccination. The duration of EAV-specific antibody detected by cELISA was over 5 years after the last vaccination. This cELISA could detect EAV-specific antibody in serum samples collected from horses infected with various EAV strains. In the field trial performed by American Association of Veterinary Laboratory Diagnosticians-accredited state laboratories and OIE laboratory, the diagnostic specificity of the cELISA was 99.5% and the diagnostic sensitivity was 98.2%. The data using various serum panels also had consistently significant positive correlation between SN titers and cELISA %I results. The results further confirm that the EAV antibody cELISA is a reliable, simple alternative to the SN assay for detecting EAV-specific antibodies in equine sera.

Comparison of an improved competitive enzyme-linked immunosorbent assay with the World Organization for Animal Health-prescribed serum neutralization assay for detection of antibody to Equine arteritis virus.

Equine arteritis virus (EAV) causes contagious equine viral arteritis, characterized by fever, anorexia, conjunctivitis, nasal discharge, dependent edema, abortion, infrequent death in foals, and establishment of the carrier state in stallions. The World Organization for Animal Health (OIE) defines a horse as seropositive if the serum neutralization (SN) antibody titer is ≥1:4 to EAV. However, determining the SN titer is time-consuming and requires specific laboratory facilities, equipment, and technical expertise to perform. Furthermore, interpretation of the SN titer of some sera can be difficult because of nonspecific cellular cytotoxicity of particular samples. Finally, the problem of interlaboratory variation also exists with SN assays. For these reasons, an alternative serologic test is desirable; however, none of the reported tests have equivalent sensitivity and specificity to the SN to be generally adopted. In an attempt to improve on a previously developed competitive enzyme-linked immunosorbent assay (cELISA) using EAV gp5-specific neutralizing monoclonal antibody (mAb) 4B2, the current study developed a modified protocol substituting the non-neutralizing mAb 17B7 for the neutralizing mAb 4B2; this along with several modifications of the test procedure improved the performance of the test. The relative specificity of the revamped cELISA was 99.8% when evaluated with 2,223 SN-negative sera. The relative sensitivity was 95.5% when evaluated with 246 SN-positive sera. This new cELISA was not affected by the presence of non-EAV-specific cytotoxicity in sera as observed in the SN assay. The results indicate that this new cELISA may be a viable alternative to the SN assay and merit additional validation.

Evidence that in vitro susceptibility of CD3+ T lymphocytes to equine arteritis virus infection reflects genetic predisposition of naturally infected stallions to become carriers of the virus.

We investigated the correlation between in vitro susceptibility of CD3(+) T lymphocytes to equine arteritis virus (EAV) infection and establishment of persistent infection among 14 stallions following natural infections. The data showed that carrier stallions with a CD3(+) T lymphocyte susceptibility phenotype to in vitro EAV infection may be at higher risk of becoming carriers than those that lack this phenotype (P = 0.0002).

Evaluation of two magnetic-bead-based viral nucleic acid purification kits and three real-time reverse transcription-PCR reagent systems in two TaqMan assays for equine arteritis virus detection in semen.

This study showed that under specifically defined conditions with respect to nucleic acid extraction method and testing reagents, a previously described real-time reverse transcription-PCR (rRT-PCR) assay (T1 assay) provides sensitivity equal to or higher than that of virus isolation for the detection of equine arteritis virus in semen.

Molecular epidemiology and genetic characterization of equine arteritis virus isolates associated with the 2006-2007 multi-state disease occurrence in the USA.

In 2006-2007, equine viral arteritis (EVA) was confirmed for the first time in Quarter Horses in multiple states in the USA. The entire genome of an equine arteritis virus (EAV) isolate from the index premises in New Mexico was 12 731 nt in length and possessed a previously unrecorded unique 15 nt insertion in the nsp2-coding region in ORF1a and a 12 nt insertion in ORF3. Sequence analysis of additional isolates made during this disease occurrence revealed that all isolates from New Mexico, Utah, Kansas, Oklahoma and Idaho had 98.6-100.0 % (nsp2) and 97.8-100 % (ORF3) nucleotide identity and contained the unique insertions in nsp2 and ORF3, indicating that the EVA outbreaks in these states probably originated from the same strain of EAV. Sequence and phylogenetic analysis of several EAV isolates made following an EVA outbreak on another Quarter Horse farm in New Mexico in 2005 provided evidence that this outbreak may well have been the source of virus for the 2006-2007 occurrence of the disease. A virus isolate from an aborted fetus in Utah was shown to have a distinct neutralization phenotype compared with other isolates associated with the 2006-2007 EVA occurrence. Full-length genomic sequence analysis of 18 sequential isolates of EAV made from eight carrier stallions established that the virus evolved genetically during persistent infection, and the rate of genetic change varied between individual animals and the period of virus shedding.

Comparison of two real-time reverse transcription polymerase chain reaction assays for the detection of Equine arteritis virus nucleic acid in equine semen and tissue culture fluid.

Two previously developed TaqMan fluorogenic probe-based 1-tube real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays (T1 and T2) were compared and validated for the detection of Equine arteritis virus (EAV) nucleic acid in equine semen and tissue culture fluid (TCF). The specificity and sensitivity of these 2 molecular-based assays were compared to traditional virus isolation (VI) in cell culture. The T1 real-time RT-PCR had a higher sensitivity (93.4%) than the T2 real-time RT-PCR (42.6%) for detection of EAV RNA in semen. However, the T1 real-time RT-PCR was less sensitive (93.4%) than the World Organization for Animal Health (OIE)-prescribed VI test (gold standard). The sensitivity of both PCR assays was high (100.0% [T1] and 95.2% [T2]) for detecting EAV RNA in TCF. In light of the discrepancy in sensitivity between either real-time RT-PCR assay and VI, semen that is negative for EAV nucleic acid by real-time RT-PCR that is from an EAV-seropositive stallion should be confirmed free of virus by VI. Similarly, the presence of EAV in TCF samples that are VI-positive but real-time RT-PCR-negative should be confirmed in a 1-way neutralization test using anti-EAV equine serum or by fluorescent antibody test using monoclonal antibodies to EAV. If the viral isolate is not identified as EAV, such samples should be tested for other equine viral pathogens. The results of this study underscore the importance of comparative evaluation and validation of real-time RT-PCR assays prior to their recommended use in a diagnostic setting for the detection and identification of specific infectious agents.

Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test.

The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5(55-98) protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5(55-98) MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP5(55-98) MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.