Equine viral arteritis (EVA) is a highly contagious disease whose etiology was defined in the early 1950s. Clinical signs of EVA vary considerably among individual horses and among outbreaks. There is a very real risk of equine arteritis virus (EAV) being transferred indirectly via personnel and fomites. Virus isolation is the gold standard for detection of EAV in samples, including semen, and the OIE (World Organization for Animal Health or Office International des Epizooties) prescribed test for international trade. The treatment of EVA is general supportive care until the acute phase passes. Abortion is a frequent outcome in naïve pregnant mares, and generally occurs 10–30 days after exposure to EAV and at any time between 3 and 10 months of gestation. For EVA and other reportable diseases, more widespread screening of stallion populations and tighter quality controls over laboratories providing diagnostics would increase the detection rate of carrier stallions.
Summary In March 2024, highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b H5N1 infections in dairy cows were first reported from Texas, USA. Rapid dissemination to more than 190 farms in 13 states followed. Here, we provide results of two independent clade 2.3.4.4b experimental infection studies evaluating (i) oronasal susceptibility and transmission in calves to a US H5N1 bovine isolate genotype B3.13 (H5N1 B3.13) and (ii) susceptibility of lactating cows following direct mammary gland inoculation of either H5N1 B3.13 or a current EU H5N1 wild bird isolate genotype euDG (H5N1 euDG). Inoculation of the calves resulted in moderate nasal replication and shedding with no severe clinical signs or transmission to sentinel calves. In dairy cows, infection resulted in no nasal shedding, but severe acute mammary gland infection with necrotizing mastitis and high fever was observed for both H5N1 genotypes/strains. Milk production was rapidly and drastically reduced and the physical condition of the cows was severely compromised. Virus titers in milk rapidly peaked at 10 8 TCID 50 /mL, but systemic infection did not ensue. Notably, adaptive mutation PB2 E627K emerged after intramammary replication of H5N1 euDG. Our data suggest that in addition to H5N1 B3.13, other HPAIV H5N1 strains have the potential to replicate in the udder of cows and that milk and milking procedures, rather than respiratory spread, are likely the primary routes of H5N1 transmission between cattle.
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.
Accurate quantitative analysis of equine insulin in blood samples is critical for assessing hyperinsulinemia in horses. Although there are various laboratory methods for evaluating equine serum insulin, different immunoassays show significant discrepancies between the determined insulin concentrations and are often not comparable. The aim of this study was to evaluate the Immulite® 1000 chemiluminescent immunoassay (CLIA) to establish independent laboratory and assay-specific cut values to provide an accurate diagnosis of hyperinsulinemia in horses. Thus, the analytical and clinical performance of Immulite® 1000 CLIA in terms of precision (intra- and inter-assay coefficient of variance, CV) and recovery upon dilution were evaluated and compared with radioimmunoassay (RIA), which has been previously validated for use in horses.Archived serum samples (n = 106) from six Quarter horse mares enrolled in the glucose phase of a Frequently Sampled Insulin and Glucose Test (FSIGT) study were used to measure blood insulin.The Immulite® 1000 CLIA had good precision with acceptable intra- and inter-assay CVs, adequate recovery on dilution, and a strong correlation with the RIA (r = 0.974, P < 0.0001), with constant bias resulting in consistently lower values.On this basis, the Immulite® 1000 Insulin Assay is valid for measuring equine serum insulin for diagnostic and monitoring purposes when cut values are appropriately adjusted.
The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of >or=10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.
Since emerging in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has repeatedly crossed the species barrier with natural infections reported in various domestic and wild animal species. The emergence and global spread of SARS-CoV-2 variants of concern (VOCs) has expanded the range of susceptible host species. Previous experimental infection studies in cattle using Wuhan-like SARS-CoV-2 isolates suggested that cattle were not likely amplifying hosts for SARS-CoV-2. However, SARS-CoV-2 sero- and RNA-positive cattle have since been identified in Europe, India, and Africa. Here, we investigated the susceptibility and transmission of the Delta and Omicron SARS-CoV-2 VOCs in cattle. Eight Holstein calves were co-infected orally and intranasally with a mixed inoculum of SARS-CoV-2 VOCs Delta and Omicron BA.2. Twenty-four hours post-challenge, two sentinel calves were introduced to evaluate virus transmission. The co-infection resulted in a high proportion of calves shedding SARS-CoV-2 RNA at 1- and 2-days post-challenge (DPC). Extensive tissue distribution of SARS-CoV-2 RNA was observed at 3 and 7 DPC and infectious virus was recovered from two calves at 3 DPC. Next-generation sequencing revealed that only the SARS-CoV-2 Delta variant was detected in clinical samples and tissues. Similar to previous experimental infection studies in cattle, we observed only limited seroconversion and no clear evidence of transmission to sentinel calves. Together, our findings suggest that cattle are more permissive to infection with SARS-CoV-2 Delta than Omicron BA.2 and Wuhan-like isolates but, in the absence of horizontal transmission, are not likely to be reservoir hosts for currently circulating SARS-CoV-2 variants.
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.
SUMMARY The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.
