Virus derived from an infectious cDNA clone of equine arteritis virus (EAV030H) was intranasally inoculated into two stallions, neither of which subsequently developed clinical manifestations of equine viral arteritis (EVA). Virus was isolated from nasal swabs and mononuclear cells collected from both stallions =14 days p.i. and from the semen of one stallion only at 7 days p.i. Similarly, viral RNA was detected by RT nested-PCR in nasal swabs and mononuclear cells for =14 days p.i. and at 7 days p.i. in the semen of the one stallion. Both stallions seroconverted to EAV by 10 days p.i. and maintained high neutralizing antibody titers thereafter. Sequence and restriction digestion analysis demonstrated that the recombinant virus present in nasal swabs, mononuclear cells, and semen from the two stallions was identical to the infectious clone-derived virus that was used to inoculate them. Furthermore analysis of multiple clones derived by RT nested-PCR amplification from several samples indicated that the recombinant EAV030H virus was stable during replication in horses. These studies document for the first time that a recombinant virus derived from an infectious cDNA clone of a member of the order Nidovirales is replication competent in animals, and the genetic stability of the recombinant virus during in vivo replication indicates that it will be useful for the characterization of genetic determinants of virulence and persistence of EAV. The genetic conservation of the cloned recombinant virus during in vivo infection is similar to that which occurs during natural horizontal and vertical transmission of EAV in horses and contrasts with the heterogeneous virus population (quasispecies) that occurs in the semen of carrier stallions.
-A prospective cohort study was used to estimate the incidence of West Nile virus (WNV) infection in a group of unvaccinated horses (n = 37) in California and compare the effects of natural WNV infection in these unvaccinated horses to a group of co-mingled vaccinated horses (n = 155). Horses initially were vaccinated with either inactivated whole virus (n = 87) or canarypox recombinant (n = 68
The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing) and 38,000×(Illumina). The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were “selected” from a pre-existing pool rather than through de novo mutation and subsequent population fixation.
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