Equine Arteritis Virus Derived from an Infectious cDNA Clone Is Attenuated and Genetically Stable in Infected Stallions

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.