Identification of an additional neutralization determinant of equine arteritis virus

We recently established an in vitro model of equine arteritis virus (EAV) persistence in HeLa cells. The objective of this study was to determine whether viral variants with novel neutralization phenotypes emerged during persistent EAV infection of HeLa cells, as occurs during viral persistence in carrier stallions. Viruses recovered from persistently infected HeLa cells had different neutralization phenotypes than the virus in the original inoculum, as determined by neutralization assays using EAV-specific monoclonal antibodies and polyclonal equine antisera raised against different strains of EAV. Comparative sequence analyses of the entire structural protein genes (ORFs 2a, 2b, and 3-7) of these viruses, coupled with construction of chimeric viruses utilizing an infectious cDNA clone of EAV, confirmed that the alterations in neutralization phenotype were caused by amino acid changes in the CP5 protein encoded by ORF5. Site-directed mutagenesis studies unequivocally confirmed that amino acid 98 in the GP5 protein was responsible for the altered neutralization phenotype of these viruses. Amino acid 98 in the GP5 protein, which has not previously been identified as a neutralization determinant of EAV, should be included in an expanded neutralization site D (amino acids 98-106). (C) 2008 Elsevier B.V. All rights reserved.