Seroanalysis of Felis catus gammaherpesvirus 1 infection in domestic cats

Abstract

We recently described a novel herpesvirus of domestic cats, Felis catus gammaherpesvirus 1 (FcaGHV1). FcaGHV1 is a member of the gammaherpesvirus subfamily, which also includes the human cancer-associated herpesviruses, Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV). To determine FcaGHV1 antigens that elicit a detectable humoral-immune response in naturally-infected domestic cats, I chose to evaluate seven FcaGHV1 proteins. These proteins are conserved across the subfamily and antigenic in other gammaherpesvirus infections. I amplified and cloned each of the seven FcaGHV1 genes into a mammalian expression vector and transfected intact clones into Crandell Rees feline kidney (CRFK) cells. I developed an immunofluorescent antibody test using transfected cells exposed to sera from nine shelter cats diagnosed as FcaGHV1-positive by quantitative PCR (qPCR) of blood-cell DNA. This analysis indicated that tegument proteins ORF52 and ORF38 reacted most consistently with serum from cats with positive FcaGHV1-qPCR reactions. Based on these results, recombinant antigens were used to develop two optimized indirect ELISAs. Genes for ORF52 and ORF38 were cloned into a mammalian expression vector. Antigens were produced in a transient transfection system and purified using immunoprecipitation. Indirect ELISA conditions were optimized using known positive and negative controls. Using the two optimized ELISAs, I screened sera from 133 shelter cats that had been previously tested by FcaGHV1-qPCR. Seroprevalence of FcaGHV1 reactive antibodies was 32%, compared to the previously published 16% prevalence evaluated by qPCR. Nineteen of twenty qPCR positive cats were also seroreactive against one or both antigens on ELISA. Sera from 24 cats were seropositive based upon ELISA testing, but negative using qPCR analysis. Risk factors identified in previous publications were confirmed by ELISA, namely geographic location, male sex, adult age, and association of FcaGHV1 with several co-infections. Based on our knowledge of gammaherpesvirus latency, this ELISA provides evidence of viral exposure, while qPCR viral DNA detection likely represents reactivation from latency or primary infection. The addition of serologic analysis as a measurement of FcaGHV1 exposure will aid in determining association of FcaGHV1 with disease and routes of transmission.