Specific detection of Alphaherpesvirus with a recombinant based gE indirect ELISA

INTRODUCTION Ruminant alphaerpesvirus related to Bovine Herpesvirus 1 (BoHV1), includes several host-adapted viruses found in bovine, bufaline, caprine, elk and cervids. Water buffalo is the primary host and reservoir of Bubaline Herpesvirus 1 (BuHV1), a virus more related to BoHV 5 but with an impact on IBR control programs difficult to establish. Both cattle and water buffalo are suscettible to heterologous infections. Moreover goat represents the natural host reservoir of Caprine herpesvirus 1 (CpHV1) even if infection can be experimentally induced in other ruminant species: cross-species infection may occur in countries where mixed cattle-goat population are reared in close contact. Serological diagnosis for the differentiation of these two infections from BoHV1 could be performed using serum cross-neutralization (SCN) test and ELISA assay (gB/gE blocking ELISAs). Aim of the study was the evaluation of the diagnostic potential of two recombinant gE based immunoassay to specifically detect and differentiate BuHV1 and CpHV1 infection from BoHV1. MATERIAL AND METHOD Glycoprotein E of CpHV1, BuHV1 and BoHV1 were obtained in secreted form using HEK293T cells transiently transfected with eukaryotic expression vector. The first indirect ELISA was optimized as a combined assay BoHV1-BuHV1. A panel of 91 bovine (BoHV1 infected cattle, marker vaccinated cattle, BoHV1 free animals and one bovine serum sperimentally infected with BuHV1) and 84 water buffalo (true negative gB-/gE-, doubt gB+/gE- and positive gB+/gE+ and two sera of sperimentally infected water buffalo, one with BoHV1 and the other with BuHV1) sera was used. For CpHV1 indirect ELISA it was used a panel of 53 bovine (BoHV1 infected cattle, marker vaccinated cattle and BoHV1 free animals) and 169 caprine sera (deriving from infected and negative herds by SN test) was used. RESULTS With BoHV1-BuHV1 indirect ELISA, all bovine sera showed reactivity against homologous antigen at least double compared to BuHV1 gE, while all buffalo sera were more reactive with similar strength ratio against BuHV1 gE, independently from the gE ELISA status. Same results were obtained for the three experimentally infected sera. In this case, SN was not able to differentiate the two infections. CpHV1 recombinant gE ELISA showed a greater reactivity versus homologous infection in goat sera: plotting the reactivity of positive samples versus BoHV1 and CpHV1 gE a clear recognition of homologous infection was evident in both animal species, according to SN test. Using SN as gold standard, sensitivity and specificity of gE indirect ELISA was 97.11% (CI95% 91.80-99.40%) and 98.57% (CI95% 92.30-99.96%). DISCUSSION The study allowed the development two new gE recombinant indirect ELISAs, able to identify CpHV1 infections and to differentiate BoHV1 from BuHV1 infection. Finally, a possible application of BoHV1 gE indirect ELISA as confirmatory test, truly independent from gE blocking ELISA could be a potential future application.