Improvement of eradication program for infectious bovine rhinotracheitis in France inferred by serological monitoring of singleton reactors in certified BoHV1-free herds.

Within the framework of the national voluntary eradication program for Bovine alphaherpesvirus 1 (BoHV1) in France, the proportion of certified-free herds which experienced no more than two positive animals (termed singleton reactors) steadily increased to reach up to 95% in 2015. The aim of this study was to collate and evaluate serological data to gain insight into these epidemiological questionable BoHV1 seropositive animals. Preliminary evaluation of the performances of BoHV1 ELISA kits using a collection of 997 field sera with well-defined status revealed a relatively low specificity of the two gB blocking ELISAs most used in France for confirmatory testing (93.2% and 97.5% for gB-IDVet and gB-Idexx, respectively). In both ELISAs, the suboptimal specificity was associated with the presence of antibodies against BoHV2. Reassessment of the cut-offs led to a specificity and a sensitivity higher than 99.3%. Consequently, a comprehensive analysis of gB-positive sera from 2551 singleton reactors was performed by using gB ELISAs with optimized cut-offs, combined with viral neutralization test (campaign 2014-2015) or gE ELISA (campaign 2015-2016). Fifty percent of the 728 sera collected in 2014-2015 reacted below the optimized cut-offs in both gB ELISAs. Analysis of new blood samples collected at a minimum 6-week interval showed that these weak-positive reactions did not increase with time and could not be confirmed by confirmatory tests. Among the 1823 sera collected in 2015-2016, only 84 samples tested positive by gE ELISA, most of them corresponding to sera with reactivity above the optimized cut-offs in gB ELISAs. Screening for BoHV2 antibodies revealed a significantly increased prevalence among herds with singleton reactors, compared with the between-herd prevalence in French cattle herds. Altogether, these results provided suitable analytical strategies to limit the occurrence of false-positive BoHV1 reactions and inappropriate withdrawal of the BoHV1-free status, without alteration of diagnostic costs and reliability of eradication programs.

Diverse host-virus interactions following caprine arthritis-encephalitis virus infection in sheep and goats.

Interspecies transmissions substantially contribute to the epidemiology of small ruminant lentiviruses (SRLVs), including caprine arthritis encephalitis virus (CAEV) and visna-maëdi virus. However, comprehensive studies of host-virus interactions during SRLV adaptation to the new host are lacking. In this study, virological and serological features were analysed over a 6 month period in five sheep and three goats experimentally infected with a CAEV strain. Provirus load at the early stage of infection was significantly higher in sheep than in goats. A broad antibody reactivity against the matrix and capsid proteins was detected in goats, whereas the response to these antigens was mostly type-specific in sheep. The humoral response to the major immunodominant domain of the surface unit glycoprotein was type-specific, regardless of the host species. These species-specific immune responses were then confirmed in naturally infected sheep and goats using sera from mixed flocks in which interspecies transmissions were reported. Taken together, these results provide evidence that SRLV infections evolve in a host-dependent manner, with distinct host-virus interactions in sheep and goats, and highlight the need to consider both SRLV genotypes in diagnosis, particularly in sheep.

Genetic and antigenic characterization of small ruminant lentiviruses circulating in Poland.

Small ruminant lentivirus (SRLV) infections are widespread in Poland, but the genetic features of sheep viruses are still lacking and limited to partial gag sequences for goat viruses. In this study, segments from the gag and env genes of Polish SRLV strains screened by heteroduplex mobility assay were subjected to genetic analyses. Subtype A1 was found in both sheep and goats, while subtypes B1 and B2 were found in goats and sheep, respectively. In addition, two novel subtypes (named A12 and A13) were found in sheep. Their close phylogenetic relatedness with SRLV strains previously isolated from Polish goats indicated that these new subtypes are predominant and circulate in both species. The antigenic relationships of subtypes A12 and A13 with other SRLV subtypes were tested in an ELISA assay based on recombinant antigens carrying the immunodominant domains of structural proteins (MA, CA and SU). Antigenic cross-reactivity in the Gag epitopes was evident among genotype A subtypes and, to a lower extent, between genotypes A and B. In contrast, a subtype-specific immunoresponse was detected in the SU epitopes. These results emphasize the broad genetic and antigenic diversity of SRLV strains circulating in Europe and confirmed the need to consider all viral genotypes to choose the antigens in serological tests in order to avoid misdiagnosis in control and eradication programs.

Interference of vaccination against bluetongue virus serotypes 1 and 8 with serological diagnosis of small-ruminant lentivirus infection.

The effects of the recent vaccinations against bluetongue virus serotype 1 (BTV-1) and BTV-8 in Europe on the reliability of enzyme-linked immunosorbent assays (ELISAs) currently used for diagnosis of small-ruminant lentivirus (SRLV) infection were examined. Primary vaccination against BTV-8 in goats induced an increase in reactivity that did not exceed 3 months in a whole-virus indirect ELISA and a competitive ELISA based on the gp135 glycoprotein. Subsequent BTV-1/8 vaccination extended the time scale of false-positive reactivity for up to 6 months. These results are of relevance for SRLV-monitoring programs.

Field evaluation of a gag/env heteroduplex mobility assay for genetic subtyping of small-ruminant lentiviruses.

Small-ruminant lentiviruses (SRLVs) display a high genetic diversity and are currently classified into five genotypes and an increasing number of subtypes. The co-circulation of subtypes in restricted geographical regions, combined with the occurrence of cross-species infection, suggests the need for development of a large-scale screening methodology for rapid monitoring of the prevalence of the various genetic subtypes and their genetic evolution. Here, a heteroduplex mobility assay (HMA) was developed for the rapid identification of group B subtypes. The assay was validated for both the p14 nucleocapsid-coding region of the gag gene and the V1-V2 region of the env gene using a panel of reference standards and was applied to the genetic subtyping of SRLV field isolates from five mixed flocks in France. Subtyping of 75 blood samples using the env HMA revealed a preferential distribution of subtypes B1 and B2 in sheep and goats, despite direct evidence for interspecies transmission of both subtypes. Adding the gag HMA to the env HMA provided evidence for dual infection and putative recombination between subtypes B1 and B2 in five goats, and between groups A and B in one sheep. Phylogenetic analysis revealed that 100 % (23/23) and 96.7 % (30/31) of samples were correctly classified using the gag and env HMAs, respectively. These results indicate that dual infection and recombination may be a significant source of new variation in SRLV and provide a useful tool for the rapid genetic subtyping of SRLV isolates, which could be relevant for the development of more accurate diagnosis of prevalent SRLV strains in different countries.