Inter-laboratory comparison of eleven quantitative or digital PCR assays for detection of proviral bovine leukemia virus in blood samples.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis and causes a persistent infection that can leave cattle with no symptoms. Many countries have been able to successfully eradicate BLV through improved detection and management methods. However, with the increasing novel molecular detection methods there have been few efforts to standardize these results at global scale. This study aimed to determine the interlaboratory accuracy and agreement of 11 molecular tests in detecting BLV. Each qPCR/ddPCR method varied by target gene, primer design, DNA input and chemistries. DNA samples were extracted from blood of BLV-seropositive cattle and lyophilized to grant a better preservation during shipping to all participants around the globe. Twenty nine out of 44 samples were correctly identified by the 11 labs and all methods exhibited a diagnostic sensitivity between 74 and 100%. Agreement amongst different assays was linked to BLV copy numbers present in samples and the characteristics of each assay (i.e., BLV target sequence). Finally, the mean correlation value for all assays was within the range of strong correlation. This study highlights the importance of continuous need for standardization and harmonization amongst assays and the different participants. The results underscore the need of an international calibrator to estimate the efficiency (standard curve) of the different assays and improve quantitation accuracy. Additionally, this will inform future participants about the variability associated with emerging chemistries, methods, and technologies used to study BLV. Altogether, by improving tests performance worldwide it will positively aid in the eradication efforts.

A retrospective evaluation of pooled serum ELISA testing in the frame of the French eradication program for infectious bovine rhinotracheitis.

Pooled serum testing using whole-virus indirect ELISA has been recently recognized as an official method for surveillance of bovine herpesvirus 1 (BoHV1) in cattle herds in Europe. In this study, a retrospective analysis of data from the French BoHV1 surveillance campaign 2018-2019, including 7434 BoHV1-free certified herds and 157 infected herds, was performed in order to evaluate the diagnostic specificity and sensitivity of two pooled serum indirect ELISAs (from IDEXX and IDVet), in comparison with individual testing by blocking ELISAs targeting the gB and gE proteins. Pooled serum testing showed a relative specificity higher than 97.5% and a detection rate of 100% since all gB+/gE+ samples were found in positive pools. At the herd level, no more than one false positive pool was observed in most of BoHV1-free certified herds, leading to a herd relative specificity of 85.1% and 86.0% for the IDEXX and IDVet pooled serum ELISAs, respectively. Among infected herds tested by pool sizes up to 10 sera (n = 122), 46% of herds were detected through pools of size 10 containing a single positive sample, 23% through pools of size 10 containing at least two positive samples, and 31% through pools of smaller sizes. A complementary study based on manually constituted pools revealed that at least one positive sample in 100% and 93.4% of herds could be detected individually by pools of size 10 with the IDEXX and IDVet ELISAs, respectively. However, pooled serum ELISAs were influenced by the level of individual reactivity, since pools composed of either one weak-positive sample or one gB+/gE- sample could yield negative results. Altogether, these results provided the first evidence that pooled serum testing (pool size up to 10) is a suitable strategy for surveillance of BoHV1-free cattle farms.

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.

Epidemiological survey in single-species flocks from Poland reveals expanded genetic and antigenic diversity of small ruminant lentiviruses.

Small ruminant lentivirus (SRLV) infections are widespread in Poland and circulation of subtypes A1, A12, A13, B1 and B2 was detected. The present work aimed at extending previous study based on the analysis of a larger number of animals from single-species flocks. Animals were selected for genetic analysis based on serological reactivity towards a range of recombinant antigens derived from Gag and Env viral proteins. Phylogenetic analysis revealed the existence of subtypes B2 and A12 in both goats and sheep and subtypes A1 and B1 in goats only. In addition, two novel subtypes, A16 and A17, were found in goats. Co-infections with strains belonging to different subtypes within A and B groups were detected in 1 sheep and 4 goats originating from four flocks. Although the reactivity of serum samples towards the recombinant antigens confirmed immunological relatedness between Gag epitopes of different subtypes and the cross-reactive nature of Gag antibodies, eleven serum samples failed to react with antigens representing all subtypes detected up-to-date in Poland, highlighting the limitations of the serological diagnosis. These data showed the complex nature of SRLV subtypes circulating in sheep and goats in Poland and the need for improving SRLV-related diagnostic capacity.

Schmallenberg virus-two years of experiences.

In autumn 2011, a novel species of the genus Orthobunyavirus of the Simbu serogroup was discovered close to the German/Dutch border and named Schmallenberg virus (SBV). Since then, SBV has caused a large epidemic in European livestock. Like other viruses of the Simbu serogroup, SBV is transmitted by insect vectors. Adult ruminants may show a mild transient disease, while an infection during a critical period of pregnancy can lead to severe congenital malformation, premature birth or stillbirth. The current knowledge about the virus, its diagnosis, the spread of the epidemic, the impact and the possibilities for preventing infections with SBV is described and discussed.

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.

Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein.

BACKGROUND: Lentiviral genomes encode multiple structural and regulatory proteins. Expression of the full complement of viral proteins is accomplished in part by alternative splicing of the genomic RNA. Caprine arthritis encephalitis virus (CAEV) and maedi-visna virus (MVV) are two highly related small-ruminant lentiviruses (SRLVs) that infect goats and sheep. Their genome seems to be less complex than those of primate lentiviruses since SRLVs encode only three auxiliary proteins, namely, Tat, Rev, and Vif, in addition to the products of gag, pol, and env genes common to all retroviruses. Here, we investigated the central part of the SRLV genome to identify new splice elements and their relevance in viral mRNA and protein expression. RESULTS: We demonstrated the existence of a new 5' splice (SD) site located within the central part of CAEV genome, 17 nucleotides downstream from the SD site used for the rev mRNA synthesis, and perfectly conserved among SRLV strains. This new SD site was found to be functional in both transfected and infected cells, leading to the production of a transcript containing an open reading frame generated by the splice junction with the 3' splice site used for the rev mRNA synthesis. This open reading frame encodes two major protein isoforms of 18- and 17-kDa, named Rtm, in which the N-terminal domain shared by the Env precursor and Rev proteins is fused to the entire cytoplasmic tail of the transmembrane glycoprotein. Immunoprecipitations using monospecific antibodies provided evidence for the expression of the Rtm isoforms in infected cells. The Rtm protein interacts specifically with the cytoplasmic domain of the transmembrane glycoprotein in vitro, and its expression impairs the fusion activity of the Env protein. CONCLUSION: The characterization of a novel CAEV protein, named Rtm, which is produced by an additional multiply-spliced mRNA, indicated that the splicing pattern of CAEV genome is more complex than previously reported, generating greater protein diversity. The high conservation of the SD site used for the rtm mRNA synthesis among CAEV and MVV strains strongly suggests that the Rtm protein plays a role in SRLV propagation in vivo, likely by competing with Env protein functions.

Distribution and heterogeneity of small ruminant lentivirus envelope subtypes in naturally infected French sheep.

Small ruminants lentiviruses (SRLV) nucleotide sequences spanning the V1V2 variable regions of the env gene were amplified by nested-PCR from 38 blood samples collected from 16 naturally infected sheep flocks in France. For the rapid SRLV group determination of field isolates, the PCR-amplified fragments were subjected to a SRLV-adapted heteroduplex mobility assay (HMA). All viral sequences were clearly assignable to the SRLV group B by HMA analysis. Twenty-seven SRLV isolates were selected for DNA sequence analysis. In each case, nucleotide comparison and phylogenetic analyses confirmed the genetic relationships inferred by HMA. Six SRLV isolates belonged to subtype B1, and 21 pertained to subtype B2, one flock being infected with both subtypes. Subtypes B1 and B2 were found with different frequencies and geographic spread, but exhibited similar genetic diversities. These results give a more complete picture of the distribution and heterogeneity of SRLV env subtypes in sheep and confirmed that multiple interspecies transmission occurred in the past. Furthermore, HMA appeared to be a rapid and reliable method to differentiate caprine arthritis encephalitis virus from maedi-visna virus.

Establishment and characterization of a goat synovial membrane cell line susceptible to small ruminant lentivirus infection.

Primary goat synovial membrane (GSM) cells are widely used to study small ruminant lentiviruses (SRLV), i.e. maedi visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV), but their limited life-span of 15-20 passages in vitro is problematic. Here, we report that ectopic expression of the catalytic subunit of human telomerase (hTERT) was sufficient to immortalize primary GSM cells. Cultures of hTERT-transfected GSM cells have been passaged for 2 years without showing any phenotypic difference from the original primary GSM cells. The hTERT-transfected cells continued to grow beyond a population doubling number of 250, while no net telomere lengthening was observed for these cells. Moreover, the immortalized GSM cells were susceptible to infection by both CAEV and MVV and were able to propagate theses viruses. Such cell line provides a useful source of standard and robust cells for both research and veterinary purposes.

Characterisation of an Irish caprine lentivirus strain--SRLV phylogeny revisited.

Small ruminant lentiviruses (SRLV), i.e. caprine arthritis-encephalitis virus (CAEV) (which infects goats) and maedi-visna virus (MVV) (which infects sheep) are two closely related lentiviruses but the relationship between goat and sheep lentiviruses has not been clearly established. To better understand their genetic relationship, we reinvestigated the phylogeny of SRLV using new sequences from an Irish and a Norwegian strain together with sequences available from databases. The phylogenetic analyses were carried out on the gag, pol and env fragments using four methods: neighbor-joining (NJ), Fitch and Margoliash (Fitch), Fitch and Wagner parsimony (Pars) and maximum likelihood (ML). The tree topologies were consistent whether derived from any of the four methods or any of the gene fragments, but the phylogenetic analyses in the pol and env regions were more informative than in the gag region. The Tamura-Nei model with variable rates across sites (described by a gamma distribution) provides a more accurate description of SRLV evolution than simple methods. The newly described Irish lentivirus strain, which was isolated from a goat, was closely related to the lentivirus that infects sheep: MVV. The novel Norwegian CAEV strain belonged to a cluster specific to the CAEV strains from Norway. Together, both data confirm the previously reported subdivision of the different SRLV strains into six clades. The caprine and ovine lentivirus sequences are interspersed in phylogenetic trees, supporting the existence of cross-species transmission. Nevertheless, the transmission of an ovine lentivirus to a goat could trigger the emergence of some goat-adapted phylums. Our new sequences confirm the complex situation in SRLV phylogeny but more sequences are needed to elucidate more precisely the relationship between SRLV.