Hans-Rudolf Vogt

Analysis of the Antibody Response to an Immunodominant Epitope of the Envelope Glycoprotein of a Lentivirus and Its Diagnostic Potential

ABSTRACT The envelope glycoprotein of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response and contains several linear B-cell epitopes. We amplified and sequenced the genomic segment encoding the SU5 antigenic site of the envelope glycoprotein of several SRLV field isolates. With synthetic peptides based on the deduced amino acid sequences of SU5 in an enzyme-linked immunosorbent assay (ELISA), we have (i) proved the immunodominance of this region regardless of its high variability, (ii) defined the epitopes encompassed by SU5, (iii) illustrated the rapid and peculiar kinetics of seroconversion to this antigenic site, and (iv) shown the rapid and strong maturation of the avidity of the anti-SU5 antibody. Finally, we demonstrated the modular diagnostic potential of SU5 peptides. Under Swiss field conditions, the SU5 ELISA was shown to detect the majority of infected animals and, when applied in a molecular epidemiological context, to permit rapid phylogenetic classification of the infecting virus.

Bovine viral diarrhea virus in free-ranging wild ruminants in Switzerland: low prevalence of infection despite regular interactions with domestic livestock

BackgroundIn the frame of an eradication program for bovine viral diarrhea (BVD) in Swiss livestock, the question was raised whether free-ranging wildlife could threaten the success of this sanitary measure. Therefore, we conducted serological and virological investigations on BVD virus (BVDV) infections in the four indigenous wild ruminant species (roe deer, red deer, Alpine chamois and Alpine ibex) from 2009 to 2011, and gathered information on interactions between wild and domestic ruminants in an alpine environment by questionnaire survey.ResultsThirty-two sera out of 1’877 (1.7%, 95% confidence interval [CI] 1.2-2.4) were seropositive for BVDV, and a BVDV1 sub genotype h virus was found in a seropositive chamois (0.05%, 95% CI 0.001-0.3). The seropositive animals originated from sub-alpine or alpine regions and significantly more seropositive red deer, chamois and ibex than roe deer were found. There were no statistically significant differences between sampling units, age classes, genders, and sampling years. The obtained prevalences were significantly lower than those documented in livestock, and most positive wild ruminants were found in proximity of domestic outbreaks. Additionally, BVDV seroprevalence in ibex was significantly lower than previously reported from Switzerland. The survey on interspecific interactions revealed that interactions expected to allow BVDV transmission, from physical contacts to non-simultaneous use of the same areas, regularly occur on pastures among all investigated ruminant species. Interactions involving cervids were more often observed with cattle than with small ruminants, chamois were observed with all three domestic species, and ibex interacted mostly with small ruminants. Interactions related to the use of anthropogenic food sources were frequently observed, especially between red deer and cattle in wintertime.ConclusionsTo our knowledge, this is the first report of BVDV RNA isolated from an Alpine chamois. Nevertheless, our results suggest that BVDV infections are only sporadic in Swiss wild ruminants, despite regular occurrence of interactions with potentially infected livestock. Overall, serological, virological and ethological data indicate that wildlife is currently an incidental spill-over host and not a reservoir for BVDV in Switzerland.

Genetic analysis of small ruminant lentiviruses following lactogenic transmission

Lactogenic transmission plays an important role in the biology of lentiviruses such as HIV and SIV or the small ruminant lentiviruses (SRLV). In this work we analyzed the characteristics of viruses that goats, naturally infected with two strains of SRLV, transmitted to their kids. The spectrum of viral genotypes transmitted was broader and the efficiency of transmission greater compared to their human and simian counterparts. The newly described A10 subgroup of SRLV was more efficiently transmitted than the B1 genotype. The analysis of a particular stretch of the envelope glycoprotein encompassing a potential neutralizing epitope revealed that, as in SIV, the transmitted viruses were positively charged in this region, but, in contrast to SIV, they tended to lack a glycosylation site that might protect against antibody neutralization. We conclude that the physiology of the ruminant neonatal intestine, which permits the adsorption of infected maternal cells, shaped the evolution of these particular lentiviruses that represent a valid model of lactogenic lentivirus transmission.

Persistent infections after natural transmission of bovine viral diarrhoea virus from cattle to goats and among goats

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle worldwide. Infection of a pregnant animal may lead to persistent infection of the foetus and birth of a persistently infected (PI) calf that sheds the virus throughout its life. However, BVD viruses are not strictly species specific. BVDV has been isolated from many domesticated and wild ruminants. This is of practical importance as virus reservoirs in non-bovine hosts may hamper BVDV control in cattle. A goat given as a social companion to a BVDV PI calf gave birth to a PI goat kid. In order to test if goat to goat infections were possible, seronegative pregnant goats were exposed to the PI goat. In parallel, seronegative pregnant goats were kept together with the PI calf. Only the goat to goat transmission resulted in the birth of a next generation of BVDV PI kids whereas all goats kept together with the PI calf aborted. To our knowledge, this is the first report which shows that a PI goat cannot only transmit BVD virus to other goats but that such transmission may indeed lead to the birth of a second generation of PI goats. Genetic analyses indicated that establishment in the new host species may be associated with step-wise adaptations in the viral genome. Thus, goats have the potential to be a reservoir for BVDV. However, the PI goats showed growth retardation and anaemia and their survival under natural conditions remains questionable.

B-cell epitopes of the envelope glycoprotein of caprine arthritis-encephalitis virus and antibody response in infected goats.

Goats infected with caprine arthritis-encephalitis virus (CAEV) develop high titres of antibodies to Env. Not only is no consistent neutralizing response found but anti-Env antibodies have even been associated with disease in infected goats. To identify the continuous antigenic determinants involved in this atypical anti-Env response, we mapped CAEV-CO Env by screening an epitope expression library with infected goat sera. In addition to the four previously described epitopes, seven novel antigenic sites were identified, of which five were located on the surface (SU) and two in the transmembrane (TM) subunits of Env. The SU antibody-binding domains located in the variable regions of the C-terminal part of the molecule (SU3 to SU5) showed the strongest reactivity and induced a rapid seroconversion in six experimentally infected goats. However, the response to these immunodominant epitopes did not appear to be associated with any neutralizing activity. The pattern of serum reactivity of naturally infected goats with these epitopes was restricted, suggesting a type-specific reaction. Interestingly, the reactivity of peptides representing SU5 sequences derived from CAEV field isolates varied with the geographical and/or breeding origin of the animals. This suggests that peptides corresponding to the immunodominant SU epitopes may well be useful in the serotyping of CAEV isolates. Furthermore, the identification of the CAEV Env epitopes will permit us to functionally dissect the antibody response and to address the role of anti-Env antibodies either in the protection from or in the pathogenesis of CAEV infection.

Virological and phylogenetic characterization of attenuated small ruminant lentivirus isolates eluding efficient serological detection.

Three field isolates of small ruminant lentiviruses (SRLVs) were derived from a mixed flock of goats and sheep certified for many years as free of caprine arthritis encephalitis virus (CAEV). The phylogenetic analysis of pol sequences permitted to classify these isolates as A4 subtype. None of the animals showed clinical signs of SRLV infection, confirming previous observations which had suggested that this particular subtype is highly attenuated, at least for goats. A quantitative real time PCR strategy based on primers and probes derived from a highly variable env region permitted us to classify the animals as uninfected, singly or doubly infected. The performance of different serological tools based on this classification revealed their profound inadequacy in monitoring animals infected with this particular SRLV subtype. In vitro, the isolates showed differences in their cytopathicity and a tendency to replicate more efficiently in goat than sheep cells, especially in goat macrophages. By contrast, in vivo, these viruses reached significantly higher viral loads in sheep than in goats. Both env subtypes infected goats and sheep with equal efficiency. One of these, however, reached significantly higher viral loads in both species. In conclusion, we characterized three isolates of the SRLV subtype A4 that efficiently circulate in a mixed herd of goats and sheep in spite of their apparent attenuation and a strict physical separation between goats and sheep. The poor performance of the serological tools applied indicates that, to support an SRLV eradication campaign, it is imperative to develop novel, subtype specific tools.

Survey of bluetongue virus infection in free-ranging wild ruminants in Switzerland

BackgroundIn 2006, bluetongue virus serotype 8 (BTV-8) was detected for the first time in central Europe. Measures to control the infection in livestock were implemented in Switzerland but the question was raised whether free-ranging wildlife could be a maintenance host for BTV-8. Furthermore Toggenburg orbivirus (TOV), considered as a potential 25th BTV serotype, was detected in 2007 in domestic goats in Switzerland and wild ruminants were considered a potential source of infection. To assess prevalences of BTV-8 and TOV infections in wildlife, we conducted a serological and virological survey in red deer, roe deer, Alpine chamois and Alpine ibex between 2009 and 2011. Because samples originating from wildlife carcasses are often of poor quality, we also documented the influence of hemolysis on test results, and evaluated the usefulness of confirmatory tests.ResultsTen out of 1,898 animals (0.5%, 95% confidence interval 0.3-1.0%) had detectable antibodies against BTV-8 and BTV-8 RNA was found in two chamois and one roe deer (0.3%, 0.1-0.8%). Seroprevalence was highest among red deer, and the majority of positive wild animals were sampled close to areas where outbreaks had been reported in livestock. Most samples were hemolytic and the range of the optical density percentage values obtained in the screening test increased with increasing hemolysis. Confirmatory tests significantly increased specificity of the testing procedure and proved to be applicable even on poor quality samples. Nearly all samples confirmed as positive had an optical density percentage value greater than 50% in the ELISA screening.ConclusionsPrevalence of BTV-8 infection was low, and none of the tested animals were positive for TOV. Currently, wild ruminants are apparently not a reservoir for these viruses in Switzerland. However, we report for the first time BTV-8 RNA in Alpine chamois. This animal was found at high altitude and far from a domestic outbreak, which suggests that the virus could spread into/through the Alps. Regarding testing procedures, hemolysis did not significantly affect test results but confirmatory tests proved to be necessary to obtain reliable prevalence estimates. The cut-off value recommended by the manufacturer for the screening test was applicable for wildlife samples.

EPIZOOTIOLOGIC INVESTIGATIONS OF SELECTED ABORTIVE AGENTS IN FREE-RANGING ALPINE IBEX (CAPRA IBEX IBEX ) IN SWITZERLAND

In the early 2000s, several colonies of Alpine ibex (Capra ibex ibex) in Switzerland ceased growing or began to decrease. Reproductive problems due to infections with abortive agents might have negatively affected recruitment. We assessed the presence of selected agents of abortion in Alpine ibex by serologic, molecular, and culture techniques and evaluated whether infection with these agents might have affected population densities. Blood and fecal samples were collected from 651 ibex in 14 colonies throughout the Swiss Alps between 2006 and 2008. All samples were negative for Salmonella spp., Neospora caninum, and Bovine Herpesvirus-1. Antibodies to Coxiella burnetii, Leptospira spp., Chlamydophila abortus, Toxoplasma gondii, and Bovine Viral Diarrhea virus were detected in at least one ibex. Positive serologic results for Brucella spp. likely were false. Overall, 73 samples (11.2%) were antibody-positive for at least one abortive agent. Prevalence was highest for Leptospira spp. (7.9%, 95% CI=5.0–11.7). The low prevalences and the absence of significant differences between colonies with opposite population trends suggest these pathogens do not play a significant role in the population dynamics of Swiss ibex. Alpine ibex do not seem to be a reservoir for these abortive agents or an important source of infection for domestic livestock in Switzerland. Finally, although interactions on summer pastures occur frequently, spillover from infected livestock to free-ranging ibex apparently is uncommon.

A synthetic peptide encompassing the G5 antigenic region of the rabies virus induces high avidity but poorly neutralizing antibody in immunized animals.

The immunization of goats with a synthetic peptide encompassing the G5 antigenic site of the rabies virus surface glycoprotein induces a strong humoral immune response in the absence of a carrier protein. The immunized animals mounted high antibody titers and showed a strong avidity maturation of the B cell immune response to both the G5-peptide and purified surface glycoprotein G. This antibody weakly neutralized rabies virus carrying the G5 epitope but failed to neutralize escape mutants carrying a single point mutation in this epitope. A putative T helper cell epitope, functional in the context of different caprine MHC haplotypes, was identified by structure analysis of the G5-peptide. This striking dichotomy between high titers and antibody of high avidity to the glycoprotein G and poor neutralizing activity strongly suggests that antibody binding assays such as ELISA cannot always reliably predict the neutralizing activity of sera as measured in functional assays.

Expression of TNFα in arthritis caused by caprine arthritis encephalitis virus

Goats infected with caprine arthritis encephalitis virus (CAEV) develop chronic arthritis sharing many features with human rheumatoid arthritis (RA). TNF is thought to be a key mediator contributing to the formation of the arthritic lesion in RA. We studied this cytokine in goats suffering from chronic arthritis. TNF alpha expressing cells were detected by in situ hybridization in synovial membranes of arthritic carpal joints. Expression of TNF alpha did not correlate with the degree of viral replication as assessed by in situ hybridization for viral RNA. In line with the lack of correlation between the degree of viral replication and TNF expression in vivo, we failed to detect increased cytokine RNA in goat macrophages infected with CAE virus in vitro and no TNF protein was found in culture supernatants. In addition, virus infection failed to prime goat macrophages for enhanced TNF expression in response to lipopolysaccharide. Taken together, these findings argue against a direct role of CAE virus in increasing the expression of TNF alpha in caprine arthritis.