Evaluation of Serological Methods and a New Real-Time Nested PCR for Small Ruminant Lentiviruses.

Small ruminant lentiviruses (SRLVs), i.e., CAEV and MVV, cause insidious infections with life-long persistence and a slowly progressive disease, impairing both animal welfare and productivity in affected herds. The complex diagnosis of SRLVs currently combines serological methods including whole-virus and peptide-based ELISAs and Immunoblot. To improve the current diagnostic protocol, we analyzed 290 sera of animals originating from different European countries in parallel with three commercial screening ELISAs, Immunoblot as a confirmatory assay and five SU5 peptide ELISAs for genotype differentiation. A newly developed nested real-time PCR was carried out for the detection and genotype differentiation of the virus. Using a heat-map display of the combined results, the drawbacks of the current techniques were graphically visualized and quantified. The immunoblot and the SU5-ELISAs exhibited either unsatisfactory sensitivity or insufficient reliability in the differentiation of the causative viral genotype, respectively. The new truth standard was the concordance of the results of two out of three screening ELISAs and the PCR results for serologically false negative samples along with genotype differentiation. Whole-virus antigen-based ELISA showed the highest sensitivity (92.2%) and specificity (98.9%) among the screening tests, whereas PCR exhibited a sensitivity of 75%.

Traces of history conserved over 600 years in the geographic distribution of genetic variants of an RNA virus: Bovine viral diarrhea virus in Switzerland.

The first records of smallpox and rabies date back thousands of years and foot-and-mouth disease in cattle was described in the 16th century. These diseases stood out by their distinct signs, dramatic way of transmission from rabid dogs to humans, and sudden appearance in cattle herds. By contrast, infectious diseases that show variable signs and affect few individuals were identified only much later. Bovine viral diarrhea (BVD), endemic in cattle worldwide, was first described in 1946, together with the eponymous RNA virus as its cause. There is general agreement that BVD was not newly emerging at that time, but its history remains unknown. A search for associations between the nucleotide sequences of over 7,000 BVD viral strains obtained during a national campaign to eradicate BVD and features common to the hosts of these strains enabled us to trace back in time the presence of BVD in the Swiss cattle population. We found that animals of the two major traditional cattle breeds, Fleckvieh and Swiss Brown, were infected with strains of only four different subgenotypes of BVDV-1. The history of these cattle breeds and the events that determined the current distribution of the two populations are well documented. Specifically, Fleckvieh originates from the Bernese and Swiss Brown from the central Alps. The spread to their current geographic distribution was determined by historic events during a major expansion of the Swiss Confederation during the 15th and 16th centuries. The association of the two cattle populations with different BVD viral subgenotypes may have been preserved by a lack of cattle imports, trade barriers within the country, and unique virus-host interactions. The congruent traces of history in the distribution of the two cattle breeds and distinct viral subgenotypes suggests that BVD may have been endemic in Switzerland for at least 600 years.

Correction to: Insemination with border disease virus-infected semen results in seroconversion in cows but not persistent infection in fetuses.

The original article [1] contained an error whereby a co-author, Sarah Züblin had their name displayed incorrectly. This error has now been corrected.

Antibody response to Influenza booster vaccination in Franches-Montagnes stallions supplemented with Equi-Strath® : a randomized trial.

Bio-Strath® is a plasmolyzed yeast product enriched with herbs, malt, honey and orange juice. In this study, the effect of Equi-Strath® , the adapted product for horses, on the equine immune system was evaluated. A routine influenza booster vaccination was used as a model to study the effects of Equi-Strath® supplementation on the immune response. Twenty healthy Franches-Montagnes stallions with pre-existing antibody levels were randomly divided into a study group (SG, n = 10) receiving 0.06 mL/kg bodyweight of Equi-Strath® , and a control group (CG, n = 10), receiving the same amount of placebo, daily. The supplement and placebo were given from week 1 until week 14 of the trial. After 10 weeks, the horses were vaccinated with a commercial vaccine containing equine influenza strains of the H3N8 subtype. Antibody titres in blood were measured at day 0 before vaccination, and 14 and 32 days after vaccination. In addition, a complete blood count (CBC) was performed on day 0 and day 32. A linear increase of haemagglutination inhibition titres in both groups was observed after vaccination, but with no difference between treatment groups. CBC components remained unaffected by treatment. In conclusion, daily Equi-Strath® supplementation did not affect the adaptive immune response in stallions after a routine commercial H3N8 influenza booster vaccination.

Insemination with border disease virus-infected semen results in seroconversion in cows but not persistent infection in fetuses.

BACKGROUND: This study examined various health variables in cows after artificial insemination with Border disease virus (BDV)-infected semen and the occurrence of persistent infection in ensuing fetuses. Five cows were inseminated (day 0) with BDV-infected semen as well as with semen from a fertile Eringer bull. One cow, inseminated with virus-free semen only, served as a control. Clinical examination, assessment of eating and rumination activities, measurement of intraruminal temperature and leukocyte count were used to monitor the health of the cows. Blood samples were collected at regular intervals for the detection of viral RNA and antibodies against BDV, and the cows were slaughtered on day 56. The uteri, placentae and fetuses were examined macroscopically, histologically, immunohistochemically and by means of molecular methods for the presence of pestiviruses. RESULTS: The demeanour, eating and rumination activities and intraruminal temperature were not affected by insemination with BDV-infected semen, whereas the total leukocyte and lymphocyte counts dropped transiently and were significantly lower on day 6 than on day 0. Seroconversion occurred by day 28 in the five infected cows but not in the control cow. The uteri, placentae and fetuses had no macroscopic or histological lesions, and immunohistochemical examination and RT-PCR were negative for pestiviruses. CONCLUSIONS: The findings showed that cows inseminated with BDV-infected semen seroconverted and fetuses thus produced were not persistently infected. Transmission of BDV to cattle through infected semen, therefore, seems to be of minor importance.

Molecular Epidemiology and Evolution of European Bat Lyssavirus 2.

Bat rabies cases in Europe are mainly attributed to two lyssaviruses, namely European Bat Lyssavirus 1 (EBLV-1) and European Bat Lyssavirus 2 (EBLV-2). Prior to the death of a bat worker in Finland in 1985, very few bat rabies cases were reported. Enhanced surveillance in the two subsequent years (1986-1987) identified 263 cases (more than a fifth of all reported cases to date). Between 1977 and 2016, 1183 cases of bat rabies were reported, with the vast majority (>97%) being attributed to EBLV-1. In contrast, there have been only 39 suspected cases of EBLV-2, of which 34 have been confirmed by virus typing and presently restricted to just two bat species; Myotis daubentonii and Myotis dasycneme. The limited number of EBLV-2 cases in Europe prompted the establishment of a network of European reference laboratories to collate all available viruses and data. Despite the relatively low number of EBLV-2 cases, a large amount of anomalous data has been published in the scientific literature, which we have here reviewed and clarified. In this review, 29 EBLV-2 full genome sequences have been analysed to further our understanding of the diversity and molecular evolution of EBLV-2 in Europe. Analysis of the 29 complete EBLV-2 genome sequences clearly corroborated geographical relationships with all EBLV-2 sequences clustering at the country level irrespective of the gene studied. Further geographical clustering was also observed at a local level. There are high levels of homogeneity within the EBLV-2 species with nucleotide identities ranging from 95.5-100% and amino acid identities between 98.7% and 100%, despite the widespread distribution of the isolates both geographically and chronologically. The mean substitution rate for EBLV-2 across the five concatenated genes was 1.65 × 10-5, and evolutionary clock analysis confirms the slow evolution of EBLV-2 both between and within countries in Europe. This is further supported by the first detailed EBLV-2 intra-roost genomic analysis whereby a relatively high sequence homogeneity was found across the genomes of three EBLV-2 isolates obtained several years apart (2007, 2008, and 2014) from M. daubentonii at the same site (Stokesay Castle, Shropshire, UK).

Safety, tolerability and efficacy of intradermal rabies immunization with DebioJect™.

In a single-center study, 66 healthy volunteers aged between 18 and 50years were randomized to be immunized against rabies with three different injection routes: intradermal with DebioJect™ (IDJ), standard intradermal with classical needle (IDS), also called Mantoux method, and intramuscular with classical needle (IM). "Vaccin rabique Pasteur®" and saline solution (NaCl 0.9%) were administered at D0, D7 and D28. Antigen doses for both intradermal routes were 1/5 of the dose for IM. Tolerability, safety and induced immunogenicity of IDJ were compared to IDS and IM routes. Pain was evaluated at needle insertion and at product injection for all vaccination visits. Solicited Adverse Event (SolAE) and local reactogenicity symptoms including pain, redness and pruritus were recorded daily following each vaccination visit. Adverse events (AE) were recorded over the whole duration of the study. Humoral immune response was measured by assessing the rabies virus neutralizing antibody (VNA) titers using Rapid Fluorescent Focus Inhibition Test (RFFIT). Results demonstrated that the DebioJect™ is a safe, reliable and efficient device. Significant decreases of pain at needle insertion and at vaccine injection were reported with IDJ compared to IDS and IM. All local reactogenicity symptoms (pain, redness and pruritus) after injection with either vaccine or saline solution, were similar for IDJ and IDS, except that IDJ injection induced more redness 30min after saline solution. No systemic SolAE was deemed related to DebioJect™ and classical needles. No AE was deemed related to DebioJect™. No Serious Adverse Event (SAE) was reported during the study. At the end of the study all participants were considered immunized against rabies and no significant difference in humoral response was observed between the 3 studied routes.

Detailed analysis of the promoter activity of an attenuated lentivirus.

In spite of an eradication campaign that eliminated clinical cases of caprine arthritis encephalitis virus-induced arthritis in the Swiss goat population, seroconversions are still observed. In the affected flocks, viruses belonging mainly to the small ruminant lentivirus A4 subtype are regularly isolated. These viruses are considered attenuated, except in the mammary gland, where high viral loads and histopathological lesions have been observed. We previously characterized and sequenced such field isolates, detecting several potentially attenuating mutations in their LTR. Here we present a detailed analysis of the promoter activity of these genetic elements, which was comparable to those of virulent isolates. An AP-1 binding site was shown to be crucial for promoter activity in reporter gene assays and also in the context of a replicating molecular clone. Other sites, such as AML(vis) and a conserved E-box, appeared to be less crucial. Analysis of a unique AP-4 site showed a clear discrepancy between results obtained with reporter gene assays and those with mutated viruses. Within the limits of this in vitro study, we did not find evidence pointing to the LTR as the genetic correlate of attenuation for these viruses. Finally, the limited replication of SRLV A4 in mammary cell culture could not explain the suggested mammary tropism. In contrast, and in view of the abundance of macrophages in the mammary gland, it is the striking replication capacity of SRLV A4 in these cells, unaffected by all LTR mutations tested, which may explain the apparent mammary tropism of these viruses.

A nationwide database linking information on the hosts with sequence data of their virus strains: A useful tool for the eradication of bovine viral diarrhea (BVD) in Switzerland.

Pestiviruses infect a wide variety of animals of the order Artiodactyla, with bovine viral diarrhea virus (BVDV) being an economically important pathogen of livestock globally. BVDV is maintained in the cattle population by infecting fetuses early in gestation and, thus, by generating persistently infected (PI) animals that efficiently transmit the virus throughout their lifetime. In 2008, Switzerland started a national control campaign with the aim to eradicate BVDV from all bovines in the country by searching for and eliminating every PI cattle. Different from previous eradication programs, all animals of the entire population were tested for virus within one year, followed by testing each newborn calf in the subsequent four years. Overall, 3,855,814 animals were tested from 2008 through 2011, 20,553 of which returned an initial BVDV-positive result. We were able to obtain samples from at least 36% of all initially positive tested animals. We sequenced the 5' untranslated region (UTR) of more than 7400 pestiviral strains and compiled the sequence data in a database together with an array of information on the PI animals, among others, the location of the farm in which they were born, their dams, and the locations where the animals had lived. To our knowledge, this is the largest database combining viral sequences with animal data of an endemic viral disease. Using unique identification tags, the different datasets within the database were connected to run diverse molecular epidemiological analyses. The large sets of animal and sequence data made it possible to run analyses in both directions, i.e., starting from a likely epidemiological link, or starting from related sequences. We present the results of three epidemiological investigations in detail and a compilation of 122 individual investigations that show the usefulness of such a database in a country-wide BVD eradication program.

Generation of a molecular clone of an attenuated lentivirus, a first step in understanding cytopathogenicity and virulence.

Small ruminant lentiviruses infect goats and sheep, inducing clinical disease in a minority of infected animals. Following an eradication campaign, clinical cases may disappear in a population. The complete elimination of these lentiviruses is however difficult to achieve and the spreading of less virulent strains often parallels the elimination of their virulent counterparts. Here, we characterized three such strains isolated from a flock in the post-eradication phase. We completely sequenced their genomes, showing that one of the isolates was most probably the product of a recombination event between the other two viruses. By comparing the sequences of these isolates with those of virulent strains, we found evidence that particular LTR mutations may explain their attenuated phenotype. Finally, we constructed an infectious molecular clone representative of these viruses, analyzing its replication characteristics in different target cells. This clone will permit us to explore the molecular correlates of cytopathogenicity and virulence.

Transmission of border disease virus from a persistently infected calf to seronegative heifers in early pregnancy.

BACKGROUND: This study describes the transmission of border disease virus (BDV) from a persistently infected calf to seronegative heifers in early pregnancy, resulting in persistently infected fetuses. On day 50 of pregnancy (= day 0 of the infection phase), six heifers were co-housed in a free stall with a bull calf persistently infected with BDV (pi BVD) for 60 days. The heifers underwent daily clinical examination, and blood samples were collected regularly for detection of pestiviral RNA and anti-pestivirus antibodies. After day 60 (= day 110 of pregnancy), the heifers were slaughtered, and the fetuses and placentae underwent post-mortem and immunohistochemical examination and RT-PCR for viral RNA detection. RESULTS: Three heifers had mild viraemia from day 8 to day 14, and by day 40 all heifers had pestivirus antibodies identified as anti-BDV antibodies in the serum neutralisation test. The placenta of the three viraemic heifers had histological evidence of inflammation, and fetal organs from these heifers were positive for pestivirus antigen by immunohistochemical examination and for BD viral RNA by RT-PCR and sequencing. Thus, co-housing of heifers in early pregnancy with a pi-BDV calf led to seroconversion in all heifers and persistent fetal infection in three. CONCLUSIONS: Considering that pi-BDV cattle can infect other cattle and lead to persistent infection of the fetus in pregnant cows, BDV should not be ignored in the context of the mandatory BVDV eradication and monitoring program. This strongly suggests that BDV should be taken into account in BVD eradication and control programs.

Characterization of small ruminant lentivirus A4 subtype isolates and assessment of their pathogenic potential in naturally infected goats.

BACKGROUND: Small ruminant lentiviruses escaping efficient serological detection are still circulating in Swiss goats in spite of a long eradication campaign that essentially eliminated clinical cases of caprine arthritis encephalitis in the country. This strongly suggests that the circulating viruses are avirulent for goats.To test this hypothesis, we isolated circulating viruses from naturally infected animals and tested the in vitro and in vivo characteristics of these field isolates. METHODS: Viruses were isolated from primary macrophage cultures. The presence of lentiviruses in the culture supernatants was monitored by reverse transcriptase assay. Isolates were passaged in different cells and their cytopathogenic effects monitored by microscopy. Proviral load was quantified by real-time PCR using customized primer and probes. Statistical analysis comprised Analysis of Variance and Bonferroni Multiple Comparison Test. RESULTS: The isolated viruses belonged to the small ruminant lentiviruses A4 subtype that appears to be prominent in Switzerland. The 4 isolates replicated very efficiently in macrophages, displaying heterogeneous phenotypes, with two isolates showing a pronounced cytopathogenicity for these cells. By contrast, all 4 isolates had a poor replication capacity in goat and sheep fibroblasts. The proviral loads in the peripheral blood and, in particular, in the mammary gland were surprisingly high compared to previous observations. Nevertheless, these viruses appear to be of low virulence for goats except for the mammary gland were histopathological changes were observed. CONCLUSIONS: Small ruminant lentiviruses continue to circulate in Switzerland despite a long and expensive caprine arthritis encephalitis virus eradication campaign. We isolated 4 of these lentiviruses and confirmed their phylogenetic association with the prominent A4 subtype. The pathological and histopathological analysis of the infected animals supported the hypothesis that these A4 viruses are of low pathogenicity for goats, with, however, a caveat about the potentially detrimental effects on the mammary gland. Moreover, the high proviral load detected indicates that the immune system of the animals cannot control the infection and this, combined with the phenotypic plasticity observed in vitro, strongly argues in favour of a continuous and precise monitoring of these SRLV to avoid the risk of jeopardizing a long eradication campaign.

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.

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.

[Bovine viral diarrhea (BVD): from biology to control]. / Bovine Virusdiarrhöe (BVD): von der Biologie zur Bekämpfung.

Bovine viral diarrhea virus (BVDV) is endemic worldwide. Together with classical swine fever and border disease viruses, it belongs to the genus Pestivirus of the family Flaviviridae. Most infections with BVDV take a transient, acute, course. Only rarely BVDV persists in its hosts. Due to the early time point of infection in utero, persistently infected (PI) animals are immunotolerant to the infecting non-cytopathic BVDV. In such animals the virus may mutate to a cytopathic biotype, causing lethal mucosal disease. In BVD-endemic regions, approximately 1% of the animals are PI. Removal of all PI animals leads to extinction of BVD. This approach to BVD eradication has been vindicated in Scandinavia. Following the same principles, regional and country-wide eradication programs are run in different parts of the world. These programs differ in the way PI animals are detected and in the role of vaccines. The Scandinavian two-step method of detecting PI animals is based on (i) the high level of seroprevalence in herds where PI animals are present and (ii) on testing all animals for virus in such herds. However, the high average herd seroprevalence in Switzerland made it impossible to define a reasonable threshold for virus testing. Therefore, all animals were directly tested for virus in the year 2008 and all newborn calves until the end of 2012, when the PI prevalence had dropped to 0.02%. Vaccination remains prohibited. Since 2013, surveillance for BVD is accomplished by serology. As a unique consequence of eradication, over 7500 viral strains are available to us for genetic studies.

Antigenic and genetic variations in European and North American equine influenza virus strains (H3N8) isolated from 2006 to 2007.

Equine influenza virus (EIV) surveillance is important in the management of equine influenza. It provides data on circulating and newly emerging strains for vaccine strain selection. To this end, antigenic characterisation by haemaggluttination inhibition (HI) assay and phylogenetic analysis was carried out on 28 EIV strains isolated in North America and Europe during 2006 and 2007. In the UK, 20 viruses were isolated from 28 nasopharyngeal swabs that tested positive by enzyme-linked immunosorbent assay. All except two of the UK viruses were characterised as members of the Florida sublineage with similarity to A/eq/Newmarket/5/03 (clade 2). One isolate, A/eq/Cheshire/1/06, was characterised as an American lineage strain similar to viruses isolated up to 10 years earlier. A second isolate, A/eq/Lincolnshire/1/07 was characterised as a member of the Florida sublineage (clade 1) with similarity to A/eq/Wisconsin/03. Furthermore, A/eq/Lincolnshire/1/06 was a member of the Florida sublineage (clade 2) by haemagglutinin (HA) gene sequence, but appeared to be a member of the Eurasian lineage by the non-structural gene (NS) sequence suggesting that reassortment had occurred. A/eq/Switzerland/P112/07 was characterised as a member of the Eurasian lineage, the first time since 2005 that isolation of a virus from this lineage has been reported. Seven viruses from North America were classified as members of the Florida sublineage (clade 1), similar to A/eq/Wisconsin/03. In conclusion, a variety of antigenically distinct EIVs continue to circulate worldwide. Florida sublineage clade 1 viruses appear to predominate in North America, clade 2 viruses in Europe.

A Gag peptide encompassing B- and T-cell epitopes of the caprine arthritis encephalitis virus functions as modular carrier peptide.

Short synthetic peptides are important tools in biomedical research permitting to generate hapten specific polyclonal sera for analytical purposes or functional studies. In this paper we provide proof of principle that a peptide located in a highly conserved portion of the Gag protein of the caprine arthritis encephalitis virus and carrying an immunodominant T helper cell epitope functions as an efficient carrier peptide, mediating a strong antibody response to a peptidic hapten encompassing a well-characterized B cell epitope of Env. The carrier and hapten peptides were collinearly synthesized permutating their molecular arrangement. While the antibody response to the hapten was similar for both constructs, the antibody response to a B cell epitope overlapping the T helper cell epitope of the Gag carrier peptide was considerably different. This permits a modular use of the carrier peptide to generate antibody directed exclusively to the hapten peptide or a strong humoral response to both carrier- and hapten-peptide. Finally, we have mapped the epitopes involved in this polarized antibody response and discussed the potential immunological implications.

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.

Genetic characterisation of attenuated SAD rabies virus strains used for oral vaccination of wildlife.

The elimination of rabies from the red fox (Vulpes vulpes) in Western Europe has been achieved by the oral rabies vaccination (ORV) of wildlife with a range of attenuated rabies virus strains. With the exception of the vaccinia rabies glycoprotein recombinant vaccine (VRG), all strains were originally derived from a common ancestor; the Street Alabama Dufferin (SAD) field strain. However, after more than 30 years of ORV it is still not possible to distinguish these vaccine strains and there is little information on the genetic basis for their attenuation. We therefore sequenced and compared the full-length genome of five commercially available SAD vaccine viruses (SAD B19, SAD P5/88, SAG2, SAD VA1 and SAD Bern) and four other SAD strains (the original SAD Bern, SAD VA1, ERA and SAD 1-3670 Wistar). Nucleotide sequencing allowed identifying each vaccine strain unambiguously. Phylogenetic analysis revealed that the majority of the currently used commercial attenuated rabies virus vaccines appear to be derived from SAD B19 rather than from SAD Bern. One commercially available vaccine virus did not contain the SAD strain mentioned in the product information of the producer. Two SAD vaccine strains appeared to consist of mixed genomic sequences. Furthermore, in-del events targeting A-rich sequences (in positive strand) within the 3' non-coding regions of M and G genes were observed in SAD-derivates developed in Europe. Our data also supports the idea of a possible recombination that had occurred during the derivation of the European branch of SAD viruses. If confirmed, this recombination event would be the first one reported among RABV vaccine strains.

Vaccination with a T-cell-priming Gag peptide of caprine arthritis encephalitis virus enhances virus replication transiently in vivo.

CD4+ T cells are involved in several immune response pathways used to control viral infections. In this study, a group of genetically defined goats was immunized with a synthetic peptide known to encompass an immunodominant helper T-cell epitope of caprine arthritis encephalitis virus (CAEV). Fifty-five days after challenge with the molecularly cloned CAEV strain CO, the vaccinated animals had a higher proviral load than the controls. The measurement of gamma interferon and interleukin-4 gene expression showed that these cytokines were reliable markers of an ongoing immune response but their balance did not account for more or less efficient control of CAEV replication. In contrast, granulocyte-macrophage colony-stimulating factor appeared to be a key cytokine that might support virus replication in the early phase of infection. The observation of a potential T-cell-mediated enhancement of virus replication supports other recent findings showing that lentivirus-specific T cells can be detrimental to the host, suggesting caution in designing vaccine candidates.