Ilona Reimann

Pathogenic, antigenic and molecular properties of rabbit haemorrhagic disease virus (RHDV) isolated from vaccinated rabbits: detection and characterization of antigenic variants

SummaryRabbit haemorrhagic disease virus (RHDV) isolates were obtained from several animals previously vaccinated with an inactivated vaccine. Seven isolates were analyzed by immunological and molecular biological methods and compared to reference strains. Antigenic characterization with monoclonal antibodies as well as haemagglutination assays demonstrated considerable differences between individual isolates. However, sequencing of the capsid protein genes revealed a high degree of homology between five of these isolates and the reference strain FRG. In contrast, two isolates specified remarkably different capsid proteins with a degree of variation not observed so far in RHDV. Amino acid alterations were found clustered between residues 301 and 328 (region C), 344 and 434 (region E) and also in the 3′ region of the capsid protein gene. Interestingly, experimental vaccination of rabbits followed by challenge with the heterologous variant strains showed restricted cross-protection against one of the strains. In summary, we found a level of antigenic variation not detected in RHDV so far, and describe two distinct new antigenic variants.

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.

New real-time reverse transcriptase polymerase chain reactions facilitate detection and differentiation of novel A/H1N1 influenza virus in porcine and human samples.

Influenza A viruses are maintained as a quasispecies cloud in several natural host reservoirs of avian as well as mammalian species. Accidental host exposure, selection and further adaptation of individual influenza A viruses during sporadic trans-species transmission may eventually lead to the establishment of new, stably circulating lineages in a new, possibly mammalian, host species. Given a high transmissibility of such a virus and a susceptible, immunologically naive population, pandemic spread of such viruses within a short time may ensue. In April 2009, a novel multi-reassortant influenza A virus of subtype H1N1 has emerged and regionally spread in humans in Mexico and the United States causing flu-like symptoms. Until June 2009 increasing levels ofa multiregional, global spread of this virus prompted the WHO to raise the pandemic alert to the highest level. Data from experimental infections in pigs as well as experience from natural outbreaks in swine farms world-wide have shown that porcine populations are fully susceptible to the new virus and are able to sustain uninterrupted transmission chains. A broad front incursion of the new human pandemic virus into the porcine population would have a significant negative impact on measures to restrict further spread of the virus in the human population.Therefore, sensitive tools for monitoring and detection of such an incursion in a timely manner are mandatory. We have developed two real-time RT PCRs which are specific for the hemagglutinin gene of the novel A/H1N1 virus and which allow detection of infected pigs with high sensitivity.These PCRs may become useful tools in future surveillance programmes.

Generation of recombinant pestiviruses using a full-genome amplification strategy

Complete genome amplification of viral RNA provides a new tool for the generation of modified viruses. We have recently reported a full-genome amplification strategy for recovery of pestiviruses (Rasmussen et al., 2008). A full-length cDNA amplicon corresponding to the Border disease virus-Gifhorn genome was generated by long RT-PCR and then RNA transcripts derived from this amplicon were used to rescue infectious virus. Here, we have now used this full-genome amplification strategy for efficient and robust amplification of three additional pestivirus strains: the vaccine strain C and the virulent Paderborn strain of Classical swine fever virus plus the CP7 strain of Bovine viral diarrhoea virus. The amplicons were cloned directly into a stable single-copy bacterial artificial chromosome generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived.

Modified live marker vaccine candidate CP7_E2alf provides early onset of protection against lethal challenge infection with classical swine fever virus after both intramuscular and oral immunization.

Due to the vast economic consequences of classical swine fever (CSF) outbreaks, emergency vaccination plans are under discussion in European Union Member States. However, animals vaccinated with the conventional C-strain vaccine are subject to trade restrictions. To ease these restrictions, potent marker vaccines are required. One promising candidate is the chimeric pestivirus CP7_E2alf. For emergency vaccination in a CSF outbreak scenario, early onset of immunity is required. Here, the studies performed with a CP7_E2alf virus stock produced under good manufacturing conditions (GMP) are reported. In challenge experiments, CP7_E2alf induced full clinical protection 1 week after intramuscular vaccination, and 2 weeks after oral immunization. Furthermore, even after application of diluted vaccine preparations complete protection could be achieved if challenge infection was carried out 4 weeks after vaccination. In conclusion, GMP-produced CP7_E2alf proved to be a suitable marker vaccine candidate - also for emergency vaccination - both after intramuscular and oral application.

Trans-complementation of autonomously replicating Bovine viral diarrhea virus replicons with deletions in the E2 coding region

Autonomously replicating Bovine viral diarrhea virus (BVDV) genomes (replicons) were constructed from the full-length BVDV cDNA clone pA/BVDV/Ins- (G. Meyers et al., J. Virol. 70, 8606-8613, 1996). The sequences coding for envelope protein E2, for E2 without the C-terminal transmembrane region, or for E2 and nonstructural protein p7 were deleted, and the resulting mutants were tested for their ability to replicate after transfection. All deletion mutants were able to replicate and to express the inserted green fluorescent protein but did not produce infectious progeny virus in bovine kidney PT cells. The replicons were also tested for their ability to be trans-complemented in the bovine cell line PT_805, which constitutively expresses BVDV structural proteins. E2-negative BVDV mutants were complemented and >10(6) infectious units were obtained at 24 h after transfection. Complementing PT_805 cells could only inefficiently be infected using trans-complemented virions, however, and low levels of virus production were observed when complemented BVDV was passaged using PT_805 cells. Similarly, infection of PT_805 cells with BVDV was highly inefficient, but transfection of full-length BVDV NCP7 RNA into PT_805 resulted in 10,000-fold higher virus titers when compared to those obtained 24 h after transfection of parental PT cells. We concluded that self-replicating E2-deleted BVDV RNAs can be efficiently trans-complemented by constitutively expressed E2, and that expression of BVDV structural proteins markedly influences susceptibility of cells to BVDV infection as well as BVDV titers after transfection of full-length BVDV RNA.

Mixed Triple: Allied Viruses in Unique Recent Isolates of Highly Virulent Type 2 Bovine Viral Diarrhea Virus Detected by Deep Sequencing

ABSTRACT In February 2013, very severe acute clinical symptoms were observed in calves, heifers, and dairy cattle in several farms in North Rhine Westphalia and Lower Saxony, Germany. Deep sequencing revealed the coexistence of three distinct genome variants within recent highly virulent bovine viral diarrhea virus type 2 (BVDV-2) isolates. While the major portion (ca. 95%) of the population harbored a duplication of a 222-nucleotide (nt) segment within the p7-NS2-encoding region, the minority reflected the standard structure of a BVDV-2 genome. Additionally, unusual mutations were found in both variants, within the highly conserved p7 protein and close to the p7-NS2 cleavage site. Using a reverse genetic system with a BVDV-2a strain harboring a similar duplication, it could be demonstrated that during replication, genomes without duplication are generated de novo from genomes with duplication. The major variant with duplication is compulsorily escorted by the minor variant without duplication. RNA secondary structure prediction allowed the analysis of the unique but stable mixture of three BVDV variants and also provided the explanation for their generation. Finally, our results suggest that the variant with duplication plays the major role in the highly virulent phenotype. IMPORTANCE This study emphasizes the importance of full-genome deep sequencing in combination with manual in-depth data analysis for the investigation of viruses in basic research and diagnostics. Here we investigated recent highly virulent bovine viral diarrhea virus isolates from a 2013 series of outbreaks. We discovered a unique special feature of the viral genome, an unstable duplication of 222 nucleotides which is eventually deleted by viral polymerase activity, leading to an unexpectedly mixed population of viral genomes for all investigated isolates. Our study is of high importance to the field because we demonstrate that these insertion/deletion events allow another level of genome plasticity of plus-strand RNA viruses, in addition to the well-known polymerase-induced single nucleotide variations which are generally considered the main basis for viral adaptation and evolution.

Deletion Mutants of Schmallenberg Virus Are Avirulent and Protect from Virus Challenge

ABSTRACT Since its emergence, Schmallenberg virus (SBV), a novel insect-transmitted orthobunyavirus which predominantly infects ruminants, has caused a large epidemic in European livestock. Newly developed inactivated vaccines are available, but highly efficacious and safe live vaccines are still not available. Here, the properties of novel recombinant SBV mutants lacking the nonstructural protein NSs (rSBVΔNSs) or NSm (rSBVΔNSm) or both of these proteins (rSBVΔNSs/ΔNSm) were tested in vitro and in vivo in type I interferon receptor knockout mice (IFNAR−/−) and in a vaccination/challenge trial in cattle. As for other bunyaviruses, both nonstructural proteins of SBV are not essential for viral growth in vitro. In interferon-defective BHK-21 cells, rSBVΔNSs and rSBVΔNSm replicated to levels comparable to that of the parental rSBV; the double mutant virus, however, showed a mild growth defect, resulting in lower final virus titers. Additionally, both mutants with an NSs deletion induced high levels of interferon and showed a marked growth defect in interferon-competent sheep SFT-R cells. Nevertheless, in IFNAR−/− mice, all mutants were virulent, with the highest mortality rate for rSBVΔNSs and a reduced virulence for the NSm-deleted virus. In cattle, SBV lacking NSm caused viremia and seroconversion comparable to those caused by the wild-type virus, while the NSs and the combined NSs/NSm deletion mutant induced no detectable virus replication or clinical disease after immunization. Furthermore, three out of four cattle immunized once with the NSs deletion mutant and all animals vaccinated with the virus lacking both nonstructural proteins were fully protected against a challenge infection. Therefore, the double deletion mutant will provide the basis for further developments of safe and efficacious modified live SBV vaccines which could be also a model for other viruses of the Simbu serogroup and related orthobunyaviruses. IMPORTANCE SBV induces only mild clinical signs in adult ruminants but causes severe fetal malformation and, thereby, can have an important impact on animal welfare and production. As SBV is an insect-transmitted pathogen, vaccination will be one of the most important aspects of disease control. Here, mutant viruses lacking one or two proteins that essentially contribute to viral pathogenicity were tested as modified live vaccines in cattle. It could be demonstrated that a novel recombinant double deletion mutant is a safe and efficacious vaccine candidate. This is the first description of a putative modified live vaccine for the complete genus Orthobunyavirus, and in addition, such a vaccine type has never been tested in cattle for any virus of the entire family Bunyaviridae. Therefore, the described vaccine also represents the first model for a broad range of related viruses and is of high importance to the field.

Direct recovery of infectious pestivirus from a full-length RT-PCR amplicon.

This study describes the use of a novel and rapid long reverse transcription (RT)-PCR for the generation of infectious full-length cDNA of pestiviruses. To produce rescued viruses, full-length RT-PCR amplicons of 12.3 kb, including a T7-promotor, were transcribed directly in vitro, and the resulting RNA transcripts were electroporated into ovine cells. Infectious virus was obtained after one cell culture passage. The rescued viruses had a phenotype similar to the parental Border Disease virus strain. Therefore, direct generation of infectious pestiviruses from full-length RT-PCR cDNA products could be a valuable instrument for virus rescue, conservation and further characterization.

Innocuousness and safety of classical swine fever marker vaccine candidate CP7_E2alf in non-target and target species

Chimeric pestivirus CP7_E2alf is a promising live marker vaccine candidate against classical swine fever. Prior to a possible application in the field, several safety aspects have to be addressed. Due to the fact that CP7_E2alf is based on a bovine viral diarrhea virus backbone, its behavior in ruminants is of particular interest. In the framework of this study, its innocuousness in non-target species was addressed by inoculation of calves, young goats, lambs, and rabbits. To this means, high titres of CP7_E2alf were applied orally to three animals of each species. Additional animals were left as unvaccinated contact controls. During the study, all animals remained clinically healthy, and neither fever nor leukopenia were observed. Virus could not be isolated from purified white blood cells or from nasal or faecal excretions. Moreover, none of the animals (inoculated or contact control) seroconverted. In the target species, innocuousness, shedding and transmission of vaccine virus was addressed in different animal trials that were carried out primarily for the purpose of efficacy, potency or duration of immunity studies. In all experiments, CP7_E2alf proved to be completely safe for the vaccinees and unvaccinated contact controls. Furthermore, no shedding or transmission was detected in any of the experiments. Even after parental vaccination, vaccine virus genome was barely detectable in blood or organ samples of vaccinated animals. Thus, CP7_E2alf can be regarded as completely safe for both target and non-target species.