Bungowannah Pestivirus Chimeras as Novel Double Marker Vaccine Strategy against Bovine Viral Diarrhea Virus.

Marker or DIVA (differentiation of infected from vaccinated animals) vaccines are beneficial tools for the eradication of animal diseases in regions with a high prevalence of the designated disease. Bovine viral diarrhea virus (BVDV)-1 (syn. Pestivirus A) is a flavivirus that infects predominantly cattle resulting in major economic losses. An increasing number of countries have implemented BVDV eradication programs that focus on the detection and removal of persistently infected cattle. No efficient marker or DIVA vaccine is yet commercially available to drive the eradication success, to prevent fetal infection and to allow serological monitoring of the BVDV status in vaccinated farms. Bungowannah virus (BuPV, species Pestivirus F), a related member of the genus Pestivirus with a restricted prevalence to a single pig farm complex in Australia, was chosen as the genetic backbone for a marker vaccine candidate. The glycoproteins E1 and E2 of BuPV were substituted by the heterologous E1 and E2, which are major immunogens, of the BVDV-1 strain CP7. In addition, the candidate vaccine was further attenuated by the introduction of a deletion within the Npro protein coding sequence, a major type I interferon inhibitor. Immunization of cattle with the chimeric vaccine virus BuPV_ΔNpro_E1E2 CP7 (modified live or inactivated) followed by a subsequent experimental challenge infection confirmed the safety of the prototype strain and provided a high level of clinical protection against BVDV-1. The serological discrimination of vaccinated cattle could be enabled by the combined detection of BVDV-1 E2- in the absence of both BVDV NS3- and BVDV Erns-specific antibodies. The study demonstrates for the first time the generation and application of an efficient BVDV-1 modified double marker vaccine candidate that is based on the genetic background of BuPV accompanied by commercially available serological marker ELISA systems.

High genetic variability of Schmallenberg virus M-segment leads to efficient immune escape from neutralizing antibodies.

Schmallenberg virus (SBV) is the cause of severe fetal malformations when immunologically naïve pregnant ruminants are infected. In those malformed fetuses, a "hot-spot"-region of high genetic variability within the N-terminal region of the viral envelope protein Gc has been observed previously, and this region co-localizes with a known key immunogenic domain. We studied a series of M-segments of those SBV variants from malformed fetuses with point mutations, insertions or large in-frame deletions of up to 612 nucleotides. Furthermore, a unique cell-culture isolate from a malformed fetus with large in-frame deletions within the M-segment was analyzed. Each Gc-protein with amino acid deletions within the "hot spot" of mutations failed to react with any neutralizing anti-SBV monoclonal antibodies or a domain specific antiserum. In addition, in vitro virus replication of the natural deletion variant could not be markedly reduced by neutralizing monoclonal antibodies or antisera from the field. The large-deletion variant of SBV that could be isolated in cell culture was highly attenuated with an impaired in vivo replication following the inoculation of sheep. In conclusion, the observed amino acid sequence mutations within the N-terminal main immunogenic domain of glycoprotein Gc result in an efficient immune evasion from neutralizing antibodies in the special environment of a developing fetus. These SBV-variants were never detected as circulating viruses, and therefore should be considered to be dead-end virus variants, which are not able to spread further. The observations described here may be transferred to other orthobunyaviruses, particularly those of the Simbu serogroup that have been shown to infect fetuses. Importantly, such mutant strains should not be included in attempts to trace the spatial-temporal evolution of orthobunyaviruses in molecular-epidemiolocal approaches during outbreak investigations.

A Synthetic Modified Live Chimeric Marker Vaccine against BVDV-1 and BVDV-2.

Bovine viral diarrhea virus (BVDV), a pestivirus which exists in the two distinct species BVDV-1 (syn. Pestivirus A) and BVDV-2 (syn. Pestivirus B), is the causative agent of one of the most widespread and economically important virus infections in cattle. For economic as well as for animal health reasons, an increasing number of national BVDV control programs were recently implemented. The main focus lies on the detection and removal of persistently infected cattle. The application of efficient marker or DIVA (differentiation of infected from vaccinated animals) vaccines would be beneficial for the eradication success in regions with a high BVDV prevalence to prevent fetal infection and it would allow serological monitoring of the BVDV status also in vaccinated farms. Therefore, a marker vaccine based on the cytopathic (cp) BVDV-1b strain CP7 was constructed as a synthetic backbone (BVDV-1b_synCP7). For serological discrimination of vaccinated from infected animals, the viral protein Erns was substituted by the heterologous Erns of Bungowannah virus (BuPV, species Pestivirus F). In addition, the vaccines were attenuated by a deletion within the type I interferon inhibitor Npro protein encoding sequence. The BVDV-2 vaccine candidate is based on the genetic sequence of the glycoproteins E1 and E2 of BVDV-2 strain CS8644 (CS), which were introduced into the backbone of BVDV-1b_synCP7_ΔNpro_Erns Bungo in substitution of the homologous glycoproteins. Vaccine virus recovery resulted in infectious cytopathic virus chimera that grew to titers of up to 106 TCID50/mL. Both synthetic chimera BVDV-1b_synCP7_ΔNpro_Erns Bungo and BVDV-1b_synCP7_ΔNpro_Erns Bungo_E1E2 BVDV-2 CS were avirulent in cattle, provided a high level of protection in immunization and challenge experiments against both BVDV species and allowed differentiation of infected from vaccinated cattle. Our study presents the first report on an efficient BVDV-1 and -2 modified live marker vaccine candidate and the accompanying commercially available serological marker ELISA system.

Single-Round Infectious Particle Production by DNA-Launched Infectious Clones of Bungowannah Pestivirus.

Reverse genetics systems are powerful tools for functional studies of viral genes or for vaccine development. Here, we established DNA-launched reverse genetics for the pestivirus Bungowannah virus (BuPV), where cDNA flanked by a hammerhead ribozyme sequence at the 5' end and the hepatitis delta ribozyme at the 3' end was placed under the control of the CMV RNA polymerase II promoter. Infectious recombinant BuPV could be rescued from pBuPV-DNA-transfected SK-6 cells and it had very similar growth characteristics to BuPV generated by conventional RNA-based reverse genetics and wild type BuPV. Subsequently, DNA-based ERNS deleted BuPV split genomes (pBuPV∆ERNS/ERNS)-co-expressing the ERNS protein from a separate synthetic CAG promoter-were constructed and characterized in vitro. Overall, DNA-launched BuPV genomes enable a rapid and cost-effective generation of recombinant BuPV and virus mutants, however, the protein expression efficiency of the DNA-launched systems after transfection is very low and needs further optimization in the future to allow the use e.g., as vaccine platform.

Autonomously Replicating RNAs of Bungowannah Pestivirus: ERNS Is Not Essential for the Generation of Infectious Particles.

Autonomously replicating subgenomic Bungowannah virus (BuPV) RNAs (BuPV replicons) with deletions of the genome regions encoding the structural proteins C, ERNS, E1, and E2 were constructed on the basis of an infectious cDNA clone of BuPV. Nanoluciferase (Nluc) insertion was used to compare the replication efficiencies of all constructs after electroporation of in vitro-transcribed RNA from the different clones. Deletion of C, E1, E2, or the complete structural protein genome region (C-ERNS-E1-E2) prevented the production of infectious progeny virus, whereas deletion of ERNS still allowed the generation of infectious particles. However, those ΔERNS viral particles were defective in virus assembly and/or egress and could not be further propagated for more than three additional passages in porcine SK-6 cells. These "defective-in-third-cycle" BuPV ΔERNS mutants were subsequently used to express the classical swine fever virus envelope protein E2, the N-terminal domain of the Schmallenberg virus Gc protein, and the receptor binding domain of the Middle East respiratory syndrome coronavirus spike protein. The constructs could be efficiently complemented and further passaged in SK-6 cells constitutively expressing the BuPV ERNS protein. Importantly, BuPVs are able to infect a wide variety of target cell lines, allowing expression in a very wide host spectrum. Therefore, we suggest that packaged BuPV ΔERNS replicon particles have potential as broad-spectrum viral vectors.IMPORTANCE The proteins NPRO and ERNS are unique for the genus Pestivirus, but only NPRO has been demonstrated to be nonessential for in vitro growth. While this was also speculated for ERNS, it has always been previously shown that pestivirus replicons with deletions of the structural proteins ERNS, E1, or E2 did not produce any infectious progeny virus in susceptible host cells. Here, we demonstrated for the first time that BuPV ERNS is dispensable for the generation of infectious virus particles but still important for efficient passaging. The ERNS-defective BuPV particles showed clearly limited growth in cell culture but were capable of several rounds of infection, expression of foreign genes, and highly efficient trans-complementation to rescue virus replicon particles (VRPs). The noncytopathic characteristics and the absence of preexisting immunity to BuPV in human populations and livestock also provide a significant benefit for a possible use, e.g., as a vector vaccine platform.

Seroprevalences of Newly Discovered Porcine Pestiviruses in German Pig Farms.

Several novel porcine pestiviruses that are linked to disease outbreaks in commercial pig farms were discovered during recent years. Bungowannah pestivirus (BuPV; new species Pestivirus F) causes sudden death in young pigs, but has only ever been isolated in the Australian region Bungowannah. Atypical porcine pestivirus (APPV; new species Pestivirus K) on the other hand has been found in multiple countries worldwide and is potentially linked to congenital tremor, a disease that causes considerable production problems in pig farms. To assess the seroprevalences of both viruses in German commercial farms during the years 2009/10 and 2018, two approaches were selected. Antibodies against Pestivirus F were detected by a traditional in-house indirect immunofluorescence test against the culture-grown virus isolate, while for the detection of Pestivirus K-specific antibodies, a newly developed test system utilizing a chimeric construct of bovine viral diarrhea virus 1 (BVDV-1; species Pestivirus A) containing the E1 and E2 encoding sequences of APPV was established. A total of 1115 samples originating from 122 farms located in seven German federal states were investigated. Antibodies against Bungowannah virus could not be detected, confirming the absence of this virus in other regions than the initially affected Australian pig farm complex. In contrast, antibodies against APPV were highly prevalent throughout Germany at both investigated time points. The seroprevalence at the state level fluctuated to some degree, but the overall percentage remained stable, as is to be expected for an endemic pestivirus lacking any form of control measures.

Bungowannah virus in the affected pig population: a retrospective genetic analysis.

Bungowannah virus, which belongs to the genus Pestivirus within the family Flaviviridae, has been associated with myocarditis and a high incidence of stillbirths in pigs. In 2003, the virus was initially detected in a large pig farming complex on two separate sites in New South Wales, Australia. Until now, it has not been detected at other locations. Despite a program of depopulation and disinfection, the virus could be only eradicated from one of the affected farm complexes, the Bungowannah unit, but became endemic on the second complex, the Corowa unit. In the present study, the genetic variability of virus isolates collected between 2003 and 2014 in the endemically infected population has been retrospectively investigated. Phylogenetic analysis carried out based on sequences of the E2 and NS5B coding regions and the full-length open-reading frame revealed that the isolates from the different farm sites are closely related, but that samples collected between 2010 and 2014 at the Corowa farm site clustered in a different branch of the phylogenetic tree. Since 2010, a high-genetic stability of this RNA virus within the Corowa farm complex, probably due to an effective adaptation of the virus to the affected pig population, could be observed.

Schmallenberg virus non-structural protein NSm: Intracellular distribution and role of non-hydrophobic domains.

Schmallenberg virus (SBV) induces fetal malformation, abortions and stillbirth in ruminants. While the non-structural protein NSs is a major virulence factor, the biological function of NSm, the second non-structural protein which consists of three hydrophobic transmembrane (I, III, V) and two non-hydrophobic regions (II, IV), is still unknown. Here, a series of NSm mutants displaying deletions of nearly the entire NSm or of the non-hydrophobic domains was generated and the intracellular distribution of NSm was assessed. SBV-NSm is dispensable for the generation of infectious virus and mutants lacking domains II - V showed growth properties similar to the wild-type virus. In addition, a comparable intracellular distribution of SBV-NSm was observed in mammalian cells infected with domain II mutants or wild-type virus. In both cases, NSm co-localized with the glycoprotein Gc in the Golgi compartment. However, domain IV-deletion mutants showed an altered distribution pattern and no co-localization of NSm and Gc.

A decade of research into classical swine fever marker vaccine CP7_E2alf (Suvaxyn® CSF Marker): a review of vaccine properties.

Due to its impact on animal health and pig industry, classical swine fever (CSF) is still one of the most important viral diseases of pigs. To control the disease, safe and highly efficacious live attenuated vaccines exist for decades. However, until recently, the available live vaccines did not allow a serological marker concept that is essentially important to circumvent long-term trade restrictions. In 2014, a new live attenuated marker vaccine, Suvaxyn® CSF Marker (Zoetis), was licensed by the European Medicines Agency. This vaccine is based on pestivirus chimera "CP7_E2alf" that carries the main immunogen of CSF virus "Alfort/187", glycoprotein E2, in a bovine viral diarrhea virus type 1 backbone ("CP7"). This review summarizes the available data on design, safety, efficacy, marker diagnostics, and its possible integration into control strategies.

The amino terminal subdomain of glycoprotein Gc of Schmallenberg virus: disulfide bonding and structural determinants of neutralization.

Orthobunyaviruses are enveloped viruses that can cause human and animal diseases. A novel and major member is the Schmallenberg virus (SBV), the etiological agent of an emerging disease of ruminants that has been spreading all over Europe since 2011. The glycoproteins Gn and Gc of orthobunyaviruses mediate the viral entry, and specifically Gc is a major target for the humoral immune response. For example, the N terminal subdomain of the SBV glycoprotein Gc is targeted by neutralizing monoclonal antibodies that recognize conformational epitopes. Here, we determined the structural features of the N terminus of Gc, and analysed its interaction with monoclonal antibodies. We were able to demonstrate that one of two N-glycosylation sites is essential for secretion and interaction with a subset of Gc-specific monoclonal antibodies. Furthermore, four disulfide bonds (S-S) were identified and the deletion of the third S-S blocked reactivity with another subset of mAbs with virus-neutralizing and non-neutralizing activity. The mutagenesis of the N-glycosylation sites and the disulfide bonds strongly indicated the independent folding of two subdomains within the SBV Gc N terminus. Further, the epitopes recognized by a panel of mAbs could be grouped into two clusters, as revealed by fine mapping using chimeric proteins. Combining the disulfide bonding and epitope mapping allowed us to generate a structural model of the SBV Gc N-terminus. This novel information about the role and structure of the amino terminal region of SBV Gc is of general relevance for the design of antivirals and vaccines against this virus.

Classical swine fever vaccines-State-of-the-art.

Due to its impact on animal health and pig industry, classical swine fever (CSF) is still one of the most important viral diseases of pigs. To control the disease, safe and highly efficacious live attenuated vaccines exist for decades. These vaccines have usually outstanding efficacy and safety but lack differentiability of infected from vaccinated animals (DIVA or marker strategy). In contrast, the first generation of E2 subunit marker vaccines shows constraints in efficacy, application, and production. To overcome these limitations, new generations of marker vaccines are developed. A wide range of approaches have been tried including recombinant vaccines, recombinant inactivated vaccines or subunit vaccines, vector vaccines, and DNA/RNA vaccines. During the last years, especially attenuated deletion vaccines or chimeric constructs have shown potential. At present, especially two new constructs have been intensively tested, the adenovirus-delivered, Semliki Forest virus replicon-vectored marker vaccine candidate "rAdV-SFV-E2" and the pestivirus chimera "CP7_E2alf". The later was recently licensed by the European Medicines Agency. Under field conditions, all marker vaccines have to be accompanied by a potent test system. Particularly this point shows still weaknesses and it is important to embed vaccination in a well-established vaccination strategy and a suitable diagnostic workflow. In summary, conventional vaccines are a standard in terms of efficacy. However, only vaccines with DIVA will allow improved eradication strategies e.g. also under emergency vaccination conditions in free regions. To answer this demand, new generations of marker vaccines have been developed and add now to the tool box of CSF control.

Chimeric Pestivirus Experimental Vaccines.

Chimeric pestiviruses have shown great potential as marker vaccine candidates against pestiviral infections. Exemplarily, we describe here the construction and testing of the most promising classical swine fever vaccine candidate "CP7_E2alf" in detail. The description is focused on classical cloning technologies in combination with reverse genetics.

A novel panel of monoclonal antibodies against Schmallenberg virus nucleoprotein and glycoprotein Gc allows specific orthobunyavirus detection and reveals antigenic differences.

A panel of monoclonal antibodies (mAbs) specific for the nucleocapsid (N) protein or the glycoprotein Gc of Schmallenberg virus (SBV), a novel member of the Simbu serogroup (genus Orthobunyavirus, family Bunyaviridae), was produced and used to analyze antigenic differences among members of this serogroup. Reactivity with various SBV-isolates and other Simbu serogroup viruses was assessed by an indirect immunofluorescence test and by immunoblotting. The Gc-specific mAbs detected different SBV isolates as well as two closely related members of the Simbu serogroup. In addition, one mAb showed a highly specific reactivity with the homologous SBV strain only. Based on their differing reactivity with different SBV-strains, these antibodies represent a valuable novel tool to rapidly determine the phenotype of new SBV isolates. In contrast, the N-specific mAbs showed a broad reactivity spectrum and detected not only all the tested SBV-isolates, but also several other viruses of the Simbu serogroup. One out of these mAbs even recognized all of the tested Simbu serogroup viruses in the indirect immunofluorescence assay. In order to further characterize the N-specific antibodies, PepScan analysis was performed and a specific epitope could be identified. In summary, the newly generated mAbs showed differing pan-Simbu virus-, pan-SBV- as well as SBV-isolate-specific reactivity patterns. Thus, they represent valuable tools for the development of novel antigen and antibody detection systems either specific for SBV or, in a broader approach, for the pan-Simbu serogroup diagnostics.

Deletion mutants of Schmallenberg virus are avirulent and protect from virus challenge.

UNLABELLED: 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.

The viral envelope is not sufficient to transfer the unique broad cell tropism of Bungowannah virus to a related pestivirus.

Bungowannah virus is the most divergent pestivirus, and both origin and reservoir host have not been identified so far. We therefore performed in vitro tropism studies, which showed that Bungowannah virus differs remarkably from other pestiviruses. Interestingly, cell lines of vervet monkey, mouse, human and even of bat origin were susceptible. This broad in vitro tropism was not observed for a chimeric bovine viral diarrhoea virus (BVDV) expressing all structural proteins of Bungowannah virus. The viral envelope was not sufficient to completely transfer the cell tropism of Bungowannah virus to another pestivirus, and viral RNA replication was either markedly reduced or not detectable in a number of different cell lines for the tested BVDV strain and the chimera. We therefore suggest that the replication machinery together with the viral envelope is responsible for the unique broad cell tropism of Bungowannah virus.

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.

Mixed triple: allied viruses in unique recent isolates of highly virulent type 2 bovine viral diarrhea virus detected by deep sequencing.

UNLABELLED: 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.

N pro of Bungowannah virus exhibits the same antagonistic function in the IFN induction pathway than that of other classical pestiviruses.

Bungowannah virus is the most divergent atypical pestivirus that had been detected up to now, and does not fit into any of the four approved species: Bovine viral diarrhea virus type 1 (BVDV-1) and type 2 (BVDV-2), Classical swine fever virus (CSFV) and Border disease virus (BDV). However, the presence of N(pro) and E(rns) coding regions, which are unique to pestiviruses, provides clear evidence of a pestivirus. Nevertheless, the amino acid identity of Bungowannah virus N(pro) and BVDV-1 N(pro) (strain CP7) is only 51.5%. By using a BVDV-1 backbone, a novel chimeric construct was generated, in which the genomic region encoding the non-structural protein N(pro) was replaced by that of Bungowannah virus (CP7_N(pro)-Bungo). In vitro studies of CP7_N(pro)-Bungo revealed autonomous replication with the same efficacy as the BVDV backbone CP7 and infectious high-titer virus could be collected. In order to compare the ability of interferon (IFN) suppression, two reporter gene assays, specific for type-I IFN, were carried out. In virus-infected cells, no significant difference in blocking of IFN expression between the parental virus CP7, Bungowannah virus and the chimeric construct CP7_N(pro)-Bungo could be detected. In contrast, an N(pro) deletion mutant showed an impaired replication in bovine cells and a marked type-I IFN response. Taken together, our findings reveal the compatibility of non-structural protein N(pro) of atypical Bungowannah virus with a BVDV type 1 backbone and its characteristic feature as an inhibitor of type-I IFN induction with an inhibitor-activity comparable to other pestiviruses.

Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA.

BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.

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.