Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR.

BACKGROUND: The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. METHODS: Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. RESULTS: All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. CONCLUSION: Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

N-terminal domain of Schmallenberg virus envelope protein Gc delivered by recombinant equine herpesvirus type 1 and modified vaccinia virus Ankara: Immunogenicity and protective efficacy in cattle.

Schmallenberg virus (SBV), which emerged in 2011 in Central Europe and subsequently spread very rapidly throughout the continent, affects predominantly ruminants. SBV is transmitted by insect vectors, and therefore vaccination is one of the major tools of disease control. Only recently, a domain connected to virus neutralization has been identified at the amino-terminal part of the viral envelope protein Gc. Here, this Gc domain delivered by recombinant EHV-1 or MVA vector viruses was tested in a vaccination-challenge trial in cattle, one of the major target species of SBV. The EHV-1-based vaccine conferred protection in two of four animals, whereas immunization using the MVA vector vaccine efficiently induced an SBV-specific antibody response and full protection against SBV challenge infection in all the vaccinated animals. Moreover, due to the absence of antibodies against SBVs N-protein, both vector vaccines enable the differentiation between vaccinated and field-infected animals making them to a promising tool to control SBV spread as well as to prevent disease in domestic ruminants.

Full genome characterization of porcine circovirus type 3 isolates reveals the existence of two distinct groups of virus strains.

BACKGROUND: The occurrence of the novel porcine circovirus type 3 (PCV3) was reported from the Americas, Asia and Europe. Although this virus was detected in association with various clinical syndromes in pigs, its role as possible swine pathogen remains unclear. PCV3 was detected with high prevalence in Polish farms, but to date no genome sequences were available from European PCV3 strains. METHODS: We collected 1060 serum samples from piglets at the age of 20-24 weeks from 53 farms distributed all over Germany. PCV3 DNA was detected using a real-time PCR and subsequently complete PCV3 genome sequences were obtained after multiply primed rolling circle amplification and sequencing of overlapping PCR products. Phylogenetic analysis was performed by neighbor-joining method and maximum likelihood method. RESULTS: We obtained 15 complete PCV3 genome sequences as well as nine partial sequences including the putative ORFs 1, 2 and 3 from PCV3 viremic animals in German pig farms. Phylogenetic analysis of these German as well as 30 full genome sequences received from GenBank divided the PCV3 strains into two main groups and several subclusters. Furthermore, we were able to define group specific amino acid patterns in open reading frame 1 and 2. CONCLUSION: PCV3 is distributed with high prevalence in German pig industry. Phylogenetic analysis revealed two clearly separated groups of PCV3 strains, which might be considered as PCV3 genotypes. Specific nucleotide and amino acid marker positions may serve for easy and fast intraspecies classification and genotyping of PCV3 strains. No correlation between PCV3 variants with their geographical origin was evident. We found the same diversity of PCV3 strains in Germany as in other countries. We hypothesize that PCV3 is not a newly emerging virus in the German pig population. Future studies will have to show, if PCV3 genotype specific biological properties are evident.

Sus scrofa papillomavirus 2 - genetic characterization of a novel suid papillomavirus from wild boar in Germany.

We identified a novel papillomavirus, Sus scrofa papillomavirus 2 (SsPV2), which is the first papillomavirus associated with papillomas in pigs. In skin alterations of a German wild boar, showing typical gross and histological appearance of papillomas, papillomavirus-like particles were demonstrated by electron microscopy. Degenerate papillomavirus-specific primers were used to amplify and sequence parts of the viral DNA. Subsequently, the complete genomic DNA was cloned and sequenced. The SsPV2 genome had a length of 8218 bp, encoded the early proteins E6, E7, E1 and E2, the late proteins L1 and L2 and contained an upstream regulatory region. Genomic characterization demonstrated papillomavirus-typical characteristics as well as unique features. For example, the E2 protein was significantly larger than in every other known papillomavirus species. Phylogenetic analysis was not able to relate SsPV2 unambiguously with other papillomavirus species or existing genera. Therefore, it might be representative of a new papillomavirus genus.