The high sequence variation of RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes in molecular diagnostic assays. We showed previously that PCR amplification in two rounds, first with long target-specific primers and then with short generic primers, followed by detection using long probes, can tolerate sequence variation. Here we demonstrate that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for the detection of norovirus genogroup II with improved sensitivity. Probe design (method of incorporating quenchers, use of Zen internal quencher or traditional quenchers) greatly affects the sensitivity of the real-time PCR assays.

ABSTRACTClassical swine fever (CSF) is a highly contagious viral disease of pigs that has a tremendous socioeconomic impact. Vaccines are available for disease control. However, most industrialized countries are implementing stamping-out strategies to eliminate the disease and avoid trade restrictions. These restrictions can be avoided through the use of marker vaccines such as CP7_E2alf. Marker vaccines have to be accompanied by reliable and robust discriminatory assays. In this context, a multiplex microsphere immunoassay for serological differentiation of infected from vaccinated animals (DIVA) was developed to distinguish CSF virus (CSFV)-infected animals from CP7_E2alf-vaccinated animals. To this end, three viral proteins, namely, CSFV E2, CSFV Erns, and bovine viral diarrhea virus (BVDV) E2, were produced in insect cells using a baculovirus expression system; they were used as antigens in a microsphere immunoassay, which was further evaluated by testing a large panel of pig sera and compared to a well-characterized commercial CSFV E2 antibody enzyme-linked immunosorbent assays (ELISAs) and a test version of an improved CSFV Ernsantibody ELISA. Under a cutoff median fluorescence intensity value of 5,522, the multiplex microsphere immunoassay had a sensitivity of 98.5% and a specificity of 98.9% for the detection of antibodies against CSFV E2. The microsphere immunoassay and the CSFV ErnsELISA gave the same results for 155 out of 187 samples (82.8%) for the presence of CSFV Ernsantibodies. This novel multiplex immunoassay is a valuable tool for measuring and differentiating immune responses to vaccination and/or infection in animals.

In this study, we show a significantly reduced assay time and a greatly increased bead recovery for a commercial Luminex-based multiplex diagnostic immunoassay by performing all liquid handling steps of the assay protocol in a non-contact acoustic trapping platform. The Luminex assay is designed for detecting antibodies in poultry serum for infectious bursal disease virus, infectious bronchitis virus, Newcastle disease virus and avian reovirus. Here, we show proof-of-concept of a microfluidic system capable of being fully automated and handling samples in a parallel format with a miniature physical footprint where the affinity beads are retained in a non-contact levitated mode in a glass capillary throughout the assay protocol. The different steps are: incubation with the serum sample, secondary antibodies and fluorescent reporters and finally washing to remove any non-specifically bound species. A Luminex 200 instrument was used for the readout. The flow rates applied to the capillary during the initial trapping event and the wash steps were optimised for maximum bead recovery, resulting in a bead recovery of 75% for the complete assay. This can be compared to a bead recovery of approximately 30% when an automatic wash station was used when the assay was performed in the conventional manual format. The time for the incubation steps for a single assay was reduced by more than 50%, without affecting assay performance, since intermediate wash steps became redundant in the continuously perfused bead trapping capillary. We analyzed seven samples, in triplicates, and we can show that the readout of the assay performed in the acoustic trap compared 100% to the control ELISAs (positive or negative readout) and resulted in comparable S/P values as the conventional manual protocol. As the acoustic trapping does not require the particles to have magnetic properties, a greater degree of freedom in selecting microparticles can be provided. In extension, this can provide an opportunity to develop cheaper and more effective microparticles.

Atypical bovine pestiviruses are related antigenically and phylogenetically to bovine viral diarrhea viruses (BVDV-1 and BVDV-2), and may cause the same clinical manifestations in animals. Glycoprotein E(rns) of an atypical bovine pestivirus Th/04_KhonKaen was produced in a baculovirus expression system and was purified by affinity chromatography. The recombinant E(rns) protein was used as an antigen in a microsphere immunoassay for the detection of antibodies against BVDV-1 and atypical bovine pestivirus. The diagnostic performance of the new method was evaluated by testing a total of 596 serum samples, and the assay was compared with enzyme-linked immunosorbent assay (ELISA). Based on the negative/positive cut-off median fluorescence intensity (MFI) value of 2800, the microsphere immunoassay had a sensitivity of 100% and specificity of 100% compared to ELISA. The immunoassay was able to detect antibodies against both BVDV-1 and the atypical pestivirus. This novel microsphere immunoassay has the potential to be multiplexed for simultaneous detection of antibodies against different bovine pathogens in a high-throughput and economical way.

To the Editor: The article by Decaro et al. (1) described an outbreak of severe respiratory disease in calves in Italy caused by an atypical bovine pestivirus. This report confirms our concern that this group of viruses is probably widespread (2) and present on >3 continents. Moreover, it demonstrates that atypical bovine pestiviruses are capable of causing disease in calves in the field and a clinical picture consistent with most bovine viral diarrhea virus (BVDV) infections that occur naturally or experimentally (3,4). Some key issues remain unknown. The origin of the bovine pestivirus and the route of introduction into the herd are unclear, although phylogenies demonstrated a close relationship between this virus strain from Italy and atypical bovine pestiviruses from Brazil. Batches of fetal bovine serum from Brazil have repeatedly been found to be contaminated with atypical bovine pestiviruses (2,3), and there is a risk for contamination of vaccines with these viruses. Animal trade and vaccines should be considered when conducting further investigations into this outbreak. The evolutionary relationship between the atypical and the recognized pestivirus species (1) needs to be clarified. Maximum-likelihood and Bayesian analyses of a concatenated dataset positioned atypical pestiviruses consistently in a clade sister to BVDV-1 and BVDV-2, and the larger clade was sister to pestivirus of giraffes (5). The same pattern was observed in complete genome phylogeny (1). The term atypical is not informative, and a new name has been proposed for these bovine pestiviruses (5). Outcomes of infections with these viruses are typical, but the viral antigens and phylogenies are unique.

The Chinese hog cholera lapinized virus (HCLV), also called the “Chinese strain” or “C-strain” of classical swine fever virus (CSFV), was developed in China in the 1950s. There are uncertainties about the genetic heterogeneity and origin of this vaccine virus. The objectives of this study were to investigate the genetic heterogeneity of the C-strain, for which nucleotide sequences have been submitted to GenBank from different laboratories, and to determine whether there is any evidence to support the hypothesis that the C-strain originated from the Shimen strain. Analysis of 150 nearly complete E2 gene sequences revealed that the C-strain clade includes several HCLV vaccine strains, cell-culture-adapted Riems strains, and viruses isolated from diseased pigs. The whole-genome phylogeny indicated that the ancestor of the C-strain was only distantly related to the Shimen strain. Therefore, there was no phylogenetic evidence to support the Shimen-origin hypothesis.

Classical swine fever (CSF) is a highly contagious disease, causing severe economic losses in the pig industry worldwide. Vaccination of pigs with lapinized Chinese vaccines is still practised in some regions of the world, where the virus is enzootic, in order to prevent and control the disease. However, a single real-time assay that can detect all lapinized Chinese vaccines used widely, namely, Lapinized Philippines Coronel (LPC), Hog Cholera Lapinized virus (HCLV) and the Riems C-strain is still lacking. This study describes a real-time RT-PCR assay, targeting the N(pro) gene region, for specific detection of these lapinized vaccine strains. The assay is highly sensitive, with a detection limit of 10 genome copies per reaction for HCLV and Riems C-strain and highly specific, as more than 100 strains of wild type CSFV representing all major genotypes were not detected. The assay is also highly repeatable: the coefficient of variation of Ct values in three runs was 2.77% for the detection of 10 copies of the vaccine viral RNA. This study provides a potentially useful tool for specific detection of the lapinized Chinese vaccines, HCLV and C-strain, and the differentiation of these vaccines from wild type CSFV.

The recently emerging atypical bovine pestiviruses have been detected in commercial foetal bovine serum (FBS) of mainly South American origin so far. It is unclear how widely the viruses are presented in commercial FBS of different geographic origins. To further investigate the possible pestivirus contamination of commercially available FBS batches, 33 batches of FBS were obtained from ten suppliers and analysed in this study for the presence of both the recognised and the atypical bovine pestiviruses. All 33 batches of FBS were positive by real-time RT-PCR assays for at least one species of bovine pestiviruses. According to the certificate of analysis that the suppliers claimed for each batch of FBS, BVDV-1 was detected in all 11 countries and BVDV-2 was detected exclusively in the America Continent. The atypical pestiviruses were detected in 13 batches claimed to originate from five countries. Analysis of partial 5'UTR sequences showed a high similarity among these atypical bovine pestiviruses. This study has demonstrated, for the first time that commercial FBS batches of different geographic origins are contaminated not only with the recognised species BVDV-1 and BVDV-2, but also with the emerging atypical bovine pestiviruses.

To investigate whether there is any phylogenetic evidence to support the hypothesis that swine is the natural host of HEV genotypes 3 and 4, Bayesian analysis of 80 full-length genomic sequences of HEV was performed. The results showed that the strains of genotypes 3 and 4 from swine are paraphyletic with regard to strains of human origin, which are thus phylogenetically nested among the swine strains. Recognition of HEV genotypes 3 and 4 as viruses from swine or swine HEV can provide an evolutionary explanation to the observation of cross-species infection by genotypes 3 and 4 HEV.

Classical swine fever (CSF) is a contagious and devastating disease, causing serious losses in the pig industry worldwide. Vaccination of pigs with the conventional C-strain vaccine has been practised in different regions of the world in order to prevent the disease. In the control programmes of CSF, rapid detection and identification of the causing agent, classical swine fever virus (CSFV) is a crucial step. This study describes a novel real-time PCR assay based on primer-probe energy transfer (PriProET) technology for improved detection of CSFV. The assay is able to detect 20 copies of viral cDNA per reaction, showing a high sensitivity. The specificity has been evaluated by testing 57 pestiviruses, representing all species and unclassified pestiviruses. The assay has been found to be highly reproducible. Following PCR amplification, melting curve analysis allows confirmation of specific amplicons, and differentiation between wild-type CSFV and certain C-strain vaccines. This study provides a new tool for the diagnosis of CSF.

This study describes a novel blocking microsphere-based immunoassay for highly sensitive and specific detection of antibodies against bovine viral diarrhoea virus (BVDV). The intra- and inter-assay variability are 4.9% and less than 7%, respectively, and variability of bead conjugations is less than 6.6%. The diagnostic performance of the assay was evaluated by testing a total of 509 serum samples. Based on a negative/positive cut-off value of 30.3%, the assay has a sensitivity of 99.4% and a specificity of 98.3% relative to ELISA. The new microsphere immunoassay provides an alternative to conventional ELISA systems and can be used for high-throughput screening in the BVD control programmes.

Phylogenetic analysis of pestiviruses is a useful tool for classifying novel pestiviruses and for revealing their phylogenetic relationships. In this study, robustness of pestivirus phylogenies has been compared by analyses of the 5′UTR, and complete Npro and E2 gene regions separately and combined, performed by four methods: neighbour-joining (NJ), maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI). The strategy of analysing the combined sequence dataset by BI, ML, and MP methods resulted in a single, well-supported tree topology, indicating a reliable and robust pestivirus phylogeny. By contrast, the single-gene analysis strategy resulted in 12 trees of different topologies, revealing different relationships among pestiviruses. These results indicate that the strategies and methodologies are two vital aspects affecting the robustness of the pestivirus phylogeny. The strategy and methodologies outlined in this paper may have a broader application in inferring phylogeny of other RNA viruses.

Bovine viral diarrhoea virus 1 (BVDV-1) and Bovine viral diarrhoea virus 2 (BVDV-2) are two recognised bovine pestivirus species of the genus Pestivirus. Recently, a pestivirus, termed SVA/cont-08, was detected in a batch of contaminated foetal calf serum originating from South America. Comparative sequence analysis showed that the SVA/cont-08 virus shares 15–28% higher sequence identity to pestivirus D32/00_‘HoBi’ than to members of BVDV-1 and BVDV-2. In order to reveal the phylogenetic relationship of SVA/cont-08 with other pestiviruses, a molecular dataset of 30 pestiviruses and 1,896 characters, comprising the 5′UTR, Npro and E2 gene regions, was analysed by two methods: maximum likelihood and Bayesian approach. An identical, well-supported tree topology was observed, where four pestiviruses (SVA/cont-08, D32/00_‘HoBi’, CH-KaHo/cont, and Th/04_KhonKaen) formed a monophyletic clade that is closely related to the BVDV-1 and BVDV-2 clades. The strategy applied in this study is useful for classifying novel pestiviruses in the future.

The genus Pestivirus comprises four established species: Bovine viral diarrhoea viruses 1 (BVDV-1) and 2 (BVDV-2), Border disease virus (BDV), and Classical swine fever virus (CSFV); and a tentative species, Pestivirus of giraffe. Additional pestiviruses have been identified and suggested for recognition as novel subgroups/species. To achieve a reliable phylogeny as the basis for classification of pestiviruses, a molecular dataset of 56 pestiviruses and 2089 characters, comprising the 5'UTR, complete N(pro) and E2 gene regions was analysed by Maximum likelihood and Bayesian approach. An identical, robust tree topology was inferred, where seven well-supported monophyletic clades and two highly divergent lineages were identified. Dating most recent common ancestor was estimated for major pestivirus lineages and their evolutionary histories were revealed. Accordingly, a new proposal is presented for the classification of pestiviruses into nine species: BVDV-1, BVDV-2, BVDV-3 (atypical bovine pestiviruses), Pestivirus of giraffe, CSFV, BDV, Tunisian sheep virus (TSV; previously termed "Tunisian isolates"), Antelope and Bungowannah.