>Bovine viral diarrhea virus(BVDV) is a positive-sense single-stranded RNA virus, belonging to the genus Pestivirus in the Flaviviridae family(Riitho et al. 2020). Currently, BVDV is divided into 3 main genotypes, BVDV-1,BVDV-2, and BVDV-3, based on the genetic differences in the 5′untranslated region(5′UTR)(Muasya et al. 2022). BVDV-1 is the dominant genotype circulating in China(Deng et al. 2020), while BVDV-2 is mainly identified in North America and occasionally in Europe and China(Liu et al. 2012). The BVDV-3 Hobby-like strain has recently been described in Brazil, but the global prevalence has not been investigated yet(Stalder et al. 2005). In addition, the BVDV exists as 2 biotypes: the non-cytopathogenic(NCP) and the cytopathogenic(CP) biotypes.

IntroductionNeonatal calf diarrhea (NCD) is one of the most common diseases in calves, causing huge economic and productivity losses to the bovine industry worldwide. The main pathogens include bovine rotavirus (BRV), bovine coronavirus (BCoV), and Enterotoxigenic Escherichia coli (ETEC) K99. Since multiple infectious agents can be involved in calf diarrhea, detecting each causative agent by traditional methods is laborious and expensive.MethodsIn this study, we developed a one-step multiplex Real-Time PCR assay to simultaneously detect BRV, BCoV, and E. coli K99+. The assay performance on field samples was evaluated on 1100 rectal swabs of diseased cattle with diarrhea symptoms and compared with the conventional gel-based RT-PCR assay detect BRV, BCoV, and E. coli K99+.ResultsThe established assay could specifically detect the target pathogens without cross-reactivity with other pathogens. A single real-time PCR can detect ~1 copy/µL for each pathogen, and multiplex real-time PCR has a detection limit of 10 copies/µL. Reproducibility as measured by standard deviation and coefficient of variation were desirable. The triple real-time PCR method established in this study was compared with gel-based PT-PCR. Both methods are reasonably consistent, while the real-time PCR assay was more sensitive and could rapidly distinguish these three pathogens in one tube. Analysis of surveillance data showed that BRV and BCoV are major enteric viral pathogens accounting for calves’ diarrhea in China. DiscussionThe established assay has excellent specificity and sensitivity and was suitable for clinical application. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and calf diarrhea research. ​

H10 subtype avian influenza viruses (AIVs) have been isolated from wild and domestic avian species worldwide and have occasionally crossed the species barrier to mammalian hosts. Fatal human cases of H10N8 infections and the recent detection of human H10N3 infections have drawn widespread public attention. In this study, 25 H10Nx viruses were isolated from wild waterfowl in China during a long-term surveillance of AIVs. We conducted phylogenetic and phylogeographic studies of the hemagglutinin (HA) genes of global H10 viruses to determine the spatiotemporal patterns of spread and the roles of different hosts in viral transmission. We found the pattern of AIV transmission from wild birds to poultry to humans, and Anatidae have acted as the seeding population in the spread of the virus. Phylogenetic incongruence indicated complex reassortment events and our isolates were divided into eight genotypes (G1-8). We also found that the HA genes of the G8 viruses belonged to the North American lineage, indicating that intercontinental gene flow has occurred. Their receptor-binding specificity showed that the G1/4/5/6/7/8 viruses bind to both human-type α2,6-linked sialic acid receptors and avian-type α2,3-linked sialic acid receptors. Mouse studies indicated that the H10Nx isolates replicated efficiently in the respiratory system without preadaptation, but showed low pathogenicity in mice. The H10Nx isolates showed no (G2/4/7) or low pathogenicity (G1/3/5/6/8) in chickens, and the G6 and G8 viruses could be transmitted to chickens through direct contact. The asymptomatic shedding of these wild-bird-origin H10Nx isolates in chickens and their good adaptation in mice should increase the ease of their transmission to humans, and they therefore pose a threat to public health. Our findings demonstrate a further understanding of wild bird-origin H10 viruses and provide information for the continuous surveillance of H10 subtype viruses.

H5N8 and H5N1 highly pathogenic avian influenza viruses (AIVs) of clade 2.3.4.4b were isolated from dead migratory birds and fecal samples collected in Tibet, China, in May 2021. Phylogenetic analyses showed that the viruses isolated in this study may have spread from wintering or stopover grounds of migratory birds in South Asia. We monitored two disparate clade 2.3.4.4b H5Nx viruses in migratory birds in Tibet during their breeding season. The data revealed that breeding grounds may exhibit a potential pooling effect among avian influenza viruses in different migratory populations. IMPORTANCE In this study, 15 H5N8 and two H5N1 highly pathogenic avian influenza viruses of clade 2.3.4.4b were isolated from dead migratory birds and fecal samples in Tibet, China. Isolates of H5N1 virus of clade 2.3.4.4b have been rarely reported in China. Our findings highlight that breeding grounds may exhibit a potential pooling effect among avian influenza viruses (AIVs) in different migratory populations. In addition to intensification of the surveillance of AIVs in migratory birds in Tibet, China, international cooperation should be strengthened.

Highly pathogenic influenza A(H5N8) viruses have caused several worldwide outbreaks in birds and are able cross the species barrier to infect humans, posing a substantial threat to public health. After the first detection of H5N8 viruses in deceased swans in Inner Mongolia, we performed early warning and active monitoring along swan migration routes in central China. We isolated and sequenced 42 avian influenza viruses, including 40 H5N8 viruses, 1 H5N2 virus, and 1 H9N2 virus, in central China. Our H5N8 viruses isolated in swan stopover sites and wintering grounds showed high nucleotide homologies in the whole genome, revealing a common evolutionary source. Phylogenetic analysis revealed that the H5 viruses of clade 2.3.4.4b prevalent in 2020 have further diverged into two sub-clades: b1 and b2. The phylogeographic analysis also showed that the viruses of sub-clade b2 most likely originated from poultry in Russia. Notably, whooper swans were found to be responsible for the introduction of sub-clade b2 viruses in central China; whooper and tundra swans play a role in viral spread in the Yellow River Basin and the Yangtze River Basin, respectively. Our findings highlight swans as an indicator species for transborder spreading and monitoring of the H5N8 virus.

During October 2020, we identified 13 highly pathogenic avian influenza A(H5N8) clade 2.3.4.4b viruses from wild ducks in Ningxia, China. These viruses were genetically related to H5N8 viruses circulating mainly in poultry in Europe during early 2020. We also determined movements of H5N8 virus‒infected wild ducks and evidence for spreading of viruses.