H9N2 avian influenza viruses are widely prevalent in birds and pose an increasing threat to humans because of their enhanced virulence and transmissibility in mammals. Active surveillance on the prevalence and evolution of H9N2 viruses in different avian hosts will help develop eradication measures. We isolated 16 H9N2 viruses from chickens, green peafowls, and wild birds in eastern China from 2017 to 2019 and characterized their comparative genetic evolution, receptor-binding specificity, antigenic diversity, replication, and transmission in chickens and mice. The phylogenetic analysis indicated that the green peafowl viruses and swan reassortant shared the same ancestor with the poultry H9N2 viruses prevalent in eastern China, while the seven wild bird viruses belonged to wild bird lineage. The chicken, peafowl, and swan H9N2 viruses that belonged to the poultry lineage preferentially recognized α-2, 6-linked sialic acids (human-like receptor), but the wild bird lineage viruses can bind both α-2, 3 (avian-like receptor) and human-like receptor similarly. Interestingly, the H9N2 viruses of poultry lineage replicated well and transmitted efficiently, but the viruses of wild bird lineage replicated and transmitted with low efficiency. Importantly, the H9N2 viruses of poultry lineage replicated in higher titer in mammal cells and mice than the viruses of wild birds lineage. Altogether, our study indicates that co-circulation of the H9N2 viruses in poultry, wild birds, and ornamental birds increased their cross-transmission risk in different birds because of their widespread dissemination.

Bovine parainfluenza virus 3 (BPIV3) is one of several viruses that contribute to bovine respiratory disease complex (BRDC). During this study, isolation of BPIV3 was attempted from 20 PCR-positive swabs by Madin-Darby Bovine Kidney (MDBK) cells. Nine samples showed obvious cytopathic lesions identified as BPIV3 by reverse-transcription polymerase chain reaction amplification and sequencing. The genomes of isolates XJ21032-1 and XJ20055-3 were sequenced using Illumina sequencing technology and determined to have lengths of 15,512 bp and 15,479 bp, respectively. Phylogenetic analysis revealed that isolate XJ21032-1 was genotype B, and isolate XJ20055-3 was genotype C. In addition, the two isolates had multiple amino acid changes in nucleocapsid protein, fusion protein, and hemagglutinin/neuraminidase, major antigenic proteins. This allows the further recognition of the presence of BPIV3 type B in Chinese cattle herds. We hope this will help trace the origin of BPIV3, improve the understanding of differences between genotypes, and provide data support for vaccine development.

Background: Classical swine fever (CSF), African swine fever (ASF), and atypical porcine pestivirus (APPV) are acute, virulent, and contagious viral diseases currently hampering the pig industry in China, which result in mummification or stillbirths in piglets and mortality in pigs. Diagnostic assays for the differentiation of infection and vaccination of CSFV, in addition to the detection of ASFV and APPV, are urgently required for better prevention, control, and elimination of these viral diseases in China. Methods: A quadruple PCR-based gene microarray assay was developed in this study to simultaneously detect wild-type and vaccine CSFV strains, ASFV and APPV according to their conserved regions. Forty-two laboratory-confirmed samples, including positive samples of 10 other swine viral diseases, were tested using this assay to confirm its high specificity. Results: This assay's limit of detections (LODs) for the wild-type and vaccine CSFV were 6.98 and 6.92 copies/µL. LODs for ASFV and APPV were 2.56 × 10 and 1.80 × 10 copies/µL, respectively. When compared with standard RT-PCR or qPCR for CSFV (GB/T 26875–2018), ASFV (MARR issue No.172), or APPV (CN108611442A) using 219 clinical samples, the coincidence was 100%. The results showed that this assay with high sensitivity could specifically distinguish ASFV, APPV, and CSFV, including CSFV infection and immunization. Conclusion: This assay provides a practical, simple, economic, and reliable test for the rapid detection and accurate diagnosis of the three viruses and may have good prospects for application in an epidemiological investigation, prevention, and control and elimination of these three diseases.

The continued diversification of the H9N2 avian influenza virus (AIV) into multiple antigenically and phylogenetically distinct lineages is promoting the emergence of strains with pandemic potential. Constant monitoring of the genetic evolution and changes in biological characteristics of the H9N2 viruses is therefore essential. In this study, we analyzed the genetic evolution of the H9N2 viruses isolated from poultry farms between 2022 and 2023 and evaluated their pathogenicity in chickens and mice. The HA genes of all ten isolates belonged to the h9.4.2.5 lineage, which is currently the predominant evolutionary lineage in China. Yet, their HA genes further divided into distinct subbranches within the h9.4.2.5 lineage. The NA genes of these viruses shared high homology with the prevalent H9N2 AIVs in recent years. However, these viruses were located in different evolutionary groups. Notably, the internal genes showed close relationships with those of recent H3, H6, and H9 subtype AIVs, suggesting active reassortment events among co-circulating viruses. Pathogenicity assessment in mice and chickens demonstrated divergent virulence between two representative isolates, FS22 and JM14, which clustered into different h9.4.2.5 subbranches. FS22 exhibited more efficient and prolonged replication in the lungs and turbinates of mice compared to JM14. Both viruses replicated efficiently in the lungs, kidneys, and trachea of chickens at 3 days post-infection (DPI), but differed in their horizontal transmission potential. Particularly, inoculated and contacted chickens all produced high antibody levels from the 5 DPI until the end of the experiment, and peak antibody titers for both viruses occurred at 7 DPI. These findings underscored the continuous evolution ofH9N2 AIV enhanced its genetic and phenotypic diversity, leading it to pose a threat to public health. Thus, continuous surveillance in poultry farms is necessary.

African horse sickness (AHS) is an arthropod-borne, severe equid disease caused by African horse sickness virus (AHSV). AHSV has high mortality and is endemic to sub-Saharan Africa. It has been classified into nine distinct serotypes (AHSV-1 to AHSV-9) based on VP2 immunogenicity. The AHS outbreak in Thailand in 2020, caused by AHSV-1, marked the first occurrence of this disease in Southeast Asia. It posed a substantial threat to the security of the equine industry in the nations across the region. To ensure the emergency reserve for AHS prevention and control, the AHSV strain imported to China from abroad over 60 years ago was characterized in this study. The strain was passaged in mice and then blind-passaged in Vero cells. The plaque purification method was then used to purify the strain and obtain its cell-adapted version, named AHSV/C. Neutralization tests confirmed that the virus belongs to AHSV-1. Whole-genome sequencing revealed that AHSV/C was highly homologous to AHSV-1 isolate 1180, with over 95% homology of major antigenic protein VP2, as compared to other AHSV-1 strains, including the prevalent strain in Thailand. In the mouse models, AHSV/C exhibited no clinical signs or histopathological lesions, suggesting low virulence and safety. This research for the first time characterized the in vitro growth characteristics and viral subtypes of the AHSV in China, determined its complete whole-genome sequence, and evaluated its safety using a mouse model. It provides crucial experimental materials and scientific foundations for the development of diagnostic methods and vaccines against AHSV-1.