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H10 subtype avian influenza viruses were endemic in wild and domestic avian species worldwide. Strikingly, it frequently crossed the species barrier to infect mammalian hosts. Human infection with H10N3 and H10N8 were reported previously. Recently, a 63-year-old woman from Anhui province of China who died from a mixed infection of H3N2 and H10N5 influenza viruses, which have drawn widespread public health attention. Here, we perform the evolutionary dynamics of H10N5 influenza viruses of bird- and human-origin worldwide, and found that wild bird-origin H10N5 influenza viruses from China did not cluster together with human-origin H10N5 influenza viruses, while grouped together with LPAIV gene pools circulating in wild birds that derived from other Eurasian countries. Human-derived H10N5 virus is a novel reassortant, which frequently reassorted with wild bird-derived influenza viruses, and in turn, spillover into humans. Collectively, our results suggested that H10 subtype influenza viruses continuously pose threat to public health.

【Objective】A disease model of fowl adenovirus serotype 2 (FAdV-2) was established to provide a refference for evaluating the immunogenicity and vaccine effectiveness of FAdV-2.【Method】The samples suspected to be infected with avian adenovirus were isolated and identified. The genetic evolution of the Fiber and Hexon gene sequences of the isolates was analyzed. The nucleotide and amino acid sequences of the isolated strain and other FAdV representative strains were analyzed using DNAStar and MEGA7.0. After determining that the isolated virus was FAdV-2, the pathogenicity of the isolated strain was analyzed in 6-week-old SPF chickens under different infection routes and different infection doses in animal experiment. The best infection model and infection dose were selected to establish the FAdV-2 pathogenesis model.【Result】One FAdV-2 strain was successfully isolated from the infected chicken tissue samples, named KM strain. Genetic evolution analysis shows that KM strain and GX01 strain belong to the FAdV-2 of avian adenovirus species D of group Ⅰ. The homology analysis shows that the nucleotide similarity of Fiber gene and Hexon gene between KM strain and GX01 strain is 98.9% and 99.3%, and the amino acid similarity is 98.9% and 98.8%, respectively. The pathogenicity of intravenous injection was higher than that of intramuscular injection, and the chickens showed obvious hepatitis hydropericardium syndrome (HHS). The infection route of the KM strain model was intravenous injection, and the infection dose was 108.0TCID50. The chickens died after infection, with a mortality rate of 50%. Obvious pathological changes were found in the dead chickens at necropsy, and the chickens were positive for cloaca virus shedding at 1 d post infection. The established KM strain disease model provides a certain reference for vaccine development and vaccine evaluation, which will effectively prevent and reduce the transmission of FAdV-2.【Conclusion】It is verified that FAdV-2 can cause hepatitis hydropericardium syndrome, and a pathogenesis model of FAdV-2 infection in SPF chickens is established for the first time.

【Objection】This study aimed to monitor the genetic variation and molecular evolutionary trends of H9N2 avian influenza virus (AIV) in Guangdong, and provide reference data for the evolutionary analysis and prevention and control of H9N2 AIV in China.【Method】The HA and NA genes of three H9N2 AIV strains isolated from Guangdong in 2022 were amplified, cloned, and sequenced. The DNAStar and PhyloSuite software were used for sequence assembly and alignment. The MEGA, Megalign, NetNGlyc 1.0 Server, and other software were used for genetic evolution, percent identity, receptor binding, protein activity, drug resistance, and glycosylation site analysis of their nucleotide and amino acid sequences.【Result】The phylogenetic tree showed that the HA gene of the three isolates belonged to the clade h9.4.2.5 and the NA gene belonged to the clade 1. However, the nucleotide homology with the early strain was only 81.6%~91.7% and 88.2%~91.2%, formed new clade named clade h9.4.2.5c and clade 1.2, respectively. The nucleotide and amino acid sequence analysis revealed that the HA cleavage site was PSRSSR ↓ GLF. The receptor binding site underwent I155T, H183N, A190T/V, T212I, Q226L, and Q227M mutations. The glycosylation site underwent 218N non-glycosylation and acquired new glycosylation site at 313N. The NA stalk showed nine nucleotides deletion at positions 187-195 (ACAGAGATA), leading to the loss of three amino acids at positions 63-65 (TEI). The absorbtion site of NA on red blood cells showed K/E/S368N and D369N mutations. No related mutations to neuraminidase activity and drug resistance were found at the E119, D151, R152, R224, E276, R292, and R371.【Conclusion】The HA and NA genes of the three H9N2 AIV isolated from Guangdong in 2022 had evolved into a new subgroup. Some mutaitons suggest that prevalent H9N2 in Guangdong may enhanc adaptability of mammals and its antigenicity has changed. Moreover, it has not yet acquired resistance to durgs oseltamivir and zanamivir.

Highly pathogenic influenza A(H5N8) viruses had caused several outbreaks among wild bird and poultry populations across the globe, and strikingly, caused human infection, posing serious public health concerns. In this study, we conducted influenza surveillance in China during 2021 to monitor the evolution of influenza viruses in poultry. A total of 35 influenza viruses were obtained in chickens, ducks, and geese, of which 30 H5N8 viruses, 3 H5N1 viruses, and 2 H5N6 viruses. Phylogenetic analysis suggested all of H5N1, H5N6, and H5N8 isolates were derived from clade 2.3.4.4b H5N8 viruses during 2020/21 season, and notably, the internal genes of H5N1 and H5N6 viruses shared different genetic heterogeneity with H5N8 viruses and had been reassorted with wild bird-origin H5N1 viruses from Europe. By contrast, almost all H5N8 viruses exhibited only one phylogenic cluster with wild bird-origin H5N8 viruses in China and Korea, indicating that H5N8 viruses in China were more stable. Besides, we found that Korea is the main output geographic location in the spread of these H5N8 viruses to northern and eastern China, and especially, the co-circulation of H5N8 viruses occurred within China, with central China acted as a seeding population during the H5N8 epidemic. The statistical support was strong for viral migration from wild birds to chickens and ducks, indicating that 2.3.4.4b poultry-origin H5N8 viruses during 2020–2021 were originated from wild birds. Our findings provide novel insights into evolution and transmission dynamics of H5 subtype influenza viruses among poultry after novel H5N8 viruses invaded China for nearly one year.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV is maintained in an enzootic cycle and causes reproductive failure in pigs. Notably, the shift in JEV genotypes is not fully protected by existing vaccines, so the development of a candidate vaccine is urgently needed. In this study, we compared pathogenicity between Japanese encephalitis virus SA14 and BJB (isolated from humans in the 1970s) strains. We found that the BJB strain was attenuated in mice and that there was no case fatality rate. The growth rate of BJB was higher than SA14 virus in BHK-21 cells. Based on the sequence alignment of the viral genome between the SA14 and BJB virus strains, some mutations at sites 248, 254, 258, and 307 were observed in the 3′ untranslated region (3′UTR). The 3′UTR of JEV plays a very important role in the viral life cycle. Furthermore, using a reverse genetic system, we conducted and rescued the parental JEV strain SA14 (T248, A254, and A258) and the mutant virus rSA14-3′UTRmut (T248C, A254G, A258G, and 307G). Through an analysis of the RNA secondary structure model of the 3′UTR, we discovered that the mutations of T248C, A254G, and A258G reduced the apiculus ring and increased the lateral ring significantly in the stem-loop structures IV (SL-IV) structure region of 3′UTR. Moreover, the insertion of 307G added a ring to the dumbbell structure 1 (DB1) structure region. Strikingly, these RNA secondary structure changes in 3′UTR of rSA14-3′UTRmut increased viral negative chain RNA production and enhanced the replication ability of the virus in BHK-21 cells. However, in vivo mouse experiments illustrated that the rSA14-3′UTRmut virus significantly decreased the neurovirulence of JEV. These results affirmed that the JEV SL-IV and DB1 regions play an important role in viral proliferation and pathogenicity. Taken together, we complement the study of RNA element function in the 3′UTR region of JEV by providing a new target for the rational design of live attenuated candidate vaccines and the increase of virus production.

In China, the broad prevalence of H6 subtype influenza viruses, increasingly detected in aquatic birds, promotes their exchange materials with other highly pathogenic human-infecting H5N1, H5N6, and H7N9 influenza viruses. Strikingly, some H6 subtype viruses can infect pigs, dogs, and humans, posing risks to public health. In this study, 9 H6N2 viruses recovered from waterfowl species in the Guangdong province of China in 2018 were isolated and sequenced. Phylogenetic analysis revealed that the genome sequences of these H6N2 viruses belonged to Group I, except for the NP gene in Group III. Coalescent analyses demonstrated that the reassortment of NA and NS genes have occurred in two independent clusters, suggesting H6 subtype viruses had been undergoing a complex reassortant. To examine the evolutionary dynamics and the dissemination of the H6 subtype viruses, a Bayesian stochastic search variable selection was performed for results showing higher viral migration rates between closer provinces, including Guangdong, Jiangxi, Guangxi, and Fujian. Notably, the transmission routes of the H6 subtype viruses were concentrated in Jiangxi Province, the most frequent location for input and output transmission and a region containing Poyang Lake, a well-known wintering site for migration birds. We also found that the aquatic birds, especially ducks, were the most common input source of the viral transmission. In addition, we also found that eight positively selected amino acid sites were identified in HA protein. Given their continuous dissemination and the broad prevalence of the H6 subtype influenza viruses, continued surveillance is warranted in the future.