In this study, suspected classical swine fever (CSF) samples from the Guangxi Province of China were obtained from pigs with acute CSF, aborted fetuses, newborn pigs that died at 1-2 days of age, tonsils of healthy pigs, and leukocytes of immunized sows during 2001-2009. About 92 of 775 samples were found to be positive by RT-PCR, and 41 isolates were obtained. Phylogenetic analysis was performed on the 31 isolates by sequencing the E2 gene, and the isolates were found to cluster into two groups: (1) isolates from aborted fetuses (except GXGZ02), deceased newborn baby pigs, tonsils of healthy pigs, and leukocytes of immunized sows belonged to group 1.1, along with vaccine strain, HCLV, and standard virulent strain, Shimen, of China, and (2) 20 isolates from pigs with acute CSF belonged to group 2.1, 13 of which were clustered into subgroup 2.1b with isolates from other provinces of China, and 7 of which were clustered into subgroup 2.1a with isolates from Italy and Germany.

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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.

No abstract

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.