Low pathogenic avian influenza A(H9N2) virus is endemic worldwide and continually recruit internal genes to generate human-infecting H5N1, H5N6, H7N9, and H10N8 influenza variants. Here we show that hemagglutinin cleavage sites (HACS) of H9N2 viruses tended to mutate towards hydrophilic via evolutionary transition, and the tribasic HACS were found at high prevalence in Asia and the Middle East. Our finding suggested that the tribasic H9N2 viruses increased the viral replication, stability, pathogenicity and transmission in chickens and the virulence of mice compared to the monobasic H9N2 viruses. Notably, the enlarged stem-loop structures of HACS in the RNA region were found in the increasing tribasic H9N2 viruses. The enlarged HACS RNA secondary structures of H9N2 viruses did not influence the viral replication but accelerated the frequency of nucleotide insertion in HACS. With the prevailing tendency of the tribasic H9N2 viruses, the tribasic HACS in H9N2 viruses should be paid more attention.

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Since it firstly emerged in China in 2013, clade 2.3.4.4 H5N6 highly pathogenic avian influenza viruses (HPAIVs) has rapidly replaced predominant H5N1 to become the dominant H5 subtype in China, especially in ducks. Not only endemic in China, it also crossed the geographical barrier and emerged in South Korea, Japan, and Europe. Here, we analyzed the genetic properties of the clade 2.3.4.4 H5N6 HPAIVs with full genome sequences available online together with our own isolates. Phylogenetic analysis showed that clade 2.3.4.4 H5N6 HPAIVs continuously reassorted with local H5, H6, and H7N9/H9N2. Species analysis reveals that aquatic poultry and migratory birds became the dominant hosts of H5N6. Adaption to aquatic poultry might help clade 2.3.4.4 H5N6 better adapt to migratory birds, thus enabling it to become endemic in China. Besides, migratory birds might help clade 2.3.4.4 H5N6 transmit all over the world. Clade 2.3.4.4 H5N6 HPAIVs also showed a preference for α2,6-SA receptors when compared to other avian origin influenza viruses. Experiments in vitro and in vivo revealed that clade 2.3.4.4 H5N6 HPAIVs exhibited high replication efficiency in both avian and mammal cells, and it also showed high pathogenicity in both mice and chickens, demonstrating high risk to public health. Considering all the factors together, adaption to aquatic poultry and migratory birds helps clade 2.3.4.4 H5N6 overcome the geographical isolation, and it has potential to be the next influenza pandemic in the world, making it worthy of our attention.

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.

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In the swine industry, porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease which causes heavy economic losses worldwide. Effective prevention and disease control is an important issue. In this study, we described the construction of a Japanese encephalitis virus (JEV) DNA-based replicon with a cytomegalovirus (CMV) promoter based on the genome of Japanese encephalitis live vaccine virus SA14-14-2, which is capable of offering a potentially novel way to develop and produce vaccines against a major pathogen of global health. This JEV DNA-based replicon contains a large deletion in the structural genes (C-prM-E). A PRRSV GP5/M was inserted into the deletion position of JEV DNA-based replicons to develop a chimeric replicon vaccine candidate for PRRSV. The results showed that BALB/c mice models with the replicon vaccines pJEV-REP-G-2A-M-IRES and pJEV-REP-G-2A-M stimulated antibody responses and induced a cellular immune response. Analysis of ELSA data showed that vaccination with the replicon vaccine expressing GP5/M induced a better antibodies response than traditional DNA vaccines. Therefore, the results suggested that this ectopic expression system based on JEV DNA-based replicons may represent a useful molecular platform for various biological applications, and the JEV DNA-based replicons expressing GP5/M can be further developed into a novel, safe vaccine candidate for PRRS.

Host factors play an essential role in virus replication and pathogenesis. Although UAF1 is well known to form complexes with ubiquitin-specific proteases, little is known about the function of the UAF1 protein itself.