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.

As a kind of human betacoronavirus, SARS‐CoV‐2 has endangered globally public health. As of January 2021, the virus had resulted in 2,209,195 deaths. By studying the evolution trend and characteristics of 265 SARS‐CoV‐2 strains in the United States from January to March, it is found that the strains can be divided into six clades, USA clade‐1, USA clade‐2, USA clade‐3, USA clade‐4, USA clade‐5, and USA clade‐6, in which US clade‐1 may be the most ancestral clade, USA clade‐2 is an interim clade of USA clade‐1 and USA clade‐3, the other three clades have similar codon usage pattern, while USA clade‐6 is the newest and most adaptable clade. Mismatch analysis and protein alignment showed that the evolution of the clades arises from some special mutations in viral proteins, which may help the strain to invade, replicate, transcribe and so on. Compared with previous research and classifications, we suggest that clade O in GISAID should not be an independent clade and Wuhan‐Hu‐1 (EPI_ISL_402125) should not be an ancestral reference sequence. Our study decoded the evolutionary dynamic of SARS‐CoV‐2 in the early stage from the United States, which give some clues to infer the current evolution trend of SARS‐CoV‐2 and study the function of viral mutational protein.