The H9N2 subtype avian influenza viruses (AIVs) have been circulating in China for more than 20 years, attracting more and more attention due to the potential threat of them. At present, vaccination is a common prevention and control strategy in poultry farms, but as virus antigenicity evolves, the immune protection efficiency of vaccines has constantly been challenged. In this study, we downloaded the hemagglutinin (HA) protein sequences of the H9N2 subtype AIVs from 1994 to 2019 in China—with a total of 5138 sequences. The above sequences were analyzed in terms of time and space, and it was found that h9.4.2.5 was the most popular in various regions of China. Furthermore, the prevalence of H9N2 subtype AIVs in China around 2006 was different. The domestic epidemic branch was relatively diversified from 1994 to 2006. After 2006, the epidemic branch each year was h9.4.2.5. We compared the sequences around 2006 as a whole and screened out 15 different amino acid positions. Based on the HA protein of A/chicken/Guangxi/55/2005 (GX55), the abovementioned amino acid mutations were completed. According to the 12-plasmid reverse genetic system, the rescue of the mutant virus was completed using A/PuertoRico/8/1934 (H1N1) (PR8) as the backbone. The cross hemagglutination inhibition test showed that these mutant sites could transform the parental strain from the old to the new antigenic region. Animal experiments indicated that the mutant virus provided significant protection against the virus from the new antigenic region. This study revealed the antigenic evolution of H9N2 subtype AIVs in China. At the same time, it provided an experimental basis for the development of new vaccines.

Emerging influenza D viruses (IDVs), the newest member in the genus Orthomyxovirus family, which can infect and transmit in multiple mammalian species as its relatives the influenza A viruses (IAVs). Additional studies of biological characteristics of IDVs are needed; here, we studied the characteristics of IDV nonstructural protein 2 (NS2), which shares the lowest homology to known influenza proteins. First, we generated reassortant viruses via reverse genetics to analyze the segment compatibility and gene interchangeability between IAVs and IDVs. Next, we investigated the locations and exact sequences of nuclear export signals (NESs) of the IDV NS2 protein. Surprisingly, three separate NES regions were found to contribute to the nuclear export of an eGFP fusion protein. Alanine scanning mutagenesis identified critical amino acid residues within each NES, and co-immunoprecipitation experiments demonstrated that their nuclear export activities depend on the CRM1-mediated pathway, particularly for the third NES (136-146aa) of IDV NS2. Interestingly, the third NES was important for the interaction of NS2 protein with CRM1. The findings in this study contribute to the understanding of IDV NS2 protein’s role during nucleocytoplasmic transport of influenza viral ribonucleoprotein complexes (vRNPs) and will also facilitate the development of novel anti-influenza drugs targeting nuclear export signals of IDV NS2 protein.

Influenza A virus (IAV) infection has traditionally been a serious problem in animal husbandry and human public health security. Recently, many studies identified that long noncoding RNAs play an important role in the antiviral immune response after the infection of the influenza virus. However, there are still lots of IAV-related lncRNAs that have not been well-characterized. Using RNA sequencing analysis, we identified a lncRNA, named Serpina3i Activation Associated lncRNA (SAAL), which can be significantly upregulated in mice after IAV infection. In this study, we found that overexpression of SAAL inhibited the replication of A/WSN/33(WSN). SAAL upregulated Serpina3i with or without WSN infection. Overexpression of Serpina3i reduced influenza virus infection. Meanwhile, knockdown of Serpina3i enhanced the replication of WSN. Furthermore, knockdown of Serpina3i abolished the SAAL-mediated decrease in WSN infection. Overexpression of SAAL or Serpina3i positively regulated the transcription of interferon β (IFN-β) and several critical ISGs after WSN infection. In conclusion, we found that the novel lncRNA SAAL is a critical anti-influenza regulator by upregulating the mRNA level of Serpina3i.

Long non-coding RNAs (lncRNAs) are defined as transcripts longer than 200 nucleotides(nt) with no protein-coding potential. Accumulating evidence indicates that the interaction between lncRNAs and influenza A virus (IAV) plays an crucial role in multiple biological processes, including host antiviral immunity and regulating viral replication. However, the mechanisms by which IAV resists antiviral immunity and enhances its replication by inhibiting lncRNA-mediated antiviral defense remain largely unexplored. In this study, we identified a lncRNA, designated LRIR (LncRNA Regulating IAV Replication), which is significantly downregulated upon IAV infection in A549 cells. Notably, LRIR knockdown significantly promotes IAV replication, whereas LRIR overexpression inhibits viral replication, suggesting that LRIR exerts its antiviral effect during IAV infection. Furthermore, the antiviral activity of LRIR primarily depends on the regions spanning from 258 to 381 nt and 38-97 nt. Mechanistically, LRIR was found to inhibit replication and transcription of the viral genome. Further studies indicated that LRIR suppresses IAV replication by downregulating the expression of transmembrane protease serine 2 (TMPRSS2). Collectively, our findings reveal that IAV infection suppresses LRIR expression, thereby weakening its negative regulation of TMPRSS2 and subsequently promoting viral replication. These results provide a theoretical foundation for the development of novel anti-IAV therapeutics.