Background In early 2020, a novel H9N2 AIV immune escape variant emerged in South China and rapidly spread throughout mainland China. The effectiveness of the current H9N2 vaccine is being challenged by emerging immune escape strains. Assessing key amino acid substitutions that contribute to antigenic drift and immune escape in the HA gene of circulating strains is critical for understanding virus evolution and in selecting more effective vaccine components. Methods In this study, a representative immune escape strain, A/chicken/Fujian/11/2020 (FJ/20), differed from current H9N2 vaccine strain, A/chicken/Anhui/LH99/2017 (AH/17) by 18 amino acids in the head domain in HA protein. To investigate the molecular determinants of antigenic drift of FJ/20, a panel of mutants were generated by reverse genetics including specific amino acids changes in the HA genes of FJ/20 and AH/17. The antigenic effect of the substitutions was evaluated by hemagglutination inhibition (HI) assay and antigenic cartography. Results Fujian-like H9N2 viruses had changed antigenicity significantly, having mutated into an antigenically distinct sub-clade. Relative to the titers of the vaccine virus AH/17, the escape strain FJ/20 saw a 16-fold reduction in HI titer against antiserum elicited by AH/17. Our results showed that seven residue substitutions (D127S, G135D, N145T, R146Q, D179T, R182T and T183N) near the HA receptor binding sites were critical for converting the antigenicity of both AH/17 and FJ/20. Especially, the combined mutations 127D, 135G, 145N, and 146R could be a major factor of antigenic drift in the current immune escape variant FJ/20. The avian influenza A (H9N2) variant virus need further ongoing epidemiological surveillance. Conclusions In this study, we evaluated the relative contributions of different combinations of amino acid substitutions in the HA globular head domain of the immune escape strain FJ/20 and the vaccine strain AH/17. Our study provides more insights into the molecular mechanism of the antigenic drift of the H9N2 AIV immune escape strain. This work identified important markers for understanding H9N2 AIV evolution as well as for improving vaccine development and control strategies in poultry.

Over the last decade, RNA-seq has produced a massive amount of plant transcriptomic sequencing data deposited in public databases. Reanalysis of these public datasets can generate additional novel hypotheses not included in original studies. However, the large data volume and the requirement for specialized computational resources and expertise present a barrier for experimental biologists to explore public repositories. Here, we introduce PlantExp (https://biotec.njau.edu.cn/plantExp), a database platform for exploration of plant gene expression and alternative splicing profiles based on 131 423 uniformly processed publicly available RNA-seq samples from 85 species in 24 plant orders. In addition to two common retrieval accesses to gene expression and alternative splicing profiles by functional terms and sequence similarity, PlantExp is equipped with four online analysis tools, including differential expression analysis, specific expression analysis, co-expression network analysis and cross-species expression conservation analysis. With these online analysis tools, users can flexibly customize sample groups to reanalyze public RNA-seq datasets and obtain new insights. Furthermore, it offers a wide range of visualization tools to help users intuitively understand analysis results. In conclusion, PlantExp provides a valuable data resource and analysis platform for plant biologists to utilize public RNA-seq. datasets.

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.

The continued emergence of human illness caused by avian influenza viruses (AIVs) demonstrates the threat of strains such as H5N1, H7N9, H10N8, and now H10N3. The genetic and biological properties of H10N3 viruses are not fully understood. In this study, three H10N3 strains isolated from live poultry markets (LPMs) were systematically studied. Genome sequencing showed that the poultry‐origin viruses are highly homologous to the human H10N3 isolate. The three avian strains were A/chicken/Jiangsu/0146/2021(abbreviated as JS146, H10N3), A/chicken/Jiangsu/0169/2021 (JS169, H10N3), and A/chicken/Jiangsu/0189/2021(JS189, H10N3). Animal studies indicated that all three viruses are highly pathogenic to mice and that all could replicate efficiently in mouse nasal turbinate and lungs despite maintaining their avian receptor binding affinity. We also found that these viruses replicated efficiently in A549 cells and chicken embryos. The strain JS146 had sensitivity to the neuraminidase‐targeting drugs oseltamivir and zanamivir, whereas JS169 and JS189 were more resistant; genetic comparison implied that a substitution at NA position 368 conferred drug resistance. Importantly, several key molecular markers associated with mammalian adaptation had been detected in both avian and human‐isolated H10N3 influenza viruses in the HA (G228S), PB2 (I292V and A588V), PB1 (M317V and I368V), and PA (A343S, K356R and S409N) protein. The above work contributes new insight into the biology of this potentially zoonotic subtype and provides evidence supporting the continued epidemiological monitoring of human infections caused by AIV subtype H10N3.

Summary Fungi form a large and heterogeneous group of eukaryotic organisms with diverse ecological niches. The high importance of fungi contrasts with our limited understanding of fungal lifestyle and adaptability to environment. Over the last decade, the high-throughput sequencing technology produced tremendous RNA-sequencing (RNA-seq) data. However, there is no comprehensive database for mycologists to conveniently explore fungal gene expression and alternative splicing. Here, we have developed FungiExp, an online database including 35,821 curated RNA-seq samples derived from 220 fungal species, together with gene expression and alternative splicing profiles. It allows users to query and visualize gene expression and alternative splicing in the collected RNA-seq samples. Furthermore, FungiExp contains several online analysis tools, such as differential/specific, co-expression network and cross-species gene expression conservation analysis. Through these tools, users can obtain new insights by re-analyzing public RNA-seq data or upload personal data to co-analyze with public RNA-seq data. Availability and implementation The FungiExp is freely available at https://bioinfo.njau.edu.cn/fungiExp. Supplementary information Supplementary data are available at Bioinformatics online.

Background: Influenza virus remains a continuous and serious threat to public health worldwide, and its prevention and treatment have always been a major international issue. Because of its ability to evade immune surveillance through rapid antigenic drift and antigenic shift, broad-spectrum vaccines seem increasingly important. Methods: A mAb named 3C12 from an immortalized hybrid cell was generated via immunizing mice with HA2 protein from A/chicken/Anhui/BRI99/2016 (AH/BRI99/16, H9N2) generated by prokaryotic expression. Then, its broad-spectrum activity was analyzed by WB and IFA. Next, the minimal linear epitope was identified via analyzing the reaction of a series of HA truncations with 3C12. Finally, the protective effects of 3C12 were evaluated in vitro and in vivo infection experiments.Results: The mAb could react with the viruses of subtypes H1, H2, H5, H8, H9, H12, H13, H16, and HA protein of H18 in group 1, but failed to react with viruses in group 2. The minimal linear epitope targeted by the mAb was 433NAELLVL439 in full length of HA and localized in the C-helix region of HA2 (residue 95-101, HA2 numbering). What’s more, the mAb 3C12 inhibited H1, H2, H5, H8, H9, H12, H13 and H16 virus-replication in vitro and also has shown effectiveness in preventing and treating disease in mice challenged with lethal dose of AH/BRI99/16 (H9N2) virus in vivo. These results suggested that the broadly reactive anti-HA stem mAb 3C12 exhibited prophylactic and therapeutic efficacy.Conclusions: Here, we have demonstrated that the linear epitope identified in this study could be a novel target for developing broad-spectrum influenza diagnostics or vaccine design, and the HA2-based monoclonal antibody is indeed a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.

H9N2 avian influenza virus (AIV), one of the predominant subtypes devastating the poultry industry, has been circulating widely in the poultry population and causing huge economic losses. In this study, two H9N2 viruses with similar genetic backgrounds but different antigenicity were isolated from a poultry farm, namely A/chicken/Jiangsu/75/2018 (JS/75) and A/chicken/Jiangsu/76/2018 (JS/76). Sequence analysis revealed that their surface genes differed in three amino acid residues (127, 183 and 212) on the head of hemagglutinin (HA). To explore the differences between the two viruses in their biological features, six recombinant viruses, including the wild‐type or mutant HA and NA of JS/75 and JS/76 were generated with A/Puerto Rico/8/1934 (PR8) backbone via reverse genetics. The chicken challenge study and HI assay data indicated that r‐76/PR8 showed the most obvious antigen escape due to 127 and 183 amino acid substitutions in HA gene. Further studies verified that the 127N site was glycosylated in JS/76 and its mutants. Receptor‐binding assays showed that all the recombination viruses were prone to bind the human‐like receptors, except for the mutants which glycosylated 127N was deleted. Growth kinetics and mice challenge experiments indicated that 127N‐glycosylated viruses showed less replication in A549 cells and lower pathogenicity in mice compared with wild‐type viruses. Therefore, the glycosylation site and two amino acid alternations in the HA globular head were responsible for the differences in antigenicity and pathogenicity between the two H9N2 isolates. This study is significant in the research of the antigenic variation and vaccine updates for the H9N2 AIV. Also, highlighted the critical functions of glycosylation in the influenza virus on the pathogenicity against mammals.

Background: In early 2020, a novel H9N2 AIV immune escape variant emerged in South China and rapidly spread throughout mainland China. The effectiveness of the current H9N2 vaccine is being challenged by emerging immune escape strains. Assessing key amino acid substitutions that contribute to antigenic drift and immune escape in the HA gene of circulating strains is critical for understanding virus evolution and in selecting more effective vaccine components. Methods: In this study, a representative immune escape strain, A/chicken/Fujian/11/2020 (FJ/20), differed from current H9N2 vaccine strain, A/chicken/Anhui/LH99/2017 (AH/17) by 18 amino acids in the head domain. To investigate the molecular determinants of antigenic drift of FJ/20, a panel of mutants were generated by reverse genetics including specific amino acids changes in the HA genes of FJ/20 and AH/17. The antigenic effect of the substitutions was evaluated by hemagglutination inhibition (HI) assay and antigenic cartography. Results: Fujian-like H9N2 viruses had changed antigenicity significantly, having mutated into an antigenically distinct sub-clade. Relative to the titers of the vaccine virus AH/17, the escape strain FJ/20 saw a 16-fold reduction in HI titer against antiserum elicited by AH/17. Our results showed that seven residue substitutions (D127S, G135D, N145T, R146Q, D179T, R182T and T183N) near the HA receptor binding sites were critical for converting the antigenicity of both AH/17 and FJ/20. Especially, the combined mutations 127D, 135G, 145N, and 146R could be a major factor of antigenic drift in the current immune escape variant FJ/20. The avian influenza A (H9N2) variant virus need further ongoing epidemiological surveillance.Conclusions: In this study, we evaluated the relative contributions of different combinations of amino acid substitutions in the HA globular head domain of the immune escape strain FJ/20 and the vaccine strain AH/17. Our study provides more insights into the molecular mechanism of the antigenic drift of the H9N2 AIV immune escape strain. This work identified important markers for understanding H9N2 AIV evolution as well as for improving vaccine development and control strategies in poultry.