Characterizing the Core Internal Gene Pool of H9N2 Responsible for Continuous Reassortment With Other Influenza A Viruses

Reassortment among avian influenza viruses is the main source of novel avian influenza virus subtypes. Studies have shown that the H9N2 virus often donates internal segments to generate novel reassortant avian influenza viruses, acting as a reassortment template. However, the characteristics of the internal pattern of reassortment remain unclear. In this article, we first defined the core gene pool of the internal segments of the H9N2 virus that provide templates for reassortment. We used genetic distance and sequence similarity to define typical clusters in the core gene pool. Then, we analyzed the phylogenetic relationships, feature vector distances, geographic distributions and mutation sites of strains related to the core gene pool. Strains in the same typical clusters have close phylogenetic relationships and feature vector distances. We also found that these typical clusters can be divided into three categories according to their main geographic distribution area. Furthermore, typical clusters in the same geographic area contain some common mutation patterns. Our results suggest that typical clusters in the core gene pool affect the reassortment events of the H9N2 virus in many respects, such as geographic distribution and amino acid mutation sites.

SUMOylation of matrix protein M1 and filamentous morphology collectively contribute to the replication and virulence of highly pathogenic H5N1 avian influenza viruses in mammals

The matrix protein (M1) of influenza A virus plays an important role in replication, assembly, and budding. A previous study found that aspartic acid (D) at position 30 and alanine (A) at position 215 of M1 contribute to the high pathogenicity of H5N1 viruses in mice, and double mutations of D to asparagine (N) at position 30 (D30N) and A to threonine (T) at position 215 (A215T) in M1 dramatically attenuate H5N1 viruses in mice. However, the underlying mechanisms by which these M1 mutations attenuate the virulence of H5N1 viruses are unknown. Here, we found that the amino acid mutation A215T eliminates the SUMOylation of M1 by reducing its interaction with the host SUMO1 protein, significantly reducing the stability of M1, slowing the export of the M1-vRNP complex from the nucleus to the cytoplasm, and reducing viral replication in MDCK cells. We further found that the D30N mutation in M1 alters the shape of progeny viruses from filamentous to spherical virions. Our findings reveal an essential role for M1 215A SUMOylation and M1 30D-related filamentous morphology in the pathogenesis of avian influenza viruses, which could be targeted in novel antiviral drug designs. Importance Identification of the pathogenic mechanism of highly pathogenic avian influenza viruses in mammals is helpful to develop novel anti-influenza virus strategies. Two amino acid mutations (D30N and A215T) in M1 were found to collectively attenuate H5N1 influenza viruses in mice, but the underlying mechanism remained unknown. This study found that the A215T mutation significantly decreases the SUMOylation of M1, which in turn attenuates the replication of H5N1 virus in mammalian cells. The D30N mutation in M1 was found to change the virion shape from filamentous to spherical. These findings are important for understanding the molecular mechanism of virulence of highly pathogenic avian influenza viruses in mammals.

A high-quality genome and comparison of short- versus long-read transcriptome of the palaearctic duck Aythya fuligula (tufted duck)

Background The tufted duck is a non-model organism that experiences high mortality in highly pathogenic avian influenza outbreaks. It belongs to the same bird family (Anatidae) as the mallard, one of the best-studied natural hosts of low-pathogenic avian influenza viruses. Studies in non-model bird species are crucial to disentangle the role of the host response in avian influenza virus infection in the natural reservoir. Such endeavour requires a high-quality genome assembly and transcriptome. Findings This study presents the first high-quality, chromosome-level reference genome assembly of the tufted duck using the Vertebrate Genomes Project pipeline. We sequenced RNA (complementary DNA) from brain, ileum, lung, ovary, spleen, and testis using Illumina short-read and Pacific Biosciences long-read sequencing platforms, which were used for annotation. We found 34 autosomes plus Z and W sex chromosomes in the curated genome assembly, with 99.6% of the sequence assigned to chromosomes. Functional annotation revealed 14,099 protein-coding genes that generate 111,934 transcripts, which implies a mean of 7.9 isoforms per gene. We also identified 246 small RNA families. Conclusions This annotated genome contributes to continuing research into the host response in avian influenza virus infections in a natural reservoir. Our findings from a comparison between short-read and long-read reference transcriptomics contribute to a deeper understanding of these competing options. In this study, both technologies complemented each other. We expect this annotation to be a foundation for further comparative and evolutionary genomic studies, including many waterfowl relatives with differing susceptibilities to avian influenza viruses.