A B S T R A C T Recombination events are known to contribute to the emergence of novel infectious bronchitis virus (IBV) genotypes. In this study, we carried out detailed phylogenetic analysis and sequence comparisons based on 74 complete nucleotide sequences of the IBV S1 gene, including strain I0636/16 and 73 representative sequences from each genotype and lineage. The results showed that strain I0636/16 represented a novel genotype, designated as lineage 1 within genotype VII (GVII-1). Further comparative genomic analysis revealed at least two recombination sites that replaced the spike gene in a lineage 18 within genotype I (GI-18)-like virus with an asyet-unidentified sequence, likely derived from another IBV strain, resulting a novel serotype with a lower affinity to the respiratory tract in chickens. To the best of our knowledge, this provides the first evidence for recombination leading to replacement of the complete spike gene and the emergence of a novel genotype/serotype with a lower affinity to the respiratory tract in chickens comparing to one of its parental virus ck/CH/LGX/ 111119. These results emphasize the importance of limiting exposure to novel IBVs that may serve as a source of genetic material for emerging viruses, as well as the importance of IBV surveillance in chicken flocks.

A B S T R A C TInfectious bronchitis viruses (IBVs) of GI-13 (793/B) and GI-19 (QX/LX4) lineages have been frequently detected in China in recent years. Naturally recombinant IBVs originating from the GI-13 and GI-19 lineages have also been isolated from chicken flocks with respiratory and renal problems in China. Thorough genetic and biological investigations of these recombinant viruses have led to speculation regarding their origin, evolution, and control. In order to confirm the previous results and further extend our understanding about the characteristics of the four recombinant IBV strains we had previously identified (I0718/17, I0722/17, I0724/17, and I0737/17), we conducted phylogenetic analysis by comparing their complete S1 gene sequences with those of 71 reference strains of different genotypes and lineages. We identified a close relationship between the S1 sequences of the four strains and those of GI-13 strains. The results of complete genome sequence analysis confirmed the previously identified recombination events in the four IBV strains and revealed additional recombination events in different genomic regions of strains I0718/17 and I0724/17, suggesting that the two strains originated from multiple recombination events between 4/91-like and YX10-like viruses. We comparatively evaluated the antigenicity, pathogenicity, and affinity of the four recombinant viruses and their deduced parental strains in the trachea and kidneys. Some of the strains showed comparable antigenic relatedness, pathogenicity, and affinity for the trachea and kidneys among each other and with their parental viruses; however, some of them showed varying biological characteristics. Point mutations observed in the receptor-binding domain and hypervariable region of the S1 subunit of the spike protein likely have an effect on these differences in biological characteristics, although the influence of other factors-such as host innate-immune responses and changes in genomic regions beyond the S1 protein-might also be responsible for such changes.

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A B S T R A C TTwo viruses were isolated in 2017 from commercial pheasants with severe clinical signs and mortality in Shandong and Anhui provinces, China, respectively. We examined the pathogenic effects of the viruses in chicken embryos and the size and morphology of the virus particles, performed phylogenetic analysis based on the S1 gene and complete genomic sequences, and examined the antibody responses against infectious bronchitis virus (IBV). The results suggested that the viruses I0623/17 and I0710/17 were avian coronaviruses and were identified as pheasant coronaviruses (PhCoV), with greatest similarity to IBV. Further investigations of the antigenicity, complete genome organization, substitutions in multiple genes, and viral pathogenicity, replication, and shedding in chickens and pheasants showed obvious differences between PhCoV and IBV in terms of antigenicity, and viral pathogenicity, replication, and shedding in chickens and pheasants. The close genetic relationship, but obvious differences between PhCoVs and IBVs suggested the IBVs could be the ancestors of PhCoVs, and that PhCoVs isolated from different outbreaks may have evolved independently from IBVs circulating in the specific region by adaption in pheasants. This hypothesis was supported by analysis of the S1 gene fragments of the two PhCoVs isolated in the current study, as well as PhCoVs isolated in the UK and selected IBV strains. Such analyses indicated different evolution patterns and different tissue tropisms between PhCoVs isolated in different outbreaks. Further studies are needed to confirm this hypothesis by studying the complete genomic sequences of PhCoVs from different outbreaks and the pathogenicity of IBVs in pheasants to compare and clarify the relationships between PhCoVs and IBVs.

Neuraminidase (NA) has multiple functions in the life cycle of influenza virus, especially in the late stage of virus replication. Both of Hemagglutinin (HA) and NA are highly glycosylated proteins. N-linked glycosylation (NLG) of HA has been reported to contribute to immune escape and virulence of influenza viruses. However, the function of NLG of NA remains largely unclear. In this study, we found that NLG is critical for budding ability of NA. Tunicamycin treatment or NLG knock-out significantly inhibited the budding of NA. Further studies showed that the NLG knock-out caused attenuation of virus in vitro and in vivo. Notably the NLG at 219 position plays an important role in budding, replication, and virulence of H1N1 influenza virus. To explore the underlying mechanism, unfolded protein response (UPR) was determined in NLG knock-out NA overexpressed cells, which showed that the mutant NA was mainly located in ER, and the UPR markers BIP and p-eIF2α were upregulated, and XBP1 was downregulated. All the results indicated that NLG knock-out NA was stacked in ER and triggered UPR, which might shut down the budding process of NA. Overall, the study shed light on the function of NLG of NA in virus replication and budding. IMPORTANCE NA is a highly glycosylated protein. Nevertheless, how the NLG affects the function of NA protein remains largely unclear. In this study, we found that NLG plays important roles in budding and Neuraminidase activity of NA protein. Loss of NLG attenuated viral budding and replication. Especially the 219 NLG site mutation significantly attenuated the replication and virulence of H1N1 influenza virus in vitro and in vivo, which suggested that NLG of NA protein is a novel virulence marker for influenza viruses.

The influenza A virus (IAV) is an important cause of respiratory disease worldwide. It is well known that alveolar epithelial cells are the target cells for the IAV, but there is relatively limited knowledge regarding the role of macrophages during IAV infection. Here, we aimed to analyze transcriptome differences in mouse lungs and macrophage (RAW264.7) cell lines infected with either A/California/04/2009 H1N1 (CA09) or A/chicken/SD/56/2015 H9N2 (SD56) using deep sequencing. The uniquely differentially expressed genes (UDEGs) were analyzed with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases; the results showed that the lungs infected with the two different viruses had different enrichments of pathways and terms. Interestingly, CA09 virus infection in mice was mostly involved with genes related to the extracellular matrix (ECM), while the most significant differences after SD56 infection in mice were in immune-related genes. Gene set enrichment analysis (GSEA) of RAW264.7 cells revealed that regulation of the cell cycle was of great significance after CA09 infection, whereas the regulation of the immune response was most enriched after SD56 infection, which was consistent with analysis results in the lung. Similar results were obtained from weighted gene co-expression network analysis (WGCNA)，where cell cycle regulation was extensively activated in RAW264.7 macrophages infected with the CA09 virus. Disorder of the cell cycle is likely to affect their normal immune regulation, which may be an important factor leading to their different prognoses. These results provide insight into the mechanism of the CA09 virus that caused a pandemic and explain the different reactivities of monocytes/macrophages infected by H9N2 and H1N1 IAV subtypes.

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Graphical AbstractPTAFR exacerbates the neuronal inflammatory microenvironment in AD brain.