Influenza viruses belong to the Orthomyxoviridae family, and both human influenza A and B viruses can cause seasonal epidemics. 1 Seasonal influenza is a highly contagious respiratory disease. About 5%-10% of adults and 20%-30% of children are infected by seasonal influenza viruses each year. Currently, there are two subtypes of influenza A (H1N1: H1pdm09 and H3N2: H3) and two lineages of influenza B virus (Yamagata: Yam and Victoria: Vic) that co-circulate in humans. The circulating human influenza A H1N1 subtype emerged

The 701 and 702 positions of influenza PB2 polymerase subunit are previously shown to have roles on host range. Limited polymorphisms at these two residues are identified in natural isolates, thereby limiting the study of their role in the polymerase. In this study, we generated 31 viable viruses by random mutagenesis at this region, indicating that these positions can tolerate a wide range of amino acids. These mutants demonstrated varying polymerase activities and viral replication rates in mammalian and avian cells. Notably, some mutants displayed enhanced polymerase activity, yet their replication kinetics were comparable to the wild-type virus. Surface electrostatic charge predication on the PB2 structural model revealed that the viral polymerase activity in mammalian cells generally increases as this region becomes more positively charged. One of the mutants (701A/702E) showed much reduced pathogenicity in mice while others had a pathogenicity similar to the wild-type level. Distinct tissue tropisms of the PB2-701/702 mutants were observed in infected chicken embryos. Overall, this study demonstrates that the PB2-701/702 region has a high degree of sequence plasticity and sequence changes in this region can alter virus phenotypes in vitro and in vivo.

Background Measures for mitigation of Coronavirus Disease 2019 (COVID‐19) were set to reduce the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2). SARS‐CoV‐2 and other respiratory viruses share similar transmission routes and some common clinical manifestations. Co‐circulation of SARS‐CoV‐2 and other common respiratory viruses is imminent. Therefore, development of multiplex assays for detecting these respiratory viruses is essential for being prepared for future outbreaks of respiratory viruses. Methods A panel of three reverse transcription droplet digital PCR (RT‐ddPCR) assays were developed to detect 15 different human respiratory viruses. Evaluations of its performance were demonstrated. A total of 100 local and 98 imported COVID‐19 cases in Hong Kong were screened for co‐infection with other common respiratory viruses. Results All detected viral targets showed distinct signal clusters using the multiplex RT‐ddPCR assays. These assays have a broad range of linearity and good intra‐/inter‐assay reproducibility for each target. The lower limits of quantification for all targets were ≤46 copies per reaction. Six imported cases of COVID‐19 were found to be co‐infected with other respiratory viruses, whereas no local case of co‐infection was observed. Conclusions The multiplex RT‐ddPCR assays were demonstrated to be useful for screening of respiratory virus co‐infections. The strict preventive measures applied in Hong Kong may be effective in limiting the circulation of other human respiratory viruses. The multiplex assays developed in this study can achieve a robust detection method for clinical and research purposes.

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