With the emergence of clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (AIV) infection of dairy cattle and its subsequent detection in raw milk, coupled with recent AIV infections affecting dairy farm workers, experiments were conducted to affirm the safety of cooked ground beef related to AIV because such meat is often derived from cull dairy cows. Specifically, retail ground beef (percent lean:fat = ca. 80:20) was inoculated with a low pathogenic AIV (LPAIV) isolate to an initial level of 5.6 log10 50% egg infectious doses (EID50)  per 300 g patty. The inoculated meat was pressed into patties (ca. 2.54 cm thick, ca. 300 g each) and then held at 4 °C for up to 60 min. In each of the two trials, two patties for each of the following three treatments were cooked on a commercial open-flame gas grill to internal instantaneous temperatures of 48.9 °C (120°F), 62.8 °C (145°F), or 71.1 °C (160°F), but without any dwell time. Cooking inoculated ground beef patties to 48.9 °C (ave. cooking time of ca. 15 min) resulted in a mean reduction of ≥2.5 ± 0.9 log10 EID50 per 300 g of ground beef as assessed via quantification of virus in embryonating chicken eggs (ECEs). Likewise, cooking patties on a gas grill to 62.8 °C (ave. cooking time of ca. 21 min) or to the USDA FSIS recommended minimum internal temperature for ground beef of 71.1 °C (ave. cooking time of ca. 24 min) resulted in a reduction to nondetectable levels from initial levels of ≥5.6 log10 EID50 per 300 g. These data establish that levels of infectious AIV are substantially reduced within inoculated ground beef patties (20% fat) using recommended cooking procedures.

The H10 subtype avian influenza virus (AIV) poses an ongoing threat to both birds and humans. Notably, fatal human cases of H10N3 and H10N8 infections have drawn public attention. In 2022, we isolated two H10N3 viruses (A/chicken/Shandong/0101/2022 and A/chicken/Shandong/0603/2022) from diseased chickens in China. Genome analysis revealed that these viruses were genetically associated with human-origin H10N3 virus, with internal genes originating from local H9N2 viruses. Compared to the H10N8 virus (A/chicken/Jiangxi/102/2013), the H10N3 viruses exhibited enhanced thermostability, increased viral release from erythrocytes, and accumulation of hemagglutinin (HA) protein. Additionally, we evaluated the pathogenicity of both H10N3 and H10N8 viruses in mice. We found that viral titers could be detected in the lungs and nasal turbinates of mice infected with the two H10N3 viruses, whereas H10N8 virus titers were detectable in the lungs and brains of mice. Notably, the proportion of double HA Q222R and G228S mutations in H10N3 viruses has increased since 2019. However, the functional roles of the Q222R and G228S double mutations in the HA gene of H10N3 viruses remain unknown and warrant further investigation. Our study highlights the potential public health risk posed by the H10N3 virus. A spillover event of AIV to humans could be a foretaste of a looming pandemic. Therefore, it is imperative to continuously monitor the evolution of the H10N3 influenza virus to ensure targeted prevention and control measures against influenza outbreaks.

The recent concurrent emergence of H5N1, H5N6, and H5N8 avian influenza viruses (AIVs) has led to significant avian mortality globally. Since 2020, frequent human-animal interactions have been documented. To gain insight into the novel H5 subtype AIVs (i.e., H5N1, H5N6 and H5N8), we collected 6102 samples from various regions of China between January 2021 and September 2022, and identified 41 H5Nx strains. Comparative analyses on the evolution and biological properties of these isolates were conducted. Phylogenetic analysis revealed that the 41 H5Nx strains belonged to clade 2.3.4.4b, with 13 related to H5N1, 19 to H5N6, and 9 to H5N8. Analysis based on global 2.3.4.4b viruses showed that all the viruses described in this study were likely originated from H5N8, exhibiting a heterogeneous evolutionary history between H5N1 and H5N6 during 2015-2022 worldwide. H5N1 showed a higher rate of evolution in 2021-2022 and more sites under positive selection pressure in 2015-2022. The antigenic profiles of the novel H5N1 and H5N6 exhibited notable variations. Further hemagglutination inhibition assay suggested that some A(H5N1) viruses may be antigenically distinct from the circulating H5N6 and H5N8 strains. Mammalian challenge assays demonstrated that the H5N8 virus (21GD001_H5N8) displayed the highest pathogenicity in mice, followed by the H5N1 virus (B1557_H5N1) and then the H5N6 virus (220086_H5N6), suggesting a heterogeneous virulence profile of H5 AIVs in the mammalian hosts. Based on the above results, we speculate that A(H5N1) viruses have a higher risk of emergence in the future. Collectively, these findings unveil a new landscape of different evolutionary history and biological characteristics of novel H5 AIVs in clade 2.3.4.4b, contributing to a better understanding of designing more effective strategies for the prevention and control of novel H5 AIVs.

Total 200 properties related to structural characteristics were employed to represent structures of 400 HA coded proteins of influenza virus as training samples. Some recognition models for HA proteins of avian influenza virus (AIV) were developed using support vector machine (SVM) and linear discriminant analysis (LDA). The results obtained from LDA are as follows: the identification accuracy (R ia) for training samples is 99.8% and R ia by leave one out cross validation is 99.5%. Both R ia of 99.8% for training samples and R ia of 99.3% by leave one out cross validation are obtained using SVM model, respectively. External 200 HA proteins of influenza virus were used to validate the external predictive power of the resulting model. The external R ia for them is 95.5% by LDA and 96.5% by SVM, respectively, which shows that HA proteins of AIVs are preferably recognized by SVM and LDA, and the performances by SVM are superior to those by LDA.

Since 2020, clade 2.3.4.4b highly pathogenic avian influenza H5N8 and H5N1 viruses have swept through continents, posing serious threats to the world. Through comprehensive analyses of epidemiological, genetic, and bird migration data, we found that the dominant genotype replacement of the H5N8 viruses in 2020 contributed to the H5N1 outbreak in the 2021/2022 wave. The 2020 outbreak of the H5N8 G1 genotype instead of the G0 genotype produced reassortment opportunities and led to the emergence of a new H5N1 virus with G1\'s HA and MP genes. Despite extensive reassortments in the 2021/2022 wave, the H5N1 virus retained the HA and MP genes, causing a significant outbreak in Europe and North America. Furtherly, through the wild bird migration flyways investigation, we found that the temporal-spatial coincidence between the outbreak of the H5N8 G1 virus and the bird autumn migration may have expanded the H5 viral spread, which may be one of the main drivers of the emergence of the 2020-2022 H5 panzootic.IMPORTANCESince 2020, highly pathogenic avian influenza (HPAI) H5 subtype variants of clade 2.3.4.4b have spread across continents, posing unprecedented threats globally. However, the factors promoting the genesis and spread of H5 HPAI viruses remain unclear. Here, we found that the spatiotemporal genotype replacement of H5N8 HPAI viruses contributed to the emergence of the H5N1 variant that caused the 2021/2022 panzootic, and the viral evolution in poultry of Egypt and surrounding area and autumn bird migration from the Russia-Kazakhstan region to Europe are important drivers of the emergence of the 2020-2022 H5 panzootic. These findings provide important targets for early warning and could help control the current and future HPAI epidemics.

BACKGROUND: The continuous evolution of drug-resistant influenza viruses highlights the necessity for repurposing naturally-derived and safe phytochemicals with anti-influenza activity as novel broad-spectrum anti-influenza medications.
METHODS: In this study, nitrogenous alkaloids were tested for their viral inhibitory activity against influenza A/H1N1 and A/H5N1 viruses. The cytotoxicity of tested alkaloids on MDCK showed a high safety range (CC50 > 200 µg/ml), permitting the screening for their anti-influenza potential.
RESULTS: Herein, atropine sulphate, pilocarpine hydrochloride and colchicine displayed anti-H5N1 activities with IC50 values of 2.300, 0.210 and 0.111 µg/ml, respectively. Validation of the IC50 values was further depicted by testing the three highly effective alkaloids, based on their potent IC50 values against seasonal influenza A/H1N1 virus, showing comparable IC50 values of 0.204, 0.637 and 0.326 µg/ml, respectively. Further investigation suggests that colchicine could suppress viral infection by primarily interfering with IAV replication and inhibiting viral adsorption, while atropine sulphate and pilocarpine hydrochloride could directly affect the virus in a cell-free virucidal effect. Interestingly, the in silico molecular docking studies suggest the abilities of atropine, pilocarpine, and colchicine to bind correctly inside the active sites of the neuraminidases of both influenza A/H1N1 and A/H5N1 viruses. The three alkaloids exhibited good binding energies as well as excellent binding modes that were similar to the co-crystallized ligands. On the other hand, consistent with in vitro results, only colchicine could bind correctly against the M2-proton channel of influenza A viruses (IAVs). This might explicate the in vitro antiviral activity of colchicine at the replication stage of the virus replication cycle.
CONCLUSIONS: This study highlighted the anti-influenza efficacy of biologically active alkaloids including colchicine. Therefore, these alkaloids should be further characterized in vivo (preclinical and clinical studies) to be developed as anti-IAV agents.

The rapid and sensitive detection of pathogenic viruses is important for controlling pandemics. Herein, a rapid, ultrasensitive, optical biosensing scheme was developed to detect avian influenza virus H9N2 using a genetically engineered filamentous M13 phage probe. The M13 phage was genetically engineered to bear an H9N2-binding peptide (H9N2BP) at the tip and a gold nanoparticle (AuNP)-binding peptide (AuBP) on the sidewall to form an engineered phage nanofiber, M13@H9N2BP@AuBP. Simulated modelling showed that M13@H9N2BP@AuBP enabled a 40-fold enhancement of the electric field enhancement in surface plasmon resonance (SPR) compared to conventional AuNPs. Experimentally, this signal enhancement scheme was employed for detecting H9N2 particles with a sensitivity down to 6.3 copies/mL (1.04 × 10-5 fM). The phage-based SPR scheme can detect H9N2 viruses in real allantoic samples within 10 min, even at very low concentrations beyond the detection limit of quantitative polymerase chain reaction (qPCR). Moreover, after capturing the H9N2 viruses on the sensor chip, the H9N2-binding phage nanofibers can be quantitatively converted into plaques that are visible to the naked eye for further quantification, thereby allowing us to enumerate the H9N2 virus particles through a second mode to cross-validate the SPR results. This novel phage-based biosensing strategy can be employed to detect other pathogens because the H9N2-binding peptides can be easily switched with other pathogen-binding peptides using phage display technology.

Avian influenza viruses (AIVs) are influenza A viruses, of which subtypes H1, H2 and H3 are highly transmissible in poultry and have the risk of transmission to human as well. It is important to establish an accurate, sensitive and convenient means of virus detection. In this study, we developed a multiplex real-time RT-PCR assay based on conserved sequences of the virus hemagglutinin and matrix, and designed primers and probes for the simultaneous and rapid detection of AIV subtypes H1, H2 and H3. We used different subtypes of AIVs and other avian respiratory viruses for evaluation of the specificity of this method. The results showed good sensitivity, specificity and reproducibility. The detection limit was 10-100 copies per reaction. The method also achieved good concordance with the virus isolation method when compared to 81 poultry samples evaluated. It provides a new method for detecting mixed infections of AIVs.

Terminal sialic acids (Sia) on soluble glycoprotein of saliva play an important role in the clearance of influenza virus. The aim of this study is to investigate the alteration of sialylation on the salivary proteins of women during the lactation period and its effect on the saliva binding ability to virus. In total, 210 saliva samples from postpartum women with and without breastfeeding were collected, and the expression level of α2-3/6-linked Sia on the whole salivary proteins and specific glycoproteins of IgA and MUC5B from different groups were tested and verified using lectin microarray, blotting analysis and ELISA based method. The H1N1 vaccine and three strains of Avian influenza virus (AIV) were used for the saliva binding assay. Results showed that the variation in salivary expression level of α2-3-linked Sia was much more obvious than the α2-6-linked Sia, which was up-regulated significantly in the breastfeeding groups compared to the non-breastfeeding groups at the same postpartum stage. Furthermore, the binding abilities of salivary glycoproteins to AIV strains and H1N1 vaccine were increased in breastfeeding groups accordingly. This finding adds new evidence for the maternal benefit of breastfeeding and provides new thinking to protect postpartum women from AIV infection.

Salivary glycoproteins are known as an important barrier to inhibit influenza infection by presenting sialic acid (Sia) ligands that can bind with viral hemagglutination. Here, to further understand why pregnant women are more vulnerable to avian influenza virus (AIV), we investigated the alteration of protein sialylation in the saliva of women during pregnancy and postpartum, and its impact on the saliva binding affinity to AIV. Totally 1200 saliva samples were collected, the expression levels of terminal α2-3/6-linked Sia on salivary proteins were tested and validated, and the binding activities of salivary proteins were assessed against 3 strains of AIV and the H1N1 vaccine. Result showed that the expression of terminal α2-3-linked Sia in the saliva of women decreased dramatically during pregnancy compared to that of non-pregnancy control, especially for women in the second or third trimester (fold change = 0.53 and 0.37, p < 0.001). And their salivary protein binding ability to AIV declined accordingly. The variation of terminal α2-3-linked Sia on salivary MUC5B and IgA was consistent with the above results. This study indicates that the decrease of terminal α2-3-linked Sia on salivary glycoproteins of pregnant women affects their binding ability to AIV, which may provide new insights into AIV prevention and control.