TRIM21 Promotes Oxidative Stress and Ferroptosis through the SQSTM1-NRF2-KEAP1 Axis to Increase the Titers of H5N1 Highly Pathogenic Avian Influenza Virus.

Tripartite motif-containing protein 21 (TRIM21) is involved in signal transduction and antiviral responses through the ubiquitination of protein targets. TRIM21 was reported to be related to the imbalance of host cell homeostasis caused by viral infection. Our studies indicated that H5N1 highly pathogenic avian influenza virus (HPAIV) infection up-regulated TRIM21 expression in A549 cells. Western blot and qPCR results showed that knockdown of TRIM21 alleviated oxidative stress and ferroptosis induced by H5N1 HPAIV and promoted the activation of antioxidant pathways. Co-IP results showed that TRIM21 promoted oxidative stress and ferroptosis by regulating the SQSTM1-NRF2-KEAP1 axis by increasing SQSTM1 K63-linked polyubiquitination under the condition of HPAIV infection. In addition, TRIM21 attenuated the inhibitory effect of antioxidant NAC on HPAIV titers and enhanced the promoting effect of ferroptosis agonist Erastin on HPAIV titers. Our findings provide new insight into the role of TRIM21 in oxidative stress and ferroptosis induced by viral infection.

The evolution, pathogenicity and transmissibility of quadruple reassortant H1N2 swine influenza virus in China: A potential threat to public health.

Swine are regarded as "intermediate hosts" or "mixing vessels" of influenza viruses, capable of generating strains with pandemic potential. From 2020 to 2021, we conducted surveillance on swine H1N2 influenza (swH1N2) viruses in swine farms located in Guangdong, Yunnan, and Guizhou provinces in southern China, as well as Henan and Shandong provinces in northern China. We systematically analyzed the evolution and pathogenicity of swH1N2 isolates, and characterized their replication and transmission abilities. The isolated viruses are quadruple reassortant H1N2 viruses containing genes from pdm/09 H1N1 (PB2, PB1, PA and NP genes), triple-reassortant swine (NS gene), Eurasian Avian-like (HA and M genes), and recent human H3N2 (NA gene) lineages. The NA, PB2, and NP of SW/188/20 and SW/198/20 show high gene similarities to A/Guangdong/Yue Fang277/2017 (H3N2). The HA gene of swH1N2 exhibits a high evolutionary rate. The five swH1N2 isolates replicate efficiently in human, canine, and swine cells, as well as in the turbinate, trachea, and lungs of mice. A/swine/Shandong/198/2020 strain efficiently replicates in the respiratory tract of pigs and effectively transmitted among them. Collectively, these current swH1N2 viruses possess zoonotic potential, highlighting the need for strengthened surveillance of swH1N2 viruses.

The genetic diversity, replication, and transmission of 2009 pandemic H1N1 viruses in China.

Background: The 2009 pandemic H1N1 influenza A virus (pdm09) continue to evolve, and few studies have systemically analyzed the evolution, replication, and transmission of pmd09 viruses in China. Methods: To better understand the evolution and pathogenicity of pdm09 viruses, we systematically analyzed viruses that were confirmed in 2009-2020 in China and characterized their replication and transmission ability. We extensively analyzed the evolution characteristics of pdm/09 in China over the past decades. The replication ability of 6B.1 and 6B.2 lineages on Madin-Darby canine kidney (MDCK) and human lung adenocarcinoma epithelial (A549) cells and their pathogenicity and transmission in guinea pigs were also compared. Results: In total, 3,038 pdm09 viruses belonged to clade 6B.1 (62% of all pdm09 viruses) and clade 6B.2 (4%). Clade 6B.1 pdm09 viruses are the predominant clade, with proportions of 54.1%, 78.9%, 57.2%, 58.6%, 61.7%, 76.3%, and 66.6% in the North, Northeast, East, Central, South, Southwest, and Northeast regions in China, respectively. The isolation proportion of clade 6B.1 pdm/09 viruses was 57.1%, 74.3%, 96.1%, 98.2%, 86.7%, and 78.5% in 2015-2020, respectively. A clear differentiation time point appeared in 2015 before which the evolution trend of pdm09 viruses in China was similar to that in North America but then showed a different trend after that point. To characterize pdm09 viruses in China after 2015, we further analyzed 33 pdm09 viruses isolated in Guangdong in 2016-2017, among which A/ Guangdong/33/2016 and A/Guangdong/184/2016 (184/2016) belonged to clade 6B.2, and the other 31 strains belonged to clade 6B.1. A/Guangdong/887/2017 (887/2017) and A/Guangdong/752/2017 (752/2017) (clade 6B.1), 184/2016 (clade 6B.2) and A/California/04/2009 (CA04) replicated efficiently in MDCK cells and A549 cells, as well as the turbinates of guinea pigs. 184/2016 and CA04 could transmit among guinea pigs through physical contact. Conclusion: Our findings provide novel insights into the evolution, pathogenicity, and transmission of pdm09 virus. The results show that enhancing surveillance of pdm09 viruses and timely evaluation of their virulence are essential.

Antiviral Effect of Ginsenosides rk1 against Influenza a Virus Infection by Targeting the Hemagglutinin 1-Mediated Virus Attachment.

Influenza A virus (IAV) infections have been a serious hazard to public health everywhere. With the growing concern of drug-resistant IAV strains, there is an urgent need for novel anti-IAV medications, especially those with alternative mechanisms of action. Hemagglutinin (HA), an IAV glycoprotein, plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a good target for developing anti-IAV drugs. Panax ginseng is a widely used herb in traditional medicine with extensive biological effects in various disease models, and its extract was reported to show protection in IAV-infected mice. However, the main effective anti-IAV constituents in panax ginseng remain unclear. Here, we report that ginsenoside rk1 (G-rk1) and G-rg5, out of the 23 screened ginsenosides, exhibit significant antiviral effects against 3 different IAV subtypes (H1N1, H5N1, and H3N2) in vitro. Mechanistically, G-rk1 blocked IAV binding to sialic acid in a hemagglutination inhibition (HAI) assay and an indirect ELISA assay; more importantly, we showed that G-rk1 interacted with HA1 in a dose-dependent manner in a surface plasmon resonance (SPR) analysis. Furthermore, G-rk1 treatment by intranasal inoculation effectively reduced the weight loss and mortality of mice challenged with a lethal dose of influenza virus A/Puerto Rico/8/34 (PR8). In conclusion, our findings reveal for the first time that G-rk1 possesses potent anti-IAV effects in vitro and in vivo. We have also identified and characterized with a direct binding assay a novel ginseng-derived IAV HA1 inhibitor for the first time, which could present potential approaches to prevent and treat IAV infections.

Overexpression of TRIM16 Reduces the Titer of H5N1 Highly Pathogenic Avian Influenza Virus and Promotes the Expression of Antioxidant Genes through Regulating the SQSTM1-NRF2-KEAP1 Axis.

Oxidative stress plays a vital role in viral replication. Tripartite motif containing 16 (TRIM16) is involved in diverse cellular processes. However, the role of TRIM16 in oxidative stress induced by infection of the highly pathogenic H5N1 avian influenza virus (HPAIV) is unclear. We found that under conditions of H5N1 HPAIV infection, reactive oxygen species (ROS) levels in A549 cells peaked at 24 h post infection (hpi), and antioxidant genes' expression levels were down-regulated. Overexpression of TRIM16 in A549 cells resulted in a decrease in the titter of H5N1 HPAIV and led to significant up-regulation of the antioxidant genes' expression levels, which indicates that TRIM16 positively regulates the sequestosome 1/Kelch-like associated enoyl-CoA hydratase 1 protein/nuclear factor erythrocyte 2-derived 2-like 2 (SQSTM1/NRF2/KEAP1) pathway. Under basal conditions, TRIM16 led to a modification of NRF2 through an increase in K63-linked poly-ubiquitination of NRF2. Collectively, our findings provide new insight into understanding TRIM16's role in anti-oxidative stress in H5N1 HPAIV infected A549 cells.

Neuroimaging biomarkers of small vessel disease in cerebral amyloid angiopathy-related intracerebral hemorrhage.

AIMS: The significance of the correlation of computed tomography (CT)-based cerebral small vessel disease (SVD) markers with the clinical outcomes in patients with cerebral amyloid angiopathy (CAA)-related intracerebral hemorrhage (ICH) remains uncertain. Thus, this study aimed to explore the relationship between SVD markers and short-term outcomes of CAA-ICH. METHODS: A total of 183 patients with CAA-ICH admitted to the Xuanwu Hospital, and Beijing Fengtai You'anmen Hospital, from 2014 to 2021 were included. The multivariate logistic regression analysis was performed to identify the correlation between SVD markers based on CT and clinical outcomes at 7-day and 90-day. RESULTS: Of the 183 included patients, 66 (36%) were identified with severe SVD burden. The multivariate analysis showed that the total SVD burden, white matter lesion (WML) grade, and brain atrophy indicator were independent risk factors for unfavorable outcomes at 90-day. The brain atrophy indicator was independently associated with mortality at 90-day. Severe cortical atrophy was significantly associated with early neurological deterioration. CONCLUSIONS: The neuroimaging profiles of SVD based on CT in patients with CAA-ICH might predict the short-term outcome more effectively. Further studies are required to validate these findings and identify modifiable factors for preventing CAA-ICH development.

Phylogenetic and pathogenic characterization of current fowl adenoviruses in China.

In recent years, fowl adenoviruses (FAdVs) continue to outbreak and cause huge economic losses to the poultry industry in China. The homologous recombination accounts for the diversity serotypes of adenovirus. However, the prevalence, recombination and pathogenicity of current FAdVs remain unclear. Herein, the prevalence, phylogenetic feature and pathogenicity of FAdVs in China in 2019 were characterized. Our findings showed that multiple species and serotypes of FAdVs currently circulate in China, including A, C, D and E species, and 1, 2, 4, 8a and 8b serotypes. Notably, the recombination occurred between FAdV-8a and FAdV-8b, and the recombination regions included Hexon, Fiber, ORF19 and ORF20. All five FAdVs replicated effectively in various chicken tissues, and viral shedding peaked at 4-8 dpi. Except CH/GDSZ/1905(FAdV-1/A), the remaining FAdVs caused obvious inclusion body hepatitis (IBH) in 3-week-old specific-pathogen-free (SPF) chickens, of which CH/JSXZ/1905(FAdV-4/C) caused hydropericardium-hepatitis syndrome (HHS) with a mortality rate of 62.5%. Taken together, our findings illustrate the prevalence, recombination and pathogenicity of current FAdVs in China and strengthen surveillance and further pathogenicity studies of FAdVs are extremely urgent.

Phylogenetic and Phylogeographic Analysis of the Highly Pathogenic H5N6 Avian Influenza Virus in China.

The clade 2.3.4.4b H5N8 avian influenza viruses (AIVs) have caused the loss of more than 33 million domestic poultry worldwide since January 2020. Novel H5N6 reassortants with hemagglutinin (HA) from clade 2.3.4.4b H5N8 AIVs are responsible for multiple human infections in China. Therefore, we conducted an epidemiological survey on waterfowl farms in Sichuan and Guangxi provinces and performed a comprehensive spatiotemporal analysis of H5N6 AIVs in China. At the nucleotide level, the H5N6 AIVs isolated in the present study exhibited high homology with the H5N6 AIVs that caused human infections. Demographic history indicates that clade 2.3.4.4b seemingly replaced clade 2.3.4.4h to become China's predominant H5N6 AIV clade. Based on genomic diversity, we classified clade 2.3.4.4b H5N6 AIV into ten genotypes (2.3.4.4bG1-G10), of which the 2.3.4.4bG5 and G10 AIVs can cause human infections. Phylogeographic results suggest that Hong Kong and Jiangxi acted as important epicentres for clades 2.3.4.4b and 2.3.4.4h, respectively. Taken together, our study provides critical insight into the evolution and spread of H5N6 AIVs in China, which indicates that the novel 2.3.4.4b reassortants pose challenges for public health and poultry.

Long-term outcome of cerebral amyloid angiopathy-related hemorrhage.

OBJECT: The long-term functional outcome of cerebral amyloid angiopathy-related hemorrhage (CAAH) patients is unclear. We sought to assess the long-term functional outcome of CAAH and determine the prognostic factors associated with unfavorable outcomes. METHODS: We enrolled consecutive CAAH patients from 2014 to 2020 in this observational study. Baseline characteristics and clinical outcomes were presented. Multivariable logistic regression analysis was performed to identify the prognostic factors associated with long-term outcome. RESULTS: Among the 141 CAAH patients, 76 (53.9%) achieved favorable outcomes and 28 (19.9%) of them died at 1-year follow-up. For the longer-term follow-up with a median observation time of 19.0 (interquartile range, 12.0-26.5) months, 71 (50.4%) patients obtained favorable outcomes while 33 (23.4%) died. GCS on admission (OR, 0.109; 95% CI, 0.021-0.556; p = 0.008), recurrence of ICH (OR, 2923.687; 95% CI, 6.282-1360730.14; p = 0.011), WML grade 3-4 (OR, 31.007; 95% CI, 1.041-923.573; p = 0.047), severe central atrophy (OR, 4220.303; 95% CI, 9.135-1949674.84; p = 0.008) assessed by CT was identified as independent predictors for long-term outcome. INTERPRETATION: Nearly 50% of CAAH patients achieved favorable outcomes at long-term follow-up. GCS, recurrence of ICH, WML grade and cerebral atrophy were identified as independent prognostic factors of long-term outcome.

Systematic Tracing of Susceptible Animals to SARS-CoV-2 by a Bioinformatics Framework.

Since the outbreak of SARS-CoV-2 in 2019, the Chinese horseshoe bats were considered as a potential original host of SARS-CoV-2. In addition, cats, tigers, lions, mints, and ferrets were naturally or experimentally infected with SARS-CoV-2. For the surveillance and control of this highly infectious disease, it is critical to trace susceptible animals and predict the consequence of potential mutations at the binding region of viral spike protein and host ACE2 protein. This study proposed a novel bioinformatics framework to systematically trace susceptible animals to SARS-CoV-2 and predict the binding affinity between susceptible animals' mutated/un-mutated ACE2 receptors. As a result, we identified a few animals posing a potential risk of infection with SARS-CoV-2 using the docking analysis of ACE2 protein and viral spike protein. The binding affinity of some of these species is weaker than that of humans but more potent than that of Chinese horseshoe bats. We also found that a few point mutations in human ACE2 protein or viral spike protein could significantly enhance their binding affinity, posing an enormous potential threat to public health. The ancestors of the Omicron may evolve rapidly through the accumulation of mutations in infecting the host and jumped into human beings. These findings indicate that if the epidemic expands, there may be a human-animal-human transmission route, which will increase the difficulty of disease prevention and control.

The Design of Adolescents' Physical Health Prediction System Based on Deep Reinforcement Learning.

According to the general recognition in the first half of the last century, hypertension was not considered a kind of disease, but was regarded as a compensatory response commonly seen in the elderly, and it would not occur to younger people. Because of this erroneous cognition, many young patients fail to pay attention to their own hypertension, fail to take correct and standardized treatment, and suffer from a series of complications caused by hypertension. This article summarizes the relevant factors that affect the patient's future blood pressure from three directions: the basic characteristics of adolescent patients, the way they lower blood pressure, and the impact of the external environment. In order to make the model better fit the continuous data in the feature set of adolescents with hypertension, the structure of the internal components of the deep confidence network is optimized. Gaussian noise is introduced into the visible and hidden layers of the internal components of the network so that the stored information of the network changes from discrete to continuous during operation and improves the prediction accuracy of the blood pressure prediction model for adolescents with hypertension.

Pathogenicity and transmissibility of current H3N2 swine influenza virus in Southern China: A zoonotic potential.

Swine are considered as 'mixing vessels' of influenza A viruses and play an important role in the generation of novel influenza pandemics. In this study, we described that the H3N2 swine influenza (swH3N2) viruses currently circulating in pigs in Guangdong province carried six internal genes from 2009 pandemic H1N1 virus (pmd09), and their antigenicity was obviously different from that of current human H3N2 influenza viruses or recommended vaccine strains (A/Guangdong/1194/2019, A/Hong Kong/4801/2014). These swH3N2 viruses preferentially bonded to the human-like receptors, and efficiently replicated in human, canine and swine cells. In addition, the virus replicated in turbinate and trachea of guinea pigs, and efficiently transmitted among guinea pigs, and virus shedding last for 6 days post-infection (dpi). The virus replicated in the respiratory tract of pigs, effectively transmitted among pigs, and virus shedding last until 9 dpi. Taken together, these current swH3N2 viruses might have the zoonotic potential. Strengthening surveillance and monitoring the pathogenicity of such swH3N2 viruses are urgently needed.

Tandem mass tag-based quantitative proteomics analysis reveals the new regulatory mechanism of progranulin in influenza virus infection.

Progranulin (PGRN) plays an important role in influenza virus infection. To gain insight into the potential molecular mechanisms by which PGRN regulates influenza viral replication, proteomic analyzes of whole mouse lung tissue from wild-type (WT) versus (vs) PGRN knockout (KO) mice were performed to identify proteins regulated by the absence vs. presence of PGRN. Our results revealed that PGRN regulated the differential expression of ALOX15, CD14, CD5L, and FCER1g, etc., and also affected the lysosomal activity in influenza virus infection. Collectively these findings provide a panoramic view of proteomic changes resulting from loss of PGRN and thereby shedding light on the functions of PGRN in influenza virus infection.

Duck TRIM29 negatively regulates type I IFN production by targeting MAVS.

The innate immune response is a host defense mechanism that induces type I interferon and proinflammatory cytokines. Tripartite motif (TRIM) family proteins have recently emerged as pivotal regulators of type I interferon production in mammals. Here, we first identified duck TRIM29, which encodes 571 amino acids and shows high sequence homology with other bird TRIM29 proteins. DuTRIM29 inhibited IFN-ß and IRF7 promoter activation in a dose-dependent manner and downregulated the mRNA expression of IFN-ß, IRF7, Mx and IL-6 mediated by duRIG-I. Moreover, duTRIM29 interacted and colocalized with duMAVS in the cytoplasm. DuTRIM29 interacted with duMAVS via its C-terminal domains. In addition, duTRIM29 inhibited IFN-ß and IRF7 promoter activation and significantly downregulated IFN-ß and immune-related gene expression mediated by duMAVS in ducks. Furthermore, duTRIM29 induced K29-linked polyubiquitination and degradation of duMAVS to suppress the expression of IFN-ß. Overall, our results demonstrate that duTRIM29 negatively regulates type I IFN production by targeting duMAVS in ducks. This study will contribute to a better understanding of the molecular mechanism regulating the innate immune response by TRIM proteins in ducks.

Molecular Characteristics, Antigenicity, Pathogenicity, and Zoonotic Potential of a H3N2 Canine Influenza Virus Currently Circulating in South China.

Canine influenza viruses (CIVs) could be a source of influenza viruses which infect humans because canine are important companion pets. To assess the potential risk of H3N2 CIVs currently circulating in southern China to public health, biological characteristics of A/canine/Guangdong/DY1/2019 (CADY1/2019) were detected. CADY1/2019 bound to both avian-type and human-type receptors. CADY1/2019 had a similar pH value for HA protein fusion to human viruses, but its antigenicity was obviously different from those of current human H3N2 influenza viruses (IVs) or the vaccine strains recommended in the North hemisphere. CADY1/2019 effectively replicated in the respiratory tract and was transmitted by physical contact among guinea pigs. Compared to human H3N2 IV, CADY1/2019 exhibited higher replication in MDCK, A549, 3D4/21, ST, and PK15 cells. Sequence analysis indicated that CADY1/2019 is an avian-origin virus, and belongs to the novel clade and has acquired many adaptation mutations to infect other mammals, including human. Taken together, currently circulating H3N2 CIVs have a zoonotic potential, and there is a need for strengthening surveillance and monitoring of their pathogenicity.

Variation and Molecular Basis for Enhancement of Receptor Binding of H9N2 Avian Influenza Viruses in China Isolates.

Currently, H9N2 avian influenza viruses (H9N2 AIVs) globally circulate in poultry and have acquired some adaptation to mammals. However, it is not clear what the molecular basis is for the variation in receptor-binding features of the H9N2 AIVs. The receptor-binding features of 92 H9N2 AIVs prevalent in China during 1994-2017 were characterized through solid-phase ELISA assay and reverse genetics. H9N2 AIVs that circulated in this period mostly belonged to clade h9.4.2. Two increasing incidents occurred in the ability of H9N2 AIVs to bind to avian-like receptors in 2002-2005 and 2011-2014. Two increasing incidents occurred in the strength of H9N2 AIVs to bind to human-like receptors in 2002-2005 and 2011-2017. We found that Q227M, D145G/N, S119R, and R246K mutations can significantly increase H9N2 AIVs to bind to both avian- and human-like receptors. A160D/N, Q156R, T205A, Q226L, V245I, V216L, D208E, T212I, R172Q, and S175N mutations can significantly enhance the strength of H9N2 AIVs to bind to human-like receptors. Our study also identified mutations T205A, D208E, V216L, Q226L, and V245I as the key sites leading to enhanced receptor binding of H9N2 AIVs during 2002-2005 and mutations S119R, D145G, Q156R, A160D, T212I, Q227M, and R246K as the key sites leading to enhanced receptor binding of H9N2 AIVs during 2011-2017. These findings further illustrate the receptor-binding characteristics of avian influenza viruses, which can be a potential threat to public health.

Genetic, Molecular, and Pathogenic Characterization of the H9N2 Avian Influenza Viruses Currently Circulating in South China.

The prevalence and variation of the H9N2 avian influenza virus (AIV) pose a threat to public health. A total of eight viruses isolated from farmed poultry in South China during 2017-2018 were selected as representative strains for further systematic study. Phylogenetic analyses indicated that these prevalent viruses belong to the Y280-like lineage and that the internal genes are highly similar to those of recently circulating human H7N9 viruses. The receptor-binding assay showed that most of the H9N2 isolates preferentially bound to the human-like receptor, increasing the risk of them crossing the species barrier and causing human infection. Our in vitro, multi-step growth curve results indicate these viruses can effectively replicate in mammalian cells. Infection in mice showed that three viruses effectively replicated in the lung of mice. Infection in swine revealed that the viruses readily replicated in the upper respiratory tract of pig and effectively induced viral shedding. Our findings suggested that the H9N2 AIVs circulating in poultry recently acquired an enhanced ability to transmit from avian to mammalians, including humans. Based on our findings, we propose that it is essential to strengthen the efforts to surveil and test the pathogenicity of H9N2 AIVs.