Role of feline ANP32 proteins in regulating polymerase activity of influenza A virus

Recently,increasing natural infection cases and experimental animal challenge studies demonstrated domestic cats are susceptible to multiple subtypes influenza A virus(IAV)infections.Notably,some subtype IAV strains could circulate in domestic cats after cross-species transmission and even infected humans,posing a threat to public health.Host factors related to viral polymerase activity could determine host range of IAV and acidic nuclear phosphoprotein 32(ANP32)is the most important one among them.However,role of cat-derived ANP32 on viral polymerase activity and host range of IAV is still unknown.In the present study,a total of 10 feline ANP32(feANP32)splice variants(including 5 feANP32A,3 feANP32B,and 2 feANP32E)were obtained from domestic cats by RT-PCR.Sequence alignment results demonstrated amino acid deletions and/or insertions occurred among feANP32 variants,but all feANP32 proteins were primarily localized to cell nucleus.Minigenome replication systems for several representative IAV strains were established and the support ability of feANP32 on IAV polymerase activity was estimated.The results indicated that most feANP32A and feANP32B splice variants were able to support all the tested IAV strains,though the support activity of a single feANP32 protein on polymerase activity varied among different IAV strains.In addition,the role of feANP32 in supporting H3N2 canine influenza virus was determined by investigating viral replication in vitro.Collectively,our study systematically investigated the support activity of feANP32 on IAV,providing a clue for further exploring the mechanism of susceptibility of cats to IAV.

Establishment and performance analysis of a new multiplex detection method for influenza an and B virus antigen

BACKGROUND Influenza A and B virus detection is pivotal in epidemiological surveillance and disease management.Rapid and accurate diagnostic techniques are crucial for timely clinical intervention and outbreak prevention.Quantum dot-encoded microspheres have been widely used in immunodetection.The integration of quantum dot-encoded microspheres with flow cytometry is a well-established technique that enables rapid analysis.Thus,establishing a multiplex detection method for influenza A and B virus antigens based on flow cytometry quantum dot microspheres will help in disease diagnosis.AIM To establish a codetection method of influenza A and B virus antigens based on flow cytometry quantum dot-encoded microsphere technology,which forms the foundation for the assays of multiple respiratory virus biomarkers.METHODS Different quantum dot-encoded microspheres were used to couple the monoclonal antibodies against influenza A and B.The known influenza A and B antigens were detected both separately and simultaneously on a flow cytometer,and the detection conditions were optimized to establish the influenza A and B antigen codetection method,which was utilized for their detection in clinical samples.The results were compared with the fluorescence quantitative polymerase chain reaction(PCR)method to validate the clinical performance of this method.RESULTS The limits of detection of this method were 26.1 and 10.7 pg/mL for influenza A and B antigens,respectively,which both ranged from 15.6 to 250000 pg/mL.In the clinical sample evaluation,the proposed method well correlated with the fluorescent quantitative PCR method,with positive,negative,and overall compliance rates of 57.4%,100%,and 71.6%,respectively.CONCLUSION A multiplex assay for quantitative detection of influenza A and B virus antigens has been established,which is characterized by high sensitivity,good specificity,and a wide detection range and is promising for clinical applications.

A Comparative Study of Myocardial Damage Caused by Novel Coronavirus Infection and Influenza A Virus Infection in Children during the COVID-19 Epidemic Period

Objective: To explore the comparative study of myocardial damage in children infected with COVID-19 and influenza A virus during the COVID-19 pandemic. Method: Retrospective analysis of myocardial injury caused by COVID-19 infection and influenza A virus infection in children during the COVID-19 from October 2022 to May 2023, including 106 cases of COVID-19 infection, that is, the COVID-19 group;And 164 cases of influenza A virus infection, namely, H1N1 group;Two groups were tested for various indicators of myocardial enzyme spectrum, and the situation of myocardial injury was compared between the two groups. Result: In the enrolled cases, there was no statistically significant difference in the prevalence rate of men and women in the COVID-19 group (P > 0.05);There was no statistically significant difference in the average age between men and women (P > 0.05);The comparison of the incidence rates between males and females in the H1N1 group showed a statistically significant difference (P 0.05);There was no statistically significant difference in the average age between the two groups of girls (P > 0.05). A comparison between two groups of various indicators of myocardial enzyme spectra showed that the results of AST, -HBDH and LDH were statistically significant (P 0.05). Conclusion: Both COVID-19 infection and influenza A virus infection in children have different degrees of myocardial damage, but COVID-19 infection causes more myocardial damage than influenza A virus infection, and influenza A virus is more prone to myocardial infarction, which deserves our attention.

Emergence of highly pathogenic avian influenza A(H5N8)clade 2.3.4.4b viruses in grebes in Inner Mongolia and Ningxia,China,in 2021

Highly pathogenic avian influenza(HPAI)subtype H5Nx viruses have spread globally and are a major concern for poultry,wild birds,mammals,and even humans(de Vries et al.2015;Zeng et al.2022).The hemagglutinin(HA)genes of H5 subtype viruses have evolved into multiple clades and some of these clades have been further divided into subclades(Cui et al.2022).Clade 2.3.4.4H5N8 HPAI viruses(HPAIVs)have caused several waves of disease outbreaks in wild birds and domestic poultry(Wang et al.2022).

A Review on 2009 Influenza A Virus

[Objective] The paper was to introduce the research progress of 2009 influenza A virus. [Method] 2009 influenza A virus was introduced from the aspects of classification and host, virology, molecular characteristics and vaccine. [Result] A novel influenza A/H1N1 virus emerged in early April 2009 quickly spread worldwide through human-to-human transmission. The virus contained a group of novel gene segments, the nearest known precursor was the virus found in swine. The virus appeared to retain the potential to infect swine again and thus continued reassort with swine viruses. All registered 2009 influenza A vaccines were tested for safety and immunogenicity in clinical trials on human volunteers, and all vaccines were found to be safe, single dose of vaccine could cause protective antibody responses. [Conclusion] The paper provided basis for further study on 2009 influenza A virus.

Structural basis for dsRNA recognition by NS1 protein of influenza A virus

Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 （NS1） protein of influenza A virus （NS1A） shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain （RBD） bound to a double-stranded RNA （dsRNA） at 1.7A. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel a-helices, dsRNA is anchored by a pair of invariable arginines （Arg38） from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.

Molecular Characterization of Avian-like H1N1 Swine Influenza A Viruses Isolated in Eastern China, 2011

Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide： H1N1, H3N2, and H1N2. European avian-Hke H1N1 viruses, which were initially detected in European pig populations in 1979, have been circulating in pigs in eastern China since 2007. In this study, six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China. Based on whole genome sequencing, molecular characteristics of two isolates were determined. Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations, especially similar to those found in China. Four potential glycosylation sites were observed at positions 13, 26, 198, 277 in the HA1 proteins of the two isolates. Due to the presence of a stop codon at codon 12, the isolates contained truncated PB1-F2 proteins. In this study, the isolates contained 591Q, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The isolates also had a D rather than E at position 92 of the NS1, a marker of mammalian adaptation. Both isolates contained the GPKV motif at the PDZ ligand domain of the 3＇ end of the NS1, a characteristic marker of the European avian-like swine viruses since about 1999, which is distinct from those of avian, human and classical swine viruses. The M2 proteins of the isolates have the mutation （S31N）, a characteristic marker of the European avian-like swine viruses since about 1987, which may confer resistance to amantadine and rimantadine antivirals. Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs, and raise more concerns about the occurrence of cross-species transmission events.

Genetic Analysis and Rescue of a Triple-reassortant H3N2 Influenza A Virus Isolated From Swine in Eastern China

One influenza H3N2 virus, A/swine/Shandong/3/2005 (Sw/SD/3/2005), was isolated from pigs with respiratory disease on a farm in eastern China. Genetic analysis revealed that Sw/SD/3/2005 was a triple-reassortant virus with a PB2 gene from human-like H1N1, NS from classical swine H1N1, and the remaining genes from human-like H3N2 virus. These findings further support the concept that swine can serve as reservoir or mixing vessels of influenza virus strains and maintain genetic and antigenic stability of viruses. Furthermore, we have successfully established a reverse genetics system based on eight plasmids and rescued Sw/SD/3/2005 through cell transfection. HI tests and RT-PCR confirmed that the rescued virus maintained the biological properties of the wild type Sw/SD/3/2005. The successful establishment of the reverse genetics system of Sw/SD/3/2005 will enable us to conduct extensive studies of the molecular evolution of H3N2 influenza viruses in swine.

Possible Impact of Global Warming on the Evolution of Hemagglutinins from Influenza A Viruses

Objective To determine if global warming has an impact on the evolution of hemagglutinins from influenza A viruses, because both global warming and influenza pandemics/epidemics threaten the world. Methods 4 706 hemagglutinins from influenza A viruses sampled from 1956 to 2009 were converted to a time‐series to show their evolutionary process and compared with the global, northern hemisphere and southern hemisphere temperatures, to determine if their trends run in similar or opposite directions. Point‐to‐point comparisons between temperature and quantified hemagglutinins were performed for all species and for the major prevailing species. Results The comparisons show that the trends for both hemagglutinin evolution and temperature change run in a similar direction. Conclusion Global warming has a consistent and progressive impact on the hemagglutinin evolution of influenza A viruses.

Generation and application of two monoclonal antibodies targeting conserved linear epitopes in the NP protein of influenza A virus

Monoclonal antibodies(mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein(NP) of influenza A virus(IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research. In this study, two mAbs against the NP protein, 10 E9 and 3 F3, were generated with recombinant truncated NP proteins(NP-1 and NP-2) as immunogens. The heavy-chain subclass of both 10 E9 and 3 F3 was determined to be IgG2α, and the light-chain type was κ. Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10 E9 and 3 F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively. We found that mAbs 10 E9 and 3 F3 reacted well with the NP protein of H1–H15 subtypes of IAV. Both 10 E9 and 3 F3 can be used in immunoprecipitation assay, and 10 E9 was also successfully applied in confocal microscopy. Furthermore, we found that the 10 E9-recognized _(84) SAGKDP_(89) epitope and 3 F3-recognized 320 ENPAH324 epitope were highly conserved in NP among all avian and human IAVs. Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.

Co-infection of SARS-COV-2 and Influenza A Virus:A Case Series and Fast Review

Coronavirus disease 2019(COVID-19)occurs in the influenza season and has become a global pandemic.The present study aimed to examine severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)co-infection with influenza A virus(IAV)in an attempt to provide clues for the antiviral interventions of co-infected patients.We described two patients who were co-infected with SARS-CoV-2 and IAV treated at Wuhan Union Hospital,China.In addition,we performed a review in PubMed,Web of Science and CNKI(from January 1 up to November 1,2020)with combinations of the following key words:“COVID-19,SARS-COV-2,influenza A and co-infection”.A total of 28 co-infected patients were enrolled in the analysis.Of the 28 patients,the median age was 54.5 years(IQR,34.25–67.5)and 14 cases(50.0%)were classified as severe types.The most common symptoms were fever(85.71%),cough(82.14%)and dyspnea(60.71%).Sixteen patients had lymphocytopenia on admission and 23 patients exhibited abnormal radiological changes.The median time from symptom onset to hospital admission was 4 days(IQR,3–6),and the median time of hospital stay was 14 days(IQR,8.5–16.75).In conclusion,patients with SARSCOV-2 and IAV co-infection were similar to those infected with SARS-COV-2 alone in symptoms and radiological images.SARS-COV-2 co-infection with IAV could lead to more severe clinical condition but did not experience longer hospital stay compared with patients infected with SARSCOV-2 alone.

Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

A quantitative real time reverse-transcription polymerase chain reaction （qRT-PCR） assay with specific primers recommended by the World Health Organization （WHO） has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin （HA） genes of the pandemic HlN1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic HIN1/2009 virus infection.

Effects of closing and reopening live poultry markets on the epidemic of human infection with avian influenza A virus

Live poultry markets（LPMs） are crucial places for human infection of influenza A（H7N9 virus）.In Yangtze River Delta,LPMs were closed after the outbreak of human infection with avian influenza A（H7N9） virus,and then reopened when no case was found.Our purpose was to quantify the effect of LPMs＇ operations in this region on the transmission of influenza A（H7N9） virus.We obtained information about dates of symptom onset and locations for all human influenza A（H7N9） cases reported from Shanghai,Jiangsu and Zhejiang provinces by May 31,2014,and acquired dates of closures and reopening of LPMs from official media.A two-phase Bayesian model was fitted by Markov Chain Monte Carlo methods to process the spatial and temporal influence of human cases.A total of 235 cases of influenza A（H7N9） were confirmed in Shanghai,Jiangsu and Zhejiang by May 31,2014.Using these data,our analysis showed that,after LPM closures,the influenza A（H7N9） outbreak disappeared within two weeks in Shanghai,one week in Jiangsu,and one week in Zhejiang,respectively.Local authorities reopened LPMs when there was no outbreak of influenza A（H7N9）,which did not lead to reemergence of human influenza A（H7N9）.LPM closures were effective in controlling the H7N9 outbreak.Reopening of LPM in summer did not increase the risk of human infection with H7N9.Our findings showed that LPMs should be closed immediately in areas where the H7N9 virus is confirmed in LPM.When there is no outbreak of H7N9 virus,LPMs can be reopened to satisfy the Chinese traditional culture of buying live poultry.In the long term,local authorities should take a cautious attitude in permanent LPM closure.

Nested RT-PCR method for the detection of European avian-like H1 swine influenza A virus

Swine influenza A virus（swine IAV） circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 influenza pandemic. Among multiple subtypes/lineages of swine influenza A viruses, European avian-like（EA） H1N1 swine IAV has been dominant since 2005 in China and caused infections in humans in 2010. Highly sensitive and specific methods of detection are required to differentiate EA H1N1 swine IAVs from viruses belonging to other lineages and subtypes. In this study, a nested reverse transcription（RT）-PCR assay was developed to detect EA H1 swine IAVs. Two primer sets（outer and inner） were designed specifically to target the viral hemagglutinin genes. Specific PCR products were obtained from all tested EA H1N1 swine IAV isolates, but not from other lineages of H1 swine IAVs, other subtypes of swine IAVs, or other infectious swine viruses. The sensitivity of the nested RT-PCR was improved to 1 plaque forming unit（PFU） m L^（-1） which was over 10~4 PFU m L^（-1） for a previously established multiplex RT-PCR method. The nested RT-PCR results obtained from screening 365 clinical samples were consistent with those obtained using conventional virus isolation methods combined with sequencing. Thus, the nested RT-PCR assay reported herein is more sensitive and suitable for the diagnosis of clinical infections and surveillance of EA H1 swine IAVs in pigs and humans.

Comparison of clinical characteristics in patients with coronavirus disease and influenza A in Guangzhou, China

BACKGROUND: This study aims to compare the epidemiological, clinical and laboratory characteristics between patients with coronavirus disease(COVID-19) and influenza A(H1N1), and to develop a differentiating model and a simple scoring system.METHODS: We retrospectively analyzed the data from patients with COVID-19 and H1N1. The logistic regression model based on clinical and laboratory characteristics was constructed to distinguish COVID-19 from H1N1. Scores were assigned to each of independent discrimination factors based on their odds ratios. The performance of the prediction model and scoring system was assessed. RESULTS: A total of 236 patients were recruited, including 20 COVID-19 patients and 216 H1N1 patients. Logistic regression revealed that age >34 years, temperature ≤37.5℃, no sputum or myalgia, lymphocyte ratio ≥20% and creatine kinase-myocardial band isoenzyme(CK-MB) >9.7 U/L were independent differentiating factors for COVID-19. The area under curves(AUCs) of the prediction model and scoring system in differentiating COVID-19 from H1N1 were 0.988 and 0.962, respectively. CONCLUSIONS: There are certain differences in clinical and laboratory features between patients with COVID-19 and H1N1. The simple scoring system may be a useful tool for the early identification of COVID-19 patients from H1N1 patients.

Integrated analysis of human influenza A(H1N1)virus infectionrelated genes to construct a suitable diagnostic model

The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analyses of the characteristic of the H1N1 virus infection-related genes,their biological functions,and infection-related reversal drugs were performed.Additionally,we used multi-dimensional bioinformatics analysis to identify the key genes and then used these to construct a diagnostic model for the H1N1 virus infection.There was a total of 169 differently expressed genes in the samples between 21 h before infection and 77 h after infection.They were used during the protein-protein interaction(PPI)analysis,and we obtained a total of 1725 interacting genes.Then,we performed a weighted gene co-expression network analysis(WGCNA)on these genes,and we identified three modules that showed significant potential for the diagnosis of the H1N1 virus infection.These modules contained 60 genes,and they were used to construct this diagnostic model,which showed an effective prediction value.Besides,these 60 genes were involved in the biological functions of this infectious virus,like the cellular response to type I interferon and in the negative regulation of the viral life cycle.However,20 genes showed an upregulated expression as the infection progressed.Other 36 upregulated genes were used to examine the relationship between genes,human influenza A virus,and infection-related reversal drugs.This study revealed numerous important reversal drug molecules on the H1N1 virus.They included rimantadine,interferons,and shikimic acid.Our study provided a novel method to analyze the characteristic of different genes and explore their corresponding biological function during the infection caused by the H1N1 virus.This diagnostic model,which comprises 60 genes,shows that a significant predictive value can be the potential biomarker for the diagnosis of the H1N1 virus infection.

Novel H1N1 influenza A virus infection in a patient with acute rejection after liver transplantation

BACKGROUND:The 2009 H1N1 influenza A virus was first identified in April 2009 and rapidly evolved into a pandemic. Recipients of solid-organ transplants have a higher risk for severe infection because of immunosuppression.There are limited reports of 2009 H1N1 influenza in liver transplant recipients,especially in China. METHODS:We present a case of a 48-year-old male liver transplant recipient with 2009 H1N1 influenza A virus.He received therapy for acute rejection after transplantation and was confirmed with H1N1 virus infection. RESULTS:The patient was started on oseltamivir(75 mg, orally twice daily)and had a benign hospital course,with defervescence and resolution of symptoms within 72 hours. The follow-up chest radiograph after discharge was normal. CONCLUSIONS:The 2009 H1N1 influenza in this hospitalized transplant recipient was relatively mild,and prolonged viral shedding was not noted.Oseltamivir can be a valid measure in immunocompromised individuals.

Clinical Characteristics of H1N1 Influenza A-Associated Mild Encephalopathy with Reversible Splenial Lesion: 4 Pediatric Cases

Objective Mild encephalopathy with reversible splenial lesion(MERS)is associated with a variety of infections and anti-epileptic drug withdrawal.Here we report the clinical characteristics of H1N1 influenza A-associated MERS based on our experience of four pediatric cases.Methods A detailed retrospective analysis of four patients with H1N1 influenza A-associated MERS was performed at Guangzhou Women and Children’s Medical Center.Results All patients exhibited mild influenza-like illness and seizures.Three patients presented with a new-onset seizure with fever after 5 years of age.75%patients had altered mental status.For all four patients,influenza A(H1N1)viral RNA was detected in throat swab specimens at least twice.Brain magnetic resonance images revealed similar ovoid lesions in the corpus callosum,mainly in the splenium and for one patient in the splenium and genu of the corpus callosum.Only one patient had an abnormal electroencephalogram tracing.Cells and protein in the cerebrospinal fluid were normal in all patients.All patients received oseltamivir and one patient received intravenous immunoglobulin.As a result,all patients fully recovered after 2 months and showed no neurologic sequelae at discharge.Conclusion This case series provides insight towards clinical features of H1N1 influenza A-associated MERS.

Knowledge and preventive measures practiced by junior high school students from Mexico City regarding influenza A (H1N1)

Background: Influenza A (H1N1) is the most recent pandemic disease that has affected the human population. Objective: To evaluate knowledge and preventive measures related with this disease one year after the epidemic of Mexico- that took place in 2009. Material and methods: An epidemiologic survey regarding influenza A (H1N1) was conducted in June 2010 among 2541 students from the second grade of all public high schools in a borough in Mexico City. The questionnaire included items on the knowledge of the disease and practice of preventive measures. Results: Most students obtained the information from television, half of them from parents and only one fifth from teachers;72% of the participants had a favorable knowledge about the disease and the measures to avoid being infected. However, only 37% practiced such preventive measures. Conclusion: Knowledge has a positive influence on practices towards health. Parents and teachers have an important role in health education, thus efforts should be directed to involve them more intensely in health education.

In silico modification of oseltamivir as neuraminidase inhibitor of influenza A virus subtype H1N1

This research focused on the modification of the functional groups of oseltamivir as neuraminidase inhibitor against influenza A virus subtype H1N1.Interactions of three of the best ligands were evaluated in the hydrated state using molecular dynamics simulation at two different temperatures.The docking result showed that AD3BF2 D ligand（N-[（1S,6R）-5-amino-5-{[（2R,3S,4S）-3,4-dihydroxy-4-（hydroxymethyl） tetrahydrofuran-2-yl]oxy}-4-formylcyclohex-3-en-1-yl]acetamide-3-（1-ethylpropoxy）-1-cyclohexene-1-carboxylate） had better binding energy values than standard oseltamivir.AD3BF2 D had several interactions,including hydrogen bonds,with the residues in the catalytic site of neuraminidase as identified by molecular dynamics simulation.The results showed that AD3BF2 D ligand can be used as a good candidate for neuraminidase inhibitor to cope with influenza A virus subtype H1N1.