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).

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.

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.

Effect of Aluminum Hydroxide Adjuvant on the Immunogenicity of the 2009 Pandemic Influenza A/H1N1 Vaccine:Multi-level Modeling of Data with Repeated Measures

Objective To evaluate the effect of the aluminum hydroxide （Al-OH） adjuvant on the 2009 pandemic influenza A/H1N1 （pH1N1） vaccine. Methods In a multicenter, double-blind, randomized, placebo-controlled trial, participants received two doses of split-virion formulation containing 15 ug hemagglutinin antigen, with or without aluminum hydroxide （N-OH）. We classified the participants into six age categories （〉61 years, 41-60 years, 19-40 years, 13-18 years, 8-12 years, and 3-7 years） and obtained four blood samples from each participant on days 0, 21, 35, and 42 following the first dose of immunization. We assessed vaccine immunogenicity by measuring the geometric mean titer （GMT） of hemagglutination inhibiting antibody. We used a two-level model to evaluate the fixed effect of aluminum Al-OH and other factors, accounting for repeated measures. Results The predictions of repeated measurement on GMTs of formulations with or without Al-OH, were 80.35 and 112.72, respectively. Al-OH significantly reduced immunogenicity after controlling for time post immunization, age-group and gender. Conclusion The Al-OH adjuvant does not increase but actually reduces the immunogenicity of the split-virion pH1N1 vaccine.

Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

Objective To evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR （M-RTPCR）, for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing （NGS） platform. Methods Virus genome copy was quantified and seria（iy diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance. Results The M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing Iow-titer virus. Conclusion The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.

Enhancement of Virus Replication in An Influenza A Virus NS1-Expresssing 293 Cell Line

The nonstructural protein 1 （NS1） of influenza A virus, which is absent from the viral particle, but highly expressed in infected cells, strongly antagonizes the interferon （IFN）-mediated antiviral response. We engineered an NS1-expressing 293 （293-NS1） cell line with no response to IFN stimulation. Compared with the parental 293 cells,

Feedforward loop profile among transcription factor, miRNA and mRNA in influenza A virus-infected mouse lungs

Purpose:In the present study,we focused on the 46 microRNAs and 719 genes in the microRNA-gene network,reported by us,and aimed to build a research blueprint of feedforward loops and reveal the key TFs in H1N1-infected mouse lung.Method:Based on microRNAs and genes in the microRNA-gene network previously reported by us,we used Jemboss software to find relationships between TFs and microRNAs(or genes),and then built a TF-microRNA-gene network exploiting the interactions between TFs and microRNAs(or genes).Next,we searched the sequences of above genes or microRNAs near the transcription start site(TSS)area,and then used the MatchTM algorithm to predict relevant TFs,and built the TF-Gene-Network.Result:We built a TF-microRNAgene network and exploreed eight key TFs,namely NF-AT1,GKLF,SRY,SOX10,AML1,MZF1,CRX and myogenin,in the network,and then constructed subgraphs of these eight TFs.Simultaneously,we predicted the possible target genes of microRNAs and identified the feedforward regulation relationship of possible TFs,microRNAs and mRNAs.The results showed that all eight factors with a score greater than 100 were TFs,namely NF-AT1,GKLF,SRY,SOX10,AML1,CRX,myogenin and MZF1.We then constructed subtables of the above eight TFs.Conclusion:In this study,TFs including NF-AT1,GKLF,SRY,SOX10,AML1,MZF1,CRX and myogenin showed the highest score(>100)not only in the TF-microRNA-gene network but also in feedforward loops,indicating that these eight TFs play the most important roles in mouse H1N1 influenza virus infection biology.

Study on the mechanism of action of Ephedra Herba Decoction against influenza A virus based on network pharmacology

Objective To predict the main bioactive components and potential mechanisms of Ephedra Herba Decoction(Chinese Pinyin abbreviated as MHT)in treating influenza A virus(IAV)based on network pharmacology.Methods Multiple online databases were used to search and screen out the active components from MHT,the related targets of active components of MHT and the genes related to IAV.Search the corresponding genes name of target through UniProt database.Cytoscape 3.7.2 was used to construct the drug-component-target network diagram.Venn diagram was used to screen the intersection genes of the active components corresponding to the target and disease-related genes,and the intersection genes were imported into the STRING Database Online platform to obtain the protein-protein interaction(PPI)network.Then,the PPI network was imported into Cytoscape 3.7.2 to obtain the core targets.Finally,Wei Sheng Xing(http://www.bioinformatics.com.cn/)was used to do GO function enrichment analysis and KEGG signaling pathway enrichment analysis for the intersection genes,and the results of GO and KEGG were visualized.Results A total of 116 active components and 253 potential targets were screened from MHT.Quercetin,Kaempferol,and Luteolin are the main active components,and AKT1,TNF,TP53,IL6,and JUN are the core targets of 253 potential targets.There are 2906 targets of influenza A,including 121 intersection genes.Enrichment analysis showed that there were 131 entries of molecular function,89 entries of cell component,1645 entries of biological process and 195 entries of signaling pathway.PI3K-Akt,MAPK,and JAK-STAT were the main signaling pathways.Conclusion MHT plays an important role in the prevention and treatment of influenza A by acting on multiple targets and multiple signaling pathways.

Morphofunctional Characteristics of Pulmonary Surfactant System and Its Effect on Immune Cells in Influenza A (H1N1) Pathogenesis

There is an annual increase of influenza-related SARI cases in winter months. Despite the high relevance of this problem, influenza pathogenesis and the role of surfactant system and its SP-A (surfactant protein A) enzyme in antiviral defense remain poorly understood. SP-A activates macrophage M1 polarization and triggers an antiviral response due to the activation of T-cells and dendritic cells. Therefore, surfactant system is an important element of infection protection and a promising therapeutic target.