Objective: To analyze the genetic characteristics of the first human infection with the G4 genotype of Eurasian avian H1N1 swine influenza virus (EA H1N1 SIV) in Shaanxi Province. Methods: The patient's throat swab samples were collected, and MDCK cells were inoculated for virus isolation to obtain the virus strain. The whole genome deep sequencing method was used to obtain the eight gene segments of the isolated strain. The nucleotide homology analysis was conducted through the Blast program in the GenBank database, and a phylogenetic tree was constructed to analyze the genetic characteristics of the virus. Results: The throat swab specimens of the case were confirmed as EA H1N1 SIV in the laboratory, and the isolated strain was named A/Shaanxi-Weicheng/1351/2022(H1N1v). Homology analysis found that the PB2, NP, HA, NA, and M genes of this isolate had the highest nucleotide homology with A/swing/Beijing/0301/2018 (H1N1), about 98.29%, 98.73%, 97.41%, 97.52%, and 99.08%, respectively. The phylogenetic tree showed that the isolate belonged to G4 genotype EA H1N1 SIV, with PB2, PB1, PA, NP and M genes from pdm/09 H1N1, HA and NA genes from EA H1N1, and NS gene from Triple-reassortant H1N1. The cleavage site of the HA protein was IPSIQSR↓G, which was the molecular characteristic of the low pathogenic influenza virus. No amino acid mutations associated with neuraminidase inhibitors were found in the NA protein. PB2 protein 701N mutation, PA protein P224S mutation, NP protein Q357K mutation, M protein P41A mutation, and NS protein 92D all indicated its enhanced adaptability to mammals. Conclusion: The patient is the first human infection with G4 genotype EA H1N1 SIV in Shaanxi province. The virus is low pathogenic, but its adaptability to mammals is enhanced. Therefore, it is necessary to strengthen the monitoring of such SIVs.
Swine ; Humans ; Animals ; Influenza A Virus, H1N1 Subtype/genetics* ; Phylogeny ; Genotype ; Influenza A virus ; China ; Birds ; Mammals

Objective: To analyze the genetic characteristics of the first human infection with the G4 genotype of Eurasian avian H1N1 swine influenza virus (EA H1N1 SIV) in Shaanxi Province. Methods: The patient's throat swab samples were collected, and MDCK cells were inoculated for virus isolation to obtain the virus strain. The whole genome deep sequencing method was used to obtain the eight gene segments of the isolated strain. The nucleotide homology analysis was conducted through the Blast program in the GenBank database, and a phylogenetic tree was constructed to analyze the genetic characteristics of the virus. Results: The throat swab specimens of the case were confirmed as EA H1N1 SIV in the laboratory, and the isolated strain was named A/Shaanxi-Weicheng/1351/2022(H1N1v). Homology analysis found that the PB2, NP, HA, NA, and M genes of this isolate had the highest nucleotide homology with A/swing/Beijing/0301/2018 (H1N1), about 98.29%, 98.73%, 97.41%, 97.52%, and 99.08%, respectively. The phylogenetic tree showed that the isolate belonged to G4 genotype EA H1N1 SIV, with PB2, PB1, PA, NP and M genes from pdm/09 H1N1, HA and NA genes from EA H1N1, and NS gene from Triple-reassortant H1N1. The cleavage site of the HA protein was IPSIQSR↓G, which was the molecular characteristic of the low pathogenic influenza virus. No amino acid mutations associated with neuraminidase inhibitors were found in the NA protein. PB2 protein 701N mutation, PA protein P224S mutation, NP protein Q357K mutation, M protein P41A mutation, and NS protein 92D all indicated its enhanced adaptability to mammals. Conclusion: The patient is the first human infection with G4 genotype EA H1N1 SIV in Shaanxi province. The virus is low pathogenic, but its adaptability to mammals is enhanced. Therefore, it is necessary to strengthen the monitoring of such SIVs.
Swine ; Humans ; Animals ; Influenza A Virus, H1N1 Subtype/genetics* ; Phylogeny ; Genotype ; Influenza A virus ; China ; Birds ; Mammals

Transglutaminase 2 (TGM2) is a ubiquitous multifunctional protein, which is related to the adhesion of different cells and tumor formation. Previous studies found that TGM2 is involved in the interaction between host cells and viruses, but the effect of TGM2 on the proliferation of influenza virus in cells has not been reported. To explore the effect of TGM2 during H1N1 subtype influenza virus infection, a stable MDCK cell line with TGM2 overexpression and a knockout cell line were constructed. The mRNA and protein expression levels of NP and NS1 as well as the virus titer were measured at 48 hours after pot-infection with H1N1 subtype influenza virus. The results showed that overexpression of TGM2 effectively inhibited the expression of NP and NS1 genes of H1N1 subtype influenza virus, while knockout of TGM2 up-regulated the expression of the NP and NS1 genes, and the expression of the NP at protein level was consistent with that at mRNA level. Virus proliferation curve showed that the titer of H1N1 subtype influenza virus decreased significantly upon TGM2 overexpression. On the contrary, the virus titer in TGM2 knockout cells reached the peak at 48 h, which further proved that TGM2 was involved in the inhibition of H1N1 subtype influenza virus proliferation in MDCK cells. By analyzing the expression of genes downstream of influenza virus response signaling pathway, we found that TGM2 may inhibit the proliferation of H1N1 subtype influenza virus by promoting the activation of JAK-STAT molecular pathway and inhibiting RIG-1 signaling pathway. The above findings are of great significance for revealing the mechanism underlying the interactions between host cells and virus and establishing a genetically engineering cell line for high-yield influenza vaccine production of influenza virus.
Animals ; Cell Proliferation ; Dogs ; Humans ; Influenza A Virus, H1N1 Subtype/genetics* ; Influenza, Human ; Madin Darby Canine Kidney Cells ; Protein Glutamine gamma Glutamyltransferase 2

In this paper, Asarum polysaccharides(AP) were extracted, and its composition was analyzed to study the activity against H1 N1 influenza virus in vitro and its intervention effect on mice with kidney Yang deficiency syndrome. AP was prepared by the strategy of water extraction and alcohol precipitation, the content was determined, and its monosaccharide composition was analyzed. The cell Real-time monitoring system and Reed-Muench model were adopted to evaluate the antiviral activity of AP in vitro. And the mouse model of kidney Yang deficiency syndrome was established in vivo to compare the efficacy of Mahuang Xixin Fuzi Decoction(MXF) and AP. MXF group and AP group were treated with clinical equivalent doses of 1.8 g·kg~(-1)·d~(-1) and 0.077 g·kg~(-1)·d~(-1) respectively, once a day for 6 consecutive days. Real-time PCR was used to detect the relative expression of M gene of H1 N1 influenza virus and cytokines in lung tissue. The content of AP in Asarum was 25.22%, and the protein content was 0.8%. And the monosaccharide composition was identified as L-rhamnose, D-arabinose, D-xylose, D-glucose, D-galactose and D-mannose. TI values of Tamiflu, MXF and AP were 30.00, 8.06 and 10.33, respectively. Three different doses of AP could significantly reduce the concentration of virus in supernatant. Compared with the model mice, lung indexes of MXF group and AP group decreased significantly(P<0.05), and the relative expression of M gene decreased significantly(P<0.05). The relative expressions of IL-10 and IFN-γ were up-regulated to varying degrees, while the relative gene expressions of IL-1β, IL-6 and MCP-1 were down-regulated to different degrees. In addition, AP could significantly enhance the expression of TNF-α(P<0.01). AP had a good anti-influenza virus activity in vitro, and could protect mice with kidney Yang deficiency syndrome by reducing the viral load in lung tissue, decreasing inflammation damage in lung tissue, and regulating the expression of inflammatory cytokines. Compared with the prescription of MXF, AP had a better antiviral activity.
Animals ; Antiviral Agents/therapeutic use* ; Asarum ; Cytokines/genetics* ; Drugs, Chinese Herbal ; Influenza A Virus, H1N1 Subtype ; Influenza, Human/genetics* ; Lung ; Mice ; Polysaccharides

BACKGROUND: Endothelial cells play a key role in the cytokine storm caused by influenza A virus. MicroRNA-155 (miR-155) is an important regulator in inflammation. Its role in the inflammatory response to influenza A infection, however, has yet to be elucidated. In this study, we explored the role as well as the underlying mechanism of miR-155 in the cytokine production in influenza A-infected endothelial cells. METHODS: Human pulmonary microvascular endothelial cells (HPMECs) were infected with the influenza A virus strain H1N1. The efficiency of H1N1 infection was confirmed by immunofluorescence. The expression levels of proinflammatory cytokines and miR-155 were determined using real-time polymerase chain reaction. A dual-luciferase reporter assay characterized the interaction between miR-155 and sphingosine-1-phosphate receptor 1 (S1PR1). Changes in the target protein levels were determined using Western blot analysis. RESULTS: MiR-155 was elevated in response to the H1N1 infection in HPMECs (24 h post-infection vs. 0 h post-infection, 3.875 ± 0.062 vs. 1.043 ± 0.013, P = 0.001). Over-expression of miR-155 enhanced inflammatory cytokine production (miR-155 mimic vs. negative control, all P < 0.05 in regard of cytokine levels) and activation of nuclear factor kappa B in infected HPMECs (miR-155 mimic vs. negative control, P = 0.004), and down-regulation of miR-155 had the opposite effect. In addition, S1PR1 was a direct target of miR-155 in the HPMECs. Inhibition of miR-155 enhanced the expression of the S1PR1 protein. Down-regulation of S1PR1 decreased the inhibitory effect of the miR-155 blockade on H1N1-induced cytokine production and nuclear factor kappa B activation in HPMECs. CONCLUSION MiR-155 maybe modulate influenza A-induced inflammatory response by targeting S1PR1.
Down-Regulation ; Endothelial Cells ; Humans ; Influenza A Virus, H1N1 Subtype/genetics* ; Influenza A virus ; Influenza, Human/genetics* ; MicroRNAs/genetics* ; Sphingosine-1-Phosphate Receptors

Preparation of maternal strain A/PR/8/34 HA antiserum for influenza virus classical reassortment. A/PR/8/34 virus was digested by bromelain after inactivation and purification. 5%-20% sucrose continuous density gradient centrifugation method was used to purify HA protein. SIRD method was used to select the target protein. SDS-PAGE method was used to identified HA protein. High Immunogenic A/PR/8/34 HA protein was successfully prepared and HI titer reached 10240. High purity HA antiserum was identified by SIRD method. The key reagent in the classical reassortment of influenza virus was prepared, and the complete set of technical methods were explored, which laid the foundation for the independent research and development of seasonal influenza vaccine strains of China.
Animals ; Antibodies, Viral ; immunology ; Electrophoresis, Polyacrylamide Gel ; Female ; Hemagglutination Inhibition Tests ; Hemagglutinin Glycoproteins, Influenza Virus ; analysis ; immunology ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; immunology ; Influenza, Human ; immunology ; virology ; Rabbits ; Reassortant Viruses ; genetics ; immunology

Rapid and accurate identification of an influenza outbreak is essential for patient care and treatment. We describe a next-generation sequencing (NGS)-based, unbiased deep sequencing method in clinical specimens to investigate an influenza outbreak. Nasopharyngeal swabs from patients were collected for molecular epidemiological analysis. Total RNA was sequenced by using the NGS technology as paired-end 250 bp reads. Total of 7 to 12 million reads were obtained. After mapping to the human reference genome, we analyzed the 3-4% of reads that originated from a non-human source. A BLAST search of the contigs reconstructed de novo revealed high sequence similarity with that of the pandemic H1N1 virus. In the phylogenetic analysis, the HA gene of our samples clustered closely with that of A/Senegal/VR785/2010(H1N1), A/Wisconsin/11/2013(H1N1), and A/Korea/01/2009(H1N1), and the NA gene of our samples clustered closely with A/Wisconsin/11/2013(H1N1). This study suggests that NGS-based unbiased sequencing can be effectively applied to investigate molecular characteristics of nosocomial influenza outbreak by using clinical specimens such as nasopharyngeal swabs.
Databases, Genetic ; Genotype ; High-Throughput Nucleotide Sequencing ; Humans ; Influenza A Virus, H1N1 Subtype/classification/*genetics/isolation & purification ; Influenza, Human/diagnosis/*virology ; Nasopharynx/*virology ; Nucleic Acid Amplification Techniques ; Phylogeny ; RNA, Viral/analysis/metabolism ; Sequence Analysis, RNA ; Viral Proteins/genetics

To analyze influenza pathogen spectrum in Yunnan province during 2009-2014 years, and analyze HA and NA genes of influenza A H1N1. Analysis was made on the monitoring date of influenza cases in Yunnan province in recent 6 years, 23 strains of influenza virus of HA and NA gene was sequenced and analyzed by MEGA 5 software to construct phylogenetic tree. 4 times of influenza AH1N1 epidemic peak were monitored from 2009-2014 years in Yunnan Province, as the nucleic acid detection results of influenza A H1N1 accounted for 28.8% of the total. The sequencing result showed that HA and NA gene were divided into 3 groups, one was detected with H275Y mutation strains. Influenza A H1N1 is one of the important subtypes in Yunnan province and their genes have divided into three branches during the period of 2009-2014 years, the vast majority of influenza a H1N1 are still sensitive to neuraminidase inhibitors.
China ; epidemiology ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; metabolism ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; enzymology ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Molecular Sequence Data ; Mutation ; Neuraminidase ; genetics ; metabolism ; Phylogeny ; Viral Proteins ; genetics ; metabolism

To study the effect of Yinghua Pinggan granule (YHPG) against influenza A/H1N1 virus in vivo and on the immunologic function of infected mice. The intranasal influenza virus infection was adopted in ICR mouse to establish the influenza virus pneumonia model. At the 3rd and 7th day after the infection, the lung index and pathologic changes in lung tissues of mice were detected. Realtime PCR and flow cytometry were employed to observe the virus load in lung tissues and the levels of CD4+, CD8+, and CD4+/CD8+ in peripheral blood. The result showed that at the 3rd and 7th day after the infection, YHPG (15, 30 g x kg(-1)) can significant decrease in the lung index and virus load in lung tissues of mice infected with influenza virus, alleviate the pathologic changes in lung tissues, significantly increase the levels of CD4+ and CD4+/CD8+ ratio and reduce the levels of CD8+ in whole blood. This indicated that YHPG can inhibit the influenza virus replication, alleviate pulmonary damage and adjust the weak immunologic function of infected mice, with a certain therapeutic effect on mice infected by H1N1 virus in vivo.
Animals ; Antiviral Agents ; administration & dosage ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; genetics ; physiology ; Influenza, Human ; drug therapy ; pathology ; virology ; Lung ; pathology ; virology ; Male ; Mice ; Mice, Inbred ICR ; Virus Replication ; drug effects

We investigated the genetic diversity and evolution of the M gene of human influenza A viruses in Hangzhou (Zhejiang province, China) from 2009 to 2013, including subtypes of A(H1N1) pdm09 strains and seasonal A(H3N2) strains. Subtypes of analyzed viruses were identified by cell culture and real-time reverse transcription-polymerase chain reaction, followed by cloning, sequencing and phylogenetic analyses of the M gene. Assessment of 5675 throat swabs revealed a positive rate for the influenza virus of 20.46%, and 827 cases were diagnosed as. infections due to influenza A viruses. Seventy-six influenza-A strains were selected randomly from nine stages during six phases of a virus epidemic. Sequences of the M gene showed high homology among six epidemics with identities of amino-acid sequences of 98.98-100%. All strains contained the adamantine-resistant mutation S31N in its M2 protein. Two of the A(H1N1)pdm09 strains had double mutants of V27A/S31N or V271/S31N. One of the seasonal A(H3N2) viruses had another form of double-mutant R45H/S31N. Evolutionary rate of the M gene was much lower than that of the HA gene and NA gene. Compared with A(H3N2) strains, higher positive pressure on the M1 and M2 proteins of A(H1N1) pdm09 viruses was observed. Separate analyses of M1 and M2 proteins revealed very different selection pressures. Knowledge of the genetic diversity and evolution of the M gene of human influenza-A viruses will be valuable for the control and prevention of diseases.
Amino Acid Substitution ; China ; epidemiology ; Evolution, Molecular ; Genetic Variation ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Phylogeny ; Selection, Genetic ; Viral Matrix Proteins ; genetics ; Viral Proteins ; chemistry ; genetics