Tripartite motif (TRIM) family proteins are important effectors of innate immunity against viral infections. Here we identified TRIM35 as a regulator of TRAF3 activation. Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon (IFN) in response to viral infection. Trim35-deficient mice were more susceptible to influenza A virus (IAV) infection than were wild-type mice. TRIM35 promoted the RIG-I-mediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1. IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3. TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2, thereby antagonizing its suppression of TRAF3 activation. Our in vitro and in vivo findings thus reveal novel roles of TRIM35, through catalyzing Lys63- or Lys48-linked polyubiquitination, in RIG-I antiviral immunity and mechanism of defense against IAV infection.
A549 Cells ; Animals ; Apoptosis Regulatory Proteins/immunology* ; DEAD Box Protein 58/immunology* ; Dogs ; HEK293 Cells ; Humans ; Influenza A Virus, H1N1 Subtype/immunology* ; Madin Darby Canine Kidney Cells ; Mice ; Mice, Knockout ; Orthomyxoviridae Infections/pathology* ; Proteolysis ; RAW 264.7 Cells ; Signal Transduction/immunology* ; THP-1 Cells ; TNF Receptor-Associated Factor 3/immunology* ; Ubiquitination/immunology* ; Viral Proteins/immunology*

The COVID-19 pandemic is still raging across the world. Everyday thousands of infected people lost their lives. What is worse, there is no specific medicine and we do not know when the end of the pandemic will come. The nearest global pandemic is the 1918 influenza, which caused about 50 million deaths and partly terminate the World War Ⅰ. We believe that no matter the virus H1N1 for the 1918 influenza or 2019-nCoV for COVID-19, they are essentially the same and the final cause of death is sepsis. The definition and diagnostic/management criteria of sepsis have been modified several times but the mortality rate has not been improved until date. Over decades, researchers focus either on the immunosuppression or on the excessive inflammatory response following trauma or body exposure to harmful stimuli. But the immune response is very complex with various regulating factors involved in, such as neurotransmitter, endocrine hormone, etc. Sepsis is not a kind of disease, instead a misbalance of the body following infection, trauma or other harmful stimulation. Therefore we should re-think sepsis comprehensively with the concept of systemic biology, i.e. inflammationomics.
Betacoronavirus ; Coronavirus Infections ; complications ; epidemiology ; immunology ; Humans ; Immune Tolerance ; Inflammation ; complications ; Influenza A Virus, H1N1 Subtype ; Influenza, Human ; complications ; epidemiology ; immunology ; Pandemics ; Pneumonia, Viral ; complications ; epidemiology ; immunology ; Sepsis ; etiology

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

To explore the impact of the history of infection by the influenza A virus subtype H1N1 on secondary infection by the influenza A virus subtype H9N2, pigs non-infected and pre-infected with H1N1 were inoculated with H9N2 in parallel to compare nasal shedding and seroconversion patterns. Unlike pigs without a background of H1N1 infection, nasal shedding was not detected in pigs pre-infected with H1N1. Both groups generated antibodies against H9N2. However, levels of H1N1 antibodies in pigs pre-infected with H1N1 increased quickly and dramatically after challenge with H9N2. Cross-reaction was not observed between H1N1 antibodies and H9N2 viruses. These findings suggest that circulation of the H1N1 virus might be a barrier to the introduction and transmission of the avian H9N2 virus, thereby delaying its adaptation in pigs.
Animals ; Antibodies, Viral ; immunology ; Cross Reactions ; Immune Sera ; immunology ; Influenza A Virus, H1N1 Subtype ; immunology ; physiology ; Influenza A Virus, H9N2 Subtype ; immunology ; Orthomyxoviridae Infections ; blood ; immunology ; Species Specificity ; Swine ; immunology ; virology

Ribavirin is a broad-spectrum antiviral agent and glycyrrhizin has activities of anti-inflammation, immunoregulation and anti-viral infections. To enhance antiviral efficacy and weaken side-effects of ribavirin, antiviral effects of the combination of glycyrrhizin and ribavirin were studied in the present study. Firstly, a mouse model of viral pneumonia was established by inoculation of influenza H1N1 virus. Protective effects of glycyrrhizin and ribavirin used alone or in combination against H1N1 virus infection in mice were evaluated based on the survival rate, lung index and virus titer in lungs of mice. Results showed that the combination of glycyrrhizin and ribavirin significantly inhibited the lung consolidation with a 36% inhibition ratio on the lung swell of infected mice. The combination of the two drugs exhibited synergetic effects on survival of infected mice. The combination of 50 mg · kg(-1) · d(-1) glycyrrhizin and 40 mg · kg(-1) · d(-1) ribavirin resulted a 100% protection for infected mice with a synergetic value of 36, which was significantly higher than the control group and each drug alone. This combination also resulted a significant drop of lung virus titer (P < 0.01), as well as inhibition on the production of proinflammatory cytokines IL-6 (P < 0.01), TNF-α (P < 0.01) and IL-1β (P < 0.05) induced by virus infection compared to the control. The treatment of ribavirin plus glycyrrhizin was more effective in influenza A infection in mice than either compound used alone, which suggested a potential clinical value of the combination of the two agents.
Animals ; Antiviral Agents ; pharmacology ; Disease Models, Animal ; Drug Synergism ; Drug Therapy, Combination ; Glycyrrhizic Acid ; pharmacology ; Inflammation ; immunology ; Influenza A Virus, H1N1 Subtype ; drug effects ; Interleukin-1beta ; immunology ; Interleukin-6 ; immunology ; Lung ; immunology ; virology ; Mice ; Orthomyxoviridae Infections ; drug therapy ; Pneumonia, Viral ; drug therapy ; Ribavirin ; pharmacology ; Tumor Necrosis Factor-alpha ; immunology

Adult ; Antibodies, Viral ; blood ; immunology ; Cross Reactions ; Hemagglutinin Glycoproteins, Influenza Virus ; immunology ; Humans ; Influenza A Virus, H1N1 Subtype ; immunology ; Influenza A Virus, H3N2 Subtype ; immunology ; Influenza B virus ; immunology ; Influenza Vaccines ; immunology ; Orthomyxoviridae ; immunology ; Seasons ; Vaccination

This study aims to investigate the characteristics of genomic variation of pandemic A/H1N1/2009 influenza virus isolated in Fujian Province, China. Complete genome sequence analysis was performed on 14 strains of pandemic A/H1N1/2009 influenza virus isolated from Fujian during 2009-2012. All virus strains were typical low-pathogenic influenza viruses, with resistance to amantadine and sensitivity to neuraminidase inhibitors. Eight genome fragments of all strains were closely related to those of A/California/07/2009 (H1N1) vaccine strain, with > or = 98.2% homology. Compared with the vaccine strain, the influenza strains from Fujian had relatively large variation, and variation was identified at 11 amino acid sites of the HA gene of A/Fujiangulou/SWL1155/2012 strain, including 4 sites (H138R, L161I, S185T, and S203T) involved inthree antigen determinants (Ca, Sa, and Sb). In conclusion, the influenza vaccine has a satisfactory protective effect on Fujian population, but the influenza strains from Fujian in 2012 has antigenic drift compared with the vaccine strain, more attention should therefore be paid to the surveillance of mutations of pandemic A/H1N1/2009 influenza virus.
Antiviral Agents ; pharmacology ; China ; epidemiology ; Drug Resistance, Viral ; genetics ; Genome, Viral ; genetics ; Genomics ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; genetics ; immunology ; physiology ; Influenza, Human ; epidemiology ; prevention & control ; Pandemics ; prevention & control ; Viral Vaccines ; immunology

OBJECTIVETo observe the anti-virus effects of andrographolide (AD) on the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) signaling pathway when immunological cells were infected with H1N1.

METHODSLeukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line (16HBE). The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium (MTT) assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunosorbent assay (ELISA) detection while immunological cells for real-time polymerase chain reaction (RT-PCR).

RESULTSThe optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-β promoter stimulator-1 (IPS-1), interferon regulatory factor (IRF)-7, IRF-3 and nuclear transcription factor κB (NF-κB) mRNA levels increased significantly (P<0.05). When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF-κB decreased significantly in each group with significant statistic differences (P<0.05).

CONCLUSIONSThe RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Antiviral Agents ; pharmacology ; Cells, Cultured ; Coculture Techniques ; DEAD Box Protein 58 ; DEAD-box RNA Helicases ; genetics ; metabolism ; Dendritic Cells ; drug effects ; immunology ; virology ; Diterpenes ; pharmacology ; Fetal Blood ; cytology ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; immunology ; Influenza, Human ; drug therapy ; immunology ; virology ; Interferon-beta ; genetics ; metabolism ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Leukocytes, Mononuclear ; drug effects ; immunology ; virology ; Macrophages ; drug effects ; virology ; NF-kappa B ; genetics ; metabolism ; Promoter Regions, Genetic ; drug effects ; immunology ; RNA, Messenger ; metabolism ; Signal Transduction ; drug effects ; genetics ; immunology

Nucleoprotein (NP) of influenza virus is highly conserved and type-specific. NP can trigger strong cell-mediated immune responses in host and is involved in the protection against the challenges with different subtype influenza viruses. Here, NP of an avian H5N1 (A/Hubei/1/2010, HB) was expressed by baculovirus surface-display technology and its immunogenicity as well as protective mechanism was investigated in mice infection model. Western blot and immunolabeled electron microscopy assay showed NP was displayed on baculovirus surface. ELISA results showed NP could induce high level of anti-NP IgG in the sera from NP-Bac-inoculated mice. Two cellular immune peptides (NP57-74 IQNSITIERMVLSAFDER and NP441-458 RTEIIKMMESARPEDLSF) were identified by IFN-gamma ELISPOT assay. NP57-66 and NP441-450 and NP protein could be able to trigger the activation of CD4+ and CD8+ T cells, and the response of CD8+ T was more predominant. The challenge study of mice-adapted virus A/PR/8/34 (H1N1) showed that NP-Bac could reduce viral load and attenuate the damage to lung tissue. 50% protection ratio against the virus could be detected.
Animals ; Antibodies, Viral ; immunology ; Baculoviridae ; genetics ; metabolism ; Cross Protection ; Enzyme-Linked Immunospot Assay ; Female ; Humans ; Immunity, Cellular ; Influenza A Virus, H1N1 Subtype ; genetics ; immunology ; Influenza A Virus, H5N1 Subtype ; genetics ; immunology ; Influenza, Human ; immunology ; virology ; Mice ; Mice, Inbred BALB C ; RNA-Binding Proteins ; genetics ; immunology ; T-Lymphocytes ; immunology ; Viral Core Proteins ; genetics ; immunology

OBJECTIVETo investigate the epidemiological features, genetic drift in the epitopes of hemagglutinin (HA) of the novel influenza A (H1N1) virus and oseltamivir-resistant variants characterized by H275Y and N295S mutations in children in Shanghai since the outbreak.

METHODBetween June 2009 and May 2012, a prospective surveillance study was carried out in Shanghainese children who attended the outpatient clinic of Children's Hospital of Fudan University for influenza-like illness. One-step real-time fluorescence quantitative RT-PCR was performed to detect seasonal influenza A and influenza B virus and the novel influenza A (H1N1) virus in the respiratory samples. Genetic drift from the vaccine strain in HA epitopes of the novel influenza H1N1 virus and the molecular markers associated with oseltamivir resistance in neuraminidase (NA) were analyzed.

RESULTOut of 3475 enrolled cases, the novel influenza A (H1N1) virus was confirmed virologically in 222 (6.4%) otherwise healthy children with 133 (59.9%) being boys and 89 (40.1%) girls. The median ages of children with the novel influenza A (H1N1) virus infection during the first wave from August 2009 to February 2010 and the second wave from December 2010 to February 2011 were 53.5 months and 32.0 months, respectively (Z = -4.601, P = 0.000); 119 (46.9%) had the close contact with persons suffering from fever or respiratory infection, of whom, 68 (57.1%) contacts were family members and 47 (39.5%) contacts were classmates. During the outbreak in 2009-2010 season, 66 (40.9%) were exposed to primary index cases, school students were the major exposure subjects, accounting for 50.0%. The nucleotide sequences of HA1 gene were highly homologous between the vaccine strain A/California/07/2009 and Shanghai circulating novel influenza A (H1N1) strains and only S83P mutation in epitope E of HA was detected inclusively in the circulating strains. The H275Y and N295S amino acid mutations associated with oseltamivir resistance were not found in the circulating novel influenza (H1N1) strains.

CONCLUSIONTwo major waves of the novel influenza A (H1N1) outbreaks occurred in Shanghainese children during 2009-2011. Institutional children were the major affected individuals during the 2009 pandemic wave. Households and schools were the main sites of transmission among children during influenza pandemic. Influenza vaccination should be enhanced in children and their close family contacts. The novel influenza A (H1N1) virus in Shanghai has not undergone significant genetic changes. Oseltamivir is effective for the treatment of the novel influenza A (H1N1) virus.

Adolescent ; Amino Acid Sequence ; Antiviral Agents ; pharmacology ; Child ; Child, Preschool ; China ; epidemiology ; Drug Resistance, Viral ; Female ; Hemagglutinins, Viral ; genetics ; Humans ; Infant ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; drug therapy ; epidemiology ; pathology ; virology ; Male ; Molecular Epidemiology ; Molecular Sequence Data ; Neuraminidase ; genetics ; Oseltamivir ; pharmacology ; Pandemics ; Viral Vaccines ; genetics ; immunology