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Interferon-induced transmembrane protein 3 (IFITM3) is an important member of the IFITM family. However, the molecular mechanisms underlying its antiviral action have not been completely elucidated. Recent studies on IFITM3, particularly those focused on innate antiviral defense mechanisms, have shown that IFITM3 affects the body's adaptive immune response. The aim of this study was to determine the contribution of IFITM3 proteins to immune control of influenza infectionWe performed proteomics, flow cytometry, and immunohistochemistry analysis and used bioinformatics tools to systematically compare and analyze the differences in natural killer (NK) cell numbers, their activation, and their immune function in the lungs ofBased on our results, we speculate that the NK cells are more readily activated in the absence of IFITM3, increasing mortality in

To recover broad-neutralizing monoclonal antibodies (BnAbs) from avian influenza A (H5N1) virus infection cases and investigate their genetic and functional features.We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients. The genetic basis, biological functions, and epitopes of the obtained BnAbs were assessed and modeled.Two BnAbs, 2-12D5, and 3-37G7.1, were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset. Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity (ADCC) activity. Albeit derived from distinct Ab lineages,Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus. The HA stem epitopes targeted by the BnAbs, and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

In several lately published studies, the association between single-nucleotide polymorphism (SNP, rs12252) of IFITM3 and the risk of influenza is inconsistent. To further understand the association between the SNP of IFITM3 and the risk of influenza, we searched related studies in five databases including PubMed published earlier than 9 November 2017. Ten sets of data from nine studies were included and data were analysed by Revman 5.0 and Stata 12.0 in our updated meta-analysis, which represented 1365 patients and 5425 no-influenza controls from four different ethnicities. Here strong association between rs12252 and influenza was found in all four genetic models. The significant differences in the allelic model (C vs. T: odds ratio (OR) = 1.35, 95% confidence interval (CI) (1.03–1.79), P = 0.03) and homozygote model (CC vs. TT: OR = 10.63, 95% CI (3.39–33.33), P < 0.00001) in the Caucasian subgroup were discovered, which is very novel and striking. Also novel discoveries were found in the allelic model (C vs. T: OR = 1.37, 95% CI (1.08–1.73), P = 0.009), dominant model (CC + CT vs. TT: OR = 1.48, 95% CI (1.08–2.02), P = 0.01) and homozygote model (CC vs. TT: OR = 2.84, 95% CI (1.36–5.92), P = 0.005) when we compared patients with mild influenza with healthy individuals. Our meta-analysis suggests that single-nucleotide T to C polymorphism of IFITM3 associated with increasingly risk of severe and mild influenza in both Asian and Caucasian populations.

To the Editor: Influenza virus subtype H6 was first isolated from a turkey in 1965 in the United States (1) and was subsequently found in other parts of the world (2). Over the past several decades, the prevalence of H6 virus has dramatically increased in wild and domestic birds (2–4). In China, highly pathogenic influenza A(H5N1), low pathogenicity influenza (H9N2), and H6 are the most prevalent avian influenza viruses among poultry (5). Although only 1 case of H6 virus infection in a human has been reported worldwide (6), several biological characteristics of H6 viruses indicate that they are highly infectious to mammals. Approximately 34% of H6 viruses circulating in China have enhanced affinity to human-like receptors (ɑ-2,6 NeuAcGal) (2). H6 viruses can also infect mice without prior adaptation (2,7), and some H6 viruses can be transmitted efficiently among guinea pigs (2). To evaluate the potential threat of H6 viruses to human health, we conducted a systematic serologic study in populations occupationally exposed to H6 viruses.

A rapid and sensitive H7 and N9 subtype-specific reverse transcription loop-mediated isothermal amplification assay was developed respectively for visual detection of human-infected influenza A (H7N9) virus. The reaction was performed in one step in a single tube at 63°C for 60 min with the addition of hydroxynaphthol blue dye before amplification. The detection limits of both subtype-specific assays were comparable to those of validated H7 and N9 real-time PCR assays respectively and no cross-detection was observed with influenza A pandemic H1N1, H3N2, H5N1, H9N2 or influenza B virus. The assays were evaluated further with H7N9 virus-infected clinical specimens.

Many viruses interact with the host cell division cycle to favor their own growth. In this study, we examined the ability of influenza A virus to manipulate cell cycle progression. Our results show that influenza A virus A/WSN/33 (H1N1) replication results in G 0 /G 1 -phase accumulation of infected cells and that this accumulation is caused by the prevention of cell cycle entry from G 0 /G 1 phase into S phase. Consistent with the G 0 /G 1 -phase accumulation, the amount of hyperphosphorylated retinoblastoma protein, a necessary active form for cell cycle progression through late G 1 into S phase, decreased after infection with A/WSN/33 (H1N1) virus. In addition, other key molecules in the regulation of the cell cycle, such as p21, cyclin E, and cyclin D1, were also changed and showed a pattern of G 0 /G 1 -phase cell cycle arrest. It is interesting that increased viral protein expression and progeny virus production in cells synchronized in the G 0 /G 1 phase were observed compared to those in either unsynchronized cells or cells synchronized in the G 2 /M phase. G 0 /G 1 -phase cell cycle arrest is likely a common strategy, since the effect was also observed in other strains, such as H3N2, H9N2, PR8 H1N1, and pandemic swine H1N1 viruses. These findings, in all, suggest that influenza A virus may provide favorable conditions for viral protein accumulation and virus production by inducing a G 0 /G 1 -phase cell cycle arrest in infected cells.

A real-time NASBA assay for the specific confirmation of influenza A H5N1 infection was developed and evaluated using proficiency panels distributed to the UK influenza network of laboratories and clinical samples received through the Chinese National Influenza Centre in Beijing. The aim of the proficiency panels was to determine the sensitivity and specificity of the assay on a range of influenza virus types and subtypes including different clades of influenza A H5 viruses. The assay was then evaluated using 19 clinical samples obtained from seven confirmed human cases of influenza A H5N1 infection in China. The assay was shown to have a level of sensitivity of 0.01 TCID50 and 10 copies/μl using RNA transcripts of the A/VietNam/1194/2004 H5N1 virus. During the evaluation the assay successfully detected H5N1 viruses known to infect humans from clades 1, 2.1, 2.2 and 2.3 as well as low pathogenic H5N3 avian influenza viruses. The clinical utility of the real-time NASBA assay was proven on a range of clinical samples from patients with confirmed H5N1 infection collected during 2005 and 2006. The real-time NASBA assay was demonstrated to be sensitive and rapid allowing for same day confirmation of a H5N1 infection direct from clinical samples.

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.Virus genome copy was quantified and serially 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.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 low-titer virus.The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.

Fragments encoding amino acids 76-130 in the linear conserved region (LCR) of A/Hubei/1/2010 (H5N1) HA2 was fused to hepatitis B core antigen (HBc) to generate a LCR-HBe virus-like particle (VLP). Results showed that the fusion protein of LCR-HBc was highly expressed in this prokaryotic expression system. The purified LCR-HBc particle stimulated high levels of IgG production in mice with a titer of1:12 800, and provided 50% cross-protection against lethal challenge by H1N1 viruses.

In March 2012, an H7N7 subtype avian influenza virus (AIV) named A/wild goose/Dongting/PC0360/2012 (H7N7) (DT/PC0360) was recovered from a wild goose in East Dongting Lake. We performed whole-genome sequencing of the isolate, and analyzed the phylogenetic and molecular characterization.RNA was extracted from environment samples (including fecal samples from wild bird or domestic ducks, and water samples) for detecting the presence of Influenza A Virus targeting Matrix gene, using realtime RT-PCR assay. The positive samples were performed virus isolation with embryonated eggs. The subtype of the isolates were identified by RT-PCR assay with the H1-H16 and N1-N9 primer set. The whole-genome sequencing of isolates were performed. Phylogenetic and molecular characterizations of the eight genes of the isolates were analyzed.Our results suggested that all the eight gene segments of DT/PC0360 belonged to the Eurasian gene pool, and the HA gene were belonged to distinct sublineage with H7N9 AIV which caused outbreaks in Mainland China in 2013. The hemagglutinin cleavage site of HA of DT/PC0360 showed characterization of low pathogenic avian influenza virus.Strengthening the surveillance of AIVs of wild waterfowl and poultry in this region is vital for our knowledge of the ecology and mechanism of transmission to prevent an influenza pandemic.

Wild birds (mainly Anseriformes and Charadriiformes) are recognized as the natural reservoir of avian influenza viruses (AIVs). The long-term surveillance of AIVs in wild birds has been conducted in North America and Europe since 1970s. More and more surveillance data revealed that all the HA and NA subtypes of AIVs were identified in the wild ducks, shorebirds, and gulls, and the AIVs circulating in wild birds were implicated in the outbreaks of AIVs in poultry and humans. Therefore, the AIVs in wild birds pose huge threat to poultry industry and human health. To gain a better understanding of the ecology and epidemiology of AIVs in wild birds, we summarize the transmission of AIVs between wild birds, poultry, and humans, the main results of surveillance of AIVs in wild birds worldwide and methods for surveillance, and the types of samples and detection methods for AIVs in wild birds, which would be vital for the effective control of avian influenza and response to possible influenza pandemic.

Five H9N2 avian influenza virus strains were isolated from the environmental samples in live poultry market in Qinghai Lake region from July to September, 2012. To evaluate the phylogenetic characteristics of these H9N2 isolates, the eight gene segments were amplified by RT-PCR and sequenced. The phylogenetic and molecular characteristics of the five strains were analyzed. The results showed that the HA genes of five strains shared 93. 2%-99. 1% nucleotide identities with each other, and the NA genes shared 94. 5%-99. 8% nucleotide identities. The HA cleavage site sequence of the A/environment/qinghai/ 017/2012 isolate was PSKSSRGLF, and the HA cleavage site sequences of the other four strains were all PSRSSRGLF. The HA receptor-binding site had the Q226L mutation. The M1 gene segment had the N30D and T215A mutations. The phylogenetic analysis showed that the five strains were similar to the virus A/chicken/Hunan/5260/2005 (H9N2) isolated in Hunan Province, China and were reassortant genotype viruses; the HA, NA, and NS genes belonged to the Y280-like lineage; the MP gene belonged to the G1-like lineage; the NP, PB1, PB2, and PA genes belonged to the F98-like lineage.

Vaccination is the primary strategy for the prevention and control of pandemic influenza. Because influenza virus is highly variable across strains, universal influenza vaccines need to be developed to address this problem. This review describes the research progress in conserved epitopes of influenza virus, the advances in the research and development of universal influenza vaccines based on the relatively conserved sequences of NP, M2e, HA2, and headless HA, the mechanisms of cross-protection, and the methods to improve cross-protection.

Thogoto virus belongs to the family Orthomyxoviridae. It is a tick-borne arbovirus that can infect both human and animals. Thogoto virus's genetic constitution, replication and transcription, and the function of the translated proteins are similar to influenza virus. The studies on Thogoto virus are important for us to better understand the conservative sites of influenza virus. Moreover, the animal model of Thogo-to virus is expected to be an alternative model for highly pathogenic influenza viruses. In the past years, Thogoto virus attracted limited public attention and few studies were engaged in this area. The classification of Thogoto virus, the genetic constitution and evolution, and viral proteins were included in this review. The functions of M protein and ML protein were emphasized, which were translated from the sixth segment and played an important role in viral replication, the interaction between Thogoto virus and host were also highlighted.

Since the first report of a swine influenza virus (SIV) infection in humans in 1958, cases have occurred continuously and increased significantly after the 2009 H1N1 pandemic. Although exposure to swine is thought to be a risk factor for human SIVs infections, approximately half of the reported cases had no known exposure to pigs. Besides, epidemiological investigation showed that several cases had limited human-to-human transmission. Based on the analyses of data on swine influenza virus infection in humans in this review, both the improved SIVs surveillance in humans and swine population and wider vaccination coverage among occupational workers are critical strategies in pandemic preparedness and response.

In order to investigate the relationship between selection pressure and the prevalence of antigenic clusters, we sequenced and analyzed the H3N2 influenza virus from China between 1992 and 2012.The H3N2 influenza virus (n = 1206) in China from 1992 to 2012 was analyzed, include global selection pressure and sites positive selection pressure analysis.Considering all the H3N2 influenza viruses during these 21 years, a total of four amino acid sites subject to positive selection. The global selection pressure varies with the variation of different antigenic clusters and three years with peak bottom selection pressure were identified.The global selection pressure rise from the peak bottom, a new antigenic clusters will appear andprevalent in the population, indicating the best time to replace the vaccine strain.

To prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China.Recombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses.The 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics.The 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.

Reverse genetics was used to construct the platform of flu pandemic strain A/California/07/2009 (H1N1).Eight genes fragements were amplified and ligated with bidirectional vector, recombinant plasmids were co transfected to the 293 T cells and rescued the virus. Gene sequencing, antigenic analysis and growth property were used to evaluate the rescued virus.Rescued virus show the genes sequence correct, keep the same antigenicity and similar growth property compared with wild type virus.The pandemic virus reverse genetics platform of A/California/07/2009 (H1N1) were built. Based on this platform, rescued virus hold the similarity of antigenicity and growth ability with wild type virus.

To develop a rapid duplex Real-time reverse transcription PCR (rRT-PCR) method to detect E119V mutation on neuraminidase (NA) of influenza A(H3N2) subtype with drug resistance to oseltamivir.Twenty-six NA genes of influenza A(H3N2) virus between 2000 and 2012 in GenBank database were selected as the target genes, and specific TaqMan-MGB probe was designed to target the E119V amino acid change in neuraminidase protein. rRT-PCR was then performed and evaluated for the sensitivity, specificity and reproducibility using virus with E119V mutation and clinical samples.This study described the validation of a highly sensitive and specific duplex rRT-PCR for detection of substitutions leading to the E119V amino acid change in NA protein of influenza A(H3N2). Fluorescence signals could be detected even when diluted a A (H3N2) virus (HA = 8) into 10(-5) and linear correlation between the logarithm of the viral titer with the Ct values was observed. In addition, the assay was highly specific in that there was no cross-react with other respiratory viruses, nor did two TaqMan-MGB probes. E119V substitution in quasispecies with both sensitive and resistant viruses could be detected as well. The limit of detection was 5% for quasispecies with high concentrations and 50% for quasispecies with low concentrations. The average coefficient of variation (CV) for within-run assays was 2.32% and 0.57% for H3N2-119E and H3N2-119V primer/probe sets separately, 1.77% and 0.97% for average CV of between-run assays, which exhibited good repeatability. Sequence analysis of twenty NA genes verified glutamic acid (E) at amino acid site 119, which was in consistent with the results from our rRT-PCR method.The assay developed in this study is highly sensitive and specific, and easy to operate; thereby it could be used for identification of A(H3N2) virus with E119V amino acid change in NA protein.

Swine influenza viruses (SIVs) are respiratory pathogens of pigs. They cause both economic bur den in livestock-dependent industries and serious global public health concerns in humans. Because of their dual susceptibility to human and avian influenza viruses, pigs are recognized as intermediate hosts for genetic reassortment and interspecies transmission. Subtypes H1N1, H1N2, and H3N2 circulate in swine populations around the world, with varied origin and genetic characteristics among different continents and regions. In this review, the role of pigs in evolution of influenza A viruses, the genetic evolution of SIVs and interspecies transmission of SIVs are described. Considering the possibility that pigs might produce novel influenza viruses causing more outbreaks and pandemics, routine epidemiological surveillance of influenza viruses in pig populations is highly recommended.

To study the prevalence and variation of influenza A(H3N2) viruses, the antigenic and genetic characteristics of influenza A(H3N2) viruses circulating in Mainland China during April 2011 to March 2012 were analyzed. The results showed that influenza A(H3N2) viruses increased gradually since 2012 and became the dominant strain since March. The viruses were antigenically closely related to the vaccine strain A/PER/16/09 (87.2%) and the representative virus A/FJ/196/09 (76.0%) in Mainland China. The genetic characteristics analysis results showed that recently isolated viruses belonged to the Vic/208 clade, and most of the low reaction strains also fell into the same clade. Crystal structure analysis of HA protein found that, compared with the vaccine strain A/PER/16/09, the recently isolated viruses had amino acid substitutions in the antigenic site A, B and C areas, in addition to gaining potential glycosylation sites at the amino acid position of 45 of HA and 367 of NA. Although the majority of circulating influenza A (H3N2) viruses in 2011-2012 season in Mainland China were antigeniclly matched by current influenza vaccine strain and the selected representative viruses, low reaction strains have increased since 2012, therefore it is necessary to strengthen the surveillance on the variation of influenza virus and to provide solid information for the vaccine strain selection.

Since 2002, H7 subtype avian influenza viruses (AIVs) have caused more than 100 human infection cases in the Netherlands, Italy, Canada, the United States, and the United Kingdom, with clinical illness ranging from conjunctivitis to mild upper respiratory illness to pneumonia. On March 31st, three fatal cases caused by infection of a novel reassortant H7N9 subtype were reported in Shanghai City and Anhui Province in China. With the ability of H7 subtype to cause severe human disease and the increasing isolation of subtype H7 AIVs, we highlighted the need for continuous surveillance in both humans and animals and characterization of these viruses for the development of vaccines and anti-viral drugs.

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.

To understand if the Neuraminidase (N1) of Influenza A virus at the surface of yeast-displaying system, eukaryotic expression system and the infected cells could be used for anti-NA Abs screening, their activities and bindings to five candidate Abs were assayed.The surface NA expression was obtained by transfecting by recombinant NA constructors with specific tag-labels or live virus infection. The functional activity was measured by the fluorescent assay. Their bindings to the Abs were detected by flow cytometry.The surface NAs presenting on the yeast-displaying system and eukaryotic expression system exhibited functional NA activities as the NA at the surface of virus-infected cells which showed affinities to Ab1, 4, and 5. The same bindings to Abl and 5 were found in the surface NA expressed by eukaryotic expression system while minor binding was observed in the yeast displayed-NA.The epitopes of yeast-displayed NA may be different from the NAs present at eukaryotic expression system and the infected cells which more likely suitable for the screening of anti-NA Abs.

To analyze the genetic composition of a novel H2N3 virus isolate identified from a duck cage swab in a live poultry market (LPM) in 2009 in Guangdong province of China.PCR-positive specimens were inoculated into embryonated chicken eggs and subtyped by conventional RT-PCR. All segments of the virus A/environment/Guangdong/2/2009 were sequenced, and phylogenetic trees were constructed and analyzed.The genes of this virus belong to Eurasian-lineage avian viruses. The virus is a reassortant with the HA gene from an H2N2 virus and the NA gene from an H5N3 virus. The PB1, PB2, and NP genes were from an H4N6 virus, the PA was from an H3N8 virus, the M gene was from an H1N3 virus, and the NS gene was from an H10N6 virus.A novel avian-origin reassortant H2N3 influenza virus was detected in a live poultry market. Its potential impacts and evolution should be closely monitored.

Pdm09 virus outbreak occurred in Mainland China in May 2009, a few months later, the prevalence of seasonal H1N1(sH1N1) influenza virus that already circulated in human for tens of years began to decline and disappeared afterwards. To identify the reason for the rapid decline of sH1N1 in mainland China, we sequenced the HA1 of sH1N1 during 2006-2011, and then analyzed the selective pressure in different phases. Our results showed before Pdm09 outbreak, the omega value was 0. 36 while after Pdm09 outbreak the omega value was 0. 28 and significant difference (t test, P0. 05) was identified. We concluded that sH1N1 obtained stronger purifying selection after Pdm09 outbreak in China. This might one of the major reasons causing the disappearance of sH1N1 in human.

Viral respiratory tract infection is among the leading causes of mortality and morbidity worldwide. Rapid screening methods for multiple detection of a wider range of pathogens become very important for diagnosis of respiratory infection. This article describes conventional detection technologies and several emerging multiplex assays that have potential applications in the diagnosis and monitoring of respiratory viral infections. These techniques include new rapid culture system, multiplex reverse transcription-PCR, real-time reverse transcription PCR, solid and suspension microarrays, mass spectrometry as well as metagenomics methods. The development and application of these techniques will not only improve the ability of rapid detection and control of viral respiratory infection, but play pivotal roles in the rapid characterization of new viral pathogens.

To determine whether it could protect mice from challenge of lethal influenza virus which group prior infected A(H1N1) pdm09 and H9N2 virus respectively.150 BALB/c mice are divided into three groups. Mice are infected A(H1N1) pdm09 virus (pCA07) and poultry H9N2 virus (GZ333) respectively. Infected mice are challenged with 10 times of lethal dose virus (PR8) then compare the viral load, antibody and survival of the two group mice before and after challenged.Both experimental group mice survived after challenge of lethal influenza virus and lung viral load are lower than that of the first infection. Antibodies derived from the infective virus and challenge virus.Prior infected A(H1N1) pdm09 and H9N2 virus could protect mice from challenge of lethal influenza virus.

In order to understand the prevalence and variation of influenza B viruses, the antigenic and genetic characteristics of influenza B viruses circulating in Mainland China during April, 2011 to March, 2012 were analyzed. The results showed the B Victoria lineage viruses were much more prevalent than B Yamagata lineage during this period, phylogenetic analysis showed vast majority of Victoria lineage viruses belong to genetic group 1, intra-clade reassortant between HA1 and NA gene was identified in a minor proportion of the viruses. 72.8% of the B/Victoria-lineage viruses were antigenically closely related to the vaccine strain B/Brisbane/60/2008. B Yamagata component was not included in the trivalent influenza vaccine in China during the study period, however vast majority of B Yamagata lineage viruses were antigenically and genetically closely related to the representative virus B/Hubei-Wujiagang/158/2009(97.8%) and B/Sichuan-Anyue/139/2011(85.2%) in China, reassortant between HA1 and NA was not identified in B Yamagata lineage viruses. Overall, the predominant circulating influenza B viruses in 2011-2012 season in China were matched by current influenza vaccine and the selected representative viruses were proved to represent the antigenic and genetic characteristics of the circulating viruses.

Influenza viruses are highly contagious for human population and result in acute respiratory infectious diseases ranging from mild to severe. Neuraminidase (NA) inhibitors (NAIs) (oseltamivir, zanamivir, peramivir and laninamivir), which target the NA glycoproteins of influenza A and B viruses are widely used in the prophylaxis and treatment of influenza virus infection. However, the substitutions of amino acids in NA or HA gene may lead to resistances to NAIs. NAI-resistance-related substitutions are typically specific to certain NA type or subtype. The sensitivity for NAI-resistance detection is affected by different assays used.

It is the first multicenter clinical study in China to investigate zanamivir use among Chinese adolescents and adults with influenza-like illness (ILI) since 2009, when inhaled zanamivir (RELENZA(®)) was marketed in China.An uncontrolled open-label, multicentre study to evaluate the antiviral activity, and safety of inhaled zanamivir (as Rotadisk via Diskhaler device); 10 mg administered twice daily for 5 days in subjects ≥ 12 years old with ILI. Patients were enrolled within 48 hours of onset and followed for eight days. Patients were defined as being influenza-positive if the real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) test had positive results.A total of 400 patients ≥ 12 years old were screened from 11 centers in seven provinces from March 2010 to January 2011. Three hundred and ninety-two patients who took at least one dose of zanamivir were entered into the safety analysis. The mean age was 33.8 years and 50% were male. Cardiovascular diseases and diabetes were the most common comorbidities. All the reported adverse events, such as rash, nasal ache, muscle ache, nausea, diarrhea, headache, occurred in less than 1% of subjects. Mild sinus bradycadia or arrhythmia occurred in four subjects (1%). Most of the adverse events were mild and did not require any change of treatment. No severe adverse events (SAE) or fatal cases were reported. Bronchospasm was found in a 38 years old woman whose symptoms disappeared after stopping zanamivir and without additional treatment. All the 61 influenza virus isolates (43 before enrollment, 18 during treatment) proved to be sensitive to zanamivir.Zanamivir is well tolerated by Chinese adolescents and adults with ILIs. There is no evidence for the emergence of drug-resistant isolates during treatment with zanamivir.

To describe the epidemiological and clinical features of hospitalized people less than 18 years old with influenza A (H1N1)-associated pneumonia and associated risk factors.Through Chinese Reporting System of Influenza A (H1N1), children aged under 18 years who were hospitalized with laboratory confirmed influenza A (H1N1), case report forms and related information on pneumonia were collected between 1 September 2009 and 4 July 2010. Epidemiological and clinical characteristics including demographics, underlying chronic diseases, treatment, complications and clinical outcome etc. were described. Hospitalized children with pneumonia were compared with those without the above mentioned features, through the univariate and multivariate analysis.There were 4240 influenza A (H1N1)-associated hospitalized children with case report forms identified. Of the 4107 influenza A (H1N1)-associated hospitalized children with related information on pneumonia shown in the case report forms, 2289 (55.7%) of them had pneumonia. Hospitalized children with influenza A (H1N1)-associated pneumonia had a younger median age (4.9 year old), when compared with those without pneumonia (13.1 year old, P0.0001). When compared with the hospitalized children without pneumonia, those hospitalized children with pneumonia were more likely to require intensive care unit care, using mechanical ventilation equipment to develop ARDS, respiratory failure or leading to death. Data from multivariate analysis showed that children aged6 months (OR=7.08, 95%CI: 4.15-12.06) between 6 and 23 months (aOR=8.26, 95%CI: 6.10-11.20) or between 2 to 4 year old (aOR=9.53, 95%CI: 7.39-12.29) were more likely to develop pneumonia than children aged 5 to 17. Factors as having asthma (OR=12.19, 95%CI: 5.18-28.72), cardiovascular disease (OR=5.19, 95%CI: 1.94-13.90), chronic renal diseases (OR=2.14, 95%CI: 1.02-4.53), chronic hepatic diseases (OR=5.26, 95%CI: 1.40-19.81) and allergy (OR=2.54, 95%CI: 1.64-3.93) were significantly associated with influenza A (H1N1)-associated pneumonia. Risk of complication with pneumonia had an increase when oseltamivir treatment was initiated2 days after the onset of illness.Pneumonia was a common complication among children hospitalized with influenza A (H1N1). Hospitalized children with influenza A (H1N1)-associated pneumonia were more likely to develop either severe clinical courses or outcomes than those without pneumonia.

Hepatitis B virus core antigen (HBcAg) is a major viral nucleocapsid protein of HBV. It is a 21-22kD protein consisting of 183-185 amino acids. Because of its easy purification, strong immunogenicity, high expression level, and self-assembles into the virus-like particles (VLP), HBcAg could be an efficient and safe VLP carrier for developing vaccines for various pathogens. Up to now, HBcAg VLP carrier has been an important system to develop novel vaccines and many antigen epitope genes from viruses, bacteria and parasites were expressed successfully using the system.

To study the epidemiological characteristics on the clustering nature of pandemic (H1N1) 2009 in China.Time and place distribution of pandemic (H1N1) 2009 on the nature of clustering through data from Public Health Emergency Management Information System were described.As of August 10, 2010, 2773 pandemic (H1N1) 2009 clusters, a total of 77 363 cases (including 20 deaths) were reported in the mainland of China. The most reported number of clusters was from schools and kindergartens with the total number of 2498 (accounted for 90.08% of the total number). Middle schools appeared the have the most clusters (1223, accounting for 48.96%). The number of clusters reported in the southern provinces (cities) accounted for 77.03% of the total, and was more than that in the northern provinces (cities). Two reported peaks in the southern provinces (cities) were in June and November, 2009, respectively. There was only one reported peak in the northern provinces in September, 2009.Time and place distribution characteristics on the clusters of pandemic (H1N1) 2009 were similar to the seasonal influenza, but the beginning of winter peak was much earlier and intensity of reporting was much higher on the clusters of pandemic (H1N1) 2009 than that of seasonal influenza.

To conduct a full genome sequence analysis for genetic characterization of an H3N8 influenza virus isolated from drinking water of a domestic duck farm in Poyang Lake area in 2011.The virus was cultivated by specific pathogen free (SPF) chicken embryo eggs and was subtyped into hemagglutinin (HA) and neuraminidase (NA) by real-time PCR method. Eight gene segments were sequenced and phylogenetic analysis was conducted.The NA gene of this virus belongs to North American lineage; other seven genes belong to Eurasian lineage. Compared with the viruses containing NA gene, the PB2 and PB1 gene came from different clades. And this indicates that the virus was a novel reassortant genotype. The HA receptor binding preference was avian-like and the cleavage site sequence showed a low pathogenic feature. There was no drug resistance mutation of M2 protein. The mutations of Asn30Asp, and Thr215Ala of the M1 protein implied the potential of pathogenicity increase in mice.The finding of novel genotype of H3N8 virus in drinking water in this duck farm near Poyang Lake highlighted the importance of strengthening the surveillance of avian influenza in this region, which could contribute to pinpointing the influenza ecological relations among avian, swine, and human.

Analyze the proliferation of different host H1N1 subtype influenza viruses in A549 and BEAS-2B cells.Human, avain and swine three hosts of the H1N1 influenza viruses infected A549 and BEAS-2B cells and analyze the characteristics of different periods after inocubation. Determine the receptor binding specificity of influenza virus by hemagglutination (HA) test with RBCs with two types of receptor. And the receptors on surfaces of A549 and BEAS-2B cells were tested by flow cytometry.The Cell Pathologic Effect (CPE) is obvious after 24 h inoculation in A549 cells by all the H1N1 influenza viruses, moreover, the peak hemagglutinin (HA) and 50% tissue culture cell infected dose (TCID50) titers was observed after 36 h of culturing in A549 cells. Otherwise, the CPE is not typical from 24 h-120 h inoculated by the same viruses and the HA, TCID50 titers were keep low all the periods in the BEAS-2B cell after inoculation. The receptor-binding preference of H1N1 viruses used in the study was screened by HA assay and some were found with 2-6-receptor binding affinity. Both SA a-2, 3Gal and SA a-2, 6Gal receptors were detected on A549 and BEAS-2B, furthermore, receptor density on A549 cells was significantly higher than that of BEAS-2B cells.A549 cells were susceptible to human, avian and swine H1N1 influenza viruses infection and permissively for viral replication. However, BEASE-2B cells with similar receptor pattern and epithelium-derived propriety as A549 cells were unsusceptible to their infection and replication. Possible host factors involved in effective viral infection and replication were needed further study.

To understand the possible origins, genetic re-assortment and molecular characterization of 4 highly pathogenic avian influenza A (H5N1) viruses isolated from humans in Hunan province, between 2006 and 2009.H5N1 PCR test-positive specimens were inoculated in embryonated eggs while H5N1 virus was isolated and genomes sequenced. Genome homology and genetic molecular characterization were analyzed by BLAST and MEGA 4.0.All gene segments of the 4 viruses were avian in origin. No re-assortment was found between avian influenza A (H5N1) viruses and human seasonal influenza viruses. Viruses that isolated from domestic poultry shared high similarity with the 4 human viruses in gene homology. Data from the whole genome phylogenetic analysis showed that the 4 viruses were in clade 2.3.4, while 2 viruses belonged to genotype V, and another 2 were new genotypes. Results from molecular characterization showed that amino acid sequences of HA cleavage site of the 4 viruses were PLRERRKR/G. All 4 viruses had A160T mutation in HA, a 20 amino acid deletion in the neuraminidase (NA) stalk at position 49 - 68, and a 5 amino acid deletion in the non-structural protein 1 (NS1). Most sites in the HA molecules showed that the viruses preferentially bound to avian influenza virus receptor. However, T192I mutation that might enhance the α 2, 6-linked sialic acid human influenza receptor binding had emerged in HN/1/09 and HN/2/09. D701N mutation of PB2 that increased the virulence in mice was found in HN/1/08. Analysis on drug resistance gene amino acid showed that all 4 viruses were sensitive to amantadine and oseltamivir.Highly pathogenic avian influenza A (H5N1) viruses isolated from humans in Hunan province from 2006 to 2009 were avian in origin, and the 4 viruses belonged to different genotypes. Some mutations that related to virulence and receptor binding positions had emerged in some of the strains.

Both the 2, 6 linkage and its topology on target cells are critical for the recognition by human influenza virus. The binding preference of avian flu virus H5N1 HA to the 2, 3-linked sialylated glycans is considered the major factor limiting its efficient infection and transmission in humans. To monitor potential adaptation of H5N1 virus in human population, the surveillance of receptor-binding specificity was undertaken in China.The binding specificity of 32 human H5N1 virus strains isolated from 2003 to 2009 was tested by 2, 3-specific sialidase-treated chicken red blood cell (CRBC) agglutination assay and a solid-phase direct binding assay with synthetic sialylglycopolymers.Dual binding preference to 2, 3 and 2, 6-glycans were found in two strains: A/Guangdong/1/06 (A/GD/1/06) and A/Guangxi/1/08 (A/GX/1/08). Though minor effect of short-2, 6-binding was detected in A/GX/1/08 at a low virus titer, both showed high affinity to the oligosaccharide at a high load. Notably both are of the long-2, 6-recognition, with the same topology as that of human H1N1 and H3N2 viruses.The findings suggest that human H5N1 virus in China likely acquired the potential human-adaptation ability. Further research and surveillance on receptor-binding specificity of H5N1 viruses are required.

To establish a multiplex RT-PCR-based reverse dot blot hybridization technique to detect influenza viruses.Obtain the HA nucleotide sequences of seasonal influenza H1N1, seasonal influenza H3N2, influenza H1N1 and human avian influenza H5N1 from GenBank. Design primers in conservative district and probes t in high variable region respectively, after analyzing the HA nucleotide sequences of influenza virus through the Vector NTI 9.0. Establish and optimize multiple RT-PCR system by comparing amplification efficiency and specificity at different primer concentrations. Establish the reverse dot hybridization system after optimizing the concentration of probes. To compare the sensitivity and specificity of this technique and the general RT-PCR Method through extracting the viral RNA of the mentioned influenza virus which are to be the reference substance.Successfully establish a multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses. This technique is 100-1000 times more sensitive than gel electrophoresis method, and it has a good specificity.Successfully established multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses.