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Human infection associated with a novel reassortant avian influenza H7N9 virus has recently been identified in China. A total of 132 confirmed cases and 39 deaths have been reported. Most patients presented with severe pneumonia and acute respiratory distress syndrome. Although the first epidemic has subsided, the presence of a natural reservoir and the disease severity highlight the need to evaluate its risk on human public health and to understand the possible pathogenesis mechanism. Here we show that the emerging H7N9 avian influenza virus poses a potentially high risk to humans. We discover that the H7N9 virus can bind to both avian-type (α2,3-linked sialic acid) and human-type (α2,6-linked sialic acid) receptors. It can invade epithelial cells in the human lower respiratory tract and type II pneumonocytes in alveoli, and replicated efficiently in ex vivo lung and trachea explant culture and several mammalian cell lines. In acute serum samples of H7N9-infected patients, increased levels of the chemokines and cytokines IP-10, MIG, MIP-1β, MCP-1, IL-6, IL-8 and IFN-α were detected. We note that the human population is naive to the H7N9 virus, and current seasonal vaccination could not provide protection.

The human respiratory tract is a major site of avian influenza A(H5N1) infection. However, many humans infected with H5N1 present with gastrointestinal tract symptoms, suggesting that this may also be a target for the virus. In this study, we demonstrated that the human gut expresses abundant avian H5N1 receptors, is readily infected ex vivo by the H5N1 virus, and produces infectious viral particles in organ culture. An autopsy colonic sample from an H5N1-infected patient showed evidence of viral antigen expression in the gut epithelium. Our results provide the first evidence, to our knowledge, that H5N1 can directly target human gut tissues.

A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of pandemic influenza A H1N1 virus infection. The reaction was performed in one step in a single tube at 65 degrees C for 60 min with the addition of hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was approximately 60 copies, and no cross-detection was observed. The assay was evaluated further with 50 clinical specimens diagnosed clinically with seasonal influenza or pandemic influenza A H1N1 virus infection. RT-LAMP with HNB dye was demonstrated to be a sensitive and easy assay for rapid detection of pandemic influenza A H1N1 virus.

Summary Background In December, 2007, a family cluster of two individuals infected with highly pathogenic avian influenza A (H5N1) virus was identified in Jiangsu Province, China. Field and laboratory investigations were implemented immediately by public-health authorities. Methods Epidemiological, clinical, and virological data were collected and analysed. Respiratory specimens from the patients were tested by reverse transcriptase (RT) PCR and by viral culture for the presence of H5N1 virus. Contacts of cases were monitored for symptoms of illness for 10 days. Any contacts who became ill had respiratory specimens collected for H5N1 testing by RT PCR. Sera were obtained from contacts for H5N1 serological testing by microneutralisation and horse red-blood-cell haemagglutinin inhibition assays. Findings The 24-year-old index case died, and the second case, his 52-year-old father, survived after receiving early antiviral treatment and post-vaccination plasma from a participant in an H5N1 vaccine trial. The index case's only plausible exposure to H5N1 virus was a poultry market visit 6 days before the onset of illness. The second case had substantial unprotected close exposure to his ill son. 91 contacts with close exposure to one or both cases without adequate protective equipment provided consent for serological investigation. Of these individuals, 78 (86%) received oseltamivir chemoprophylaxis and two had mild illness. Both ill contacts tested negative for H5N1 by RT PCR. All 91 close contacts tested negative for H5N1 antibodies. H5N1 viruses isolated from the two cases were genetically identical except for one non-synonymous nucleotide substitution. Interpretation Limited, non-sustained person-to-person transmission of H5N1 virus probably occurred in this family cluster. Funding Chinese Ministry of Science and Technology; US National Institute of Allergy and Infectious Diseases, National Institutes of Health; China-US Collaborative Program on Emerging and Re-emerging Infectious Diseases.

We report the use of ResPlex III for genotyping influenza A viruses. The performance characteristics of the assay with regard to H5N1 are further evaluated. The ResPlex system incorporates a novel multiplex PCR technology, target-enriched multiplex PCR, to simultaneously amplify multiple molecular targets in one reaction. The ResPlex III assay targets the H1, H2, H3, H5, H7, H9, N1, and N2 genes from the influenza A virus as well as the NS genes from influenza A (NSA) and B (NSB) viruses, providing detection and genotyping of influenza A and B viruses. The analytical sensitivities for detecting the H5, N1, and NSA genes were 1, 10 −1 , and 10 50% tissue culture infectious doses/200 μl/reaction, respectively. A total of 217 sequential clinical samples including 14 samples with human H5N1 infections were tested by the ResPlex III assay, and the results were compared to a reference standard combined with results of viral culture and conventional reverse transcriptase and real-time PCR. The clinical sensitivity and specificity for detecting H5N1 were 93.3% and 100%, respectively, indicating that different subtypes of influenza A virus can be quickly and correctly identified using the ResPlex III genotyping approach.

Human infections of H5N1 highly pathogenic avian influenza virus have continued to occur in China without corresponding outbreaks in poultry, and there is little conclusive evidence of the source of these infections. Seeking to identify the source of the human infections, we sequenced 31 H5N1 viruses isolated from humans in China (2005 to 2010). We found a number of viral genotypes, not all of which have similar known avian virus counterparts. Guided by patient questionnaire data, we also obtained environmental samples from live poultry markets and dwellings frequented by six individuals prior to disease onset (2008 and 2009). H5N1 viruses were isolated from 4 of the 6 live poultry markets sampled. In each case, the genetic sequences of the environmental and corresponding human isolates were highly similar, demonstrating a link between human infection and live poultry markets. Therefore, infection control measures in live poultry markets are likely to reduce human H5N1 infection in China.

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.

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.

Background Reverse transcription PCR (RT-PCR) and real time RT-PCR (rRT-PCR) have been indispensable methods for influenza surveillance, especially for determination of avian influenza. The movement of testing beyond reference lab introduced the need of quality control, including the implementation of an evaluation system for validating personal training and sample proficiency testing. Methods We developed a panel with lysates of seasonal influenza virus (H1N1, H3N2 and B), serials of diluted H5N1 virus lysates, and in-vitro transcribed H5 hemaglutinin (HA) and an artificial gene RNAs for RT-PCR and rRT-PCR quality control assessment. The validations of stability and reproducibility were performed on the panel. Additionally, the panel was implemented to assess the detection capability of Chinese human avian influenza networks. Results The panel has relatively high stability and good reproducibility demonstrated by kappa's tests. In the implementation of panel on Chinese human avian influenza networks, the results suggested that there were a relatively low number of discrepancies for both concise and reproducibility in Chinese avian influenza virus net works. Conclusions A quality control panel of RT-PCR and real-time RT-PCR for avian influenza A (H5N1) surveillance network was developed. An availably statistical data, which are used to assess the detection capability of networks on avian influenza virus (H5N1), can be obtained relatively easily through implementation of the panel on networks.

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.

To express and purify H5N1 influenza virus (A/Anhui/1/2005) NP in prokaryotic system and to explore the NP-interacting proteins of human bronchial epithelial cells BEAS-2B in vitro.The full length H5N1 NP gene fragment was amplified by PCR, inserted into prokaryotic expression vector (pET30a) to generate NP expression plasmid pET30a-NP. After transforming pET30a-NP into E. coli (BL21), the expression of soluble NP protein was induced by IPTG. The expressed NP protein was purified by two steps with metal chelation chromatography and ion exchange chromatography. Then the total proteins of BEAS-2B cells was extracted for screening the components which have protein-protein interaction with purified NP by pull-down and LC-MS/MS methods.The expression of H5N1 NP protein could be induced by IPTG in bacterial system using expression plasmid pET30a-NP. The soluble NP was purified. Twenty proteins were found by pull-down and LC-MS/MS, the further experiments may be needed to prove protein-protein interaction between them.The soluble H5N1 NP fusion protein with high purity was obtained and twenty proteins were found which could interact with it by pull-down and LC-MS/MS.

Background Adamantane-derived drugs have been used for treatment and prophylaxis of influenza A virus infection for many years worldwide. Rapid surveillance of antiviral drug resistance is important for appropriate clinical guideline development. Here, we retrospectively assessed adamantane resistance among different influenza A subtypes (H1N1, H3N2 and H5N1) over 53 years (1956–2009) in mainland China. Methods A total of 1,451 viruses, including 773 H3N2 viruses, 647 H1N1 viruses and 31 human H5N1 viruses, were analysed by matrix gene sequencing and assayed for drug resistance. Results Our results show that the prevalence of adamantane- resistant H3N2 viruses was low between 1956 and 2002, but substantially increased in 2003 to the extent that since 2006 all H3N2 viruses have been drug resistant. The percentage of adamantane- resistant H1N1 viruses also increased from 50.0% in 2004 to 98.7% in 2007; however, this decreased to 46.7% in 2009. Only three adamantane-resistant H5N1 viruses have been detected since 2003, when the first case of human H5N1 virus infection was detected in mainland China. Phylogenetic analysis demonstrated that the increase of adamantane-resistant isolates was caused by point mutations or intrasubtype reassortment instead of intersubtype reassortment. Conclusions Because of the high percentage of adamantane- resistant H3N2 and H1N1 viruses in mainland China, the use of amantadine and rimantadine drugs for prophylaxis and treatment of current seasonal influenza A infection is not recommended.

To analyse the correlation between the virus isolation and the specimen collection of the H5N1 human high pathogenic avain influenza cases in Mainland China.The specimens were collected in Mainland China from 2005.10 to 2009.3 and the H5N1 viruses were isolated by passage in embryonated chicken eggs.Most specimens were obtained within 14 days after disease onset. For the specimens collected within 7 days, the isolation rate was relatively high and the difference of the positive rate between different years was lower than those specimens collected after 7 days. Most of the samples in our study were collected from the upper or lower respiratory tract with few from blood, feces, et al. The isolation rate of lower respiratory specimens was higher and the difference of the positive rate between different years was relatively lower than those from upper respiratory specimens.We suggest that the samples should be collected from lower respiratory tract during the acute phase to get the higher isolation rate.

A new flu caused by a novel influenza A(H1N1) virus has spread over the United States, Mexico and more than 40 other countries. And because of the immediate global concern, WHO has announced that the current level of influenza pandemic alert is raised to phase 5, indicating approaching of an influenza pandemic. As patients suffering from the influenza A (H1N1) have the similar symptoms as patients with seasonal influenza, differential detection and identification of the influenza virus have to depend on specific laboratory tests. We have successfully developed a RT-PCR based method for detection of the influenza A (H1N1) virus, and had applied the method to detection of clinical samples.

To provide a technology platform for vaccine development as well as the research on transmission and pathogenesis, the reverse genetic system for H9N2 avian influenza virus was established.Eight full-length cDNAs of avian influenza virus A/Guangzhou/333/99 (H9N2) were amplified by RT-PCR and separately cloned into the transcription/expression vector, pCI-polI. The 8 plasmids DNA was cotransfected into 293T cell, the cell supernatant was collected and inoculated into embryonated eggs, the rescued virus from the allantoic fluid was identified by hemagglutinination assay.The avian influenza H9N2 virus was successfully rescued by 8 plasmids co-transfection in 293T cells. The hemagglutinination titer of the rescued virus is up to 2(9)/50 microl and its growth curve remained relatively as to the wild-type virus.The reverse genetic for avian influenza H9N2 subtype virus has been established successfully.

To establish the DNA microarray to detect influenza viruses and avian influenza viruses, and identify their virulence.Hemagglutinin (HA), neuramidinase (NA) and nucleoprotein(NP) genes were chosen simultaneously as targets for designing a microarray used for detection of viruses and identification virulence. The nucleic acid were amplified by single primer amplication (SPA). And then its specificity,sensitivity and reproducibility were evaluated.The microarray was able to specially detect H1N1, H3N2, B influenza viruses and H5N1, H9N2 avian influenza viruses. Their limits were 8HAU, 16HAU, 32HAU, and 8HAU, 8HAU respectively. The limit for virulence was 32HAU. When samples were analyzed by both RT-PCR and microarray in parallel, the results agreed in 83.9% (47/56).The microarray can detect and distinguish five tested viruses, and especially identify virulence. It not only supplies an assistant tool for clinical diagnosis and control of infectious disease, but also is valuable for controlling and preventing outbreak of avian influenza epidemic.

To investigate the cause of death of a pregnant woman with undefined pneumonia reported from the People's Hospital of Tongling City in Anhui Province on November 8, 2005, the patient's tracheal aspirates and serum samples were collected and tested by RT-PCR and Real-time PCR to detect viral nucleic acids of HA of A/H5N1, A/H7N7, A/H9N1 and A/M. Tracheal aspirates were inoculated into special pathogen free (SPF) embryonated eggs for cultivation and identification of virus. The HA gene of the virus was sequenced and analyzed. Serum samples were tested by HI assay to detect antibody of H5N1. The results showed that HA gene of A/H5N1 virus and A/M were positive in tracheal aspirates by both PCR tests. The serum sample collected on Nov. 9 was A/M gene positive by Real-time PCR. The analysis of HA gene of A/AnHui/1/2005 sequence showed that the receptor specificity and the connecting peptide between HA1 and HA2 were still avian influenza origin. The HI antibody of H5N1 was negative at 7th, 8th, 9th d of disease onset. This undefined pneumonia case was confirmed as the first pregnant woman case of avian influenza (H5N1) virus infection by etiology in the mainland of China.

To study the correlation of human influenza A/H3N2 hemagglutinin gene variation and the epidemic from 1995 to 2005 in China, 550 HA1 sequences of H3N2 viruses isolated in China were analyzed with phylogenetic tree. The results showed that the evolution of HA1 represented a long trunk with short side branches. The animo acid changes of HA1 mostly located at the antigenic sites or aside of them, but also may occur at the other sites simultaneously. The analysis also showed three possibilities of HA1 variation to cause H3N2 epidemic, the first is multiple site mutations happened simultaneously; the second is mutation sites happened gradually and then accumulated to multiple sites; the third is a single antigenic site mutation occurred simultaneously with the receptor binding site variation.

The NA genes of 395 strains of human H3N2 influenza virus isolated from 1996 to 2005 in China were sequenced, analyzed with bioinformatics tools. The NA nucleotide sequence of phylogenetic tree showed a main evolution branch with multiple short side branches. The strains in the same year may be divided into several branches. There was an obvious lag between vaccine strains recommended by WHO and the Chinese circulating strains in phylogenetic tree of the NA nucleotide. The result also showed no amino acid deletion and insertion in the NA. In NA antigen sites, where including residues 197-199 aa, 431-434 aa and 339-347aa the mutation was higher, in contrast, the residues including 153 aa, 328-336 aa, 367-370aa and 400-403 aa, the mutation was lower. Besides the antigenic determinant sites, there also had the other amino acid mutated highly, such as 18, 23, 30, 93, 143, 208, 216, 221, 249, 265, 267, 307, 385 and 437 aa, among them 143 and 267 mutation were higher than that in antigenic determinant sites, their biological significance are not clear yet. The neuraminidase active-site residues in NA were highly conservative and the same were the disulphide bond and the glycosylation sites in NA. In conclusion, our analysis provides some information for influenza prevention and control and the NA inhibitor medicine application.

To analyze the genetic and antigenic characteristics of hemagglutinin (HA) gene of human influenza B virus isolated from the mainland of China since 2004-2005.The single-way hemagglutinin inhibition (HI) tests were used to test the antigenic characteristics, and the HA1 gene was sequenced based on the antigenic results.The Yamagata-like and Victoria-like viruses co-circulated in 2004-2005. For the Yamagata-like virus, the single-way HI results showed that 3.7% and 4.5% of the viruses had 4-fold greater HI titer difference compared with B/Shanghai/361/02 in 2004 and 2005, respectively. The HA1 sequence data showed that the virus had amino acid mutation, and there was one more glycosylation site at 196th site. For the Victoria-like virus, the single-way HI results showed that 8.5% and 20.6% of the viruses had 4-fold greater HI titer difference compared with B/Hong kong/330/01 in 2004 and 2005, respectively. The HA1 sequence data showed that the virus had replacement of 9 amino acids, and there was one more glycosylation site at 197th site.The results showed that influenza B viruses had changed antigenic and genetic characteristics compared with B/Shanghai/361/02, B/Hong kong/330/01 in 2004-2005.

To analyze the genetic and antigenic characteristics of hemagglutinin (HA) gene of human influenza (H1N1) virus isolated from the mainland of China since 2004 to 2005.The single-way hemagglutination inhibition (HI) tests were used to test the antigenic characteristics, and the HA1 gene was sequenced based on the antigenic results.The single-way HI results showed that no virus isolates had 4-folds greater HI titer compared with A/Shanghai/1/1999 (H1N1) in 2004, but there was 6.3% virus had 4-fold greater difference in 2005. The HA1 sequence data showed that the H1N1 virus had the following amino acid mutations such as 54 KR, 90 TK, 101 YH, 149 RK, 169 VA, 190 DN, 212 RK, 219 KR, 245 WR, 246 YF, 258 TN, 318 VA and the 54 and 190 amino acids located in antigenic group of HA1.The H1N1 virus was changing in antigenic and genetic characteristics.

To study the incidence of adamantane resistance among influenza A (H3N2) viruses isolated from the mainland of China since 1989 through our influenza surveillance system, and to provide more information for the clinical usage of adamantane drugs.Totally 584 influenza A (H3N2) virus strains were randomly selected from our surveillance network since 1989, the adamantane drug resistance related gene M2 of all 584 strains was sequenced, and the drug sensitivity of viruses was also assayed by using biological methods in cells.No adamantane resistant strains were detected among the strains isolated from 1989 to 1999, but there was a surprisingly increased resistance rate of 56% in 2003 compared with 3.4% in 2002, and in 2005 the resistance rate increased to 77.6%.Over 50% of virus among the strains isolated showed adamantane resistance since 2003, and the incidence rate is increasing.

To establish a method for H5N1 RNA detection and laboratory diagnosis of suspected human avian influenza (H5N1) virus infected cases.The typing and sub-typing primers were designed according to M and H5 and N1 gene respectively, and the RT-PCR and real-time PCR were developed using these primers.The RT-PCR and real-time PCR could be used for H5N1 detection specifically, and there was no cross reaction with other influenza subtypes such as H1 and H3. The sensitivity for RT-PCR and real-time PCR was 1 TCID50 and 0.01 TCID50 respectively. Thirteen laboratory confirmed human H5N1 cases were detected from 42 suspected cases by using these methods.The established RT-PCR and real-time PCR methods can be used for laboratory detection of suspected human H5N1 cases.

To describe the clinical, laboratory and radiological presentation of a human case infected by influenza A (H5N1), and to understand its management and prognosis.The clinical and autopsy data of the first human case infected by influenza A (H5N1) in Jiangxi Province were collected and analyzed.The first case infected by influenza A (H5N1) in Jiangxi Province was confirmed by laboratory findings with reverse transcription-polymerase chain reaction (RT-PCR) and influenza A (H5N1) isolation. The patient had been healthy in the past and exposed to the environment of bird flu before illness. The initial symptoms included high fever with influenza-like symptoms, and then cough and purulent sputum mixed with blood appeared. The clinical situation deteriorated progressively with occurrence of diarrhea and dyspnea. Laboratory abnormalities included decrease of peripheral white blood cells and lymphocytes, urine protein, dramatic increase of enzymes associated with hepatic injury and myocarditis and decrease of serum albumin. Six days later, penicillin-resistant streptococcus pneumoniae was isolated from multiple sputum cultures. With the deterioration of clinical situation, several other bacteria and fungi were found in sputum culture. Pulmonary infiltrates were evident in right middle and lower lobe at day 5 after illness, and rapidly progressed to involve bilateral lungs as acute respiratory distress syndrome (ARDS)-like changes. The patient was treated with antiviral, antibacterial, and antifungal reagents, and corticosteroids and invasive mechanical ventilation were also administered, but without any improvement. The patient died 27 days after the onset of symptoms and an autopsy was performed. Pathologically, the lungs exhibited diffuse alveolar damage. The lymphocytes in the spleen, the lymph nodes and the tonsils were depleted prominently with histiocytic hyperplasia and hemophagocytic phenomena. Edema and degeneration of myocytes in the heart and extensive acute tubular necrosis in the kidney were observed.The prognosis was very poor if influenza A (H5N1) infected human cases was developed as ARDS with multiple organ damage or failure.

To study the antigenic and genetic characteristics of influenza (H3N2) virus circulated in China in 2004.Single-way and cross-way hemagglutination inhibition (HI) tests were firstly used to determine the reactivity with the reference serum of virus isolates. Based on the serological results, virus isolates were selected according to the different time and location in China in 2004. The HA1 domain of HA gene of those virus isolates were then sequenced in order to analyze the gene characterization.Single-way HI test results showed that 52.3% of isolates showed 4 folds or more HI titer difference compared to A/Fujian/411/2002 (H3N2) itself (international reference strain in 2004). Cross-way HI test results showed that the antigenic ratio was 4. The nucleic acid and amino acid sequence data of HA1 domain showed that the mutated virus appeared in early February of 2004, and became the dominant circulating strain gradually. There were four important mutant positions, they were 159 YF, 189 SN, 145 KN, 226 VI, respectively. The results also indicated that the mutated viruses originated from southern China, then transmitted to northern China, according to the analysis of time and location distribution.The HA1 domain of HA gene of influenza virus (H3N2) isolated from 2004 in China showed mutation and antigenic drift, and the mutated viruses were becoming the dominant circulating strain in China, and showed amino acid sequence difference compared to A/Fujian/411/2002 (H3N2) A/Wellington/1/2004 (H3N2), the vaccine components pronounced by WHO for 2004-2005 northern hemisphere and 2005 southern hemisphere respectively, which suggested that further surveillance should be conducted to monitor the virus mutation in circulation.

To understand whether pigs play a role in human infection with avian influenza A H9N2 viruses.The target gene was amplified by RT-PCR, and the PCR product was linked to PGEM-T Vector (Promega, USA) at 4 degrees C, the recombined plasmid was transferred into dH5a bacteria, and the positive colonies were selected and identified with restriction endonuclease. Afterwards, they were sent to Liu He Tong Company in Beijing for nucleotide sequencing. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseq (Version 3.69) software.The genomic characterizations of A/Swine/Shandong /5/2002(H9N2) and A/Swine/Shandong/10/2002(H9N2) viruses were different from those of H9N2 viruses which were isolated either from men or from chickens. The genomic characteristics of H9N2 viruses isolated from humans in China mainland were similar to those of H9N2 viruses isolated from chickens. Whereas, the genomes of H9N2 viruses isolated from men in Hong Kong, China were closely related to those of H9N2 viruses isolated from quails. Avian influenza A H9N2 viruses not only have wide range of host, but their genomes are also diverse.Avian influenza A H9N2 viruses can directly infect human. Avian influenza A H9N2 viruses did not require to pass through the pigs as mixing vessels prior to infecting man.

To understand the origin of HA and NA genes of swine influenza A (H9N2) viruses isolated from pigs in the mainland of China and on basis of these to reveal the pathogenicity of them in pigs.The target gene was amplified by PCR, the PCR product was ligated with PGEM-T Easy Vector (Promega company, USA) at 4 degrees, the recombined plasmid was transferred into DH-10-beta bacteria; positive colonies were selected and identified then digested with restriction enzyme. Afterwards,the nucleotide sequence was determined. Finally,phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseg (Version 3.69) softwares.Two strains of swine influenza A(H9N2) virus isolated in the mainland had an amino acid residue, leucine (L) at position 226 (H3 numbering) on HA protein molecule found in H9N2 viruses isolated either in pigs or humans previously; the amino acid sequence at HA connecting peptide of isolates possessed R-L-S-R, whereas the other H9N2 viruses with virulence in poultry had R-S-S-R at HA connecting peptide. The two pig H9N2 isolates shared the same three-amino-acids deletion in the NA stalk at 62.64 position found in A/Shaoguan/408/98 and A/Swine/Hong Kong/9/98, as well as A/Duck/Hong Kong /Y280/97(H9N2) viruses. The analysis of the phylogenetic tree indicated that the HA and NA genes of new isolates were closely related to those of A/Chicken/Hong Kong/G23/97 and A/Chicken/Hong Kong/G9/97 and A/Shaoguan/408/98 viruses, respectively.The HA and NA genes of swine influenza A(H9N2) viruses isolated in the mainland of China probably were derived from those of avian influenza A(H9N2) virus. The occurrence of substitution of amino acid sequence at HA connecting peptide, could result in the H9N2 virus from non pathogenic to pathogenic in pigs. However, avian influenza A(H9N2) virus had deletion in the stalk of the NA that resulted in host range transmission. Therefore they could infect pigs directly.

To complete the full-length sequencing of the human adenovirus type 7 vaccine strain (Ad7v) for novel vector constructing.The Ad7v DNA was digested with SalI and the 17.5-68.0 map unit (mu) fragment was cloned and sequenced. The homology of encoding sequence of Ad7v hexon to those of group A,C,D,E,F and other numbers of group B was accomplished with the software CLUSTAL.V. The three-dimensional structure of the Ad7v hexon was predicted with the RasMo12.71.The fragment contains 17,596 bp, part of E2 and late gene L1, L2 and L3 were encoded by this region. Polypeptide encoded by hexon gene lies in L3 region, which is composed of 934 amino acids. Multiple sequence alignment with the other nine known hexon protein sequences suggested that the variable sequences are mainly concentrated on seven regions, namely hypervariable regions (HVRs). The seven HVRs are related to type-specificity and group-specificity. The three-dimensional structure of the Ad7v hexon revealed that the variable regions are located in the I1 and I2 loops of the molecule mostly on the tower of the hexon.The full-length genome sequencing of Ad7v was accomplished at last. Since the deduced amino acid sequence of Ad7v hexon was quite different from other adenoviral vectors such as Ad5 and Ad2, this virus can be potentially used for the construction of novel gene delivery vectors to counterpart the immunity to the vectors widely used at present.

To understand the origin of hemagglutinin (HA), and neuraminidase (NA) gene of swine influenza A (H1N1) viruses isolated in pigs in mainland China in 2002 and reveal the reason of pathogenesis of them in pigs.The target gene amplified by PCR,PCR product was linked with PGEM-T Easy Vector(Promega company, USA) at 4? degrees C, the recombined plasmid was transferred into DH 10B bacteria, positive colonies were selected and identified them with restriction enzyme. Afterwards, they were sent to Liu He Tong company in Beijing for testing nucleotide sequence. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03)and Editseq (Version 3.69) software.The HA and NA genes of three strains of swine influenza A (H1N1) viruses isolated from pigs in China were closely related to those of swine influenza A (H1N1) virus, but different from those of avian or human influenza A (H1N1) virus. The swine strain of influenza A (H1N1) virus isolated in 2002 was derived from swine influenza A (H1N1) virus circulated in pigs in China in 1991. Since the antigenic drifts of HA and NA proteins of the new isolates occurred, their activity in pigs is increasing and they can cause disease in pigs.The HA and NA genes of three strains of influenza A (H1N1) virus tested were identified to be derived from those of swine influenza A (H1N1) virus. The increased activity and pathogenesis of them in pigs were most likely due to antigenic drifts of HA and NA proteins of the new isolates.

To understand the characteristics of internal genes of two strains of influenza A(H9N2) virus isolated from men and on the basis of these to reveal the origin of these two strains of influenza A(H9N2)virus.The target gene was amplified by RT-PCR,the PCR product was ligated with P GEM-T Vector (Promega Company, USA) at 4 degrees, the recombined plasmid was transferred into dH5a bacteria, and the positive colonies were selected and identified with restriction enzyme. Afterwards, they were sent to Liu He Tong Company in Beijing for nucleotide sequencing. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03)and Editseq (Version 3.69) softwares.Internal genes of the two strains of H9N2 virus were G9 lineage. There was a slight difference in nucleotide sequence in PA gene between the two strains, whereas another five gene segments were identical to each other.The genomes of the two strains of influenza A(H9N2)virus were G9 lineage.They were transmitted to men separately from different avian sources with different characteristics of gene of influenza A(H9N2) virus, respectively.

To understand the antigenic and genetic characteristics of influenza A H3N2 viruses circulated in man in China from 2000 to 2002.Embryonated chicken eggs inoculated with virus for amplification of viral yield. The harvesting egg allantoic fluids with influenza viruses were provided for testing antigen and RNA extraction. Virion RNA was transcribed into cDNA by reverse transcriptase, cDNA amplified by PCR, and the product of PCR was purified. Afterward RNA sequence analysis was performed by the dideoxynucleotide chain termination method using synthetic oligodeoxynucleotide primers. Finally the phylogenetic tree was analyzed with MegAlign software.The H3N2 viruses isolated during 2000-2002 were different in amino acid sequences on HA1 domain protein molecule from those of A/Wuhan359/1995 H3N2 as well as those of A/Sydney/7/1997 H3N2 strains. There were four different positions of amino acid sequence on HA1 domain protein molecule among the H3N2 viruses isolated in 2000 and during 2001-2002. They located at 83, 186, 202, 222 and 225 position, respectively. Of them 83 and 186 were in antigenic determinant E and B, respectively. The others located at left wall of the receptor binding site (RBS).From the end of 2001 to the beginning of 2002, the influenza epidemic in Northern China caused by H3N2 virus was due to occurrence of antigenic and genetic changes of influenza A(H3N2) virus.

To clarify the features of gene variation among epidemic strains of human rotavirus NSP4 in China.SP4 genes from 27 epidemic strains of human rotavirus isolated in different area of China in recent years were amplified with RT-PCR, the resulted cDNAs were cloned and sequenced. The sequences of full length cDNAs were compared with 10 rotavirus NSP4 sequences available in the GenBank using the Clustal x 1.8 TreeView32 and DNA Star softwares. The G serotype of VP7 was analysed by PCR.The homology of the amino acid among the 27 rotavirus strains isolated in China was 81.7%-99.4%. Based on the variation of amino acid sequence, the virus strains can be divided into two groups, represented by Wa and KUN with the homology of 92.0%-99.4% and 92.0%-98.9% within each group, respectively. The diversity between the two groups were 16.6%-21.0%. The Wa group could further be separated into three subgroups, according to the diversity between those strains and the characterization in the highly variable domain. The association between VP7 serotype and NSP4 genotype was not strong.The NSP4 gene of human rotavirus epidemic strains in China can be divided into Wa and KUN two groups, Wa group is the main group and contains three subgroups possessing characteristic amino acid sites. Samples isolated in the same year but not in the same area shared higher homology.

To understand the epidemics and antigenic drift of influenza viruses in China from 2000 to 2001.The viruses were grown in embryonated hen eggs with 9 - 10 days old. The egg allantoic fluids with influenza viruses were used. Virion RNA was transcribed into cDNA by reverse transcriptase while cDNA amplified by PCR. Products of PCR were purified. RNA sequence analysis was then performed. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseq (Version 3.69) software.Data from comparison of amino acid sequence on HA1 domain of HA protein molecule between H1N1 viruses isolated in 2001 and A/Shanghai/7/99 (H1N1) strain indicated that there was only one difference of amino acid located at 190 position (antigenic determinant D). However, phylogenetic analysis showed that there were two distinguishable genetically lineages of H1N1 viruses co-circulating in men in China in 2001. Two antigenically distinct genetic lineages of influenza B viruses were still existing in men in China. Most of influenza B viruses were Yamagata-like strain and there were two different amino acid sequences located at 197 and 199 position on HA1 domain of HA protein molecule, between Victoria-like virus isolated and B/Shandong/7/97 strain. When comparing amino acid sequences on HA1 protein domain of H3N2 viruses isolated in 2000 with those of A/Sydeney/5/97 (H3N2) virus, it was revealed that there were 7 - 8 differences of amino acid sequences between them. However, there were four differences related to amino acid sequences on HA1 protein domain between H3N2 viruses isolated in 2000 and in 2001. These results were further demonstrated by analysis of phylogenic tree.Influenza was not prevalent in China from 2000 to 2001. The antigenic drifts of H3N2 and B/Victoria-like viruses occurred. Two antigenically distinct genetic lineages of influenza B viruses were still co-circulating in men in China. Two genetically distinct lineages of influenza A (H1N1) virus were also co-circulating in men in China.

BackgroundMainland China experienced pandemic influenza H1N1 (2009) virus (pH1N1) with peak activity during November-December 2009. To understand the geographic extent, risk factors, and attack rate of pH1N1 infection in China we conducted a nationwide serological survey to determine the prevalence of antibodies to pH1N1.Methodology/principal findingsStored serum samples (n = 2,379) collected during 2006-2008 were used to estimate baseline serum reactogenicity to pH1N1. In January 2010, we used a multistage-stratified random sampling method to select 50,111 subjects who met eligibility criteria and collected serum samples and administered a standardized questionnaire. Antibody response to pH1N1 was measured using haemagglutination inhibition (HI) assay and the weighted seroprevalence was calculated using the Taylor series linearization method. Multivariable logistic regression analyses were used to examine risk factors for pH1N1 seropositivity. Baseline seroprevalence of pH1N1 antibody (HI titer ≥40) was 1.2%. The weighted seroprevalence of pH1N1 among the Chinese population was 21.5%(vaccinated: 62.0%; unvaccinated: 17.1%). Among unvaccinated participants, those aged 6-15 years (32.9%) and 16-24 years (30.3%) had higher seroprevalence compared with participants aged 25-59 years (10.7%) and ≥60 years (9.9%, PConclusions/significanceThe Chinese population had low pre-existing immunity to pH1N1 and experienced a relatively high attack rate in 2009 of this virus. We recommend routine control measures such as vaccination to reduce transmission and spread of seasonal and pandemic influenza viruses.

Autopsy studies have shown that human highly pathogenic avian influenza virus (H5N1) can infect multiple human organs other than just the lungs, and that possible causes of organ damage are either viral replication and/or dysregulation of cytokines and chemokines. Uncertainty still exists, partly because of the limited number of cases analysed. In this study, a full autopsy including 5 organ systems was conducted on a confirmed H5N1 human fatal case (male, 42 years old) within 18 hours of death. In addition to the respiratory system (lungs, bronchus and trachea), virus was isolated from cerebral cortex, cerebral medullary substance, cerebellum, brain stem, hippocampus ileum, colon, rectum, ureter, aortopulmonary vessel and lymph-node. Real time RT-PCR evidence showed that matrix and hemagglutinin genes were positive in liver and spleen in addition to positive tissues with virus isolation. Immunohistochemistry and in-situ hybridization stains showed accordant evidence of viral infection with real time RT-PCR except bronchus. Quantitative RT-PCR suggested that a high viral load was associated with increased host responses, though the viral load was significantly different in various organs. Cells of the immunologic system could also be a target for virus infection. Overall, the pathogenesis of HPAI H5N1 virus was associated both with virus replication and with immunopathologic lesions. In addition, immune cells cannot be excluded from playing a role in dissemination of the virus in vivo.