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Objective: To establish chemiluminescence enzyme immunoassay (CLEIA) for quantitative detection of procollagen III N-terminal peptide (P III NP) in serum., Methods: A sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of P III NP as the catalytic enzyme and the luminol as the luminescence reagent. Several reactions liquid's concentration and reaction conditions were optimized. The method was evaluated in all aspects such as linear range, sensitivity, specificity, stability and so on. The CLEIA was compared with imported ELISA kits, by detecting clinical serum., Results: The linear range was 0.8-85 ng/ml. The detection limit was 0.5 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 96.2%, 91.2% and 101.1%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0.99 and RSD lower than 6%. The detected results of clinical sera with CLEIA closely corresponded to those with imported ELISA., Conclusion: Established CLEIA for quantity determination of serum P III NP has high accuracy, sensitivity and repeatability.

Objective: To establish enzyme-linked immunosorbent assay (ELISA) for quantitative detection of Golgi protein73 (GP73) in serum., Methods: A sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of GP73 as the catalytic enzyme. Several reactions liquid's concentration and reaction conditions were optimized. The method was evaluated in all aspects such as linear range, sensitivity, specificity, stability and so on., Results: The linear range was 25-500 ng/ml. The detection limit was 18.5 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 95.3%, 92.6% and 103.7%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0.98 and RSD lower than 10%., Conclusion: Established ELISA for quantity determination of serum GP73 has high accuracy, sensitivity and repeatability.

Objective: To establish enzyme-linked immunosorbent assay (ELISA) for detection of hepatitis B virus large surface protein(HBV-LP) in serum., Methods: A sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of HBV-LP as the catalytic enzyme. Several reactions liquid's concentration and reaction conditions were optimized. The method was evaluated in all aspects such as sensitivity, specificity, stability and so on., Results: The detection limit was 5 ng/ml. Interassay and intra-assay RSD were both less than 10%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0.98 and RSD lower than 10%., Conclusion: Established ELISA for determination of serum HBV-LP has high sensitivity and repeatability. Enzyme-linked immunosorbent assay;

Objective: To clone and express human Golgi glycoprotein73 protein, and prepare the monoclonal antibody (mAb) against the protein., Methods: GP73 gene was amplified from HepG2 cells by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-GP73 and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot was used to detect specificity of mAbs., Results: The prokaryotic plasmid expressing the recombinant protein was constructed, and the GP73 recombinant protein was expressed and purified. Five hybridoma cell lines that secreted anti-GP73 mAbs were obtained. 2 of 5 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with GP73 protein., Conclusion: The GP73 recombinant protein is highly purified and has strong antigenicity. The anti-GP73 mAbs were prepared successfully.

Objective: To establish microplate chemiluminescence enzyme immunoassay (CLEIA) for quantitative analysis of tissue inhibitor of metalloproteinases I (TIMP I) in human serum., Methods: A sandwich reaction was preformed with horseradish peroxidase(HRP) labeled monoclonal antibody of TIMP I as the catalytic enzyme and the H2O2-luminol as the luminescence reagent. Several physical and chemical parameters were studied and optimized such as immunoreaction conditions, the dilution ratio of TIMP I-HRP, luminescence reaction time and so on. In order to evaluate the method, recovery test, heat stabilization test and comparison test were carried out., Results: The linear range was 0. 2-12 ng/ml with r = 0.996. The detection limit was 0.12 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 100.6%, 96.5% and 106.5%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0. 998 and RSD lower than 6%. The detected results with CLEIA closely corresponded to those with imported ELISA in 60 patients sera with liver fibrosis., Conclusion: Established CLEIA for quantity determination of serum TIMP I has high accuracy, sensitivity and repeatability.

Objective: To prepare the monoclonal antibody (mAb) against tissue inhibitor of metalloproteinases I (TIMP-I) fusion protein., Methods: TIMP-I gene was amplified from fibrotic human liver tissue by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-TIMP-I and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot were used to detect specificity of mAbs., Results: The prokaryotic plasmid expressing the recombinant protein was constructed, and the TIMP-I recombinant protein was expressed and purified. Four hybridoma cell lines that secreted anti-TIMP-I mAbs were obtained. 3 of 4 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with TIMP-I protein., Conclusion: The TIMP-I recombinant protein is highly purified and has strong antigenicity. The anti- TIMP-I mAbs were prepared successfully.

Objective: To establish a purificatory method of alpha-fetoprotein variant (AFP-L3) based on microspincolumn with lens culinaris agglutinin (LCA)., Methods: LCA was isolated by ammonium sulfate precipitation method from lens culinaris. AFP-L3 affinity adsorption microspincolumns which were made from LCA coupled with activated Sepharose 4B were prepared. By adding into the centrifuge column, serum was absorbed and eluted to purify AFP-L3. The results of purified AFP-L3 detection of 10 cases AFP positive sera by electro-chemiluminescence immunoassay were compared with traditional crossed affinity immunoelectrophoresis., Results: 8 of 10 cases AFP-L3 concentration were greater than 5 ng/ml in purified sera. Six cases show positive reaction in affinity immune cross electrophoresis experiment., Conclusion: Successfully established purification method of AFP-L3 by affinity absorption based on microspincolumn. The method was more conducive to clinical laboratory applications due to its high sensitive and easy operation.

Objective: To estimate the consistency of two VITROS 3600 chemiluminescent analyzers according to the requirement of ISO15189., Methods: Verification tests were made for precision and accuracy of anti-HCV in two instruments. While 40 serum samples including Anti-HCV negative (10 cases) , positive (10 cases) , and weakly positive (20 cases). and the test results were statistical analised., Results: Two instruments negative and positive control samples intra-batch precision and coefficients of variation were 5% , 4% and 7. 14% , 7. 23% , inter-batch precision and coefficients of variation were 9. 47% , 7. 7% and 8.04%, 7. 6%, are less than requirement CV (15%) by ISO15189. The accuracy of two instrument were 100% , The test results of the control samples showed no significant difference (P < 0. 05). The correlation analysis of the test results of clinical samples R2 =0. 9984, with good consistency., Conclusion: Test results of two Vitros 3600 has good consistency and comparability.

Objective: Evaluated the chemiluminescence and enzyme-linked immunosorbent assay (ELISA) to detect HIV antibodies, and compared the results, to provide a reference for the selection and clinical application of HIV screening., Methods: 3000 cases of our hospital patients were measured by enzyme-linked immunosorbent assay and chemiluminescence immunoassay, using comfirmming experimental results as gold standards. Comparing sensitivity, specificity and other Indicators., Results: In the diagnosis of HIV infection, enzyme-linked immunosorbent assay and chemiluminescence immunoassay had no significant difference. The positive rate of enzyme-linked immunosorbent assay was 0.93%, while the sensitivity and specificity were 89.66%, 99.93%, the positive rate of chemiluminescence immunoassay was 1.03%, while the sensitivity and specificity were 100%, 99.93%, respectively., Conclusion: Both methods are suitable for screening of HIV, having high specificity, and chemiluminescence has greater sensitivity than ELISA.

Objective: Establish a confirmatory test based on ELISA, and use to verify the authenticity of HBsAg weak positive samples, pick and get rid of the false result, and avoid the mistake diagnosis., Method: The particles (reagent A) coated by streptavidin and biotinylated HBsAb (reagent B) were mixed in different proportions, then neutralized with serum whose the COI of HBsAg > 20 by ELISA in order to identify the activity of HBsAb in confirmatory reagent. 30 pieces of HBsAg weak positive serum neutralized with the confirmatory reagent, the serum were considered to be positive if rate of decline of HBsAg COI > 50%. The results were compared to Roche confirmatory Kit., Result: Confirmatory reagent was able to neutralized with HBsAg. 24 of 30 pieces of HBsAg weak positive samples were judged to be positive, while 6 poeces were negative. The ELISA comfirm method is fully consistent with Roche confirmatory Kit., Conclusion: The ELISA confirmatory test for suspicious HBsAg positive samples is a simple, accurate and low cost initial validation method, After further clinical trials, should be widely applied.

Objective: To investigate the clinical efficacy and safety of adefovir dipivoxil (ADV) in combination with bicyclol for the treatment of chronic hepatitis B (CHB) in seniors., Methods: 96 senior patients with CHB were randomly divided into two groups, the treatment group and the control group. On the basis of routine liver protective treatment, patients in the treatment group received ADV (10 mg/d) and bicyclol tablets (25 mg, tid.) orally, and those in the control group were orally administrated ADV tablets (10 mg/d) only. The treatment course for both groups was 24 weeks. Serum ALT, AST, and alterations of virological parameters were observed before and after the treatment., Results: Before and at the end of the 24 weeks treatment, ALT level for the treatment group was (208.44 +/- 94.22) and (34.47 +/- 12.79) U/L, and those for the control group was (205.73 +/- 96.48) and (44.20 +/- 21.96) U/L, respectively (difference between groups P < 0.01). At the end of the 24 weeks treatment, ALT normalization rates for the treatment group and the control group were 76.6% and 54.5%, respectively, and AST normalization rates for them were 76.6% and 54.5%, respectively (both differences between groups P < 0.05); HBV DNA loads for the treatment group and the control group were decreased by (3.1 +/- 1.40) lgIU/ml and (2.98 +/- 1.17) lgIU/ ml, respectively (difference between groups P > 0.05). The incidence rates of adverse events between two groups were not statistically significant., Conclusion: It suggested that the treatment of ADV in combination with bicyclol for senior patients with CHB is effective and safe.

Objective: Establish a kind of confirmation method based on ELISA, and use to verify authenticity of HBsAb + in HBsAg + HBsAb + serum, pick and get rid of the false masculine gender the result, and avoid the mistake diagnosis., Method: Collect 60 pieces of serum whose thick degree of HBsAg at 1000 COI above tested by ECLIA as confirm serum, mixed the confirm serum of different dilution with HBsAb positive serum to screen and verify best thick degree of HBsAg. Collected 40 pieces of HBsAg + HBsAb + serum, ELISA tested the descend rate of HBsAb COI after neutralized with confirm serum in order to confirm authenticity of HBsAb + in pieces of HBsAg + HBsAb + serum., Result: When thick degree of HBsAg is 2000 COI, the performance of neutralization to HBsAb is best. The ELISA confirmatory test is fully consistent with the ECLIA method with true positive of 37 pieces of HBsAg + HBsAb + serum while false-positive of 3 pieces of serum., Conclusion: The ELISA confirm method is a simple, accurate and low cost initial validation method.

Objective: To evaluate the clinical value of an enzyme-linked immunosorbent assay by the Helicobacter pylori stool antigen (HpSA) test for the detection of H. pylori infection., Methods: 328 patients were measured upper gastrointestinal endoscopic examination which as gold standards and HpSA test in the meantime, comparing accuracy, sensitivity, specificity and other Indicators., Results: In the diagnosis of Hp infection, HpSA test had no significant difference comparing with gold standards and had a P value of over 0.5. The sensitivity of HpSA test was 94.6%, while the specificity, veracity, expected positive value, and expected negative value were 96.9%, 96.3%, 89.7% and 98.4%, respectively., Conclusion: The H. pylori stool antigen test is a simple, non-invasive method for accurate diagnosis of H. pylori infection.

Objective: To under stand influenza A viruses epidemic, antigenicity and genetic characteristics variation between the vaccine and Circulation strains during 2004-2008 year in China., Methods: The influenza A viruses (H1N1, H3N2) isolated from 2004-2008 year were under took antigenic and sequence analysis. Influenza A virus antigenicity and genetic characteristics were analyzed thought amino acid variation compassion of HA1 protein of influenza A virus isolates., Results: The antigenicity of influenza H1N1 subtype viruses isolated from 2004 to 2007 is very similar with vaccine strain A/New Caledonia/20/1999 (HIN1)-like virus. The influenza H1N1 viruses circulated in 2008 year had similar antigenic characteristics with A/Brisben/59/2007 (H1N1) which is component of influenza vaccines for northern hemisphere 2008-2009 year. The influenza H3N2 subtype viruses of 2004-2005 year had antigenic variation comparatively with vaccine strain A/Fujian/411/12002 (H3N2), The antigenicity of 2006-2007 H3N2 viruses and 2008 year's is A/Wiscansin/67/2006(H3N2) and A/ Brisben/10/2006(H3N2) respectively., Conclusion: There is change of influenza A viruses (H1N1, H3N2) antigenic and genetic characteristics during 2004-2008 in China.

Objective: 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., Methods: 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., Results: 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., Conclusion: The reverse genetic for avian influenza H9N2 subtype virus has been established successfully.

Objective: To establish the DNA microarray to detect influenza viruses and avian influenza viruses, and identify their virulence., Methods: 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., Results: 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)., Conclusion: 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.

Objective: To investigate clinical features of the patients with hepatitis B superinfected with acute hepatitis E (AHE)., Methods: Totally 625 consecutive patients enrolled from Dec 2002 to Dec 2006 were studied retrospectively. All of the patients were subclassified into acute hepatitis E group (AHE=437 cases) and Superinfected Group (S=188 cases), and S group was further divided into the group of chronic hepatitis B superinfected with acute hepatitis E (CHB+AHE, 130 cases) and the group of liver cirrhosis and hepatitis B superinfected with acute hepatitis E (LCB+AHE, 58 cases). In 32 of the 188 superinfected patients the effects of HEV on HBV were observed by comparing the levels of HBV DNA in acute vs. convalescence stages., Results: Compared with the patients with AHE, the superinfected patients had a higher level of total bilirubin (TBil), an elevated frequency of fulminate hepatitis, mortality and a longer period of the mean hospital stay for the cured patients but significantly lower levels of alanine aminotransferase (ALT), serum albumin and prothrombin activity (PA). Furthermore, the group of LCB+AHE had a higher level of TBil and higher incidences of complications such as ascites, peritonitis, hepatic encephalopathy and disturbance in glycometabolism than the group of CHB+AHE. The follow-up for the superinfected patients showed that 20 of 32 patients (62.5 percent) had decreased copies of HBV DNA during the recovery phase compared with the acute phase, and the mean decrease of HBV DNA was 2.1 log10. The HBV DNA was in a persistently undetectable level in 6 of 32 (18.8 percent) superinfected patients. However, 4 of 32 patients (12.5 percent) showed an unchanged levels of HBV DNA and 2 cases (6.2 percent) had a slightly increased HBV DNA levels., Conclusion: Superinfection with AHE in patients with chronic hepatitis B leads to a more severe hepatic damage and the replication of HBV DNA can be transiently inhibited.

Objective: To analyse seasonal influenza epidemic situation in 2006, and to analyse the genetic and antigenic characteristics of viral hemagglutinin (HA) gene., Methods: The single-way hemagglutination inhibition (HI) tests were used to test the antigenic characteristics of these viruses from influenza surveillance network, and the HA1 genes were sequenced based on the antigenic test results according to different isolation times and sites., Results: The influenza virus types A and B co-circulated in 2006. influenza A H1N1 subtype and Victoria-like B influenza circulated preponderantly during this epidemic season. The HA1 gene sequence of H1N1 viruses showed that 192, 193, 196, 198 positions (located at antigenic site B) have an amino acid substitute, compared with the last circulating strain A/Hubeihongshan/53/2005(H1N1). Two amino acid changes at 142 and 144 positions compared with A/Yunnan/1145/2005 (H3N2). There was no change in influenza B viruses either Victoria-like B or Yamagata-like B virus, i.e . antigenic characteristics is analogous to B/shenzhen/155/2005 and B/tianjin/144/2005, respectively., Conclusion: The H1N1 and H3N2 influenza viruses had changing antigenic and genetic characteristics in 2006. Influenza virus types B did not change in 2006.

Background: 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., Methods: 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., 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 K > R, 90 T > K, 101 Y > H, 149 R > K, 169 V > A, 190 D > N, 212 R > K, 219 K > R, 245 W > R, 246 Y > F, 258 T > N, 318 V > A and the 54 and 190 amino acids located in antigenic group of HA1., Conclusion: The H1N1 virus was changing in antigenic and genetic characteristics.

Background: To establish a method for H5N1 RNA detection and laboratory diagnosis of suspected human avian influenza (H5N1) virus infected cases., Methods: 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., Results: 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., Conclusion: The established RT-PCR and real-time PCR methods can be used for laboratory detection of suspected human H5N1 cases.

Background: 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., Methods: 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., Results: 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%., Conclusion: Over 50% of virus among the strains isolated showed adamantane resistance since 2003, and the incidence rate is increasing.

Background: 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., Methods: 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., 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., Conclusion: 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.

Background: To analyze the genetic and antigenic characteristics of human H5N1 virus isolated from the mainland of China., Methods: The hemagglutinin (HA) gene of human H5N1 virus were sequenced and analyzed., Results: The results of HA gene sequencing showed that all the virus isolates belong to the same group because of the high similarity, but they were different from the virus isolated from Thailand and Vietnam. The sequence data also showed that the receptor specificity and the connecting peptide between HA1 and HA2 are still avian influenza origin., Conclusion: The virus isolates from mainland of China until now belong to the same group and are different from the virus isolated from Thailand and Vietnam, and there is no evidence showing the human-avian influenza reassortant and recombination.

Background: To determine the etiologic agent of an atypical pneumonia human case admitted to Xiangtan City hospital, Hunan Province in Oct. 2005., Methods: The patient's respiratory tract samples and serum were collected. Throat swabs were tested by microneutralization and hemagglutination-inhibition assays., Results: The results of nucleic acid detection of all respiratory samples were negative and virus isolation was also negative. The H5-specific antibodies of convalescence showed a 4-fold greater rise than acute phase., Conclusion: The atypical pneumonias case was confirmed as the first human case of avian influenza A (H5N1) infection in the mainland of China.

Background: To understand the optimal condition of single radial hemolysis (SRH) for diagnosis of avian influenza A (H5N1) virus in order that SRH could be performed in general laboratories., Methods: The effect of different concentration of virus and species of red blood cells, as well as kind and concentration of agarose on testing sensitivity of SRH was determined. Meanwhile the sensitivity and specificity of this method were compared with those of micro-neutralization test., Results: The optimal condition of SRH included the viral concentration of 1000 HA units per 0.1 ml packed chicken red blood cells, the agarose concentration of 1.0%, the compliment added into agarose-virus-rbc slides after diffusion of sera. The sensitivity and specificity of SRH were very similar to those of micro-neutralization test. Meanwhile, no cross reaction between antibodies, especially antibodies against N1 antigens, H5N1 and H1N1 viruses was detected., Conclusion: The sensitivity and specificity of SRH were very similar to those of micro-neutralization assay. SRH could be performed in normal laboratories and be used for testing large scale serum samples.

Background: To study the antigenic and genetic characteristics of influenza (H3N2) virus circulated in China in 2004., Methods: 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., Results: 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 Y>F, 189 S>N, 145 K>N, 226 V>I, 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., Conclusion: 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.

Background: Through the analysis of internal genes of viruses to understand whether the internal genes of two strains of swine influenza A(H1N1) virus contain the gene segment deriving from avian influenza A viruses, and whether the reassortment of the internal genes occurred between swine H1N1 and swine H9N2 viruses., Methods: Viruses were passaged in embryonated hen eggs and virion RNA was extracted from allantoic fluids and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards, RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03)., Results: All the six, but PB2 gene segment internal gene segments were the same between A/swine/Beijing/1/2002(H1N1) and A/Swine/Beijing/3/2002(H1N1) viruses. Whereas all the six internal gene segments in two trains of swine (H1N1) virus were similar to those of swine H1N1 viruses, but different from those of classical strain of swine (H1N1) virus., Conclusion: Two strains of Beijing swine H1N1 influenza A virus were not reassortant, All the six internal gene segments were closely related to swine influenza A (H1N1) viruses.

Objective: To understand whether pigs play a role in human infection with avian influenza A H9N2 viruses., Methods: 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., Results: 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., Conclusion: 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.

Background: To investigate the effect of Oxymatrine (OM) on serum cholinesterase (ChE) during the treatment of viral hepatitis and the relationship between the change of ChE and the change of albumin (ALB), prothrombin activity (PTA) and other liver function tests., Methods: A total of 98 patients with viral hepatitis were divided into four groups. Group A consisted of 31 patients and were treated with OM intravenous infusion; Group B consisted of 30 patients, treated with OM orally; Group C consisted of 7 patients and were treated with OM intramuscular injection while Group D consisted of 30 patients, and were not treated with OM. ChE, ALB, PTA, liver function, renal function, soluble complement receptor-1 (sCR1) and erythrocyte innate immune adhesion function (EIIAF) were regularly determined., Results: ChE in Group A,B,C was dropped obviously during the treatment (P less than 0.001, less than 0.001, 0.023=. But there were no change in ALB, PTA, sCR1, EIIAF (P greater than 0.05), and remarkable improvement of ALT, AST, TBiL was seen during the treatment in Groups A, B, C. After the treatment with OM, the level of ChE recovered soon., Conclusion: Serum level of ChE significantly declined during the treatment of viral hepatitis with OM, but no change was found in ALB, PTA, sCR1, EIIAF while liver function tests showed better results. So the drop of ChE does not mean deprivation of patient's liver disease.

Objective: To understand the antigenic and genetic characteristics of influenza A H3N2 viruses circulated in man in China from 2000 to 2002., Methods: 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., Results: 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)., Conclusion: 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.

Objective: 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., Methods: 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., Results: 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., Conclusion: 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.

Objective: 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., Methods: 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., Results: 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., Conclusion: 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.

Background: 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., Methods: 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., Results: 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., Conclusion: 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.

Background: To understand the antigenic and genetic characteristics of Victoria like strain of influenza B virus isolated recently and to provide a scientific evidence for influenza surveillance and monitoring of influenza epidemic in future., Methods: Viruses were passed in embryonated hen eggs and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign., Results: B/Sichuan/63/2001 and B/Zhejiang/2/2001 viruses were antigenically different from B/Shandong/7/97 strain. The substitution of nucleotide sequences of HA1genes of them compared with those of B/Shandong/7/97strain resulted in the change of amino acid sequences in antigenic determinants on HA1 protein domain. The phylogenetic analysis also indicated that strains isolated recently were genetically different from B/Shandong/7/97/strain. However, there was neither differences on the antigenicity nor genetic partern between these two isotates., Conclusions: The antigenic drift of Victoria-like strain of influenza B virus isolated recently in China has further occurred.