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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.

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 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 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.