OBJECTIVETo establish and evaluate a single-tube multiplex RT-real time PCR assay for detecting novel influenza A H1N1, influenza A and influenza B viruses (called "IV" for short) simultaneously.

METHODSA total of 213 clinical specimens of influenza-like patient's throat swab were collected during October 2010 and April 2011. 152 bp fragment in HA gene of novel influenza A H1N1 virus, 128 bp fragment in M gene of influenza A virus and 107 bp fragment in NP gene of influenza B virus were chosen as the target genes for multiplex RT-real time PCR, a specific primers and probes labeled with different fluoresceins were designed. The standard plasmid was constructed using in vitro transcription assay, and the standard curve was established. The reproducibility, specificity and sensitivity of the assay were evaluated. Furthermore, RNA extracted from 213 clinical specimens of throat swab was detected and verified by sequencing.

RESULTSThe corresponding standard curves of novel influenza A H1N1 virus, influenza A virus and influenza B virus were Y = - 3.46 lgX + 46.985, Y = - 3.49 lgX + 37.709, Y = - 3.51 lgX + 38.889, respectively; Y was cycle threshold (Ct), and lgX was logarithm value of virus replication number. The standard curve coefficient was 0.998. The detection limit of this assay was 10(2) copies/microl in one reaction. The specificity was strong. 39 (18.3%), 63 (29.6%) and 23 (10.8%) of 213 clinical specimens detected were positive for novel influenza A H1N1 virus RNA,influenza A virus RNA and influenza B virus RNA respectively. The positive samples were verified by sequencing.

CONCLUSIONThe single-tube multiplex RT-real time PCR assay developed in this study for detecting and identifying novel influenza A H1N1, influenza A and influenza B viruses simultaneously was rapid, specific and sensitive.

Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; isolation & purification ; Influenza A virus ; genetics ; isolation & purification ; Influenza B virus ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods

Animals ; China ; Influenza A virus ; genetics ; isolation & purification ; Influenza in Birds ; virology ; Nucleic Acids ; genetics ; Poultry ; virology

OBJECTIVETo analyze the genetic composition of a novel H2N3 virus isolate identified from a duck cage swab in a live poultry market (LPM) in 2009 in Guangdong province of China.

METHODSPCR-positive specimens were inoculated into embryonated chicken eggs and subtyped by conventional RT-PCR. All segments of the virus A/environment/Guangdong/2/2009 were sequenced, and phylogenetic trees were constructed and analyzed.

RESULTSThe genes of this virus belong to Eurasian-lineage avian viruses. The virus is a reassortant with the HA gene from an H2N2 virus and the NA gene from an H5N3 virus. The PB1, PB2, and NP genes were from an H4N6 virus, the PA was from an H3N8 virus, the M gene was from an H1N3 virus, and the NS gene was from an H10N6 virus.

CONCLUSIONA novel avian-origin reassortant H2N3 influenza virus was detected in a live poultry market. Its potential impacts and evolution should be closely monitored.

Animals ; China ; Ducks ; virology ; Genome, Viral ; Influenza A virus ; genetics ; isolation & purification ; Influenza in Birds ; virology ; Phylogeny

To set up a new rapid method for the rapid determination of influenza virus H5N1 and H7N9 basing on the Multi-Analyte Suspension Array (MASA) technology. Sequence analysis and design of degenerate primers and specific probes were set in the comparison and analysis of H5, N1, H7 and N9 genes. In combination with MASA technology, these primers and probes were used for the determination of samples of H5N1 and H7N9 and other subtypes ( H1N1, PH1N1, H5N2, H3N2 and H9N2). We developed a rapid determination method. This method had high specificity and sensitivity that could detect H5N1 and H7N9 at one time, and could detect samples that containing 10 copies of H5N1 and H7N9. This determination method could be used for rapid determination of influenza virus H5N1 and H7N9 at one time.
Humans ; Influenza A Virus, H5N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza A Virus, H7N9 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; virology ; Oligonucleotide Array Sequence Analysis ; methods

Global Health ; History, 20th Century ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; history ; therapy ; virology ; Russia ; epidemiology

Global Health ; History, 20th Century ; Humans ; Influenza A Virus, H2N2 Subtype ; genetics ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; history ; mortality ; virology

OBJECTIVETo analyse seasonal influenza epidemic situation in 2006, and to analyse the genetic and antigenic characteristics of viral hemagglutinin (HA) gene.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONThe H1N1 and H3N2 influenza viruses had changing antigenic and genetic characteristics in 2006. Influenza virus types B did not change in 2006.

Amino Acids ; analysis ; China ; Hemagglutination Inhibition Tests ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; Influenza A Virus, H1N1 Subtype ; immunology ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; immunology ; isolation & purification ; Influenza B virus ; immunology ; isolation & purification ; Time Factors

OBJECTIVETo investigate the epidemiological characteristics of influenza B viruses and explore the genetic evolution characteristics of the hemagglutinin(HA) and neuraminidase(NA) genes of local isolated strains in Ningbo, Southeast China, during 2010 to 2012.

METHODSRespiratory specimens from 3440 cases of patients with influenza-like illness(ILI) during 2010 to 2012 were collected in for virus isolation. And the 628 sera samples were collected in 2010 from the healthy community population to detect the HI antibody level against the local isolated virus.For phylogenetic analysis, the fragments of HA and NA genes were amplified and sequenced from strains isolated in different years. The association between evolution of HA and epidemiological characteristics were analyzed.

RESULTSA total of 109 strains of influenza B virus were isolated, including 102 (93.6%) Victoria-lineage strains and 7 (6.4%) Yamagata-lineage strains. Positive rates of HI antibody against Victoria-lineage strains and Yamagata-lineage strains were 51.1% (321) and 47.8% (300), respectively (χ(2) = 1.405, P > 0.05) among the 628 sera samples. The phylogenetic analysis showed that all HA fragments of isolated strains clustered the same branch with Malaysia/2506/2004 while the NA genes formed different branches. Compared with Brisbane/60/2008 strain, there were 1 to 5 Amino acid mutations in HA domain, and more mutations were detected in NA domain, ranged from 6 to 16 sites. The genetic evolution of NA in Victoria-lineage strains were faster compared with HA.

CONCLUSIONThe genetic evolution rates of NA genes were faster than that of HA genes in the local circulated Victoria-lineage viruses during 2010 to 2012;The comprehensive analysis of HA and NA fragments were more reliable and sensitive on surveillance of genetic evolution of influenza B viruses.

China ; epidemiology ; Genetic Variation ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza B virus ; classification ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Neuraminidase ; genetics ; Phylogeny ; RNA, Viral

OBJECTIVETo isolate and identify the influenza virus that caused four influenza-like-illness outbreaks in Jining city of Shandong Province in 2009 and analyze the genetic characteristics of hemagglutinin (HA) and neuraminidase (NA) gene, the variation of these genes were studied.

METHODS34 nasopharyngeal swabs from fever patients of four influenza-like-illness outbreaks were collected and diagnosed by real time quantitative RT-PCR method. The positive samples were incubated and cultured for virus. HA and NA genes of isolated pandemic influenza A (H1N1) virus were sequenced, the homology analysis was done with DNAStar software and the genetic evolution and amino acid substitutions were performed with Mega 4.0 software. The sequences were compared with WHO recommended vaccine virus, native reference virus.

RESULTSSeventeen of 34 nasopharyngeal swabs were positive, 11 pandemic influenza A (H1N1) viruses were isolated and HA and NA genes of 7 strains were sequenced. Phylogenetic analysis for hemagglutinin and neuraminidase gene of Shandong outbreak strains showed that there were 98.4% - 99.6% and 99.2% - 100.0% sequence identity. Compared with WHO-recommended vaccine strain, the reference virus in mainland China strain, eleven amino acids were changed for HA protein, including position 38, 40, 56, 90, 100, 145, 172, 173, 220, 303 and 338, and 38, 40, 303 of HA protein were located in the antigenic determination C cluster, 172, 173 in the D cluster, 56 in the E cluster, site 40 of HA protein were glycosylated. In NA protein, seven amino acids were changed, including position 80, 106, 241, 248, 351, 369 and 386, site 40 of NA protein were glycosylated. No mutations of 275 in NA protein were found.

CONCLUSIONThe HA and NA genes of the epidemic strains showed high homology, some mutations in the HA and NA proteins were found, the antigenic site and glycosylation site of some strains were changed during the epidemic process.

China ; epidemiology ; Disease Outbreaks ; Glycosylation ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Neuraminidase ; genetics

Replication of duck plague virus(DPV) in artificially infected ducks were detected by in situ hybridization (ISH) which employed a 37bp oligonucleotide as probe designed according to DPV DNA sequence in GenBank. The results indicated that DPV DNA was detected in liver, intestine and bursa Fabricius at 4 h, in spleen and esophagus at 6h, in thymus at 12h post infection; DPV DNA in lung and kidney was detected only in dead ducks and no positive signal was detected in muscle, heart, cerebrum and pancreas. DPV DNA was distributed in cell nucleus and cytoplasm. Hepatocytes, sinus endodermal cells and Kuffer's cells were the mainly infected cell types in liver. DPV DNA was mainly detected in epithelium of villi, in lamina propria of intestinal villi of duodenum, in stratum spinosum of esophagus, and in epithelium, cortex, medulla of bursa Fabricius. The positive signals were mainly detected in medulla of thymus, lymphocytes and macrophages of spleen. The research suggests that ISH is a direct and specific method in detecting DPV DNA in paraffin sections and it's also a good method for virus diagnosis and DNA location of DPV.
Animals ; DNA, Viral ; analysis ; Ducks ; virology ; In Situ Hybridization ; Influenza A virus ; genetics ; isolation & purification ; physiology ; Virus Replication