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Objective To analyze the genetic characteristics of the hemagglutinin （HA) and neuraminidase （NA) genes of influenza B viruses isolated in Yancheng City from 2015 to 2017. Methods The throat swab specimens of influenza-like illness( ILI) from sentinel surveillance hospital and outbreak sites were collected and sent to Yancheng CDC for virus nucleic acids and virus isolation testing. After validation with serological tests, eighteen strains of influenza B virus isolates were selected to amplify their HA1 and NA genes through RT-PCR assay. Their molecular characteristics of the obtained viral HA1 and NA gene sequences were analyzed using bioinformation software from three aspects, including nucleic acid level, amino acid level and molecular evolution level. Results Basically, the clustering relationships and the branche patterns between HA1 and NA genes from the 18 Yancheng influenza B virus strains were similar. The Yamagata lineage strains in 2015 were distributed in the Yamagata Clade 3 branch, belonging to Phuket/3073 strains. The Victoria lineage strains in 2016-2017 were distributed in the Victoria Clade 1A branch, belonging to Brisbane/60 strains. D196N substitution was detected on HA1 protein in all of Yamagata lineage strains at 190-helix epitope; Amino acid substitutions of victoria lineage strains involved two antigenic epitopes, 117 and 129 sites of 120-loop epitope and 197 and 199 sites of 190-helix epitope. No Intra-lineage or inter-lineage rearrangements occurred in Yancheng strains. Eighteen influenza B strains had no mutations in catalytic residues and drug resistant sites of NA genes. Conclusion The Yamagata strains well matched with vaccine strain B/Phuket/3073/2013. The HA1 and NA genes of victoria lineage strains circulated in Yancheng City during 2016 to 2017 are changing gradually. The accumulation of these mutations will result in antigenic drift of victoria lineage strains and increase the mismatch of the IFV field stains with the available vaccine strains, which may reduce the protective effect of flu vaccine.
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Objective To understand the genetic variations of neuraminidase (NA) genes of avian influenza virus H9N2 in Weining,Guizhou Province,and to provide the scientific evidence for the prevention and control of avian influenza virus.Methods Ribonucleic acids (RNA) were extracted and NA genes were amplified and sequenced from 13 randomly selected H9N2 positive samples from the live poultry market (LPM)environments in north of Weining Yi and Hui and Miao autonomous county (Weining),Guizhou Province during 2015 to 2017.Then the homology,genetic evolution,and sites of stalk deletion areas,potential N-glycosylation,receptor binding regions and drug resistance of H9N2 subtype avian influenza viruses were analyzed by a series of bioinformation software.Results Homology analysis revealed that there were 93.0％-100.0％ and 92.1％-100.0％ similarity among 13 strains H9N2 avian influenza viruses in nucleotide and amino acid of the NA gene,respectively.All strains belonged to DK/HK/Y280/97 sub-lineage,but their genetic sources were complex and diverse.Thirteen strains had a stalk deletion of 3 amino acid residues TEI at positions 63-65 and 3 isolates had mutation QN to QK at positions 39-40.The potential N-glycosylation sites at amino acid residues 86,146,200,and 234 of the NA protein of all strains were highly conserved,while other N-glycosylation sites had quantity and site mutations.There were different mutation types at the three sialic acid binding site areas,especially at 399-404 area.All NA protease activity sites and key sites of the 13 strains had no mutations associated with resistance to the neuraminidase inhibitor drugs.Conclusions All 13 strains H9N2 viruses belongs to DK/HK/Y280/97 sub-lineage in Weining,Guizhou Province during 2015-2017,and their genetic sources are complex and diverse.The mutations on sites of stalk areas,potential N-glycosylation and sialic acid binding site areas are presented at different degrees.Hence,enhancing surveillance and controlling H9N2 avian influenza virus is necessary.
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Objective To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province.Methods RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017.Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package.Results Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8％-99.2％ and 99.2％ similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO,respectively.Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2％-99.3％ and 97.6％-98.8％ similarities with vaccine strain A/Hunan/02650/2016,respectively.Other 6 stains detected in 2017 shared 99.1％-99.4％ and 98.9％-99.3％ similarities with strain A/Guangdong/17SF003/2016,respectively.Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch,but they were derived from different small branch.PEVPKRKRTAR ↓ GLF was found in 6 of 24 strains cleavage site sequences of HA protein,indicating the characteristic of highly pathogenic avian influenza virus.Mutations A134V,G186V and Q226L at the receptor binding sites were found in the HA.All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein,and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/ 18980/2017.In addition,potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains.Condusions HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017.The mutations of key sites might enhance the virulence of the virus,human beings are more susceptible to it.Hence,the risk of infection is increasing.
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Objective To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province.Methods RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017.Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package.Results Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8％-99.2％ and 99.2％ similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO,respectively.Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2％-99.3％ and 97.6％-98.8％ similarities with vaccine strain A/Hunan/02650/2016,respectively.Other 6 stains detected in 2017 shared 99.1％-99.4％ and 98.9％-99.3％ similarities with strain A/Guangdong/17SF003/2016,respectively.Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch,but they were derived from different small branch.PEVPKRKRTAR ↓ GLF was found in 6 of 24 strains cleavage site sequences of HA protein,indicating the characteristic of highly pathogenic avian influenza virus.Mutations A134V,G186V and Q226L at the receptor binding sites were found in the HA.All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein,and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/ 18980/2017.In addition,potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains.Condusions HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017.The mutations of key sites might enhance the virulence of the virus,human beings are more susceptible to it.Hence,the risk of infection is increasing.
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Objective@#To analyze the genetic characteristics of the hemagglutinin(HA) and neuraminidase(NA) genes of the influenza A/H1N1(09pdm) viruses isolated in the city of Yancheng in 2014-2017.@*Methods@#The throat swab specimens of patients with influenza-like illness (ILI) from sentinel surveillance hospitals and outbreak sites were detected using the method of real time RT-PCR. The influenza A/H1N1(09pdm) viruses were isolated using MDCK cell culture method in 2014-2017. The strains in 2014-2017 were selected randomly and their sequences of the HA1 and NA genes were amplified through one step RT -PCR method and the PCR products were sequenced. The mutations of genes and acid locus were analyzed and the evolutional trees were generated using bioinformatics software.@*Results@#The clustering relationships of the respective branches of HA1 and NA genes of seventeen A/H1N1(09pdm) strains isolated in Yancheng area were basically the same and the phylogenetic trees of HA1 and NA genes were respectively clustered into four evolutionary branches. Compared with the vaccine strain A/California/07/2009(H1N1pdm)in the Northern Hemisphere, a total of three antigen epitopes (Ca, Sa, Sb) in HA1 genes of strains in Yancheng area were involved in six antigenic sites (K154R, S162N, K163Q, S185T, L191I, S203T); there were three mutations (D222G/N, G223R, E224K) in the 220 ring and one locus (L191I) in the 190 helix of the receptor binding sites; the two strains (A/Jiangsu-YC/SWL1540/2017, A/Jiangsu-YC/SWL1545/2017) isolated in 2017 increased the 162NQS glycosylation site. Because the strains of the antigen epitopes, receptor binding sites and glycosylation sites in the HA1 genes had a certain degree of variations in Yancheng area in 2014-2017, the protective effects of vaccine strain A/California/07/2009 (H1N1pdm) was limited at the gene level. The two strains (A/Jiangsu-YC/SWL1540/2017 and A/Jiangsu-YC/SWL1545/2017) isolated in 2017 were clustered with vaccine strain A/Michigan/45/2015(H1N1pdm) and had better protective effects. Seventeen A/H1N1(09pdm) strains had no mutations in catalytic residues and drug resistant sites of NA genes, but a part of strains had a certain degree of variations in glycosylation sites of NA genes.@*Conclusions@#These results indicated the HA1 and NA genes of influenza A/H1N1(09pdm) viruses circulated in Yancheng area in 2014-2017 changed gradually. The accumulation of these mutations would result in antigenic drift of influenza A/H1N1(09pdm) viruses.
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Objective To characterize the neuraminidase (NA) genes of influenza B virus in Shangrao.Methods The specimens of nasopharyngeal swabs were collected from patients with influenza-like symptoms in influenza sentinel hospital.Seven strains of influenza B virus were randomly selected for culture and isolation in Madin-Darby Canine Kidney Epithelial Cells (MDCK Line).Viral RNA was extracted.Fragments of NA genes were amplified by one-step RT-PCR and then were sequenced.The data obtained were analyzed with software DNAStar 6.0 and Mage 5.0.The deduced amino acid sequences were examined to explore the features ofNA gene.Results The NA gene showed high homology ofnucleotides between the 7 strains of influenza B virus.No amino acid substitution was found in catalytic or framework residues of the deduced amino acid sequences of NA gene.Conclusions All the 7 strains of influenza B virus were sensitive to neuraminidase inhibitors.However,ongoing resistance surveillance is necessary for control and prevention of influenza.
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We analyzed genetic evolution characteristics of avian influenza A (H7N9) virus isolated in Zhaoqing,China,2014-2016.Nucleic acid were extracted and sequenced from 17 samples of H7N9 positive cases in Zhaoqing.Genetic characteristics of homology and important amino acid sites were analyzed by using BioEdit5.0 and MEGA6.0.The evolutionary trees were constructed by Neighbor-Joining and the referenced sequences were downloaded from GenBank,Eight nucleic acid fragments from 7 strains of H7N9 viruses were successfully generated.The highest homology was found in HA gene with A/chicken/Dongguan/695/2014(H7N9),and NA gene with A/chicken/Dongguan/1075/2014(H7N9).The internal genes were high homology with avian H7N9 and H9N2 virus from Dongguan and Shenzhen in Guangdong,China.The HA and NA genes were directly evolved in the Pearl River Delta evolution branch with the H7N9 sequences from the cities of Dongguan,Guangzhou and Shenzhen,while the sequences from the provinces of Anhui,Zhejiang,and Jiangsu were in the Yangtze River Delta evolution branch.There were 2 alkaline amino acids in cleavage site of HA,2 mutations (G186V and Q226L) in the crucial sites related with the receptor of HA protein,1 mutation (E627K) in PB2 protein,and 1 drug resistance mutation (S31N) in M2 protein.And no evidence of neuraminidase resistance in NA protein was found.In conclusion,the H7N9 virus for human infection in Zhaoqing may originate from avian H7N9 and H9N2 viruses,which circulated in the Pearl River Delta region of Guangdong from 2013 to 2014.The mutations of G186V,Q226L and E627 K might be related with high susceptibility to human beings.
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Objective@#To establish a TaqMan-MGB probe-based real-time fluorescence RT-PCR assay for avian influenza H5N6 virus used in rapid diagnosis for suspected cases and surveillance for outer environment of live poultry markets.@*Methods@#Based on the conservative sequences of avian influenza H5N6 virus for HA and NA gene published on GenBank, specific primers and TaqMan-MGB probes were designed to develop and optimize for the dual real-time RT-PCR assay. Specificity, sensitivity, repeatability and comparison tests were carried out.@*Results@#This dual real-time RT-PCR detection can be completed within 80 minutes. There was no cross-reaction with other subtypes of influenza virus and common respiratory pathogens. The minimum detection limit could be up to 10 copies/reaction. The correlation coefficient of standard curve for the gene of H5 and N6 were 0.999 and 0.993, and the coefficients of variation for cycle threshold were range from 0.151%-0.549%and 0.213%-0.575%, respectively. The positive and negative coincidence rates of the validation test were 100%.@*Conclusions@#This TaqMan-MGB probe-based dual real-time RT-PCR for avian influenza H5N6 virus was rapid, specific and sensitive. It will have a good use in early emergency detection of suspected cases and continuous monitoring of external environment in live poultry trade market.
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Introducción: El virus de influenza pandémica A (H1N1), cuya circulación se inició en abril del año 2009 en México y Estados Unidos, se constituyó en el último virus pandémico desde los casos detectados en Hong Kong en 1968. El genoma del virus de influenza A está formado por 8 segmentos ARN de cadena simple (polaridad negativa), que codifican para 10 proteínas. Los genes hemaglutinina y neuraminidasa codifican para dos proteínas de superficie y son los utilizados en los análisis de variabilidad genética. Objetivos: a) Detectar la circulación del virus pandémico en pacientes con sospecha clínica de infección por influenza, y b) Diseñar una estrategia para amplificar de forma completa los genes hemaglutinina y neuraminidasa. Materiales y Métodos: Fueron analizados por Real-Time RT-PCR (transcripción reversa y reacción en cadena de la polimerasa en tiempo real) un total de 181 muestras de hisopado faríngeo, colectadas o remitidas al Hospital de Clínicas, del 6 de agosto al 11 de octubre de 2009. Para el diseño de amplificación de los genes hemaglutinina y neuraminidasa, se han utilizado herramientas bioinformáticas y reacción en cadena de la polimerasa. Resultados: Del total de muestras analizadas, 27 (14.9 %) dieron resultado positivo para el nuevo virus pandémico. Por otra parte, la amplificación completa de ambos genes proporcionó los resultados esperados: 1678-pares de bases (pb) para la hemaglutinina, y 1427-pb para la neuraminidasa. Conclusiones: La implementación de esta tecnología de amplificación permitirá posteriormente la secuenciación de estos genes a fin de determinar las variaciones genéticas del virus que podrían tener un impacto en la salud humana.
Introduction: The pandemic influenza A (H1N1) virus, whose circulation was detected in April 2009 in Mexico and the United States, is the latest pandemic virus since the cases reported in Hong Kong in 1968. The genome of the influenza A virus consists of 8 segments of single-stranded RNA of negative polarity, coding for 10 proteins. The hemagglutinin and neuraminidase genes encode for two surface proteins and are used in the analysis of genetic variability. Objectives: a) to detect circulation of the pandemic virus in patients with clinical suspicion of influenza infection and b) design a strategy to fully amplify the hemagglutinin and neuraminidase genes.Materials and Methods: A total of 181 pharyngeal swabs were collected and sent to the Hospital de Clínicas for analysis using Real-Time RT-PCR (reverse transcription and polymerase chain reaction in real time) between 6 August and 11 October 2009. To design the amplification of hemagglutinin and neuraminidase genes, we used bioinformatic tools and polimerase chain reaction. Results: Of the samples analyzed, 27 (14.9%) were positive for the new pandemic virus. Moreover, the complete amplification of both genes provided the expected results: 1678-base pairs (bp) for the hemagglutinin, and 1427-bp for neuraminidase. Conclusions: The use of this technology for amplification will eventually allow sequencing to identify genetic variations of the virus that could have an impact on human health.
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Humans , HN Protein , Influenza A Virus, H1N1 Subtype , Pediatrics , HN ProteinABSTRACT
[Objective]This study was designed to investigate the genetic evolution of the neuraminidase(NA)gene of seasonal A/H1N1 and 2009 novel A/H1N1 inflilenza virus,and discuss the genetic variation of influenza A virus.[Methods]The virus strains were separately isolated from the clinical samples collected in 2006 and 2009,and then identified as seasonal A/H1N1 and novel A/H1N1.The full length of the NA gene of these strains was amplified by RT-PCR.Then the genetic evolution and mutations of important functional sites were analyzed.[Results]The homology of NA gene between the 2009 novel A/H1N1 isolates and 2006 seasonal A/H1N1 isolates was low(77.9%～78.8%),so was the homology of NA gene between the 2009 novel A/H1N1 isolates and representative strains of different periods and 1979-2001 WHO recommended vaccine strains(78.1%～79.3%).But compared with the WHO recommended vaccine strains of 2009 novel A/H1N1,the homology reached more than 99%.The genetic evolution analysis revealed that NA gene of 2009 novel A/H1N1 had the closest genetic relationship with the swine influenza A virus(A/swine/Belgium/1/1983)from Eurasian Iineage,and some of the antigenic sites and neuraminidase active sites of NA gene of seasonal A/H1N1 were mutated after 2005.[Conclusion]The NA gene of 2009 novel A/H1N1 may originate from Eurasian Iineage of swine influenza virus.The variation of NA gene of seasonal A/H1N1 has occurred in a certain degree.Hence,it is very necessary to continuously monitor the variant of influenza A virus.
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Twenty Newcastle disease virus(NDV)strains were isolated from diseased chicken and geese in field outbreaks during 2005 and 2006 in some regions of Jiangsu and Guangxi,and the antigenic analysis of the all NDV isolates had been done based on the reaction spectrum with a panel of monoclonal antibodies to the HN glycoprotein.The entire ORFs encoding HN protein of these NDV isolates were amplified by RT-PCR successfully,cloned and sequenced.The resultant sequences of HN genes of 13 isolates of chicken origin and 7 isolates of goose origin were gained and analyzed.The results of reaction spectrum showed that there were some distinct differences in the antigenic epitopes among the 20 NDV isolates.And the sequences revealed that the coding regions of the HN genes of these isolates all consisted of 1716 nt characteristic of virulent strains of NDV,coding for 571 amino acids.Neucleotides sequence homology were found to be from 94.8%to 100%among 18 NDV isolates of genotypeⅦ,and the neucleotides sequence homology between all the isolates and the other genotypeⅦstrains of recent years in China ranged from 92.1%to 99.6%.The deduced amino acid sequences and the receptor-binding regions of HN proteins between the NDV isolates of chicken origin and of goose origin were compared and analyzed.The results showed that some unique amino acid substitutions were found in the genome of the NDV isolates,and the close genetic similarity provided evidence for epidemiological linkage between the NDV isolates of chicken origin and of goose origin in the same period.
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Objective To construct an eukaryotic expression plasmid containing gene coding for the hemagglutinin-neuraminidase(HN)of newcastle disease virus(NDV)oncolytic strain Italien,and then to express the protein in eukaryotic cell.Method The HN cDNA was synthesized from viral RNA by RT-PCR,and the eukaryotic expression vector of HN gene(named pcDNA3.1-HN)was constructed.The vector pcDNA3.1-HN was transfected into CHO-K1 cell by liposome,and G418 was used to select stable clones expressing HN gene.The expression of HN protein was visualized by Western blot and Immunofluorescence microscopy.Results Restriction analysis and DNA sequencing proved that HN gene was correctly cloned into expression vector.Western blot analysis and immunofluorescence showed that the HN was expressed in CHO-K1 cells.Conclusion The HN cDNA of NDV was successfully cloned into eukaryotic vector which showed good expression of HN protein in CHO-K1 cells.