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Objective:To investigate the phylogenetic and antigenic characteristics of hemagglutinin (HA) gene of influenza B/Victoria lineage (BV) viruses in Beijing during the 2021-2022 influenza surveillance season, and to analyze whether the circulating BV viruses match the vaccine strain.Methods:Pharyngeal swab specimens from influenza like-illness (ILI) cases in the 2021-2022 influenza surveillance season were collected from surveillance network labs in Beijing and cultured in MDCK cells and chicken embryo to isolate BV viruses. Nucleic acids of the viruses were extracted, and the HA gene was amplified and sequenced. The nucleotide and amino acid sequence identity of the HA gene was analyzed using MEGA5.0 software. A phylogenetic tree of HA gene was constructed using the maximum likelihood method. The N-glycosylation sites in HA were predicted online. Three-dimensional structure of HA was constructed using SWISS-MODEL homologous modeling. Hemagglutination inhibition (HI) test was performed to analyze the antigenicity of BV viruses.Results:A total of 402 BV viruses were collected and 58 strains with full-length HA gene sequences were chosen for further analysis. Compared with the HA gene of this year′s vaccine strain (B/Washington/02/2019), there were 27 amino acid mutations, 11 of which were located in four different antigenic determinants. The phylogenetic analysis revealed that three subgroups of 1A.3, 1A.3a1, and 1A.3a2 co-circulated in Beijing with 54 strains (54/58, 93.10%) clustered to the Clade 1A.3a2, two strains (2/58, 3.45%) clustered to the Clade 1A.3a1, and two strains (2/58, 3.45%) in the same subgroup (Clade 1A.3) as the vaccine component BV strain in 2021-2022. Compared with the vaccine strain (B/Washington/02/2019), two BV strains had an additional N-glycosylation site at residue 197, while the other 56 strains showed no change in N-glycosylation sites. Antigenic analysis showed that 35 BV strains (35/58, 60.34%) were antigenically similar to the vaccine strain and 23 strains (23/58, 39.66%) were low-response strains.Conclusions:Three subgroups of BV viruses co-circulated in Beijing during the 2021-2022 influenza surveillance season. The predominant subgroup was Clade 1A.3a2 (93.10%), showing a certain genetic distance with the vaccine strain (B/Washington/02/2019). Nearly 40% (39.66%) of the viruses were low-response strains. This study indicated that continuous monitoring of the variations of influenza epidemic strains and timely providing laboratory basis for screening vaccine component strains were the basic technical guarantee for coping with influenza pandemic.
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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 investigate the molecular characteristics of H5 subtype avian influenza viruses (AIV) in Weining, Guizhou Province. Methods Nine representative strains were randomly select-ed from H5 subtype AIV that were identified by real-time PCR in Weining, Guizhou Province from 2015 to 2017. Nucleic acid was extracted from each sample and hemagglutinin (HA) genes were amplified and then sequenced. Homology, genetic evolution and the sites related to pathogenicity, receptor binding regions as well as potential glycosylation of H5 AIV were analyzed by bioinformation software. Results Homology analysis revealed that there was 96. 1%-99. 9% and 95. 7%-100% similarity among the nine strains in nu-cleotide and amino acid of HA gene, respectively. These strains belonged to two branches, H5-1 and H5-2. The cleavage site motifs were PLREKRRKR↓GLF for five strains in H5-1 branch and PQRERRRKR↓GLF for four strains in H5-2 branch, which made them high pathogenic. QSG and QRG at the key receptor bind-ing sites were found in H5-1 and H5-2 branch strains, respectively. They were responsible for receptor bind-ing specificity of AIV. Mutations of 138Q, 139G and 53K were all detected in the nine strains. 129K, 189T, 140K and 282V mutations were discovered in the five strains of H5-1 branch, while 189N, 140M and 282I mutations were found in the four strains of H5-2 branch. Results of the glycosylation motif analysis showed that six sites were conservative, but there was an addition of 124NHT site in two strains of H5-2 branch isolated in 2017. Conclusion Two high pathogenic H5 subtypes of AIV could be epidemic in Wein-ing, Guizhou Province during 2015 to 2017. Although H5 subtype AIV did not possess specific receptor binding regions like human influenza viruses, they were in continuous variation with an increase in glycosyla-tion motifs, which might enhance their virulence and pathogenicity to human beings. Hence, surveillance and study on the molecular properties of H5 subtype AIV should be strengthened.
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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 investigate the molecular characteristics and tracing of the hemagglutinin (HA) gene, and to analyze the risk of human infection with influenza virus A (H7N9) in Guizhou Province, so that to provide evidence for the prevention and control of highly pathogenic avian influenza A (H7N9).@*Methods@#Nucleic acids of 5 strains of H7N9 including 1 sample of the patient′s nasopharyngeal swab and 4 samples of the live poultry market (LPM) environment were extracted and HA genes were amplified and sequenced. Then the homology, genetic evolution and the pivotal sites related to receptor binding regions, pathogenicity and potential glycosylation of the avian influenza A (H7N9) viruses were analyzed by a series of bioinformatics softwares.@*Results@#Homology analysis revealed that the homologies of nucleotide and amino-acid of the HA gene of H7N9 strains from the patient and LPM in Weining County, Guizhou Province were 99.8% and 99.6%, respectively, while those of 4 strains from LPM were both 100%. The homologies of nucleotide and amino-acid of the HA gene of H7N9 strains were the highest with the strain of A/Guangxi/5/2017 isolated from a Guangxi infected patient (99.7%-99.9% and 99.4%-99.8%, respectively), while those with the strain isolated from LPMs environment at the end of 2016 (A/Environment/Guangdong/C16283222/2016) were 99.0%-99.2% and 98.9%-99.2%, respectively. However, the homologies of nucleotide and amino-acid of the HA gene of H7N9 strains with A/Shanghai/2/2013 recommended by world health organization and the candidate vaccine strain A/Anhui/1/2013 were 96.8%-97.0% and 95.8%-96.2%, respectively. Phylogenetic analysis showed that the 5 strains had the nearest genetic distance to the strain A/Guangxi/5/2017. All the 5 strains cleavage site sequences of HA protein showed mutation of PEVPKRKRTAR↓GLF, and they were highly pathogenic avian influenza viruses mutant strains, which all had mutation of G186V at the receptor binding sites of HA gene, while no Q226L mutation was found. All 5 strains had new mutation of A363S, and new mutations of R56K and I297V were only found in the strain isolated from the patient. Among the five potential glycosylation motifs in the HA, only 421NWT and 493NNT had variation of the position post shift.@*Conclusions@#All the 5 H7N9 strains isolated in Weining County, Guizhou Province are highly pathogenic avian influenza mutative viruses. The current candidate vaccine may not provide a very good protection. The mutations of cleavage site of HA protein, G186V as well as other new mutation sites of HA may enhance the susceptibility and pathogenicity to human beings.
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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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O vírus da cinomose canina (CDV), um Morbillivirus da família Paramyxoviridae, é o agente etiológico de doença neurológica e sistêmica em cães. O diagnóstico laboratorial da infecção requer o isolamento viral ou detecção do material genético do vírus em secreções ou tecidos de cães com suspeita clínica da doença. A diversidade genética entre os isolados de CDV pode ser aferida pelo sequenciamento efilogenia molecular do gene que codifica a hemaglutinina viral (gene H), havendo atualmente um especial interesse em comparar as amostras circulantes a campo com o genogrupo América-1, que abrange as cepas presentes nas vacinas disponíveis no mercado. No presente estudo, foi realizada a detecção molecular do gene H de CDV a partir de amostras biológicas colhidas ante- e post- -mortem de 15 cães com sinais clínicos sugestivos de cinomose na região metropolitana de Campinas, São Paulo. Dez dos 15 cães analisados tiveram ao menos um órgão positivo na detecção molecular e os amplicons obtidos foram submetidos ao sequenciamento nucleotídico seguido de análise filogenética molecular. De forma semelhante ao que já foi reportado para estudo analisando a diversidade do gene H em outros países, a reconstrução filogenética obtida para as amostras de casos de cinomose da região de Campinas demonstrou as mesmas foram agrupadas junto a amostras norte-americanas, europeias e japonesas recentes, em um grupo genético distinto do grupo de amostras clássicas de CDV, nomeado America-1, o qual engloba as estirpes vacinais Snyder Hill, Onderstepoort e Lederle.
Canine distemper virus (CDV), a Morbillivirus of the family Paramyxoviridae, is the etiological agent of neurological and systemic disease in dogs. The laboratory diagnosis of infection requires viral isolation or detection of genetic material of the virus in secretions or tissues of dogs with clinical suspicion of the disease. The genetic diversity among isolates of CDV can be assessed by sequencing and phylogenetic analysis of the gene that encodes the viral hemagglutinin (H gene), and there is currently a special interest in comparing the strains currently circulating in the field with the genogroup America-1, which comprises strains present in vaccines available in the market. In this study, the molecular detection of CDV gene H was performed from biological samples harvested ante-and post-mortem from 15 dogs with clinical signs suggestive of canine distemper in the metropolitan region of Campinas, São Paulo. Ten of the 15 dogs examined had at least one positive organ under molecular detection and the obtained amplicons were sequenced and further analyzed by molecular phylogenetic analysis. Similarly to what has already been reported on previous studies regarding the diversity of the gene H in other countries, the phylogenetic reconstruction obtained for the samples of cases of distemper from Campinas region showed they were grouped with the North American, European and Japanese newly described samples, a genetic group distinguished from classical samples of CDV, named America-1, which encompasses the vaccine strains Snyder Hill, Onderstepoort and Lederle.
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Animals , Dogs , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Distemper Virus, Canine/isolation & purification , Distemper Virus, Canine/pathogenicity , Molecular Sequence Annotation , PhylogenyABSTRACT
Influenza A virus (IAV) infections are endemic in pork producing countries around the world. The emergence of the pandemic 2009 human H1N1 influenza A virus (pH1N1) raised questions about the occurrence of this virus in Brazilian swine population. During a 2009-2010 swine influenza virus research project at Embrapa Swine and Poultry (CNPSA), an outbreak of a highly transmissible H1N1 influenza A virus disease was detected in a pig herd in Santa Catarina State, Brazil. The virus caused a mild disease in growing pigs and sows without mortality. Three clinically affected piglets were euthanized. Gross lesions included mild to moderate consolidation of cranioventral areas of the lung. Microscopically, the lesions were characterized by necrotizing obliterative bronchiolitis and bronchointerstitial pneumonia. Immunohistochemistry using a monoclonal antibody against type A influenza virus nucleoprotein revealed positive staining in the nuclei of the bronchiolar epithelial cells. Lung tissue from three piglets and nasal swabs from five sows and four piglets were positive for influenza A by RT-PCR. Influenza virus was isolated from one lung, later confirmed by the hemagglutination test (HA titer 1:128) and RT-PCR. Sequence analyses of Hemmaglutinin (HA) and Matrix (M) genes revealed that the virus was consistent with the pandemic (A/H1N1) 2009 influenza virus strain that circulated in humans. This is the first report of an outbreak of pandemic A/H1N1 influenza virus in pigs in Brazil.
A infecção causada pelo vírus Influenza A (IAV) é endêmica em suínos no mundo inteiro. O surgimento da pandemia de influenza humana pelo vírus A/H1N1 (pH1N1) em 2009 levantou dúvidas sobre a ocorrência deste vírus em suínos no Brasil. Durante o desenvolvimento de um projeto de pesquisa do vírus de influenza suína em 2009-2010, na Embrapa Suínos e Aves (CNPSA), foi detectado em um rebanho de suínos em Santa Catarina, Brasil, um surto de influenza altamente transmissível causado pelo subtipo viral H1N1. Este vírus causou uma doença leve em suínos em crescimento e em fêmeas adultas, sem mortalidade. Tres leitões clinicamente afetados foram eutanasiados. As lesões macroscópicas incluiam consolidação leve a moderada das áreas cranioventrais do pulmão. Microscopicamente, as lesões foram caracterizadas por bronquiolite necrosante obliterativa e pneumonia broncointersticial. A imunohistoquímica, utilizando um anticorpo monoclonal contra a nucleoproteína do vírus influenza A, revelou marcação positiva no núcleo das células epiteliais bronquiolares. O tecido pulmonar de três leitões e os suabes nasais de cinco fêmeas e quatro leitões foram positivos para influenza A pela RT-PCR. O vírus influenza foi isolado de um pulmão, mais tarde sendo confirmado pelo teste de hemaglutinação (título HA 1:128) e por RT-PCR. A análise das seqüências de nucleotídeos dos genes da hemaglutinina (HA) e proteína da matriz (M) revelou que o vírus isolado foi consistente com o vírus pandêmico A/H1N1/2009 que circulou em humanos no mesmo período. Este é o primeiro relato de um surto de influenza causado pelo vírus pandêmico A/H1N1 em suínos no Brasil.
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Animals , Swine/virology , Influenza A Virus, H1N1 Subtype/isolation & purification , Disease Outbreaks/veterinary , Infections , Lung/virologyABSTRACT
Objective To understand the evolutionary characterization of hemagglutinin (HA)gene of pandemic H1N1 influenza virus in Guangdong during 2009-2011. MethodsWe selected 83 pandemic H1N1 strains isolated in Guangdong during 2009-2011. The HA1 genes were sequenced and analyzed comparatively by Bioedit 7.0 and MEGA 4.0. ResultsThe evolutionary rate of Hal gene of pandemic H1N1 and seasonal H1N1 viruses was 5.2×10-3 substitutions/site/year, higher than that of seasonal H1N1 viruses. Most amino acid changes in HA1 molecules accumulated on the surface of the molecule and were partly located in antigenic sites. Two fatal infections were detected with a mutation at HA residue 222, in one virus with a change D222G, and in one virus D222N. ConclusionThe phylogenetic analysis demonstrates that the influenza epidemic in Guangdong at the beginning of 2011 are due to occurrence of genetic changes of pandemic H1N1 virus. The amino acid change at residue 222 of the HA1 are likely to be associated with severe or even fatal illness.
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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.
Subject(s)
Humans , HN Protein , Influenza A Virus, H1N1 Subtype , Pediatrics , HN ProteinABSTRACT
Objective: To investigate the evolutionary relationship of the hemagglutinin (HA) gene of novel influenza virus A/H1N1 in 2009 pandemic with the HA genes of A/H1N1 viruses isolated in different parts of the world previously. Methods: The sequences of the HA gene of the novel A/H1N1 strain and the reference sequences of human, swine, and avian influenza A viruses were retrieved from NCBI. MEGA 4.0 software was employed to align, blunt nucleotide sequences, and construct phylogenetic tree. The deduced amino acid sequences of the HA genes of novel influenza virus A/H1N1 were compared with those of the A/H1N1 isolates in North America, Europe, and Asia. Results: Phylogenetic tree of the HA genes of A/H1N1 strains worldwide showed that the HA genes of novel influenza virus A/H1N1 in 2009 shared a high homology with those of the 7 human A/H1N1 influenza viruses isolated in North America during 1976 to 2007, and shared a low homology with those of the human influenza viruses A/H1N1 isolated in Europe and Asia. Phylogenetic tree of the HA gene between different species showed that the HA genes of novel influenza virus A/H1N1 in 2009 had a close evolutionary relationship with those of the two swine A/H1N1 strains isolated in 1998 and 2007 in North America, but a distant evolutionary relationship with those of swine and avian A/H1N1 isolated in Europe and Asia. Alignment of amino acid at important antigenic sites showed that the HA gene of the novel A/H1N1 strains shared important antigen sites with the swine A/H1N1 influenza viruses isolated in North America, and did not share with the swine A/H1N1 influenza viruses isolated in Europe and Asia or the human A/H1N1 influenza vaccine strains. Conclusion: The HA genes of the novel influenza virus A/H1N1 might originate from swine A/H1N1 influenza viruses in North America after a long time evolution and the reassortment with fragments of human A/H1N1 in the area, and the current A/human/H1N1 influenza vaccine may not be effective for the novel A/H1N1 virus.