RESUMO
We performed Ebola virus disease diagnosis and viral load estimation for Ebola cases in Sierra Leone during the late stage of the 2014-2015 outbreak (January-March 2015) and analyzed antibody and cytokine levels and the viral genome sequences. Ebola virus disease was confirmed in 86 of 1001 (9.7%) patients, with an overall case fatality rate of 46.8%. Fatal cases exhibited significantly higher levels of viral loads, cytokines, and chemokines at late stages of infection versus early stage compared with survivors. The viruses converged in a new clade within sublineage 3.2.4, which had a significantly lower case fatality rate.
Assuntos
Ebolavirus/genética , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/imunologia , Carga Viral , Anticorpos Antivirais/sangue , Citocinas/sangue , Surtos de Doenças , Genoma Viral , Humanos , Serra Leoa/epidemiologia , SobreviventesRESUMO
OBJECTIVE: To prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China. METHODS: Recombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses. RESULTS: The 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics. CONCLUSION: The 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.
Assuntos
Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Influenza Aviária/virologia , Animais , Embrião de Galinha , Galinhas , China , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Neuraminidase/genética , Neuraminidase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vacinas Sintéticas/imunologiaRESUMO
OBJECTIVE: To 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. METHODS: PCR-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. RESULTS: The 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. CONCLUSION: A novel avian-origin reassortant H2N3 influenza virus was detected in a live poultry market. Its potential impacts and evolution should be closely monitored.
Assuntos
Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , Animais , China , Patos/virologia , Genoma Viral , Vírus da Influenza A/genética , FilogeniaRESUMO
The proportion of human isolates with reduced neuraminidase inhibitors (NAIs) susceptibility in highly pathogenic avian influenza (HPAI) H7N9 virus was high. These drug-resistant strains showed good replication capacity without serious loss of fitness. In the presence of oseltamivir, R229I substitution were found in HA1 region of the HPAI H7N9 virus before NA R292K appeared. HPAI H7N9 or H7N9/PR8 recombinant viruses were developed to study whether HA R229I could increase the fitness of the H7N9 virus bearing NA 292K. Replication efficiency was assessed in MDCK or A549 cells. Neuraminidase enzyme activity and receptor-binding ability were analyzed. Pathogenicity in C57 mice was evaluated. Antigenicity analysis was conducted through a two-way HI test, in which the antiserum was obtained from immunized ferrets. Transcriptomic analysis of MDCK infected with HPAI H7N9 24hpi was done. It turned out that HA R229I substitution from oseltamivir induction in HA1 region increased (1) replication ability in MDCK(P < 0.05) and A549(P < 0.05), (2) neuraminidase enzyme activity, (3) binding ability to both α2,3 and α2,6 receptor, (4) pathogenicity to mice(more weight loss; shorter mean survival day; viral titer in respiratory tract, P < 0.05; Pathological changes in pneumonia), (5) transcriptome response of MDCK, of the H7N9 virus bearing NA 292K. Besides, HA R229I substitution changed the antigenicity of H7N9/PR8 virus (>4-fold difference of HI titre). It indicated that through the fine-tuning of HA-NA balance, R229I increased the fitness and changed the antigenicity of H7N9 virus bearing NA 292K. Public health attention to this mechanism needs to be drawn.
Assuntos
Antivirais , Subtipo H7N9 do Vírus da Influenza A , Neuraminidase , Infecções por Orthomyxoviridae , Oseltamivir , Replicação Viral , Animais , Oseltamivir/farmacologia , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/efeitos dos fármacos , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Subtipo H7N9 do Vírus da Influenza A/imunologia , Subtipo H7N9 do Vírus da Influenza A/fisiologia , Neuraminidase/genética , Neuraminidase/metabolismo , Cães , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Humanos , Camundongos , Infecções por Orthomyxoviridae/virologia , Células Madin Darby de Rim Canino , Células A549 , Camundongos Endogâmicos C57BL , Farmacorresistência Viral/genética , Substituição de Aminoácidos , Influenza Humana/virologia , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Feminino , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
OBJECTIVE: To conduct a full genome sequence analysis for genetic characterization of an H3N8 influenza virus isolated from drinking water of a domestic duck farm in Poyang Lake area in 2011. METHODS: The virus was cultivated by specific pathogen free (SPF) chicken embryo eggs and was subtyped into hemagglutinin (HA) and neuraminidase (NA) by real-time PCR method. Eight gene segments were sequenced and phylogenetic analysis was conducted. RESULTS: The NA gene of this virus belongs to North American lineage; other seven genes belong to Eurasian lineage. Compared with the viruses containing NA gene, the PB2 and PB1 gene came from different clades. And this indicates that the virus was a novel reassortant genotype. The HA receptor binding preference was avian-like and the cleavage site sequence showed a low pathogenic feature. There was no drug resistance mutation of M2 protein. The mutations of Asn30Asp, and Thr215Ala of the M1 protein implied the potential of pathogenicity increase in mice. CONCLUSION: The finding of novel genotype of H3N8 virus in drinking water in this duck farm near Poyang Lake highlighted the importance of strengthening the surveillance of avian influenza in this region, which could contribute to pinpointing the influenza ecological relations among avian, swine, and human.
Assuntos
Vírus da Influenza A Subtipo H3N8/genética , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Poluentes da Água/isolamento & purificação , Sequência de Aminoácidos , Criação de Animais Domésticos , Animais , Sequência de Bases , China , DNA Viral/genética , Água Potável , Patos , Lagos , Filogenia , RNA Viral/genética , Análise de Sequência de DNA , Microbiologia da ÁguaRESUMO
Influenza-like illness (ILI) is one of the most important public health problems globally, causing an enormous disease burden. Influenza infections are the most common cause of ILI. Bacterial and virus co-infection is common yet the data of co-infection with influenza A and B viruses are scarce. To identify the epidemiological patterns of and co-infection of influenza A and B in Anhui province, China, we analyzed the surveillance data of 5 years from 2009 to 2014 collected by the Chinese National influenzas network. The results showed that the weekly ratio of ILI was 3.96 ± 1.9% (95% CI 3.73-4.2%) in outpatients and the highest affected population was children under 5 years old. The epidemic of influenza viruses was highest during 2009-2010. For the other 4 surveillance years, school-aged people (5-14 years) were the most highly affected population. Influenza B and H3N2 viruses were more prevalent than H1N1pdm09 virus after 2010. In addition, a significant co-circulation of influenza A (H1N1pdm09 and H3N2) and influenza B virus was detected with 0.057% PCR positive rate during 2009-2014 in Eastern China, yet isolated only in pediatric patients. Our data reveals school-aged population would be the main vulnerable population and a distinct seasonality for influenza. In addition, the co-infection of influenza A and B were found in Anhui Province, China. Ongoing surveillance is critical to understand the seasonality variation and make evidence-based vaccination recommendations. Information on the epidemiological patterns and co-infections of influenza A and B can help us to implement different strategies for selecting vaccine formulations and monitoring new emerging influenza strains. In addition, the identification of the susceptible population can help us to develop more precise protection measures.
Assuntos
Coinfecção , Vacinas contra Influenza , Influenza Humana , Criança , Pré-Escolar , China/epidemiologia , Coinfecção/epidemiologia , Humanos , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza B , Influenza Humana/epidemiologiaRESUMO
OBJECTIVE: Interferon-induced transmembrane protein 3 (IFITM3) is an important member of the IFITM family. However, the molecular mechanisms underlying its antiviral action have not been completely elucidated. Recent studies on IFITM3, particularly those focused on innate antiviral defense mechanisms, have shown that IFITM3 affects the body's adaptive immune response. The aim of this study was to determine the contribution of IFITM3 proteins to immune control of influenza infection in vivo. METHODS: We performed proteomics, flow cytometry, and immunohistochemistry analysis and used bioinformatics tools to systematically compare and analyze the differences in natural killer (NK) cell numbers, their activation, and their immune function in the lungs of Ifitm3-/- and wild-type mice. RESULTS: Ifitm3-/- mice developed more severe inflammation and apoptotic responses compared to wild-type mice. Moreover, the NK cell activation was higher in the lungs of Ifitm3-/- mice during acute influenza infection. CONCLUSIONS: Based on our results, we speculate that the NK cells are more readily activated in the absence of IFITM3, increasing mortality in Ifitm3-/- mice.
Assuntos
Influenza Humana/virologia , Proteínas de Membrana/genética , Infecções por Orthomyxoviridae/veterinária , Doenças dos Roedores/virologia , Doença Aguda , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/virologiaRESUMO
To construct eucaryotic expression recombinant vector containing vivo truncated region of UL83 gene of human cytomegalovirus, realize its steady expression in Hep-2 cell, and study sheltered effect of the eucaryotic expression recombinant vector as DNA vaccine. A vivo truncated UL83 gene fragment encoding for truncated HCMV pp65 was obtained by PCR from human cytomegalovirus AD169 stock genome. By gene recombinant ways, the truncated UL83 gene fragment was cloned into eucaryotic expression vector pEGFP-C1 with reported gene coding GFP to construct recombinant vector pEGFP-C1-UL83. The recombinant vector pEGFP-C1-UL83 was tested by different methods including PCR, restriction digestion and gene sequencing. Test results showed the recombinant vector was constructed successfully. After pEGFP-C1-UL83 was transfected into Hep-2 cell by lipofectin mediation, expression of GFP and truncated pp65 fusion protein in Hep-2 cell was observed at different time points by fluorescence microscope. Results showed that quantity of fusion protein expression was the highest at 36h point. Then, Hep-2 cell was cultured selectively by RPMI-1640 containing G418 (200 microg/mL) to obtain a new cell stock of expressing truncated UL83 Gene fragment steadily. RT-PCR and Western blot results showed the truncated fragment of UL83 gene could be expressed steadily in Hep-2 cell. The result showed a new cell stock of expressing Tpp65 was established. This cell stock could be useful in some HCMV research fields, for example, it could be a tool in study of pp65 and HCMV infection, and it could provide a platform for the research into the therapy of HCMV infection. Immune sheltered effect of pEGFP-C1-UL83 as DNA vaccine was studied in vivo of HCMV congenital infection mouse model. The mouse model was immunized solely by pEGFP-C1-UL83, and was immunized jointly by pEGFP-C1-UL83 and its expression product. When the mouse was pregnant and brought to bed, differential antibody of anti-HCMV pp65 was tested by indirect ELISA in mother mouse, the infectious virus was separated with the method of virus separation, and pp65 antigen was checked up by indirect immunofluorescence staining in fetal mouse. Results showed differential antibody of anti-HCMV pp65 was produced in mouse model. Tilter of the antibody was from 1:2.51 to 1:50.79. Results of virus separation and pp65 checkup of fetal mouse brain tissue were negative. So the conclusion can be reached that pEGFP-C1-UL83 as DNA vaccine in vivo has sheltered effect which can prevent HCMV vertical transmission from mother mouse to her fetus.
Assuntos
Citomegalovirus/genética , DNA Recombinante/genética , Células Eucarióticas/metabolismo , Deleção de Genes , Vetores Genéticos/genética , Fosfoproteínas/genética , Vacinas de DNA/genética , Proteínas da Matriz Viral/genética , Animais , Anticorpos/análise , Anticorpos/imunologia , Linhagem Celular Tumoral , Citomegalovirus/imunologia , Citomegalovirus/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos/metabolismo , Humanos , Camundongos , Fosfoproteínas/deficiência , Fosfoproteínas/imunologia , Gravidez , Transfecção , Vacinação , Vacinas de DNA/imunologia , Proteínas da Matriz Viral/deficiência , Proteínas da Matriz Viral/imunologiaRESUMO
BACKGROUND: Avian influenza viruses have caused human infection and posed the pandemic potential. Live poultry markets are considered as a source of human infection with avian influenza viruses. Avian influenza routine surveillance of live poultry markets is taken annually in China. We isolated the 2 H11N9 influenza virus from the surveillance program. To better understand the risk caused by these new viruses, we characterize the genetic and pathogenicity of the two viruses. METHODS: Viral isolation was conducted with specific pathogen-free (SPF) embryonated chicken eggs. Whole genome was sequenced, and phylogenetic analysis was conducted. RESULTS: Two H11N9 viruses were identified, with all 8 segments belonging to the Eurasian lineage. The HA, NA, M, NS and PA genes were similar to virus isolates from ducks, and the NP, PB2 and PB1 gene segments were most similar to those viruses from wild birds, indicating that the H11N9 viruses might represent reassortant viruses from poultry and wild birds. The HA receptor binding preference was avian-like, and the cleavage site sequence of HA showed low pathogenic. The NA gene showed 94.6 % identity with the novel H7N9 virus that emerged in 2013. There was no drug resistance mutation in the M2 protein. The Asn30Asp and Thr215Ala substitutions in the M1 protein implied a potentially increased pathogenicity in mice. Both viruses were low-pathogenic strains, as assessed by the standards of intravenous pathogenicity index (IVPI) tests. CONCLUSION: Two reassortant H11N9 avian influenza viruses were detected. These viruses showed low pathogenicity to chickens in the IVPI test. Public health concern caused by the reassortant H11N9 viruses should be emphasized during the future surveillance.
Assuntos
Galinhas , Patos , Genoma Viral , Vírus da Influenza A/genética , Influenza Aviária/epidemiologia , Doenças das Aves Domésticas/epidemiologia , Vírus Reordenados/genética , Animais , China/epidemiologia , Microbiologia Ambiental , Vírus da Influenza A/classificação , Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Filogenia , Doenças das Aves Domésticas/virologia , Vírus Reordenados/classificação , Vírus Reordenados/patogenicidade , Análise de Sequência de RNA/veterináriaRESUMO
Five H9N2 avian influenza virus strains were isolated from the environmental samples in live poultry market in Qinghai Lake region from July to September, 2012. To evaluate the phylogenetic characteristics of these H9N2 isolates, the eight gene segments were amplified by RT-PCR and sequenced. The phylogenetic and molecular characteristics of the five strains were analyzed. The results showed that the HA genes of five strains shared 93. 2%-99. 1% nucleotide identities with each other, and the NA genes shared 94. 5%-99. 8% nucleotide identities. The HA cleavage site sequence of the A/environment/qinghai/ 017/2012 isolate was PSKSSRGLF, and the HA cleavage site sequences of the other four strains were all PSRSSRGLF. The HA receptor-binding site had the Q226L mutation. The M1 gene segment had the N30D and T215A mutations. The phylogenetic analysis showed that the five strains were similar to the virus A/chicken/Hunan/5260/2005 (H9N2) isolated in Hunan Province, China and were reassortant genotype viruses; the HA, NA, and NS genes belonged to the Y280-like lineage; the MP gene belonged to the G1-like lineage; the NP, PB1, PB2, and PA genes belonged to the F98-like lineage.
Assuntos
Genoma Viral , Vírus da Influenza A Subtipo H9N2/classificação , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/virologia , Filogenia , Doenças das Aves Domésticas/virologia , Animais , China , Genótipo , Vírus da Influenza A Subtipo H9N2/genética , Dados de Sequência Molecular , Aves Domésticas , Proteínas Virais/genéticaRESUMO
Since 2002, H7 subtype avian influenza viruses (AIVs) have caused more than 100 human infection cases in the Netherlands, Italy, Canada, the United States, and the United Kingdom, with clinical illness ranging from conjunctivitis to mild upper respiratory illness to pneumonia. On March 31st, three fatal cases caused by infection of a novel reassortant H7N9 subtype were reported in Shanghai City and Anhui Province in China. With the ability of H7 subtype to cause severe human disease and the increasing isolation of subtype H7 AIVs, we highlighted the need for continuous surveillance in both humans and animals and characterization of these viruses for the development of vaccines and anti-viral drugs.
Assuntos
Vírus da Influenza A/fisiologia , Influenza Aviária/virologia , Influenza Humana/virologia , Doenças das Aves Domésticas/virologia , Animais , Galinhas , Patos , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/patogenicidade , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Aviária/imunologia , Influenza Aviária/prevenção & controle , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/prevenção & controle , PerusRESUMO
A real-time NASBA assay for the specific confirmation of influenza A H5N1 infection was developed and evaluated using proficiency panels distributed to the UK influenza network of laboratories and clinical samples received through the Chinese National Influenza Centre in Beijing. The aim of the proficiency panels was to determine the sensitivity and specificity of the assay on a range of influenza virus types and subtypes including different clades of influenza A H5 viruses. The assay was then evaluated using 19 clinical samples obtained from seven confirmed human cases of influenza A H5N1 infection in China. The assay was shown to have a level of sensitivity of 0.01 TCID50 and 10copies/µl using RNA transcripts of the A/VietNam/1194/2004 H5N1 virus. During the evaluation the assay successfully detected H5N1 viruses known to infect humans from clades 1, 2.1, 2.2 and 2.3 as well as low pathogenic H5N3 avian influenza viruses. The clinical utility of the real-time NASBA assay was proven on a range of clinical samples from patients with confirmed H5N1 infection collected during 2005 and 2006. The real-time NASBA assay was demonstrated to be sensitive and rapid allowing for same day confirmation of a H5N1 infection direct from clinical samples.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Humana/diagnóstico , Neuraminidase/genética , Replicação de Sequência Autossustentável/métodos , Animais , Aves/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/isolamento & purificação , Humanos , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/diagnóstico , Influenza Aviária/virologia , Influenza Humana/virologia , Neuraminidase/isolamento & purificação , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Alinhamento de SequênciaRESUMO
A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.
Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Colorimetria/métodos , Primers do DNA/genética , Eletroforese em Gel de Ágar , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/virologia , Naftalenossulfonatos/química , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , TemperaturaRESUMO
A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of pandemic influenza A H1N1 virus infection. The reaction was performed in one step in a single tube at 65 degrees C for 60 min with the addition of hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was approximately 60 copies, and no cross-detection was observed. The assay was evaluated further with 50 clinical specimens diagnosed clinically with seasonal influenza or pandemic influenza A H1N1 virus infection. RT-LAMP with HNB dye was demonstrated to be a sensitive and easy assay for rapid detection of pandemic influenza A H1N1 virus.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/diagnóstico , Influenza Humana/virologia , Naftalenossulfonatos/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Coloração e Rotulagem/métodos , Virologia/métodos , Humanos , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To construct pseudovirus bearing H5N1 HA based on a lentivirus vector system. Then we study the biological feature of the pseudovirus. With the newly established viral particles, we performed the serological tests. METHODS: H5N1 avian influenza virus that isolated from human case was cloned to construct pLP-HA, then pLP-HA co-transfected with lentivirus vector plasmids pLP1, pLP2 and pEmGFP into 293T cells. The supernatant was harvested 48h post-transfection. Concentrated by super centrifuge, the pseudotyped viruses were analyzed by infection test, HA test and micro-neutralization test. At the same time, optimized HA gene and a Vietnam H5N1 HA gene were used to construct pseudotyped virus for comparison. RESULTS: Pseudotyped virus particles can be observed with electronic microscope. Western-blot revealed that HA glycoprotein can be expressed in the virions. Our neutralization assay by using the pseudoviruses was comparable with the conventional microneutralization assay with wild-type viruses. A high degree of correlation was detected. CONCLUSION: Pseudotyped Viruses coated with HA of H5N1 High Pathogenic Avian Influenza were successfully constructed; it can be used to for the microneutralization assay. The HA gene from different sources affect the efficiency of the packaging of the pseudovirus. But the optimized HA gene can not obviously improve packaging efficiency of the pseudovirus.
Assuntos
Engenharia Genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Virus da Influenza A Subtipo H5N1/fisiologia , Vírion/fisiologia , Animais , Linhagem Celular , Cães , Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/ultraestrutura , Influenza Humana/virologia , Lentivirus/genética , Lentivirus/metabolismo , Vírion/genética , Vírion/ultraestruturaRESUMO
OBJECTIVE: To analyse the correlation between the virus isolation and the specimen collection of the H5N1 human high pathogenic avain influenza cases in Mainland China. METHODS: The specimens were collected in Mainland China from 2005.10 to 2009.3 and the H5N1 viruses were isolated by passage in embryonated chicken eggs. RESULTS: Most specimens were obtained within 14 days after disease onset. For the specimens collected within 7 days, the isolation rate was relatively high and the difference of the positive rate between different years was lower than those specimens collected after 7 days. Most of the samples in our study were collected from the upper or lower respiratory tract with few from blood, feces, et al. The isolation rate of lower respiratory specimens was higher and the difference of the positive rate between different years was relatively lower than those from upper respiratory specimens. CONCLUSION: We suggest that the samples should be collected from lower respiratory tract during the acute phase to get the higher isolation rate.
Assuntos
Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Humana/virologia , Animais , Embrião de Galinha , China/epidemiologia , Humanos , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/genética , Influenza Humana/epidemiologia , Sistema Respiratório/virologiaRESUMO
A new flu caused by a novel influenza A(H1N1) virus has spread over the United States, Mexico and more than 40 other countries. And because of the immediate global concern, WHO has announced that the current level of influenza pandemic alert is raised to phase 5, indicating approaching of an influenza pandemic. As patients suffering from the influenza A (H1N1) have the similar symptoms as patients with seasonal influenza, differential detection and identification of the influenza virus have to depend on specific laboratory tests. We have successfully developed a RT-PCR based method for detection of the influenza A (H1N1) virus, and had applied the method to detection of clinical samples.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Humanos , Vírus da Influenza A Subtipo H1N1/genéticaRESUMO
The clinical throat swab specimen of an imported suspected case of influenza A (H1N1) was detec ted with real-time PCR, RT-PCR and subsequently confirmed by gene sequencing. The presence of influ enza A (H1N1) virus confirmed the first case with A (H1N1) infection in Mainland China.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/virologia , China , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/genética , Dados de Sequência Molecular , FilogeniaRESUMO
The tracheal aspirates and serum samples of a suspected human case of high-pathogenic avian influenza (firstly found in Shenzhen, China) were collected and tested by a series of assays. The results showed that the RNA extracted from the tracheal aspirate specimens of the patient was confirmed positive for H5N1 avian influenza virus by Real-time PCR. The H5N1 avian influenza virus was isolated from patient's tracheal aspirates on MDCK cell and was named A/Guangdong/2/06(H5N1). The viral load of tracheal aspirates collected at different time points were detected by Real-time PCR. The virus microneutralization and the antigenic ratio of human H5N1 isolated were also assayed. It was found that when the virus load decreased gradually after the disease onset, the serum neutralizing antibody titer in the patient increased to 1 : 160 and subsequently decreased gradually. By molecular analysis, the eight gene segments of A/Guangdong/2/06 revealed to be similar to that of H5N1 avian influenza viruses isolated from south China in 2005-2006. However, there were obvious differences in the gene sequence of the detected H5N1 viral RNA as compared with that of the strains isolated from Vietnam, Thailand and Indonesia.