RESUMO
The global outbreak of novel A/H1N1 spread in human population worldwide has revealed an emergency need for producing a vaccine against this virus. Current influenza vaccines encounter problems with safety issues and weak response in high-risk population. It has been established that haemagglutinin is the most important viral antigen to which antibody responses are directed, and recombinant subunit vaccines, haemagglutinin of influenza A and B viruses, have been considered in order to facilitate vaccine production. In the present study, we have focused on construction of a recombinant baculovirus encoding the large subunit of novel influenza virus A/H1N1 haemagglutinin. The full genome of haemagglutinin was cloned into pGEM-TEasy vector and sequenced. The large subunit of the haemagglutinin gene was amplified by PCR using specific primers and cloned into pFast- BacHTc donor plasmid, which was then confirmed by restriction enzyme analysis and sequencing and transformed into E. coli DH10Bac competent cells. The bacmid DNA was transfected into insect cells to produce recombinant baculovirus. Expression of recombinant haemagglutinin in insect cells was determined by SDS-PAGE and immunoblotting. It has been shown that the recombinant haemagglutinin (rHA) obtained from the baculovirus insect cell expression system has suitable immunogenicity in human and can be considered as a candidate flu vac- cine. Here we produced large amounts of the HA1 protein of novel influenza A/H1N1 (Iranian isolate) in insect cells. The immunogenicity and efficacy of the recombinant HA1 will be evaluated as a vaccine candidate and compared to the recombinant HA1 produced in a prokaryotic system.
Assuntos
Epitopos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Linhagem Celular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Immunoblotting , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Spodoptera/genética , Spodoptera/virologia , TransfecçãoRESUMO
PURPOSE: Influenza virus is a major cause of human respiratory infections and responsible for pandemics and regional outbreaks around the world. This investigation aims to determine the prevalent influenza genotypes during 2005-2007 outbreaks in Shiraz, the capital city of Fars province, southern Iran and compare the results obtained with those of previous study. MATERIALS AND METHOD: Of the 300 pharyngeal swabs collected from influenza patients, 26 were found to be positive by culture and hemagglutination (HA) assays. Typing and subtyping of the isolates carried out by using multiplex RT-PCR and phylogenetic analysis performed on isolated HA genes using neighbour-joining method. RESULT: Out of 26 positive isolates 12 and 14 were H1N1 and H3N2 respectively. The phylogenetic and amino acid sequence analyses of our H1N1 isolates showed 99-100% genetic resemblance to A/NewCaledonia/20/99 (H1N1) vaccine strain. Most of the Iranian H3N2 isolates varied form A/California/7/2004 vaccine strain in 20 amino acids of which positions 189,226 and 227 were located in antigenic sites of HA1 molecule. These substitutions were not observed in any of the H3N2 subtypes from the same region reported previously. CONCLUSION: The H3N2 subtype strains prevalent during the 2005/7 influenza outbreak in southern Iran demonstrated a drastic antigenic variation and differed from A/California/7/2004 vaccine strain. The H1N1 subtypes showed a notable resemblance to A/NewCaledonia/20/99 vaccine strain and therefore were predicted to be capable of conferring sufficient immunity against H1N1 subtypes.
Assuntos
Variação Antigênica , Vírus da Influenza A/classificação , Vírus da Influenza A/imunologia , Influenza Humana/epidemiologia , Influenza Humana/virologia , Adolescente , Adulto , Idoso , Substituição de Aminoácidos/genética , Criança , Pré-Escolar , Genótipo , Testes de Hemaglutinação , Hemaglutininas Virais/genética , Humanos , Lactente , Irã (Geográfico)/epidemiologia , Pessoa de Meia-Idade , Epidemiologia Molecular , Dados de Sequência Molecular , Faringe/virologia , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Cultura de Vírus , Adulto JovemRESUMO
BACKGROUND: To date there are no reports of molecular and phylogenetic analyses of human influenza virus in Tehran, Iran. OBJECTIVES: We isolated and characterized circulating influenza viruses in a sample of patients in Tehran. METHODS: Nasal and pharyngeal swabs were collected from 57 individuals who were suspected of having influenza between October 2005 and January 2007. These samples were cultured and subsequently genotyped by RT-PCR and sequencing analyses. RESULTS: Twelve of 57 samples (21%) were positive for human influenza virus. Out of the 12 positive samples, 7 were A/H3N2 (58%), 3 were A/H1N1 (25%) and 2 were B subtypes (17%). The phylogenetic analysis of the hemagglutinin gene showed that the H1N1 isolates were close to the A/New Caledonia/20/99 and the H3N2 isolates were close to the A/Panama/2007/99 and A/Moscow/10/99 vaccine strains. CONCLUSION: In a sample of clinical patients in Tehran, Iran, the predominant subtype of human influenza virus was determined to be A/H3N2, followed by A/H1N1 and B. In addition, phylogenetic analysis on H1 showed some genetic drifts from vaccine strains, but the phylogeny of H3 demonstrated that these isolates were from the previous vaccine strains.