| Among children,acute respiratory tract infections(ARTIs) are a leading cause of morbidity and mortality worldwide.The main infectious agents responsible for ARTI include respiratory syncytial virus(RSV),influenza viruses,adenoviruses, parainfluenza viruses,rhinoviruses,coronaviruses,and the recently discovered human metapneumovirus,human bocavirus,human coronaviruses(SARS,HKU1,NL63),and all these likely account for a majority of respiratory tract illnesses.However,for a substantial proportion of respiratory tract illnesses in children,the pathogens can not be identified from the respiratory samples.In 2001,Van den Hoogen et al discovered a new kind of virus in respiratory secretions obtained from individuals with clinical features of respiratory tract illness. Sequence and phylogenetic analysis demonstrated that this new virus was a paramyxovirus and closely related to avian metapneumovirus which are members of the genus Metapneumovirus of the family Paramyxoviridae,subfamily Pneumoviranae, named human metapneumovirus(hMPV).To determine the prevalence and clinical characteristics of hMPV,we investigated the presence and genotype of this virus strains isolated from children with respiratory tract infection in 2005 in Hunan province,China.The membrane protein sequences of hMPV strains isolated were compared with hMPV sequences available in NCBI.Two DNA candidate vaccines and two adenovirus vector candidate vaccines were constructed with hMPV(B genotype) fusion protein(F△TM) and matrix protein(M) isolated from China,respectively.The immunization effects of these vaccines single and combined were evaluated also in this study.The major results are followed:1.Human metapneumovirus prevalence in children with acute respiratory tract illness in Hunan province,ChinaWe screened nasopharyngeal aspirates(NPAs) collected from hospitalized children with ARTI in 2005 in Hunan province.Overall,232 nasopharyngeal aspirates specimens from 232 hospitalized children were tested.17 hMPV positive(7.3%) were identified by RT-PCR and sequencing.Phylogenetic analysis shows that there are four genotypes(A1,A2,B1,and B2).Among the hMPV-positive patients,11 were male and 6 were female.The age of the infected patients ranged from 2 months to 5 years.The most common clinical presentations were fever,wheezing,and all these children with hMPV infection had diagnosis as pneumonia,bronchiolitis and bronchopneumonia. Among these hMPV positive cases,there are co-infections which included 5 human respiratory syncytial viruses and 1 human bocavirus.The co-infection rate is 35%.2.The polymorphism of membrane protein genes from different human metapneumovirus strainsWe amplified the complete coding sequence of F,G and M genes by using RT-PCR. Bioinformatics soft-wares were employed to analyze these sequence variations of hMPV.The results show that these viruses can be divided into two groups A and B based on their G genes.There are four types of sequence lengths of hMPV G glycoprotein in Hunan province isolated in 2005,which are 711nt,675nt,660nt,696nt, respectively.Differences in the G protein lengths were caused by the usage of alternative termination codons(TGA,TAG,TAA),the presence of in-frame duplications,deletions and insertions of bases,frame-shift mutation.Most of the substituted amino acids in these hMPVs G protein located at the extra-cellular domains. This reveals that the genetic variation degree is high and the character of geographic epidemic differences is distinct.The highly glycosylated character of hMPV G protein suggested that its function in infection and immunity is similar to that of the G protein of human respiratory syncytial virus(hRSV).Homology of the complete coding sequence of each individual gene of hMPV shows that the F and M genes are the most conserved regions.The homology of F and M amino acid sequences between A and B genotype reached 94%and 95%, respectively.As the major protective antigen,F is usually acted as the target gene of hMPV vaccine.M gene is the most conserved among the whole virus genome.Because of this,we usually designed primers based on M to detect hMPV.M protein is associated with virus assembling and budding and it can interact with membrane protein and nucleocapsid protein.We select M as the target gene of hMPV candidate vaccine too.3.Research on hMPV Candidate VaccinesBased on sequences analysis and data available,we establish the target gene F△TM(F without transmembrane region) and M to act as candidate vaccines.Then we prepare two kinds of candidate vaccines:DNA vaccine and adenoviral vector vaccine constructed with inserted F△TM or M respectively.These two kinds of candidate vaccines were combined to immunize mice,which could contribute their own advantages.The immunization strategy adopts priming with DNA vaccine to immunize BALB/c mice intramuscularly and boosting with recombinant adenoviral vector intramuscularly or intranasal and trachea twice,which can induce stronger immune responses comparing to single vaccine alone.Two weeks since the last immunization, immunized mice were sacrificed for immune response assay.The results show that combined immunizations induce stronger specific cellular and humoral immune responses than single immunization does,and which is consistent with the relevant study results.The specific IgG responses and IgG2a/IgG1 ratios are tested by ELISA, specific CD8~+ T lymphocytes which secret IFN-γ,is determined by ELISPOT.Our results indicate the immunization inclines to Th1-bias immune responses.The immune strategy that priming with(pcDNA3.1-F△TM+ pcDNA3.1-M) intramuscularly once and boosting with(r-Ad-F△TM+ r-Ad-M) intranasal and trachea twice can induce strong specific system immune responses and mucosal immune responses than that F△TM vaccine alone immunization.Maybe there is some kind of interaction between F△TM and M antigen,so it enhances the antigenicity of vaccines and optimizes immunity effects.The mechanism should be studied further to optimize the candidate vaccine immunization strategy.
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