| Objective: Determine the gene sequence of hantavirus (HV)in HFRS patients and the rat carried in Hebei hemorrhagic fever with renal syndrome (HFRS) epidemic areas, comparative analysis by the DNASTAR software, compare and contrast the HV pathogen's characters from rats and patients, provide a reasonable scientific basis for controlling of HFRS.Methods:1 Collecting the patient or the border line case of acute-phase serum specimens through epidemiology survey data;investigate the epidemic areas, capture rats and take it's lungs in sterile condition;2 Before extracting the HV RNA, using the assay of indirect immuno-fluorescence (IFA) screening the positive samples; the positive lungs were sliced with freezing microtome. Then positive lungs were screened with IFA after they were fixed by cold acetone;3 Through the assay of RT-nested PCR amplifying the virus nucleic acids, genotyping; RNA of HV was extracted from positive lungs and cDNA was synthesized, RT-nested PCR was conducted to identify the genotype of HV by using primers of SEO type and HTN type, which were designed according to the standard strains of HV;4 Choose the representative patients'serum samples and lung-positive samples in different areas by PCR amplification products purified before the sequencing;5 Comparing the sequence of HV from patients and rats and construct phylogenetic tree (by DNASTAR software) . And compare the HV isolated from the patients and rats of the same area.Results:1 The experiment collected 158 serum specimens of HFRS, tested by IFA, and the positive cases was 101 and the positive rate was 63.92%. Positive serum specimens for RT-PCR amplification, we got 17 positive results, the positive rate was 16.83%.2 The HV positive serum specimens were amplified by RT-nested PCR using primers of the types of SEO and HTN separately. The results indicated that the specimens are all SEO type positive and HTN type is negative.3 In this experiment, we got 43 positive rat lungs out of 1630, positive rate is 2.64%. the rate of the population of the rodents from the whole province and the distribution of HV, the province HFRS rat infected foci mainly in residential areas. The HFRS is transmitted mainly by Rattus norvegicus in residential area, followed by Mus musculus, the wild rat virus is not the main source of infection.4 HV positive lungs were all amplified by the primers of SEO and HTN types of G1 segment. The results are all SEO positive and HTN type is negative.5 Comparing separately the HV's G1and G2 segment which patients infected and rats infected, and analysis the genetic variation.5.1 Comparing G1 fragment nucleotide homology and genetic variation of HV infected in people and ratThe ressults of campairison of HV G1 fragment between that people infected and that rats carried indicate that G1 fragment nucleotide homology up to 95.0% ~ 99.7%. The homology of SJZ and ffTS is 95.0%, CD2,BD1,BD2,BD3 and ffSJZ,QHD2,TS and ffQHD,CZ1,CZ2 and ffCZ are 99.7%,The remaining are between 95.2% ~ 99.4%. The two specimens which from the same area were: Baoding 95.5%~96.4%, Chengde 95.5%~ 96.6%, Cangzhou 99.7%, Qinhuangdao 95.8%~99.7%, Shi jiazhuang 95.2%,Tangshan 99.4%. The highest nucleotide homology are CZ1 and ffCZ of Cangzhou ; QHD2 and ffQHD of Qinhuangdao, it is 99.7%, the minimum is 95.5% among BD1 and ffBD1 of Baoding ; CD1 and ffCD1 of Chengde. No matter its regions are the same or not, the HV that patients and rats have high level homologus.5.2 Comparingthe homology and genetic variation of G2 fragment that people infected and rats carriedThe G2 fragment nucleotide homology between 9 people,s serums and 7 rat lungs specimens is up to 92.7% ~ 99.7%.The CZ2 and ffBD1 is 92.7%,CD2 and ffCZ,BD3,BD1 and ffSJZ,QHD and ffTS Separately is 99.7%.The others is between 93.7%~99.3%. The two specimens of same area:Baoding is 95.7%~98.7%,Chengde is 98.7%~99.3%,Cangzhou is 97.3% ~98.3%,Shijiazhuang is 94.7%,Tangshan is 99.4%. The highest nucleotide homology are CD2 and ffCD1 of Chengde ; TS and ffTS of Tangshan, they are all 99.3%, the minimum is SJZ,ffSJZ of Shijiazhuang, which is 94.7%.6 The phylogenetic tree of G1 (217-573nt) and G2 (2002-2301nt) segments were constructed by DNASTAR software package. The phylogenetic tree of G1 segment displayed that QHD,ffQHD,TS,ffTS had the closer genetic relationship, ffBD1,SJZ had the closer genetic relationship, CZ1,ffCZ had the closer genetic relationship.The phylogenetic tree of G2 segment displayed that ffSJZ,BD1,BD3 had the closer genetic relationship, QHD2,TS,ffTS had the closer genetic relationship, SJZ,ffBD1 had the closer genetic relationship, G2 is consistent with G1 fragment.This indicate that two places have approximate homology. Other sequences have far distance.7 Genotype and phylogenetic tree analysis showed that Hebei Province is a typical SEO-type epidemic area. According to G1, G2 fragment, their genetic subtypes are divided into S1,S3 subtypes.Results of sub-type are consistent, Their subtype are mainly S3. No new subtype found. An exception carried out in Chengde , carried by one of Mus musculus.According to the phylogenetic tree,the HV G1 fragment belong to S4 subtype, G1 and G2 fragments classification basically have the same results.Conclusion:1 Whether it is the same area or not, the HV which people infected with and rat carried are high homology.HV of Rattus carried is the most important source of infection in Hebei Province.2 On the basis of genotype which we tested out and phylogenetic trees analysised, Hebei province is a typical epidemic area of SEO. The subtypes of S1 and S3 were coexisting but the S3 was the major subtype. SEO type was the dominant genotype carried by host animals in epidemic areas in Hebei. S3 was the major subtype and it was widespread in Hebei province.3 Geographical proximity in region, virus genome nucleotide sequence homology is at relatively high level Phylogenetic evolution relationship is close and the distribution of hantavirus has a certain regional character.
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