河北省HFRS疫区鼠携带汉坦病毒与人感染汉坦病毒的基因特征对应性研究
发布时间：2018-09-01 17:44
【摘要】： 目的:对河北省肾综合征出血热(HFRS)疫区人感染和鼠携带汉坦病毒(HV)进行基因序列测定,通过DNASTAR软件对两者进行比较分析,阐明同一地区鼠携带HV与人感染HV二者的病原学特征及联系,为HFRS的防控提供科学依据。 方法: 1通过流行病学调查,收集病人或疑似病人急性期血清标本;并在相同区域开展夹夜法捕鼠,无菌取鼠肺; 2采用间接免疫荧光法(IFA)筛选阳性血清标本,从中提取HV RNA;用冷冻切片机切鼠肺,冷丙酮固定后,IFA筛选HV阳性鼠肺; 3通过反转录套式聚合酶链反应(RT-nested PCR)扩增病毒核酸,进行基因分型;从阳性鼠肺中提取HV RNA,逆转录合成cDNA,根据已知HV标准毒株的基因序列设计SEO型和HTN型型特异性引物,用RT-nested PCR扩增,进行基因分型; 4选择代表性疫区血清标本以及相同地区鼠肺标本PCR扩增产物经纯化回收后进行序列测定; 5利用DNASTAR软件对人感染的HV及鼠携带的HV进行同源比较并构建系统发生树,并对同一地区人感染的HV及鼠携带的HV进行比较。 结果: 1本实验共收集病人或疑似病人血清标本158份,经IFA检测阳性101份,阳性率为63.92%。对阳性血清标本进行RT-PCR扩增,得到17份阳性结果,阳性率为16.83%。 2对河北省HV阳性病人血清标本同时用SEO型和HTN型分型引物进行RT-nested PCR扩增,显示阳性标本所携带HV均为SEO型,未发现HTN型。 3本实验从1630份鼠肺中共获得阳性鼠肺43份,带毒率为2.64%。从全省各地鼠密度和带毒率分布情况看,我省HFRS疫源地带毒鼠主要以宅区褐家鼠为主,其次为宅区小家鼠,野外带毒鼠不是主要传染源。 4对阳性鼠肺标本总RNA同时用G1片段SEO型和HTN型分型引物分别进行扩增,显示阳性标本所携带HV均为SEO型,未发现HTN型。 5人感染HV与鼠携带HV的G1、G2片段基因变异分析 5.1人感染HV与鼠携带HV的G1片段核苷酸同源性比较及基因变异情况 全省各地整体的人感染HV与鼠携带HV的G1片段核苷酸同源性比较结果显示G1片段核苷酸序列同源性高达95.0%~99.7%。其中SJZ和ffTS为95.0%,CD2、BD1、BD2、BD3和ffSJZ,QHD2、TS和ffQHD,CZ1、CZ2和ffCZ都是99.7%,其余的为95.2%~99.4%之间。同一地区两种标本的分别为:保定为95.5%~96.4%,承德95.5%~96.6%,沧州为99.7%,秦皇岛为95.8%~99.7%,石家庄为95.2%,唐山为99.4%。其中核苷酸同源性最高的为沧州的CZ1和ffCZ,秦皇岛的QHD2和ffQHD都是99.7%,核苷酸同源性最低为保定的BD1和ffBD1以及承德的CD1和ffCD1,各为95.5%。提示无论是同一地区的鼠和人之间,还是不同地区的鼠和人之间,HV都是具有较高的同源性。 5.2人感染HV与鼠携带HV的G2片段核苷酸同源性比较及基因变异情况 从9份人血清和7份鼠肺中提取得标本两者之间G2片段核苷酸同源性高达92.7%~99.7%。其中CZ2和ffBD1之间的同源性为92.7%,CD2和ffCZ,BD3、BD1和ffSJZ,QHD和ffTS都分别为99.7%,其余的都在93.7%~99.3%之间。同一地区两种标本的分别为:保定为95.7%~98.7%,承德为98.7%~99.3%,沧州为97.3%~98.3%,石家庄为94.7%,唐山为99.3%。其中核苷酸同源性最高的为承德的CD2和ffCD1以及唐山的TS和ffTS都是99.3%,核苷酸同源性最低石家庄的SJZ和ffSJZ为94.7%。 6根据G1(217-573nt)和G2(2002-2301nt)片段用DNASTAR软件包构建系统进化树。G1片段构建的系统进化树显示QHD、ffQHD、TS、ffTS株亲缘关系接近。ffBD1,SJZ株亲缘关系接近, CZ1,ffCZ株亲缘关系较近。G2片段构建的系统进化树显示ffSJZ,BD1,BD3株亲缘关系接近, QHD2、TS、ffTS株亲缘关系接近,SJZ,ffBD1株亲缘关系接近,这与G1片断的比较一致,说明两地的同源性比较接近。其他序列同源性相对远些。 7基因分型及系统进化树分析表明,河北省是典型的SEO型疫区。其基因亚型根据G1、G2片段分为S1、S3二个亚型,分型结果一致,均以S3亚型为主,未发现新亚型。除承德一小家鼠所携带HV根据G1片段构建进化树分型为S4亚型, G1和G2片段的分型结果基本一致。 结论: 1无论是同一区域,还是不同地区,河北省人感染HV与鼠携带HV高度同源,褐家鼠携带的HV是HFRS病人感染的最主要来源。 2河北省人感染HV以SEO型为主,共包括S1、S3两个亚型,以S3亚型为主。疫源地宿主动物所携带的HV基因型以SEO型为主,基因亚型以S3亚型为主,且在河北省广泛分布。 3在地理位置相近的地区,病毒基因组核苷酸序列同源性相对较高,系统进化关系较近,HV的分布具有一定的地理区域性。
[Abstract]:Objective: To determine the genetic sequence of human and Mouse-borne Hantavirus (HV) in the epidemic area of hemorrhagic fever with renal syndrome (HFRS) in Hebei Province, and to clarify the pathogenic characteristics and relationship between HV carried by mice and human infection by DNA STAR software, so as to provide scientific basis for the prevention and control of HFRS.
Method:
1. To collect serum samples from patients or suspected patients in acute phase through epidemiological investigation, and to catch rats by trapping in the same area at night, and to collect rat lungs aseptically.
The positive serum samples were screened by indirect immunofluorescence assay (IFA), and the HV RNA was extracted from them.
3. Viral nucleic acid was amplified by RT-nested polymerase chain reaction (RT-nested PCR) for genotyping; HV RNA was extracted from the lungs of positive mice, and the DNA was synthesized by reverse transcription. The specific primers of SEO and HTN genotypes were designed according to the genomic sequence of known HV standard strains, and the genotypes were amplified by RT-nested PCR.
4. The PCR products of serum samples from representative epidemic areas and rat lung samples from the same areas were purified and recovered for sequencing.
5. The homologous comparison between human-infected HV and Mouse-borne HV was carried out by using DNA STAR software, and phylogenetic tree was constructed.
Result:
A total of 158 serum specimens from patients or suspected patients were collected, 101 of which were positive by IFA, and the positive rate was 63.92%. Seventeen positive serum specimens were amplified by RT-PCR, and the positive rate was 16.83%.
2 Serum samples of HV-positive patients in Hebei Province were amplified by RT-nested PCR using SEO and HTN typing primers. The results showed that all the positive samples were of SEO type and no HTN type was found.
In this study, 43 positive rat lungs were obtained from 1 630 rat lungs with a virus-carrying rate of 2.64%. According to the distribution of rat density and virus-carrying rate in the whole province, the rodents in HFRS focus areas were mainly residential Rattus norvegicus, followed by residential Rattus norvegicus, and the rodents in the field were not the main source of infection.
Four pairs of positive lung specimens were amplified with G1 segmental SEO and HTN typing primers, respectively. The results showed that all positive lung specimens were SEO type, and no HTN type was found.
Genetic variation of G1 and G2 fragments in 5 infected HV and mice carrying HV
Homology comparison and genetic variation of 5.1 human infected HV and HV G1 fragments
The nucleotide homology of G1 fragment was 95.0% ~ 99.7% between human and mouse infected with HV in the whole province. Among them, SJZ and ffTS were 95.0%, CD2, BD1, BD2, BD3 and ffSJZ, QHD2, TS and ffQHD, CZ1, CZ 2 and ffCZ were 99.7% and 95.2% ~ 99.4% respectively. The results were as follows: Baoding 95.5%~96.4%, Chengde 95.5%~96.6%, Cangzhou 99.7%, Qinhuangdao 95.8%~99.7%, Shijiazhuang 95.2% and Tangshan 99.4%. The highest nucleotide homology was CZ1 and ffCZ in Cangzhou, QHD 2 and ffQHD in Qinhuangdao 99.7%, and the lowest nucleotide homology was BD1 and ffBD1 in Baoding and CD1 and CD1 and CD1 and CD1 in Chengde 95.5% respectively. HV is highly homologous to both mice and humans in the same area and between mice and humans in different areas.
Homology comparison and genetic variation of 5.2 human infected HV and HV G2 fragments
The homology of G2 fragment between CZ2 and ffBD1 was 92.7%~99.7%. The homology of CD2 and ffCZ, BD3, BD1 and ffSJZ, QHD and ffTS were 99.7%~99.3%. The homology of G2 fragment between CZ2 and ffBD1 was 92.7%. The homology of CD2 and ffCZ, BD3, BD1 and ffSJZ, QHD and ffTS was 99.7%~99.3%. The homology of the two samples from the same area was 95.7%~98.7% respectively. The nucleotide homology of CD2 and ffCD1 in Chengde, TS and ffTS in Tangshan was 99.3%, and that of SJZ and ffSJZ in Shijiazhuang was 94.7%.
6 The phylogenetic tree constructed from G1 (217-573nt) and G2 (2002-2301nt) fragments was constructed by DNA STAR software package. The phylogenetic tree constructed from G1 fragments showed that QHD, ffQHD, TS and ffTS strains were close to. ffBD1, SJZ strains were close to each other, CZ1 and ffCZ strains were close to each other. The phylogenetic tree constructed from G2 fragments showed that ffSJZ, BD1 and BD3 strains were close to each other, and Q was close to each other. HD2, TS, ffTS strains are close to each other, SJZ, ffBD1 strains are close to each other, which is consistent with G1 fragment, suggesting that the homology between the two places is close. Other sequences are relatively distant.
Genotyping and phylogenetic tree analysis showed that Hebei Province was a typical epidemic area of SEO. The genotypes were divided into S1 and S3 subtypes according to G1 and G2 fragments. The results of genotyping were consistent. S3 subtype was the main subtype and no new subtype was found. The fruit is basically the same.
Conclusion:
No matter in the same region or in different regions, human infection with HV in Hebei Province is highly homologous to that in mice, and the most common source of HFRS infection is that carried by Rattus norvegicus.
2 The main HV subtype in Hebei Province was SEO, including S1 and S3 subtypes, and the main subtype was S3 subtype.
3. In geographically similar areas, the nucleotide sequence homology of viral genome is relatively high, and the phylogenetic relationship is relatively close. The distribution of HV has certain geographical region.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R512.8;R181.3
[Abstract]:Objective: To determine the genetic sequence of human and Mouse-borne Hantavirus (HV) in the epidemic area of hemorrhagic fever with renal syndrome (HFRS) in Hebei Province, and to clarify the pathogenic characteristics and relationship between HV carried by mice and human infection by DNA STAR software, so as to provide scientific basis for the prevention and control of HFRS.
Method:
1. To collect serum samples from patients or suspected patients in acute phase through epidemiological investigation, and to catch rats by trapping in the same area at night, and to collect rat lungs aseptically.
The positive serum samples were screened by indirect immunofluorescence assay (IFA), and the HV RNA was extracted from them.
3. Viral nucleic acid was amplified by RT-nested polymerase chain reaction (RT-nested PCR) for genotyping; HV RNA was extracted from the lungs of positive mice, and the DNA was synthesized by reverse transcription. The specific primers of SEO and HTN genotypes were designed according to the genomic sequence of known HV standard strains, and the genotypes were amplified by RT-nested PCR.
4. The PCR products of serum samples from representative epidemic areas and rat lung samples from the same areas were purified and recovered for sequencing.
5. The homologous comparison between human-infected HV and Mouse-borne HV was carried out by using DNA STAR software, and phylogenetic tree was constructed.
Result:
A total of 158 serum specimens from patients or suspected patients were collected, 101 of which were positive by IFA, and the positive rate was 63.92%. Seventeen positive serum specimens were amplified by RT-PCR, and the positive rate was 16.83%.
2 Serum samples of HV-positive patients in Hebei Province were amplified by RT-nested PCR using SEO and HTN typing primers. The results showed that all the positive samples were of SEO type and no HTN type was found.
In this study, 43 positive rat lungs were obtained from 1 630 rat lungs with a virus-carrying rate of 2.64%. According to the distribution of rat density and virus-carrying rate in the whole province, the rodents in HFRS focus areas were mainly residential Rattus norvegicus, followed by residential Rattus norvegicus, and the rodents in the field were not the main source of infection.
Four pairs of positive lung specimens were amplified with G1 segmental SEO and HTN typing primers, respectively. The results showed that all positive lung specimens were SEO type, and no HTN type was found.
Genetic variation of G1 and G2 fragments in 5 infected HV and mice carrying HV
Homology comparison and genetic variation of 5.1 human infected HV and HV G1 fragments
The nucleotide homology of G1 fragment was 95.0% ~ 99.7% between human and mouse infected with HV in the whole province. Among them, SJZ and ffTS were 95.0%, CD2, BD1, BD2, BD3 and ffSJZ, QHD2, TS and ffQHD, CZ1, CZ 2 and ffCZ were 99.7% and 95.2% ~ 99.4% respectively. The results were as follows: Baoding 95.5%~96.4%, Chengde 95.5%~96.6%, Cangzhou 99.7%, Qinhuangdao 95.8%~99.7%, Shijiazhuang 95.2% and Tangshan 99.4%. The highest nucleotide homology was CZ1 and ffCZ in Cangzhou, QHD 2 and ffQHD in Qinhuangdao 99.7%, and the lowest nucleotide homology was BD1 and ffBD1 in Baoding and CD1 and CD1 and CD1 and CD1 in Chengde 95.5% respectively. HV is highly homologous to both mice and humans in the same area and between mice and humans in different areas.
Homology comparison and genetic variation of 5.2 human infected HV and HV G2 fragments
The homology of G2 fragment between CZ2 and ffBD1 was 92.7%~99.7%. The homology of CD2 and ffCZ, BD3, BD1 and ffSJZ, QHD and ffTS were 99.7%~99.3%. The homology of G2 fragment between CZ2 and ffBD1 was 92.7%. The homology of CD2 and ffCZ, BD3, BD1 and ffSJZ, QHD and ffTS was 99.7%~99.3%. The homology of the two samples from the same area was 95.7%~98.7% respectively. The nucleotide homology of CD2 and ffCD1 in Chengde, TS and ffTS in Tangshan was 99.3%, and that of SJZ and ffSJZ in Shijiazhuang was 94.7%.
6 The phylogenetic tree constructed from G1 (217-573nt) and G2 (2002-2301nt) fragments was constructed by DNA STAR software package. The phylogenetic tree constructed from G1 fragments showed that QHD, ffQHD, TS and ffTS strains were close to. ffBD1, SJZ strains were close to each other, CZ1 and ffCZ strains were close to each other. The phylogenetic tree constructed from G2 fragments showed that ffSJZ, BD1 and BD3 strains were close to each other, and Q was close to each other. HD2, TS, ffTS strains are close to each other, SJZ, ffBD1 strains are close to each other, which is consistent with G1 fragment, suggesting that the homology between the two places is close. Other sequences are relatively distant.
Genotyping and phylogenetic tree analysis showed that Hebei Province was a typical epidemic area of SEO. The genotypes were divided into S1 and S3 subtypes according to G1 and G2 fragments. The results of genotyping were consistent. S3 subtype was the main subtype and no new subtype was found. The fruit is basically the same.
Conclusion:
No matter in the same region or in different regions, human infection with HV in Hebei Province is highly homologous to that in mice, and the most common source of HFRS infection is that carried by Rattus norvegicus.
2 The main HV subtype in Hebei Province was SEO, including S1 and S3 subtypes, and the main subtype was S3 subtype.
3. In geographically similar areas, the nucleotide sequence homology of viral genome is relatively high, and the phylogenetic relationship is relatively close. The distribution of HV has certain geographical region.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R512.8;R181.3
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