河北省HFRS疫區(qū)鼠攜帶漢坦病毒與人感染漢坦病毒的基因特征對(duì)應(yīng)性研究
發(fā)布時(shí)間：2018-09-01 17:44
【摘要】： 目的:對(duì)河北省腎綜合征出血熱(HFRS)疫區(qū)人感染和鼠攜帶漢坦病毒(HV)進(jìn)行基因序列測(cè)定,通過(guò)DNASTAR軟件對(duì)兩者進(jìn)行比較分析,闡明同一地區(qū)鼠攜帶HV與人感染HV二者的病原學(xué)特征及聯(lián)系,為HFRS的防控提供科學(xué)依據(jù)。 方法: 1通過(guò)流行病學(xué)調(diào)查,收集病人或疑似病人急性期血清標(biāo)本;并在相同區(qū)域開(kāi)展夾夜法捕鼠,無(wú)菌取鼠肺; 2采用間接免疫熒光法(IFA)篩選陽(yáng)性血清標(biāo)本,從中提取HV RNA;用冷凍切片機(jī)切鼠肺,冷丙酮固定后,IFA篩選HV陽(yáng)性鼠肺; 3通過(guò)反轉(zhuǎn)錄套式聚合酶鏈反應(yīng)(RT-nested PCR)擴(kuò)增病毒核酸,進(jìn)行基因分型;從陽(yáng)性鼠肺中提取HV RNA,逆轉(zhuǎn)錄合成cDNA,根據(jù)已知HV標(biāo)準(zhǔn)毒株的基因序列設(shè)計(jì)SEO型和HTN型型特異性引物,用RT-nested PCR擴(kuò)增,進(jìn)行基因分型; 4選擇代表性疫區(qū)血清標(biāo)本以及相同地區(qū)鼠肺標(biāo)本PCR擴(kuò)增產(chǎn)物經(jīng)純化回收后進(jìn)行序列測(cè)定; 5利用DNASTAR軟件對(duì)人感染的HV及鼠攜帶的HV進(jìn)行同源比較并構(gòu)建系統(tǒng)發(fā)生樹(shù),并對(duì)同一地區(qū)人感染的HV及鼠攜帶的HV進(jìn)行比較。 結(jié)果: 1本實(shí)驗(yàn)共收集病人或疑似病人血清標(biāo)本158份,經(jīng)IFA檢測(cè)陽(yáng)性101份,陽(yáng)性率為63.92%。對(duì)陽(yáng)性血清標(biāo)本進(jìn)行RT-PCR擴(kuò)增,得到17份陽(yáng)性結(jié)果,陽(yáng)性率為16.83%。 2對(duì)河北省HV陽(yáng)性病人血清標(biāo)本同時(shí)用SEO型和HTN型分型引物進(jìn)行RT-nested PCR擴(kuò)增,顯示陽(yáng)性標(biāo)本所攜帶HV均為SEO型,未發(fā)現(xiàn)HTN型。 3本實(shí)驗(yàn)從1630份鼠肺中共獲得陽(yáng)性鼠肺43份,帶毒率為2.64%。從全省各地鼠密度和帶毒率分布情況看,我省HFRS疫源地帶毒鼠主要以宅區(qū)褐家鼠為主,其次為宅區(qū)小家鼠,野外帶毒鼠不是主要傳染源。 4對(duì)陽(yáng)性鼠肺標(biāo)本總RNA同時(shí)用G1片段SEO型和HTN型分型引物分別進(jìn)行擴(kuò)增,顯示陽(yáng)性標(biāo)本所攜帶HV均為SEO型,未發(fā)現(xiàn)HTN型。 5人感染HV與鼠攜帶HV的G1、G2片段基因變異分析 5.1人感染HV與鼠攜帶HV的G1片段核苷酸同源性比較及基因變異情況 全省各地整體的人感染HV與鼠攜帶HV的G1片段核苷酸同源性比較結(jié)果顯示G1片段核苷酸序列同源性高達(dá)95.0%~99.7%。其中SJZ和ffTS為95.0%,CD2、BD1、BD2、BD3和ffSJZ,QHD2、TS和ffQHD,CZ1、CZ2和ffCZ都是99.7%,其余的為95.2%~99.4%之間。同一地區(qū)兩種標(biāo)本的分別為:保定為95.5%~96.4%,承德95.5%~96.6%,滄州為99.7%,秦皇島為95.8%~99.7%,石家莊為95.2%,唐山為99.4%。其中核苷酸同源性最高的為滄州的CZ1和ffCZ,秦皇島的QHD2和ffQHD都是99.7%,核苷酸同源性最低為保定的BD1和ffBD1以及承德的CD1和ffCD1,各為95.5%。提示無(wú)論是同一地區(qū)的鼠和人之間,還是不同地區(qū)的鼠和人之間,HV都是具有較高的同源性。 5.2人感染HV與鼠攜帶HV的G2片段核苷酸同源性比較及基因變異情況 從9份人血清和7份鼠肺中提取得標(biāo)本兩者之間G2片段核苷酸同源性高達(dá)92.7%~99.7%。其中CZ2和ffBD1之間的同源性為92.7%,CD2和ffCZ,BD3、BD1和ffSJZ,QHD和ffTS都分別為99.7%,其余的都在93.7%~99.3%之間。同一地區(qū)兩種標(biāo)本的分別為:保定為95.7%~98.7%,承德為98.7%~99.3%,滄州為97.3%~98.3%,石家莊為94.7%,唐山為99.3%。其中核苷酸同源性最高的為承德的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莊的SJZ和ffSJZ為94.7%。 6根據(jù)G1(217-573nt)和G2(2002-2301nt)片段用DNASTAR軟件包構(gòu)建系統(tǒng)進(jìn)化樹(shù)。G1片段構(gòu)建的系統(tǒng)進(jìn)化樹(shù)顯示QHD、ffQHD、TS、ffTS株親緣關(guān)系接近。ffBD1,SJZ株親緣關(guān)系接近, CZ1,ffCZ株親緣關(guān)系較近。G2片段構(gòu)建的系統(tǒng)進(jìn)化樹(shù)顯示ffSJZ,BD1,BD3株親緣關(guān)系接近, QHD2、TS、ffTS株親緣關(guān)系接近,SJZ,ffBD1株親緣關(guān)系接近,這與G1片斷的比較一致,說(shuō)明兩地的同源性比較接近。其他序列同源性相對(duì)遠(yuǎn)些。 7基因分型及系統(tǒng)進(jìn)化樹(shù)分析表明,河北省是典型的SEO型疫區(qū)。其基因亞型根據(jù)G1、G2片段分為S1、S3二個(gè)亞型,分型結(jié)果一致,均以S3亞型為主,未發(fā)現(xiàn)新亞型。除承德一小家鼠所攜帶HV根據(jù)G1片段構(gòu)建進(jìn)化樹(shù)分型為S4亞型, G1和G2片段的分型結(jié)果基本一致。 結(jié)論: 1無(wú)論是同一區(qū)域,還是不同地區(qū),河北省人感染HV與鼠攜帶HV高度同源,褐家鼠攜帶的HV是HFRS病人感染的最主要來(lái)源。 2河北省人感染HV以SEO型為主,共包括S1、S3兩個(gè)亞型,以S3亞型為主。疫源地宿主動(dòng)物所攜帶的HV基因型以SEO型為主,基因亞型以S3亞型為主,且在河北省廣泛分布。 3在地理位置相近的地區(qū),病毒基因組核苷酸序列同源性相對(duì)較高,系統(tǒng)進(jìn)化關(guān)系較近,HV的分布具有一定的地理區(qū)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R512.8;R181.3
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