河北省汉坦病毒宿主动物携带病毒特性与种群遗传特性分析研究
发布时间：2018-08-12 18:39
【摘要】： 目的:了解河北省肾综合征出血热疫源地鼠种构成、带毒率及宿主动物所携带汉坦病毒与种群遗传特性间的关系,指导对本病的预防和控制。 方法: 1疫源地调查,夹夜法捕鼠,无菌取鼠肺; 2用冷冻切片机切鼠肺,冷丙酮固定,间接免疫荧光法筛选汉坦病毒阳性鼠肺; 3从阳性和阴性鼠肺中提取鼠基因组DNA; 4根据文献以及GENEBANK中已知的微卫星位点查找特异性引物,用聚合酶链反应(PCR)进行扩增,并进行电泳和观察结果; 5根据电泳结果显示,比较同一地区和不同地区阴性阳性标本间的差异,并根据相似系数计算不同地区间种群的亲缘关系。 结果: 1 2008年的鼠间疫情资料显示褐家鼠占总捕获鼠种的70.39%,是河北省的优势鼠种。河北省居民区鼠种主要为褐家鼠和小家鼠,分别占居民区总捕获鼠种的73.38%和24.10%;野外鼠种主要为褐家鼠、大仓鼠和小家鼠,分别占野外总捕获鼠种的54.72%、20.75%和13.21%。河北省居民区鼠密度为2.16%,高于野外鼠密度(0.53%)。 2将331份鼠肺冷冻切片,冷丙酮固定后用间接免疫荧光法检测,阳性鼠肺共1份,阳性率为0.30%。提示我省肾综合征出血热疫源地鼠带毒率较低,带毒鼠为宅区褐家鼠,野外及其它鼠种未检出。 3通过对同一地区携带与未携带汉坦病毒的褐家鼠间微卫星扩增结果比较得出,D4mit20、D16mit4和D18mit1三个位点均不稳定,但D16mit4位点扩增出的条带在唐山地区稳定存在,D18mit1位点仅可在沧州地区的阳性标本上扩增出条带,D2mit12、D3mit15、D3mit22、D7mit26、D12wox10、D12wox11和D14mit2这7个位点上阳性与阴性标本的扩增结果未显现出明显差别。 4通过对同一地区未携带汉坦病毒的褐家鼠间微卫星扩增结果比较得出,D4mit20、D16mit4和D18mit1三个位点极不稳定,但偶尔会出现,不能作为遗传特征的标记位点;D2mit12、D7mit26和D12wox10三个位点在各地区内比较稳定且发生变异小;D3mit15、D3mit22、D12wox11和D14mit2四个位点在各地区内遗传变异大,表现出多态性,鼠个体间存在遗传差别。 5通过对不同地区未携带汉坦病毒的褐家鼠间微卫星扩增结果比较得出,褐家鼠在D3mit15、D12wox10、D12wox11和D14mit2位点上存在地区间差异,表明不同地区的褐家鼠在遗传上存在差别,其余微卫星位点未显示出差别。经过比较相似系数发现,邯郸与保定、石家庄与唐山、沧州与邯郸的褐家鼠种群间亲缘关系较远,遗传距离较大。沧州与唐山的相似系数为0.8,亲缘关系较近。 6通过对不同地区携带汉坦病毒的褐家鼠间微卫星扩增结果比较得出,D16mit4位点在唐山地区的标本上扩增出条带;D18mit1位点在沧州标本上也可以扩增出条带,但不稳定;D2mit12、D3mit22、D12wox10和D14mit2位点上各地区标本扩增出的条带电泳位置一致,呈单态性,这三个地区间的阳性标本在这些位点上无差异;D7mit26、D12wox11和D3mit15位点在三个地区扩增结果稳定,但表现出多态性;D4mit20位点扩增结果稳定,但与预期结果不符。经过比较相似系数发现,唐山、沧州和保定三个地区携带有汉坦病毒的褐家鼠在这十个位点上的遗传距离不大,亲缘关系较近。 结论: 1褐家鼠、小家鼠是河北省居民区主要鼠种,褐家鼠、大仓鼠和小家鼠是野外主要鼠种。褐家鼠是河北省的优势鼠种,并且是肾综合征出血热的主要传染源。 2经过对携带汉坦病毒和未携带汉坦病毒的褐家鼠在十个微卫星位点的比较,未发现两者之间存在明显差别,还不能说明褐家鼠感染汉坦病毒与其遗传因素有关。 3通过计算相似系数得出,沧州与唐山的褐家鼠亲缘关系较近;邯郸与保定、邯郸与沧州、石家庄与唐山间的褐家鼠亲缘关系较远,遗传距离大。不同地区的褐家鼠存在遗传上的差别。 4在所研究微卫星位点中,D16mit4、D18mit1和D4mit20三个位点极不稳定;D2mit12、D7mit26和D12wox10位点表现出单态性,遗传差异小;D3mit15、D3mit22、D12wox11和D14mit2位点表现出多态性,遗传差异大。
[Abstract]:Objective: To understand the species composition, virulence rate and the relationship between Hantavirus carried by host animals and the genetic characteristics of the population in the epidemic focus of hemorrhagic fever with renal syndrome (HFRS) in Hebei Province, and to guide the prevention and control of the disease.
Method:
1 the investigation of the focus of the epidemic.
The lungs of mice were cut by frozen section machine, fixed by cold acetone, and screened by indirect immunofluorescence.
3 extract genomic DNA from the lungs of positive and negative mice.
According to the literature and the known microsatellite sites in GENEBANK, specific primers were searched and amplified by polymerase chain reaction (PCR), and the results of electrophoresis and observation were carried out.
5. The results of electrophoresis showed that the difference between the negative and positive samples in the same region and different regions was compared, and the genetic relationship among the populations in different regions was calculated according to the similarity coefficient.
Result:
The epidemic data in 2008 showed that Rattus norvegicus accounted for 70.39% of the total captured species, and it was the dominant species in Hebei Province. %, 20.75% and 13.21%. of rodent density in Hebei province were 2.16%, higher than that of rodent density (0.53%).
2. 331 frozen sections of rat lungs were fixed with cold acetone and detected by indirect immunofluorescence method. The positive rate was 0.30%. It was suggested that the rodents in the epidemic focus of hemorrhagic fever with renal syndrome in our province had a low virulence rate, and the rodents were Rattus norvegicus in the residential area, but not found in the field and other rodents.
The results of microsatellite amplification showed that D4mit20, D16mit4 and D18mit1 were unstable, but the amplified bands of D16mit4 were stable in Tangshan area. D18mit1 could only be amplified in positive samples of Cangzhou area, D2mit12, D3mit15, D3. There was no significant difference in the amplification results of positive and negative specimens at mit22, D7mit26, D12wox10, D12wox11 and D14mit2 loci.
The results of microsatellite amplification showed that D4mit20, D16mit4 and D18mit1 loci were very unstable, but occasionally appeared and could not be used as marker loci for genetic characteristics; D2mit12, D7mit26 and D12wox10 loci were stable and had little variation in each region; D3mit15, D3mit2 were stable and the mutation was small. 2. The genetic variations of D12wox11 and D14mit2 loci in different regions were large, showing polymorphism, and there were genetic differences among individuals.
5 Comparing the results of microsatellite amplification among Rattus norvegicus without Hantaan virus in different areas, we found that there were regional differences in D3mit15, D12wox10, D12wox11 and D14mit2 loci in Rattus norvegicus, indicating that there were genetic differences among Rattus norvegicus in different areas, and the other microsatellite loci did not show differences. The genetic distance between the populations of Rattus norvegicus in Danzhou and Baoding, Shijiazhuang and Tangshan, Cangzhou and Handan was relatively large, and the similarity coefficient between Cangzhou and Tangshan was 0.8.
6. Comparing the results of microsatellite amplification of Hantavirus in Rattus norvegicus from different regions, we found that the D16mit4 locus amplified a band in Tangshan region, the D18mit1 locus amplified a band in Cangzhou region, but it was not stable; the band electrophoresis sites of D2mit12, D3mit22, D12wox10 and D14mit2 loci amplified in different regions. The results of D7mit26, D12wox11 and D3mit15 loci amplification were stable but polymorphic in the three regions. The results of D4mit20 loci amplification were stable but not consistent with the expected results. The genetic distance between the ten loci of norvegicus with hantavirus was not large and the genetic relationship was close.
Conclusion:
Rattus norvegicus and Mus musculus are the main rodent species in residential areas of Hebei Province. Rattus norvegicus, hamster and Mus musculus are the main rodent species in the field. Rattus norvegicus is the dominant rodent species in Hebei Province and the main source of infection of hemorrhagic fever with renal syndrome.
After comparing the Hantavirus-carrying and non-Hantavirus-carrying Rattus norvegicus at 10 microsatellite loci, no significant difference was found between them. It is not clear that Hantavirus-carrying Rattus norvegicus is related to its genetic factors.
3. By calculating the similarity coefficients, it is concluded that the relationship between Rattus norvegicus in Cangzhou and Tangshan is close, while that between Handan and Baoding, Handan and Cangzhou, Shijiazhuang and Tangshan is far, and the genetic distance is large.
Among the microsatellite loci studied, D16mit4, D18mit1 and D4mit20 were highly unstable; D2mit12, D7mit26 and D12wox10 loci were monomorphic and had little genetic difference; D3mit15, D3mit22, D12wox11 and D14mit2 loci were polymorphic and had great genetic difference.
【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】：R181.3
本文编号：2179948
[Abstract]:Objective: To understand the species composition, virulence rate and the relationship between Hantavirus carried by host animals and the genetic characteristics of the population in the epidemic focus of hemorrhagic fever with renal syndrome (HFRS) in Hebei Province, and to guide the prevention and control of the disease.
Method:
1 the investigation of the focus of the epidemic.
The lungs of mice were cut by frozen section machine, fixed by cold acetone, and screened by indirect immunofluorescence.
3 extract genomic DNA from the lungs of positive and negative mice.
According to the literature and the known microsatellite sites in GENEBANK, specific primers were searched and amplified by polymerase chain reaction (PCR), and the results of electrophoresis and observation were carried out.
5. The results of electrophoresis showed that the difference between the negative and positive samples in the same region and different regions was compared, and the genetic relationship among the populations in different regions was calculated according to the similarity coefficient.
Result:
The epidemic data in 2008 showed that Rattus norvegicus accounted for 70.39% of the total captured species, and it was the dominant species in Hebei Province. %, 20.75% and 13.21%. of rodent density in Hebei province were 2.16%, higher than that of rodent density (0.53%).
2. 331 frozen sections of rat lungs were fixed with cold acetone and detected by indirect immunofluorescence method. The positive rate was 0.30%. It was suggested that the rodents in the epidemic focus of hemorrhagic fever with renal syndrome in our province had a low virulence rate, and the rodents were Rattus norvegicus in the residential area, but not found in the field and other rodents.
The results of microsatellite amplification showed that D4mit20, D16mit4 and D18mit1 were unstable, but the amplified bands of D16mit4 were stable in Tangshan area. D18mit1 could only be amplified in positive samples of Cangzhou area, D2mit12, D3mit15, D3. There was no significant difference in the amplification results of positive and negative specimens at mit22, D7mit26, D12wox10, D12wox11 and D14mit2 loci.
The results of microsatellite amplification showed that D4mit20, D16mit4 and D18mit1 loci were very unstable, but occasionally appeared and could not be used as marker loci for genetic characteristics; D2mit12, D7mit26 and D12wox10 loci were stable and had little variation in each region; D3mit15, D3mit2 were stable and the mutation was small. 2. The genetic variations of D12wox11 and D14mit2 loci in different regions were large, showing polymorphism, and there were genetic differences among individuals.
5 Comparing the results of microsatellite amplification among Rattus norvegicus without Hantaan virus in different areas, we found that there were regional differences in D3mit15, D12wox10, D12wox11 and D14mit2 loci in Rattus norvegicus, indicating that there were genetic differences among Rattus norvegicus in different areas, and the other microsatellite loci did not show differences. The genetic distance between the populations of Rattus norvegicus in Danzhou and Baoding, Shijiazhuang and Tangshan, Cangzhou and Handan was relatively large, and the similarity coefficient between Cangzhou and Tangshan was 0.8.
6. Comparing the results of microsatellite amplification of Hantavirus in Rattus norvegicus from different regions, we found that the D16mit4 locus amplified a band in Tangshan region, the D18mit1 locus amplified a band in Cangzhou region, but it was not stable; the band electrophoresis sites of D2mit12, D3mit22, D12wox10 and D14mit2 loci amplified in different regions. The results of D7mit26, D12wox11 and D3mit15 loci amplification were stable but polymorphic in the three regions. The results of D4mit20 loci amplification were stable but not consistent with the expected results. The genetic distance between the ten loci of norvegicus with hantavirus was not large and the genetic relationship was close.
Conclusion:
Rattus norvegicus and Mus musculus are the main rodent species in residential areas of Hebei Province. Rattus norvegicus, hamster and Mus musculus are the main rodent species in the field. Rattus norvegicus is the dominant rodent species in Hebei Province and the main source of infection of hemorrhagic fever with renal syndrome.
After comparing the Hantavirus-carrying and non-Hantavirus-carrying Rattus norvegicus at 10 microsatellite loci, no significant difference was found between them. It is not clear that Hantavirus-carrying Rattus norvegicus is related to its genetic factors.
3. By calculating the similarity coefficients, it is concluded that the relationship between Rattus norvegicus in Cangzhou and Tangshan is close, while that between Handan and Baoding, Handan and Cangzhou, Shijiazhuang and Tangshan is far, and the genetic distance is large.
Among the microsatellite loci studied, D16mit4, D18mit1 and D4mit20 were highly unstable; D2mit12, D7mit26 and D12wox10 loci were monomorphic and had little genetic difference; D3mit15, D3mit22, D12wox11 and D14mit2 loci were polymorphic and had great genetic difference.
【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】：R181.3
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