褐家鼠群体MHC遗传多态性与SEOV感染相关性的研究

Background: Hemorrhagic fever with renal syndrome (HFRS) in China is caused by Hantaan virus（HTNV）and Seoul virus (SEOV) belonging to Hantavirus genus. SEOV mainly carried by Rattus norvegicus has been found in all HFRS areas of endemicity in China. Recently, HFRS caused by SEOV occurred in the areas where HFRS had never been reported before, suggesting that both the virus and the disease are spreading further. MHC genes polymorphism influences the ability of humans and rodents to control hantavirus replication. The MHC polymorphism of R. norvegicus wild population and the variability of R. norvegicus susceptibility to SEOV infection are still unclear...Aim: To investigate association between class II MHC gene polymorphism and SEOV infection in the wild R. norvegicus population. ..Methodology: Wild R. norvegicus are sampled in different geographic areas in Hubei, China. SEOV antigen in rat lungs and IgG antibodies against hantavirus are screened. The viral load in rat lungs are detected with SYBR Green based real-time RT-PCR. The genes of SEOV are amplified with RT-PCR and sequenced. The MHC-II DQA-exon2 and DRB-exon2 of R. norvegicus are amplified with PCR and analyzed with SSCP and sequencing. The followings are statistically analyzed. 1) The distribution and frequencies of MHC alleles in R. norvegicus population from different locations. 2) The distribution and frequencies of MHC alleles in R. norvegicus with and without SEOV infections. 3) The relationship between MHC single nucleotide polymorphism and the SEOV load. 4) The comparision of infection rates among R. norvegicus belonging to different phylogenetic branches. ..Significance: This study will reveal the possible variability of R. norvegicus susceptibility to SEOV infection, the distribution of MHC alleles in different R. norvegicus populations, and the association between the MHC polymorphism and SEOV infection among the R. norvegicus populations. It may help to explain the differences of hantavirus susceptibility among different host populations. It is important for the study of temporal and special changes in the HFRS incidence, and the mechanism of HFRS.

Seoul 病毒(SEOV)是我国肾综合征出血热（HFRS）的重要病原体，其宿主褐家鼠在HFRS流行中起关键作用。研究目的：探讨自然褐家鼠群体MHC基因多态性与SEOV感染的相关性。方法：在湖北省不同地形地貌的HFRS流行区采集褐家鼠，检测褐家鼠携带汉坦病毒抗原、特异性抗体；SEOV基因扩增、载量检测、测序和分析；褐家鼠MHC-II DQA-exon2和DRB-exon2基因检测与分析。统计学分析：不同采样地点褐家鼠群体MHC等位基因的分布频率；SEOV阳性和阴性褐家鼠群体MHC等位基因的分布频率；褐家鼠MHC单核苷酸多态性位点与SEOV病毒载量之间的关联；属于不同进化分支的褐家鼠SEOV感染率的差异。此研究有助于阐明MHC等位基因在不同地理环境褐家鼠群体中的分布，揭示不同褐家鼠群体对SEOV易感性差异与MHC基因多态性的关系。这对于研究HFRS发病率的时空变化及发病机制具有重要意义。

目的：研究湖北省褐家鼠汉城病毒（Seoul virus, SEOV）感染与MHC-II DQA-exon2 基因多态性的关系。方法：2014-2015年在湖北的襄阳、宜昌、荆州、武汉四个地区采用隔夜法捕鼠。间接免疫荧光法（IFA）检测鼠血清SEOV-IgG抗体。应用褐家鼠、黄胸鼠线粒体DNA 细胞色素b特异性PCR鉴别鼠种。建立巢式PCR扩增SEOV S片段方法。褐家鼠MHC-II DQA- exon2 特异性PCR，产物纯化测序。对MHC-II DQA- exon2杂合子进行TA克隆并测序。卡方检验比较湖北省不同地区的SEOV阳性率。分析湖北褐家鼠MHC-II DQA-exon2单倍型和杂合度。卡方检验比较湖北不同地区褐家鼠SEOV感染与MHC-II DQA- exon2单倍型以及杂合度之间关系。结果：2014-2015年在湖北的襄阳、宜昌、荆州、武汉这四个地区共捕获鼠245只，其中褐家鼠215只，黄胸鼠30只。IFA法检测啮齿动物SEOV-IgG的阳性率为5.71%，巢式RT-PCR检测SEOV S片段的阳性率为8.57%。以上地区啮齿动物SEOV的阳性率有显著性差异（P<0.05）。对204只褐家鼠的MHC-II DQA-exon2进行测序，分析出有10种单倍型。褐家鼠MHC-II DQA- exon2杂合度，在不同地区之间有显著性差异（P<0.05）；在SEOV-IgG阴性和阳性褐家鼠之间不具有显著性差异（P >0.05）；在SEOV S片段巢式RT-PCR阴性和阳性褐家鼠之间不具有显著性差异（P >0.05）。褐家鼠MHC-II DQA- exon2基因单倍型比较，在不同地区之间有显著性差异（P<0.05）；在SEOV-IgG阴性和阳性褐家鼠之间不具有显著性差异（P >0.05）；在SEOV S片段巢式RT-PCR阴性和阳性褐家鼠之间不具有显著性差异（P >0.05）。结论：2014-2015年湖北省襄阳、宜昌、荆州、武汉这四个地区褐家鼠感染SEOV阳性率有显著性差异。褐家鼠MHC-II DQA-exon2基因杂合度在以上地区有显著性差异，在SEOV阳性或阴性褐家鼠之间无显著性差异。褐家鼠MHC-II DQA- exon2基因单倍型在湖北以上地区有显著性差异，在SEOV阳性或阴性褐家鼠之间无显著性差异。该研究为汉坦病毒感染的防控提供了理论依据。