人偏肺病毒标准株病毒适合度与致病性关系的探讨

发布时间：2018-06-01 13:13

本文选题：人偏肺病毒 + 标准株　；参考：《遵义医学院》2012年硕士论文

【摘要】：第一部分关于人偏肺病毒标准株病毒适合度的研究目的人偏肺病毒(Human metapneumovirus hMPV)是2001年首次被分离的呼吸道病毒病原,可导致各年龄组人群呼吸道感染,以儿童感染为主,严重者威胁生命造成死亡,不仅如此,hMPV感染还与哮喘发生、发展有关。但目前针对hMPV感染,尚无可靠疫苗和治疗药物。病毒适合度(virai fitness)信息的获得,能够为疫苗的设计,病毒感染后病情、病程评估,药物选择及疾病的预后,提供有重要参考价值的信息,然而目前世界范围内对hMPV适合度的研究未见报道。本研究通过hMPV两亚型代表株NL/1/00(A亚型)与NL/1/99(B亚型)的体内外竞争性复制实验,检测不同标准株的相对适合度,对hMPV标准株适合度进行评价,提供国内外首份hMPV标准株适合度的分析报告。方法1.将分别代表A、B两亚型的病毒株NL/1/00与NL/1/99以20：80、50：50、80：20混合成3个比例不同的混合病毒株。2个单独病毒株及3个混合株以相同的感染量分别接种于生长于6孔板中的各孔单层Vero-E6细胞,设置复孔及空白对照孔。病毒连续培养十几代,每代培养4-5天后将病毒液均放置于-80℃冰箱速冻保存,部分用于传代,部分用于提取病毒RNA,反转录成cDNA后,Real-Time PCR及核苷酸序列图谱法检测两个病毒株所占比例变化。2.将6-8周,雌性BALB/c小鼠随机分为6组,其中5组以滴鼻法分别接种上诉2个单独病毒株及3个混合株,每组每只小鼠接种病毒量均为50ul1×109copies/ul,剩余1组设为空白对照组。于感染后第1,2,4,6,8,13,17,20天处死小鼠取肺组织,提取病毒RNA后,反转录成用cDNA,用Real-Time PCR与核苷酸序列图谱法检测两个病毒株所占比例变化. 结果在动物实验中,hMPV感染BALB/C小鼠后,可在小鼠体内复制存活约3周,于第4天到达复制高峰后病毒量逐渐减少,3周后检测不到病毒；20天内3个混合组均以NL/1/99所占比例为高,表现出较强的生长趋势；空白对照组中未检测到hMPV感染。在细胞体系中,病毒共传代培养至12代,1-5代3组混合组均以NL/1/99占较高比例,6-8代以NL/1/00为主,第9代开始NL/1/99消失,只剩NL/1/00单独生长；空白孔未检测到hMPV感染。在单独感染NL/1/00和NL/1/99的动物及细胞体系中,均未检测到除此病毒株外的其余hMPV类型。结论动物实验中NL/1/99表现出较强的竞争复制优势,与细胞系中前期趋势一致。细胞体系中NL/1/00和NL/1/99生长优势出现交替,与近些年来广泛报道的hMPV两亚型在人群中交替流行的现象相吻合。第二部分人偏肺病毒A与B基因型对小鼠致病性的观察目的通过观察人偏肺病毒(human metapneumovirus, hMPV)A和B亚型感染BALB/c小鼠后,小鼠在体重、病毒载量、病理及气道反应性等方面的改变,探讨两亚型病毒致病性上的差异,为hMPV的进一步研究奠定基础。方法hMPV感染BALB/c动物模型后,通过使用Real-time PCR方法检测鼠肺内病毒载量的变化,肺功能检测仪监测小鼠气道反应性的变化及组织系统评分判定病理改变情况,来观测两亚型致病性的差异。结果hMPV两亚型感染组,在体重的动态监测、病毒载量、肺组织病理改变及气道反应性上均无统计学意义的差异。结论hMPV两基因型感染BALB/c小鼠的致病性无差异。
[Abstract]:The first part is about the fitness of human standard strain of human partial Lev virus.
Human metapneumovirus hMPV is the first pathogen of respiratory tract virus isolated in 2001. It can cause respiratory infection in all age groups, mainly children infection, and serious people threaten life cause death. Not only that, but hMPV infection is also related to the development of asthma, but there is no reliable vaccine for hMPV infection at present. The acquisition of Virai fitness information can provide important reference information for the design of the vaccine, the condition of the virus infection, the course evaluation, the drug selection and the prognosis of the disease. However, the research on the fitness of hMPV has not been reported worldwide. This study represents the NL/1/00 by the hMPV two subtype. In vivo and in vitro competitive replication (A subtype) and NL/1/99 (B subtype), the relative fitness of different standard strains was detected, the fitness of hMPV standard strain was evaluated, and the analysis report on the fitness of the first hMPV standard strain at home and abroad was provided.
Method 1. A, B two subtype virus strain NL/1/00 and NL/1/99 were mixed with 20:80,50:50,80:20 into 3 separate virus strains and 3 mixed strains of mixed virus strains and 3 mixed strains were inoculated separately in the monolayer Vero-E6 cells, which grew in 6 orifice plates, and set up the compound holes and blank control holes. For more than ten generations, 4-5 days after each generation, the virus was stored in -80 C fridge for quick freezing, partly used for passage and part used to extract virus RNA. After reverse transcription to cDNA, Real-Time PCR and nucleotide sequence atlas were used to detect the proportion of two virus strains for 6-8 weeks, and female BALB/c mice were randomly divided into 6 groups, of which 5 groups were treated with nasal drip method. 2 separate virus strains and 3 mixed strains were inoculated respectively. Each group was inoculated with 50ul1 x 109copies/ul, the remaining 1 groups were set as blank control group. The lung tissue was killed on day 1,2,4,6,8,13,17,20 after infection. After the virus RNA was extracted, the reverse transcription was cDNA. Real-Time PCR and nucleotide sequence mapping method were used to detect two. A change in the proportion of the virus strains.
Results in the animal experiment, in the animal experiment, hMPV infected BALB/C mice for about 3 weeks. After the fourth day of replication peak, the amount of virus gradually decreased and the virus was not detected in 3 weeks. The proportion of the 3 mixed groups in the 20 days was high in the proportion of NL/1/99, showing a strong growth trend, and the hMPV infection was not detected in the blank control group. In the cell system, the virus was co cultured to 12 generations, and the 1-5 generation 3 groups of mixed groups accounted for a high proportion of NL/1/99, the 6-8 generation was mainly NL/1/00, the ninth generation began to disappear, only the NL/1/00 alone was left, and the blank hole was not detected by hMPV infection. The virus strains were not detected in the animals and cell systems of NL/1/00 and NL/1/99 alone. The rest of the hMPV type.
Conclusion in animal experiments, NL/1/99 showed a strong competitive replication advantage, which was consistent with the early trend of cell lines. The growth advantages of NL/1/00 and NL/1/99 appeared alternately in the cell system, which coincided with the phenomenon of the alternately popular hMPV two subtype in the population in recent years.
Observation on pathogenicity of second human partial lung viruses A and B genotypes in mice
Objective to explore the difference in the pathogenicity of the two subtype virus by observing the changes in weight, viral load, pathology and airway responsiveness of the human metapneumovirus (hMPV) A and B subtypes in BALB/c mice, and to lay a foundation for further research on hMPV.
Methods after hMPV infection of BALB/c animal model, the changes of viral load in the lungs were detected by the Real-time PCR method. The pulmonary function detector monitored the changes of airway responsiveness and the histological grading of the mice to determine the pathological changes of the two subtypes.
Results there was no significant difference in the dynamic monitoring of body weight, viral load, pathological changes of lung tissue and airway responsiveness in hMPV two subtype infection group.
Conclusion there is no difference in pathogenicity of hMPV two genotype infected BALB/c mice.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.6

【相似文献】