【作者】 蒋桃珍；

【导师】 毕丁仁；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2010， 博士

【摘要】 马流行性感冒是由马流感病毒(Equine influenza virus, EIV)引起的马属动物的一种急性呼吸道传染病,世界动物卫生组织(OIE)将其列为法定报告动物疫病。目前马流感病毒分为两个不同的亚型,分别是H7N7亚型病毒和H3N8亚型病毒,由这两个亚型引起的马流感广泛分布于世界各地。近30年来,世界各国所暴发的马流感均由H3N8亚型马流感病毒所引起。H3N8亚型马流感的频繁暴发,对养马业,尤其是赛马业危害严重,已在世界范围内造成了巨大的经济损失。2007年,许多国家包括澳大利亚、日本等暴发了马流感疫情,我国新疆以及华北地区也暴发了类似马流感的疫情,在流行病学调查的基础上,我们从华北地区分离到了一株H3N8亚型马流感病毒。本研究在病毒分离鉴定的基础上,为进一步探讨马流感病毒的致病机理,马流感病毒跨种间传播的可能性以及疫苗对马流感的预防效果,开展了如下研究工作。(1)对2007年华北地区马群中暴发的H3N8亚型马流感病毒进行了分离鉴定；测定了不同代次的鸡胚传代病毒对马致病性和致病性差异分子机制的探讨与分析。从发病赛马中分离到1株H3N8亚型马流感病毒,分离毒株为低致病性流感病毒,属于美洲谱系病毒,为马源H3N8亚型流感病毒流行毒株,命名为A/Equine/Huabei/1/07(H3N8)。利用F2代分离病毒,采用喷雾的方式以约5×106.7EID50/匹的剂量感染12个月～20个月龄的马,建立起以持续性发烧和流水样鼻汁为主要症状的马流感发病模型。F3、F4、F5和F10代病毒对马的致病性逐渐减弱,不能用于发病模型的建立。采用RT-PCR和基因序列测定技术,成功地获得F2、F3、F4、F5和F10代毒株的基因组序列。F2代-F5代毒株之间HA基因的核苷酸和氨基酸的同源率均为100%,而F2代～F5与F10代核苷酸的同源率均为99.90%,氨基酸的同源率均为99.80%；变异位点在核苷酸第539位,在此位点上F2代～F5代毒株核苷酸序列为A(腺嘌呤),而F10代毒株为G(鸟嘌呤),这一核苷酸位点的变异导致氨基酸159位(阅读框176位)抗原位点的氨基酸发生改变,由F2代～F5代毒株的N(天冬酰胺)变为F10代毒株的酸性氨基酸D(天冬氨酸)。HA基因的变异初步解释了毒株致病性减弱的主要分子机制。(2)H3N8亚型马流感病毒分离毒株对不同品种犬的感染性研究,对马流感病毒的跨种间传播提供了试验数据；用分离的A/Equine/Huabei/1/07(H3N8)毒株,经滴鼻感染途径分别以1.0mL或2.0mL(106.7EID50/条或2×106.7EID50/条)的接种剂量人工感染灵缇犬和比格犬。结果表明试验用H3N8亚型马流感病毒仅能引起犬轻度的上呼吸道病变,多数感染犬排毒且血清学检测抗体阳转,但感染犬无异常临床症状。此研究证实目前我国分离的马源H3N8亚型流感病毒能够感染犬,但可能暂时不会对犬类的健康构成威胁。(3)利用分离的H3N8亚型马流感病毒流行毒株,初步研制成功H3亚型马流感全病毒灭活疫苗。利用铝胶佐剂制备了H3N8亚型马流感全病毒灭活疫苗,经豚鼠安全性评价试验结果表明铝胶佐剂副反应轻。分别用F6、F8和F10代次的病毒制备了马流感灭活疫苗,经豚鼠和马的免疫试验证实,疫苗具有良好的免疫效果,马和豚鼠免疫后均能产生较高的HI抗体,而且豚鼠和马之间的HI抗体效价具有较好的平行关系,豚鼠可以作为马流感灭活疫苗效力检验的替代动物。更多还原

【Abstract】 Equine influenza is one kind of acute contagious respiratory disease in Equus animals caused by the equine influenza virus, and also it is in notifiable form of OIE. There are currently two subtypes of equine influenza viruses in circulation, the outbreaks of equine influenza caused by Equine -1 influenza virus (H7N7) or Equine-2 influenza virus (H3N8) is very common in the world. The equine influenza occurred in the world was caused only H3N8 subtype EIV in the last 30 years. Frequent outbreaks of H3N8 subtype equine influenza results in interruption of horse industry, and in some cases, particularly in the racing industry, with significant financial impact.In 2007, Outbreaks of equine influenza have been reported in Australia, Mongolia, Japan and other countries, an outbreak of respiratory disease with the similar influenza symptoms occurred in racehorses in Xinjiang and Northern China. According to the results of epidemiological investigation, we got one H3N8subtype equine influenza virus. Based on the data of isolation and identification, in order to further exploring pathogenesis, interspecies spread and the inactivated vaccine against Equine influenza, following experiments have been conducted in this study:(1) Isolation and identification of H3N8 EIV occurred in Northern China, determination the pathogenicity, exploring and analysis the molecular mechanism of attenuation with embryo propagations of virus seed lot of H3N8 EIV strains.One isolates denominated A/Equine/Huabei/1/07(H3N8) of lower pathogenic Equine influenza virus was isolated from nasal discharge of sick racing horse, it belongs to the American lineage and it is original horse strain.Twelve to 20-month-day-old Mongolia Horses were experimentally inoculated with the F2, F3, F4, F5 and F10 propagation of EIV H3N8 by spraying routes at the dose of 5×106.7EID50 per horse. All inoculated horses with F2 propagation virus exhibited the typical clinical signs such as continuity pyrexia and watery nasal discharge of EI. In contrast, all the horses inoculated with other propagation virus showed mild and subclinical signs of EI, the data from the experiment clearly suggest that the F10 propagation virus seed lot may not serve as a challenge virus for develop the model of El.By means of RT-PCR with specific primers from the noncoding sequence of each gene segment and sequencing, full coding sequences of eight viral genes of the F2, F3, F4, F5 and F10 propagation of EIV H3N8 were amplified and sequenced. Phylogenetic analysis based on HA gene and deduced amino acid showed that (1) the four propagation of virus (F2, F3, F4, F5) possessed an HA cleavage site sequence identical to each other with HA homology of 100% and 99.90%,99.80% to F10 respectively.The variation site is located in the 539, the nucleotide of F2, F3, F4, F5 propagation is Adenine and Guanine from F10 propagation, corresponding the amino acid of Asparagine (A) is changed to acidity Aspartic Acid (D), the variation of HA gene primarily interpret the mechanism of pathogenicity of 5 propagations of EIV.(2) Infectivity analysis of canine to isolates of EIV H3N8, providing the experimental data for further exploring interspecies spread regarding the infectivity of canine to EIV H3N8.Seven greyhounds and 12 beagles were experimentally inoculated with F2 propagation isolates of EIV H3N8 by intranasal (IN) routes at the dose of 106.7 EID50 per dog or 2×106.7 EID50 per dog. Of the 19 inoculated canines, none of them exhibited any clinical signs of illness and all remained healthy during the observation period (on days 14-21 post inoculation) and showed no gross lesions. While the trachea exhibited very mild lesions such as falling off of cilia. Both viruses were recovered and HI antibody was detected from most of the experimentally infected canines. Results from the experiment clearly suggest that the H3N8 subtype EIV prevalence now may not threaten to the dogs.(3) Inactivated vaccine against Equine influenza was primarily developed with isolated strain of H3N8 EIV.The inactivated whole virus vaccine against equine influenza containing Aluinium Hydroxide adjuvant was manufactured, the data of safety test indicated that the guinea pigs vaccinated with killed vaccine exhibited very little adverse reaction. Both horses and guinea pigs which received different vaccines containing 64 or 128 HA unit from F6, F8 and F10 propagation antigen had high levels of HI antibody on the 28 days post vaccination, furthermore, the serum HI titers from horse and guinea pigs showed direct correlation, guinea pigs is one of the ideal experimental animals available for equine influenza vaccine efficacy.更多还原