猪血凝性脑脊髓炎病毒呼吸道感染模型的建立及其在体内的动态分布
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摘要
猪血凝性脑脊髓炎(Porcine hemagglutinating encephalomyelitis)是由血凝性脑脊髓炎病毒(Hemagglutinating encephalomyelitis virus, HEV)引起的仔猪一种以神经症状或者呕吐、衰竭为临床症状的急性、高度接触性传染病,主要感染1-3周龄的仔猪,死亡率可高达20-100%。血清学调查表明HEV感染是呈世界性分布的,给养猪业造成了很大的经济损失,但是迄今为止对HEV的致病性研究不够深入,对HEV在猪体内的动态分布更不清楚,影响了该病毒致病机制的研究,因此,建立一种能模拟感染发病动物临床症状及病理变化的动物模型,以研究该病毒在猪体内的动态分布及感染机制,模型的建立对于HEV的研究和防制具有重要意义。
本试验选用经HEV抗体检测为阴性的一日龄的仔猪作为实验动物,采用HEV-67N标准毒株为攻毒株,通过滴鼻接种感染仔猪,建立猪血凝性脑脊髓炎病毒感染仔猪的动物模型,结果显示猪血凝性脑脊髓炎病毒可人工感染仔猪,症状比较明显,其临床症状和病理变化与自然发病猪基本一致,病理学组织观察,肺脏、脑出现了明显的病理变化,与HEV典型的病理变化是一致的,模型重复性良好。仔猪接种病毒后分阶段处死,采集病料并做相应处理,然后应用荧光定量RT-PCR和免疫组织化学方法技术来检测病毒在感染动物后不同时间段的分布特征及移行路径。
根据GenBank报道中HEV-67N毒株S基因序列(AY078417)保守型区域设计了一对特异性引物,通过对反应条件的优化,建立了检测HEV的荧光定量RT-PCR,应用建立的荧光定量RT-PCR检测了各组织病毒的含量,结果显示病毒主要存在于呼吸系统和神经系统中,其他的脏器也有病毒的存在,但是含量相对较少,说明该病毒可以通过呼吸系统传播和具有嗜神经性。
应用免疫组织化学技术检测了病毒在组织中的动态分布分布情况,结果表明,不同的感染时间各组织脏器内病毒分布是不相同的,结果发现大脑中病毒的阳性信号主要位于大脑皮质部的锥体细胞内,小脑中病毒的阳性信号位于普肯野氏细胞内,肺脏中病毒的阳性信号位于肺泡上皮细胞内,其它组织样本中都没有检测到阳性信号。
Porcine hemagglutinating encephalomyelitis is a kind of piglets disease caused by Hemagglutinating encephalomyelitis virus (HEV), showed neurological symptoms or vomiting and failure as the clinical symptoms of acute and highly contagious disease, mainly affects pigs from 1 to 3 weeks, the mortality rate was up to 20-100%. Serological survey showed that HEV infection was distributed by worldwide, and has caused great economic losses to the pig industry,but until now the studies on the pathogenicity of HEV was not deep enough, the dynamic distribution of the HEV in pigs was not clear,which impact the research of the HEV pathogenesis. So, we establish an animal model which can simulate the clinical symptoms and pathological changes, so that we can study the dynamic distribution and infection mechanisms in pigs.The establishment of this model is very important to HEV research and prevention.
According to GenBank reported that HEV-67N strain of S gene sequences (AY078417)of conservative region designed a pair of specific primers, established quantitative fluorescence detection HEV RT-PCR, through the optimization of reaction conditions, using the established fluorescence quantitative RT-PCR to detect the virus content of each organization. The result shows that the virus mainly exited in the respiratory system and the nervous system, other organs also have the virus, but the levels was low, indicating that the virus is mainly transmitted through the respiratory system and have neurotropic nature.
Using the Immunohistochemical technique to detect the distribution of the virus of the organizations, the results showed that within different infection time the distribution of virus in organs of the organizations was different, we also found that the positive signals in the virus of the brain were mainly located in the brain cortex of the pyramidal cells, the positive signals in the cerebellum virus located in the Purkinje cells,the positive signals in lung virus located in the alveolar epithelial cells, and we not found positive signals in other tissue samples.
本试验选用经HEV抗体检测为阴性的一日龄的仔猪作为实验动物,采用HEV-67N标准毒株为攻毒株,通过滴鼻接种感染仔猪,建立猪血凝性脑脊髓炎病毒感染仔猪的动物模型,结果显示猪血凝性脑脊髓炎病毒可人工感染仔猪,症状比较明显,其临床症状和病理变化与自然发病猪基本一致,病理学组织观察,肺脏、脑出现了明显的病理变化,与HEV典型的病理变化是一致的,模型重复性良好。仔猪接种病毒后分阶段处死,采集病料并做相应处理,然后应用荧光定量RT-PCR和免疫组织化学方法技术来检测病毒在感染动物后不同时间段的分布特征及移行路径。
根据GenBank报道中HEV-67N毒株S基因序列(AY078417)保守型区域设计了一对特异性引物,通过对反应条件的优化,建立了检测HEV的荧光定量RT-PCR,应用建立的荧光定量RT-PCR检测了各组织病毒的含量,结果显示病毒主要存在于呼吸系统和神经系统中,其他的脏器也有病毒的存在,但是含量相对较少,说明该病毒可以通过呼吸系统传播和具有嗜神经性。
应用免疫组织化学技术检测了病毒在组织中的动态分布分布情况,结果表明,不同的感染时间各组织脏器内病毒分布是不相同的,结果发现大脑中病毒的阳性信号主要位于大脑皮质部的锥体细胞内,小脑中病毒的阳性信号位于普肯野氏细胞内,肺脏中病毒的阳性信号位于肺泡上皮细胞内,其它组织样本中都没有检测到阳性信号。
Porcine hemagglutinating encephalomyelitis is a kind of piglets disease caused by Hemagglutinating encephalomyelitis virus (HEV), showed neurological symptoms or vomiting and failure as the clinical symptoms of acute and highly contagious disease, mainly affects pigs from 1 to 3 weeks, the mortality rate was up to 20-100%. Serological survey showed that HEV infection was distributed by worldwide, and has caused great economic losses to the pig industry,but until now the studies on the pathogenicity of HEV was not deep enough, the dynamic distribution of the HEV in pigs was not clear,which impact the research of the HEV pathogenesis. So, we establish an animal model which can simulate the clinical symptoms and pathological changes, so that we can study the dynamic distribution and infection mechanisms in pigs.The establishment of this model is very important to HEV research and prevention.
According to GenBank reported that HEV-67N strain of S gene sequences (AY078417)of conservative region designed a pair of specific primers, established quantitative fluorescence detection HEV RT-PCR, through the optimization of reaction conditions, using the established fluorescence quantitative RT-PCR to detect the virus content of each organization. The result shows that the virus mainly exited in the respiratory system and the nervous system, other organs also have the virus, but the levels was low, indicating that the virus is mainly transmitted through the respiratory system and have neurotropic nature.
Using the Immunohistochemical technique to detect the distribution of the virus of the organizations, the results showed that within different infection time the distribution of virus in organs of the organizations was different, we also found that the positive signals in the virus of the brain were mainly located in the brain cortex of the pyramidal cells, the positive signals in the cerebellum virus located in the Purkinje cells,the positive signals in lung virus located in the alveolar epithelial cells, and we not found positive signals in other tissue samples.
引文
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