【作者】 张瑞竹；

【导师】 刘正飞；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2011， 硕士

【摘要】 牛疱疹病毒1型和5型同属于α疱疹病毒属,二者的氨基酸相似性高达82%以上,而且其宿主动物主要是牛。但引起的临床症状却截然不同：由牛疱疹病毒1型(BHV-1)引起的传染病俗称牛传染性鼻气管炎,是一种牛的急性、热性、接触性传染病,主要引起呼吸道和生殖道疾病等,侵染途径主要是通过鼻腔到达三叉神经节最后形成潜伏感染；而BHV-5是一种嗜神经性病毒,经鼻腔到达嗅球或直接到三叉神经节形成潜伏感染,病毒会通过顺式转运侵染中枢神经系统,从而引起癫痫等神经症状。由于这两种传染病最终都可以在三叉神经节形成潜伏性感染而导致终身带毒,还有该病毒还有很多基因的功能以及二者在致病机理和临床表现的联系与区别还没有研究清楚,因此给该病的防治带来诸多困难。通过基因组比较,发现UL0.5基因只在BHV-1中编码。因此,本文对BHV-1中UL0.5基因在BHV-5中的功能进行研究,初步探究二者在临床症状不同的原因、病毒与宿主互作的分子机制以及在神经系统的感染机理。我们首先从BHV-1基因组中克隆UL0.5基因,然后利用同源重组的方法整合到BHV-5的基因组中形成嵌合病毒,然后分别通过体外实验和体内试验来对比亲本病毒和重组病毒的差异。1、体外实验本文首先通过反转录试验证明了UL0.5基因可以转录为RNA,然后用磷酸钙共转染的方法构建了BHV-5 UL0.5嵌合病毒以及其回复突变株,并且分别用PCR测序和Southern blotting对重组病毒进行验证。在UL0.5基因成功嵌合到BHV-5的基因组后,我们分别设计空斑大小比较、一步法增殖曲线和电镜试验来验证其在增殖能力上的区别。通过试验我们发现在相同的条件下相对于BHV-5野毒和回复突变株,嵌合病毒在细胞上形成的空斑明显减小,但其病毒粒子的大小却没有明显区别。然后取不同的时间点来验证病毒的增殖趋势,发现嵌合病毒在早期的增殖能力也明显的低于亲本病毒。Real-timePCR试验表明UL0.5基因的插入影响了BICP0基因的正常表达,但其下游同源臂ORF2基因的表达没有受其影响。通过在体外进行的一系列试验,我们对UL0.5基因的功能有了初步的认识。UL0.5基因可以作用于BHV-5基因组中其他基因,从而抑制了病毒在细胞上的增殖能力,并且降低了病毒的滴度。2、体内实验将试验兔随机分成4组：空白对照组、BHV-5 wt组、BHV-5 UL0.5组和BHV-5UL0.5R组。用鼻腔攻毒的方法对新西兰白兔进行攻毒试验,将不同攻毒组分开饲养,连续注射3天地塞米松并连续观察14天,到攻毒后第5天,野毒组和回复突变组开始出现明显的神经症状,到第10天恢复正常；而嵌合病毒从急性感染期到潜伏感染,没有出现过典型的神经症状,只有一些轻度的反应。然后选取第3、6、9天的试验动物,麻醉后做磁共振成像试验(MRI)。MRI试验表明,当BHV-5亲本毒和回复突变株病毒进入神经中枢后,会引起脑膜发炎出血,并且导致脑脊液的大量分泌；而正常对照动物的脑脊液本身很少而且变化不大,但嵌合病毒可以观察到少量的脑脊液的分布。通过鼻眼拭子和脑组织的分毒试验,发现嵌合病毒的分毒量明显低于亲本毒和回复突变株。最后我们通过免疫组化试验,更进一步的证实了三种不同的病毒在脑组织分布的区别,感染亲本毒和回复突变组的神经元数目比嵌合病毒的多.也就是说,UL0.5基因抑制了BHV-5在中枢神经系统中的传播和神经毒性。总之,通过体内外试验研究表明,UL0.5基因插入BHV-5基因组后,嵌合病毒与野毒相比,增殖能力下降,并抑制了BHV-5的神经毒性。本研究对理解BHV-1和BHV-5神经致病性差异的分子机理提供了一定的证据。更多还原

【Abstract】 Bovine herpesvirus 1 (BHV-1) and 5 (BHV-5) are two closely related alphaher-pesvirus for which the primary host are mainly cattle. The BHV-1 and BHV-5 exhibit an average of 82% amino acid identity amongst different proteins, but the clinical diseases are completely different:Bovine infectious rhinotracheitis caused by BHV-1 is known as an acute, pyrexic and contagious disease that caused abortion, respiratory, and genital infections. The path of invading is that virus establishing latency in the trigeminal ganglion through the nasal cavity. But BHV-5 is a highly neurovirulent viru. Virus could reach olfactory bulb or trigeminal ganglia directly from nasal cavity, it is transported to the central nervous system by anterograde transport. At last it leads the neurological symptoms such as epilepsy. Because both BHV-1 and BHV-5 can establish latency in the trigeminal (TG), the animals carried the virus lifelong. So it brings many difficulties to the prevention and cure. Furthermore, the function of many genes, pathogenic mechanism and clinical manifestations between two virus are still not very clear. On the basis of genomic comparisons, BHV-5 encodes the full complement of genes in BHV-1 except for one coding region named UL0.5. Therefore, the objective of this study is to explore the funcation of UL0.5 gene in BHV-5, preliminary inquiry the reason of difference in clinical symptoms, molecular mechanism between virus and host each other and the infection mechanism in nervous system.First, the UL0.5 gene was cloned from the BHV-1 genome to integrate into the BHV-5 genome by homologus recombination. Then we compared the recombinant virus with parental virus in vivo and in vitro to make clear the difference.In vitroThis study showed that the UL0.5 gene could transcript to mRNA by RT-PCR. Then we constructed the BHV-5 UL0.5 chimeric mutant and BHV-5 UL0.5 revertant mutant by co-transfected and verified the recombinant virus through PCR and Southern Blotting.After the UL0.5 gene integrated into the BHV-5 genome successfully, we disigned comparison of plaque size, one-step growth curve and electron microscope tests to analyze the difference of proliferation. Compared to the BHV-5 wt and BHV-5 UL0.5R, we found that the size of plaque was smaller in BHV-5 UL0.5+, but there was no significant difference in virus particles. Then we observed the proliferation trends in different time, the proliferation in BHV-5 was lower than parental virus in early times. After Real-time PCR, we found that the insertion of UL0.5 would influence the expression of BICPO and no effect to ORF2. The UL0.5 gene acted on other genes in BHV-5 genome, to inhibit virus proliferating on cells and reduce the virus titer. In vivoTwenty New Zealand white rabbits were randomly divided into four groups: control group, BHV-5 wt, BHV-5 UL0.5 and BHV-5 UL0.5R. The animals were challenged by nasal administration, feeding separately. Followed a 3 days continuous injection of dexam-ethasone and watching 14 days continually, rabbits infected with BHV-5 wild-type and BHV-5 ULO.5-reverted showed asevere neurological signs at 5 days postinfection and returned to normal at 10 days. But none of five rabbits infected BHV-5 UL0.5-inserted virus showed neurological signs between acute infection and letent infection. The animals at 3,6,9 days postinfection were tested by Magnetic Resonance Imaging. When BHV-5 wild-type and BHV-5 UL0.5-reverted virus invaded the Central Nervous System, the meningeal appeared inflammation and bleeding with cerebrospinal abnormal fluid secretion. Although there was little cerebrospinal in control group and no significant change, we can notice a few cerebrospinal distributed. From the virus isolation, significant amounts of virus were detected in the brain tissues and nasal secretions of rabbits infected with BHV-5 ULO.5, which was comparable to levels in rabbits infected with BHV-5 wild-type and BHV-5 UL0.5-reverted virus. Based on immunohistochemistry, BHV-5 wild-type and BHV-5 ULO.5-reverted virus spraed to all the areas of brain, but fewer BHV-5 UL0.5-inserted virus were detected by immunohistochemistry in the olfactory bulb and other olfactory pathway structures. As discussed above, the ULO.5 gene inhibited the neuroinvasiveness in BHV-5. From the study in vivo and vitro, compared to the BHV-5 wild type, the insertion of ULO.5 could reduce multiplication capacity and inhibit the neuropathogenesis of BHV-5. This study showed some evidence to explain the difference on pathogenicity of molecular mechanism between BHV-1 and BHV-5.更多还原