【作者】 池雪林；

【导师】 汪世华； 罗树红；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2015， 博士

【摘要】 羊传染性脓疱病,俗称羊口疮(Orf),是由羊口疮病毒(Orf virus,ORFV)引起绵羊、山羊的一种接触性、嗜上皮性的传染病,也能感染人和其他动物。Orf临床上以在口唇、鼻、舌、乳房等部位形成丘疹、水疱、脓疱和痂皮为特征。福建近年来山羊养殖发展很快,偶尔有Orf的案例报道,但缺乏病原的分离鉴定及生物学特性方面的资料。因此,研究Orf在福建省的流行状况、分子遗传特征、不同毒株的致病性,研制安全有效的疫苗等问题成为亟需解决的问题。本研究的内容和结果如下:1.首先在2011年和2012年从福建省多个地区收集疑似羊口疮发病羊的痂皮,利用羊胚胎鼻上皮细胞(Ovine fetal turbinate,OFTu)细胞从8个羊场羔羊唇部痂皮中分离获得8株病毒,并通过PCR扩增ORFV011和ORFV059基因对这8株分离株进行鉴定,分别命名为:OV-GO、OV-SL、OV-YX、OV-SJ1、OV-NP、OV-FQ、OV-DS和OV-GT。对其中的OV-YX分离株进行电镜负染观察、病理组织学和免疫组化等方法进一步鉴定。病理组织学显示,ORFV感染后典型的组织学变化包括表皮过度增生、角化细胞气球样变性和皮肤炎性细胞浸润;免疫组化结果显示,ORFV主要分布于皮肤基底层棘细胞中。并测定其中4株病毒的半数细胞培养物感染量(TCID50),测得 OV-YX P3、OV-GO P4、OV-SJI P3、OV-NP P4 的TCID50分别为:10-6.5/0.1 mL、10-7.6/0.1 mL、10-6.1/0.1 mL和 10-6.7/0.1 mL。2.收集福建省4个地区15个山羊养殖场的349份血清,检测ORFV抗体;并对获得的这一地区10个羊场11株ORFV的ORFV011和ORFV059基因序列分别构建进化树,确定这一地区不同分离株与国内外不同地区ORFV的遗传进化关系。结果发现,基于ORFV011和ORFV059,只有采样点相距1 km的两羊场分离的两株ORFV(OV-GO和OV-GS)具有100%核苷酸同源性,其余9株显示出遗传多样性,地理距离和ORFV011、ORFV059及ORFV011-059遗传距离之间并没有相关性;这一地区ORFV遗传进化上的差异主要与分离株种间差异有关。建立了检测ORFV抗体检测的间接ELISA方法,对福建省15个羊场共349份血清样本检测ORFV抗体,阳性率达64.47%,表明福建省羊群中ORFV感染非常普遍。免疫印迹实验结果表明,ORFV感染后优势抗原为40 kDa的蛋白。3.利用福建ORFV分离株YX株感染OFTu细胞为模型,系统地对ORFV的形态发生学和感染细胞的超微结构变化进行了观察。结果显示,ORFV感染后细胞超微结构变化较大的细胞器有内质网、线粒体、高尔基体和细胞核。感染12 h后,首次在内质网观察到不成熟病毒粒子;感染18 h后,胞浆中可观察到发育不同阶段的病毒粒子,且病毒粒子在高尔基体获得囊膜。病毒粒子一经成熟即朝细胞膜移动,主要通过出芽方式释放病毒粒子。本试验选用OFTu细胞增殖ORFV,确定最佳观察时间是在感染后24 h之内。4.基于ORFV011和ORFV059基因核苷酸序列的进化树及分离株的地域分布,我们选择了4株典型的ORFV毒株(OV-GO、OV-YX、OV-NP和OV-SJ1),通过二代测序技术测定这4株ORFV的全基因组序列,进一步在全基因组水平上分析这些毒株的遗传进化多样性。这4株病毒基因组长度为132～139 kb,编码124至132个开放阅读框,G+C含量约64%,GC含量在整个基因组分布是不均匀的。不同ORFV毒株之间序列差异很大,尤其是头尾两端的序列变异更大,分布在末端的基因同源性较低。病毒株OV-SJ]、OV-GO和OV-YX全基因组序列中,有一个基因001/134基因完全处于基因组的ITR中,但OV-NP的001基因只有部分序列位于ITR,对应的134基因位于非ITR区域启动子发生突变,不形成有编码意义的开放阅读框。基于全基因组序列比较发现,OV-NP缺失了 114到120的基因,共缺7个基因(约5.5 kb),而OV-SJ1缺失了 1 17,118和119基因3个基因(约1.5 kb)。用ORFV编码的每个基因推导的氨基酸序列分别构建进化树,在132个进化树中,基于16个基因的氨基酸的进化树可以很容易鉴定毒株宿主起源于绵羊还是山羊,其中008基因的氨基酸序列具有最高的分辨率。本研究在国内首次完成了宿主为山羊的ORFV全基因组序列,有助于我们理解这一地区ORFV毒株遗传进化的多样性。5.本文还通过动物接种试验比测定全基因组序列的4株ORFV的毒力。结果显示,从临床病变及病程持续时间,还是造成的病理组织学损伤程度,都表明福建这4株ORFV毒力从大到小依次为:OV-GO>OV-YX>OV-SJ1>OV-NP,其中OV-NP和OV-SJ1显示出非常弱的毒性。这4株ORFV基因组比较时,发现OV-NP缺失了 114～120的7个基因,OV-SJ1缺失了 117～119的3个基因,基因缺失的2个毒株显示低毒性,基因完整的2个毒株显示较强的毒性,推测基因缺失导致对山羊的毒力减弱。这2株弱毒株的发现,为进一步研制ORFV弱毒疫苗奠定基础。更多还原

【Abstract】 Contagious ecthyma,also known as Orf,is caused by the Orf virus(ORFV)and is a contagious skin disease of goats,sheep,humans and other animals.Clinically,the disease is characterized by the formation of papules,vesicles,pustules and scabs onthe skin,and in the mucosa of the lips,noses,tongues and breasts.In recent years,goat raising has developed rapidly.There have been occasional reports of cases of orf,but they lack information regarding pathogen isolation and identification and ORPV biological characteristics.It is therefore imperative to illuminate the current epidemiological status of Orf in the Fujian province and to develop a vaccine against ORFV infection.The main results of our research were detailed as:1.First,scab samples were collected from suspected ORFV infected goat herds in several regions of Fujian Province(China)from 2011 to 2012.The 8 novel ORFV isolates were successfully isolated by use of primary ovine fetal turbinate(OFTu cell),and identified as ORPV by PCR amplification of the ORFV011 and ORFV059 genes.The 8 isolates were named as OV－GO,OV－SL,OV-YX,OV-SJ1,OV-NP,OV-FQ,OV-DS and OV-GT,respectively.The OV－YX isolate was further identified by transmission electron microscopy,pathologic histology and immunohistochemistry.Pathological findings in this case revealed excessive hyperplasia of skin keratosis,ballooning degeneration in keratinocytes and inflammatory cell infiltration.Irnmunohistochemistry results revealed that the virus antigen was localized in the spinous cells in the stratum spinosum.Tissue culture infective dose(TCID50)of four ORFV isolates of the cell cultures were detemined,and the TCID50 of OV-YX P3,oV-GO P4,oV-SJ1 P3,oV－NP P4 were 10-6.5/0.1 mL,10-7.6/0.1 mL,10-6.1/0.1mL and 10.6 7/0.1mL,respectively.2.In this study,phylogenetic trees based on the ORFV0ll and ORFV059 genes of 11 Fujian ORFV isolates and other isolates from GenBank were constructed to determine the relationship of ORPV isolates from Fujian province with other isolates from China and abroad.We found high heterogeneity between isolates from 10 farms in Fujian Province.Only two isolates from locations within 1 km to each other had the same viral genes.There is no correlation between the geographical distance between the corresponding collection sites and the phylogenetic distance in ORFV011,ORV059 or ORV011-059 sequences for any pairing of two isolates.The genetic variety was mainly due to interspecies difference.Anti-ORFV serum antibodies were detected by indirect ELISA for 349 serum samples collected from 15 goat farms in four regions of Fujian Province,and the results showed that the positive rate was 64.47%,indicating that ORFV infection was common in the goat herds in Fujian Province.Western blot results showed that a 40 kDa protein was the dominant antigen in ORFV infection.3.The morphogenesis of orf virus(ORFV)virulent strain YX in OFTu cells and the ultrastructural changes of host cells were observed and analyzed using transmission electron microscopic techniques.This approach was effective,and we determined that the best observation time was within 24 h of infection.Results revealed that the endoplasmic reticulum,mitochondria,golgi apparatus and the nucleus showed greater ultrastructure changes in ORFV infected cells.At 12 h post-infection,immature virus particles were first observed in the endoplasmic reticulum.At 18 h post-infection,virus particles in different developmental stages were observed,and the envelop of virions was presumably derived from the golgi network.Once the virus particles matured,they immediately moved towards the cell membrane,releasing virus particles through budding.4.Based on phylogenetic trees of ORFV01 1 and ORfV059 nucleotide sequences and the geographical distribution,4 typical ORFV isolates(OV-GO,OV-YX,OV-NP and OV-SJ1)were sequenced by next generation sequencing to determine the whole genome sequence for analysis of the genetic diversity at the genome level.The four isolates ere 132 to 139 kb in length,each containing 124 to 132 genes and about 64%G+C content.Great variation appeared in different isolates,and the most notable differences between the four isolates were found near the genome termini.Genes at the end of the genome shared low identity among different isolates.In the GO,YX,and SJ1 genomes,one predicted gene appeared twice completely located within the inverted terminal repeat(001/134).However,in the NP genome,only a partial sequence of one predicted gene was located in the inverted terminal repeat.In the NP genome,gene 001 is intact,but the corresponding 134 gene was disrupted due to the lack of initiation coding.By comparison of whole genome sequences,it was surprising to find no sequence corresponding to genes 114 to 120 genes(7 genes in total,about 5.5 kb)in the OV-NP genome.Genes 117,118 and 119 were deleted in OV-SJ1 genome(3 genes in total,about 1.5 kb).The phylogenetic trees based on the deduced amino acid sequences of each ORFV gene from this study and GenBank were constructed by Neighbor-joining Algorithm Method using MEGA 5.0 software.Among 132 genes of ORFV,16 genes showed great variations between goat ORFVs and sheep ORFVs with gene 008 showing the greatest variation.This study provided the first complete sequence of goat ORFV isolates from China,which helped us to understand the genetic diversity of ORFV in the region.5.An animal experiment was set up to investigate the virulence of OV-YX,OV－SJI,OV-GO and OV-NP.Clinical symptoms and histopathology indicated differential virulence of OV-GO,OV-YX,OV-SJ1 and OV-NP,in descending order.OV-NP and OV-SJ1 with several gene deletions showed low virulence while OV-GO and OV-YX without gene deletions showed strong virulence,indicating gene deletion resulted in an attenuated disease phenotype in the natural host.The 2 attenuated ORPV isolates may lay the foundation for the further research of an ORFV vaccine.更多还原