【作者】 陈汉阳；

【导师】 陈焕春； 郭爱珍；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2007， 博士

【摘要】 鸡传染性支气管炎(Avian Infectious Bronchitis，IB)是由鸡传染性支气管炎病毒(Infectious Bronchitis Virus，IBV)引起的一种高度接触性的病毒性传染病。主要引起鸡的呼吸系统疾病、肾炎并伴随产蛋量和蛋品质的下降。IBV属于冠状病毒科(Coronaviridae)，为单股正链RNA病毒。大部分冠状病毒具有宿主特异性和组织嗜性，只能侵染天然宿主和一些亲缘关系极其相近的宿主。以前的研究认为IBV只能感染鸡，但近两年相继在其他一些鸡形目的动物体内分离到IBV样病毒，如孔雀、几内亚鸡和鹧鸪等，这些分离的毒株在基因结构上与IBV很相似，基因序列的同源性甚至达到99％以上；而在非鸡形目动物如鸭、鸽子和灰雀等的体内分离到的病毒，通过基因组序列比对发现，虽然它们在氨基酸同源性上与IBV存在一定的差异，但依然属于第3群冠状病毒的范畴。IBV这种从感染单一宿主延伸到更多的其他动物的现象，让人联想到2002年爆发的“严重急性呼吸系统综合症”(Severe acute respiratory syndrome，SARS)，它的病原是一种新的冠状病毒(严重急性呼吸系统综合症冠状病毒，SARS-CoV)，而在多种动物体内分离到的SARS-CoV样冠状病毒说明SARS-CoV极有可能最初起源于动物。因此，研究动物冠状病毒与SARS-CoV的关系及冠状病毒跨越物种屏障的机制很有必要。基于此，本研究以IBV为模式病毒，将其跨种感染人源细胞HeLa，探讨动物冠状病毒跨种感染的机制，同时对IBV跨种感染过程中起到关键作用的因子进行了研究，另外也鉴定了IBV的天然受体。主要研究内容如下：1、不同IBV毒株跨种感染HeLa细胞的研究7个不同的IBV毒株，包括M41、H52、H120、Gray、Holte、Connecticut和M52-19(Beaudette52-19)，分别接种单层和刚刚消化的HeLa细胞。结果显示这些毒株均不能在单层的HeLa细胞上增殖；而M41、H52、H120和Gray可以在刚刚新鲜消化的HeLa细胞上增殖，并会导致HeLa细胞发生明显的病变，剩余的3个毒株Holte、Connecticut和M52-19却不能。通过观察是否产生细胞病变及中和实验、S1基因的RT-PCR扩增、血凝及血凝抑制、病毒N蛋白的Westem blotting检测等方面来证实感染的真实性。2、S1基因的变异在IBV跨种感染过程中的作用M41毒株在新鲜消化的HeLa细胞上持续传26代，RT-PCR扩增第1代、第5代和第21代病毒的S1基因，结果显示第1代跟第5代S1基因的序列完全一致，而第21代与第1代相比也仅仅只有一个碱基(A728G)的改变，导致一个氨基酸的变异。然而这唯一的一个点突变对IBV的跨种感染似乎没有决定性的作用。3、病毒血凝价的改变与跨种感染的关系据报道IBV经胰酶或磷酸脂酶C处理后可以凝集0.5％的新鲜制备的鸡红细胞。本研究发现在上述7个IBV的毒株中，M41、H52、H120和Gray经磷酸脂酶处理后可以凝集鸡红细胞，而另外3个毒株Holte、Connecticut和M52-29则不能凝集；另一方面，M41在HeLa细胞上持续传代会导致M41的血凝价逐渐下降；但这些传代的细胞毒(低或无血凝性)接种鸡胚后，尿囊液中的病毒会重新获得较高的血凝价。可见具有血凝性的IBV毒株更易于适应异源细胞。4、唾液酸糖蛋白(脂)在IBV跨种感染过程中的作用某些冠状病毒可以利用细胞表面的唾液酸糖蛋白(脂)作为进入细胞的受体，而神经氨酸酶(NA)可以降解细胞表面的唾液酸糖蛋白(脂)，这样就会降低病毒感染的几率。根据这一原理，将IBV感染经NA处理和未处理的HeLa细胞，对比发现IBV在NA处理过的HeLa细胞的感染滴度明显降低，说明唾液酸糖蛋白(脂)在IBV感染HeLa细胞的过程中起到重要的作用。5、氨基肽酶N、胰酶和EDAT对IBV跨种感染的作用氨基肽酶(APN)是很多冠状病毒的受体，通过封闭细胞表面APN的受体功能可以达到阻断病毒感染细胞的目的。用鼠抗人氨基肽酶(hAPN)的单克隆抗体(WM15)封闭HeLa细胞表面的氨基肽酶的活性，结果发现这种封闭并没有阻断IBV感染HeLa细胞，这说明hAPN不是IBV感染HeLa细胞的途径。以前的研究证实胰酶可以提高某些病毒的滴度。先确定胰酶的安全浓度，然后在IBV接种后将此安全浓度胰酶添加到的细胞培养液里，结果显示添加了胰酶的IBV的感染力与单独使用IBV是一样的；另外，IBV对经EDTA消化HeLa细胞与经胰酶消化的HeLa细胞的感染滴度是相同的。这说明胰酶、EDTA不是IBV跨种感染所必需的。6、鉴定IBV的天然受体研究证实IBV的一个毒株(Ark99)可以利用猫的氨基肽酶(fAPN)作为细胞受体。本研究通过克隆鸡的APN(gAPN)，探讨gAPN作为IBV天然受体的可能性。RT-PCR扩增到全长的gAPN基因，分别在原核和真核表达载体中进行表达。将原核表达的gAPN蛋白进行的ELISA及Westem blotting试验证实原核表达的gAPN蛋白在体外可以与IBV结合，而且这种结合可以被针对gAPN的抗体阻断。将gAPN转染IBV的非敏感细胞(PK-15、HeLa)，构建真核表达体系。通过间接免疫荧光、流式细胞术、半定量RT-PCR等试验证实IBV可以在这些转染后细胞上增殖。首次证明gAPN是IBV的天然受体之一。更多还原

【Abstract】 Avian Infectious Bronchitis （IB） is a highly contacting viral infection of the domesticfowl （Gallus gallus; more commonly known as the chicken） by infectious bronchitis virus（IBV）. It can cause respiratory disease, kidney inflammation with low production andpoor quality of eggs. IBV is a positive-stranded RNA virus, belonging to Coronaviridae.The previous researches indicated IBV replicated only in chicken, like most coronavirusesnaturally infecting only one animal species or, at most, a limited number of closely relatedspecies. In recent years, there is increasing evidence that IBV can infect species of birdother than the chicken. Coronaviruses have been isolated from peafowl （Pavo）, guineafowl （Nurnida meleagris）, partridage （Alectoris）, all of which were being reared in thevicinity of domestic fowl. These viruses were closely related in genome organization toIBV, and the homology in gene sequences exceeded 99% in some strains. Moreover, somecoronaviruses have been isolated from non-gallinaceous birds, the teal （Anas） and greylaggoose. These coronaviruses are not closely related in terms of gene sequences to IBV. Andas a result, they are clearly not simply isolates of IBV, but represent new coronavirusspecies.The phenomenon of IBV is not limited to replicating in a single host species, whichwas highlighted by the spread of SARS-coronavirus from mammals, like civet cat andbat.Individual coronaviruses had often been described as infecting their hosts in aspecies-species manner. The emergence of SARS-CoV and fragments of somecoronaviruses cross-species infecting make it clear that we should not assume that a givencoronavirus species is limited to replication in a single host. It is necessary to probe intorelationship between animal coronaviruses and SARS-CoV, and the mechanism ofcoronavirus carrying out cross-species infection.Most IBV strains grow poorly in cell culture except in primary chicken kidney cells.Studies on IBV growth in cell culture would be essential to understand the interactionbetween the coronaviruses and cells, and mechanism of coronavirus cross-speciesinfection. In this thesis, the investigation was undertaken to elucidate the potential factorsthat involve in IBV infection of HeLa cells, and identify the natural receptor of IBV. Themain projects are:1: IBV propagation in HeLa cellsAllantoic fluid of IBV strains including M41, H52, H120, Gray, Holte, Connecticutand M52-19 （a Beaudette52-19 strain） containing IBV 5×10³⁰⁴ EID50 was inoculated intoconfluent HeLa monolayers or freshly digested HeLa with 0.25% trypsin. All of them could not propagate in HeLa cell monolayers. However, the strains M41, H52, H120 andGray, but not Holte, Connecticut and M52-19, could propagate in freshly digested HeLacells. Cytopathic effect was characteristic of cell rounding, congregating and detachingfrom the cellular sheets, was produced in infected cells. The infection were verified fromneutralization test, alignment sequences of S1 gene from several IBV passages,haemagglutination and haemagglutination-inhibition test, detection of viral N protein byWestern blotting and so on.2: Mutation in S1 gene contributing to the infection of IBVReverse transcription PCR （RT-PCR） was performed to amplify the spike （S1） gene（1025bp） of IBV after different passages. S1 sequences for AY561712 （GenBank）, P0,and P5 remained the same. However, one nucleotide mutation was found in P21 S1leading to one amino acid replacement. In our test, the mutation in S1 gene seemed notthe major reasons responsible for IBV tropism switch.3: Relationship between HA activity and cross-species infection of IBVAfter treated with lecithinase C, the strains M41, H52, H120 and Gray, but notHolte, Connecticut and M52-19, could haemagglutinate 0.5% chicken erythrocytes.We also found the viral HA activity to chicken erythrocytes was significantlydecreased with increased passages in HeLa cells. When the HeLa adapted IBV wasinoculated back to the chicken embryo, IBV HA activity was restored. It seemed that thestrains, which have HA activity, were easier to be adapted in more species cells.4: Sialic acid was involved into IBV infecting HeLa cellsSialic acid can serve as a receptor for Group 2 coronavirus, e.g. BCoV andHCoV-OC43. Neuraminidase could remove sialic acid from cellular surface and thusblock the attachment of these viruses to host cells. Our results agreed with these findings.When the freshly dispersed HeLa cells were treated with NA, the virus TCID₅₀ wasreduced by 13 fold compared with those untreated HeLa. Furthermore, the HA activity ofIBV is determined by the capability of the virus to attach to sialic acid on the surface ofred blood cells. These results denoted that binding to sialic acid may be useful for IBV toinfect cells.5: The function of Aminopeptidase N and trypsin in IBV cross-species infectionA number of group 1 coronaviruses require the aminopeptidase N （APN） for entryinto their target cells, and trypsin was reported to be able to markedly enhance infectivityof various viruses in vitro. In our experiments, a monoclonal antibody, WM15, could notblock IBV infection of human HeLa ceil line. The trypsin also could not raise the titer ofIBV. 6: Identify natural receptor of IBVFeline APN had been reported to serve as a receptor for Ark99 strain of IBV. Wecloned full length chicken （Gallus gallus） APN （gAPN） gene, and approached thepossibility of gAPN acting as a receptor for IBV.The gAPN were cloned into prokaryotic （pET-28a） and eukaryotic （PCI-neo）expression vectors. The recombinant protein were purled from prokaryotic bacteria hadactivity of binding to IBV in ELISA and Western blotting test, which could be blocked byantiserum against gAPN.HeLa and PK-15 cell monolayers that do not permit natural infection by IBV weretransfected with gAPN-neo plasmid. The transfected and non-transfected cells wereinoculated with IBV and viral binding and replication were determinded by indirectflurescent assay, flow cytometry and semi-quantity RT-PCR. The results indicated thatthe insensitive cells were permissive to IBV after transfection with a gAPN cDNA,suggesting the gAPN plays an essential role in IBV entry.更多还原