SARS病毒与动物冠状病毒相关性的研究进展

2003年4月世界卫生组织宣布，严重急性呼吸系统综合症(SARS)的病原是一种新的冠状病毒变种，从而引起了世界各国对人类和动物冠状病毒的极大关注。作者对SARS病毒与动物冠状病毒相关性的研究进展进行了综述。     

动物"身上"的冠状病毒

人类与SARS经历一场搏杀之后,科学家们揭开了SARS的层层面纱,宣告SARS的元凶是冠状病毒的一个变种,一时间冠状病毒成了沸点话题,动物身上的冠状病毒惊醒人类。动物身上到底有多少种冠状病毒?它与SARS病毒有着怎样的联系呢? 冠状病毒(Coronavirus)属于正股单链RNA病     

动物冠状病毒的抗原性、病原性、生态学和公共卫生意义的研究进展

作者从动物冠状病毒与SARS病毒相关性的角度，对动物冠状病毒属成员的抗原性、病原性、生态学和公共卫生意义进行了综述，认为SARS的发生与流行可能对传统的冠状病毒病原性理论提出了挑战，很有必要重新评估动物冠状病毒对人类的潜在危害性。．     

冠状病毒基因组及其编码蛋白

冠状病毒宿主范围广泛 ,不同种动物(禽、火鸡、猪等 )分离株又有众多血清型 ,最重要的特征之一是具有高度的变异性 ,所以对冠状病毒的系统研究一直是个难点。对其中某些病原体 (如犬、猫冠状病毒等 )的研究也相对较少。但最近出现的严重急性呼吸系统综合征( SARS)病原。使人认识到冠状病毒不仅能给畜禽业带来重大的经济损失 ,也可能会威胁到人类自身 ,甚至带来非常严重的后果。到目前还不清楚 SARS病毒的真正起源。是早已存在 ,不为我们所认识 ,还是现有某种病毒的变异体 ?因此 ,有必要对该属病原体的一些特性进行了解。文章对动物冠状病毒编码蛋白的研究进展进行了概述 ,为更好地对 SARS病原体的特性、进化和演变进行研究提供借鉴和参考。     

冠状病毒研究进展

本文对冠状病毒的分类、形态、囊膜糖蛋白、基因组和复制过程进行了综述，并对SARS冠状病毒的研究进展作了简要介绍，包括SARS冠状病毒的临床表现、与已知冠状病毒的同源性比对、系统发生学分析、基因突变、检测和防治等，以期使大家对冠状病毒有一个更清晰、快捷的了解。     

SARS冠状病毒的流行演化分析

严重急性呼吸综合症(Severe acute respiratory syndrome,SARS)是21世纪初暴发的一种重症病毒感染疾病.病原体为一种以前未在人类或动物中发现过的新的冠状病毒--SARS相关冠状病毒(SARS-CoV).尽管SARS-CoV基因组结构与其他的冠状病毒相似,但SARS-CoV与其他冠状病毒的潜在关系仍未明确.文章简要地介绍了其生物学特性,回顾了SARS发生和发展过程,并根据SARS的流行传播特点和SARS-CoV的进化分析,推测了SARS-CoV可能的发源和衍变.     

SARS冠状病毒纤突蛋白的分子生物学研究进展

从纤突蛋白的结构分析、受体及其表位分析三方面论述了SARS冠状病毒纤突蛋白的研究进展，并与部分其他动物冠状病毒进行了比较，从分子水平上阐述了纤突蛋白的结构、受体和表位研究概况，旨在为SARS冠状病毒的诊断、疫苗设计及基础研究提供理论依据。     

猪冠状病毒的经验是否有助于防治SARS

美国国家过敏与传染病中心主任Anthony Fauci向美国国会陈述疫苗用于治疗猪病毒性呼吸道疾病的经验证明疫苗可能有助于治疗人严重流行性呼吸综合症(SARS)。 SARS病例的数量在全世界以很快的速度增长,明显地是以空气传播。许多实验室从SARS病例分离出一种新的冠状病毒。通常人冠状病毒引起中度的上呼吸道感染,但是这种新病毒引起严重的肺炎症状,并伴随着很高的致死率。 Fauci认为防治普通的动物冠状病毒     

非典型肺炎冠状病毒与其他动物冠状病毒的亲缘关系

非典型肺炎即重症急性呼吸综合征(severe acute respiratory syndromes，SARS)，其病原目前被普遍认为是一种冠状病毒。本试验利用分子生物学计算机软件，首先对已在GenBank中发表的17株SARS病毒的主要结构蛋白基因S、M、N进行了定位，然后分别与已发表的其他冠状病毒的S、M、N基因进行同源性分析。结果发现，我国内地的5个SARS毒株的主要结构蛋白基因与其他冠状病毒的相对应基因的同源性普遍较低，而且变化范围较大。香港及国外的SARS毒株的情况恰好相反。从总体上看，SARS病毒与已发表的其他冠状病毒的亲源关系均较远，相对而言，与牛冠状病毒(BCV)、小鼠肝炎冠状病毒(MHV)的亲缘关系最近。这一结果对于SARS病毒的起源及流行病学研究乃至该病的预防和控制研究具有一定的参考价值。     

东北三省冠状病毒流行病学调查

冠状病毒(coronavirus)是属于巢状病毒目、冠状病毒科、冠状病毒属的一种正义单链RNA病毒,因形似王冠而得名.该病毒在世界范围内广泛存在,感染多发于冬春季,是鸡的传染性支气管炎、猪呼吸道冠状病毒病/传染性胃肠炎、牛冠状病毒病、鼠肝炎病毒病、猫传染性腹膜炎等多种动物疾病的病原,但引起人的严重疾病在传染性非典型肺炎(Severe Acute Respiratory Syndrome,SARS)的爆发前未见报道.2002-2003年冬春之交,SARS肆虐全球,后经确认为冠状病毒感染引起,并且确定了引起SARS的病原为一种未知的冠状病毒.由于冠状病毒感染的广泛存在,病毒的细胞分离或SPF鸡胚分离、RT-PCR等技术对东北三省的冠状病毒感染情况作了血清学和病原学的流行病学调查.     

Complete diphallia, imperforate ani (type 2 atresia ani), and an accessory scrotum in a 5-day-old calf.

A 5-day-old Angus calf was submitted to the necropsy service at the University of Kentucky Livestock Disease Diagnostic Center. At birth, the calf was noted to have 2 scrota. Five days post-calving the calf developed severe tenesmus and an umbilical infection. Clinical examination revealed atresia ani. At necropsy, complete diphallus, imperforate ani (type 2 atresia ani), and remnants of 2 scrota were identified. This report describes the gross pathologic appearance of a rare case of complete diphallia, imperforate ani (type 2 atresia ani), and an accessory scrotum in a 5-day-old calf.     

Negative contrast electron microscopic techniques for diagnosis of viruses of veterinary importance

Negative contrast electron microscopy (NCEM) was utilized as a routine tool in the diagnosis of viral infections of domestic and wild animals. Viruses identified by this technique were observed in infected culture systems or clinical specimens from several species including horses, cattle, sheep, dogs, cats, pigs, deer, Rocky Mountain bighorn sheep, antelope, and several avian species. Viruses were identified by NCEM based on their size, morphology, and symmetry and consisted of adenoviruses, herpesviruses, paramyxoviruses, myxoviruses, picornaviruses, parvoviruses, coronaviruses, reoviruses, rotaviruses, and poxviruses. Mixed populations were also readily demonstrable by this technique: the most common mixed infections consisted of coronaviruses and rotaviruses, and picorna- or parvo-viruses with coronaviruses, rotaviruses, herpesviruses, or adenoviruses. Immunoelectron microscopy was also used to serotype viral agents present in the specimens examined. Viruses identified by this technique were bovine rotaviruses, coronaviruses, and herpesviruses, and bovine and equine adenoviruses.     

Identification of coronaviruses by the use of indirect protein A-gold immunoelectron microscopy.

Concentration by airfuge and protein A-colloidal gold immunoelectron microscopy (PAG-IEM) offered a rapid and sensitive method for detection and identification of coronaviruses from various species. The method was applied to partially purified tissue culture-adapted or egg-adapted mammalian and avian coronaviruses and to clarified fecal samples from diarrheic calves and turkey poults for detection of enteric coronaviruses. Aggregates of virus coated with specific antibody were seen in virus samples mixed with homologous antiserum but not in control samples containing preexposure serum. At least a 10-50-fold enhancement of the sensitivity of direct EM for virus detection was obtained using protein A-colloidal gold complex as an electron-dense marker. The PAG-IEM method demonstrated low nonspecific background labeling and permitted detection of soluble and particle-associated antigen. Reciprocal cross-reactivity was detected among the subgroup of mammalian hemagglutinating coronaviruses, and antisera to 4 members of other subgroups only recognized their homologous virus.     

黑麦草有害动物群落动态研究

对合肥地区多年生黑麦草Lolium perenne有害动物群落进行了研究,共记录有害动物13种,分属3个动物门、4个纲,以节肢动物门为主,占总数量的95.7%。主要优势种为麦圆叶爪螨Penthaleus major、黑草蚁Lasius fuliginosus、麦杆蝇Meromyza saltatrix、鼠妇Armadillidium unlgare、麦长管蚜Macrosiphmavenae和大青叶蝉Cicadella viridis;优势种中麦长管蚜、大青叶蝉和麦杆蝇主要分布于上部和中部,黑草蚁和鼠妇主要分布于下部和中部,麦圆叶爪螨主要集中在中部。群落均匀度和多样性变化一致,均以群落形成初期较高。聚类分析结果表明,黑麦草从早春萌芽到抽穗期间有害动物群落比较稳定。     

7种动物冠状病毒特异基因的克隆与序列分析

为了比较冠状病毒基因相关性,获得特异基因克隆制备冠状病毒基因芯片,根据发布的基因序列,每种病毒设计4~17对引物,利用火鸡冠状病毒(TCV)原毒和蔗糖密度梯度离心纯化浓缩的犬冠状病毒(CCV)、猫冠状病毒(FCV)、猫传染性腹膜炎病毒(FIPV)、猪传染性胃肠炎病毒(TGEV)、猪呼吸道冠状病毒(PRCV)、牛冠状病毒(BCV)细胞毒,提取总RNA并反转录和PCR扩增。回收PCR产物连接pGEM-T-easy载体并转化大肠杆菌TGI,经PCR鉴定后测序。将所有基因片段的核苷酸序列和推导的氨基酸序列,分别与GenBank有关病毒相关基因片段的核苷酸序列进行分析比较,确定它们的同源性。通过对不同冠状病毒不同基因片段的克隆和测序,发现同一群冠状病毒核苷酸序列间具有较高的同源性。     

Rectovaginal fistula with anal atresia in 5 dogs

Five dogs with rectovaginal fistula and atresia ani that had been treated by surgical correction of the malformations were studied retrospectively. Ages at presentation varied from 1 to 3 months and weight from 350 g to 7.5 kg. The histories included voiding of feces through the vulva, with or without tenesmus, usually observed after weaning. Atresia ani, presence of feces in the vaginal canal, abdominal distention, and discomfort on abdominal palpation were observed during clinical examination. Also, 3 dogs had partial tail agenesis. In all dogs, the rectovaginal fistula was isolated and transected, the vulvar and rectal defects were closed separately, and the atresia ani was repaired. Normal defecation was restored, but 1 dog had fecal incontinence that subsequently resolved. One dog died 2.5 months postoperatively, and follow-up was done on the others for periods ranging from 1.6 year to 7.7 years. Surgical correction in dogs with rectovaginal fistula and atresia ani may result in a favorable outcome, if it is done early.     

The relationship of severe acute respiratory syndrome coronavirus with avian and other coronaviruses

In February 2003, a severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in humans in Guangdong Province, China, and caused an epidemic that had severe impact on public health, travel, and economic trade. Coronaviruses are worldwide in distribution, highly infectious, and extremely difficult to control because they have extensive genetic diversity, a short generation time, and a high mutation rate. They can cause respiratory, enteric, and in some cases hepatic and neurological diseases in a wide variety of animals and humans. An enormous, previously unrecognized reservoir of coronaviruses exists among animals. Because coronaviruses have been shown, both experimentally and in nature, to undergo genetic mutations and recombination at a rate similar to that of influenza viruses, it is not surprising that zoonosis and host switching that leads to epidemic diseases have occurred among coronaviruses. Analysis of coronavirus genomic sequence data indicates that SARS-CoV emerged from an animal reservoir. Scientists examining coronavirus isolates from a variety of animals in and around Guangdong Province reported that SARS-CoV has similarities with many different coronaviruses including avian coronaviruses and SARS-CoV-like viruses from a variety of mammals found in live-animal markets. Although a SARS-like coronavirus isolated from a bat is thought to be the progenitor of SARS-CoV, a lack of genomic sequences for the animal coronaviruses has prevented elucidation of the true origin of SARS-CoV. Sequence analysis of SARS-CoV shows that the 5' polymerase gene has a mammalian ancestry; whereas the 3' end structural genes (excluding the spike glycoprotein) have an avian origin. Spike glycoprotein, the host cell attachment viral surface protein, was shown to be a mosaic of feline coronavirus and avian coronavirus sequences resulting from a recombination event. Based on phylogenetic analysis designed to elucidate evolutionary links among viruses, SARS-CoV is believed to have branched from the modern Group 2 coronaviruses, suggesting that it evolved relatively rapidly. This is significant because SARS-CoV is likely still circulating in an animal reservoir (or reservoirs) and has the potential to quickly emerge and cause a new epidemic.     

动物冠状病毒通用PCR方法的建立及基因序列分析

参考GenBank中公布的冠状病毒相关序列,根据动物冠状病毒聚合酶基因的保守区段设计一对通用引物。用这对引物对犬冠状病毒、猫冠状病毒等8种动物冠状病毒的cDNA模板进行通用PCR扩增和通用PCR反应条件的优化,结果均得到与试验设计相符的449 bp条带;特异性试验结果表明犬冠状病毒、猫冠状病毒等均扩增出449 bp目的条带,而阴性对照没有。敏感性试验结果表明,其敏感程度与常规PCR方法相同。将扩增的聚合酶基因与冠状病毒的参考毒株进行同源性比对,同源率在56.69%～99.55%之间。进化树分析结果与文献中对冠状病毒根据血清学与基因学角度分类报道相一致。