7种动物冠状病毒基因芯片同步检测的研究

根据发布的7种动物冠状病毒相关基因的序列,给每种病毒设计了4～17对引物,同时依据RNA聚合酶基因序列合成了l对兼并引物,利用TCV原毒和纯化浓缩的CCV、FCV、FIPV、TGEV、PRCV、BCV细胞毒,构建了89个基因片段的克隆.采用煮沸裂解法制备质粒DNA,进行特异引物PCR扩增,回收纯化产物,点制冠状病毒基因芯片.抽提病毒总RNA,利用Cy3-dCTP随机渗入反转录PCR标记,同时对BCV、CCV利用Cy3-dCTP随机渗入反转录标记,与芯片进行杂交检测.结果显示,BCV和TCV基因克隆间未见交叉,CCV、FCV、FIPV、TGEV、PRCV则存在广泛交叉.选择无交叉的特异克隆片段点制最终基因芯片,并与病毒多重PCR扩增产物杂交,芯片信号未见交叉,结果判断明确.表明,基因芯片检测法比传统PCR法敏感1000倍.     

犬猫3种冠状病毒基因克隆的构建与芯片检测技术

蔗糖密度梯度离心纯化浓缩犬冠状病毒(CCV)、猫冠状病毒(FCV)和猫传染性腹膜炎病毒(FIPV)的细胞培养物,分别设计7、17、11对引物,对病毒RNA进行反转录和PCR扩增,回收PCR产物连接pGEM-T载体并转化大肠杆菌TGI,构建35个基因片段的克隆。煮沸裂解法制备质粒DNA,回收PCR扩增产物,点制冠状病毒基因芯片。抽提病毒总RNA,利用Cy3-dCTP随机渗入反转录PCR标记,与芯片进行杂交检测,淘汰交叉的克隆片段,每种病毒只选择其4对引物克隆的基因片段。将特异克隆扩增片段重新点制基因芯片,与病毒样品PCR扩增产物杂交,未发现交叉现象。基因芯片检测比传统PCR敏感1000倍,可有效应用于3种病毒的检测与区分。     

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。     

猫冠状病毒和猫传染性腹膜炎病毒RT-PCR检测方法的建立

本文利用A-72细胞繁殖猫冠状病毒(FCV)和猫传染性腹膜炎病毒(FIPV),用蔗糖密度梯度离心纯化,根据病毒S基因序列设计引物,初步建立了检测FCV和FIPV的RT-PCR方法。采用此方法对人工攻毒猫组织进行检测,结果特异灵敏。     

RT-PCR检测和区分猪传染性胃肠炎病毒和猪呼吸道冠状病毒的研究

根据猪传染性胃肠炎病毒（TGEV）和猪呼吸道冠状病毒（PRCV）的基因组核苷酸序列，在S基因（纤突蛋白基因）5′端保守区设计了一对引物P3／P4，该对引物在TGEV扩增跨幅约为2.4kb；而PRCV由于在此区域存在一约0.6kb碱基缺失，扩增跨幅约为1.8kb。用引物P3／P4对TGEV Miller株、Purdue株和PRCV AR310株分别进行RT－PCR，根据RT－PCR扩增片段大小可以直接区分TGEV和PRCV。用引物P3／P4与引物P1／P2作Nested-PCR,提高了该RT－PCR的特异性和敏感性，建立的RT－PCR可为临床上诊断TGEV及调查我国是否存在PRCV感染提供可靠的鉴别手段。     

猪冠状病毒通用荧光定量PCR检测方法的建立

已知感染猪群的冠状病毒有猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪德尔塔冠状病毒(PDCoV)、猪急性腹泻综合征冠状病毒(SADS-CoV)、猪呼吸道冠状病毒(PRCV)、猪血凝性脑脊髓炎病毒(PHEV);本研究拟建立针对这6种病毒的通用荧光定量PCR检测方法。依据NCBI公布的这6种猪冠状病毒序列，利用MEGA软件进行全基因序列比对，通过分析在ORF1b中找到1段长为196 bp的保守序列，设计兼并引物，建立了用于检测猪冠状病毒的通用荧光定量PCR检测方法；结果显示，该检测方法能扩增出6种猪冠状病毒，对PDCoV、PRCV、PHEV、SADS-CoV、TGEV的灵敏性均可达到10¹拷贝/μL,对PEDV的灵敏性可达到10⁰拷贝/μL;与CSFV、PRRSV、PPV、PCV、PRV无交叉反应，特异性良好；组内和组间变异系数均小于2.2%,重复性良好。利用建立的检测方法对62份病料进行检测，检测出27份样品呈现猪冠状病毒阳性，而利用普通RT-PCR方法共检测出24份阳性样品。选择2份猪冠状病毒阳性样品进行克隆测序，在NCB...     

猪呼吸道冠状病毒研究进展

综述了猪呼吸道冠状病毒(PRCV)的病原学意义、分子生物学特性、与传染性胃肠炎病毒TGEV的鉴别诊断、PRCV对TGEV的免疫保护作用等四个方面的研究进展。     

巢式RT-PCR在猪传染性胃肠炎病毒检测中的应用

建立对猪传染性胃肠炎病毒(TGEV)的巢式RT-PCR(RT-nested PCR)检测方法,为该病的诊断和流行病学调查提供更为可靠和敏感的手段。根据GenBank中收录的TGEV,纤突蛋白S基因的核酸序列设计了2对引物,以mRNA为模板,通过RT-PCR、RT-nested PCR方法,在优化RT-PCR反应条件的基础上,建立了快速检测TGEV的RT-nested PCR诊断方法。试验结果成功扩增出长度为886 bp和690 bp的TGEV目的片段,但未扩增出猪传染性腹泻病毒(PEDV)片段。表明RT-nested PCR方法可用于检测猪传染性胃肠炎病毒,而且此法简单省时、灵敏性高。     

猪繁殖与呼吸综合征病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立

为建立猪繁殖与呼吸综合征病毒(PRRSV)SYBR GreenⅠ实时荧光定量PCR检测方法,采用RT-PCR扩增PRRSV N基因266bp片段,并克隆到pMD18-T载体中,以纯化的重组质粒为模板进行荧光定量PCR扩增,建立了PRRSV荧光定量PCR检测方法,该方法检测灵敏度可达1.3×101拷贝/μL,与猪瘟病毒(CSFV)、猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)和猪日本脑炎病毒(JEV)均不发生交叉反应,具有很好的特异性和重复性。结果表明,建立的PRRSV实时荧光定量PCR具有特异、敏感、快速、定量、重复性好等优点,可用于临床猪繁殖与呼吸综合征病毒病(PRRS)的诊断。     

猪呼吸道冠状病毒(PRCV)研究概况

猪呼吸道冠状病毒病(PorcineDespiratoryCorouavirus,PRC)是由于感染猪呼吸道冠状病毒(PRCV)引起的一种呼吸道疾病。PRCV由猪传染性胃肠炎病毒(TGEV)演化而来的。本文综述了猪呼吸道冠状病毒(PRCV)的特性、基因组结构及主要病毒蛋白、诊断方法、免疫学上的作用等方面在国内外的研究情况。     

Differentiation between transmissible gastroenteritis virus and porcine respiratory coronavirus using a cDNA probe.

A plasmid, pG3BS, containing a cDNA clone from the 5' coding region of the peplomer glycoprotein gene appears to be specific for enteric transmissible gastroenteritis virus (TGEV) strains and for live-attenuated TGEV vaccines. This cDNA probe is used to differentiate porcine respiratory coronavirus (PRCV) isolates from TGEV field and vaccine strains by a slot blot hybridization assay. Probe pG3BS also hybridizes to canine coronavirus (CCV) RNA but does not hybridize to antigenically related feline infectious peritonitis virus (FIPV) RNA. The RNAs of 13 enteric TGEV isolates from the United States, Japan, and England, 4 US-licensed live-attenuated TGEV vaccines, and antigenically closely related CCV were detected by pG3BS. The RNAs of FIPV and 3 US isolates of PRCV did not react with pG3BS but were detected by a TGEV-derived plasmid, pRP3. Pigs infected with either PRCV or TGEV test serologically positive for TGEV antibody by the serum neutralization test. Characterization of the virus circulating in a swine herd by the pG3BS probe will differentiate between an enteric TGEV and a respiratory PRCV infection.     

Differentiation of canine coronavirus and porcine transmissible gastroenteritis virus by neutralization with canine,porcine and feline sera

Monospecific antisera were prepared in rabbits against canine coronavirus (CCV) and transmissible gastroenteritis virus of pigs (TGEV), and in 24 pigs and 3 cats against TGEV alone. Neutralizing antibody titres were higher for the immunizing than the heterologous virus, although cross-neutralization usually was detected. This confirmed that CCV and TGEV are distinct, but antigenically related coronaviruses. In sera from 41 dogs, CCV-neutralizing titres were on average 2.7 fold higher than TGEV-neutralizing titres, suggesting that CCV was the causal agent. Sera from 29 cats in colonies with feline infectious peritonitis (FIP) and known to contain TGEV-neutralizing antibody, were found to have titres 12.3 fold higher against CCV. The FIP virus (FIPV) is probably more closely related to CCV than TGEV as judged by antigens involved in virus neutralization.Antisera to two isolates of bovine coronavirus, three isolates of haemagglutinating encephalomyelitis virus, seven strains of avian infectious bronchitis virus and the 229E strain of human coronavirus all failed to neutralize CCV and TGEV. Thus CCV, TGEV and probably FIPV fall into a group of antigenically related agents, separable from other members of the family Coronaviridae, by both virus neutralization and immunofluorescence tests.     

Studies of enteric coronaviruses in a feline cell line

Development is reported of a feline cell line which can support the growth of coronaviruses from canine (CCV), feline (FIPV) and porcine (TGEV) species. The cell culture has been serially transferred over 100 times and has retained its initial growth requirements, proliferative capacity and morphologic features. Each virus had specific growth characteristics in this cell culture although all produced a similar CPE and plaques under agar. Cross neutralization studies demonstrated a two-way relationship between TGEV and CCV and between TGEV and FIPV, whereas a one-way relationship was demonstrated between CCV and FIPV.     

Antigenic analysis of feline coronaviruses with monoclonal antibodies (MAbs): preparation of MAbs which discriminate between FIPV strain 79-1146 and FECV strain 79-1683.

We prepared 31 monoclonal antibodies (MAbs) against either FIPV strain 79-1146 or FECV strain 79-1683, and tested them for reactivity with various coronaviruses by indirect fluorescent antibody assay (IFA). Sixteen MAbs which reacted with all of the 11 strains of feline coronaviruses, also reacted with canine coronavirus (CCV) and transmissible gastroenteritis virus (TGEV). In many of them, the polypeptide specificity was the recognition of transmembrane (E1) protein of the virus. We succeeded in obtaining MAbs which did not react with eight strains of FIPV Type I viruses (showing cell-associated growth) but reacted with FIPV Type II (79-1146, KU-1) and/or FECV Type II (79-1683) (showing non-cell associated growth). These MAbs also reacted with CCV or TGEV. These MAbs recognized peplomer (E2) glycoprotein, and many antigenic differences were found in this E2 protein. These results suggest that FIPV Type II and FECV Type II viruses are antigenically closer to TGEV or CCV than to FIPV Type I viruses. Furthermore, the MAb prepared in this study has enabled discrimination between FIPV strain 79-1146 and FECV strain 79-1683, which was thought to be impossible by the previous serological method.     

Lesions in the small intestine of newborn pigs inoculated with porcine, feline, and canine coronaviruses

The infectivity and pathogenicity to newborn pigs of antigenically related coronaviruses from pigs (transmissible gastroenteritis virus; TGEV), cats (feline infectious peritonitis virus; FIPV), and dogs (canine gastroenteritis virus; CGEV) were studied by light, scanning electron, and immunofluorescence microscopy. Hysterectomy-derived, 12-hour-old pigs were orally given tissue culture or frozen preparations of 6 coronavirus strains (3 porcine, 2 feline, and 1 canine). The pigs were killed at regular intervals between 24 and 144 hours after exposure. Virulent TGEV and virulent FIPV produced necrosis of villous epithelium, resulting in villous atrophy in the jejunum and the ileum. Similar, but less extensive and severe lesions, were produced by the 4 other viruses. Coronaviral antigens were identified by immunofluorescence in villous epithelial cells of pigs that had been inoculated with virulent TGEV, attenuated TGEV, virulent FIPV, and tissue culture-adapted FIPV. In contrast, coronaviral antigens were not induced by the small plaque variant TGEV and virulent CGEV in the villous epithelium, but rather in cells of the lamina propria and crypt epithelium.     

应用套式PCR检测和区分猪传染性胃肠炎病毒和猪呼吸道冠状病毒的试验

根据猪传染性胃肠炎病毒和猪呼吸道冠状病毒的S基因核苷酸序列，设计合成了2对引物，以猪传染性胃肠炎病毒和猪呼吸道冠状病毒细胞培养物为模板，进行PCR特异性片段扩增，猪传染性胃肠炎病毒扩增片段大小为886bp，猪呼吸道冠状病毒扩增片段大小为214bp。建立的套式PCR经过特异性、敏感性试验及对临床送检样品检测，证明本法具有快速、特异和高度敏感的特点。     

Induction and enhancement of feline infectious peritonitis by canine coronavirus.

Preexisting antibody to feline infectious peritonitis virus (FIPV) causes acceleration and enhancement of disease on subsequent infection of cats with FIPV. Other workers have shown that canine coronavirus (CCV) can infect cats subclinically, but have found no evidence of enhancement of, or protection against, subsequent FIPV infection. With various isolates of CCV, we determined that 1 strain of CCV can induce transient mild diarrhea in cats and, furthermore, that previous infection with CCV causes acceleration and enhancement of subsequent infection with FIPV. In addition, sequential inoculation of cats with another strain of CCV caused lesions indistinguishable from those of FIP, without exposure at any time to FIPV.     

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。