犬冠状病毒小膜蛋白基因的分子克隆与序列分析

首次对犬冠状病毒(CCV)V1株和DXMV株小膜蛋白(SM)基因进行了克隆和序列测定.用RT-PCR对分离的CCV V1和DXMV野毒株SM基因进行了扩增,并将其克隆到pMD18-T中进行核苷酸序列测定.结果两毒株SM基因ORF全长均为246bp,编码82个氨基酸;与GenBank中CCV标准毒株Insavc-1 SM基因相比,核苷酸和推导的氨基酸序列同源性分别为100%、92.1%和100%、90.9%.DXMV株与Insavc-1株SM基因相比有9个碱基发生了改变,导致4个氨基酸发生变化.对冠状病毒科中不同的冠状病毒SM基因及其推导的氨基酸序列聚类分析表明,冠状病毒可分为4个群,群内不同冠状病毒SM基因及其蛋白显示出很高的同源性,而不同群之间的同源性却较低.     

大熊猫犬冠状病毒部分S基因的克隆与全S基因序列分析

从重庆某动物园死亡的大熊猫肝脏中分离到一株病毒，经鉴定为犬冠状病毒（命名为CCV DXMV）。本实验根据CCV K378和CCV Insavc-1株基因序列设计合成了普通PCR引物SF1-SR1和SF2-SR2，以及半套式引物SF3-SR3、SR13、SF13、SF4-SR4和SF14，分段扩增了CCVDXMV株部分S基因，得到了368bp、792bp、737bp、1178bp和985bp5个基因片段。将纯化的RCR产物分别克隆到pGEM—T载体中，通过筛选和鉴定，获得了5个阳性重组质粒。将重组质粒送生物公司测序，将测得的基因序列与先前测定的该株病毒部分S基因序列，拼接成全S基因序列，并与其它株CCV及TGEV HN2002和FIPV79—1146株的S基因序列进行分析比较，绘制进化树。结果表明，该株病毒与CCV K378株的同源性最高，达到99．4％；而与CCV 23／03株的同源性最低，为56．9％；与FIPV和TGEV分别有90．4％和82．1％的同源性。     

犬冠状病毒TN449株M基因的克隆与转移载体的构建

用蔗糖密度梯度离心纯化犬冠状病毒TN449株病毒液，提取总RNA并反转录，同时设计1对5’端加有BamHⅠ和HindⅢ内切酶位点的引物，对病毒cDNA进行了PCR扩增，回收PCR产物将其连接入pGEM-T Easy载体并转化大肠埃希氏菌。并对阳性重组质粒菌测序和序列分析。结果，犬冠状病毒与牛冠状病毒、猫冠状病毒、猫传染性腹膜炎病毒、人冠状病毒、鸡传染性支气管炎病毒、鼠传染性肝炎病毒、猪呼吸道冠状病毒、人重症急性呼吸道综合征病毒和猪胃肠炎病毒的同源性分别是39．39％、85．41％、83．98％、36．80％、26．64％、37．23％、93．51％、29．009／5和94．81％；犬冠状病毒M蛋白有3个疏水性结构域。同时构建了pET28a-CCV-M表达质粒。     

RT-PCR检测犬粪便中的冠状病毒

根据犬冠状病毒(caninecoronavirus, CCV) 的S基因序列, 用计算机设计并合成了1对引物P1、P2, 以此引物及以从美国进口的CCV疫苗的反转录产物为模板, 初步建立了检测CCV的RT PCR。将纯化的PCR产物成功地克隆到pGEM T easy载体中, 鉴定、测序并进行同源性分析。结果表明, 与1 71和UCD 1株同源性分别为91 5%和94 3%。该RT PCR能对疫苗中CCV及CCV参考株USCV B1的S基因进行特异性地扩增,长度为575bp, 对犬的其他病毒及CRFK细胞未能扩增出任何片段, 该RT PCR能检测出0 5μLCCV培养液/2g粪便。用建立的RT PCR在上海对50条犬粪便进行检测, 结果表明CCV阳性6例。本研究建立了检测犬粪便中的CCV的RT PCR, 能用于犬冠状病毒流行病学调查。     

犬冠状病毒YS1株细胞培养弱毒的实验免疫研究

应用纯化后的犬冠状病毒 (CCV)YS1株细胞培养致弱的弱毒 (CCVYS1V60 ) ,经口鼻和肌肉接种CCV ,SN抗体小于 1∶2的易感犬作安全试验 ,结果未见任何CCV临床症状与病理解剖学改变 ;用不同剂量的该CCV弱毒分组免疫CCV易感犬 ,经SN抗体测定与用CCV强毒攻毒试验 ,结果SN抗体达 1∶60以上的犬 90 %以上可获得免疫保护 ;应用该CCV弱毒分别免疫母源抗体达 1∶4和 1∶8的 2组试验犬 ,第 1次免疫 1 4d后SN抗体均未见升高 ,追加 2～ 3次免疫后 ,SN抗体才逐渐上升至 1∶60以上的免疫保护水平 ;用CCV易感犬和猫肾传代细胞 (CRFK)分别将该CCV细胞培养弱毒连续传 5代和 2 0代 ,结果对犬仍然安全 ,免疫原性也未见下降 ;与犬瘟热病毒 (CDV)、犬传染性肝炎病毒 (ICHV)和犬细小病毒(CPV)弱毒作免疫互扰试验 ,结果与各弱毒的单独免疫结果未见差异 ;该毒的免疫期在 1年以上 ,- 2 0℃冻结保存的保存期为 9个月。     

核酸探针检测犬冠状病毒方法的建立和初步应用

以^32P标记犬冠状病毒特异性RT—PCR及产物制成核酸探针，与CCV NL-18株、犬瘟热病毒、犬细小病毒、犬副流感病毒、犬腺病毒、狂犬病病毒反转录产物、正常CRFK细胞以及做10～10000倍稀释的CCV NL-18株反转录产物杂交，进行核酸探针的敏感性和特异性试验。结果表明，该探针仅与CCV HLl8株反转录产物呈阳性杂交，与对照病毒和正常细胞反转录产物均呈阴性反应。初步应用试验结果，该探针可与国内CCV分离病毒YSl、CI1株杂交，且可从8份腹泻犬粪便中检出5份CCV阳性病料。本研究为在我国开展犬冠状病毒感染的分子流行病学调查和临床诊断提供了一种敏感、特异的检测方法。     

Molecular characterization of canine coronavirus strains circulating in Brazil

To characterize canine coronavirus (CCoV) circulating in diarrheic puppies in Brazil, 250 fecal samples collected between 2006 and 2012 were tested. By using RT-PCR to partially amplify the M gene, CCoV RNA was detected in 30 samples. Sequence analysis of the M protein grouped eight strains with CCoV-I and another 19 with CCoV-II prototypes. To genotype/subtype the CCoV strains and assess the occurrence of single or multiple CCoV infections, RT-PCR of the S gene was performed, and 25/30 CCoV-positive strains amplified with one or two primer pairs. For 17/25 samples, single infections were detected as follows: six CCoV-I, nine CCoV-IIa and two CCoV-IIb. Eight samples were positive for more than one genotype/subtype as follows: seven CCoV-I/IIa and one CCoV-I/IIb. Sequence analysis revealed that the CCoV-I and IIa strains shared high genetic similarity to each other and to the prototypes. The Brazilian strains of CCoV-IIb displayed an aminoacid insertion that was also described in CCoV-IIb-UCD-1 and TGEV strains. Among the 25 CCoV-positive puppies, five had a fatal outcome, all but one of which were cases of mixed infection. The current study is the first reported molecular characterization of CCoV-I, IIa and IIb strains in Brazil.     

Prevalence of canine coronavirus (CCoV) in dog in Japan: detection of CCoV RNA and retrospective serological analysis

We collected rectal swabs from dogs in Japan during 2011 to 2014, and canine coronavirus (CCoV) nucleocapsid gene was detected by RT-PCR. The relationship between CCoV infection and the manifestation of diarrhea symptoms was investigated, and a correlation was noted (df=1, χ²=8.90, P<0.005). The types of CCoV detected in samples from CCoV-infected dogs were CCoV-I in 88.9% and CCoV-II in 7.4%, respectively. We retrospectively investigated the seroprevalence of CCoV-I in dogs in Japan during 1998 to 2006. The sera were tested with a neutralizing antibody test. In the absence of CCoV-I laboratory strain, we used feline coronavirus (FCoV)-I that shares high sequence homology in the S protein with CCoV-I. 77.7% of the sera were positive for neutralizing anti-FCoV-I antibodies.     

北京地区犬瘟热病毒分离株H基因的分型

根据发表的犬瘟热病毒(CDV)参考毒株Onderstepoort的序列设计了一对引物,以感染犬瘟热病毒的犬的粪便总RNA为模板,RT-PCR扩增出17株CDV的H全基因1877bp的片段,将这个片段连接到pGEM-Teasy载体上,经酶切鉴定得到阳性克隆,将阳性质粒进行序列测定,并与国内外毒株进行同源性比较和系统发育分析,结果显示17株均为Asia-1型。北京地区流行的毒株主要是America-1型和Asia-1型,其中2007年主要流行America-1(疫苗型),而2008年主要是Asia-1型。     

Isolation and identification of a canine coronavirus strain from giant pandas (Ailuropoda melanoleuca)

Two giant pandas (Ailuropoda melanoleuca) died of unknown causes in a Chinese zoo. The clinical disease profile suggested that the pandas may have suffered a viral infection. Therefore, a series of detection including virus isolation, electron microscopy, cytobiological assay, serum neutralization and RT-PCR were used to identify the virus. It was determined that the isolated virus was a canine coronavirus (CCV), on the basis of coronavirus, neutralization by canine anti-CCV serum, and 84.3% to 100% amino acid sequence similarity with CCV. The results suggest that the affected pandas had been infected with CCV.     

犬瘟热病毒蓝狐分离株融合蛋白基因的序列分析

根据已发表的犬瘟热病毒(Canine distemper virus,CDV)Onderstepoort株序列设计1对引物,RT-PCR法扩增出约1 000 bp的融合蛋白基因片段,通过T-A克隆技术,将PCR产物克隆至pMD18-T载体。序列分析表明CDV蓝狐分离株F蛋白基因片段由1 044 bp组成,编码348个氨基酸,与其它CDV25259株、2544-Han95株、A75-17株、DOGDK91C株、5804P株、ONP株、PDV-2株核苷酸序列同源性分别为94.5%、94.1%、94.7%、94.2%、94.2%、97.6%和94.9%;氨基酸序列的同源性分别为98.6%、97.1%、98.0%、98.0%、98.3%、97.7%和98.6%;与其它CDV毒株相比,蓝狐分离株存在核苷酸缺失突变(1 030位～1 038位)和氨基酸缺失突变(344位～348位)。     

The seroprevalence of canine respiratory coronavirus and canine influenza virus in dogs in New Zealand

Abstract

AIM: To determine whether canine respiratory coronavirus (CRCoV) and canine influenza virus (CIV) are present in dogs in New Zealand.

METHODS: Serum samples from 251 dogs of varying age, breed and clinical histories were tested for the presence of antibodies to CRCoV and CIV, using indirect fluorescent antibody (IFA) analysis. The population sampled represented a wide geographic area but principally encompassed the central and lower North Island of New Zealand.

RESULTS: Seventy-three of the 251 samples (29%) were seropositive for CRCoV. Dogs <2 years old were less likely to be seropositive for CRCoV than older dogs. None was seropositive for CIV.

CONCLUSIONS: This study revealed the presence of antibodies to CRCoV in dogs in New Zealand. Young dogs are less likely to be seropositive than older dogs, probably due to increased opportunity for exposure to CRCoV over time. Serum antibodies to CIV were not detected in any of the dogs sampled, suggesting that this virus is unlikely to be present in dogs in New Zealand.

CLINICAL RELEVENCE: Canine respiratory coronavirus is present in New Zealand. Although the role of this virus in canine infectious tracheobronchitis has not been fully elucidated, evidence suggests that it may have a causal role in this disease. Veterinarians should consider CRCoV as a differential diagnosis in cases of respiratory disease in dogs in New Zealand. While CIV appears not to be currently present in New Zealand, veterinarians should consider infection with this virus as a differential diagnosis in dogs presenting with respiratory signs.     

犬瘟热病毒H基因的序列分析与表达

以临床疑似犬瘟热病犬外周血总RNA为模板,采用RT-PCR方法扩增犬瘟热病毒(CDV)血凝素(H)基因。序列测定和分析表明该病料犬瘟热病毒H基因序列与国内其他地区分离到的毒株同源性较高(94.6%~99.2%),而与Onderstepoort株、Convac株等疫苗毒株的同源性较低(90.4%~91.2%)。分子遗传进化分析显示所有毒株可分为7个大的分支,且各分支间具有一定的地域性,其中待检病料中CDV与大多数中国分离株一样处于Ⅰ型。进一步以H全基因为模板,截短表达其3′端816 bp、459 bp两个片段,并克隆入原核表达载体pET30 a进行表达。结果显示它们分别能表达大小约为36.4 ku和22.2 ku的融合蛋白。免疫转印表明该纯化蛋白均可与犬瘟热病毒抗血清发生阳性反应,说明重组蛋白具备抗原性,可用于进一步的血清学研究。     

63株IBV分离株M基因的遗传变异与系统进化分析

对1993－2010年从中国不同地区分离的63株传染性支气管炎病毒(infectious bronchitis virus,IBV)野毒株,采用RT-PCR方法克隆测定所分离野毒株的M基因核苷酸序列,并与GenBank中公布的部分国内外IBV毒株的M基因序列进行比较分析,研究中国IBV的分子流行病学特点和分子遗传变异规律。结果显示,所测毒株M基因具有6种不同长度的开放阅读框,这些长度的差异是由于5'端的核苷酸插入或缺失造成的;N端含有1~2个N-糖基化位点,其中1个糖基化位点"Asn-Cys-Thr"(NCT)是高度保守的。63个IBV分离株间氨基酸序列相似性为88.9%~100%,分离株与参考株间相似性为87.2%~100%。系统进化分析结果显示,本研究的63个IBV分离株可分为9个基因型,2005－2010年IBV中国流行株大部分与Mass型疫苗株处于不同的基因型,而且氨基酸序列相似性都小于94%。     

M gene evolution of canine coronavirus in naturally infected dogs

Two stray pups (A and B), three and five months old, respectively, both naturally infected with canine coronavirus (CCoV), were studied for 180 days. The virus was detected intermittently in the pups' faeces by PCR for periods of 156 and 146 days, respectively. Sequence analysis of a fragment of the gene encoding the M protein revealed that the viruses detected at the onset of the infection were very similar to typical strains of CCoV, whereas from 42 days after infection in pup A and 40 days after infection in pup B the viruses had nucleotide and amino acid mutations resembling sequences in feline coronavirus.     

三株广西狂犬病病毒NS基因和M基因的克隆与序列分析

本研究设计了一对特异性引物NSM1/NSM2,对三株广西狂犬病病毒NS和M基因同时进行了RT_PCR扩增、克隆和测序。同源性分析表明,三株广西野毒NS基因核苷酸同源性为87.2%~98.4%,M基因核苷酸同源性为90.1%~99.7%;与固定毒和狂犬病相关病毒比较,NS基因分别为79.9%~82.8%和69.7%~71.0%;M基因的分别为82.8%~87.8%和75.0%~77.8%。三株野毒NS基因氨基酸同源性为93.3%~98.7%,M基因氨基酸同源性分别为97.5%~100%。表明广西各地毒株之间亲缘关系不同,但最为相近;与狂犬病固定毒株亲缘关系较远;与狂犬病相关病毒亲缘关系最远。     

鸡传染性支气管炎病毒地方分离株核蛋白基因的遗传变异分析

对2006-2009年从山西各地疑似传染性支气管炎的病例中分离到的7株IBV地方分离株的核蛋白基因片段进行序列测定及分析。结果发现,7株IBV地方分离株核蛋白基因有5株含有一个长1 230 bp的ORF,其余2株含有1 227 bp,编码410/409氨基酸残基组成的多肽,与常用疫苗株H120推导的氨基酸序列比对发现,存在基因突变现象。与GenBank中的34株国内外分离毒株核蛋白基因推导的氨基酸序列进行比较和分析,系统进化关系显示41株IBV毒株分属于4个群,至少有3个群在我国流行,7个分离株分布在第Ⅳ群中,第IV群大多来自我国的北方地区地方分离毒株,第Ⅱ群来自我国南方地区的部分地方分离毒株,从N基因推导氨基酸进化树上分析可见,我国的IBV地方分离毒株主要分布在第Ⅱ和第Ⅳ群中,具有较明显的地理区域性,可见IBV地方分离株在基因进化关系上形成了自己较为独立的进化群。     

Canine coronavirus in Australian dogs

OBJECTIVE: To estimate the frequency of serum antibodies (IgG and IgM) to canine coronavirus (CCV) in the Australian dog population and evaluate the role of CCV as a causative agent of gastroenteritis. DESIGN: A serological survey of antibodies to CCV among different dog populations. PROCEDURE: The development and characterisation of an indirect ELISA for the detection of antibodies (IgG and IgM) to CCV was undertaken. Sera collected from both diarrhoeal and non-diarrhoeal dogs from various populations throughout Australia were tested for these antibodies to CCV. RESULTS: Serum samples (1396) collected from 1984 to 1998 were tested for the presence of IgG antibodies to CCV. Samples were divided into two categories on the basis of the number of dogs housed together. The groups were either an open population containing dogs housed as groups of three or less, or kennel populations. Sera from 15.8% of the open population and 40.8% of kennelled dogs were positive for CCV antibodies. The prevalence of antibodies varied from zero to 76% in kennelled dogs. About 23% of 128 dogs positive for IgG antibodies to CCV were also positive for IgM antibodies to CCV, indicating recent CCV infection. Of those dogs that were presented with clinical signs of gastroenteritis such as diarrhoea and vomiting (n = 29), 85% were positive in the IgM ELISA and 85.7% in the IgG ELISA for antibodies to CCV. In comparison, for those dogs presented without any history of gastroenteritis only 15% were positive for IgM and 30% positive for IgG. CONCLUSION: Serological evidence indicates that infection with CCV in dogs is widespread throughout the Australian mainland. The prevalence of antibodies varies greatly among different populations, with an average of 40.8% positive in kennelled populations and 15.8% in the open population.     

2011年河南省猪流行性腹泻病毒流行株的遗传进化分析

2011年前后,猪流行性腹泻(PED)在河南省爆发流行.为探明该病再次流行的原因,本研究对2011年收集的7份来自河南省不同地区PED病毒(PEDV)流行病毒株M基因进行RT-PCR扩增、克隆、测序及分析.序列分析显示,7株PEDV河南流行株M基因部分核苷酸及氨基酸发生变异,不同于以往参考病毒株.基于M基因的遗传进化分析显示,PEDV河南流行株和参考株可以分为3个群(G1、G2、G3),7株PEDV河南流行株均属于第3群,与2010年中国BJ2010株、2007年韩国PFF188株及2008年泰国08RB03株亲缘关系密切,而与欧洲病毒株(CV777、Br1/87)、1995年和2004年泰国株(M_NIAH1795_04、M_NIAH2013_95)及2006年中国LZC株亲缘关系较远.结果表明,2011年在中国河南省及其它地区流行的PEDV是一个新的基因型,可能起源于韩国和泰国.