RT-PCR快速诊断禽流感

根据禽流感病毒ＮＰ基因的序列分析结果,设计了一对ＮＰ基因特异的引物。采用该对引物,不经病毒分离,直接从禽流感病毒感染鸡的气管、泄殖腔棉拭子和组织样品中提取核酸, ＲＴ～ＰＣＲ可以扩增出 ３２６ｂｐ的 ＮＰ基因片段。采用该技术对１４个亚型禽流感病毒标准参考株,４个亚型１２株国内分离野毒株,ＲＴ－ＰＣＲ检测的结果都呈阳性;对新城疫病毒、传染性法氏囊病毒、传染性支气管炎病毒、传染性喉气管炎病毒以及减蛋综合症病毒,ＲＴ－ＰＣＲ扩增结果都呈阴性。禽流感病毒 Ａ／Ｇｏｏｓｅ／Ｇｕａｎｇｄｏｎｇ（Ｈ５Ｎ１）和 Ａ／Ａｆｒｉｃａｎ　Ｓｔａｒｌｉｎｇ／Ｅｎｇｌａｎｄ（Ｈ７Ｎ１）实验感染鸡样品 ＲＴ－ＰＣＲ检测与鸡胚病毒分离阳性率分别为３４／４２、３２／４２; ２４／５５、２４／５５, 二者符合率大于９５％。 ＲＴ－ＰＣＲ最少可检测到１０ｐｇ的病毒核酸。对山东某地发病鸡场样品进行ＲＴ－ＰＣＲ检测,只用６个小时就可得出准确的诊断结果,证明ＲＴ－ＰＣＲ检测方法敏感特异,可用于禽流感的快速诊断。     

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。     

Serological comparison and antigenic relationships of seven serotypes of infectious bronchitis virus using the hemagglutination-inhibition test

The antigenic relationships, antigenic spectrum, and immunogenicity of seven isolates of infectious bronchitis virus (IBV) were examined using the hemagglutination-inhibition (HI) test. Because there was a discontinuity of antigenic relationships and a high degree of cross-reactivity among serotypes of IBV in cross-hemagglutination-inhibition tests, the range of antigenic spectrum used to group the serotypes with the HI test should be wider than the limits suggested by the plaque-reduction test. The HI test may provide valuable information in monitoring the immune status of a flock following vaccination when the area has a history of infectious bronchitis infection. It may also be used as a rapid diagnostic test if a flock is experiencing an outbreak of a disease caused by emergence of a new type of IBV. Interpretation of HI titers in evaluating immune status of chickens following infection with IBV depends on further cross-challenge and cross-protection studies of various types of IBV.     

Sensitivity and specificity of the fluorescent antibody technique for detection of infectious laryngotracheitis virus.

The specificity of a fluorescent conjugate to infectious laryngotracheitis virus was examined using chick trachea organ culture or tissue sections infected with other avian viruses (adenovirus, infectious bronchitis, poxvirus, reovirus, Newcastle disease virus, Marek's disease virus, avian encephalomyelitis and infectious bursal agent) or Mycoplasma gallisepticum. Confirmation of virus replication in these preparations was obtained by either 1) demonstration of virus titre increase or 2) demonstration of fluorescence when using the homologous conjugate. Once either of these criteria had been satisfied, negative results with the infectious laryngotracheitis conjugate were taken to indicate that the conjugate would not present false positive results in differentiated cells infected with these heterologous viruses. The spectrum of reactivity of the infectious laryngotracheitis conjugate was then examined on organ cultures infected with several infectious laryngotracheitis isolates from across Canada. Finally, the conjugate was applied to experimental and natural cases of infectious laryngotracheitis and its efficiency was compared to routine virus isolation methods.     

Localization of linear B-cell epitopes on infectious bronchitis virus nucleocapsid protein

The nucleocapsid (N) protein of many viruses is highly conserved, immunogenic, and abundantly expressed during infection. These features make it a suitable candidate for diagnostic applications. The nucleocapsid protein of infectious bronchitis virus (IBV) was dissected into 12 fragments and expressed in Escherichia coli. Sera against Australia T, China Ch5, Singapore P4, USA M41 and China T3 isolates were used to study the conservation and localization of the antigenic region on the IBV nucleocapsid protein. Our results show linear immunodominant epitopes, which were found in three fragments covering amino acid residues 175-241, 310-370 and 360-409.     

Determination of the antigenic relationships within the Massachusetts (M41) type of infectious bronchitis virus using the hemagglutination-inhibition test

The antigenic relationships, antigenic spectrum, and immunogenicity of seven IBV-Massachusetts-41 isolates were investigated using the hemagglutination-inhibition (HI) test. HI titers equal to 32 are considered suspicious, titers lower than 32 are considered negative, and titers higher than 32 are considered positive immune responses to infectious bronchitis virus (IBV). Some isolates of Massachusetts-41 ( M41 ) were capable of inducing large quantities of antibodies in chickens following inoculation and demonstrated a wider antigenic spectrum than others. Variations in antigenic spectrum observed within M41 isolates in this study are in agreement with previous reports. This variation is of importance in selecting a proper vaccine strain for a successful immunization program for IBV.     

鸡4种病毒抗原液的浓缩及其四联油佐剂灭活苗的研制

本研究通过超滤浓缩技术对鸡新城疫病毒、传染性支气管炎病毒、产蛋下降综合征病毒和传染性法氏囊病病毒的尿囊液进行了10倍或10倍以上的浓缩处理，并按一定的比例配比研制成四联油乳剂灭活疫苗，对鸡的最小免疫剂量是0.25ml，免疫接种二周后，鸡新城疫和产蛋下降综合征病毒的HI抗体效价分别达到8log     

Vaccine efficacy against Ontario isolates of infectious bronchitis virus

Infectious bronchitis (IB) is an economically important viral disease with worldwide distribution. Every country with an intensive poultry industry has infectious bronchitis virus (IBV). The virus rapidly spreads from bird to bird through horizontal transmission by aerosol or ingestion. Sentinel bird studies were carried out in southern Ontario and IBV has been isolated from layer flocks. Genetic analysis of the S1 region of the strains showed that they were not vaccine related. The pathogenicity of selected Ontario variants of IBV isolates was studied and the subsequent work was to determine the degree of protection against field isolates provided by a commonly used vaccine MILDVAC-Ma5 in Ontario. The protection was evaluated by challenging immunized chickens with the respiratory (IBV-ON1) and nephropathogenic (IBV-ON4) viruses. The mean vaccine efficacy for IBV-ON1 was 66.7% indicating that a Massachusetts serotype vaccine would provide some protection against IBV field isolates.     

Serological differentiation of avian infectious bronchitis field isolates using an enzyme immunoassay: presence of Dutch strains in West Germany

Six field virus isolates were identified as the etiologic agent of avian infectious bronchitis (IB) in West Germany. A serological comparison was carried out using the M-41 strain from the Massachusetts serotype and the Dutch variant strains D-207, D-1466, D-3128, and D-3896 in a cross indirect immunoperoxidase test. Three isolates demonstrated similarity to the M-41 strain; the remainder showed a closer antigenic relationship to the Dutch variant strains. These results correspond to differences observed in a hemagglutination-inhibition test. The enzyme immunoassay is proposed as another possible method for differentiating IB virus strains.     

SOME IMMUNOLOGICAL ASPECTS OF A RECENT AUSTRALIAN ISOLATE OF INFECTIOUS BRONCHITIS VIRUS

An infectious bronchitis virus, designated G48, isolated from birds during an outbreak of nephritis in a previously vaccinated broiler flock, overcame the resistance induced in birds vaccinated with 2 commercially available vaccines. Birds vaccinated with the A isolate of infectious bronchitis resisted challenge with this new virus. Cross neutralisation studies revealed that the new virus was serologically distinct from the 4 viruses tested. Homologous antiserum to G48 did not neutralise the other viruses and only antiserum to the A virus completely neutralised the new virus.     

Genetic and antigenic diversity in avian infectious bronchitis virus isolates of the 1940s

In order to verify a commonly held assumption that only Massachusetts (Mass) serotype of infectious bronchitis virus (IBV) was prevalent in the United States between the 1930s (when IBV was first isolated) and the 1950s (when the use of commercial IBV vaccines began), we examined 40 IBV field isolates from the 1940s. Thirty-eight of those isolates were recognized as Mass serotype viruses based on their reactivity to Mass-specific monoclonal antibody (Mab) and neutralization by Mass-specific chicken serum. The remaining two isolates, N-M24 and N-M39, that did not react with Mass-specific Mab, resisted neutralization by Mass-specific chicken serum, and were neutralized only by homologous chicken antibody were identified as non-Mass IBV. When the first 900 nucleotides (nt) from the 5'-end of the spike (S1) glycoprotein gene and their deduced amino acid (aa) sequences were compared, the two non-Mass isolates differed from each other by 24% and 28%, respectively. In a similar comparison, the non-Mass viruses N-M24 and N-M39 differed from M28, a Mass-type isolate from the 1940s, by 21% and 22% (nt) and 28% and 27% (aa), respectively. These data indicate that antigenic and genetic diversity among IBV isolates existed even in the 1940s. Interestingly, when the N-terminal region of the S1 of M28 was compared to that of M41, a prototype Mass virus that has undergone countless number of in vivo and in vitro host passages, the two viruses differed by only 2% (nt) and 4% (aa). This finding suggests that frequent genetic changes are not inherent in all IBV genomes.     

Molecular characterization of avian infectious bronchitis virus strains isolated in Colombia during 2003

Sixteen infectious bronchitis virus (IBV) isolates were recovered from broilers and layers from five geographic poultry regions in Colombia. The viruses were isolated from tracheas, lungs, and cecal tonsils of birds, previously vaccinated with the Massachusetts strain, that were showing respiratory signs. Further analysis of the IBV isolates was achieved by phylogenetic analysis comparing their deduced amino acid sequences in the hypervariable region 1 of the S1 gene with reference strains. Four unique genotype clusters containing isolates with indigenous genotypes were observed. One isolate was found to be the Connecticut genotype and three isolates were found to be the Massachusetts genotype.     

鸡传染性支气管炎病毒广西分离株血清型的分析

应用病毒感染的鸡胚材料免疫新西兰兔的方法制备抗鸡传染性支气管炎病毒(IBV)单因子血清,然后在鸡胚气管环培养(Tracheal organ cultures,TOC)上对广西分离的7个IBV代表性毒株和3个常用疫苗株进行交叉病毒中和试验。结果显示,10个毒株被分为6个血清型。根据试验所得的R值,应用聚类分析法分析了各血清型毒株之间的亲缘关系,显示目前在广西流行的IBV野毒株之间以及其与疫苗株间的抗原性存在很大程度的差异,分属不同的血清型。同时还对IBV基因分型和血清分型之间的关系进行了探讨。     

Isolation of cytopathogenic strains of infectious bursal virus of chickens in cell cultures

Five virus strains with cytopathogenic properties for the cell cultures of chick embryo fibroblasts and embryo kidney cells were isolated from chickens clinically suspected of suffering from infectious bursitis. According to the form of cytopathic effect (CPE) on cell cultures, according to chloroform and thermal resistance, nature of nucleic acid (RNA), according to the absence of the production of intracellular inclusions, negative haemagglutination, and according to antigenic identity with the reference strain, the isolates were deemed to belong to the viruses of infectious bursitis. This conclusion was also corroborated by histological and serological studies in isolates of infected experimental animals.     

Antigenic relationship of turkey coronavirus isolates from different geographic locations in the United States

The purpose of the present study was to examine the antigenicity of turkey coronavirus (TCV) isolates from various geographic areas with antibodies to different viruses. Seventeen isolates of TCV were recovered from intestinal samples submitted to Animal Disease Diagnostic Laboratory, Purdue University, from turkey farms located in different geographic areas. The prototype TCV Minnesota isolate (TCV-ATCC) was obtained from the American Type Culture Collection. Intestinal sections were prepared from turkey embryos infected with different TCV isolates and reacted with polyclonal or monoclonal antibodies to TCV, infectious bronchitis virus (IBV), bovine coronavirus (BCV), transmissible gastroenteritis virus (TGEV), reovirus, rotavirus, adenovirus, or enterovirus in immunofluorescent antibody staining. All 18 TCV isolates have the same antigenic reactivity pattern with the same panel of antibodies. Positive reactivity was seen with polyclonal antibodies to the TCV Indiana isolate, the TCV Virginia isolate, TCV-ATCC, and the IBV Massachusetts strain as well as monoclonal antibodies to the TCV North Carolina isolate or the membrane protein of IBV. Antibodies to BCV or TGEV were not reactive with any of the TCV isolates. Reactivity of antibodies to unrelated virus, rotavirus, reovirus, adenovirus, or enterovirus with different TCV isolates was all negative, except positive response was seen between enterovirus antibody and a TCV western North Carolina isolate, suggesting coinfection of turkeys with TCV and enterovirus in that particular case. The results indicated that the TCV isolates from these geographic locations in the U.S. shared close antigenicity and were antigenically related to IBV.     

Characterization of infectious laryngotracheitis viruses, antigenic comparison by kinetics of neutralization and immunization studies.

Five isolates of infectious laryngotracheitis virus were compared by pock formation on the chorioallantoic membrane of embryonated eggs, plaque size in chicken embryo kidney tissue culture, and antigenic relationship using reciprocal kinetics of neutralization. The A4557-5 strain of infectious laryngotracheitis virus, which causes mild respiratory disease, produced pocks with a zone of edema on the chorioallantoic membrane. A virulent virus (Virus 1), isolated from an outbreak of severe disease characterized by a diphtheritic laryngotracheitis, produced the largest plaques in chicken embryo kidney cell culture. Other virulent viruses (Viruses 2, 3 and V154) did not have unique growth characteristics when grown on the chorioallantoic membrane or in chicken embryo kidney cell culture. All viruses were closely related antigenically as shown by kinetics of neutralization but viruses 2 and 3 were not homogeneous with the other three viruses when neutralized by anti-V154 chicken serum. Following aerosol infection, chickens infected with the A4557-5 virus were immune to challenge with virulent V154 virus. However, in comparison to SA-2 virus, this virus was a less effective immunizing agent when administered by the vent or drinking water methods.     

Australian infectious bronchitis viruses: identification of nine subtypes by a neutralisation test

The antigenic relationships between 17 Australian infectious bronchitis viruses, including six vaccine viruses, were studied by a neutralisation test using a plaque reduction method in chick embryo kidney cell monolayers. The 17 viruses formed nine distinct subtypes. Antiserum to each subtype had a high titre to viruses of the same subtype and a lower titre to viruses of different subtypes. The heterologous titres of sera varied widely.     

传染性法氏囊病病毒广西分离株血清亚型的确定

应用兔制备的抗传染性法氏囊病病毒(IBDV)高免血清,在鸡胚成纤维细胞(CEF)上对从广西发病鸡群分离的4个IBDV代表性流行毒株和2个常用疫苗株进行交叉中和试验。结果表明6个毒株被分为2个血清亚型;根据试验所得的R值,应用聚类分析法分析了各亚型毒株之间的亲缘关系,结果显示目前在广西流行的IBDV野毒株之间以及其与疫苗株间的抗原性存在一定的差异。研究结果对及时掌握广西IBDV流行毒株的抗原变异并为研制更有效的适合本地使用的IBD疫苗提供了科学依据。     

Identification and analysis of the Georgia 98 serotype, a new serotype of infectious bronchitis virus

Twenty-five field infectious bronchitis viruses (IBVs) similar to, but genetically distinct from, the DE072 serotype were isolated from several states in the United States from 1990 through 1999 and were examined molecularly and antigenically. A 421-bp sequence in the hypervariable region of the S1 gene was examined, and phylogenetic analysis on that region indicated that these viruses are closely related but fall into unique groups. Cross-virus neutralization testing and entire S1 sequence analysis on selected isolates further confirmed that fact, and we divided the viruses into the DE072 serotype and two other unique groups. In a vaccine protection trial, the commercially available DE072 vaccine showed less than 50% protection against viruses in one of the groups. The majority of the recent isolates belong to that group and share very low antigenic relatedness to the DE072 strain as well as other serotypes of IBV. Consequently, we designated this group as a new serotype, Georgia 98. We developed a restriction fragment length polymorphism analysis that can differentiate this new serotype from all other serotypes of IBV.