用气管环交叉中和试验鉴定IBV山东分离株的血清型

利用气管环组织培养 ,通过交叉中和试验 ,以IBVM41 及T株作为对照 ,对从山东省分离的 4株传染性支气管炎病毒进行了血清型的初步分型。结果表明 ,4株中有 1株与T株为同一血清型 ,其余 3株既不属于M41 株 ,也不属于T株     

Antigenic characterization of foot-and-mouth disease virus serotype Asia1 field isolates using polyclonal and monoclonal antibodies

Foot-and-mouth disease virus (FMDV) serotype Asia1 field isolates (n = 100) were compared using a panel of 11 monoclonal antibodies (Mab) in sandwich ELISA. The majority (over 89%) of the isolates showed either homologous (76% and above reactivity) or reduced affinity (20-75% reactivity) for the Mabs 2A, 13, 40, 34 and 81, suggesting that these Mab binding epitopes are conserved, whereas a more variable reactivity was observed for the Mabs B3, 1A, 24, 72, 82 and 89. Polyclonal relationship ('r' value) of the field isolates in liquid phase blocking (LPB) ELISA was examined, and the mean 'r' value was 0.62 relative to vaccine virus IND 63/72. Some of the field isolates (n = 34) were tested in virus neutralization test (VNT) and showed an 'r' value of >0.40. Although a minor antigenic difference was observed in the Mab profiling study, there has not been large antigenic divergence between reference virus and field viruses, thereby providing evidence of wide antigenic coverage of the vaccine strain.     

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.     

Infectious bronchitis virus serotypes in poultry flocks in Jordan

Infectious bronchitis virus (IBV) causes respiratory disease in chickens all over the world. IBV has many serotypes that do not confer cross protection against each other. Hemagglutination inhibition (HI) test has been used to determine the serotypes of IBV as a substitute to the more laborious virus neutralization test and the more sophisticated restriction endonuclease digestion or sequencing of the S1 gene. In Jordan, no previous studies have been carried out to determine the involvement of IBV in respiratory disease in chickens, or the serotypes of IBV that possibly exist. In this study, serum from different chicken flocks (n = 20) that suffered from respiratory disease were tested for IBV antibodies using commercial IBV antibody ELISA at time of the initial signs of the respiratory disease and repeated on serum samples from the same flocks 10–14 days later. ELISA titer for IBV increased in 14 out of 20 flocks (70%) after 10–14 days of the initial signs of the respiratory disease and this indicates a recent exposure to IBV. The second serum samples from these 14 flocks were further examined against a panel of five IBV antigens (Ark, Conn, DE-072, JMK, and Mass) by HI test to determine the serotype(s) of IBV they have been exposed to. The HI test results indicated that the exposure of some of these flocks were to Ark, DE-072, and Mass like serotypes. However, the HI titers against the antigens used in this study were relatively similar in 10 out of the 14 flocks (71%) and the serotype of IBV that these flocks were exposed to could not be determined and the possible causes of this are discussed.     

Variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens.

Twenty infectious bronchitis virus (IBV) field isolates obtained from commercial layer and broiler chickens in 1987 and 1988 were serotyped using the virus-neutralization (VN) test. Six different previously unrecognized variant serotypes were identified from a total of seven isolates from layer chickens. Only two isolates, both from Maine, were the same variant serotype. Variant serotypes also were recovered from layer flocks in Illinois and Washington and the province of Ontario, Canada. Two different variants were isolated from the same multi-age layer complex in Connecticut. Only one of 13 broiler chicken isolates was found to be a new variant serotype, that being from birds reared in Delaware. Cross-protection studies in specific-pathogen-free chickens indicated that vaccines containing the Holland, L-1, or Connaught strains of Massachusetts (Mass) combined with Arkansas produced a broader spectrum of immunity against challenge with the layer variants than Mass (Holland) alone or Mass (L-1) + Connecticut. All vaccines tested produced solid immunity (greater than or equal to 80% protection) against the broiler variant virus.     

腺胃病变型鸡传染性支气管炎病毒变异株的比较研究

腺胃病变型鸡传染性支气管炎病毒（ＩＢＶ）变异株Ｈ９５提纯样品经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ,证明Ｈ９５株单抗ＤＥ７和Ｍ４１株单抗６ＤＨ８均能识别Ｈ９５株５４０００蛋白多肽。采用单抗、多抗介导间接ＥＬＩＳＡ试验表明,Ｈ９５毒株能与抗ＩＢＶＭ４１Ｎ蛋白单抗６ＤＨ８和ＩＢＶＭ蛋白单抗ＭＣ发生反应,也能与抗Ｍ４１、Ｇｒａｙ、Ｈｏｌｔｅ、Ｔ、Ｈ５２、Ｎ１１５和分离株Ｃ９００１、ＤＬＺ９１１１的多抗血清反应,同时抗Ｈ９５株的４株单抗、多抗血清也能与上述毒株反应。卵磷脂酶Ｃ处理的Ｈ９５株的血凝活性能被相应的Ｈ９５株的单克隆抗体和高免血清所抑制,也能被Ｍ４１单抗和高免血清所抑制。通过ＲＴ－ＰＣＲ获得了Ｈ９５毒株的免疫原基因Ｓ１,经Ｓｏｕｔｈｅｒｎｂｌｏｔｉｎｇ和ＩＢＶ的Ｓ探针检测呈阳性。     

Serological and molecular characterization of three enteric isolates of infectious bronchitis virus of chickens

Three coronaviruses isolated from the intestines of laying chickens were partially characterized. Serological and molecular assays indicated that the enteric coronaviruses are infectious bronchitis virus (IBV) isolates. Although the three isolates were recovered from three unrelated chicken flocks, their RNase T1-resistant oligonucleotide fingerprints were almost identical. The three isolates were not neutralized by antisera specific to IBV serotype Connecticut, but their RNase T1-resistant oligonucleotide fingerprints closely matched the fingerprints of strain Conn-46, a Connecticut serotype. This and the co-fingerprinting data suggested that the three field isolates may have emerged from the Connecticut virus through mutation(s). The mutation(s) apparently involved the S1 protein gene that determines the virus serotype.     

Characterization of fowl adenoviruses isolated in Ontario and Quebec, Canada

Fowl adenoviruses (FAdV) are generally considered ubiquitous, but certain serotypes and strains are known to be associated with primary diseases, such as inclusion body hepatitis (IBH). Fifty-two FAdV isolates were collected from the provinces of Ontario and Quebec over a 4-year period. These 2 provinces have the largest poultry industries in Canada. Except for one virus, which originated from a guinea fowl, all other viruses were isolated from chicken samples. Most of these were from broilers, although some were from broiler breeders, and one was from layer pullets. Thirty-four isolates were from clinical IBH cases with the final laboratory diagnosis of IBH; however, for 18 isolates, the varied case diagnosis was seemingly unrelated to FAdV. All IBH-associated viruses had deoxyribonucleic acid (DNA) profiles compatible with FAdV species E (28 cases) or species D (6 cases), and the DNA fragment profiles of 26 species E viruses were indicative of serotype 8. Two viruses were serotype 6, as confirmed by virus neutralization. All species D viruses had a DNA profile similar to that of FAdV-2. The number of serotype 8 virus isolations has increased over the years, and by 2001 serotype 8 had become the dominant serotype in Ontario, and continues to be so. Moreover, this virus (FAdV-8) has shown a strong association with IBH.     

Detection of avian infectious bronchitis viral infection using in situ hybridization and recombinant DNA

A recombinant DNA probe with specificity for the 3' end of genomic RNA from the Ark 99 strain of infectious bronchitis virus (IBV) was found to hybridize with extracted RNA of three strains with the Ark serotype, as well as the Mass41, Holl52, Gray, JMK, Conn, Fla and SE17 strains of IBV. Viral infection was detected in the cytoplasm of chicken embryo kidney cells inoculated with Mass41, Ark99, SE17 or two recent field isolates of IBV using in situ cytohybridization and a biotinylated probe. In vivo infections were detected in individual cells of tracheas and lungs 2,4, and 6 days after inoculation of chicks with Mass41 and Ark99. In situ hybridization of Ark99 infected tissue sections using 32P-dATP labelled probe indicated that more viral replication was present in the trachea on day 4 than either days 2 or 6; whereas more viral RNA was found in the lungs on day 6 than days 2 or 4 after inoculation.     

Infectious bronchitis virus in testicles and venereal transmission

Even though males represent only 8%-12% of the birds of a breeder flock, their role in infectious bronchitis virus (IBV) dissemination is largely unknown. We first assessed the effect of IBV replication in the chicken testes. Ten-week-old males were inoculated with Arkansas (Ark) or Massachusetts (Mass) IBV virulent strains. Seven days postinoculation (DPI) IBV RNA was detected in testicles of 100% of M41- and in 96% of Ark-infected males. Marginal nonsynonymous variation was detected in spike (S) gene of the predominant population of IBV replicating in the testes compared to the S gene of the predominant population of viruses prior to inoculation. IBV M41 and Ark were detected in spermatogonia and Sertoli cells of testicles of infected roosters by immunofluorescence, without evident histopathological changes. We next assessed venereal transmission of IBV by artificially inseminating 54-wk-old hens either with semen from IBV-infected roosters or with IBV suspended in na?ve semen. IBV RNA was detected in the trachea of all hens inseminated with IBV-spiked semen and in 50% of hens inseminated with semen from IBV-infected males. The egg internal and external quality was negatively affected in hens inseminated with semen containing IBV. These results provide experimental evidence for IBV venereal transmission.     

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 and characterization of a novel antigenic subtype of infectious bronchitis virus serotype DE072.

Three infectious bronchitis virus (IBV) isolates, CU82792, CU82805, and CU82808, were recovered from sentinel chickens placed with three different layer flocks of a large commercial poultry farm in New York State. The three isolates were classified as members of the DE072 serotype on the basis of 1) their S1 genes could be amplified with only a modified primer designed for the DE072 serotype and 2) their restriction fragment length polymorphism patterns, after digestion with endonucleases HaeIII, BstyI and XcmI, were indistinguishable from that of DE072 virus. Additional characterization of one of the isolates, CU82805, revealed that its S1 gene bears approximately 96% identity at the nucleotide level and 94% identity at the amino acid level with DE072. Yet, in an in vitro reciprocal virus neutralization test, only a one-way neutralization was observed, i.e., antiserum to CU82805 neutralized DE072, whereas CU82805 was not neutralized by DE072 antiserum. Implications of these findings with regard to IBV diagnosis and immunization are discussed.     

鸡传染性支气管炎病毒沈阳分离株生物学特性及其免疫原S1基因的克隆与序列分析

本研究对分离自沈阳地区的一株传染性支气管炎病毒（ＳＹ毒株）进行了生物学特性的研究,同时成功地对其免疫原Ｓ１基因进行了ＲＴ－ＰＣＲ扩增、克隆与序列分析。 通过电镜观察、动物回归试验、血凝特性研究等试验验证分离自沈阳地区的ＳＹ毒株确实为一株传染性支气管炎病毒。气管环组织培养交叉中和试验结果表明,分离株ＳＹ株不同于参考毒株澳大利亚Ｔ、Ｈ５２、Ｍ４１,且不同于国内其它流行株ＨＤ、ＨＢ、ＸＢ、ＤＢ等,是一个新的变异株。 利用ＩＢＶ Ｓ１基因特异性寡聚核苷酸引物,经ＲＴ－ＰＣＲ扩增ＳＹ毒株的Ｓ１基因,得到预期的约１．７Ｋｂ片段;并将扩增所得ｃＤＮＡ插入克隆质粒ｐＵＣ１９的ＥｃｏＲⅠ／ＢａｍＨⅠ位点,在大肠杆菌ＤＨ５ａ中实现目的基因的克隆。经限制性核酸内切酶分析及ＰＣＲ鉴定,证实为阳性重组质粒,利用末端双脱氧链终止法对其测序,得到Ｓ１基因全长１６４０ｂｐ,包括整个开放阅读框。通过序列分析软件ＤＮＡＳＩＳ、ＰＲＯＳＩＳ、ＭＥＧＡ等软件对Ｓ１基因核苷酸序列及推导的氨基酸序列进行分析,结果表明：分离株ＳＹ与７株参考株和国内流行株ＨＤ株相比,无论是核苷酸序列同源百分率还是氨基酸序列同源百分离都较低,均未达到８０％,这就提示我们ＳＹ毒     

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.     

Phenotypic and molecular characterization of isolates of Ornithobacterium rhinotracheale from chickens and pigeons in Taiwan

Forty Ornithobacterium rhinotracheale (ORT) strains were isolated from 28 chickens and 12 pigeons for the first time in Taiwan. All isolates reacted positively in the p-nitrophenyl-beta-D-galactopyranoside (PNPG) and oxidase tests, showing an API 20NE identification system biocode 0-0-2-0-0-0-4. All the pigeon isolates and 85.7% (24 of 28) of the chicken isolates belonged to serotype A. Compared to the ORT ATCC 51464 strain, 14.3% (4 of 28) of chicken isolates and 58.3% (7 of 12) of pigeon isolates showed smaller colonies after 72 hr incubation. Most of the chicken isolates (22 of 28), but none of the pigeon isolates, could agglutinate chicken and pigeon red blood cells. There appears to be a correlation that ORT isolates with a larger colony size tend to be more able to agglutinate red blood cells than the ORT isolates with a smaller colony size. A majority of isolates was sensitive to amoxicillin, ampicillin, ceftiofur, penicillin, and oxytetracycline. The 16S ribosomal RNA (rRNA) sequences of 23 Taiwanese ORT isolates showed high identity (98%-100%) to sequences in GenBank. Phylogenetic analysis of these sequences showed that pigeon isolates formed a distinctive cluster, while chicken isolates and all other 16S rRNA sequences obtained from GenBank belonged to another two clusters. The results indicate that pigeon ORT isolates are different from most chicken isolates in regard to a number of phenotypic and molecular traits.     

鸡肾型IBV分离株M基因的克隆与特性分析

依据NCBI所登录的鸡传染性支气管炎病毒的M基因序列设计了一对引物,应用TRIzol试剂盒对8个肾型鸡传染性支气管炎病毒陕西地方分离株进行总RNA的提取,以所得到的RNA为模板,用RT-PCR对M基因进行扩增;其目的条带回收提纯后,与PMD18-T克隆载体进行连接,转化到DH5α宿主菌,并经药物抗性筛选、PCR及酶切鉴定,将所筛选鉴定出的阳性质粒进行测序,最后对测序结果进行分析。结果表明：陕西地方8个毒株（BJ2、FF2、YL2、B01、G5、YX、WH、WG）M基因长约为650 bp,其中YX、WH株本身无BamH 1酶切位点。WH与其它分离株的核苷酸同源性为91.8%～92.6%,而其它的分离株间的同源性为98.8%～99.8%。8个毒株与参考株的核苷酸同源性为74.9%～99.8%。其N端90 aa肽段与对照株相比,不同之处表现为高亲水性氨基酸取代低亲水性氨基酸,这会使其具有更好的抗原性。