Strain specific antibodies revealed by immunoabsorption studies with avian infectious bronchitis virus

Rabbit antisera prepared against the Massachusetts 41 (M41) strain of avian infectious bronchitis virus (IBV) and absorbed with chick embryo immunoabsorbent produced multiple precipitin lines in immunodouble-diffusion (IDD) tests with homologous or heterologous strains of virus. These precipitin lines were all removed by absorption with concentrated M41 virus preparations, but repeated absorption with concentrated, purified preparations of IBV strains: T, Holte, Connecticut, Beaudette or H120 failed to remove all precipitin lines produced to M41 virus, although all those to the heterologous viruses were removed. The remaining line(s) produced with M41 virus by sera absorbed with different heterologous viruses showed identity in IDD tests and was associated with the surface projections of the virus.     

Heparan sulfate is a selective attachment factor for the avian coronavirus infectious bronchitis virus Beaudette

The avian coronavirus infectious bronchitis virus (IBV) strain Beaudette is an embryo-adapted virus that has extended species tropism in cell culture. In order to understand the acquired tropism of the Beaudette strain, we compared the S protein sequences of several IBV strains. The Beaudette strain was found to contain a putative heparan sulfate (HS)-binding site, indicating that the Beaudette virus may use HS as a selective receptor. To ascertain the requirements of cell-surface HS for Beaudette infectivity, we assayed for infectivity in the presence of soluble heparin as a competitor and determined infectivity in mutant cell lines with no HS or glycosaminoglycan expression. Our results indicate that HS plays a role as an attachment factor for IBV, working in concert with other factors like sialic acid to mediate virus binding to cells, and may explain in part the extended tropism of IBV Beaudette.     

Study of protection by recombinant fowl poxvirus expressing C-terminal nucleocapsid protein of infectious bronchitis virus against challenge.

A stable recombinant fowl poxvirus (rFPV) expressing the C-terminal region (119 amino acids) of the nucleocapsid (N) protein of an infectious bronchitis virus (IBV) strain Ch3 was constructed by inserting the coding sequence within the thymidine kinase gene of fowl poxvirus (FPV) by homologous recombination. The N protein was expressed under control of the vaccinia virus promoter P7.5 in chicken embryo fibroblast cell cultures as seen in immunofluorescence assay and in rFPV-inoculated specific-pathogen-free (SPF) chickens by detecting antibodies with enzyme-linked immunosorbent assay (ELISA). A homologous IBV strain (Ch3) and two heterologous IBV strains (Ch5 and H4) were used to inoculate SPF chickens in a challenge to examine the protective efficacy of the rFPV. When the chickens were challenged with IBV Ch3 or Ch5, the control birds had respiratory signs of infections bronchitis, whereas all the vaccinated birds were clinically normal although low levels of the IBV infection were detected by a differential ELISA. In contrast, in the chickens challenged with IBV H4, all control birds and vaccinated birds suffered from the highly lethal IBV H4 infection. Our results suggest that the C-terminal 119 amino acid of the nucleocapsid expressed by FPV is a host-protective antigen and may induce cross-protective immunity against illness among some IBV strains.     

重组鸡α-干扰素的表达及其生物活性的初步研究

为获得重组鸡α-干扰素并对其进行生物活性研究,本试验对重组大肠杆菌进行诱导表达,收集重组鸡α-干扰素包涵体后进行复性纯化及免疫印迹试验,并通过测定病毒EID₅₀,在接种H9亚型禽流感病毒、新城疫病毒和传染性支气管炎病毒的鸡胚中进行干扰素抗病毒活性试验,运用微变量细胞病变抑制法进行重组鸡α-干扰素抗病毒效价的测定。结果显示复性后重组鸡α-干扰素的免疫印迹试验成功获得了预期条带,在鸡胚中重组鸡α-干扰素具有显著的抗H9亚型禽流感病毒、新城疫病毒和传染性支气管炎病毒等活性作用。在CEF/VSV细胞系上测定重组鸡α-干扰素的抗病毒效价为1.5×2¹⁰ U/mg左右,高浓度重组鸡α-干扰素具有一定的细胞毒性。结果表明重组鸡α-干扰素蛋白具有免疫原性和抗原性,在鸡胚内具有较好的抑制或杀灭H9亚型禽流感病毒、新城疫病毒和传染性支气管炎病毒的效果,呈广谱性,抗病毒效价较高,为下一步抗病毒制品的创制奠定基础。     

Use of a constant-virus diluting-serum microneutralization technique for the detection and quantification of infectious bronchitis virus antibodies

A constant-virus diluting-serum microneutralization test (CVMNT) for avian infectious bronchits virus (IBV) was evaluated for both reliability and repeatability. The virus used in the assay was a chick kidney (CK) cell-adapted strain, the Beaudette strain (IBV 42). Sera tested were from 24-week-old broiler-breeder chickens that had been vaccinated 3 times from a combination vaccine of Newcastle disease virus (NDV) and IBV. Test results were not repeatable or comparable when the same sera were tested on different days, but test results were repeatable and comparable when the sera were tested on the same day. Differences in virus titer at the different times that tests were performed appeared to cause the variation in test results. A comparison was made between the CVMNT and a constant-serum diluting-virus microneutralization test (CSMNT). The CVMNT was able to detect differences in flock antibody titers that the CSMNT could not.     

Phylogenetic analysis of S1 gene of infectious bronchitis virus isolates from China

Between 2006 and 2009, seven strains of infectious bronchitis (IB) virus (IBV) were isolated from vaccinated chicken flocks on different chicken farms in China. The pathogenic characters of seven IBV strains were assessed. Each of the seven strains was infective to the test chickens and could induce an immune response. The results from chicken embryo cross-neutralization assays showed that these strains were antigenically distinct from classic IBV strains of H120, M41, Conn, and Gray. Compared to H120 vaccine strain, point mutation, short insertion, and deletion occurred at many positions in the S1 protein of the seven strains. Five of the seven strains had the motif (HRRRR), which was identical to that of the epidemic IBV strains in China. Two new motifs (HRLRR and RRIRR) emerged in the isolated strains. The homology of the nucleotide and amino acid sequences of the S1 gene among the seven isolates was 81.7%-99.7% and 79.0%-99.4%, respectively. These seven strains were also genetically different from the vaccine strains and non-China IBV strains but closely related to large numbers of Chinese strains. The seven isolates and 36 reference IBV strains were clustered into six distinct groups (I-VI). The seven strains were categorized into groups I, II, and III, forming a big phylogenetic branch, which is closely related to Chinese IBVs, whereas the vaccine strains belonging to group VI are genetically distant from groups I, II, and III. The results from this study indicate that different IBV strains cocirculate in the chicken population in China.     

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     

Observations on the preparation and stability of infectious bronchitis virus hemagglutination antigen from virus propagated in chicken embryos and chicken kidney cell cultures

A total of 166 infectious bronchitis virus (IBV) hemagglutination (HA) antigen preparations were made during a 30-month period from allanto-amnionic fluid (AAF) from chicken embryos inoculated with 10 different IBV strains (Mass 41, Conn 46, H52, Florida 18288, Ark 99, JMK, T, Holte, EF, SE17). Antigens were prepared by inoculating 9- or 10-day-old embryos with 10(5.0) to 10(6.5) EID50 IBV, harvesting AAF after a 30-hour-postinoculation incubation, and phospholipase C (PLC) treatment of virus concentrated by pelleting from the AAF. Longer (48 hr) incubation times were tried, but production of H52 HA antigen was successful only from AAF harvested after 30 hours of incubation. AAF from JMK-infected embryos had lower infectivity titers and frequently yielded lower HA antigen titers than the other strains. The treatment of AAF with fluorocarbon did not enhance or diminish HA activity but did yield clearer antigens by removing extraneous material. Polyethylene glycol precipitation of virus was an acceptable alternative to pelleting virus at 39,000 X g. Inactivation of IBV with 0.1% betapropiolactone did not affect HA activity, whereas inactivation with 0.1% formalin caused a marked reduction in HA titer. Different buffer formulations including phosphate, tris, or HEPES were tried to optimize the conditions for PLC treatment of virus concentrate, but there were no apparent differences in the antigens prepared in the different buffers. The HA antigen preparations were stored and were stable at 4 C. Antigen titers of greater than or equal to 64 after storage for 20 months or longer were not uncommon. Addition of merthiolate as a preservative had no deleterious effect on HA activity. Antigen stability appeared to be enhanced by incorporating EDTA in buffer for virus pellet recovery and during enzyme treatment. Attempts to produce HA antigens from cell-culture-adapted virus propagated in chicken kidney cells were less satisfactory. An acceptable HA antigen was prepared from only two (Mass 41, SE17) of the seven strains that were tried. Virus propagation in chicken embryos is the better method of the two for IBV HA antigen production. Aside from the need to concentrate virus and treat the concentrate with PLC, there appeared to be considerable latitude in the procedures that can be used to make acceptable IBV HA antigens.     

核酸探针检测鸡传染性支气管炎病毒方法的建立及应用

以地高辛(DIG)标记鸡传染性支气管炎病毒(IBV)pol基因的保守片段制成核酸探针,与IBV参考株、新城疫病毒、传染性法氏囊病病毒、禽流感病毒、正常鸡胚尿囊液及正常鸡肾组织等的RT-PCR产物进行斑点杂交,以检测探针的特异性,结果该探针仅与IBV毒株的RT-PCR产物杂交呈阳性,与对照病毒和组织的RT-PCR产物杂交呈阴性.敏感性试验显示,探针最低能检出约3.4pg的IBV RT-PCR产物.用该方法检测了38份疑似IBV临床病料,31份阳性;而用RT-PCR法扩增IBV S2基因确诊为阳性的只有29份.对人工接种IBV H52弱毒苗鸡咽喉和肛门拭子32份进行检测,检出15份阳性.结果表明,利用DIG探针检测IBV的RT-PCR产物,特异性和敏感性强,可重复,能克服RT-PCR非特异性反应和探针Northern杂交的不稳定性.     

IBV安徽分离株AH1-99M基因克隆及序列分析

利用RT-PCR技术成功扩增出IBV分离株AH1-99 M基因全长cDNA,并将其克隆到pMD18-T载体上,获得该分离株M基因的重组质粒.序列分析结果表明,该分离株M基因全长共678个核苷酸,编码225个氨基酸;同其他IBV的核苷酸序列的同源性为87.2%～92.5%,氨基酸的同源性为89.8%～95.1%;在IBV M基因核苷酸进化关系树中,AH1-99株M基因与H120、M41、Beaudette以及多数国内分离株亲源关系较近.     

鸡传染性支气管炎病毒SC株N基因序列测定与同源性分析

从我国四川省一疑似鸡传染性支气管炎(Avianinfectious bronchitisvirus,IB)的雏鸡病料中成功分离到一株鸡传染性支气管炎病毒(Infectious Bronchitis Virus,IBV),命名为SC。病毒经鸡胚传代、血凝试验监测和负染电镜检查证实为IBV。自接毒鸡胚尿囊液中提取RNA后应用反转录-聚合酶链反应(RT-PCR)扩增得到了IBVSC株mRNA6 cDNA(编码N蛋白)。应用DNAstar5．06,Clustal1．8分析软件将克隆测序的N蛋白基因与Genbank中11株国内外参考毒株进行序列比较分析和同源性分析,发现IBV SC株变异独特,明显不同于国内外参考毒株。     

Preparing hemagglutinating antigen from isolates of infectious bronchitis virus

The hemagglutinating (HA) activity of 14 strains of infectious bronchitis virus (IBV) was investigated. The optimal conditions for IBV antigen preparation include inoculation of 10- or 11-day-old specific pathogen-free embryonated eggs and incubation for 30 hours at 37 C. Embryos were inoculated via the allantoic cavity with 0.1 ml of a low embryonic passage of the virus (10(7) to 10(8) EID50/ml). Allantoic fluid was harvested and pooled, and a 100-fold concentration of virus particles was achieved by centrifugation for 3 hours at 30,000 x g. Virus pellets were resuspended in Tris-hydrochloride buffer containing 3 units of phospholipase-C (type-1) enzyme/ml and incubated for 2 hours at 37 C. All IBV strains tested demonstrated positive HA activity with chicken red blood cells. The antigen was stored in liquid state or lyophilized at 4 C.     

鸡传染性支气管炎病毒CQ/01/2004株的分离与鉴定

2004年从重庆某肉用鸡场疑似鸡传染性支气管炎的病鸡中采集病料，按常规处理后接种9～10日龄鸡胚，通过鸡胚连续传代培养3代，并对该分离毒株的鸡胚致病性、血凝性和NDV的干扰特性进行检测．同时进行了动物回归试验。结果表明，该分离株具有IBV感染特征，可使鸡胚胚体出血、蜷缩、矮化；该分离毒株无直接血凝性，对NDV有明显的干扰作用；动物回归试验中有75％的感染鸡在10d内发病或死亡。剖检病死鸡可见肾苍白、肿胀，肾小管内充塞大量尿酸盐，支气管有出血点、有大量粘液。采用反转录．聚合酶链式反应（RT-PCR）技术对CQ／01／2004的纤突蛋白S1基因进行扩增、克隆和序列测定，结果表明该基因具有IBVSl基因的共有分子特征，将测序结果提交GenBank进行同源性检索，发现分离株CQ／01／2004和J株S1的同源性最高，核苷酸同源性为94％，氨基酸同源性为89．4％，与M41的核苷酸同源性为80．6％，氨基酸同源性为78．0％。试验结果表明，分离的病毒株CQ／01／2004为鸡传染性支气管炎病毒。     

Characterization of infectious bronchitis virus using monoclonal antibodies

Three monoclonal antibodies (MABs) reactive against two structural proteins--the nucleoprotein (NP) or the surface (S) protein--of avian infectious bronchitis virus (IBV) were produced and characterized. The MABs did not neutralize virus infectivity or inhibit hemagglutination. Their reactivity patterns with the homologous strain and eight heterologous strains of IBV were determined using the indirect immunoperoxidase test, the indirect immunofluorescent test, transfer-immunoblotting of separated proteins, and a dot-immunoblotting assay (DIA). Two MABs, NP- or S-protein-specific, reacted with all nine strains; one (NP-specific) reacted with only two strains. The two MABs reacting with all nine strains of IBV also detected 18 IBV field isolates of unknown serotype in the DIA. The MAB detecting only two strains did not react in the DIA. The diagnostic application of these MABs appears promising.     

检测鸡传染性支气管炎病毒抗体的三种血清学方法的比较

应用酶联免疫吸附试验（ＥＬＩＳＡ）、琼脂扩散沉淀试验（ＡＧＰ）和鸡气管环培养中和试验（ＳＮｉｎＴＯＣｓ）三种常规血清学方法对实验鸡血样的鸡传染性支气管炎病毒抗体进行了检测。从实验鸡血样的检测结果表明，ＥＬＩＳＡ和气管环中和试验的灵敏度较好，而ＡＧＰ的灵敏度相对较差，但三者均有较好的特异性。对田间送检血样的检测结果表明，ＥＬＩＳＡ与气管环中和试验、ＥＬＩＳＡ与ＡＧＰ的一致性均较好，而气管环中和试验与ＡＧＰ的一致性较差。ＥＬＩＳＡ效价与气管环中和试验效价的相关系数为０．８４     

Isolation and identification of four infectious bronchitis virus strains in China and analyses of their S1 glycoprotein gene

Between 2003 and 2005, four strains of infectious bronchitis virus (IBV) were isolated from the vaccinated chicken flocks in China. The results from chicken embryo cross-neutralization assays showed that all the four isolates were relative to strain A2 of IBV, which was isolated in 1996 in Beijing and related to strain 4/91. The S1 gene of the spike protein was amplified and sequenced. The nucleotide and amino acid sequence of the S1 gene had a similar degree of identity (88.98-99.28%) among the four Chinese IBV isolates. The identity of the S1 protein gene between the four Chinese IBV isolates and 14 strains of other IBVs varied from 70.06 to 81.59%. Phylogenetic analysis suggested that there are at least four groups of IBVs circulating in China and the disease outbreaks might have been caused by infection of multiple strains of IBV.     

鸡传染性支气管炎病毒HN99株S1基因的克隆与序列测定

根据基因库中收录的鸡传染性支气管炎病毒 (IBV)S1基因的序列 ,设计了一对引物并采用RT -PCR扩增了鸡传染性支气管炎病毒HN99株的S1基因 ,扩增产物进行了克隆、测序 ,获得了IBVHN99株S1基因片段 ,其大小为1 739bp(含前导序列 ) ,其核苷酸序列与H1 2 0、H52、M41、Gray、Holte的S1基因核苷酸序列同源性较低 ,分别为 79.1 %,79.2 %,77.3%,77.8%,79 .4%,有大量的点突变并伴有基因插入和缺失 ;IBVHN99株的S1基因推导的氨基酸与H1 2 0、H52、M41、Gray、Holte株氨基酸的同源性分别为80 .1 %,79.9%,79.5%,78.5%,78.5%,经S1基因系统进化分析 ,提示IBVHN99株与其它各毒株的亲缘关系较远 ,初步证实IBVHN99株为一新的IBV毒株