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1.
一步法RT—PCR检测禽呼肠孤病毒方法的建立与应用（摘要） 总被引：2，自引：0，他引：2
禽呼肠孤病毒 (ＡＲＶ)是呼肠孤病毒属的成员 ,在临床上主要引起鸡病毒性关节炎 /腱鞘炎、吸收障碍综合症等多种疾病。禽呼肠孤病毒感染导致鸡群的饲料报酬降低、生产能力下降、屠宰废弃率高和引起免疫抑制而造成其它疾病的并发或继发感染 ,使鸡群死亡率升高 ,其危害相当严重。目前 ,对于禽呼肠孤病毒的检测已建立起了常规的病毒分离鉴定、ＥＬＩＳＡ和荧光抗体检测方法 ,近年来 ,我们也建立了ＲＴ ＰＣＲ、半套式ＰＣＲ和核酸探针等技术。本研究的目的是建立一种更加简便的一步法ＲＴ ＰＣＲ检测禽呼肠孤病毒的技术。禽呼肠孤病毒为双链Ｒ… 相似文献
2.
我国鸡群免疫抑制病感染现状与防控 总被引：1，自引：0，他引：1
一、鸡群免疫抑制病的感染现状
1．经对鸡群中禽网状内皮增生病病毒、鸡传染性贫血病毒、禽呼肠孤病毒3种病毒感染的血清学调查发现,绝大多数鸡场中鸡传染性贫血病毒、禽呼肠孤病毒的母源抗体阳性率都非常高,在经过1～3周母源抗体消失后不久,从5周龄开始出现自然感染引发的抗体阳性率逐渐升高;到20周龄时,不论什么遗传品系和地理分布,绝大数鸡群中鸡传染性贫血病毒、禽呼肠孤病毒的抗体阳性率可达到95％～100％。 相似文献
3.
番鸭呼肠孤病毒非结构基因的克隆和序列分析 总被引：8，自引：1，他引：8
参考GenBank禽呼肠孤病毒(Avian Reovirus,ARV)和番鸭呼肠孤病毒(Muscovy Duck Reovirus,MDRV)非结构基因(NS)序列设计合成一对引物,对番鸭呼肠孤病毒S14和C4株NS基因进行RT-PCR扩增,克隆到pMD18-T载体中,并对克隆产物进行酶切鉴定和测序;番鸭呼肠孤病毒NS基因由1 291 bp核苷酸组成,与禽呼肠孤病毒NS基因相比,在非编码区第1155位少一个碱基,本文第一次证实1291bp是番鸭呼肠孤病毒NS基因特有的长度;番鸭呼肠孤病毒S14和C4株NS基因的5'末端和3'末端分别为5‘GCTTTT和TCATC-3',是禽类呼肠孤病毒基因末端特有的碱基序列,S14和C4株NS基因的的有效阅读框(24～1127bp)编码367个氨基酸组成的蛋白,分子量约为40kDa;番鸭呼肠孤病毒S14和C4株NS蛋白等电点分别是7.3和7.0,GC含量分别为54.26%和53.71%,番鸭呼肠孤病毒S14和C4株NS基因间核苷酸同源性为99.3%,仅有4个氨基酸差异,S14和C4与法国番鸭呼肠孤病毒89026株NS基因核苷酸同源性分别为87.8%和87.9%,与鸡关节炎病毒S1133 NS基因同源性分别为79.0%和79.3%;进化树分析表明本研究中的两株番鸭呼肠孤病毒非结构基因(NS)与番鸭呼肠孤病毒的亲缘关系比禽呼肠孤病毒近的多,建议番鸭呼肠孤病毒应归属为正呼肠孤病毒属第二个亚群中不同于禽和内尔森贝海湾呼肠病毒独立基因群. 相似文献
4.
禽呼肠孤病毒（Avianreovirus,ARV）属于呼肠孤病毒科,呼肠孤病毒属的成员,是一种能引起关节炎、心包炎、肠炎、肝炎、法氏囊及胸腺萎缩、骨骼异常、产蛋下降和营养吸收障碍等疾病的双链RNA病毒。1957年Olson等首次分离到禽呼肠孤病毒,此后英国、美国、 相似文献
5.
将增殖的禽呼肠孤病毒S1733毒株进行浓缩,然后进行蔗糖梯度纯化,测定纯化后病毒的浓度,按每只鼠100 μg的病毒用量免疫BALB/c小鼠,免疫5次后取其脾细胞与骨髓瘤细胞SP2/0 按5∶1进行融合。对融合后的杂交瘤细胞进行筛选,阳性孔经3次有限稀释法克隆。通过间接ELISA方法测定抗体效价,并通过Western blotting、Dot-ELISA、直接免疫荧光和中和反应等方法对2株单克隆抗体特性进行检测。试验结果表明,纯化的病毒含量为43 mg/mL,用纯化的病毒免疫BALB/c小鼠后与骨髓瘤细胞融合,通过克隆筛选成功获得2株能稳定传代并分泌抗禽呼肠孤病毒单克隆抗体的杂交瘤细胞株SF6-3K3和SB2-1K3。2株单克隆抗体的腹水经间接ELISA测定效价达105以上,其抗体为IgG1亚类,特异性试验表明其与其他病毒株没有交叉反应,具有良好的特异性,并且这2株单克隆抗体没有中和禽呼肠孤病毒的能力,具有识别禽呼肠孤病毒的能力,可以用于禽呼肠孤病毒的特异性检测。 相似文献
6.
用反转录聚合酶链反应检测禽呼肠孤病毒的研究 总被引：7，自引：0，他引：7
本文报告了建立反转录聚合酶链反应（ Ｒ Ｔ Ｐ Ｃ Ｒ）检测禽呼肠孤病毒（ Ａ Ｒ Ｖ）的方法,根据禽呼肠孤病毒 Ｓ１１３３ 毒株 Ｓ１ 基因序列,设计合成两对引物,用 Ｒ Ｔ Ｐ Ｃ Ｒ 技术对 ６ 株禽呼肠孤病毒国际标准株进进行了检测。结果两对引物对 ６ 株 Ａ Ｒ Ｖ 均可扩增出与预期大小相符 ５３２ｂｐ 和 ４３５ｂｐ 的 Ｒ Ｔ Ｐ Ｃ Ｒ 产物,而对其它 ６ 种禽病病原核酸的扩增结果均为阴性;该 Ｒ Ｔ Ｐ Ｃ Ｒ 可以检测出 １ｐｇ 的 Ａ Ｒ Ｖ Ｒ Ｎ Ａ 模板。 相似文献
7.
将禽呼肠孤病毒ZJS株细胞毒(弱毒)在鸡胚成纤维细胞(CEF)上连续传代,对其CI、C2、C3代种毒进行了无菌和支原体污染检验,对C5、C8和C10代种毒进行了外源病毒污染检验,对C3、C8和C10代种毒还进行了特异性鉴定,以确定禽呼肠孤病毒ZJS株细胞毒(弱毒)作为种毒的可行性.结果显示,禽呼肠孤病毒ZJS株细胞毒(... 相似文献
8.
中国禽(番鸭)呼肠孤病毒YB株S4基因序列分析 总被引：5，自引：0，他引：5
应用非免疫番鸭胚增殖中国禽(番鸭)呼肠孤病毒YB分离株,用LSTRIAZOL提取病毒RNA,反转录-聚合酶链反应(RT-PCR)扩增中国禽(番鸭)呼肠孤病毒YB分离株S4基因节段cDNA.将S4cDNA克隆到PMDl8-T载体上,并进行了鉴定和核苷酸序列测定.序列分析结果,克隆的S4cDNA共1 124bp,包括非编码区和完整的阅读框架.分子进化系统分析表明该毒株与禽呼肠孤病毒(鸡呼肠孤病毒)的亲缘关系较远,DRV-YB与DRV-89330同缘率为93.3%. 相似文献
9.
通过对疑似感染鹅呼肠孤病毒的病鹅进行实验室检测,有利于对该病的早期确诊,及时采取防控措施。取有典型病理变化的病鹅肝或脾脏组织,采用RT-PCR检测鹅呼肠孤病毒和禽流感病毒,采用I群禽腺病毒通用型二温式PCR方法检测腺病毒,对呼肠孤病毒阳性者进行病毒扩增片段测序。结果鹅呼肠孤病毒测序结果为阳性。I群禽腺病毒和禽流感病毒检测无特异性条带,为阴性。本病例确诊为鹅新型呼肠孤病毒感染。 相似文献
10.
3种禽类呼肠孤病毒血清学相关性及致细胞病变差异分析 总被引：1，自引：0，他引：1
本研究旨在研究3种不同疾病型禽类呼肠孤病毒间的抗原性关系及病毒的培养特性。作者通过血清中和试验测定了禽呼肠孤病毒(ARV S1133株)、番鸭呼肠孤病毒(MDRV 9710株)、新型鸭呼肠孤病毒(NDRV NP01株)3种禽类呼肠孤病毒的血清学相关性,统计抗原相关性R值;并应用部分禽胚原代细胞及哺乳动物传代细胞对这3种病毒的培养特性进行了初步研究。结果表明,3种病毒株之间的R值很小,抗原相关性较低;三者具有广泛的细胞亲嗜性,能在多种细胞中增殖,并产生细胞病变,但病毒致细胞病变特征有所差异,ARV和NDRV均以巨融合为主,而MDRV则以细胞圆缩坏死为主。上述结果表明导致禽类不同疾病的ARV、MDRV和NDRV三者之间的抗原相关性较低,病毒的细胞培养特性也不同,细胞病变类型的差别提供了一种初步鉴别禽类呼肠孤病毒的方法。 相似文献
11.
中国禽(番鸭)呼肠孤病毒分离株S1基因全序列分析 总被引：14，自引：0，他引：14
采用RT-PCR技术,分别以DRV-YH、DRV-YJL两株中国禽(番鸭)呼肠孤病毒RNA为模板,扩增了S1全基因的cDNA片段.将S1 cDNA克隆到T载体后进行序列测定,测序结果表明所扩增的cDNA片段长1643个核苷酸,包含了完整的S1基因的三个开放阅读框架(ORF1,ORF2和ORF3)和基因两端的非编码区.核苷酸序列比较分析结果表明:DRV-YH与ARV-S1133,176分别有6个,10个核苷酸的差异,DRV-YJL与ARV-S1133,176分别有8个,12个核苷酸的差异,DRV-YH与DRV-YJL有4个核苷酸的差异. 相似文献
12.
The antigenic interrelatedness of 3 clone-purified turkey reoviruses (NG-Turkey, 82-88, and NC-TEV) to each other and to 4 clone-purified chicken reoviruses (S1133, Co8, Fahey-Crawley, and avian type 2) was determined in reciprocal cross-neutralization tests, using polyclonal antisera and the plaque-reduction technique. The morphologic features of plaques formed under agar were studied for all 7 reoviruses, and size comparisons for turkey vs chicken isolates were made. All 3 turkey reoviruses (with the exception of NG-Turkey vs Fahey-Crawley chicken reovirus) formed plaques significantly (P less than 0.05) smaller than plaques produced by their chicken counterparts. The 3 turkey reoviruses were closely related to each other and to chicken reovirus CO8. The antigenic differences between turkey reoviruses 82-88 and NC-TEV and chicken reovirus S1133 were slight (minor subtype); however, the latter and NG-Turkey were serotypically distinct. The NG-Turkey and 82-88 turkey reoviruses were more related (minor subtype) to the Fahey-Crawley and avian type 2 chicken reoviruses, than was NC-TEV turkey reovirus (major subtype). 相似文献
13.
犬呼肠孤病毒3型（AMMS株）的分离与鉴定 总被引：3，自引：0，他引：3
从 １ 只临床表现呼吸道症状的濒死病犬肺脏分离到 １ 株病毒,经理化特性、血凝性、核酸型、血凝抑制试验和电镜观察,鉴定为犬呼肠孤病毒 ３ 型。这是国内首次分离到此病毒。 相似文献
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15.
The open reading frame of the S3 segment encoding the sigma2 protein of four turkey reovirus field isolates was analyzed for sequence heterogeneity. The turkey reoviruses we present here have a 97% amino acid identity to turkey NC 98. The S3 nucleotide and amino acid sequence similarity was < or =61% and 78%-80%, respectively, when compared to the chicken reovirus isolates. Comparison of amino acid sequences from chickens and turkeys with that of a duck isolate revealed a 53% and 55% similarity, respectively. Phylogenetic analyses, based on both nucleotide and amino acid sequence, resulted in three major groups among the avian reoviruses; these groups were clearly separated by species. The results of this study provide further evidence, based on the deduced sigma2 sequence, that turkey reoviruses form a distinct, separate group relative to chicken and duck isolates. In addition, as a result of the limited sequence identity with their avian counterparts, turkey reoviruses could potentially be considered a separate virus species within subgroup 2 of the Orthoreovirus genus. 相似文献
16.
J. MEANGER R. WICKRAMASINGHE CE ENRIQUEZ GE WILCOX 《Australian veterinary journal》1997,75(6):428-432
Objectives To assess the efficacy of the vaccination procedure and the effect of the transfer of maternal antibodies to progeny chickens on reovirus pathogenicity.
Design To vaccinate chickens and challenge progeny chickens with high doses of homologous and heterologous viruses.
Procedure High doses of reovirus strains RAM-1, 1091 and 724 were used to induce tenosynovitis lesions. High doses were produced by concentration of viruses grown in cell culture. Then similar doses of viruses were used to challenge immunised chickens progeny.
Result Vaccination of breeding hens with the RAM-1 strain of avian reovirus, which resulted in the passive transfer of neutralising antibody to progeny chickens, completely prevented the development of tenosynovitis in 80% of progeny chickens infected with the homologous virus. Even though multiple injection of hens resulted in broadening of the normal type-specificity of the neutralising antibody response against heterologous serotypes of avian reovirus, only marginal protection against strains of two heterologous serotypes of avian reovirus was obtained.
Conclusions A model for assessing the efficacy of vaccination against avian reovirus strains on clinical signs such as tenosynovitis was developed that overcome the normal low virulence of Australian strains of avian reovirus. Breeding hens can be immunised with Australian strain of avian reovirus with passive transfer of antibody via the yolk to the progeny chickens. Although the neutralising antibody response to three injections of inactivated virus decreased the specificity of the neutralising antibody response against antigenically heterologous strains of avian reovirus, the protective immunity appeared to retain type-specificity. 相似文献
Design To vaccinate chickens and challenge progeny chickens with high doses of homologous and heterologous viruses.
Procedure High doses of reovirus strains RAM-1, 1091 and 724 were used to induce tenosynovitis lesions. High doses were produced by concentration of viruses grown in cell culture. Then similar doses of viruses were used to challenge immunised chickens progeny.
Result Vaccination of breeding hens with the RAM-1 strain of avian reovirus, which resulted in the passive transfer of neutralising antibody to progeny chickens, completely prevented the development of tenosynovitis in 80% of progeny chickens infected with the homologous virus. Even though multiple injection of hens resulted in broadening of the normal type-specificity of the neutralising antibody response against heterologous serotypes of avian reovirus, only marginal protection against strains of two heterologous serotypes of avian reovirus was obtained.
Conclusions A model for assessing the efficacy of vaccination against avian reovirus strains on clinical signs such as tenosynovitis was developed that overcome the normal low virulence of Australian strains of avian reovirus. Breeding hens can be immunised with Australian strain of avian reovirus with passive transfer of antibody via the yolk to the progeny chickens. Although the neutralising antibody response to three injections of inactivated virus decreased the specificity of the neutralising antibody response against antigenically heterologous strains of avian reovirus, the protective immunity appeared to retain type-specificity. 相似文献
17.
禽呼肠孤病毒C-98分离株L1基因的克隆与序列分析 总被引：2，自引：2，他引：0
参考禽呼肠孤病毒S1133株（GenBank）L1基因序列设计3对特异性引物,采用RT-PCR方法对禽呼肠孤病毒内蒙古分离株C-98的L1基因进行了克隆和序列测定。结果表明：获得的C-98 L1基因全长3959 bp,其中包括21 bp的5’非编码区和56 bp的3’非编码区,阅读框位于5’端第22位核苷酸与3’端第3903位核苷酸之间。将C-98株与国内外禽呼肠孤病毒群和哺乳动物呼肠孤病毒群的不同分离株进行序列比较,结果显示：C-98与台湾地区分离株919和美国分离株1733、2408、S1133亲缘关系最近,其核苷酸同源性分别为99.8%、99.7%、99.7%、99.4%;与哺乳动物呼肠孤病毒L3基因核苷酸及其编码的氨基酸同源性较低,为43%～45%。 相似文献