Pathogenicity of avian leukosis viruses

Three methods were used in attempts to obtain non-oncogenic avian leukosis virus for possible use as an immunoprophylactic agent for the control of lymphoid leukosis in chickens. These were: 1) isolate a nononcogenic virus from commercial breeder flocks experiencing very little or no lymphoid leukosis; 2) obtain a non-oncogenic recombinant from mixed infection of a strain with low oncogenicity, Rous-associated virus-60 (RAV-60), with RAV-1 or RAV-2 in cell culture; and 3) attempt to attenuate subgroup A avian leukosis virus by serial passage in avian cell culture. Of 43 isolates obtained from field sources, all were pathogenic except one, and its pathogenicity was questionable because of the low amount of virus tested. All 42 clones from mixed infection of highly oncogenic and poorly oncogenic virus and all clones passaged serially in cell culture were oncogenic.     

Heterophil function and resistance to staphylococcal challenge in broiler chickens naturally infected with avian leukosis virus subgroup J

Avian leukosis virus subgroup J has a high tropism for myeloid lineage cells and frequently induces neoplastic transformation of myelocytes. The impact of congenital avian leukosis virus subgroup J infection on the function of circulating heterophils and susceptibility to staphylococcal infection was investigated. Six-week-old broiler chickens negative for exogenous avian leukosis viruses or congenitally infected with avian leukosis virus subgroup J were inoculated intravenously with 10(6) colony-forming units of Staphylococcus aureus, and pre- and postinoculation heterophil function was assessed. All chickens developed a leukocytosis with heterophilia after inoculation, but total leukocyte and heterophil counts were significantly higher in leukosis-negative chickens than in virus-infected chickens. Tenosynovitis was more severe in leukosis-negative chickens, and 2/10 (20%) of the virus-infected chickens had no histologic evidence of tenosynovitis. Osteomyelitis in the tibiotarsus or tarsometatarsus developed in 5/10 (50%) of the chickens in each group. S. aureus was recovered from the hock joint of 6/10 (60%) of the chickens in each group. Heterophils from all chickens exhibited similar phagocytic ability pre- and postinoculation. Heterophils from virus-infected chickens exhibited less bactericidal ability preinoculation than did heterophils from leukosis-negative chickens. However, postinoculation bactericidal ability was similar in both groups. Avian leukosis virus subgroup J provirus was present in heterophils isolated from congenitally infected chickens. Heterophils isolated from broiler chickens congenitally infected with avian leukosis virus subgroup J exhibit no significant functional deficits, and infected and uninfected chickens exhibit similar susceptibility to staphylococcal infection.     

Testing and management of a specific pathogen free chicken flock with special reference to avian leukosis virus infections.

A specific pathogen free (SPF) chicken flock was reared in isolation under laboratory conditions during five years and continuously tested for presence of specified avian pathogens. The potential occurrence of avian leukosis virus (ALV) was most thoroughly examined. The RIF and neutralization tests were unequivocally negative. Radioimmunoassay was used for detecting the presence of the major protein (gs-a) of the group-specific antigen of avian onoorna viruses. This test seemed to he well suited for checking ALV infections in chicken flocks whereas the COFAL (complement fixation avian leukosis) test was considered unreliable for this purpose. Yolk and serum from SPF chickens were negative for anti-gs-a antibodies measured by the radioimmunoassay; immunized or naturally infected birds showed anti-gs-a amounts correlating with the neutralizing titre. Besides, the flock was regularly tested for presence of seven other contagious avian pathogens. There was no evidence of infection.SPF chicken flock; avian leukosis; laboratory diagnosis of avian leukosis virus infections.     

部分禽源生物制品外源病毒的检测

进行了部分禽用生物制品外源病毒检测的鸡胚检查法、细胞检查法和鸡检查法。鸡胚检查法应用SPF鸡胚检验;细胞检查法主要通过鸡红细胞吸附试验、禽白血病病毒ELISA和禽网状内皮组织增生症病毒IFA检验疫苗是否含有外源鸡红细胞吸附因子、禽白血病病毒和禽网状内皮组织增生症病毒,并提出疫苗检验的判定标准,为各兽用生物制品生产企业新城疫疫苗的外源病毒检验提供参考。     

散养淮南王土鸡J亚群禽白血病的诊断

对湖北省某农户散养的12周龄淮南王土鸡病鸡进行剖检,发现病鸡肝脏有白色肿瘤;心肌增厚,有疑似肿瘤赘生物;脾脏萎缩,切面呈暗色。用J亚群禽白血病病毒（ALV-J）特异性引物进行PCR检测,扩增出了约545 bp的条带,病鸡组织内含有ALV-J病毒核酸序列,表明该病例为ALV-J感染。     

我国禽白血病病毒生物学特点及其控制

综述了我国流行的禽白血病病毒（ALV）,总结了J亚群禽白血病病毒的流行病学、临床症状、组织病理学和病毒生物学特性,并阐述了ALV-J常用的分离鉴定方法,提出了控制和根除ALV的方法。     

单管套式PCR检测外源性禽白血病病毒

从贵州省4个养鸡场采集出现肿瘤病变或疑似肿瘤病料样本17份,以单管套式PCR方法检测禽白血病病毒长末端重复序列(LTR),15份病料均扩增出一条213 bp的DNA带,与预期扩增长度相符合,外源性禽白血病病毒的检出率达到88.2%.同时针对禽白血病病毒的囊膜糖蛋白基因(gp85)进行PCR扩增,在被检的17份病料中,7份检出J亚群特异性的DNA带(545 bp),占41.1%;9份检出A亚群特异性的DNA带(691 bp),占52.9%;其中2份病料同时检出J亚群与A亚群特异性的病原核酸.A、J亚群的PCR检出率低于LTR的套式PCR扩增.     

用ALV-J gp85单克隆抗体证明蛋鸡存在J亚群禽白血病

采用免疫组化法，对病理学初步诊断为蛋用型鸡J亚群白血病的自然发病鸡的肿瘤、骨髓、肝脏、脾脏、肾脏、肺脏、心脏、胰脏、输卵管、卵巢、腺胃、骨骼肌、大脑、坐骨神经，用特异性抗J亚群禽白血病病毒(ALV—J)囊膜糖蛋白gp85的单克隆抗体进行检测，待检的组织切片中均检出阳性抗原，免疫组化的研究结果与病理学诊断结果相一致。在国内外首次发现并报道蛋用型鸡J亚群禽白血病的自然病例。     

J亚群禽白血病毒gp85基因克隆表达与初步应用

在本实验室分离鉴定J亚群禽白血病病毒的基础上,用PCR方法扩增gp85基因,长度为921 bp的DNA片段,与J亚群禽白血病标准毒株序列比对同源率为96.4%。将该片段连接到pET28a表达载体上,构建了重组表达质粒pET28a-S1-Jgp85,对质粒测序后分析后,转化大肠杆菌BL21(DE3),用IPTG诱导表达,经SDS-PAGE分析,表达出约为40 kD的融合蛋白。Western blot结果表明,表达产物可以与ALV-J阳性血清发生特异性反应。以纯化的His-Jgp85重组蛋白为包被抗原,建立间接ELISA方法,用于J亚群禽白血病的临床诊断。本研究对抗原包被浓度,待检血清稀释浓度,酶标抗体浓度,特异性试验及临界值等条件进行优化,对优化后的ELISA检测方法进行了临床应用试验。本研究建立了能特异性检测J亚群禽白血病血清抗体的ELISA方法。     

Avian leukosis subgroup J in broiler breeders in Switzerland

Since a new envelope subgroup (J) of the avian leukosis-sarcomatosis-complex was isolated for the first time from broiler breeders in the United Kingdom in 1989 and was characterized and associated with myeloid leukosis (syn. myelocytomatosis) the emergence of this subgroup was reported from all over the world. Thus the first known case of subgroup J avian leukosis in Switzerland in four imported broiler breeder flocks will be described. A total of 53 broiler breeder birds from four flocks showing reduced performance and increased mortality were submitted for postmortem examination. Approximately 20 blood samples from each flock were monitored serologically for antibodies against avian leukosis virus subgroup J (ALV-J). On necropsy myeloid leukosis (ML) was diagnosed in all four flocks. Furthermore the blood samples of three flocks showed significant ELISA-titres for ALV-J.     

重庆市禽白血病及禽网状内皮组织增生症的血清流行病学调查

禽白血病和禽网状内皮组织增生症均为禽的肿瘤性免疫抑制疾病,是危害养鸡业的两种非常重要的病毒性传染病。为调查重庆市肉鸡中禽白血病及禽网状内皮组织增生症的流行情况,在北碚区、涪陵区、开县、垫江县、潼南县五个区（县）的8个活禽交易市场采集260份血清样本,采用酶联免疫吸附试验检测了所有血清中禽白血病病毒（ALV）和禽网状内皮组织增生病毒（REV）的抗体。检测结果显示：禽白血病病毒和禽网状内皮组织增生病毒抗体阳性率分别为16.5%（43/260）和5%（13/260）,双抗体阳性率为4.61%（12/260）。与全国其他地区相比,重庆市肉鸡中这两种病原的感染率相对要低,但仍应重视这两种疾病的防控,以控制病原的进一步传播。     

蛋鸡J亚群禽白血病的流行病学调查

本试验对蛋鸡养殖密集地区发生的禽白血病疫情进行了流行病学调查,结果显示,此次疫情由禽白血病病毒J亚群感染引起,主要发生在海兰褐品种的商品代蛋鸡,损失比较严重。采用ELISA方法对18个鸡场采集的665份血清、688份泄殖腔棉拭子及180枚种蛋进行了实验室检测,结果表明,湖北省内蛋鸡养殖密集地区的蛋鸡尤其是商品代蛋鸡感染情况相当严重。     

J亚型禽白血病研究进展

J亚型禽白血病（AL-J）是由外源性J亚型禽白血病病毒（ALV-J）引起的致瘤性和免疫抑制性传染病。该病既可水平传播又可垂直传播,在中国鸡群中污染极为普遍且日趋复杂化,严重危害养鸡业的健康发展。由于该病迄今尚无疫苗和有效治疗药物,目前所采取的主要防控措施是对种鸡群净化。作者就其病原学、流行病学、诊断与防控等方面的研究近况进行了综述。     

J 亚群禽白血病病毒gp85单抗1E3株抗原识别表位的初步分析

J 亚型禽白血病是由 J 亚群禽白血病病毒引起的一种以成髓细胞增生、免疫抑制、生长发育受阻为特点的传染性肿瘤疾病,于1988年首次发现,其病毒囊膜表面蛋白gp85决定了病毒亚群特异性.本试验将原核表达质粒pET-28a-J gp85中的gp85基因分段亚克隆至重组表达质粒pGEX-6p-1上并在大肠杆菌中成功地表达,用ALV-J Sl gp85单克隆抗体1E3株对分段表达的gp85蛋白进行初步的免疫印迹分析.结果表明:ALV-J Sl gp85单抗1E3株所识别的抗原表位位于蛋白N端的第69至112个氨基酸之间.这将为ALV-J gp85抗原决定簇所在位点及其免疫学功能的研究奠定基础.     

禽白血病病毒p27蛋白在大肠杆菌中的表达和多克隆抗体的制备

将禽白血病病毒p27基因克隆并构建重组表达质粒pET28a-p27,在大肠杆菌BL21中经IP TG诱导后产生可溶性表达蛋白。表达的蛋白用Western blot进行活性检测,其能与辣根过氧化物酶标记的兔抗p27抗体发生特异性反应;采用金属螯和层析对表达蛋白进行纯化,其纯度约为95％;用纯化的表达蛋白p27免疫家兔制备抗血清,ELISA抗体效价可达1∶25600。研究结果表明,大肠杆菌表达的p27蛋白具有良好的抗原性,可以替代纯化病毒蛋白用于禽白血病病毒检测。     

某海兰褐鸡场祖代、父母代及商品代鸡血管瘤型ALV-J的分离与鉴定

从山东省某海兰褐鸡场祖代、父母代种鸡和商品代蛋鸡中获得疑似血管瘤型禽白血病(Avian leukosis,AL)病料.采用病理剖检、IFA、分子生物学检测,确定为J亚群禽白血病.从祖代、父母代病料中各分离到1株J亚群禽白血病病毒(J subgroup of avian leukosis virus,ALV-J),从商品代蛋鸡中分离到4株ALV-J.根据原型毒株HPRS103设计1对gp85基因引物,获得gp85基因序列.获得的gp85基因序列与各亚群参考毒株序列核苷酸同源性比对,结果显示:分离自商品代蛋鸡的Commercial03株、Commercial04株、Commercial06株和父母代分离株Parent02株位于同一分支,同源性在97.2％～97.9％,与HPRS103株同源性94.7％～95.2％;Commercial05株与祖代分离株Grandparent01株在同一分支,与HPRS103株同源性为98.3％,4株分离自商品代的ALV-J同源性为95.0％～99.9％.表明商品代蛋鸡中的ALV-J可能来自父母代或祖代种鸡的垂直传播,也可能来自于其他来源的水平传播.从同一鸡场祖代、父母代及商品代鸡中分离得到ALV-J,这在我国还是首次.对后续研究其基因突变、致瘤机制等奠定了良好的基础.     

Characteristics of two new reticuloendotheliosis virus isolates of turkeys.

Reticuloendotheliosis (RE) virus strains MN81 and MN67 isolated from epiornithics of RE in turkeys were partially characterized. Strains MN81 and MN67 replicated in chicken embryo fibroblast,duck embryo fibroblast and turkey embryo-fibroblast cultures and produced syncytial cytopathic effects in duck embryo fibroblast and turkey embryo fibroblast cultures. The virions of MN81 and MN67 measured approximately 100 nm in diameter, resembled RE virus strain T, and could be distinguished from avian leukosis viruses morphologically. The buoyant density of strain MN81 was found to be 1.15 g/cm3 in sucrose gradients. Strains MN81 and MN67 were inactivated by heat, acid pH, ether, and chloroform treatments. These strains were serologically unrelated to avian leukosis virus but were related to RE virus strains T, CS, DIA, and SN.     

黄羽肉鸡J亚群禽白血病病毒分离鉴定与全基因组序列分析

为了解J亚群禽白血病在黄羽肉鸡中的流行状况,采用病料研磨液接种DF-1细胞、ELISA p27抗原检测、PCR扩增等方法,从广东某鸡场送检疑似禽白血病的黄羽肉鸡病料中分离鉴定出1株J亚群禽白血病病毒,命名为GDLZ0715。为进一步了解该病毒分子学特性,对其进行全基因组测序,并与其他ALV-J毒株进行比较。结果表明,GDLZ0715分离株整个基因组中gag、pol、env基因和LTR相对保守,与各参考ALV-J毒株序列同源性分别达93.5%~95.9%、96.8%~97.3%、89.6%~94.6%和90.8%~95.1%;3′UTR变异较大,与各ALV-J参考毒株序列同源性仅为80.5%~93.4%,其中rTM和E元件大量碱基缺失;进一步分析表明3′UTR中rTM区和E元件大量碱基缺失正成为我国肉鸡ALV-J毒株的变异趋势。     

J亚群与E亚群禽白血病自然重组病毒的全基因组序列分析

为了解我国东北地区部分养鸡场禽白血病病毒(ALV)的基因组序列特征及其变异情况,本研究从具有典型血管瘤病变的禽白血病发病鸡中分离到一株J亚群ALV(ALV-J)命名为JL0901,并进行了全基因测序.将该序列与已发表的ALV-J毒株序列进行比较研究,结果表明JL0901基因组的gag和pol基因相对保守,而env基因和3'端非编码区(3'UTR)的变异较大.对JL0901的env基因核苷酸序列进一步分析发现,在其gp85基因和gp37基因交界位置发生J亚群和E亚群ALV重组现象.本研究证实国内鸡群中存在J亚群和其他亚群ALV的自然重组现象,并表明国内ALV已出现新的变异趋势.