Development of a polymerase chain reaction to differentiate avian leukosis virus (ALV) subgroups: detection of an ALV contaminant in commercial Marek's disease vaccines

Avian leukosis viruses (ALVs) are common in many poultry flocks and can be detected using an enzyme-linked immunosorbent assay or any other test designed to identify p27, the group-specific antigen located in gag. However, endogenous retroviruses expressing p27 are often present and can be confused with exogenous ALVs. A more specific and informative assay involves targeting the variable envelope glycoprotein gene (gp85) that is the basis for dividing ALVs into their different subgroups. We designed polymerase chain reaction (PCR) primers that would specifically detect and amplify viruses from each of the six ALV subgroups: A, B, C, D, E, and J. Subgroup B and D envelopes are related, and our B-specific primers also amplified subgroup D viruses. We also designed a set of common primers to amplify any ALV subgroup virus. To demonstrate the usefulness of these primers, we obtained from the Center for Veterinary Biologics in Iowa culture supernatant from chicken embryo fibroblasts infected with an ALV that was found to be a contaminant in two commercial Marek's disease vaccines. Using our PCR primers, we demonstrate that the contaminant was a subgroup A ALV. We cloned and sequenced a portion of the envelope gene and confirmed that the ALV was a subgroup A virus. Unlike typical subgroup A viruses, the contaminant ALV grew very slowly in cell culture. We also cloned and sequenced a portion of the long terminal repeat (LTR) from the contaminant virus. The LTR was found to be similar to those LTRs found in endogenous ALVs (subgroup E) and very dissimilar to LTRs normally found in subgroup A viruses. The E-like LTR probably explains why the contaminant grew so poorly in cell culture.     

禽白血病病毒B、E和J亚群基因芯片检测方法的建立

目的基于多重PCR技术,建立禽白血病病毒(ALV)的B、E、J亚群的基因芯片分型和检测方法。方法根据NCBI已收录的ALV三个亚群的参考毒株cDNA序列,在各亚群特异性基因突变区两端选取其保守区域,设计合成三个亚群的通用上游引物1条,以及B、E亚群的通用下游引物和J亚群下游引物各1条,将上述引物用Cy3标记,建立多重PCR体系;参考靶序列内部的三个亚群各自的保守区域,选择亚群之间基因突变位点多的区域,设计合成5条寡核苷酸探针,制作寡核苷酸探针基因检测芯片;以寄主细胞DF-1中提取传代ALV的cDNA,以及合成NCBI收录的各亚群参考毒株的cDNA序列作为检测模板;利用Cy3标记的PCR扩增产物,与基因芯片进行杂交反应,扫描结果。结果芯片准确检测并分型三个亚群的参考毒株,其检测灵敏度能够达到102个基因拷贝,且与禽类常见的四种病毒均无交叉反应。结论本研究结果证明,基因芯片技术是一种ALV的B、E和J亚群进行检测和分型的有效方法,且具有较高的特异性和灵敏度,为今后在临床应用中快速鉴别诊断ALV等免疫抑制病提供可行性。     

禽白血病病毒贵州流行株env基因克隆与序列分析

为了解禽白血病病毒（ALV）贵州流行株的遗传变异情况及分子特征,本试验基于ALV env基因设计合成引物对禽白血病贵州临床病例进行目的基因扩增、克隆和序列分析。结果显示,从临床病例中筛选获得3份阳性样本,PCR扩增均获得大小约921 bp的目的基因片段,将其命名为：GZ-ALV-1株、GZ-ALV-2株和GZ-ALV-3株。序列分析结果显示,3株ALV贵州流行株之间核苷酸同源性在97.2%~97.6%之间,与国内外ALV-J的同源性相对较高,为93.1%~99.3%;而与A、B、C、D、E、K亚群ALV同源性仅为51.4%~53.2%。系统进化分析显示,3株ALV贵州流行株与ALV-J亚群参考株处于同一分支,表明本试验所检测的ALV毒株均为ALV-J亚群;与A、B、C、D、E、K亚群处于不同进化分支。基因变异分析显示,3株流行株37处相同核苷酸变异导致17处氨基酸发生位点变异,其中9个可变点在高变区hr1和hr2,1个可变点在低变区vr3。结果表明,3株ALV贵州流行株均为ALV-J亚群,env基因存在位点发生了变异,且可变位点位于序列高变区。本研究结果为明确贵州禽白血病流行概况及ALV的防控与净化提供基础数据。     

家禽疱疹病毒CRISPR/Cas9基因编辑最新研究进展

基于CRISPR/Cas9系统的基因编辑是最新一代的基因组编辑技术，在向导RNA （gRNA）的介导下几乎可以靶向编辑任何一种基因，实现基因组的定点突变、敲除或插入。近年来将CRISPR/Cas9基因编辑技术应用于大基因组DNA病毒的研究，尤其是用于疱疹病毒的基因编辑已成为病毒学研究领域的最新国际热点。自2016年首次报道利用CRISPR/Cas9系统改造家禽疱疹病毒如马立克病病毒（MDV）基因组以来，短短5年时间已全面应用于家禽疱疹病毒的蛋白编码基因和非编码RNA基因的编辑、基因缺失疫苗和重组疫苗研发、抗病毒治疗以及抗病育种等领域。本文详细综述了当前CRISPR/Cas9基因编辑技术在家禽疱疹病毒中的应用进展和最新成果，并对其面临的问题和前景进行了展望，以期为后续研究提供重要参考。     

七彩山鸡F亚群禽白血病病毒的分离与鉴定

为了解广东某七彩山鸡种禽场禽白血病流行情况,通过接种DF-1细胞、ELISA抗原检测、聚合酶链式反应(PCR)、囊膜基因克隆测序等方法分离并鉴定出两株ALV-F,分别命名为FGD1801和FGD1802。为进一步了解分离株遗传进化特点,利用PCR方法扩增出env基因并测序,同时与各亚群参考毒株的gp85核苷酸序列进行对比。结果显示:这两个分离株env基因gp85片段长度都为1080 bp,预计编码360个氨基酸,FGD1801和FGD1802分离株gp85基因核苷酸序列的相似性为92.8%,与A、B、E、J和K亚群共26株ALV参考毒株相似性在47.0%~64.5%之间,而与F亚群参考株的相似性在92.0%~92.5%,显著高于其他亚群,且位于同一进化分支上。研究表明,从七彩山鸡分离的FGD1801和FGD1802属于ALV-F,是我国华南地区新发现的ALV亚群。     

CRISPR/Cas9基因编辑技术在绵羊中的应用研究进展

随着基因编辑技术的迅速发展,研究者利用基因编辑技术在越来越多的领域中取得了许多突破性的进展。目前,众多的基因编辑工具中CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)系统应用最为广泛。CRISPR/Cas9及其衍生出的碱基编辑器(base editor, BE)和先导编辑器(prime editor, PE)系统使研究者可以在目标基因组中简单、高效地完成碱基的删除、插入和替换等修饰。CRISPR/Cas9相关基因编辑技术在生产具有特定遗传特征的动物方面有巨大的潜在价值。绵羊作为具有许多重要经济性状的家畜,是理想的基因工程研究大动物模型。CRISPR/Cas9相关基因编辑技术已经用于绵羊基因组工程研究,如生产具有优良特性的品种,利用乳腺生产疾病治疗药物,构建用于人类疾病和再生医学研究的动物模型。作者从CRISPR/Cas9基因编辑技术的原理出发,着重阐述了利用CRISPR/Cas9系统进行基因编辑的具体流程,并概述了CRISPR/Cas9系统...     

Avian leukosis virus (ALV) infection, shedding, and tumors in maternal ALV antibody-positive and -negative chickens exposed to virus at hatching

Chickens highly susceptible to avian leukosis virus (ALV) infection and tumors, with and without ALV subgroup A maternal antibody (MAB), were infected with a field strain of ALV subgroup A at hatching. Viremia, antibody development, cloacal and albumen shedding, and tumors in chickens with MAB (MAB+) were compared with those in chickens lacking MAB (MAB-). At 18 weeks of age, the incidence of viremia was significantly lower in MAB+ chickens than in MAB- chickens; further, MAB significantly reduced the proportion of tolerantly infected (viremic antibody-negative) chickens. Cloacal shedding of ALV at 22 weeks of age and shedding of ALV group-specific (gs) antigen in albumen of eggs from all laying hens at 30-32 weeks of age were significantly lower in MAB+ hens than in MAB- hens. The incidence of ALV-induced tumors was lower in MAB+ chickens than in MAB- chickens, significantly so in one of three trials conducted. These results suggest that MAB may influence the development of viremia, antibody, and shedding of ALV following massive exposure to virus at hatching.     

广西地方禽类品种ALV和REV感染情况的调查及ALV分离株gp85基因序列分析

为了解广西钦州地区主要地方品种商品禽类中禽白血病病毒(ALV)、禽网状内皮组织增殖症病毒(REV)的感染情况,本试验随机采集了广西钦州地区代表饲养场的麻鸭、狮头鹅、铁脚麻鸡、火鸡、鸽子共5个品种的蛋清、肛拭子及血清样品共953份,使用ELISA商品试剂盒进行检测;然后对部分ALV p27阳性的个体采集其血清样品接种DF-1细胞进行病毒分离并测定其gp85基因的序列。结果发现,除铁脚麻鸡外其他4个非鸡禽类品种的ALV检测均为阴性;除鸽子和狮头鹅外,麻鸭、铁脚麻鸡、火鸡均检测到REV抗体阳性;获得的两株铁脚麻鸡ALV分离株gp85基因之间核苷酸同源性为94.5%,与参考株之间核苷酸同源性为86.9%~94.9%;两株分离株gp85基因之间氨基酸同源性为91.5%,与参考株之间氨基酸同源性为84.0%~91.6%。高变区hr1和hr2区存在较多可变位点,可变区vr2和vr3相对较保守。系统进化树分析结果表明这两个毒株与参考株SCAU11-XG的亲缘关系最近。     

Molecular and biological characterization of a naturally occurring recombinant subgroup B avian leukosis virus with a subgroup J-like long terminal repeat

Infection of broiler chickens with subgroup J avian leukosis virus (ALV) results in the induction of myeloid tumors. However, although egg-type chickens are susceptible to infection with ALV-J, the tumor incidence is very low, and on rare occasions the tumors observed are of the myeloid lineage. We recently described the isolation of an ALV (AF115-4) from commercial egg-type chickens suffering from myeloid leukosis. AF115-4 was initially identified as an ALV-J isolate based on PCR analysis of the long terminal repeat (LTR). However, further characterization of the viral envelope indicated that the virus is recombinant with subgroups B envelope and J LTR. Here we further characterize this recombinant virus at both the molecular and biological levels. We show that the AF115-4 isolate expresses a recombinant envelope glycoprotein encoded by a subgroup B gp85 region and a subgroup E gp37 region. The host range ofAF115-4 was analyzed using cells resistant to infection by subgroups A/B, J, or E; this shows that no ALV-J was present in the isolates obtained from the affected chickens. Additional antigenic characterization of AF115-4 using chicken sera specific for subgroups B or J indicated that no ALV-J was present in the samples examined. Inoculation of AF 115-4 into ALV-susceptible 1515 X 71 chickens resulted in the induction of lymphoid leukosis but not the expected myeloid leukosis affecting the commercial chickens. These results suggest that differences in the genetic makeup of the chickens from which AF115-4 was isolated and the line 1515 X 71 used in the present experiments may be responsible for the observed differences in pathogenicity. In addition, the results suggest that ALV-J continues to evolve by recombination, generating new viruses with different pathological properties.     

Infections Investigation of ALV and REV in Guangxi Local Poultry Breeds and Sequence Analysis of gp85 Gene of ALV Isolates

In order to investigate the infection status of avian leukosis virus (ALV) and avian reticuloendotheliosis virus (REV) in the major local breeds of Qinzhou,Guangxi,totally 953 samples of egg white,cloaca swab and serum of Ma duck,Shitou goose,Tiejiao-Ma chicken,turkey and pigeon were collected from the representing flocks and detected by the commercial ELISA kits.ALV was isolated for the ALV p27 positive samples by culturing on DF-1 cells,and gp85 gene was sequenced.The results showed that the detections of ALV were negative in the samples except those of Tiejiao-Ma chicken,while REV antibody was found positive in Ma duck,Tiejiao-Ma chicken and turkey.The nucleotide sequences of gp85 gene of two isolates shared 94.5% identity with each other,and shared 86.9% to 94.9% with reference strains.The amino acid sequences of gp85 gene of two isolates shared 91.5% identity with each other,and shared 84.0% to 91.6% with reference strains.There were many variable sites in the hyper variable region hr1 and hr2,and the vr2 and vr3 variable regions were relatively conservative.Phylogenetic tree analysis showed that the two isolates shared the highest homology with SCAU11-XG strain.     

Characterization of monoclonal antibodies to avian leukosis viruses

Hybridoma cell lines secreting monoclonal antibody (MCA) to avian leukosis virus (ALV) structural proteins p27 and p19 have been established. In an indirect enzyme-linked immunosorbent assay (ELISA), MCA 6AL20 (IgG1 isotype) reacted with RPL-40 (ALV subgroup A), avian myeloblastosis virus (AMV) (a mixture of subgroups A and B), Rous-associated virus (RAV)-2 (subgroup B), and Carr-Zilber strain of Rous sarcoma virus (CZ-RSV) (subgroup D) but not with Prague strain of RSV (PrC-RSV) (subgroup C) or the endogenous virus RAV-0 (subgroup E). MCA 6AL22 reacted as above and also reacted marginally with PrC-RSV. Both MCAs immunoprecipitated p19 from 35S-methionine-labeled chicken embryo fibroblasts (CEFs) infected with RPL-40 or RAV-1, but not from CEFs infected with RAV-0, thus identifying the viral structural protein p19 as a polypeptide with subgroup-specific epitopes. Both MCAs can be used to differentiate RPL-40 from RAV-0 infection either in an indirect antibody ELISA or by immunoprecipitation. A third MCA, 6AL42 (IgG2a isotype), reacted with the above viruses of subgroups A, B, C, and D at an antibody titer up to 1000-fold higher than with subgroup E RAV-0 virus in indirect ELISAs. MCA 6AL42 immunoprecipitated p27 from cells infected with RPL-40, RAV-1, or RAV-0. These MCAs are potentially useful in developing immunological tests for differentiation of ALV strains.     

ARV、REV与ALV三重RT-PCR检测方法的建立

根椐GenBank中已发表的禽呼肠孤病毒（ARV）、禽网状内皮增生病病毒（REV）、禽白血病病毒（ALV）等3种病毒基因组序列,设计了3对分别与ARV、REV和ALV某段基因序列互补的引物。在建立各病毒单项RT-PCR技术的基础上,优化三重RT-PCR反应条件,建立了3种病毒的三重RT-PCR技术。结果表明,用这3对引物对同一样品中的ARV、REV、ALV核酸模板进行三重RT-PCR扩增,可同时扩增ARV的247bp,ALV的675bp,REV的467bp的特异性片段,而对其他6种禽病病原的PCR扩增结果均为阴性。敏感性测定结果表明,该三重RT-PCR技术能检出10pg的ALV、1pg的ARV和10pg的REV模板。用42份临床病料对本研究多重RT-PCR技术和单项RT-PCR技术进行对比验证,结果显示,两者的总符合率为92%以上。表明建立的多重RT-PCR检测方法,具有特异、快速、准确的特点,可用于对这3种病毒的同时检测和鉴别诊断。     

Development and characterization of monoclonal antibodies to subgroup A avian leukosis virus

Avian leukosis virus subgroup A (ALV‐A) is a retrovirus which infects egg‐type chickens and is the main pathogen of lymphoid leukosis (LL) and myeloid leukosis (ML). In order to greatly enhance the diagnosis and treatment of clinical avian leukemia, two monoclonal antibodies (MAbs) to ALV‐A were developed by fusion between SP2/0 and spleen cells from mice immunized with expressed ALV‐A env‐gp85 protein. Using immunofluorescence assay (IFA), two MAbs reacted with ALV‐A, but not with subgroups B and J of ALV. Western blot tests showed that molecular weight of ALV‐A envelope glycoprotein recognized by MAbs was about 53 kD. Isotyping test revealed that two MAbs (A5C1 and A4C8) were IgG1 isotypes. These MAbs can be used for diagnosis and epidemiology of ALV‐A.     

Isolation and characterization of an adventitious avian leukosis virus isolated from commercial Marek's disease vaccines

Commercial Marek's disease (MD) vaccines produced by two manufacturers were tested for possible contamination with avian leukosis virus (ALV). Samples of MD vaccines manufactured by two companies (A and B) were received from a breeder company; samples were also received directly from vaccine company B. Using virus isolation tests, samples initially tested positive for subgroup E (endogenous) ALV. However, upon repassage, the vaccines also tested positive for exogenous ALV. The isolated exogenous ALV proved to be a subgroup A virus, as determined by flow cytometry using polyclonal chicken antibodies specific for various subgroups of ALV, and by DNA sequencing of the envelope glygoprotein (gp85). The exogenous ALV isolated from MD vaccines was inoculated in chickens from ADOL lines 15I(5) x 7(1) and 0 to determine its pathogenicity and compare it with that of Rous-associated-virus-1 (RAV-1), the prototype strain of ALV-A. Each chicken from each line was inoculated with approximately 10,000 infectious units of RAV-1 or the ALV-A isolated from vaccines termed B-39 virus at 7th day of embryonation. At hatch, and at 4, 8, and 16 wk of age, chickens were tested for viremia and cloacal shedding; chickens were also observed for ALV-induced tumors within 16 wk of age. Viremia and cloacal shedding results suggest that chickens from both lines were susceptible to infection with either virus. Within 16 wk of age, the proportion of ALV tumors induced by strain B-39 in line 0 and line 15I5 x 7(1) chickens was 0% and 12%, respectively, compared with 62% and 67% in chickens inoculated with RAV-1. The data indicate that commercial MD vaccines produced by two manufacturers were contaminated with endogenous subgroup E and an exogenous subgroup A ALV. Further, data from biological characterization suggest that the ALV-A isolated from commercial MD vaccines is of low oncogenicity, compared with that of RAV-1. GenBank accession numbers: The gp85 gene sequences of ALV isolated from commercial Marek's disease vaccines have been deposited in GenBank and assigned the following accession numbers: A46 subgroup A, DQ412726 ; B53 subgroup A, DQ412727; A46 subgroup E, DQ412728; B53 subgroup E, DQ412729.     

Identification and characterization of hens transmitting avian leukosis virus (ALV) to their embryos by ELISAs for detecting infectious ALV, ALV antigens and antibodies to ALV.

A total of 72 White Leghorn grandparent hens was examined by ELISA for avian leukosis virus (ALV), ALV antigens and anti-ALV antibodies to identify and characterize the hens transmitting ALV to their embryos (transmitters) by using fertilized eggs. These hens were divided into 3 groups as no antibody and non-viremic (NANV) (49 hens), antibody-positive and non-viremic (APNV) (21 hens) and no antibody and viremic (NAV) (2 hens) by testing the sera for the presence of ALV and anti-ALV antibody. Egg albumen and embryos were tested for the presence of ALV and ALV antigens. As a result, no ALV was detected in both albumen and embryos in the NANV group. On the other hand, all albumen samples collected repeatedly from 3 hens of the APNV group and 2 hens of the NAV group contained infectious ALV, although the infectivity differed with the individual. Also, these 5 hens produced infected embryos at varying frequencies. However, on AP hen which shed neither ALV nor ALV antigens into the albumen produced an infected embryo at a lower rate. These results indicate that testing for infectious ALV in albumen from a newly laid egg per hen is effective to identify the transmitters to some extent. When virus titers in each of 8 tissue samples from the 6 transmitting hens were determined, the highest virus titers were found in washing from the ampulla of the oviducts in most of the shedders, suggesting that embryo infection is closely correlated with ALV produced at the oviduct, but not with ALV transferred from the other parts of the body.     

An enzyme-linked immunosorbent assay for detection of antibodies to exogenous avian leukosis virus

A microplate enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to avian leukosis virus (ALV) of subgroups A and B in infected chickens was developed with the use of Rous-associated virus (RAV)-1 (subgroup A) and RAV-2 (subgroup B) antigens purified by sucrose-gradient centrifugation. The antigen was used for ELISA after treatment with Triton X-100. In the ELISA, the subgroup viral antigen reacted strongly with homologous antiserum but also reacted with heterologous antiserum. Tests with serum absorbed with purified homologous and heterologous virus and tests for antigen-blocking by group-specific antibodies to ALV revealed that the reaction was caused mainly by subgroup-specific antibodies. The ELISA was 8 to 32 times more sensitive than the virus-neutralization (VN) test and detected antibodies to ALV earlier than the VN test in chickens infected experimentally with RAV-1 and RAV-2. In field application of the ELISA, 44.2% of 484 chicken sera were positive for RAV-1 and/or RAV-2 antigen, and 80.4% of flocks were positive. These findings indicate that ELISA is superior to the VN test in sensitivity, simplicity, rapidity, and applicability for large-scale field surveys for ALV infection.     

广西凭祥斗鸡禽白血病病毒检测及分离株env基因分析

为了解广西凭祥市特有家禽品种斗鸡禽白血病病毒(ALV)的感染情况,采集了该市3个鸡场斗鸡的肛拭子、血清、血浆样品共344份,用禽白血病ELISA检测试剂盒进行检测。结果显示,斗鸡ALV感染情况严重,其中肛拭样品ALV-p27抗原阳性率高达39.13%,血清样品病毒分离阳性率为12.97%,ALV-J和ALV-A/B抗体阳性率分别为22.39%和7.46%;对从2只斗鸡获得的病毒分离株DJ-3-18和DJ-45进行病毒囊膜蛋白基因env的扩增、序列测定及比较分析,结果显示2株病毒的gp85基因与ALV-A亚群参考株之间氨基酸的同源性为88.2%~96.5%,gp37基因与ALV-A亚群参考株之间氨基酸的同源性为91.4%~98.0%,其中与台湾A亚群蛋鸡源分离株TW-3577的亲缘关系最近,而与ALV其他亚群毒株的同源性则较低。结果表明,首次获得的2株斗鸡源ALV分离株属A亚群。     

抗J亚群禽白血病病毒囊膜糖蛋白特异性单克隆抗体的研制及其特性

J亚群禽白血病病毒（ALV-J）是一种主要感染肉用型鸡的反转录病毒。本研究用表达ALV-J囊膜蛋白基因产物的Sf9细胞免疫Balb/c小鼠，取其脾脏细胞与骨髓瘤细胞NS1进行融合，获得了4株特异性抗ALV-J的单克隆抗体。免疫荧光分析结果表明，3株单克隆抗体仅与所试验的ALV-J毒株反应，而不能与ALV的A、B、C、D和E亚群的毒株反应。有趣的是，有一株单克隆抗体可以与所有试验的外源性ALV毒株反应，但不与内源性的E亚群反应。Western Blot和免疫沉淀试验结果表明，单克隆抗体识别的ALV-J囊膜糖蛋白的分子量为90-94kD，识别未糖基化的囊膜蛋白分子量约为53kD。用这些单克隆抗体能检测出ALV-J病毒感染鸡胚成纤维细胞中的病毒抗原。这些结果提示这些单克隆抗体可用于ALV-J疾病的诊断和流行病学调查。     

CRISPR/Cas9基因编辑技术在家畜育种新材料创制中的研究进展

规律性成簇间隔的短回文重复序列/CRISPR相关蛋白（clustered regularly interspaced short palindromic repeats/CRISPR-associated,CRISPR/Cas）系统是最新一代能对细胞或生物体基因组进行精准编辑的基因工程技术,与前两代基因编辑技术ZFN和TALEN相比,CRISPR/Cas具有应用成本低、适用编辑范围广、打靶效率高、操作简单、可支持多位点操作等诸多优点。近年来,CRISPR/Cas系统尤其是Type II类、A型的CRISPR/Cas9系统已经作为最新一代基因编辑技术被广泛应用于提高家畜繁殖效率、生产性能、抗病性以及动物模型构建等研究中,并创制了一批基因编辑牛羊育种新材料。本文就其发展历程、技术改造和优化最新进展以及在家畜繁殖性状、生产性状和抗病性状等方面的研究应用进行综述,重点介绍了该系统在家畜育种学研究中已取得的最新进展,并就CRISPR/Cas9基因编辑技术在家畜育种应用中现存的问题及其应用前景进行简要论述。     

Development and characterization of monoclonal antibodies to subgroup J avian leukosis virus.

In an attempt to develop a specific diagnostic test for avian leukosis virus (ALV) subgroup J (ALV-J) strain Hc1, four monoclonal antibodies (MAbs), JE9, G2, 145, and J47, were generated that are specific for ALV-J envelope glycoprotein, gp85. Polymerase chain reaction (PCR) was used to amplify genomic pro-viral DNA of Avian Disease and Oncology Laboratory (ADOL)-Hc1 and ADOL-4817 envelope genes. Both open reading frames encoding glycoproteins gp85 and gp37 were cloned into baculoviruses. Abundant expression of gp85 and gp37 was detected in the recombinant viruses with specific antibody to Hc1 strain of the ALV-J. The expressed proteins were used for immunization of mice to produce hybridoma cell lines secreting MAbs specific to ALV-J envelope protein. A panel of MAbs was generated by fusing NS1 myeloma cells and spleen cells from mice immunized with the recombinant baculoviruses. With the use of an immunofluorescence assay, three MAbs (JE9, G2, 145) reacted with ALV-J but not with subgroups A, B, C, D, or E of ALV. MAb J47 reacted with all exogenous subgroups of ALV including A, B, C, D, and J but not with endogenous subgroup E viruses. Western blot analysis was performed with all four MAbs against recombinant baculovirus and Hc1-infected chicken embryo fibroblast (CEF) lysates. A major band with a molecular weight about 90 kD corresponding to the size of ALV-J envelope was consistently obtained. With these MAbs, we detected the Hc1 antigen in CEFs infected with several ALV-J viruses isolated in the United States and also in tissue sections from chickens infected with Hc1 strain of ALV-J. These MAbs will be useful reagents for the diagnosis of ALV-J infection because they recognize a common antigenic epitope in six isolates tested thus far.