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.     

Isolation and some characteristics of a subgroup J-like avian leukosis virus associated with myeloid leukosis in meat-type chickens in the United States.

Several subgroup J-like avian leukosis viruses (ALV-Js) were isolated from broiler breeder (BB) and commercial broiler flocks experiencing myeloid leukosis (ML) at 4 wk of age or older. In all cases, diagnosis of ML was based on the presence of typical gross and microscopic lesions in affected tissues. The isolates were classified as ALV-J by 1) their ability to propagate in chicken embryo fibroblasts (CEF) that are resistant to avian leukosis virus (ALV) subgroups A and E (C/AE) and 2) positive reaction in a polymerase chain reaction with primers specific for ALV-J. The prototype strain of these isolates, an isolate termed ADOL-Hc1, was obtained from an adult BB flock that had a history of ML. The ADOL-Hc1 was isolated and propagated on C/AE CEF and was distinct antigenically from ALV of subgroups A, B, C, D, and E, as determined by virus neutralization tests. Antibody to ADOL-Hc1 neutralized strain HPRS-103, the prototype of ALV-J isolated from meat-type chickens in the United Kingdom, but antibody to HPRS-103 did not neutralize strain ADOL-Hc1. On the basis of both viremia and antibody, prevalence of ALV-J infection in affected flocks was as high as 87%. Viremia in day-old chicks of three different hatches from a BB flock naturally infected with ALV-J varied from 4% to 25%; in two of the three hatches, 100% of chicks that tested negative for virus at hatch had evidence of viremia by 8 wk of age. The data document the isolation of ALV-J from meat-type chickens experiencing ML as young as 4 wk of age. The data also suggest that strain ADOL-Hc1 is antigenically related, but not identical, to strain HPRS-103 and that contact transmission of ALV-J is efficient and can lead to tolerant infection.     

Pathogenicity of two recombinant avian leukosis viruses

We have recently described the isolation and molecular characteristics of two recombinant avian leukosis subgroup J viruses (ALV J) with an avian leukosis virus subgroup A envelope (r5701A and r6803A). In the present study, we examined the role of the subgroup A envelope in the pathogenesis of these recombinant viruses. Chickens of line 151(5) x 7(1) were inoculated at 1 day of age with r5701A, r6803A, Rous-associated virus type 1 (RAV-1), or strain ADOL-Hcl of ALV-J. At 2, 4, 10, 18, and 32 wk postinoculation (PI), chickens were tested for avian leukosis virus (ALV)-induced viremia, shedding, and neutralizing antibodies. All except one chicken inoculated with the recombinant viruses (98%) developed neutralizing antibodies by 10 wk PI compared with only 16% and 46% of the ADOL-Hcl and RAV-1-inoculated birds, respectively. ALV-induced tumors and mortality in the two groups inoculated with recombinant viruses were different. The incidence of tumors in groups inoculated with r5701A or RAV-1 was 100% compared with only 9% in the groups inoculated with r6803A or ADOL-Hcl. The data suggest that differences in pathogenicity between the two recombinant viruses might be due to differences in the sequence of the 3' untranslated region (presence or absence of the E element), and, therefore, not only the envelope but also other elements of the viral genome play an important role in the pathogenesis of ALV.     

Molecular characteristics and pathogenicity of an avian leukosis virus isolated from avian neurofibrosarcoma

Peripheral nerve sheath tumors (PNSTs) are rare in chickens and their etiology remains to be elucidated. In this study, a naturally occurring PNST in a Japanese native fowl (Gallus gallus domesticus) was pathologically examined and the strain of avian leukosis virus (ALV) isolated from the neoplasm was characterized by molecular biological analysis. The fowl presented with a firm subcutaneous mass in the neck. The mass, connected to the adjacent spinal cord (C9-14), was microscopically composed of highly cellular tissue of spindle cells arranged in interlacing bundles, streams, and palisading patterns with Verocay bodies and less cellular tissue with abundant collagen. Immunohistochemically, neoplastic cells were divided into two types: perineurial cells positive for vimentin, glucose transporter 1 (GLUT1), and claudin1; and Schwann cells positive for vimentin, occasionally positive for S-100 alpha/beta but negative for GLUT1. Based on these findings, a diagnosis of neurofibrosarcoma was made. The complete nucleotide sequence of an ALV strain, CTS_5371, isolated from the neoplasm was determined and phylogenetic analysis indicated that the strain was a novel recombinant virus from avian leukosis/sarcoma viruses previously reported. Additionally, experimental infection revealed that CTS_5371 induced the proliferation of Schwann cells and perineurial cells. These results suggest that this ALV strain has the ability to induce PNSTs in chickens.     

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。     

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.     

禽淋巴细胞性白血病的诊断与病毒亚群鉴定

采用病理组织学方法对某商品蛋鸡场送检的发病鸡进行实验室诊断,在肿胀的肝、脾、肾和法氏囊组织切片中观察到成淋巴细胞增生病变。使用禽白血病A、B亚群抗体检测试剂盒对血清样本进行间接ELISA试验,抗体阳性率达到44.4%(12/27)。采集12只病死鸡的肝脏材料提取DNA样本,11份样本中扩增出ALV-A亚群特异性的病原核酸(691bp),检出率达到91.6%(11/12)。将扩增的目的基因克隆与测序,截取gp85基因片段可变区序列与ALV-A、B、C、D和E亚群参考毒株的相应序列进行比较,同源性分别达到89.0-89.6%、67.1-67.7%、69.1-70.1%、69.1-69.5%和70.9-72.0%。结果证实本次疫病是由ALV-A亚群病毒引起的淋巴细胞性白血病。     

荧光定量RT-PCR检测禽白血病病毒方法的建立及应用

gag基因是禽白血病病毒(ALV)的群特异性基因,根据GenBank中登录的gag基因的保守序列(E46390),设计合成了1对引物,建立了基于SYBR GreenI模式的检测禽白血病病毒的实时荧光定量RT-PCR方法。该方法只对目的基因有扩增,对其它无关病毒核酸无特异性扩增;扩增效率为94.6%;在8.2×102～8.2×107拷贝的范围内有很好的线性关系(Y=3.46X+31.8,r=0.9995);最低可检测82个拷贝的病毒核酸,与普通PCR方法相比,灵敏度高出1000倍。组内CV为0.28%～1.45%;组间CV为2.13%～3.84%;对151例临床疑似样本的检出率为54.9%(83/151)。随机选择15份阳性样本PCR产物克隆测序,结果证明扩增片断为ALV的特异序列,与ALVgag基因核苷酸同源性为97.8～98.9%;对重庆14个县的14个健康商品蛋鸡群的134份粪便棉拭子进行检测,ALV的检出率为38.1%,鸡群的阳性率为14/14。同时用实时RT-PCR方法和ELISA方法对从种鸡场随机采集的30份100日龄蛋鸡的血清进行检测,实时RT-PCR检测率为5/30,ELISA方法检测率为4/30。本方法的建立为禽白血病的快速诊断、大规模检疫、监测、流行病学调查以及药物筛选等提供了新的检测方法。     

Diagnosis of myeloid leukosis induced by a recombinant avian leukosis virus in commercial white leghorn egg laying flocks

Commercial white leghorn egg layer flocks being used to produce fertile eggs for human vaccine production exhibited dramatically low peaks in egg production, two to four times higher than normal weekly mortality, and high numbers of cull, nonlaying birds after the onset of sexual maturity. These lower production characteristics could not be associated with management-related problems. Gross lesions of cull and fresh dead birds necropsied showed approximately 60% lacked ovarian activity and had lesions of a bacterial bursitis or synovitis, whereas the other 40% had tumors of the viscera but not of the bursa of Fabricius. Histologic examination of tumor-containing tissues showed lesions typical of myelocytomatosis. The diagnosis of myeloid leukosis was confirmed by the isolation of a recombinant avian leukosis virus (ALV) containing the LTR of subgroup J and the envelope of subgroup B ALV. A positive polymerase chain reaction with primers specific for the 3' untranslated region LTR confirmed the presence of LTR of ALV-J. The source of infection with this recombinant ALV was not determined; however, it is likely that commingling of the day-old egg-type chicks with ALV-J-infected meat-type chicks in a common hatchery had contributed to this outbreak.     

禽呼肠孤病毒与禽白血病病毒双重RT-PCR检测方法的建立及应用

根椐GenBank中禽呼肠孤病毒(ARV)、禽白血病病毒(ALV)基因序列,设计2对引物,在建立鉴别各病毒单项RT-PCR技术的基础上,优化双重RT-PCR反应条件,建立2种病毒的双重RT-PCR。对同一样品中的ARV、ALV核酸模板进行双重RT-PCR扩增,结果可同时扩增ARV 485 bp、ALV 673 bp的特异性片段,而对其他5种禽病病原的PCR扩增结果均为阴性。敏感性试验结果表明,该双重RT-PCR技术能检出10 pg的ALV和10 pg的ARV模板。用31份临床病料对本研究双重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100%。结果表明建立的双重RT-PCR检测方法,具有特异、快速、准确的特点,可用于对这2种病毒的同时检测和鉴别诊断。     

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。     

J亚群禽白血病病毒套式PCR检测方法的建立

本试验根据GenBank中登录的禽白血病病毒(ALV)基因组序列,设计合成了2对引物,外部引物的扩增片段大小为478 bp,内部引物的扩增片段大小为314 bp,建立了适合ALV快速检测的套式PCR方法(nested-PCR)。采用该方法对ALV毒株进行了检测,试验结果表明,能扩增到314 bp的条带,禽流感病毒(H9亚型)、新城疫病毒、传染性法氏囊病病毒、减蛋综合征病毒、禽网状内皮增生病病毒、禽呼肠孤病毒、马立克氏病病毒的扩增结果均为阴性。该方法第1次扩增的敏感性是100 pg,第2次扩增的敏感性是1 fg,第2次比第1次扩增的敏感性高10⁵倍。所建立的套式PCR方法具有敏感性高、重复性好、特异性强等优点,可用于禽白血病病毒(ALV)的临床诊断和分子流行病学调查等。     

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.     

Sequential disruption of ALV host receptor genes reveals no sharing of receptors between ALV subgroups A,B, and J

Background: Previously, we showed that targeted disruption of viral receptor genes in avian leukosis virus(ALV)subgroups using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9))-based genome editing confers resistance to ALV subgroups B and J. Here, we used the same strategy to target the receptor expressed by ALV subgroup A(TVA) and generate chicken cells resistant to infection by this virus.Results: CRISPR/Cas9-based disruption of exon 2 within the tva gene of DF-1 fibroblasts conferred resistance to infection by ALV subgroup A regardless of whether frameshift mutations were introduced during editing. Conversely,overexpression of the wild-type TVA receptor(wtTVA) by tva-modified DF-1 clones restored susceptibility to ALV subgroup A. The results confirm that exon 2, which contains the low-density lipoprotein receptor class A domain of TVA, is critical for virus entry. Furthermore, we sequentially modified DF-1 cells by editing the tva, tvb, and Na~+/H~+ exchange 1(chNHE1) genes, which are the specific receptors for ALV subgroups A, B, and J, respectively.Conclusions: Simultaneous editing of multiple receptors to block infection by different subgroups of ALV confirmed that ALV subgroups A, B, and J do not share host receptors. This strategy could be used to generate cells resistant to multiple viral pathogens that use distinct receptors for cell entry.     

抗禽白血病p27抗原单克隆抗体的制备与鉴定

检测p27抗原的检测试剂在禽白血病的研究和防治方面有着极其重要的作用.为研究国产的禽白血病诊断试剂,利用禽白血病各亚群病毒的p27蛋白很保守的特点,我们将原核表达的禽白血病p27蛋白作为抗原,免疫8周龄的BALB/C小鼠,利用淋巴细胞杂交瘤技术,获得三株能稳定分泌特异性单克隆抗体的杂交瘤细胞株.通过Western-blotting和ELISA的结果分析证明,三株单抗与原核表达得p27蛋白和禽白血病各亚群病毒反应,而不与禽流感病毒等反应.可以看出,这三株单抗在ALV的抗原分析、血清学诊断和疫苗质量监测以及禽白血病污染细胞检测等方面有着极其重要的应用价值.     

鸡白血病的流行现状和防制对策

鸡白血病普遍存在于商品鸡群中,呈渐进性发生和持续的低死亡率,白血病毒感染鸡群,导致鸡群生产性能下降,尤其是产蛋率和蛋的品质下降。介绍了鸡白血病病毒和该病在鸡群中的流行现状,分析了病毒间的谱系关系和发病原因,指出鸡白血病与禽网状内皮组织增生症的鉴别诊断方法,科学地提出对鸡白血病的预防和控制措施。     

Avian leukosis virus subgroup J: a rapidly evolving group of oncogenic retroviruses.

A strain of avian leukosis virus (ALV) belonging to a new envelope subgroup J was isolated in the UK in 1988 from meat-type chickens. The disease caused by the members of this subgroup has since spread very rapidly worldwide and has become one of the major problems facing the broiler meat industry. Molecular characterisation of HPRS -103, the prototype of subgroup J, has shown that it has a structure of a typical ALV with gag, pol and env genes. However the env gene was distinct from that of other ALV s and was closely related to that of novel endogenous retroviral elements designated EAV - HP. As other regions of the genome were closely related to ALV s, it is believed that ALV-J has evolved by recombination with the env sequences of EAV - HP. ALV-J has a tropism for myeloid cells, a feature that may be associated with its ability to induce myeloid leukosis. Recent data show that ALV -J isolates evolve rapidly resulting in sequence changes within the variable regions of the env gene leading to antigenic variation. Eradication programmes established for other subgroups are proving to be effective in eradicating ALV-J from infected flocks.     

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.     

禽白血病抗原快速检测试纸条的研制及初步应用

禽白血病给养鸡业带来极大损失,目前检测该病病原的方法主要有ELISA、PCR及RT-PCR、原位杂交(ISH)、间接免疫荧光(IFA)等。本研究应用2株抗禽白血病病毒(ALV)p27蛋白特异性单克隆抗体5D3和4F12研制了ALV快速检测试纸条。结果证明该试纸条具有良好的特异性,与H9亚型禽流感病毒、新城疫病毒、马立克氏病病毒、小鹅瘟病毒、鸡贫血病病毒没有交叉反应;采用p27表达蛋白检测试纸条灵敏性,检测下限达到70ng/mL;应用该试纸条检测71份临床样品,与商业ELISA试剂盒检测结果比较,二者符合率达到93%。该试纸条的研制,为基层快速检测ALV提供了条件。     

J亚群禽白血病病毒与禽网状内皮增生症病毒共感染对肉鸡生长和免疫功能的抑制作用

1日龄网鸡感染J亚群禽白血病病毒(ALV-J)以及共感染禽网状内皮增生症病毒(REV)后,肉鸡生长发育明显受阻,体重增重明显下降(P＜0.05),法氏囊、胸腺明显萎缩(P＜0.05),在用新城疫疫苗免疫后,感染组血清中新城疫抗体效价显著低于对照组(P＜0.05);在ALV-J和REV共感染后,这种抑制作用更为明显(P＜0.01).ALV J单独感染后,鸡对传染性法氏囊病病毒(IBDV)弱毒疫苗免疫后的抗体反应与对照组没有明显差别,但ALV-J与REV的共感染可明显延缓鸡对IBDV弱毒疫苗免疫的抗体反应.