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.     

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.     

2009年我国部分地区禽白血病分子流行病学调查

为了解自2009年年初以来国内一些地区禽白血病流行情况及流行毒株的分子特征,我们从湖北、黑龙江、山东、辽宁、吉林、广东、宁夏、安徽8个省区39个鸡场采集疑似禽白血病病料样品178份,用ALV-A、ALV-B和ALV-J特异性引物,通过PCR方法进行检测。结果表明,8个省的35个鸡场的124份病料中检出了ALV-J(69.7%);25份病料中检出了ALV-A(13.9%);7份病料中检出了ALV-B(3.9%)。14个分离毒株env基因氨基酸同源性为84.3%～99%;与J亚群原型毒株HPRS-103的氨基酸序列同源性为87.3%～98.2%;与其它J亚群env基因氨基酸序列同源性为83%～97.4%。遗传进化分析表明,14个ALV-J分离株分别分属于不同的分支。其中,LJL09DH02分离株与其它分离株及参考毒株的的亲缘关系最远,与HPRS-103的氨基酸同源性仅为87.3%。另外4个分离株的env基因与HPRS-103的氨基酸同源性低于93%,其余9株与HPRS-103的同源性较高(96.6%以上)。该调查结果表明,我国目前ALV的感染主要以J亚群为主,ALV-A和B同时存在。     

Detection and localization of naturally transmitted avian leukosis subgroup J virus in egg-type chickens by in situ PCR hybridization

Avian leukosis virus (ALV) subgroup J (ALV-J) is an exogenous ALV and causes myeloid leukosis in meat-type chickens. We have previously reported the isolation and identification of ALV-J in commercial layer flocks from 12 farms in northern China. In this report, we further characterized this virus by in situ polymerase chain reaction (PCR) hybridization in various affected organs of chickens from six of the 12 farms. A routine method for hybridization of nucleic acid uses radioactive probe, such as a P32-labelled probe. We found that the non-radioactive digoxigenin (DIG) probe is sensitive enough to detect the nucleic acid of virus in chicken tissues. We used a pair of published primers (H5/H7) specific to the gp85 envelope gene and 3' region of pol gene of prototype ALV-J strain HPRS-103. The total RNA extracted from tumour, bone marrow, oviduct, liver and spleen of the diseased chickens from six commercial flocks, and cDNA was successfully amplified. Using the primers and cDNA, we obtained an ALV-J-specific cDNA probe of 545 bp in length by PCR. In situ PCR with H5/H7 primers was carried out in the paraffin sections from tissues of the diseased chickens, followed by in situ hybridization using the DIG-labelled cDNA probe. Positive hybridization signals were detected in the cytoplasm of paraffin sections of tumours and other organ tissues. The intensity of the signals was documented using an image analysis system measuring integral optical density (IOD). The IOD values for tissue sections treated by in situ PCR hybridization are significantly higher than that by in situ hybridization alone (P < 0.01). These data taken together suggest that in situ PCR hybridization is a more sensitive technique for detection of ALV-J in tissue sections.     

Susceptibility of various parental lines of commercial white leghorn layers to infection with a naturally occurring recombinant avian leukosis virus containing subgroup B envelope and subgroup J long terminal repeat

Chickens from seven different parental lines of commercial White Leghorn layer flocks from three independent breeders were inoculated with a naturally occurring avian leukosis virus (ALV) containing an ALV-B envelope and an ALV-J long terminal repeat (LTR) termed ALV-B/J. Additional groups of chickens from the same seven parental lines were inoculated with ALV-B. Chickens were tested for ALV viremia and antibody at 0, 4, 8, 16, and 32 wk postinfection. Chickens from all parental lines studied were susceptible to infection with ALV-B with 40%-100% of inoculated chickens positive for ALV at hatch following embryo infection. Similarly, infection of egg layer flocks with the ALV-B/J recombinant virus at 8 days of embryonation induced tolerance to ALV with 86%-100% of the chickens viremic, 40%-75% of the chickens shedding virus, and only 2/125 (2%) of the chickens producing serum-neutralizing antibodies against homologous ALV-B/J recombinant virus at 32 wk postinfection. In contrast, when infected with the ALV-B/J recombinant virus at hatch, 33%-82% of the chickens were viremic, 28%-47% shed virus, and 0%-56% produced serum-neutralizing antibodies against homologous ALV-B/J recombinant virus at 32 wk postinfection. Infection with the ALV-B/J recombinant virus at embryonation and at hatch induced predominately lymphoid leukosis (LL), along with other common ALV neoplasms, including erythroblastosis, osteopetrosis, nephroblastomas, and rhabdosarcomas. No incidence of myeloid leukosis (ML) was observed in any of the commercial White Leghorn egg layer flocks infected with ALV-B/J in the present study. Data suggest that the parental line of commercial layers may influence development of ALV-B/J-induced viremia and antibody, but not tumor type. Differences in type of tumors noted in the present study and those noted in the field case where the ALV-B/J was first isolated may be attributed to differences in the genetics of the commercial layer flock in which ML was first diagnosed and the present commercial layer flocks tested in the present study.     

Comparison of Chinese field strains of avian leukosis subgroup J viruses with prototype strain HPRS-103 and United States strains

Eight Chinese field strains of subgroup J avian leukosis viruses (ALV-J) were isolated from broilers or parent stocks during January 1999 to April 2001. One strain, SD9902, was an acute transforming virus, able to induce typical myelocytomatosis in 22-38 days after inoculation of 1-day-old meat-type chicks. The envelope protein and 3'-untranslated region (UTR) of the eight field strains were compared with the U.K. prototype HPRS-103 and several U.S. field strains isolated in 1993-97. All Chinese strains shared an almost identical deletion with the U.S. strain 4817 in the E element region of 3'-UTR when compared with the prototype HPRS-103, indicating that they have a very close phylogenic relationship. Every year, China has to import grandparent stocks of meat-type chickens, mainly from the United States. Chinese isolates should represent a part in the phylogenic tree of U.S. ALV-J evolution. Envelope protein gp85 amino acid sequence analysis demonstrated that, interestingly, all recent Chinese isolates were more closely related to HPRS-103 and the earliest U.S. isolates but not to the late U.S. isolates. The result implies that envelope gp85 may not have diverged from prototype and older strains. It is also possible that some recently imported birds could have been infected by the older viruses that were introduced in the late 1990s.     

Detection of subgroup J avian leukosis virus infection in Australian meat-type chickens

OBJECTIVE: To determine the extent of avian leukosis virus subgroup J (ALV-J) infection in Australian broiler breeder flocks, using virus isolation and molecular biological detection. Any resultant ALV-J viral isolates to be characterised by neutralisation cross testing in order to determine antigenic relationships to overseas isolates of ALV-J. STUDY DESIGN: Samples of blood, feather pulp, albumen and tumours were obtained from broiler breeder flocks which represented four genetic strains of meat chickens being grown in Victoria, South Australia, NSW and Queensland. Dead and ailing birds were necropsied on farm and samples were collected for microscopic and virological examinations. Virus isolation was carried out in C/O and DF-1 CEF cultures and ALV group specific antigen was detected in culture lysates using AC-ELISA. Micro-neutralisation assay was used for antigenic characterisation of selected isolates. Genomic DNA was isolated from cultured cells, tumours and feather pulp. ALV-J envelope sequences were amplified by PCR using specific ALV-J primers while antibodies against ALV-J were detected by ELISA. RESULTS: A total of 62 ALV-J isolates were recovered and confirmed by PCR from 15 (31.3%) of 48 breeder flocks tested. Antibody to ALV-J was detected in 20 (47.6%) of the 42 flocks tested. Characteristic lesions of myeloid leukosis caused by ALV-J were found in affected flocks. The gross pathological lesions were characterised by skeletal myelocytomas located on the inner sternum and ribs, neoplastic enlargement of the liver, and in some cases gross tumour involvement of the spleen, kidney, trachea, skeletal muscles, bone marrow, skin and gonads. Microscopically, the tumours consisted of immature granulated myelocytes, and were present as focal or diffuse infiltrations in the affected organs. Virus micro-neutralisation assays demonstrated antigenic variation among Australian isolates and to overseas strains of ALV-J. CONCLUSION: ALV-J infection was prevalent in Australian broiler breeder flocks during 2001 to 2003. Australian isolates of ALV-J show a degree of antigenic variation when compared to overseas isolates.     

血管瘤相关J亚群禽白血病病毒ZH-08株的分离与全基因组序列测定

本研究从临床表现为典型血管瘤型禽白血病病例的广东某肉种鸡场的病鸡中,分离到1株J亚群禽白血病病毒(ALV-J),命名为ZH-08。利用ELISA抗原检测、PCR和间接免疫荧光试验对分离株进行鉴定,结果都呈阳性。依据ALV-J原型株HPRS-103前病毒全基因组序列设计并合成3对引物,采用分段扩增的方法完成了分离株的全基因组序列测定。结果显示该分离株基因组序列全长7 597 bp,与已公开的全基因组序列大小比较略有差异,但符合典型的复制完全型反转录病毒的基因组结构,基因序列中不含已知致癌基因。将该分离株的亚群特异性gp85基因序列与国内外各参考株相应序列进行相似性比较,发现ZH-08与YZ9901株相似性最高(93.7%)。基于gp85核苷酸序列的系统进化分析表明:ZH-08株与SD07LK1株的亲缘关系最近。本研究为该毒株的生物学特性以及致病机制研究奠定了基础。     

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.     

IMC10200株ALV-J实验诱发禽骨髓性白血病的研究

对分离自肉种鸡群亚临床感染的J亚群禽白血病病毒IMC1o200株进行了实验感染诱发禽骨髓性白血病的病理学研究.IMC10200株J亚群禽白血病病毒尿囊腔接种11日龄肉鸡胚和SPF蛋鸡胚,孵出后跟踪观察.9周龄时随机抽检感染鸡,感染肉鸡特异引物PCR检测全部为阳性;组织病理学观察感染鸡无明显病变.至21周龄时感染肉鸡出现第一例典型骨髓性白血病病例;感染SPF蛋鸡未见明显J亚群禽白血病相关病变.     

Response of white leghorn chickens of various genetic lines to infection with avian leukosis virus subgroup J

In Experiment 1, chickens from various white leghorn experimental lines were inoculated with strain ADOL-Hcl of subgroup J avian leukosis virus (ALV-J) either as embryos or at 1 day of age. At various ages, chickens were tested for ALV-J induced viremia, antibody, and packed cell volume (PCV). Also, at 4 and 10 wk of age, bursal tissues were examined for avian leukosis virus (ALV)-induced preneoplastic lesions with the methyl green-pyronine (MGP) stain. In Experiment 2, chickens harboring or lacking endogenous virus 21 (EV21) were inoculated with strain ADOL-Hcl of ALV-J at hatch. All embryo-inoculated chickens in Experiment 1 tested positive for ALV-J and lacked antibody throughout the experimental period of 30 wk and were considered viremic tolerant, regardless of line of chickens. By 10 wk of age, the incidence of ALV-J viremia in chickens inoculated with virus at hatch varied from 0 (line 0 chickens) to 97% (line 1515); no influence of ALV-J infection was noted on PCV. Results from microscopic examination of MGP-stained bursal tissues indicate that ALV-J can induce typical ALV-induced transformation in bursal follicles of white leghorn chickens. Lymphoid leukosis and hemangiomas were the most common ALV-J-induced tumors noted in chickens in Experiment 1. At termination of Experiment 2 (31 wk of age), 54% of chickens harboring EV21 were viremic tolerant compared with 5% of chickens lacking EV21 after inoculation with ALV-J at hatch. The data indicate that genetic differences among lines of white leghorn chickens, including the presence or absence of EV21, can influence response of chickens to infection with ALV-J.     

Response of white leghorn chickens to infection with avian leukosis virus subgroup J and infectious bursal disease virus

The effects of viral-induced immunosuppression on the infectious status (viremia and antibody) and shedding of avian leukosis virus (ALV) were studied. Experimental white leghorn chickens were inoculated with ALV subgroup J (ALV-J) and infectious bursal disease virus (IBDV) at day of hatch with the ALV-J ADOL prototype strain Hcl, the Lukert strain of IBDV, or both. Appropriate groups were exposed a second time with the Lukert strain at 2 wk of age. Serum samples were collected at 2 and 4 wk of age for IBDV antibody detection. Samples for ALV-J viremia, antibody detection, and cloacal shedding were collected at 4, 10, 18, and 30 wk of age. The experiment was terminated at 30 wk of age, and birds were necropsied and examined grossly for tumor development. Neoplasias detected included hemangiomas, bile duct carcinoma, and anaplastic sarcoma of the nerve. Control birds and IBDV-infected birds were negative for ALV-J-induced viremia, antibodies, and cloacal shedding throughout experiment. By 10 wk, ALV-J-infected groups began to develop antibodies to ALV-J. However, at 18 wk the incidence of virus isolation increased in both groups, with a simultaneous decrease in antibody levels. At 30 wk, 97% of birds in the ALV-J group were virus positive and 41% were antibody positive. In the ALV-J/IDBV group, 96% of the birds were virus positive at 30 wk, and 27% had antibodies to ALV-J. In this study, infection with a mild classic strain of IBDV did not influence ALV-J infection or antibody production.     

禽白血病病毒J亚群自然感染商品蛋鸡致瘤性新特征

2009年8月,山东省邹城市某海兰褐蛋鸡群,160日龄发病,死亡率为7％.患鸡经大体剖检、病理组织学、PCR和免疫组织化学等检测,确诊为禽白血病病毒J亚群(ALV-J)感染.病理组织学检测发现,病鸡单独患血管瘤,或髓细胞瘤和纤维肉瘤多发性出现,由ALV-J自然感染引起同一鸡体出现髓细胞瘤和纤维肉瘤尚属国内外首次报道.肝脏研磨接种DF-1细胞培养7d后传3代,细胞无病变,ELISA检测感染细胞上清ALV p27抗原阳性,进一步确诊此鸡群为ALV感染.对病变严重的鸡进行病毒分离及ALV-J gp85基因同源性比较显示与原型株HPRS-103的同源性最高,达94.1％.本研究丰富了ALV-J感染的临床诊断依据,并为ALV-J在我国蛋鸡群中多潜能致瘤机制的研究提供了科学基础.     

Genetic diversity and phylogenetic analysis of glycoprotein GP85 of ALV-J isolates from Mainland China between 1999 and 2010: coexistence of two extremely different subgroups in layers

Avian leukosis virus subgroup J (ALV-J), first isolated in 1989, preferentially infects meat-type birds. However, layer flocks in China have experienced outbreaks of this virus since 2008. To understand the genetic diversity of ALV-J in Chinese layers, we compared and analyzed the GP85 gene sequences of 106 ALV-J isolates that were isolated between 1999 and 2010 in Mainland China. The GP85 gene sequences of 41 layer isolates collected from 9 provinces of China between 2008 and 2010 belonged to two separate, highly diverse subgroups and were differentiated from meat-type chicken isolates. When compared to all meat-type isolates from China, Subgroup 1 exclusively contained current layer isolates and seemed to be dominant; all the isolates in this subgroup exhibited gene diversity, and many unique amino acid mutations were present. In contrast, the viruses in Subgroup 2 were perfectly conserved and shared high identity with the prototype meat-type chicken ALV-J strain HPRS-103. The two subgroups contained only two concurrent mutations at the same position. Moreover, most of the isolates in Subgroup 1 had two additional glycosylation sites (at positions 101 and 191) when compared with those in Subgroup 2. Our study provides evidence for the coexistence of two extremely different ALV-J subgroups in Chinese layers from 2008 to 2010, supporting the need for vaccine development and purification measures to prevent ALV-J infection in layers in China.     

J亚群禽白血病病毒安徽分离株的鉴定及其gp85基因序列分析

从安徽省的黄羽肉鸡和罗曼蛋鸡中各分离鉴定出1株J亚群禽白血病病毒,克隆获得了2条相应的gp85基因序列,并与参考毒株进行序列比对。结果表明,两分离毒株与J亚群参考毒株同源性为82.1%~99.4%,分离毒株之间同源性为85.4%。其中肉鸡分离毒株与J亚群原型毒株HPRS-103同源性为97.1%,与J亚群国内毒株SD09TA04、SDYC02J同源性均为99.4%;蛋鸡分离毒株与HPRS-103的同源性为89.0%,与SD09TA04和SDYC02J同源性仅为88.6%。两分离毒株的gp85氨基酸序列出现突变和缺失,在高变区hr1、hr2变异明显。进化分析进一步表明,2个分离毒株亲缘关系较远,可能来源于不同的原始病毒株。     

应用组织芯片免疫组化法检测ALV-J

禽白血病J亚群（ALV-J）是英国的Payne和他的同事们在20世纪90年代初从肉鸡中分离出来的新亚群，主要引起肉鸡的骨髓瘤白血病。自1999年，我国一些肉用型种鸡场陆续发生了禽白血病J亚群。并且近几年来，ALV-J已从最初只引起肉种鸡发病开始向蛋鸡及中国地方种鸡蔓延。组织芯片技术是一种新型特殊的生物芯片技术，它能明显提高工作效率，减少实验误差。本研究将组织芯片技术和免疫组化染色结合起来，用特异性抗ALV-J囊膜蛋白gp85的单克隆抗体来检测发病鸡只的各组织器官的组织切片。在肝脏、脾脏、肾脏、卵巢、腺胃、骨髓、髓细胞瘤组织均检出病毒阳性抗原。结果表明组织芯片技术和免疫组化染色相结合为临床诊断ALV-J提供了一个高通量、敏感的检测方法。     

Avian leukosis virus subgroup J infection profiles in broiler breeder chickens: association with virus transmission to progeny

Profiles of infection with avian leukosis virus subgroup J (ALV-J) and factors that predict virus transmission to progeny were studied. Eggs from an infected broiler breeder flock were hatched at the laboratory. The flock was reared in a floor pen, transferred to laying cages at 22 wk, and inseminated to produce fertile eggs. A cohort of 139 chickens was tested at frequent intervals over a 62-wk period for virus, viral antigens, or antibodies in plasma, cloacal swabs, egg albumen, and embryos. Virus was detected in 7% of chicks at hatch but spread rapidly so that virtually all chicks became infected between 2 and 8 wk of age. Mortality due to myeloid leukosis and related tumors was 22%. Over 40% of the chicks developed persistent infections, whereas the remainder experienced transient infections. Five types of infection profiles were recognized. Novel responses included hens that were positive for virus intermittently or started late in life to shed viral antigens into the cloaca. ALV-J was isolated from 6% of 1036 embryos evaluated between 26 and 62 wk. However, over 90% of the virus-positive embryos were produced between 29 and 34 wk of age. Of 80 hens that produced embryos, 21 produced at least one infected embryo and were identified as transmitters. All but one transmitter hen would have been detected by a combination of viremia, cloacal swab, and albumen tests conducted between 18 and 26 wk. However, virus was transmitted to embryos from hens that were not persistently viremic or that rarely shed viral group-specific antigen into the albumen of their eggs. Intermittent patterns of both antigen shedding and virus transmission to embryos were observed in some hens. These results validate current screening procedures to identify potential transmitter hens and provide some suggestions for improvement but also show that identification of all transmitter hens by such procedures is unlikely. Thus, eradication programs based solely on dam testing may be less effective than those where dam testing is combined with procedures to mitigate early horizontal transmission in progeny chicks.     

Haemangiomas, leiomyosarcoma and myeloma caused by subgroup J avian leukosis virus in a commercial layer flock

An outbreak of simultaneously occurring haemangiomas, leiomyosarcoma and myeloma was observed in a commercial layer flock in China. The sick chickens were extremely thin and dehydrated. Scattered haemangiomas were found on the claws, breast and wings. At necropsy, haemangiomas and some other nodular tumours were also found in the internal organs. In addition, diffuse enlargement of the liver and spleen appeared in some birds. Histopathologically, haemangiomas were typically cavernous haemangiomas and haemangioendothelioma. In the diffusely swollen liver and spleen, multifocal or widespread marrow tumour cells filled with ball-like acidophilic particles in cytosol were observed, which are the characteristic pathological changes of avian myelocytomatosis. The nodular tumour cells formed by muscle bundles were of variable size, irregular shape, poorly differentiated and malaligned. Immunohistochemistry for vimentin, cytokeratin, actin (smooth muscle) and actin (sarcomeric) and Masson's staining confirmed the different cell lineage of the nodular tumour, thus leading to the diagnosis of leiomyosarcoma. The seroprevalence of avian leukosis subgroup J (ALV-J) antibodies was 13.46% (7/52), while ALV-A/B and reticuloendotheliosis virus (REV) antibodies were not detectable. The DF-1 cells inoculated by virus extracted from liver samples from 24 infected chickens were cultured and the group-specific antigen (GSA) was identified by ELISA. All samples were positive for ALV, which was further identified as ALV-J by immunofluorescence assay (IFA). PCR analysis revealed that three isolates of ALV-J proviral sequence were close to the HPRS-103 prototype strain and other Chinese field strains isolated in recent years, while one isolate (DP01) had a lower homology with them. This is the first report that ALV-J infection caused the simultaneous occurrence of haemangiomas, leiomyosarcoma and myeloma in a commercial layer flock.