Avian Leukosis Virus Subgroup J Isolates Research Articles

Full-length cDNA sequence analysis of 85 avian leukosis virus subgroup J strains isolated from chickens in China during the years 1988–2018: coexistence of 2 extremely different clusters that are highly dependent upon either the host genetic background or the geographic location

During the process of transmission and spread of avian leukosis virus subgroup J (ALV-J) in chickens worldwide, the viral genome is constantly changing. A comprehensive and systematic study of the evolutionary process of ALV-J in China is needed. In this study, we amplified the full-length viral cDNA sequences of 16 ALV-J isolates of Yellow-chicken origin and analyzed and compared these sequences with another 69 ALV-J strains isolated during the years 1988–2018. These isolates were then sorted into 2 clusters: cluster I included isolates that mainly originated from the layers and White-feather broilers from northern China; cluster II included isolates mainly from the Yellow-chicken, most of them being from southern China. According to the sequence homologies of the whole genome and gag, pol, gp85, and gp37 genes, the ALV-J strains are more likely to randomly change in different directions from the original strain HPRS-103 as time passes. The results of entropy analysis of the sequences of gag, pol, and env revealed that the env gene had the largest variation, and the gag gene nonconserved sites are mainly concentrated in p19, p10, and p12. In addition, 84.71% (72/85) of the isolates had the 205-nucleotide (nt) deletion in the 3′UTR region, and 30.59% (26/85) of the isolates had the 125-nt to 127-nt deletion in the E element. Our study provides evidence for the coexistence of 2 extremely different clusters of ALV-J prevailing in China and in some other countries during the period of 1988–2018 and implies that the clusters are highly dependent on the host genetic background and the geographic location.

Characterization of avian leukosis virus subgroup J isolated between 1999 and 2013 in China

Avian leukosis virus subgroup J (ALV-J) has successively infected white feather chickens, layer hens, cultivated yellow chickens, and indigenous chickens; infection rates and tumorigenicity have attracted increasingly extensive attention in China. To clarify the correlation of the epidemiological phenomenon of ALV-J with the evolution of envelope protein gp85, 140 strains of ALV-J isolated from chickens with different genetic backgrounds from 1999 to 2013 were compared. The homology of the gp85 protein and genetic genealogical relationships between 140 strains of ALV-J and the prototype strain HPRS-103, as well as between the same ALV-J strains and 8 American isolates, were analyzed and compared. The results showed that there was no significant difference in the variation range of homology of the gp85 protein between the prototype HPRS-103 and ALV-J isolates from different genetic backgrounds and different years. However, genetic pedigree analysis showed that virus strains that isolated from the same type of chickens remained close to each other on the phylogenetic tree, which means that there was a correlation between the genetic background of infected chickens and virus strains. Further analysis of amino acid sequences also found similar results and revealed that unique amino acid sites were formed in chickens with different genetic backgrounds, which proved that ALV-J could adapt to the new host through amino acid variation. Genetic sequence phylogenetic tree analysis was more representative than sequence homology comparisons for assessing ALV-J correlations. These conclusions contributed to the control and prevention of ALV infection. ALV-J is still prevalent in Chinese indigenous chickens, more attentions should be given to fulfill the purification.

Gp85 genetic diversity of avian leukosis virus subgroup J among different individual chickens from a native flock

To compare the genetic diversity and quasispecies evolution of avian leukosis virus (ALV) among different individuals, 5 chickens, raised in Shandong Provice of China, were randomly selected from a local chicken flock associated with serious tumor cases. Blood samples were collected and inoculated into chicken embryo fibroblast and DF-1 cell lines for virus isolation and identification, respectively, of Marek's disease virus (MDV), reticuloendotheliosis virus (REV), and ALV. Five strains of ALV subgroup J (ALV-J) were identified, and the gp85 gene from each strain was amplified and cloned. For each strain, about 20 positive clones of gp85 gene were selected for sequence analyses and the variability of the quasispecies of the 5 strains was compared. The results showed that the nuclear acid length of gp85 gene of 5 ALV-J isolates is 921 bp, 921 bp, 924 bp, 918 bp, and 912 bp respectively, and amino acid homologies of different gp85 clones from the 5 ALV-J strains were 99.3 to 100%, 99.3 to 100%, 99.4 to 100%, 98.4 to 100%, 99.0 to 100%, respectively. The proportions of dominant quasispecies were 65.0%, 85.0%, 85.0%, 50.0%, 84.2%, respectively, and homology of the gp85 among these dominant quasispecies was 89.2 to 92.5%. These data demonstrated the composition of the ALV-J quasispecies varied among infected individuals even within the same flock, and the dominant quasispecies continued to evolve both for their proportion and gene mutation.

Molecular epidemiology of J-subgroup avian leukosis virus isolated from meat-type chickens in southern China between 2013 and 2014.

Members of avian leukosis virus subgroup J (ALV-J) cause various diseases associated with tumor formation and decreased fertility, resulting in major economic losses in the poultry industry worldwide. To assess the status of ALV-J infection in meat-type chickens in southern China, the molecular epidemiology of ALV-J strains was investigated. A total of 265 clinical samples collected from southern China from 2013 to 2014 were investigated in this study for the presence of ALV-J, which resulted in 12 virus isolates. Phylogenetic analysis showed that 91.7% (11/12) of the ALV-J isolates have possessed high homology to Chinese layer isolates and belong to one subgroup. One of the ALV isolates (designated GD1411-1) was relatively closely related to the ALV-J broiler isolates, indicating that the GD1411-1 isolate might be a transition strain. Several unique nucleotide substitutions in gp85 and the U3 region were detected in all 12 ALV-J isolates. This study provides some interesting information on the molecular characterization of ALV-J isolates. These findings will be beneficial for understanding of the pathogenic mechanism of ALV-J infection.

Detection and molecular characterization of J subgroup avian leukosis virus in wild ducks in China.

To assess the status of avian leukosis virus subgroup J (ALV-J) in wild ducks in China, we examined samples from 528 wild ducks, representing 17 species, which were collected in China over the past 3 years. Virus isolation and PCR showed that 7 ALV-J strains were isolated from wild ducks. The env genes and the 3′UTRs from these isolates were cloned and sequenced. The env genes of all 7 wild duck isolates were significantly different from those in the prototype strain HPRS-103, American strains, broiler ALV-J isolates and Chinese local chicken isolates, but showed close homology with those found in some layer chicken ALV-J isolates and belonged to the same group. The 3′UTRs of 7 ALV-J wild ducks isolates showed close homology with the prototype strain HPRS-103 and no obvious deletion was found in the 3′UTR except for a 1 bp deletion in the E element that introduced a binding site for c-Ets-1. Our study demonstrated the presence of ALV-J in wild ducks and investigated the molecular characterization of ALV-J in wild ducks isolates.

Isolation, identification, and phylogenetic analysis of two avian leukosis virus subgroup J strains associated with hemangioma and myeloid leukosis

Cases of myeloid leukosis and hemangioma associated with avian leukosis virus subgroup J (ALV-J) are becoming more frequent in China in commercial layer chickens and breeders of egg-type chickens. In this study, two strains of ALV-J (SCAU11-H and SCAU11-XG) associated with hemangioma and myelocytoma were isolated from commercial broiler breeder animals in 2011. Their full-length proviral sequences were analyzed, revealing several unique genetic differences between the two isolates, and suggesting that the two viruses were derived from two distinct lineages. Strain SCAU11-H showed high sequence homology to early Chinese isolates associated with hemangioma, while strain SCAU11-XG was genetically closer to the prototype strain, HPRS-103. The complete genomic nucleotide sequences of SCAU11-H and SCAU11-XG were 7471 bp and 7727 bp in length, respectively. They shared 94.8% identity with each other, and had 94.0-96.8% nucleotide identity to ALV-J reference isolates. Homology analysis of the env, pol, and gag genes of the two isolates and other references strains showed that the gag and pol genes of the two viruses were more conserved than the env gene. In addition, the two isolates had significant deletions and substitutions in their 3'-UTR regions, compared to HPRS-103. These results suggest that the env gene and the 3'-UTR regions in these ALV-J isolates have evolved rapidly, and might be involved in the oncogenic spectrum of ALV-J. The results of this study contribute to our further study of the relationship between ALV integration patterns and multi-pathotypes associated with ALV-J.

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.

Avian Leukosis Virus Subgroup J in Layer Chickens, China

To the Editor: In recent years, cases of avian leukosis virus subgroup J (ALV-J) infection and tumors in commercial layer chickens and breeders of egg-type chickens have been emerging in the People’s Republic of China. ALV-J was first isolated from meat-type chickens with myeloid leukosis in 1988. Although egg-type chickens have been experimentally infected with ALV-J to induce tumors (1), field cases of ALV-J infection and tumors in commercial layer chickens were not found worldwide until 2004 (2). ALV-J has recently been found to have induced various tumors and caused production problems in commercial layer flocks and local chicken breeds in China (2,3). Many field cases of ALV-J infection and tumors have occurred in 15- to 29-week-old egg-type chickens in several provinces. Affected flocks had dramatically reduced egg production and hemorrhage in the skin surrounding the phalanges and feather follicles. Some birds had gray-white nodules in the liver, spleen, or kidneys, and liver and spleen were enlarged up to several times their normal size. Morbidity rates for some flocks reached 60%, and mortality rates for some flocks were >20%. Clinical samples from livers, spleens, whole blood, and tumors were collected from chickens in different provinces and sent for laboratory diagnosis. Results showed that the predominant virus in the samples was ALV-J. During 2007–2009, we conducted an epidemiologic investigation of ALV in layer flocks in China. All virus isolation was performed in DF-1 cells. Briefly, 233 clinical samples were collected from 44 layer flocks in different provinces and used to inoculate subconfluent cell cultures containing Dulbecco modified essential medium supplemented with 10% (vol/vol) fetal bovine serum and antimicrobial drugs. After a 7–9 day incubation period, the cells were frozen and thawed 3×. A group-specific antigen-capture ELISA was used to identify ALV. After proviral DNA was extracted directly from infected cell culture or tumors, PCR with strain-specific primers was used to detect ALV-A, ALV-B, or ALV-J (4). Of these samples, 150 (64.4%) were ALV-J positive, 28 (12.1%) were ALV-A positive, and 8 (3.4%) were ALV-B positive. Phylogenetic analysis showed an 87.3%–98.2% aa sequence identity of env genes in all ALV isolates compared with the HPRS-103 strain (5). All isolates had complete repeated transmembrane deletion and partial direct repeat–1 deletion but contained an intact E element. A mutation was found in the enhancer and promoter region of the U3 region in the 3′ long terminal repeat; this mutation is not found in ALV-J isolated from broiler chickens (6). The newly isolated ALV-J strain from layer chickens was used to examine the pathogenicity in 1-day-old White Leghorn specific pathogen–free chicks soon after hatching in separate incubators and rooms in the experimental animal house facilities at Harbin Veterinary Research Institute, Harbin, China. The chicks were inoculated intraabdominally with a 1,000-unit 50% tissue-culture infective dose of ALV-J propagated in the DF-1 cells. Blood samples were collected to check for viremia at 10 weeks of age. Experimental birds were reared until 27–30 weeks of age. Prolonged viremia developed in 15 (50%) of 30 chicks; hemangiomas developed in the skin surrounding phalanges and in the liver of 3 (10%); and myeloid leukosis, detected by gross or histologic examination, developed in 10 (30.3%). A previous study showed that meat-type birds infected with ALV-J retained a high level of viremia over their lifetime (7) but that layer chickens cleared the infection within a few weeks. Our study demonstrated that ALV-J infection can cause disease in layer chickens and can induce tumors and long-lasting viremia. For this reason, disease caused by ALV-J in layer chickens in China should be further investigated. Because ALV-J is vertically transmitted from dam to progeny by the embryo, it represents a potential threat for humans who receive vaccines that are produced in chicken embryonic fibroblasts or embryonated eggs (e.g., yellow fever vaccine and measles and mumps vaccine) (8). An effective vaccine against ALV is not available. Eradication of ALV-J has been difficult because of substantial genetic and antigenic variation among ALV-J isolates as well as high levels of vertical and horizontal transmission (9,10). Therefore, effective prevention and elimination measures should be developed as soon as possible.

Recombinant avian leukosis viruses of subgroup J isolated from field infected commercial layer chickens with hemangioma and myeloid leukosis possess an insertion in the E element

BackgroundFive isolates (JS09GY2, JS09GY3, JS09GY4, JS09GY5, and JS09GY6) of avian leukosis virus subgroup J (ALV-J) were isolated from six infected commercial layer flocks displaying both hemangioma and myeloid leukosis (ML), which shared the same parental line, in China in 2009.ResultsAll six of the commercial layer chickens examined showed hemangiomas on their body surface or feet. Some developed hemangiomas in their internal organs, causing hepatorrhexis and blood loss. Histopathologically different stages of hemangiomas with ML in the liver, heart, and spleen, were observed. Five viral isolates were obtained from infected DF1 cells incubated with the spleen tissue or serum of the birds from the six flocks. By full genome sequences analysis, a 19-nucleotide repeat sequence was identified in the primer binding site (PBS)-leader region of isolates JS09GY3 and JS09GY6, located between sites 249 and 250 according to the sequence of reference strain HPRS103, and also present in Rous sarcoma virus strain Schmidt–Ruppin B (RSV-SRB), Rous associated virus type 1 (RAV-1), and Rous associated virus type 2 (RAV-2). The predicted Gp85 proteins of isolates JS09GY2, JS09GY3, JS09GY5, and JS09GY6 were highly variable. Interestingly, the E elements of these four examined isolates showed a key deletion at site 30, which produced a new c-Ets-1 binding site. An 11-bp insertion was also found in the E element of isolate JS09GY3 located between bp 66 and 67 according to the sequence of reference strain HPRS103, while almost all previously reported Chinese strains showed an almost identical deletion of 127 bp in the same region.ConclusionsFive ALV-J isolates were obtained from six field infected commercial layer chickens. Coexistence of hemangioma and ML were observed in these infected cases both macro- and microscopically. Complete proviral genome sequences of two isolates (JS09GY3 and JS09GY6) and the partial sequences of the other two isolates (JS09GY2 and JS09GY5) were determined. The isolates were found to be recombinants of ALV-J with a PBS-leader sequence originating from other retroviruses. The Gp85 protein with an amino acid deletion, a contiguous 11-bp insertion mutation in the E element, and a novel binding site, were noted in the proviral genomes.

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

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. 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. 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. 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.

The effects of avian leukosis virus subgroup J on broiler chicken performance and response to vaccination.

The effects of avian leukosis virus subgroup J (ALV-J) infection on meat-type chickens reared in a simulated commercial setting were evaluated. Each of three ALV-J isolates was evaluated with both simulated horizontal transmission (SHT) and simulated vertical transmission (SVT). Mortality, morbidity, disease condemnations, and feed conversions were increased and body weights at processing were decreased in ALV-J infected birds as compared to sham inoculated hatch mates. The adverse effects of ALV-J infection were more severe in birds exposed by SVT than in birds exposed by SHT. At 8 weeks of age response to vaccination for infectious bronchitis virus and Newcastle disease virus or prior exposure to a pathogenic reovirus was assessed in the ALV-J and sham inoculated broiler chickens by challenge studies. Although not statistically significant, an overall trend of decreased protection to challenge after vaccination, or prior exposure, was observed in the ALV-J inoculates as compared to sham inoculated hatch mates. Differences in vaccine response were most evident in groups inoculated with ALV-J by the SVT route.

Polymerase chain reaction for detection of avian leukosis virus subgroup J in feather pulp.

Feather pulp from experimentally infected chickens was used as a source of DNA for polymerase chain reaction (PCR) amplification of avian leukosis virus subgroup J (ALV-J) proviral DNA. A primer set that produces a large amplicon (approximately 2,125) was used to detect ALV-J proviral DNA. This primer set was used in lieu of previously published primers because it allows for sequencing of the entire envelope gene and because it was able to detect diagnostically a number of North American ALV-J isolates that could not be detected with previously published primers and PCR conditions. ALV-J proviral DNA was detected in feather pulp at 7 days of age in more than 90% of birds infected as embryos and 7 days postinoculation in over 50% of chickens infected at 3 days of age. The results obtained with PCR on feather pulp were compared with those of virus isolation. In the embryo-inoculated birds, the percentages of agreement between PCR and virus isolation were 92.5% at 7 days of age and 100% at 28, 42, 49, and 56 days of age. However, the overall sensitivity of virus isolation in embryo-infected birds was higher, particularly at 7 and 56 days of age. In chickens inoculated at 3 days of age, the percentages of agreement of detection between PCR and virus isolation ranged from 75% at 10 days of age to 100% at 42 days of age. Agreement of negative results of ALV-J detection by PCR and virus isolation in chickens infected posthatch ranged between 66.6% and 100% between the ages of 10 and 42 days. Virus isolation requires chicken embryo fibroblasts of specific genetic lines, and the process takes onaverage 7-9 days. Aseptic collection of blood and tissues for virus isolation and molecular detection of ALV-J requires sterile necropsy instruments as well as syringes and needles for each individual chicken, whereas sterile microcentrifuge tubes and gloves are the only equipment necessary for aseptic feather pulp collection for ALV-J detection by PCR. PCR-based detection of ALV-J in feather pulp is especially suitable when ALV-J infection must be diagnosed rapidly and unequivocally without killing the chicken(s) and in situations where crucial reagents or suitable virus propagation substrates are not readily available for isolation and propagation of ALV-J in cell culture.

A Genetically Engineered Cell Line Resistant to Subgroup J Avian Leukosis Virus Infection (C/J)

A cell line (DF-1\\J) expressing the envelope protein isolated from the ADOL-Hc1 strain of the avian leukosis virus subgroup J (ALV-J) was used to analyze receptor interference to six different isolates of ALV-J as well as ALV subgroups A–D. The traditional gag-specific enzyme-linked immunosorbent assay (ELISA) as well as flow cytometry was used to evaluate viral infection. The parental cell line (DF-1) was susceptible to all ALV subgroups tested while the DF-1\\J cell line was selectively resistant to the subgroup J isolates. The DF-1\\J cell line was resistant to infection by all six ALV-J isolates as determined using the gag-specific ELISA. There was no interference with the other ALV subgroups (A–D) induced by the expression of the ADOL-Hcl envelope. The ALV-J isolates used in this analysis are serologically distinct when analyzed by flow cytometry. Convalescent sera to ADOL-Hcl cross-reacts with all of the ALV-J isolates tested; however, sera to HPRS-103 did not bind to four of the six isolates. Based on the intensity and differential binding of these antisera using flow cytometry, the six ALV-J isolates used can be grouped into four categories. Thus the DF-1\\J cell line is resistant to infection by a serologically and genetically diverse group of ALV-J isolates and should be useful as a diagnostic tool.