Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China.
ABSTRACT: In recent years, cases of avian leukosis virus (ALV) infection have become more frequent in China. We isolated 6 ALV strains from yellow feather broiler breeders in south China from 2014 to 2016. Their full genomes were sequenced, compared, and analyzed with other reference strains of ALV. The complete genomic nucleotide sequences of GD150509, GD160403, GD160607, GDFX0601, and GDFX0602 were 7482 bp in length, whereas GDFX0603 was 7480 bp. They shared 99.7% to 99.8% identity with each other. Homology analysis showed that the gag, pol, long terminal repeats (LTRs), and the transmembrane region (gp37) of the env genes of the 6 viruses were well conserved to endogenous counterpart sequences (>97.8%). However, the gp85 genes displayed high variability with any known chicken ALV strains. Growth kinetics of DF-1 cells infected with the isolated ALV showed viral titers that were lower than those infected with the GD13 (ALV-A), CD08 (ALV-B), and CHN06 (ALV-J) on day 7 post-infection. The infected Specific-pathogen-free (SPF) chickens could produce continuous viremia, atrophy of immune organs, growth retardation and no tumors were observed. These subgroup ALVs are unique and may be common in south China. The results suggested that updating the control and eradication program of exogenous ALV for yellow feather broiler breeders in south China needs to be considered because of the emergence of the new subgroup viruses.
Project description: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.
Project description:Subgroup J avian leukosis virus (ALV-J) isolate GDKP1202 was isolated from a 50-day-old local yellow commercial broiler in the Guangdong province of China in 2012. Here we report the complete genomic sequence of the GDKP1202 isolate, which caused high mortality, serious growth suppression, thymic atrophy, and liver enlargement in commercial broilers. A novel potential binding site (5'-GGCACCTCC-3') for c-myb was identified in the GDKP1202 genome. These findings will provide additional insights into the molecular characteristics in the genomes and pathogenicity of ALV-J.
Project description:The feed restriction applied during rearing of broiler breeders inflicts chronic hunger, and frustration due to unfulfilled behavioural needs for feeding. To alleviate the welfare problems associated with feed restriction, qualitative feed restriction allows a larger amount of feed to be provided without increasing the energy intake. In the present study, the aim was to investigate the effect of scatter-fed qualitative feed restriction on a range of welfare indicators in broiler breeders at the end of the rearing period. In total, 1,200 female breeder chicks of the genotype Ross 308 were housed in 24 pens: six pens of initially 50 birds per dietary treatment. The treatments were: (1) standard feed (Control), (2) standard feed diluted with oat hulls (Insoluble), (3) standard feed diluted with oat hulls and sugar beet pulp (Mixed), and (4) standard feed plus maize silage (Roughage). At 15 weeks of age, a blood sample was taken from 40 birds (10/treatment) five times within 24 h. The plasma was analysed for corticosterone concentration. At 19 weeks of age, a clinical welfare assessment was performed on all birds before they were sacrificed. From each bird, three feathers were plucked and macroscopically examined for the presence of fault bars. Feather length and weight were also recorded. Mortality was registered on occurrence throughout the rearing period. Treatment affected the plumage condition, footpad dermatitis, plumage dirtiness, vent pasting, and number of severe fault bars (P ? 0.05) but not plasma corticosterone concentration, hock burns, hyperkeratosis and mortality (P ? 0.17). There was an effect of the interactions between treatment and feather type on the total number of fault bars per feather, average position of the fault bars relative to the base of the feather, and growth rates of feather mass and length (P < 0.0001). Overall, the results showed improved welfare of Roughage birds and reduced welfare of Mixed birds, whereas the welfare of Insoluble birds did not seem to differ noticeably from that of Control birds. We recommend to further develop a feeding strategy that includes daily allocation of roughage to broiler breeders during the rearing period.
Project description:From 2014 to 2015 in China, many broiler breeder and layer hen flocks exhibited a decrease in egg production and some chickens developed hepatitis syndrome including hepatomegaly, hepatic necrosis and hemorrhage. Avian hepatitis E virus (HEV) and avian leucosis virus subgroup J (ALV-J) both cause decreasing in egg production, hepatomegaly and hepatic hemorrhage in broiler breeder and layer hens. In the study, the seroprevalence of avian HEV and ALV-J in these flocks emerging the disease from Shandong and Shaanxi provinces were investigated.A total of 1995 serum samples were collected from 14 flocks with hepatitis syndrome in Shandong and Shaanxi provinces, China. Antibodies against avian HEV and ALV-J in these serum samples were detected using iELISAs. The seroprevalence of anti-avian HEV antibodies (35.09%) was significantly higher than that of anti-ALV-J antibodies (2.16%) (p?=?0.00). Moreover, the 43 serum samples positive for anti-ALV-J antibodies were all also positive for anti-avian HEV antibodies. In a comparison of both provinces, Shandong chickens exhibited a significantly higher seroprevalence of anti-avian HEV antibodies (42.16%) than Shaanxi chickens (26%) (p?=?0.00). In addition, the detection of avian HEV RNA and ALV-J cDNA in the liver samples from the flocks of two provinces also showed the same results of the seroprevalence.In the present study, the results showed that avian HEV infection is widely prevalent and ALV-J infection is endemic in the flocks with hepatitis syndrome from Shandong and Shaanxi provinces of China. These results suggested that avian HEV infection may be the major cause of increased egg drop and hepatitis syndrome observed during the last 2 years in China. These results should be useful to guide development of prevention and control measures to control the diseases within chicken flocks in China.
Project description:Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-type chickens in 1988. No field cases of ALV-J infection or tumors in layer chickens were observed worldwide until 2004. However, layer flocks in China have experienced outbreaks of this virus in recent years. The molecular epidemiology of ALV-J strains isolated from layer flocks was investigated. The env genes of 77.8% (21/27) of the ALV-J layer isolates with a high degree of genetic variation were significantly different from the env genes of the prototype strain of ALV-J (HPRS-103) and American and Chinese strains from meat-type chickens (designated ALV-J broiler isolates). A total of 205 nucleotides were deleted from the 3' untranslated region of 89.5% (17/19) of the ALV-J layer isolates. Approximately 94.7% (16/17) of the layer isolates contained a complete E element of 146 to 149 residues. The U3 sequences of 84.2% (16/19) of the ALV-J layer isolates displayed less than 92.5% sequence homology to those of the ALV-J broiler isolates, although the transcriptional regulatory elements that are typical of avian retroviruses were highly conserved. Several unique nucleotide substitutions in the env gene, the U3 region, and the E element of most of the ALV-J layer isolates were detected. These results suggested that the env gene, E element, and U3 region in the ALV-J layer isolates have evolved rapidly and were significantly different from those of the ALV-J broiler isolates. These findings will contribute to a better understanding of the pathogenic mechanism of layer tumor diseases induced by ALV-J.
Project description:In 2010, sporadic cases of avian leukosis virus (ALV)-like bursal lymphoma, also known as spontaneous lymphoid leukosis (LL)-like tumors, were identified in two commercial broiler breeder flocks in the absence of exogenous ALV infection. Two individual ALV subgroup E (ALV-E) field strains, designated AF227 and AF229, were isolated from two different breeder farms. The role of these ALV-E field isolates in development of and the potential joint impact in conjunction with a Marek's disease virus (MDV) vaccine (SB-1) were further characterized in chickens of an experimental line and commercial broiler breeders. The experimental line 0.TVB*S1, commonly known as the rapid feathering-susceptible (RFS) line, of chickens lacks all endogenous ALV and is fully susceptible to all subgroups of ALV, including ALV-E. Spontaneous LL-like tumors occurred following infection with AF227, AF229, and a reference ALV-E strain, RAV60, in RFS chickens. Vaccination with serotype 2 MDV, SB-1, in addition to AF227 or AF229 inoculation, significantly enhanced the spontaneous LL-like tumor incidence in the RFS chickens. The spontaneous LL-like tumor incidence jumped from 14% by AF227 alone to 42 to 43% by AF227 in combination with SB-1 in the RFS chickens under controlled conditions. RNA-sequencing analysis of the LL-like lymphomas and nonmalignant bursa tissues of the RFS line of birds identified hundreds of differentially expressed genes that are reportedly involved in key biological processes and pathways, including signaling and signal transduction pathways. The data from this study suggested that both ALV-E and MDV-2 play an important role in enhancement of the spontaneous LL-like tumors in susceptible chickens. The underlying mechanism may be complex and involved in many chicken genes and pathways, including signal transduction pathways and immune system processes, in addition to reported viral genes.IMPORTANCE Lymphoid leukosis (LL)-like lymphoma is a low-incidence yet costly and poorly understood disease of domestic chickens. The observed unique characteristics of LL-like lymphomas are that the incidence of the disease is chicken line dependent; pathologically, it appeared to mimic avian leukosis but is free of exogenous ALV infection; inoculation of the nonpathogenic ALV-E or MDV-2 (SB-1) boosts the incidence of the disease; and inoculation of both the nonpathogenic ALV-E and SB-1 escalates it to much higher levels. This study was designed to test the impact of two new ALV-E isolates, recently derived from commercial broiler breeder flocks, in combination with the nonpathogenic SB-1 on LL-like lymphoma incidences in both an experimental egg layer line of chickens and a commercial broiler breeder line of chickens under a controlled condition. Data from this study provided an additional piece of experimental evidence on the potency of nonpathogenic ALV-E, MDV-2, and ALV-E plus MDV-2 in boosting the incidence of LL-like lymphomas in susceptible chickens. This study also generated the first piece of genomic evidence that suggests host transcriptomic variation plays an important role in modulating LL-like lymphoma formation.
Project description: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.
Project description:In spite of the purification of the laying hens and broilers of avian leukosis virus (ALV) has made remarkable achievements, the infection of ALV was still serious in Chinese indigenous chickens.In order to assess the epidemic state of avian leukosis virus in indigenous chickens in China, 10 novel strains of ALV subgroup J (ALV-J), named JS16JH01 to JS16JH10, were isolated and identified by virus isolation and immunofluorescence antibody assays from a Chinese local breed farm with a sporadic incidence of tumors. To understand their virological characteristics further, the proviral genome of ENV-LTR was sequenced and compared with the reference strains.The homology of the gp85 gene between the ten ALV-J strains and NX0101 was in the range from 89.7-94.8% at the nuclear acid level. In addition, their gp85 genes were quite varied, with identities of 92-98% with themselves at the nuclear acid level. There were several snp and indel sites in the amino acid sequence of gp85 genes after comparison with other reference strains of ALV. Interestingly, a novel insertion in the gp85 region was found in two strains, JS16JH01 and JS16JH07, compared with NX0101 and HPRS-103.At present, owing to the large-scale purification of ALV in China, laying hens and broiler chickens with ALV infection are rarely detected, but ALVs are still frequently detected in the local chickens, which suggests that more efforts should be applied to the purification of ALV from indigenous chickens.
Project description:To survey avian leukosis virus subgroup J (ALV-J) integration in myeloid leukosis (ML) of chicken, we developed an ALV-J insertional identification platform based on hybrid-capture target enrichment and next-generation sequencing (NGS). In addition, we used gene expression profiling and bioinformatics to associate integration sites to transcriptional activity and to genetic features of the tumor cell genome. We selected six cases of ALV-J positive and diagnosed as ML for integration sites identify from commercial broiler breeder flocks in Guangdong Province of China between November 2011 and March 2012. All tumors were diagnosed on the basis of characteristic gross and microscopic lesions. Furthermore, PCR tests on the genomic DNA of tissues and virus isolation assay only showed ALV-J-specific positive results in previously study. We randomly chose 4 independent liver samples from the six cases for gene expression profile analysis. And 3 ALV-negative tissue samples from specific-pathogen-free (SPF) chickens at the same age were use as negative controls. Thus a total of 7 samples were hybridized, three representing control.
Project description:Avian leukosis virus subgroup J (ALV-J) is an immunosuppressive virus that causes considerable economic losses to the chicken industry in China. However, there is currently no effective vaccine to prevent ALV-J infection. In order to reduce the losses caused by ALV-J, we constructed two effective ALV-J vaccines by inserting the ALV-J (strain JL093-1) env or gag+env genes into the US2 gene of the Marek's disease herpesviruses (MDV) by transfection of overlapping fosmid DNAs, creating two recombinant MDVs, rMDV/ALV-gag+env and rMDV/ALV-env. Analysis of cultured chicken embryo fibroblasts infected with the rMDVs revealed that Env and Gag were successfully expressed and that there was no difference in growth kinetics in cells infected with rMDVs compared with that of cells infected with the parent MDV. Chickens vaccinated with either rMDV revealed that positive serum antibodies were induced. Both rMDVs also effectively reduced the rate of positive viremia in chicken flocks challenged with ALV-J. The protective effect provided by rMDV/ALV-env inoculation was slightly stronger than that provided by rMDV/ALV-gag+env. This represents the first study where a potential rMDV vaccine, expressing ALV-J antigenic genes, has been shown to be effective in the prevention of ALV-J. Our study also opens new avenues for the control of MDV and ALV-J co-infection.