A recombinant avian leukosis virus subgroup j for directly monitoring viral infection and the selection of neutralizing antibodies.
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ABSTRACT:
SUBMITTER: Wang Q
PROVIDER: S-EPMC4270768 | biostudies-literature | 2014
REPOSITORIES: biostudies-literature
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A recombinant avian leukosis virus subgroup j for directly monitoring viral infection and the selection of neutralizing antibodies.
Wang Qi Q Li Xiaofei X Ji Xiaolin X Wang Jingfei J Shen Nan N Gao Yulong Y Qi Xiaole X Wang Yongqiang Y Gao Honglei H Zhang Shide S Wang Xiaomei X
PloS one 20141218 12
Avian leukosis virus subgroup J (ALV-J) has induced serious clinical outbreaks and has become a serious infectious disease of chickens in China. We describe here the creation of a recombinant ALV-J tagged with the enhanced green fluorescent protein (named rHPRS-103EGFP). We successfully utilize the rHPRS-103EGFP to visualize viral infection and for development of a simplified serum-neutralization test. ...[more]
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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.
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Project description: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.
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Project description:The integration of retroviruses into the host genome following nonrandom genome-wide patterns may lead to the deregulation of gene expression and oncogene activation near the integration sites. Slow-transforming retroviruses have been widely used to perform genetic screens for the identification of genes involved in cancer. To investigate the involvement of avian leukosis virus subgroup J (ALV-J) integration in myeloid leukosis (ML) in chickens, we utilized an ALV-J insertional identification platform based on hybrid capture target enrichment and next-generation sequencing (NGS). Using high-definition mapping of the viral integration sites in the chicken genome, 241 unique insertion sites were obtained from six different ALV-J-induced ML samples. On the basis of previous statistical definitions, MYC, TERT, and ZIC1 genes were identified as common insertion sites (CIS) of provirus integration in tumor cells; these three genes have previously been shown to be involved in the malignant transformation of different human cell types. Compared to control samples, the expression levels of all three CIS genes were significantly upregulated in chicken ML samples. Furthermore, they were frequently, but not in all field ML cases, deregulated at the mRNA level as a result of ALV-J infection. Our findings contribute to the understanding of the relationship between multipathotypes associated with ALV-J infection and the molecular background of tumorigenesis.ALV-Js have been successfully eradicated from chicken breeding flocks in the poultry industries of developed countries, and the control and eradication of ALV-J in China are now progressing steadily. To further study the pathogenesis of ALV-J infections, it will be necessary to elucidate the in vivo viral integration and tumorigenesis mechanism. In this study, 241 unique insertion sites were obtained from six different ALV-J-induced ML samples. In addition, MYC, TERT, and ZIC1 genes were identified as the CIS of ALV-J in tumor cells, which might be a putative "driver" for the activation of the oncogene. In addition, the CIS genes showed deregulated expression compared to nontumor samples. These results have potentially important implications for the mechanism of viral carcinogenesis.
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Project description:The expression of env proteins that bind to viral cell receptors on avian leukosis virus (ALV)-susceptible cells can block ALV infection. In this study, we constructed a cell line (DF-1/B) by expressing the ALV-B env protein in DF-1 cells. PCR, immune fluorescence assay, Western blot, and immune electron microscopy results showed that the env gene can be stably expressed in DF-1cells and the env protein could be detected on the DF-1 cell membrane. An antiviral experiment concluded that the DF-1/B cell line could be resistant to 1 × 104 TCID50 ALV-B virus infection, but had no inhibitory effect on other subgroup ALV. This means that the DF-1/B cell line is specifically resistant to ALV-B and can be used as a tool for ALV-B diagnosis.
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Project description: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.
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Project description:Cell killing by avian leukosis virus subgroup B (ALV-B) in cultures has been extensively studied, but the molecular basis of this process has not been established. Here we show that superinfection, which has been linked to cell killing by ALV-B, plays no crucial role in cell death induction. Instead, we show that signaling by the ALV-B receptor, TVB(S3), a member of the tumor necrosis factor receptor family, is essential for ALV-B-mediated cell death. TVB(S3) activated caspase-dependent apoptosis during ALV-B infection. Strikingly, apoptosis induction occurred predominantly in uninfected cells, while ALV-B-infected cells were protected against cell death. This bystander killing phenomenon was reproduced in a virus-free system by cocultivating ALV-B Env-expressing cells with TVB(S3)-expressing cells. Taken together, our results indicated that ALV-B-mediated apoptosis is triggered by ALV-B Env-TVB(S3) interactions.
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Project description:The chicken reference genome contains 2 endogenous avian leukosis virus subgroup E (ALVE) insertions, but gaps and unresolved repetitive sequences in previous assemblies have hindered their precise characterization. Detailed analysis of the most recent reference genome (GRCg6a) now shows both ALVEs within contiguous chromosome assemblies for the first time. ALVE6 (ALVE-JFevA) and ALVE-JFevB are both located on chromosome 1, with ALVE6 close to the p-arm telomere. ALVE-JFevB is a structurally intact element containing the ALVE gag, pol, and env genes and is capable of forming replication competent viruses. In contrast, ALVE6 contains a 3,352 bp 5' truncation and lacks the entire 5' long terminal repeat and gag gene. Despite this, ALVE6 remains able to produce intact envelope protein, likely due to a mutation in the recognition site for a known inhibitory miRNA (miR-155). Whole genome resequencing data sets from layers, broilers, and 3 independent sources of wild-caught red junglefowl were surveyed for the presence of each of these reference genome ALVEs. ALVE-JFevB was found in no other chicken or red junglefowl genomes, whereas ALVE6 was identified in some layers, broilers, and native breeds but not within any other red junglefowl genome. Improved assembly contiguity has facilitated better characterization of the 2 ALVEs of the chicken reference genome. However, both the limited ALVE content and unique presence of ALVE-JFevB suggests that the reference individual is unrepresentative of ancestral Gallus gallus ALVE diversity.
| S-EPMC7597685 | biostudies-literature