Precise CRISPR/Cas9 editing of the NHE1 gene renders chickens resistant to the J subgroup of avian leukosis virus.

Avian leukosis virus subgroup J (ALV-J) is an important concern for the poultry industry. Replication of ALV-J depends on a functional cellular receptor, the chicken Na+/H+ exchanger type 1 (chNHE1). Tryptophan residue number 38 of chNHE1 (W38) in the extracellular portion of this molecule is a critical amino acid for virus entry. We describe a CRISPR/Cas9-mediated deletion of W38 in chicken primordial germ cells and the successful production of the gene-edited birds. The resistance to ALV-J was examined both in vitro and in vivo, and the ΔW38 homozygous chickens tested ALV-J-resistant, in contrast to ΔW38 heterozygotes and wild-type birds, which were ALV-J-susceptible. Deletion of W38 did not manifest any visible side effect. Our data clearly demonstrate the antiviral resistance conferred by precise CRISPR/Cas9 gene editing in the chicken. Furthermore, our highly efficient CRISPR/Cas9 gene editing in primordial germ cells represents a substantial addition to genotechnology in the chicken, an important food source and research model.

dPRLR causes differences in immune responses between early and late feathering chickens after ALV-J infection.

To understand the differences in immune responses between early feathering (EF) and late feathering (LF) chickens after infection with avian leukosis virus, subgroup J (ALV-J), we monitored the levels of prolactin, growth hormone and the immunoglobulins IgG and IgM in the serum of LF and EF chickens for 8 weeks. Moreover, we analysed the expression of immune-related genes in the spleen and the expression of PRLR, SPEF2 and dPRLR in the immune organs and DF-1 cells by qRT-PCR. The results showed that ALV-J infection affected the expression of prolactin, growth hormone, IgG and IgM in the serum. Regardless of whether LF and EF chickens were infected with ALV-J, the serum levels of the two hormones and two immunoglobulins in EF chickens were higher than those in LF chickens (P < 0.05). However, the expression of immune-related genes in the spleen of positive LF chickens was higher than that in the spleen of positive EF chickens. In the four immune organs, PRLR and SPEF2 expression was also higher in LF chickens than in EF chickens. Furthermore, the dPRLR expression of positive LF chickens was higher than that of negative LF chickens. After infection with ALV-J, the expression of PRLR in DF-1 cells significantly increased. In addition, overexpression of PRLR or dPRLR in DF-1 cells promoted replication of ALV-J. These results suggested that the susceptibility of LF chickens to ALV-J might be induced by dPRLR.

High-frequency and activation of CD4+CD25+ T cells maintain persistent immunotolerance induced by congenital ALV-J infection.

Congenital avian leukosis virus subgroup J (ALV-J) infection can induce persistent immunotolerance in chicken, however, the underlying mechanism remains unclear. Here, we demonstrate that congenital ALV-J infection induces the production of high-frequency and activated CD4+CD25+ Tregs that maintain persistent immunotolerance. A model of congenital infection by ALV-J was established in fertilized eggs, and hatched chicks showed persistent immunotolerance characterized by persistent viremia, immune organ dysplasia, severe imbalance of the ratio of CD4+/CD8+ T cells in blood and immune organs, and significant decrease in CD3+ T cells and Bu-1+ B cells in the spleen. Concurrently, the mRNA levels of IL-2, IL-10, and IFN-γ showed significant fluctuations in immune organs. Moreover, the frequency of CD4+CD25+ Tregs in blood and immune organs significantly increased, and the frequency of CD4+CD25+ Tregs was positively correlated with changes in ALV-J load in immune organs. Interestingly, CD4+CD25+ Tregs increased in the marginal zone of splenic nodules in ALV-J-infected chickens and dispersed to the germinal center. In addition, the proliferation and activation of B cells in splenic nodules was inhibited, and the number of IgM+ and IgG+ cells in the marginal zone significantly decreased. We further found that the mRNA levels of TGF- ß and CTLA-4 in CD4+CD25+ Tregs of ALV-J-infected chickens significantly increased. Together, high-frequency and activated CD4+CD25+ Tregs inhibited B cells functions by expressing the inhibitory cytokine TGF-ß and inhibitory surface receptor CTLA-4, thereby maintaining persistent immunotolerance in congenital ALV-J-infected chickens.

Geese not susceptible to virulent subgroup J avian leukosis virus isolated from chickens.

To determine whether geese are susceptible to infection by avian leukosis virus (ALV), 702 serum samples from domestic and foreign goose breeds were screened for p27 antigen as well as being inoculated into DF-1 cell cultures to isolate ALV. Although 5.7% of samples were positive for p27 antigen, reactivity appeared to be non-specific because no ALV was detected in the corresponding DF-1 cultures. To further determine whether geese are susceptible to ALV-J isolated from chickens, ALV-J strain JS09GY7 was artificially inoculated into 10-day-old goose embryos, with one-day-old hatched goslings then screened for p27 antigen and the presence of ALV. In all cases, the results of both tests were negative. Liver tissues from the 1-day-old goslings were screened using a polymerase chain reaction-based assay, which failed to amplify ALV-J gene fragments from any of the samples. Further, no histopathological damage was observed in the liver tissues. ALV-J was further inoculated intraperitoneally into one-day-old goslings, with cloacal swabs samples and plasma samples then collected every 5 days for 30 days. All samples were again negative for the presence of p27 antigen and ALV, and liver tissues from the challenged geese showed no histopathological damage and were negative for the presence of ALV-J gene fragments. Furthermore, p27 antigen detection, PCR-based screening, and indirect immunofluorescence assays were all negative following the infection of goose embryo fibroblasts with ALV-J. Together, these results confirm that virulent chicken-derived ALV-J strains cannot infect geese, and that p27 antigen detection in goose serum is susceptible to non-specific interference.

A balanced game: chicken macrophage response to ALV-J infection.

Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression in infected chickens. Macrophages play a central role in host defense against invading pathogens. In this study, we discovered an interesting phenomenon: ALV-J replication is weakened from 3 hours post-infection (hpi) to 36 hpi, which was verified using Western blotting and RT-PCR. To further investigate the interaction between ALV-J and macrophages, transcriptome analysis was performed to analyze the host genes' function in chicken primary monocyte-derived macrophages (MDM). Compared to the uninfected control, 624 up-regulated differentially expressed genes (DEG) and 341 down-regulated DEG at 3 hpi, and 174 up-regulated DEG and 87 down-regulated DEG at 36 hpi were identified in chicken MDM, respectively. ALV-J infection induced strong innate immune responses in chicken MDM at 3 hpi, instead of 36 hpi, according to the analysis results of Gene Ontology and KEGG pathway. Importantly, the host factors, such as up-regulated MIP-3α, IL-1ß, iNOS, K60, IRG1, CH25H, NFKBIZ, lysozyme and OASL were involved in the host defense response during the course of ALV-J infection. On the contrary, up-regulated EX-FABP, IL4I1, COX-2, NFKBIA, TNFAIP3 and the Jak STAT pathway inhibitors including CISH, SOCS1 and SOCS3 are beneficial to ALV-J survival in chicken macrophages. We speculated that ALV-J tropism for macrophages helps to establish a latent infection in chicken MDM from 6 to 36 hpi. The present study provides a comprehensive view of the interactions between macrophages and ALV-J. It suggests the mechanisms of defense of chicken macrophages against ALV-J invasion and how ALV-J escape the host innate immune responses.

A novel Bursin-like peptide as a potential virus inhibitor and immunity regulator in SPF chickens infected with recombinant ALV.

BACKGROUND: Avian leukosis viruses (ALVs) are important contagious suppressive factors of chicken immunity and growth performance, resulted in enormous economic loss. Although virus eradication programs are applied in breeder flocks, ALVs are still widespread globally. Therefore, other valuable adjunct to reduce the negative effect of ALVs should be considered. Bursin-like peptide (BLP) showed remarkable immunomodulatory effects, whereas their influence on ALV-infected avian groups has not been reported. Here, a designed hybrid BLP was expressed in E. coli. The purified BLP was injected subcutaneously weekly in SPF chickens congenitally infected with a natural ALV strain. Then the influences of this BLP on the growth performance, immune response and virus titer of ALV-infected chickens were determined. RESULTS: This BLP injection significantly improved the body weights of ALV-infected birds (P < 0.05). BLP injection significantly enhanced organ index in the BF in ALV-infected birds (P < 0.05). The weekly injection of BLP significantly lengthened the maintenance time of antibodies against Newcastle disease virus (NDV) attenuated vaccine of ALV-infected birds (P < 0.05) and boosted the antibody titer against avian influenza virus (AIV) H5 inactive vaccine of mock chicken (P < 0.05). BLP injection in mock chickens enhanced the levels of serum cytokines (IL-2, IL-4 and interferon-γ) (P < 0.05). Surprisingly, the novel BLP significantly inhibited expression of the ALV gp85 gene in the thymus (P < 0.05), kidney (P < 0.05) and bursa of Fabricius (BF) (P < 0.01) of ALV-infected chickens. Both viral RNA copy number and protein level decreased significantly with BLP (50 µg/mL) inoculation before ALV infection in DF1 cells (P < 0.05). CONCLUSIONS: This is the first report investigating the influence of BLP on the growth and immunity performance of chickens infected by ALV. It also is the first report about the antiviral effect of BLP in vivo and in vitro. This BLP expressed in E. coli showed potential as a vaccine adjuvant, growth regulator and antiretroviral drug in chickens to decrease the negative effects of ALV infection.

Avian leukosis virus subgroup - J as a contaminant in live commercially available poultry vaccines distributed in Nigeria.

Globally, vaccines are used to prevent and control the menace of infectious diseases in livestock with some reported to be inadvertently contaminated with extraneous agents (EAs). With the aim of screening and characterizing for some selected EAs, 44 live viral poultry vaccines were randomly selected based on availability. The vaccines comprised 14 manufacturers in 10 different countries including Nigeria were screened by Polymerase Chain Reaction. In 9% (4/44) of the vaccines, contamination with only avian leukosis virus (ALV) subgroup J (ALV-J) was recorded. Other exogenous ALV subgroups, chicken infectious anemia and infectious laryngotracheitis viruses were absent. The EAs was found in infectious bursal disease (n = 1), Fowlpox (n = 2) and Mareks disease (n = 1) vaccines. Phylogenetic analysis of the ALV-J env gene showed clustering with contemporary group I and II. The result underscores the importance of screening vaccines to avoid the introduction and spread of EAs that could pose a threat to poultry production.

Taishan Pinus massoniana Pollen Polysaccharides Enhance Immune Responses in Chickens Infected by Avian Leukosis Virus Subgroup B.

Immunosuppressive virus, which can cause suppressed immunity and vaccination failure, frequently occurs in chicken flocks and seriously destroys the poultry industry. Our previous studies have reported that Taishan Pinus massoniana pollen polysaccharide (TPPPS) possess immunomodulatory effects and improve the immune effects of vaccines. In this study, avian leukosis virus subgroup B (ALV-B) was chosen as immunosuppressive virus to artificially establish immunosuppressive models in chickens, and the immune modulatory ability of TPPPS on the immune response of chickens was evaluated. Four randomly assigned groups (Group I-IV) of these immunosuppressed chickens were administered with TPPPS at doses of 0, 100, 200, and 400 mg/kg (every kilogram chick), respectively. Group V was administered with saline as control. At seven day old, 10 chickens randomly selected from Group I-V were inoculated with the attenuated Newcastle disease (ND) vaccine. The results showed that during the monitoring period, TPPPS significantly enhanced weight of immune organs, peripheral lymphocyte proliferation, the percentage of CD4+ and the ratio of CD4+/CD8+, IL-2 and IFN-γ production, and ALV-B antibody positive rate of chickens in a dose-dependent manner, with 400 mg/kg TPPPS being the most effective. In addition, the antibody titer against Newcastle disease virus (NDV) in Group IV with 400 mg/kg was significantly higher than those in other groups. We observed the stronger immunity in the TPPPS group, which indicates that TPPPS could be used as an immunoenhancer to relieve immunosuppression caused by ALV-B in the poultry industry.

Identification of novel B-cell epitope in gp85 of subgroup J avian leukosis virus and its application in diagnosis of disease.

BACKGROUND: The gp85 is the main envelope protein of avian leukosis subgroup J (ALV-J) involved in virus neutralization. Here, we mapped the epitope in ALV-J gp85 by ELISA using synthetic peptides and developed epitope based diagnostic methods for ALV-J infection. RESULTS: The results revealed that monoclonal antibody (mAb) JE9 recognized 83WDPQEL88 motif, which was highly conserved in gp85 among different ALV-J strains by homology analysis. Moreover, after evaluation with two hundred and forty sera samples obtained from different chicken farms, the epitope-based peptide ELISA had much higher sensitivity than commercial ELISA kit for antibody detection of ALV-J. CONCLUSIONS: A novel B-cell epitope recognized by the mAb JE9 was identified. The developed peptide-ELISA based on this novel B-cell epitope could be useful in laboratory viral diagnosis, routine surveillance in chicken farms, and also in understanding the pathogenesis of ALV-J.

Acquisition of resistance to avian leukosis virus subgroup B through mutations on tvb cysteine-rich domains in DF-1 chicken fibroblasts.

Avian leukosis virus (ALV) is a retrovirus that causes tumors in avian species, and its vertical and horizontal transmission in poultry flocks results in enormous economic losses. Despite the discovery of specific host receptors, there have been few reports on the modulation of viral susceptibility via genetic modification. We therefore engineered acquired resistance to ALV subgroup B using CRISPR/Cas9-mediated genome editing technology in DF-1 chicken fibroblasts. Using this method, we efficiently modified the tumor virus locus B (tvb) gene, encoding the TVB receptor, which is essential for ALV subgroup B entry into host cells. By expanding individual DF-1 clones, we established that artificially generated premature stop codons in the cysteine-rich domain (CRD) of TVB receptor confer resistance to ALV subgroup B. Furthermore, we found that a cysteine residue (C80) of CRD2 plays a crucial role in ALV subgroup B entry. These results suggest that CRISPR/Cas9-mediated genome editing can be used to efficiently modify avian cells and establish novel chicken cell lines with resistance to viral infection.

Vertical transmission of avian leukosis virus subgroup J (ALV-J) from hens infected through artificial insemination with ALV-J infected semen.

BACKGROUND: Avian leukosis virus (ALV) is one of the main causes of tumour development within the poultry industry in China. The subgroup J avian leukosis viruses (ALV-J), which induce erythroblastosis and myelocytomatosis, have the greatest pathogenicity and transmission ability within this class of viruses. ALV can be transmitted both horizontally and vertically; however, the effects of ALV infection in chickens-especially roosters-during the propagation, on future generations is not clear. Knowing the role of the cock in the transmission of ALV from generation to generation might contribute to the eradication programs for ALV. RESULTS: The results showed that two hens inseminated with ALV-J-positive semen developed temporary antibody responses to ALV-J at 4-5 weeks post insemination. The p27 antigen was detected in cloacal swabs of six hens, and in 3 of 26 egg albumens at 1-6 weeks after insemination. Moreover, no viremia was detected at 6 weeks after insemination even when virus isolation had been conducted six times at weekly intervals for each of the 12 females. However, ALV-J was isolated from 1 of their 34 progeny chicks at 1 week of age, and its gp85 had 98.4%-99.2% sequence identity with the gp85 of ALV-J isolated from semen samples of the six cocks. CONCLUSIONS: Our findings indicated that females that were late horizontally infected with ALV-J by artificial insemination might transmit the virus to progeny through eggs, which amounts to vertical transmission.

Identification of a novel B-cell epitope specific for avian leukosis virus subgroup J gp85 protein.

Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that has caused severe economic losses in China. Gp85 protein is the main envelope protein and the most variable structural protein of ALV-J. It is also involved in virus neutralization. In this study, a specific monoclonal antibody, 4A3, was produced against the ALV-J gp85 protein. Immunofluorescence assays showed that 4A3 could react with different strains of ALV-J, including the British prototype isolate HPRS103, the American strains, an early Chinese broiler isolate, and layer isolates. A linear epitope on the gp85 protein was identified using a series of partially overlapping fragments spanning the gp85-encoding gene and subjecting them to western blot analysis. The results indicated that (134)AEAELRDFI(142) was the minimal linear epitope that could be recognized by mAb 4A3. Enzyme-linked immunosorbent assay (ELISA) revealed that chicken anti-ALV-J sera and mouse anti-ALV-J gp85 sera could also recognize the minimal linear epitope. Alignment analysis of amino acid sequences indicated that the epitope was highly conserved among 34 ALV-J strains. Furthermore, the epitope was not conserved among subgroup A and B of avian leukosis virus (ALV). Taken together, the mAb and the identified epitope may provide valuable tools for the development of new diagnostic methods for ALV-J.

[Detection of fps tumor antigen with mono-specific anti-fps serum in tumors induced by acute transforming ALV].

OBJECTIVE: To prepare anti-fps mono-specific serum, and detect the fps antigen in tumors induced by acute transforming avian leukosis/sarcoma virus containing v-fps oncogene. METHODS: Two part of v-fps gene was amplified by RT-PCR using the Fu-J viral RNA as the template. Mono-specific serum was prepared by immuning Kunming white mouse with both two recombinant infusion proteins expressed by the prokaryotic expression system. Indirect immunofluorescent assay was used to detect fps antigen in tumor tissue suspension cells and CEF infected by sarcoma supernatant. Immunohistochemical method was used to detect fps antigen in tumor tissue. RESULTS: The mouse mono-specific serum was specific as it had no cross reaction with classical ALV-J strains. The result reveals that the tumor tissue suspension cells, the CEF infected by sarcoma supernatant, and the slice immunohistochemistry of the sarcoma showed positive results. CONCLUSION: The anti-fps mono-specific serum was prepared, and the detection method was established, which laid the foundation for the study of viral biological characteristics and mechanism of tumourgenesis of acute transforming avian leukosis/sarcoma virus containing v-fps oncogene.

Reversion to subgroup J avian leukosis virus viremia in seroconverted adult meat-type chickens exposed to chronic stress by adrenocorticotrophin treatment.

Chickens infected with subgroup J avian leukosis virus (ALV J) early in posthatch life develop viremia followed by a neutralizing antibody (Nab) response that may or may not be able to clear the viremia. Occasionally, chickens that do clear viremia by developing an efficient Nab response revert to viremia, and the factors responsible for this reversion are not clear. In this study, it was hypothesized that stress can cause seroconverted viremia-free chickens to revert to viremia. Adult (52-wk-old) male commercial meat-type chickens that were exposed to ALV J at hatch and had since cleared viremia and remained viremia-free for up to 40 wk, when subjected to chronic stress (for 14 days) induced by porcine adrenocorticotrophin (ACTH), reverted to viremia and cloacal shedding (2/6 [33%]). However, chickens that were contact-exposed to ALV J at 32 wk of age and had seroconverted failed to revert to viremia when subjected to similar chronic stress. Stress did not increase the susceptibility of adult meat-type chickens to ALV J infection by contact exposure. The lack of statistical significance due to the small sample size is a limitation of this study. However, in general, the results suggest that treatment of chickens with ACTH can cause reversion of viremia and cloacal shedding in ALV J-seroconverted adult male chickens that had been exposed to the virus at hatch, but not in chickens that were contact-exposed at 32 wk of age. The results warrant further studies with greater sample size to examine the role of stress in ALV J epidemiology.

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.

[Mono-specific serum preparation and specificity of gp85 gene of subgroup A avian leukosis virus].

OBJECTIVE: In order to get a rapid specific diagnostic reagent for subgroup A Avian Leukosis Virus detection. METHODS: The Avian Leukosis Virus Subgroup A (ALV-A) SDAU09E1 strain was inoculated into DF1 cells, an ALV-A- gp85 DNA fragment of 1023 bp was amplified from infected cells and inserted into PET-32a(+) plasmid at the location between restriction endonucleases BamH I and Not I sites. The recombinant plasmid PET-SDAU09E1 -gp85 was transformed into E coli. BL21 (Rosetta) for gp85 gene expression. Then we used the purified recombinant fusion protein to immunize 6 weeks old Kunming white mice, and the antiserum were prepared. RESULTS: The recombinant ALV-A gp85 fusion protein with a molecular weight of 52.8 kDa demonstrated a good antigenecity. Mon-specific serum produced by vaccinated mice came out reactive with subgroups A and B ALV (ALV-A and ALV-B but not subgroup J ALV) by the indirect immunofluorescence (IFA) method. CONCLUSION: This was the first time to demonstrate a mono-specific antiserum specific to ALV-A and ALV-B, it could be used for differential diagnosis of exogenous ALV infections in CEF cultures when in complement with ALV-J specific monoclonal antibodies. Chickens in our country are now distressed by both classic ALV-A/B and emerging ALV-J, making differential diagnosis necessary, so studying this reagent has high practical value.

Subgroup J avian leukosis virus neutralizing antibody escape variants contribute to viral persistence in meat-type chickens.

We have previously demonstrated a high incidence of chickens with persistent viremia even in the presence of neutralizing antibodies (V+A+) against the inoculated parental virus in commercial meat-type chickens inoculated at hatch with subgroup J avian leukosis virus (ALV J) field isolates. In this study, we used an ALV J molecular clone, ADOL pR5-4, to determine the role of neutralizing antibody (NAb) escape mutants in maintaining a high incidence of viral persistence, namely, V+A+ infection profile in commercial meat-type chickens. Chickens were housed as a flock in a pen or housed in isolation in solitary Horsfall-Bauer units for testing for NAb escape variants. The emergence of NAb escape variants was evaluated by sequential autologous virus neutralization (VN) (between virus and antibody from the same sampling period) and heterologous VN (between virus and antibody from preceding and succeeding sampling periods). Sequential virus isolates and corresponding antisera from 18 chickens were examined by VN matrix. In all chickens, autologous virus isolates were not neutralized by corresponding antisera. However, some of these resilient autologous virus isolates were neutralized by antibodies from subsequent sampling intervals. Nucleotide sequence analysis of consecutive isolates from three individually housed chickens with V+A+ infection profile revealed distinct changes within the envelope region, suggesting viral evolution to escape the host immune response. These results demonstrate that the emergence of antibody escape variants in commercial meat-type chickens contributes to ALV J persistence.

Identification of a novel epitope specific for Gp85 protein of avian leukosis virus subgroup K.

During the past two decades, avian leukosis virus (ALV) caused tremendous economic losses to poultry industry in China. ALV-K as a newly found subgroup in recent years, which made the control and eradication of ALV more difficult as they were originated from the recombination of different subgroups. To date, specific rapid detection methods refer to ALV-K are still missing. Gp85 is the main structural protein of the virus, which mediates the invasion of host cells by the virus and determinates the classification of subgroups. In this study, we prepared a monoclonal antibody (Mab) named Km3 against Gp85 of ALV-K. Immunofluorescence assay showed that Km3 specifically recognized the strains of ALV-K rather than the strains of ALV-A or ALV-J. To explain the subgroups specificity of Km3, the epitope cognized by the Mab was identified by Western blotting using 15 overlapping fragments spanning the Gp85. Finally, the peptide 129AFGPRSIDTLSDWSRPQ145 was identified as the minimal linear epitope recognized by Km3. Alignment of Gp85 from different subgroups showed that the epitope was highly conserved among ALV-K strains, which was quite different from that of the strains from ALV -A, -B and -J. In conclusion, the Mab Km3 may serve as a useful reagent for ALV-K detection and diagnosis in the future.

Recombinant subgroup B avian leukosis virus combined with the subgroup J env gene significantly increases its pathogenicity.

The differences among different sub-groups of the avian leukosis virus (ALV) genome are mainly concentrated in the env gene, which binds to cell-specific receptors and determines the characteristics of viral tropism and pathogenicity. In this study, two rescued viruses rGX15MM6-2 (ALV of subgroup J, ALV-J) and rGX14FF03 (ALV of subgroup B, ALV-B) and a recombinant virus rALV-B-Jenv (ALV-B's backbone with ALV-J's env) were generated and tested utilizing both in vitro and in vivo experiments. The results showed that the replication ability of the viruses released in DF-1 cell cultures was listed in order as rGX15MM6-2 > rALV-B-Jenv > rGX14FF03. rGX15MM6-2 caused the most serious suppression of body weight gain, exhibited a significant negative effect on the development of immune organs (P < 0.05) and lower antibody responses to vaccinations with the commercial oil-emulsion vaccines (OEVs) (P<0.05) in the challenged chickens. The viral detection showed that the positive rate in blood from the birds infected with rALV-B-Jenv were respectively higher than those from the birds infected with rGX14FF03 (P < 0.05). At 25 wpi, similar tumors were found in the abdominal cavity of the birds in rGX15MM6-2 and rALV-B-Jenv groups. The results demonstrated that the ALV-J env gene significantly increases the pathogenicity of the recombinant ALV-B. With the increasing incidence of co-infections of different subgroups of ALV in the field, the possibility of viral recombination is increasing and demands further study.

Screening of immune biomarkers in different breeds of chickens infected with J subgroup of avian leukemia virus by proteomic.

Avian leucosis (AL) is a disease characterized by tumors and is caused by the avian leukosis virus (ALV). Because of the high variability of viruses and complex pathogenic mechanisms, screening and breeding J subgroup of ALV (ALV-J) resistant avian breeds is one of the strategies for prevention and treatment of AL, thus screening of significant immune markers is needed to promote the development of disease-resistant breeds. In this study, data-independent acquisition (DIA) technology was used to detect the DEPs of three breeds of chicken according to different comparison to investigate the potential markers. Results showed special DEPs for spleen development of each breed were detected, such as PCNT, DDB2, and ZNF62. These DEPs were involved in intestinal immune network used in production of IgA signaling pathways and related to immune response which can be used as potential markers for spleen development in different breeds. The DEPs such as RAB44 and TPN involved in viral myocarditis, transcriptional misregulation in cancer, and tuberculosis can be used as potential markers of spleen immune response after ALV-J infection in chickens. Pair-wise analysis was performed for the three breeds after the infection of ALV-J. The proteins such as RFX1, TAF10, and VH1 were differently expressed between three breeds. These DEPs involved in antigen processing and expression, acute myelogenous leukemia, and viral carcinogenesis can be used as potential immune markers after ALV-J infection of different genetic backgrounds. The screening of potential markers at protein level provides a strong theoretical research basis for disease resistance breeding in poultry.