Avian Leukosis Virus Infection Research Articles

Advances on ALV-J in China over the past two decades

Avian leukosis is an important tumorigenic disease caused by the avian leukosis virus (ALV) in poultry. ALVs belong to the retroviral family and are classified into 11 subgroups (ALV-A to ALV-K). Among them, ALV-J was first introduced into China in 1999, spreading widely and evolving from infecting meat-type chickens to layer chickens and Chinese local chickens. ALV-J typically induces myeloid leukosis in infected chickens, but also induces a high proportion of hemangiomas in infected layer chickens, posing a serious threat to poultry breeds in China. As a retrovirus, the genome of ALV-J has undergone significant mutations, which may be related to the expansion of the infection host range and increased pathogenicity of ALV-J. Over the last two decades, the introduction and spread of ALV-J in China have caused substantial losses to the poultry industry. Specialized detection assays have been developed to combat ALV-J infections in China. Additionally, ongoing research aims to employ gene-editing technology as a novel antiviral strategy to control the spread of ALV infections. This review highlights the importance of understanding the impact of ALV-J on the Chinese poultry industry and emphasizes the need for ongoing research and innovation to safeguard poultry health and promote sustainable poultry farming practices in China.

Rapid and sensitive visual detection of avian leukosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow immunochromatographic strip assay.

Considering that avian leukosis virus (ALV) infection has inflicted massive economic losses on the poultry breeding industry in most countries, its early diagnosis remains an important measure for timely treatment and control of the disease, for which a rapid and sensitive point-of-care test is required. We established a user-friendly, economical, and rapid visualization method for ALV amplification products based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with an immunochromatographic strip in a lateral flow device (LFD). Using the ALVp27 gene as the target, five RT-LAMP primers and one fluorescein-isothiocyanate-labeled probe were designed. After 60 min of RT-LAMP amplification at 64 °C, the products could be visualized directly using the LFD. The detection limit of this assay for ALV detection was 102 RNA copies/μL, and the sensitivity was 100 times that of reverse transcription polymerase chain reaction (RT-PCR), showing high specificity and sensitivity. To verify the clinical practicality of this assay for detecting ALV, the gold standard RT-PCR method was used for comparison, and consistent results were obtained with both assays. Thus, the assay described here can be used for rapid detection of ALV in resource-limited environments.

Gp85 protein encapsulated by alginate-chitosan composite microspheres induced strong immunogenicity against avian leukosis virus in chicken.

Avian leukosis, a viral disease affecting birds such as chickens, presents significant challenges in poultry farming due to tumor formation, decreased egg production, and increased mortality. Despite the absence of a commercial vaccine, avian leukosis virus (ALV) infections have been extensively documented, resulting in substantial economic losses in the poultry industry. This study aimed to develop alginate-chitosan composite microspheres loaded with ALV-J Gp85 protein (referred to as aCHP-gp85) as a potential vaccine candidate. Sodium alginate and chitosan were utilized as encapsulating materials, with the ALV-J Gp85 protein serving as the active ingredient. The study involved 45 specific pathogen-free (SPF) chickens to evaluate the immunological effectiveness of aCHP-gp85 compared to a traditional Freund adjuvant-gp85 vaccine (Freund-gp85). Two rounds of vaccination were administered, and antibody levels, mRNA expression of immune markers, splenic lymphocyte proliferation, and immune response were assessed. An animal challenge experiment was conducted to evaluate the vaccine's efficacy in reducing ALV-J virus presence and improving clinical conditions. The results demonstrated that aCHP-gp85 induced a significant and sustained increase in antibody levels compared to Freund-gp85, with the elevated response lasting for 84 days. Furthermore, aCHP-gp85 significantly upregulated mRNA expression levels of key immune markers, notably TNF-α and IFN-γ. The application of ALV-J Gp85 protein within the aCHP-gp85 group led to a significant increase in splenic lymphocyte proliferation and immune response. In the animal challenge experiment, aCHP-gp85 effectively reduced ALV-J virus presence and improved clinical conditions compared to other groups, with no significant pathological changes observed. The findings suggest that aCHP-gp85 elicits a strong and prolonged immune response compared to Freund-gp85, indicating its potential as an innovative ALV-J vaccine candidate. These results provide valuable insights for addressing avian leukosis in the poultry industry, both academically and practically.

Diagnosis of Avian Leukosis Virus subgroup J in asymptomatic commercial layers using qPCR

Avian Leukosis Virus (ALV), one of the dangerous retroviruses threatens the poultry industry, is oncogenic and can easily transmit. Over the past three decades, the poultry industry has been significantly affected by subgroup J (ALV-J) in particular, known for its ability to spread both horizontally and vertically. The fact that a commercial vaccine has not yet been developed against ALV increases the risk potential. For these reasons, positive cases should be identified to control ALV infections and minimize infection. A prompt and accurate diagnosis is vital to detecting infected birds. For this purpose, sampling was performed in total 153 blood samples from 14 different asymptomatic commercial layer farm located in Afyonkarahisar, T?rkiye. Herein, the presence, density and prevalence of ALV-J strain and whether it has an oncogenic effect was investigated by qPCR and western blot for the first time in T?rkiye. The average contamination rate of ALV-J was calculated as 70.91% over all samples. However, p27 antigen that is the most abundant polypeptide encoded by the gag gene of ALV could not be detected. High prevalence of ALV-J suggests that ALV strains can be found asymptomatically without showing any symptoms.

Global Maps of Avian Leukosis Viruses: Research Trends and Themes Based on Networking.

Avian leukosis virus (ALV) has a tremendous adverse impact on the poultry industry. Since its discovery, research on different aspects of ALV have been published. Due to the vast academic emphasis and economic importance of the ALV infection in poultry worldwide, this bibliometric analysis explored the scientific output associated with ALV utilizing the Web of Science (Core Collection) database. The relevant data were collected using the search query "AVIAN LEUKOSIS VIRUS", further refined by document types (article, book chapter, and proceedings paper). Finally, 1060 items with full records were imported in Plaintext and tab-delimited formats. The data analysis was carried out using MS Excel, VOS viewer, and R (Biblioshiny) software. Chinese and American research institutions produced the majority of papers during study time period. The Journal of Virology and Avian Diseases appeared as the favorite journal/source for publications. Apart from the avian leukosis virus and the ALV-J, the important keywords mentioned included avian leukosis virus subgroup j, chicken, and retrovirus. The analysis revealed substantial findings on ALV research, with a strong research response from the USA and China.

Transcription analysis of chicken embryo fibroblast cells infected with the recombinant avian leukosis virus isolate GX14FF03.

Infection with recombinant avian leukosis virus (ALV) has previously been linked to malignancies and immunosuppression. However, the processes behind the unique pathophysiology of recombinant ALV are poorly understood. In this study, we analyzed gene expression patterns in chicken fibroblast cells (CEFs) infected with the recombinant ALV isolate GX14FF03 and used the RNA-seq technique to perform a complete analysis of the transcribed mRNAs. A total of 907 significant differentially expressed genes (SDEGs) were identified. Among these SDEGs, the most significantly upregulated gene was interleukin 8-like 1 (IL8L1), while the most significantly downregulated gene was fibroblast growth factor 16 (FGF16). The 907 SDGEs were highly enriched (p < 0.05) for 252 Gene Ontology (GO) terms, including 197 BP, 3 CC, and 52 MF. According to KEGG data analysis, SDEGs are implicated in eight significant pathways (p < 0.05). Furthermore, protein-protein interaction (PPI) network analysis revealed that IL8L1 interacts with 17 genes. These findings shed light on the molecular mechanisms involved in recombinant ALV infection by showing the mRNA expression profile in CEFs infected with GX14FF03 virus.

Avian Leucosis Virus-Host Interaction: The Involvement of Host Factors in Viral Replication

Avian leukosis virus (ALV) causes various diseases associated with tumor formation and decreased fertility. Moreover, ALV induces severe immunosuppression, increasing susceptibility to other microbial infections and the risk of failure in subsequent vaccination against other diseases. There is growing evidence showing the interaction between ALV and the host. In this review, we will survey the present knowledge of the involvement of host factors in the important molecular events during ALV infection and discuss the futuristic perspectives from this angle.

Whole-genome analysis reveals possible sources of ALV-J infection in an anyi tile-like gray chicken flock

Avian leukosis virus (ALV) induces multiple tumors in chicken and is still prevalent in a lot of local flocks in China. In this study, we analyzed the ALV infection status in an Anyi tile-like gray chicken flock by DF1-cells isolation, virus identification, and genome sequencing. Results showed a 29% (29/100) ALV positive rate in this flock. Homology analysis based on env genes illustrated that all these stains belong to subgroup J (92–100% identities) and can be further divided into 5 batches, suggesting a higher diversity of ALV-J within the same flock. The whole-genome analysis of representative stains from each batch confirmed the close relationship between these isolated strains with previously reported strains from different regions (Guangxi, Shandong, and Heilongjiang), revealing the enrichment of different strains in Anyi tile-like grey chickens. This study provides the epidemiological data of ALV-J in a special chicken flock and a reference for the further eradication of ALV in China.

Chicken hepatomegaly and splenomegaly associated with novel subgroup J avian leukosis virus infection

BackgroundSubgroup J avian leukosis virus (ALV-J) is an oncovirus which can induce multiple types of tumors in chicken. In this report, we found novel ALV-J infection is closely associated with serious hepatomegaly and splenomegaly in chicken.Case presentationThe layer chickens from six flocks in Jiangsu province, China, showed serious hemoperitoneum, hepatomegaly and splenomegaly. Histopathological results indicated focal lymphocytic infiltration, cell edema and congestion in the liver, atrophy and depletion of lymphocyte in the spleen. Tumor cells were not detected in all the organs. avian hepatitis E virus (aHEV), which is thought to be the cause of a very similar disease, big liver and spleen disease (BLS), was not detected. Other viruses causing tumors or liver damage including Marek’s disease virus (MDV), reticuloendotheliosis virus (REV), fowl adenovirus (FAdV) and chicken infectious anemia virus (CIAV) were also proved negative by either PCR or RT-PCR. However, we did detect ALV-J in those chickens using PCR. Only novel ALV-J strains were efficiently isolated from these chicken livers.ConclusionsThis is the first report that chicken hepatomegaly and splenomegaly disease was closely associated with novel ALV-J, highlighting the importance of ALV-J eradication program in China.

Recombinant invasive Lactobacillus plantarum expressing the J subgroup avian leukosis virus Gp85 protein induces protection against avian leukosis in chickens.

Avian leukosis, caused by avian leukosis virus (ALV), is an infectious tumor disease and severely hinders the development of the poultry industry. The use of Lactobacillus plantarum (L. plantarum) could effectively alleviate viremia in the early period of J subgroup ALV (ALV-J) infection. In this study, an invasive L. plantarum NC8 expressing Gp85 protein of ALV-J was constructed. After chickens were orally administered the recombinant invasive NC8, the levels of expression of CD4+ and CD8+ T lymphocytes in peripheral blood and spleen by flow cytometry and the proliferation ability of splenocytes by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were examined, and the contents of cytokines, the anti-ALV-J antibody in serum, and mucosal antibody sIgA in intestinal lavage fluid were detected by enzyme-linked immunosorbent assay (ELISA). The immunoprotective efficiency was evaluated by monitoring the infection rate, the percent of cloacal swabs and survival, body weight gain, the organ indexes, and relative virus loads after challenge with ALV-J. The results showed that the recombinant invasive strain (FnBPA-gp85) could promote the expression levels of the CD8+T cells in peripheral blood and spleen, the proliferation of splenocytes, the secretions of cytokines interleukin 2 (IL-2) and γ-interferon (IFN-γ), and the production of IgG and sIgA compared with the PBS and FnBPA control groups in chickens. The FnBPA-gp85 group was exhibited the highest immune protection against ALV-J infection. The above results indicated that the recombinant invasive NC8 could promote the cellular immunity, humoral immunity, and mucosal immunity responses in chicken and provide a new method for exploring the live vaccine against ALV-J.Key points• The FnBPA-gp85 strain could enhance cellular immunity response.• The FnBPA-gp85 strain could improve the immune protection against ALV-J infection.

Antibody profiles of avian leukosis virus subgroups A/B and J In layer flocks suspected to have Marek’s disease in Nigeria

Abstract Previous reports indicate high seroprevalence of avian leukosis virus (ALV) p72 antigen in layer flocks suspected to have Marek’s disease (MD) in Kaduna and Plateau States. However, the specific subgroups responsible for ALV infection in layers in the States are still unknown, hence the need for this study. Therefore, the objective of this study was to determine the antibody profiles of ALV subgroups A/B and J in layer flocks suspected to have MD in Kaduna and Plateau States. Sera from 7 and 16 layer flocks suspected to have MD in Kaduna and Plateau States respectively, were screened for the presence of antibodies to ALV subgroups A/B and J using IDEXX enzyme linked immunosorbent assay (ELISA) kits. Out of the seven layer flocks screened in Kaduna State, antibodies to ALV subgroup A/B was detected in six of the flocks (85.7%), while antibodies to ALV subgroup J was detected in only one flock (14.3%). Antibodies to both ALV subgroups A/B and J were detected in one flock (14.3%), which suggests co-infection of the two ALV subgroups. Out of the 16 flocks screened in Plateau State, antibodies to ALV subgroup A/B were detected in 15 flocks (93.8%), while antibodies to ALV subgroup J were detected in six flocks (37.5%). Antibodies to both ALV subgroups A/B and J were detected in five flocks (31.3%). The high detection of antibodies to ALV A/B suggests that ALV infection in layers is mostly due to ALV subgroup A or B in the study areas.

ACSL1 Inhibits ALV-J Replication by IFN-Ⅰ Signaling and PI3K/Akt Pathway.

J subgroup avian leukosis virus (ALV-J) infection causes serious immunosuppression problems, leading to hematopoietic malignancy tumors in chicken. It has been demonstrated that interferon-stimulated genes (ISGs) could limit ALV-J replication; nevertheless, the underlying mechanisms remain obscure. Here, we demonstrate that Long-chain Acyl-CoA synthetase 1 (ACSL1) is an interferon (IFN)-stimulated gene that specifically restricts the replication of ALV-J due to the higher IFN-I production. More importantly, ACSL1 induces primary monocyte-derived macrophages (MDMs) to pro-inflammatory phenotypic states during ALV-J infection, and ACSL1 mediates apoptosis through the PI3K/Akt signaling pathway in ALV-J-infected primary monocyte-derived macrophages (MDMs). Overall, these results provide evidence that ACSL1 contributes to the antiviral response against ALV-J.

Molecular characteristic and pathogenicity analysis of a novel multiple recombinant ALV-K strain

Avian leukosis virus (ALV) can induce various tumors and cause serious production problems. ALVs isolated from chickens were divided into six subgroups (A–J). In 2012, a strain of a putative novel subgroup of ALVs was isolated from Chinese native chickens in Jiangsu Province and named as ALV-K. In this study, three ALV-K strains (JS14LH01, JS13LH14, and JS15SG01) were isolated from chickens with suspected ALV infection in Jiangsu Province. Their complete genomes were amplified, sequenced, and analyzed systematically. The results showed that JS14LH01 and JS13LH14 were ALV-K and ALV-E recombinant strains. Whereas JS15SG01 is an ALV-K, ALV-E, and ALV-J multiple recombinant strain containing the U3 region of ALV-J. The pathogenicity test of JS15SG01 revealed that, compared with previous ALV-K strains, the viremia and viral shedding level of JS15SG01-infected chickens were significantly increased, reaching 100 % and 59 %, respectively. More important, JS15SG01 induced significant proliferation of gliocytes in the cerebral cortex of infected chickens, accompanied by the neurotropic phenomenon. This is the first report about a multiple recombinant ALV-K strain that could invade and injure the brain tissue of chickens in China. Our findings enriched the epidemiologic data of ALV and helped to reveal the evolution of ALV strains prevalent in chicken fields.

Polyostotic osteosarcoma associated with avian leukosis virus infection in a captive bare\u2010faced curassow (Crax fasciolata)

BackgroundOsteosarcoma is a malignant mesenchymal bone tumor. Although it is a common tumor in the appendicular skeleton of dogs and cats, it is rarely reported in birds. Retroviruses are usually associated with solid tumor development in different avian species.Case presentation:This report aims to describe a case of osteosarcoma associated with the avian leukosis virus in a captive bare-faced curassow (Crax fasciolata). A captive adult female bare-faced curassow presented with lameness, hyporexia, and a non-ulcerative and firm tumor in the right femur. The bird was euthanized due to the poor prognosis. Histopathology revealed an infiltrative mesenchymal neoplasm consisting of spindle cells with moderate cell pleomorphism, organized in bundles and interspersed by marked deposition of the osteoid matrix, which was compatible with osteosarcoma affecting both femur and tibiotarsus, with renal metastasis. Immunohistochemistry of the primary and metastatic tumor demonstrated vimentin expression by neoplastic cells. Samples of the neoplasm, bone marrow, and spleen were processed for PCR, which enabled the demonstration of proviral avian leukosis virus (ALV) DNA.ConclusionsTo the best of our knowledge, this is the first report of an osteosarcoma in a bare-faced curassow with an unusual polyostotic manifestation and associated with ALV infection.

Molecular characteristics of subgroup J avian leukosis virus isolated from yellow breeder chickens in Guangdong, China, during 2016-2019.

Since 2005, subgroup J avian leukosis virus (ALV-J) infection has been present in yellow chickens in Guangdong, China, causing severe economic losses to the local poultry industry. ALV-J is a rapidly evolving retrovirus. To investigate the molecular characteristics of ALV-J isolates from yellow breeder chickens in Guangdong, 17 virus strains were isolated from 6549 anticoagulants from clinically healthy birds between 2016 and 2019, and completely sequenced and phylogenetically analyzed. Phylogenetic analysis of the gp85 gene showed that all isolated viruses were divided into three different branches. Notably, 41.2% (7/17) of the isolates shared a novel G2598A nucleotide mutation in the pol gene and caused the stop codon to be advanced by 8 positions. Nearly 200 nucleotides were deleted from the redundant TM (rTM) region in all strains, but all retained an intact direct repeat (DR1). 82.4% (14/17) of isolates contained a complete E element. Additionally, 29.4% (5/17) of isolates detected an 11bp deletion in U3 region, and the AIB REP1 transcription factor is missing. The study indicated that ALV-J infection had still been prevalent in the yellow breeder chicken farms in Guangdong, and the genetic background of the strains is diverse. This study provides the latest data on the molecular characteristics of ALV-J, which will help to reveal the evolution trend of ALV-J and develop relevant prevention and control measures.

Marek’s disease virus as a CRISPR/Cas9 delivery system to defend against avian leukosis virus infection in chickens

The CRISPR/CRISPR-associated protein 9 (Cas9) system is a powerful gene-editing tool originally discovered as an integral mediator of bacterial adaptive immunity. Recently, this technology has been explored for its potential utility in providing new and unique treatments for viral infection. Marek’s disease virus (MDV) and avian leukosis virus subgroup J (ALV-J), major immunosuppressive viruses, cause significant economic losses to the chicken industry. Here, we evaluated the efficacy of using MDV as a CRISPR/Cas9-delivery system to directly target and disrupt the reverse-transcribed products of the ALV-J RNA genome during its infection cycle in vitro and in vivo. We first screened multiple potential guide RNA (gRNA) target sites in the ALV-J genome and identified several optimized targets capable of effectively disrupting the latently integrated viral genome and providing efficient defense against new infection by ALV-J in cells. The optimal single-gRNAs and Cas9-expression cassettes were inserted into the genome of an MDV vaccine strain. The results indicated that engineered MDV stably expressing ALV-J-targeting CRISPR/Cas9 efficiently resisted ALV-J challenge in host cells. These findings demonstrated the CRISPR/Cas9 system as an effective treatment strategy against ALV-J infection. Furthermore, the results highlighted the potential of MDV as an effective delivery system for CRISPR/Cas9 in chickens.

Detection of new antibiotic resistance gene profile in Escherichia coli associated with avian leukosis virus infection from broiler chickens

The Escherichia coli (E. coli) is prevailing worldwide, but the epidemiology of E. coli infections feature regional distribution characteristics to some extent. E. coli, as a zoonotic pathogen, can be transferred from animals to humans through food chain or via contact with wounds, causing a public health risk. We reported the swelling of proventriculus and tracheal bleeding following the death in two broiler chickens (Gallus gallus domesticus) from Beijing, China. To investigate whether a virus was involved in the infection, Madin Darby Bovine Kidney (MDCK) cells were co-cultured with supernatants of proventriculus, trachea and spleen homogenates. The avian leucosis virus was detected in the samples of proventriculus and trachea, but the avian influenza virus, the Newcastle disease virus and the avian infectious laryngotracheitis virus were not detected. E. coli isolates were resistant to almost all the antimicrobial as tested except for the combinations of amoxicillin/clavulanic acid and sulfamethoxazole/trimethoprim. PCR tests demonstrated the presence of antibiotic resistance genes in these E. coli isolates and further research revealed a novel gene profile with the presence of CTX-M-1, gyrA, gyrB, oqxA, oqxB, parC and Sul2 antibiotic resistance genes in a strain isolated from a proventriculus sample. These results demonstrated that the presence of antibiotic resistant E. coli would not necessarily cause outbreak of large-scale disease. However, when the bacteria carrying new antibiotic resistance genes enter the environment, it may result in the development of more virulent strains which will potentially impact human and animal health.

A cell line resistant to avian leukosis virus subgroup B infection

ABSTRACTThe 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.

Oral Immunization of Chickens With Recombinant Lactobacillus plantarum Vaccine Against Early ALV-J Infection.

In this study, a novel oral vaccine of recombinant Lactobacillus plantarum (L. plantarum) containing the gp85 protein was explored, and the effects of this vaccine on the prevention of subgroup J Avian Leukosis Virus (ALV-J) infection were assessed. In the current study, the gp85 protein of ALV-J was expressed on the surface of L. plantarum with the surface-display motif, pgsA, by constructing a shuttle vector pMG36e:pgsA:gp85. Surface localization of the fusion protein was verified by western blotting and flow cytometry. Subsequently, Specific Pathogen Free Hy-Line Brown layer chickens were orally vaccinated with the recombinant L. plantarum and presented with high levels of serum immunoglobulin G (IgG) and secretory immunoglobulin A (sIgA) titers in bile and duodenal-mucosal fluid. After challenged with ALV-J of a 3 × 103 50% tissue culture infective dose (TCID50), serum samples of the chickens were collected and viremia was analyzed. Results showed that, compared to the L. plantarum and PBS control group, the recombinant L. plantarum group showed a significant rise in antibody levels after inoculation, and provide improved protection against ALV-J according to viremia detection. These results indicate that oral immunization with the recombinant L. plantarum provided an effective means for eliciting protective immune response against early ALV-J infection.

CCCH-type zinc finger antiviral protein is specifically overexpressed in spleen in response to subgroup J avian leukosis virus infection in chicken

The CCCH-type zinc finger antiviral protein (ZAP), a host antiviral factor, plays an important role in innate defenses. Although the anti-viral mechanism of ZAP has been elucidated, however, the tissue specificity and the viral infection correlativity have not been fully understood. Here, we tested the dynamic distribution and localization of chicken ZAP (chZAP) before and after avian leukosis virus subgroup J (ALV-J) infection. The results showed that chZAP was highly expressed in adrenal gland and testis before ALV-J infection, and significantly upregulated in liver, kidney and bursa of Fabricius, and extremely overexpressed in spleen after ALV-J infection. The results indicated that chZAP is an inducible protein and showed specific overexpression in spleen after ALV-J infection. Furthermore, we demonstrated that chZAP, as a host intracytoplasmic factor, accumulated and migrated to the periphery of nucleus in DF-1 cells post-infection with ALV-J. Taken together, chZAP characterized as an inducible antiviral protein and specifically overexpressed in spleen after ALV-J infection.