Immunopathological investigation and genetic evolution of Avian leukosis virus Subgroup-J associated with myelocytomatosis in broiler flocks in Egypt.

BACKGROUND: Avian leukosis virus Subgroup-J (ALV-J) is a rapidly oncogenic evolving retrovirus infecting a variety of avian species; causing severe economic losses to the local poultry industry. METHODS: To investigate ALV-J, a total of 117 blood samples and 57 tissue specimens of different organs were collected for virological, and pathological identification, serological examinations, molecular characterization, and sequencing analysis. To the best of our knowledge, this is the first detailed report recorded in broiler flocks in Egypt. The present study targets the prevalence of a viral tumor disease circulating in broiler flocks in the El-Sharqia, El-Dakahliya, and Al-Qalyubiyya Egyptian governorates from 2021 to 2023 using different diagnostic techniques besides ALV-J gp85 genetic diversity determination. RESULT: We first isolated ALV-J on chicken embryo rough cell culture; showing aggregation, rounding, and degeneration. Concerning egg inoculation, embryonic death, stunting, and curling were observed. Only 79 serum samples were positive for ALV-J (67.52%) based on the ELISA test. Histopathological investigation showed tumors consist of uniform masses, usually well-differentiated myelocytes, lymphoid cells, or both in the liver, spleen, and kidneys. Immunohistochemical examination showed that the myelocytomatosis-positive signals were in the spleen, liver, and kidney. The PCR assay of ALV-J gp85 confirmed 545 base pairs with only 43 positive samples (75.4%). Two positive samples were sequenced and submitted to the Genbank with accession numbers (OR509852-OR509853). Phylogenetic analysis based on the gp85 gene showed that the ALV-J Dakahlia-2 isolate is genetically related to ALV-EGY/YA 2021.3, ALV-EGY/YA 2021.4, ALV-EGY/YA 2021.14, and ALV-EGY/YA 2021.9 with amino acid identity percentage 96%, 97%; 96%, 96%; respectively. Furthermore, ALV-J Sharqia-1 isolate is highly genetically correlated to ALV-EGY/YA 2021.14, and ALV-EGY/YA 2021.9, ALV-J isolate QL1, ALV-J isolate QL4, ALV-J isolate QL3, ALV-EGY/YA 2021.4 with amino acid identity percentage 97%, 97%; 98%, 97%, 97%, 95%; respectively. CONCLUSIONS: This study confirmed that ALV-J infection had still been prevalent in broilers in Egypt, and the genetic characteristics of the isolates are diverse.

UBE2J1 promotes ALV-A proviral DNA synthesis through the STAT3/IRF1 signaling pathway.

The ubiquitin-binding enzyme E2J1 is located on the endoplasmic reticulum membrane. It plays a role in transport throughout the process of ubiquitination. In mammals, UBE2J1 can promote RNA virus replication. However, the biological function of chicken UBE2J1 is unclear. In this study, chicken UBE2J1 was cloned for the first time, and UBE2J1 overexpression and shRNA knockdown plasmids were constructed. In chicken embryo fibroblasts, overexpression of UBE2J1 promoted the replication of subtype A avian leukosis virus, while knockdown of UBE2J1 inhibited the replication of ALV-A virus. In addition, we divided virus replication into virus adsorption and invasion into DF-1 cells, synthesis of proviral DNA, and release of viral particles. UBE2J1 promoted the replication of ALV-A virus by promoting the synthesis of proviral DNA. This result was caused by UBE2J1 inhibiting the production of interferon by inhibiting the STAT3/IRF1 pathway. We mutated ser at position 184 of UBE2J1 to Gly and found that this site plays a role as the phosphorylation site of UBE2J1. We confirmed that UBE2J1 promotes ALV-A replication in chicken embryo fibroblasts by inhibiting the STAT3/IRF1 pathway. This study provides new ideas and insights into ubiquitin-related proteins and antiviral immunity.

N123I mutation in the ALV-J receptor-binding domain region enhances viral replication ability by increasing the binding affinity with chNHE1.

The subgroup J avian leukosis virus (ALV-J), a retrovirus, uses its gp85 protein to bind to the receptor, the chicken sodium hydrogen exchanger isoform 1 (chNHE1), facilitating viral invasion. ALV-J is the main epidemic subgroup and shows noteworthy mutations within the receptor-binding domain (RBD) region of gp85, especially in ALV-J layer strains in China. However, the implications of these mutations on viral replication and transmission remain elusive. In this study, the ALV-J layer strain JL08CH3-1 exhibited a more robust replication ability than the prototype strain HPRS103, which is related to variations in the gp85 protein. Notably, the gp85 of JL08CH3-1 demonstrated a heightened binding capacity to chNHE1 compared to HPRS103-gp85 binding. Furthermore, we showed that the specific N123I mutation within gp85 contributed to the enhanced binding capacity of the gp85 protein to chNHE1. Structural analysis indicated that the N123I mutation primarily enhanced the stability of gp85, expanded the interaction interface, and increased the number of hydrogen bonds at the interaction interface to increase the binding capacity between gp85 and chNHE1. We found that the N123I mutation not only improved the viral replication ability of ALV-J but also promoted viral shedding in vivo. These comprehensive data underscore the notion that the N123I mutation increases receptor binding and intensifies viral replication.

The coinfection of ALVs causes severe pathogenicity in Three-Yellow chickens.

The coinfection of ALVs (ALV-J plus ALV-A or/and ALV-B) has played an important role in the incidence of tumors recently found in China in local breeds of yellow chickens. The study aims to obtain a better knowledge of the function and relevance of ALV coinfection in the clinical disease of avian leukosis, as well as its unique effect on the pathogenicity in Three-yellow chickens. One-day-old Three-yellow chicks (one day old) were infected with ALV-A, ALV-B, and ALV-J mono-infections, as well as ALV-A + J, ALV-B + J, and ALV-A + B + J coinfections, via intraperitoneal injection, and the chicks were then grown in isolators until they were 15 weeks old. The parameters, including the suppression of body weight gain, immune organ weight, viremia, histopathological changes and tumor incidence, were observed and compared with those of the uninfected control birds. The results demonstrated that coinfection with ALVs could induce more serious suppression of body weight gain (P < 0.05), damage to immune organs (P < 0.05) and higher tumor incidences than monoinfection, with triple infection producing the highest pathogenicity. The emergence of visible tumors and viremia occurred faster in the coinfected birds than in the monoinfected birds. These findings demonstrated that ALV coinfection resulted in considerably severe pathogenic and immunosuppressive consequences.

miR-155 facilitates the synergistic replication between avian leukosis virus subgroup J and reticuloendotheliosis virus by targeting a dual pathway.

IMPORTANCE: The synergy of two oncogenic retroviruses is an essential phenomenon in nature. The synergistic replication of ALV-J and REV in poultry flocks increases immunosuppression and pathogenicity, extends the tumor spectrum, and accelerates viral evolution, causing substantial economic losses to the poultry industry. However, the mechanism of synergistic replication between ALV-J and REV is still incompletely elusive. We observed that microRNA-155 targets a dual pathway, PRKCI-MAPK8 and TIMP3-MMP2, interacting with the U3 region of ALV-J and REV, enabling synergistic replication. This work gives us new targets to modulate ALV-J and REV's synergistic replication, guiding future research on the mechanism.

A Multiplex Quantitative Polymerase Chain Reaction for the Rapid Differential Detection of Subgroups A, B, J, and K Avian Leukosis Viruses.

Avian leukosis (AL), caused by avian leukosis virus (ALV), is a contagious tumor disease that results in significant economic losses for the poultry industry. Currently, ALV-A, B, J, and K subgroups are the most common in commercial poultry and cause possible coinfections. Therefore, close monitoring is necessary to avoid greater economic losses. In this study, a novel multiplex quantitative polymerase chain reaction (qPCR) assay was developed to detect ALV-A, ALV-B, ALV-J, and ALV-K with limits of detection of 40, 11, 13.7, and 96 copies/µL, respectively, and no cross-reactivity with other ALV subtypes and avian pathogens. We detected 852 cell cultures inoculated with clinical samples using this method, showing good consistency with conventional PCR and ELISA. The most prevalent ALV strain in Hubei Province, China, was still ALV-J (11.74%). Although single infections with ALV-A, ALV-B, and ALV-K were not found, coinfections with different subgroup strains were identified: 0.7% for ALV-A/J, 0.35% for ALV-B/J, 0.25% for ALV-J/K, and 0.12% for ALV-A/B/K and ALV-A/B/J. Therefore, our novel multiplex qPCR may be a useful tool for molecular epidemiology, clinical detection of ALV, and ALV eradication programs.

Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/ß-catenin signaling via interaction with P27.

Avian leukemia virus subgroup J (ALV-J) causes various diseases associated with tumor formation and decreased fertility and induced immunosuppressive disease, resulting in significant economic losses in the poultry industry globally. Virus usually exploits the host cellular machinery for their replication. Although there are increasing evidences for the cellular proteins involving viral replication, the interaction between ALV-J and host proteins leading to the pivotal steps of viral life cycle are still unclear. Here, we reported that ribonucleoside-diphosphate reductase subunit M2 (RRM2) plays a critical role during ALV-J infection by interacting with capsid protein P27 and activating Wnt/ß-catenin signaling. We found that the expression of RRM2 is effectively increased during ALV-J infection, and that RRM2 facilitates ALV-J replication by interacting with viral capsid protein P27. Furthermore, ALV-J P27 activated Wnt/ß-catenin signaling by promoting ß-catenin entry into the nucleus, and RRM2 activated Wnt/ß-catenin signaling by enhancing its phosphorylation at Ser18 during ALV-J infection. These data suggest that the upregulation of RRM2 expression by ALV-J infection favors viral replication in host cells via activating Wnt/ß-catenin signaling. IMPORTANCE Our results revealed a novel mechanism by which RRM2 facilitates ALV-J growth. That is, the upregulation of RRM2 expression by ALV-J infection favors viral replication by interacting with capsid protein P27 and activating Wnt/ß-catenin pathway in host cells. Furthermore, the phosphorylation of serine at position 18 of RRM2 was verified to be the important factor regulating the activation of Wnt/ß-catenin signaling. This study provides insights for further studies of the molecular mechanism of ALV-J infection.

Proteomic profiling of purified avian leukosis virus subgroup J particles.

While the presence of host cell proteins in virions and their role in viral life cycles have been demonstrated in various viruses, such characteristics have remained largely unknown in avian leukosis virus (ALV). To investigate whether this is the case in ALV, we purified high-integrity and high-purity virions from the avian leukosis virus subgroup J (ALV-J) and subjected them to proteome analysis using nano LC-MS/MS. This analysis identified 53 cellular proteins that are incorporated into mature ALV-J virions, and we verified the reliability of the packaged cellular proteins through subtilisin digestion and immunoblot analysis. Functional annotation revealed the potential functions of these proteins in the viral life cycle and tumorigenesis. Overall, our findings have important implications for understanding the interaction between ALV-J and its host, and provide new insights into the cellular requirements that define ALV-J infection.

Targeted Ablation of Exon 2 of the Avian Leukosis Virus-A (ALV-A) Receptor Gene in a Chicken Fibroblast Cell Line by CRISPR Abrogates ALV-A Infection.

The U.S. Department of Agriculture Avian Disease and Oncology Laboratory currently relies on live birds of specific genetic backgrounds for producing chicken-embryo fibroblasts that are used for the diagnosis and subtyping of field isolates associated with avian leukosis virus (ALV) outbreaks. As an alternative to maintaining live animals for this purpose, we are currently developing cell lines capable of achieving the same result by ablation of the entry receptors utilized by ALV strains. We used CRISPR-Cas9 on the cell fibroblast-derived cell line DF-1 to disrupt the tva gene, which encodes the receptor required for binding and entry of ALV-A into cells. We ultimately identified seven DF-1 clones that had biallelic and homozygous indels at the Cas9 target site, exon 2 of tva. When tested in vitro for their ability to host ALV-A, the five clones that had frameshift mutations that disrupted the Tva protein were unable to support ALV-A replication. This result clearly demonstrates that modified cell lines can be used as part of a battery of tests to determine ALV subtype for isolate characterization, thus eliminating the need for live birds.

Nota de investigación- La ablación dirigida del exón 2 del gene del receptor del virus de la leucosis aviar A (ALV-A) en una línea celular de fibroblastos de pollo mediante CRISPR anula la infección por ALV-A. El Laboratorio de Oncología y Enfermedades Aviares del Departamento de Agricultura de los Estados Unidos. actualmente depende de aves vivas con antecedentes genéticos específicos para producir fibroblastos de embrión de pollo que se utilizan para el diagnóstico y la subtipificación de aislamientos de campo asociados con brotes del virus de la leucosis aviar (ALV). Como alternativa al mantenimiento de animales vivos para este propósito, actualmente se están desarrollando líneas celulares capaces de lograr el mismo resultado mediante la ablación de los receptores de entrada utilizados por las cepas ALV. Se utilizó el método repeticiones palindrómicas cortas agrupadas y regularmente interespaciadas o CRISPR-Cas9 en la línea celular DF-1 derivada de fibroblastos para interrumpir el gene Tva, que codifica el receptor requerido para la unión y entrada de ALV-A en las células. Finalmente, se identificaron siete clones de DF-1 que tenían inserciones y deleciones (indeles) bialélicos y homocigóticos en el sitio blanco Cas9, exón 2 del gene tva. Cuando se probó in vitro su capacidad para albergar ALV-A, los cinco clones que tenían mutaciones que involucraban al marco de lectura y que interrumpieron la proteína Tva no pudieron admitir la replicación de ALVA. Este resultado demuestra claramente que las líneas celulares modificadas se pueden utilizar como parte de una batería de pruebas para determinar el subtipo de ALV para la caracterización de los aislamientos, eliminando así la necesidad de aves vivas.

Avian retroviral cardiomyopathy induced by infectious molecular clones of avian leukosis viruses (fowl glioma-inducing virus variants).

We previously described cardiomyocyte abnormality caused by Km_5666 strain, a variant of fowl glioma-inducing virus (FGV) prototype, which is an avian leukosis virus (ALV). However, the cardiac involvement appeared to be eradicated from the flock after a few years. An epidemiological survey from 2017 to 2020 was performed to elucidate the current prevalence of the cardiopathogenic strains in this flock. Four of the 71 bantams pathologically examined showed both glioma and cardiomyocyte abnormality, from which three ALV strains were detected. DNA sequencing revealed that several different ALV strains coexisted in each bantam and that the conserved Km_5666 virus fluid also contained at least two different ALV strains. We generated three infectious molecular clones from these samples, named KmN_77_clone_A, KmN_77_clone_B, and Km_5666_clone. The envSU of KmN_77_clone_A shared high sequence identity with that of Km_5666 (94.1%). In contrast, the envSU of KmN_77_clone_B showed >99.2% nucleotide similarity with that of an FGV variant without cardiopathogenicity. Furthermore, Km_5666_clone experimentally reproduced both gliomas and cardiomyocyte abnormality in chickens. From these results, it is suggested that the pathogenic determinant of cardiomyocyte abnormality is located in envSU similar to that of Km_5666. The cloning technique described here is beneficial for evaluating the viral pathogenicity in cases where affected birds are coinfected with several different ALV strains.

Molecular characteristics and pathogenicity of a Tibet-origin mutant avian leukosis virus subgroup J isolated from Tibetan chickens in China.

Tibetan chicken is found in China Tibet (average altitude; ˃4500 m). However, little is known about avian leukosis virus subgroup J (ALV-J) found in Tibetan chickens. ALV-J is a typical alpharetrovirus that causes immunosuppression and myelocytomatosis and thus seriously affects the development of the poultry industry. In this study, Tibet-origin mutant ALV-J was isolated from Tibetan chickens and named RKZ-1-RKZ-5. A Myelocytomatosis outbreak occurred in a commercial Tibetan chicken farm in Shigatse of Rikaze, Tibet, China, in March 2022. About 20% of Tibetan chickens in the farm showed severe immunosuppression, and mortality increased to 5.6%. Histopathological examination showed typical myelocytomas in various tissues. Virus isolation and phylogenetic analysis demonstrated that ALV-J caused the disease. Gene-wide phylogenetic analysis showed the RKZ isolates were the original strains of the previously reported Tibetan isolates (TBC-J4 and TBC-J6) (identity; 94.5% to 94.9%). Furthermore, significant nucleotide mutations and deletions occurred in the hr1 and hr2 hypervariable regions of gp85 gene, 3'UTR, Y Box, and TATA Box of 3'LTR. Pathogenicity experiments demonstrated that the viral load, viremia, and viral shedding level were significantly higher in RKZ-1-infected chickens than in NX0101-infected chickens. Notably, RKZ-1 caused more severe cardiopulmonary damage in SPF chickens. These findings prove the origin of Tibet ALV-J and provide insights into the molecular characteristics and pathogenic ability of ALV-J in the plateau area. Therefore, this study may provide a basis for ALV-J prevention and eradication in Tibet.

TRIM25 participates in the fibrous tissue hyperplasia induced by ALV-J infection in chickens by targeting 14-3-3σ protein.

ALV-J-SD1005 strain was subcutaneously inoculated into the necks of 1-day-old HY-Line Brown chickens and caused severe growth retardation, viremia and subcutaneous fibrosarcomas in the necks of all infected chickens from 14 days post inoculation (DPI) to 21 DPI, and also significantly increased the expressions of TRIM25, P53, etc., but significantly decreased the expressions of 14-3-3σ, etc. Overexpression of chicken TRIM25 (chTRIM25) significantly promoted cell proliferation and improved the expressions of P53, CDC2, and CDK2 tumor factors; and significantly inhibited the expression of 14-3-3σ in ALV-J-SD1005-infected DF1 cells; but knockdown of chTRIM25 caused the opposite effects. The results of co-immunoprecipitation (Co-IP) and confocal microscopy confirmed that chTRIM25 can recognize and bind 14-3-3σ protein in ALV-J-SD1005-infected cells, and they were co-located in the cytoplasm. It can be concluded that chTRIM25 participates in the fibrous tissue hyperplasia induced by ALV-J-SD1005 infections in chickens by binding 14-3-3σ protein and regulating the expressions of 14-3-3σ, P53, CDC2, and CDK2.

Simultaneous Detection of Three Subgroups of Avian Leukosis Virus Using the Nanoparticle-Assisted PCR Assay.

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid detection of pathogens. A sensitive and specific multiple nanoPCR assay was developed for simultaneous detection of avian leucosis virus (ALV) subgroups A, B and J. In this study, three pairs of primers were designed, based on the conserved region of the gp85 gene. An exploration of the optimal primer concentration and annealing temperature were carried out, for better performance of the nanoPCR assay. According to the results, the multiple nanoPCR assay amplified 336 pb, 625 bp and 167 bp fragments of ALV-A, -B and -J, respectively, and showed no cross-reactivity with irrelevant pathogens, suggesting the excellent specificity of the assay. The constructed standard DNA templates were used to estimate the limit of detection. As shown by the results, the detection limit of the nanoPCR assay was nearly 10 copies/µL. To further evaluate the detection ability of the assay, 186 clinical samples were detected using the nanoPCR assay, among which, 14 samples were confirmed as ALV positive; the results were further confirmed by sequencing. In conclusion, a highly specific and sensitive nanoPCR assay was successfully developed, which could be a useful tool for clinical diagnosis as well as for the discrimination of ALV-A, -B and -J.

Current Epidemiology and Co-Infections of Avian Immunosuppressive and Neoplastic Diseases in Chicken Flocks in Central China.

The avian immunosuppressive and neoplastic diseases caused by Marek's disease virus (MDV), avian leucosis virus (ALV), and reticuloendotheliosis virus (REV) are seriously harmful to the global poultry industry. In recent years, particularly in 2020-2022, outbreaks of such diseases in chicken flocks frequently occurred in China. Herein, we collected live diseased birds from 30 poultry farms, out of 42 farms with tumour-bearing chicken flocks distributed in central China, to investigate the current epidemiology and co-infections of these viruses. The results showed that in individual diseased birds, the positive infection rates of MDV, ALV, and REV were 69.5% (203/292), 14.4% (42/292), and 4.7% (13/277), respectively, while for the flocks, the positive infection rates were 96.7% (29/30), 36.7% (11/30), and 20% (6/30), respectively. For chicken flocks, monoinfection of MDV, ALV, or REV was 53.3% (16/30), 3.3% (1/30), and 0% (0/30), respectively, but a total of 43.3% (13/30) co-infections was observed, which includes 23.3% (7/30) of MDV+ALV, 10.0% (3/30) of MDV+REV, and 10.0% (3/30) of MDV+ALV+REV co-infections. Interestingly, no ALV+REV co-infection or REV monoinfection was observed in the selected poultry farms. Our data indicate that the prevalence of virulent MDV strains, partially accompanied with ALV and/or REV co-infections, is the main reason for current outbreaks of avian neoplastic diseases in central China, providing an important reference for the future control of disease.

A20 Enhances the Expression of the Proto-Oncogene C-Myc by Downregulating TRAF6 Ubiquitination after ALV-A Infection.

Hens infected with avian leukosis virus subgroup A (ALV-A) experience stunted growth, immunosuppression, and potentially, lymphoma development. According to past research, A20 can both promote and inhibit tumor growth. In this study, DF-1 cells were infected with ALV-A rHB2015012, and Gp85 expression was measured at various time points. A recombinant plasmid encoding the chicken A20 gene and short hairpin RNA targeting chicken A20 (A20-shRNA) was constructed and transfected into DF-1 cells to determine the effect on ALV-A replication. The potential signaling pathways of A20 were explored using bioinformatics prediction, co-immunoprecipitation, and other techniques. The results demonstrate that A20 and ALV-A promoted each other after ALV-A infection of DF-1 cells, upregulated A20, inhibited TRAF6 ubiquitination, and promoted STAT3 phosphorylation. The phosphorylated-STAT3 (p-STAT3) promoted the expression of proto-oncogene c-myc, which may lead to tumorigenesis. This study will help to further understand the tumorigenic process of ALV-A and provide a reference for preventing and controlling ALV.

Synergistic Immunosuppression of Avian Leukosis Virus Subgroup J and Infectious Bursal Disease Virus Is Responsible for Enhanced Pathogenicity.

In recent years, superinfections of avian leukosis virus subgroup J (ALV-J) and infectious bursal disease virus (IBDV) have been frequently observed in nature, which has led to the increasing virulence in infected chickens. However, the reason for the enhanced pathogenicity has remained unclear. In this study, we demonstrated an effective candidate model for studying the outcome of superinfections with ALV-J and IBDV in cells and specific-pathogen-free (SPF) chicks. Through in vitro experiments, we found that ALV-J and IBDV can establish the superinfection models and synergistically promote the expression of IL-6, IL-10, IFN-α, and IFN-γ in DF-1 and CEF cells. In vivo, the weight loss, survival rate, and histopathological observations showed that more severe pathogenicity was present in the superinfected chickens. In addition, we found that superinfections of ALV-J and IBDV synergistically increased the viral replication of the two viruses and inflammatory mediator secretions in vitro and in vivo. Moreover, by measuring the immune organ indexes and blood proportions of CD3+, CD4+, and CD8α+ cells, our results showed that the more severe instances of immunosuppression were observed in the superinfected chickens. In the present study, we concluded that the more severe immunosuppression induced by the synergistic viral replication of ALV-J and IBDV is responsible for the enhanced pathogenicity.

Isolation, identification, and pathogenicity of a ALV-K strain from Chinese indigenous chicken breed.

Subgroup K avian leukosis virus (ALV-K) is a new subgroup of avian leukosis virus (ALV) first identified in Chinese indigenous chickens in recent years. In this study, an ALV-K strain was isolated from Luhua chicken in Shandong province, China, and designated SD20LH01. The full-length genomic sequence of SD20LH01 was 7491 bp, which had the highest homology with ALV-K reference strains GDFX0601, GDFX0602 and GDFX0603. The nucleotide homology of env gene of SD20LH01 with reference strains of subgroup A, B, C, D, E, and J was ranged from 57.1 to 93.2%, while 94.1 to 99.4% with other ALV-K reference strains. The nucleotide difference of SD20LH01 mainly clustered with gp85 gene and U3 sequence when compared with the reference strain of ALV-K. In order to investigate the pathogenicity of SD20LH01, SPF chicken embryos were infected by yolk sac inoculation, and 1-day-old chickens were infected by intraperitoneal inoculation of SD20LH01. The results showed that yolk sac inoculation of SD20LH01 could induce persistent viremia, growth retardation and reduce the immune response to NDV and AIV-H9 vaccines. However, intraperitoneal inoculation in 1-day-old chickens could only induce a low level of viremia. In addition, no tumors were found in infected chickens during the animal experiments. This study enriched the genomic sequence data of ALV-K isolated in Chinese indigenous chickens, and laid a foundation for further study on the pathogenesis and prevention of ALV-K.

Residues L55 and W69 of Tva Mediate Entry of Subgroup A Avian Leukosis Virus.

The receptor of the subgroup A avian leukosis virus (ALV-A) in chicken is Tva, which is the homologous protein of human CD320 (huCD320), contains a low-density lipoprotein (LDL-A) module and is involved in the uptake of transcobalamin bound vitamin B12/cobalamin (Cbl). To map the functional determinants of Tva responsible for ALV-A receptor activity, a series of chimeric receptors were created by swapping the LDL-A module fragments between huCD320 and Tva. These chimeric receptors were then used for virus entry and binding assays to map the minimal ALV-A functional domain of Tva. The results showed that Tva residues 49 to 71 constituted the minimal functional domain that directly interacted with the ALV-A gp85 protein to mediate ALV-A entry. Single-residue substitution analysis revealed that L55 and W69, which were spatially adjacent on the surface of the Tva structure, were key residues that mediate ALV-A entry. Structural alignment results indicated that L55 and W69 substitutions did not affect the Tva protein structure but abolished the interaction force between Tva and gp85. Furthermore, substituting the corresponding residues of huCD320 with L55 and W69 of Tva converted huCD320 into a functional receptor of ALV-A. Importantly, soluble huCD320 harboring Tva L55 and W69 blocked ALV-A entry. Finally, we constructed a Tva gene-edited cell line with L55R and W69L substitutions that could fully resist ALV-A entry, while Cbl uptake was not affected. Collectively, our findings suggested that amino acids L55 and W69 of Tva were key for mediating virus entry. IMPORTANCE Retroviruses bind to cellular receptors through their envelope proteins, which is a crucial step in infection. While most retroviruses require two receptors for entry, ALV-A requires only one. Various Tva alleles conferring resistance to ALV-A, including Tvar1 (C40W substitution), Tvar2 (frame-shifting four-nucleotide insertion), Tvar3, Tvar4, Tvar5, and Tvar6 (deletion in the first intron), are known. However, the detailed entry mechanism of ALV-A in chickens remains to be explored. We demonstrated that Tva residues L55 and W69 were key for ALV-A entry and were important for correct interaction with ALV-A gp85. Soluble Tva and huCD320 harboring the Tva residues L55 and W69 effectively blocked ALV-A infection. Additionally, we constructed gene-edited cell lines targeting these two amino acids, which completely restricted ALV-A entry without affecting Cbl uptake. These findings contribute to a better understanding of the infection mechanism of ALV-A and provided novel insights into the prevention and control of ALV-A.

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.

The Emergence, Diversification, and Transmission of Subgroup J Avian Leukosis Virus Reveals that the Live Chicken Trade Plays a Critical Role in the Adaption and Endemicity of Viruses to the Yellow-Chickens.

The geographical spread and inter-host transmission of the subgroup J avian leukosis virus (ALV-J) may be the most important issues for epidemiology. An integrated analysis, including phylogenetic trees, homology modeling, evolutionary dynamics, selection analysis and viral transmission, based on the gp85 gene sequences of the 665 worldwide ALV-J isolates during 1988-2020, was performed. A new Clade 3 has been emerging and was evolved from the dominating Clade 1.3 of the Chinese Yellow-chicken, and the loss of a α-helix or ß-sheet of the gp85 protein monomer was found by the homology modeling. The rapid evolution found in Clades 1.3 and 3 may be closely associated with the adaption and endemicity of viruses to the Yellow-chickens. The early U.S. strains from Clade 1.1 acted as an important source for the global spread of ALV-J and the earliest introduction into China was closely associated with the imported chicken breeders in the 1990s. The dominant outward migrations of Clades 1.1 and 1.2, respectively, from the Chinese northern White-chickens and layers to the Chinese southern Yellow-chickens, and the dominating migration of Clade 1.3 from the Chinese southern Yellow-chickens to other regions and hosts, indicated that the long-distance movement of these viruses between regions in China was associated with the live chicken trade. Furthermore, Yellow-chickens have been facing the risk of infections of the emerging Clades 2 and 3. Our findings provide new insights for the epidemiology and help to understand the critical factors involved in ALV-J dissemination. IMPORTANCE Although the general epidemiology of ALV-J is well studied, the ongoing evolutionary and transmission dynamics of the virus remain poorly investigated. The phylogenetic differences and relationship of the clades and subclades were characterized, and the epidemics and factors driving the geographical spread and inter-host transmission of different ALV-J clades were explored for the first time. The results indicated that the earliest ALV-J (Clade 1.1) from the United States, acted as the source for global spreads, and Clades 1.2, 1.3 and 3 were all subsequently evolved. Also the epidemiological investigation showed that the early imported breeders and the inter-region movements of live chickens facilitated the ALV-J dispersal throughout China and highlighted the needs to implement more effective containment measures.