RESUMO
Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Galinhas , Animais , Vírus da Leucose Aviária/genética , Vírus da Leucose Aviária/fisiologia , Galinhas/virologia , Leucose Aviária/virologia , Leucose Aviária/genética , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/genética , Resistência à Doença/genética , Sistemas CRISPR-Cas , Edição de Genes , Embrião de Galinha , Evolução Molecular , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Fibroblastos/virologia , Fibroblastos/metabolismoRESUMO
Apoptosis plays a crucial role in host antiviral defense. The avian leukosis virus subgroup J (ALV-J), an avian oncogenic retrovirus, has been shown to suppress apoptosis while promoting its own replication. ALV-J induces myeloid tumors and hemangiomas in chickens resulting in significant economic losses for commercial layer and meat-type chicken production. B-cell lymphoma/leukemia 11B (Bcl11b) encodes a C2H2-type zinc finger protein-BCL11B, that exerts critical functions in cell proliferation, differentiation, and plays an essential role in the immune system. Previous study has been shown that Bcl11b is associated with ALV-J infection. In this study, we further investigated the pathological changes in ALV-J infected cells and examined the role and expression regulation of chicken Bcl11b. Our results demonstrate that Bcl11b, as an interferon-stimulated gene (ISG), encodes C2H2-type zinc finger protein BCL11B that promotes apoptosis to inhibit ALV-J infection. Additionally, gga-miR-1612 and gga-miR-6701-3p regulate apoptosis and are involved in ALV-J infection by targeting Bcl11b, thus revealing immune response strategies between the host and ALV-J. Although the underlying mechanisms require further validation, Bcl11b and its regulatory miRNAs are the first to demonstrate inhibition of ALV-J replication via apoptosis. BCL11B can a valuable target for treating diseases triggered by ALV-J infection.
Assuntos
Apoptose , Vírus da Leucose Aviária , Leucose Aviária , Galinhas , Replicação Viral , Vírus da Leucose Aviária/fisiologia , Animais , Leucose Aviária/virologia , MicroRNAs/genética , MicroRNAs/metabolismo , Dedos de Zinco , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Regulação da Expressão GênicaRESUMO
Avian leukosis virus (ALV) is an enveloped retrovirus with a single-stranded RNA genome, belonging to the genus Alpharetrovirus within the family Retroviridae. The disease (Avian leukosis, AL) caused by ALV is mainly characterized by tumor development and immunosuppression in chickens, which increases susceptibility to other pathogens and leads to significant economic losses in the Chinese poultry industry. The government and poultry industry have made lots of efforts to eradicate ALV, but the threat of which remains not vanished. This review provides a summary of the updated understanding of ALV in China, which mainly focuses on genetic and molecular biology, epidemiology, and diagnostic methods. Additionally, promising antiviral agents and ALV eradication strategies performed in China are also included.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Galinhas , Doenças das Aves Domésticas , Animais , Vírus da Leucose Aviária/fisiologia , Leucose Aviária/prevenção & controle , Leucose Aviária/virologia , Leucose Aviária/epidemiologia , China/epidemiologia , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/epidemiologiaRESUMO
Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs' eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Sistemas CRISPR-Cas , DNA Viral , Provírus , Vírus da Leucose Aviária/genética , Vírus da Leucose Aviária/isolamento & purificação , Vírus da Leucose Aviária/classificação , Animais , Provírus/genética , Provírus/isolamento & purificação , Leucose Aviária/virologia , Leucose Aviária/diagnóstico , DNA Viral/genética , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/diagnóstico , Galinhas/virologia , Sensibilidade e Especificidade , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismoRESUMO
Given the high prevalence of avian leukosis virus subgroup K (ALV-K) in chickens in China, the positive rate of ALV-K in local chickens in Henan province was investigated, and the genetic region encoding the glycoprotein gp85 of isolates from positive chickens was analyzed. The positive rate of ALV-K in local chickens in Henan was found to be 87.2% (41/47). Phylogenetic analysis of gp85 sequences revealed six clusters that differed in their host range regions (hr1 and hr2) and variable regions (vr1, vr2, and vr3). Evidence of recombination of hr1, hr2, vr1, vr2, and vr3 was observed between the different clusters. The isolate HN23LS02 appears to have obtained its hr1 and hr2 regions from separate lineages via recombination but without having a significant affect on the replication capacity of the virus.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Galinhas , Especificidade de Hospedeiro , Filogenia , Doenças das Aves Domésticas , Recombinação Genética , Proteínas do Envelope Viral , Animais , Vírus da Leucose Aviária/genética , Vírus da Leucose Aviária/classificação , Vírus da Leucose Aviária/isolamento & purificação , Galinhas/virologia , Leucose Aviária/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Doenças das Aves Domésticas/virologia , ChinaRESUMO
The tumor suppressor p53, primarily functioning as a transcription factor, has exhibited antiviral capabilities against various viruses in chickens, including infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). Nevertheless, the existence of a universal antiviral mechanism employed by chicken p53 (chp53) against these viruses remains uncertain. This study conducted a comprehensive comparison of molecular networks involved in chp53's antiviral function against IBDV, ALV-J, and ILTV. This was achieved through an integrated analysis of ChIP-seq data, examining chp53's genome-wide chromatin occupancy, and RNA-seq data from chicken cells infected with these viruses. The consistent observation of chp53 target gene enrichment in metabolic pathways, confirmed via ChIP-qPCR, suggests a ubiquitous regulation of host cellular metabolism by chp53 across different viruses. Further genome binding motif conservation analysis and transcriptional co-factor prediction suggest conserved transcriptional regulation mechanism by which chp53 regulates host cellular metabolism during viral infection. These findings offer novel insights into the antiviral role of chp53 and propose that targeting the virus-host metabolic interaction through regulating p53 could serve as a universal strategy for antiviral therapies in chickens.IMPORTANCEThe current study conducted a comprehensive analysis, comparing molecular networks underlying chp53's antiviral role against infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). This was achieved through a combined assessment of ChIP-seq and RNA-seq data obtained from infected chicken cells. Notably, enrichment of chp53 target genes in metabolic pathways was consistently observed across viral infections, indicating a universal role of chp53 in regulating cellular metabolism during diverse viral infections. These findings offer novel insights into the antiviral capabilities of chicken p53, laying a foundation for the potential development of broad-spectrum antiviral therapies in chickens.
Assuntos
Vírus da Leucose Aviária , Galinhas , Herpesvirus Galináceo 1 , Vírus da Doença Infecciosa da Bursa , RNA-Seq , Proteína Supressora de Tumor p53 , Animais , Galinhas/virologia , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Vírus da Leucose Aviária/genética , Vírus da Leucose Aviária/fisiologia , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/fisiologia , Herpesvirus Galináceo 1/genética , Sequenciamento de Cromatina por Imunoprecipitação , Antivirais/farmacologia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/genética , Regulação da Expressão GênicaRESUMO
Exosome-mediated horizontal and vertical transmission of subgroup J avian leukosis virus (ALV-J) in poultry flocks can lead to growth inhibition and severe immunosuppression. However, there are few reports on the early infection of chicken embryonic stem cells (cESCs) with ALV-J. In this study, we confirmed that early infection with ALV-J can accelerate the differentiation of cESCs and promote the secretion of exosomes. To investigate the modulation strategy of ALV-J in cESCs, circRNA sequencing was performed for further analysis. A total of 305 differentially expressed circRNAs (DECs) were obtained, including 71 upregulated DECs. Circ-CCDC7 was found to be the most upregulated DEC and was assessed by qRT-PCR, with the result consistent with the result of circRNA-seq. Based on qRT-PCR, gga-miR-6568-3p was found to be the target of the top 3 DECs, including circ-CCDC7, and the stem cell marker gene Pax7 was identified as the target gene of gga-miR-6568-3p. This study demonstrated that exosomal circ-CCDC7/gga-miR-6568-3p/Pax7 accelerates the differentiation of cESCs after early infection with ALV-J.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Diferenciação Celular , Galinhas , Exossomos , MicroRNAs , RNA Circular , Animais , Vírus da Leucose Aviária/fisiologia , Exossomos/metabolismo , Exossomos/virologia , Exossomos/genética , RNA Circular/genética , RNA Circular/metabolismo , Leucose Aviária/virologia , MicroRNAs/genética , MicroRNAs/metabolismo , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/genética , Células-Tronco Embrionárias/virologia , Células-Tronco Embrionárias/fisiologia , Embrião de Galinha , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismoRESUMO
Avian leukemia virus subgroup J (ALV-J) and chicken infectious anemia virus (CIAV) can be vertically transmitted; however, the pathogenicity of vertically transmitted coinfection with these 2 pathogens has not been studied. In this study, we created a model of chick morbidity in which chicks carried either ALV-J, CIAV, or both viruses via embryo inoculation. Thereafter, we analyzed the effects of vertically transmitted coinfection with CIAV and ALV-J on the pathogenicity of ALV-J and performed a purification assay based on hatching, mortality viremia positivity, and detection of fecal ALV-p27 antigen rates, and body weight. The hatching rate of the ALV-J+CIAV group was 68.57%, lower than those of the single infection and control groups. The survival curve showed that the mortality rates of the CIAV and ALV-J coinfection groups were higher than those of the single infection and control groups. Body weight statistics showed that coinfection aggravated the 7-d growth inhibition effect. The results of ALV-p27 antigen detection in cell culture supernatants showed that the positivity rates of the ALV-J and ALV-J+CIAV groups were 100% at all ages and 0% in the control group. The results of ALV-p27 antigen detection by anal swabs showed that the positivity rates of the ALV-J group were 92.86, 90.90, 88.89, and 93.33% at all ages, and that the ALV-J p27 positivity detection rate of anal swabs was lower than that of plasma virus isolation. The immune organ index of the ALV-J+CIAV group was significantly or very significantly lower than those of the single infection and control groups. The immune organ viral load showed that coinfection with CIAV and ALV-J promoted the proliferation of ALV-J and CIAV in immune organs. Coinfection with ALV-J and CIAV reduced chicken embryo hatchability and increased chick mortality and growth inhibition relative to their respective single infections. Additionally, coinfection with ALV-J + CIAV was even more detrimental in inducing immune organ atrophy (e.g., the thymus, spleen, and bursa), and promoted individual virus replication during coinfection.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Vírus da Anemia da Galinha , Galinhas , Infecções por Circoviridae , Coinfecção , Transmissão Vertical de Doenças Infecciosas , Doenças das Aves Domésticas , Animais , Vírus da Leucose Aviária/fisiologia , Vírus da Leucose Aviária/patogenicidade , Galinhas/virologia , Leucose Aviária/virologia , Coinfecção/veterinária , Coinfecção/virologia , Doenças das Aves Domésticas/virologia , Vírus da Anemia da Galinha/fisiologia , Vírus da Anemia da Galinha/patogenicidade , Infecções por Circoviridae/veterinária , Infecções por Circoviridae/virologia , Transmissão Vertical de Doenças Infecciosas/veterinária , Virulência , Embrião de GalinhaRESUMO
Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which cluster separately from the well-characterized subgroups A, B, C, D, E, and J in sequence analysis, and exhibits a specific host range and a unique pattern of superinfection interference. Avian leukosis virus subgroup K replicate more slowly in avian cells than other ALV strains, leading to escaped detection during ALV eradication, but the underlying mechanism are largely unknown. In our previous study, we have reported that JS11C1 and most of other suspected ALV-K strains possessed unique mutations in the U3 region. Here, we selected 5 mutations in some important transcriptional regulation elements to explore the possible factor contributing for the lower activity of LTR, including CA-TG mutation in the CAAT box, 21 nt deletion in the CAAT box, A-G and A-T mutations in the CArG boxes, 11 nt insertion in the PRE boxes, and C-T mutation in the TATA box. On the basis of infectious clone of JS11C1, we demonstrated that the 11 nt fragment in the PRE boxes was associated with the transcription activity of LTR, the enhancer ability of U3, and the replication capacity of the virus. Notably, we determined the differential U3-protein interaction profile of ALVs and found that the 11 nt fragment specifically binds to cellular SERPINE1 mRNA binding protein 1 (SERBP1) to increase the LTR activity and enhance virus replication. Collectively, these findings reveal that a 11 nt fragment in the U3 gene contributed to its binding ability to the cellular SERBP1 to enhance its transcription and the infectious virus productions in avian cells. This study highlighted the vital role of host factor in retrovirus replication and thus provides a new perspective to elucidate the interaction between retrovirus and its host and a molecular basis to develop efficient strategies against retroviruses.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Galinhas , Vírus da Leucose Aviária/fisiologia , Vírus da Leucose Aviária/genética , Animais , Leucose Aviária/virologia , Doenças das Aves Domésticas/virologia , Transcrição Gênica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , Linhagem Celular , MutaçãoRESUMO
N6-methyladenosine (m6A) methylation in transcripts has been suggested to influence tumorigenesis in liver tumors caused by the avian leukosis virus subgroup J (ALV-J). However, m6A modifications during ALV-J infection in vitro remain unclear. Herein, we performed m6A and RNA sequencing in ALV-J-infected chicken fibroblasts (DF-1). A total of 51 differentially expressed genes containing differentially methylated peaks were identified, which were markedly enriched in microRNAs (miRNAs) in cancer cells as well as apoptosis, mitophagy and autophagy, RNA degradation, and Hippo and MAPK signaling pathways. Correlation analysis indicated that YTHDC1 (m6A-reader gene) plays a key role in m6A modulation during ALV-J infection. The env gene of ALV-J harbored the strongest peak, and untranslated regions and long terminal repeats also contained peaks of different degrees. To the best of our knowledge, this is the first thorough analysis of m6A patterns in ALV-J-infected DF-1 cells. Combined with miRNA profiles, this study provides a useful basis for future research into the key pathways of ALV-J infection associated with m6A alteration.
Assuntos
Adenosina , Vírus da Leucose Aviária , Leucose Aviária , Galinhas , MicroRNAs , Doenças das Aves Domésticas , Transcriptoma , Animais , Vírus da Leucose Aviária/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Leucose Aviária/virologia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Fibroblastos/virologiaRESUMO
Avian leukosis virus subgroup J (ALV-J) is a retrovirus that can cause immunosuppression and tumors in chicken. However, relative pathogenesis is still not clear. At present, metabolomics has shown great potential in the screening of tumor metabolic markers, prognostic evaluation, and drug target design. In this study, we utilize an untargeted metabolomics approach based on ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) to analyze the metabolic changes in chicken embryo fibroblast (CEF) cells infected by ALV-J. We found that ALV-J infection significantly altered a wealth of metabolites compared with control group. Additionally, most of the differentially expressed metabolites belonged to lipid metabolism, purine nucleotide metabolism and amino acid metabolism. Among them, the proportion of lipid metabolites account for the highest proportion (around 31%). Results suggest that these changes may be conductive to the formation of virion, thereby promoting the replication of ALV-J. These data provided metabolic evidence and potential biomarkers for the cellular metabolic changes induced by ALV-J, and provided important insight for further understanding the replication needs and pathogenesis of ALV-J.
Assuntos
Vírus da Leucose Aviária , Fibroblastos , Metabolômica , Doenças das Aves Domésticas , Animais , Vírus da Leucose Aviária/fisiologia , Metabolômica/métodos , Embrião de Galinha , Fibroblastos/virologia , Cromatografia Líquida de Alta Pressão/veterinária , Doenças das Aves Domésticas/virologia , Espectrometria de Massas em Tandem/veterinária , Leucose Aviária/virologia , Galinhas , MetabolomaRESUMO
Avian leukemia is an infectious tumorous disease of chickens caused by subgroup A of the avian leukemia virus (ALV-A), which mainly causes long-term viremia, slow growth, immune suppression, decreased production performance, multi-tissue tumors, and even death. The infection rate of this disease is very high in chicken herds in China, causing huge economic losses to the poultry industry every year. We successfully expressed the specific antigen protein of ALV (P27) through recombinant protein technology and screened a pair of highly sensitive monoclonal antibodies (mAbs) through mouse immunity, cell fusion, and antibody pairing. Based on this pair of antibodies, we established a dual antibody sandwich ELISA and gold nanoparticle immunochromatographic strip (AuNP-ICS) detection method. In addition, the parameters of the dual antibody sandwich ELISA and AuNP-ICS were optimized under different reaction conditions, which resulted in the minimum detection limits of 0.2 ng mL-1 and 1.53 ng ml-1, respectively. Commonly available ELISA and AuNP-ICS products on the market were compared, and we found that our established immune rapid chromatography had higher sensitivity. This established AuNP-ICS had no cross-reactivity with Influenza A (H1N1), Influenza A (H9N2), respiratory syncytial virus (RSV), varicella-zoster virus (VZV), Listeria monocytogenes listeriolysin (LLO), and Staphylococcal enterotoxin SED or SEC. Finally, the established AuNP-ICS was used to analyze 35 egg samples, and the results showed 5 positive samples and 30 negative samples. The AuNP-ICS rapid detection method established by our group had good specificity, high sensitivity, and convenience, and could be applied to the clinical sample detection of ALV-A.
Assuntos
Vírus da Leucose Aviária , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Ouro , Nanopartículas Metálicas , Ouro/química , Nanopartículas Metálicas/química , Animais , Vírus da Leucose Aviária/isolamento & purificação , Vírus da Leucose Aviária/imunologia , Cromatografia de Afinidade/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Antígenos Virais/imunologia , Antígenos Virais/análise , Clara de Ovo/química , Fitas Reagentes , Galinhas , Limite de Detecção , Camundongos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/químicaRESUMO
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.
Assuntos
Vírus da Leucose Aviária , Técnicas de Diagnóstico Molecular , Transcrição Reversa , Animais , Vírus da Leucose Aviária/genética , Sensibilidade e Especificidade , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
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.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Doenças das Aves Domésticas , Embrião de Galinha , Animais , Galinhas , Leucose Aviária/patologia , Vírus da Leucose Aviária/genética , Egito/epidemiologia , Filogenia , Evolução Molecular , Aminoácidos/genéticaRESUMO
Avian leukosis virus Subgroup J (ALV-J) exhibits high morbidity and pathogenicity, affecting approximately 20% of poultry farms. It induces neoplastic diseases and immunosuppression. Phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), a proapoptotic mitochondrial protein in the B-cell lymphoma-2 (Bcl-2) family, plays a role in apoptosis in cancer cells. However, the connection between the PMAIP1 gene and ALV-J pathogenicity remains unexplored. This study investigates the potential impact of the PMAIP1 gene on ALV-J replication and its regulatory mechanisms. Initially, we examined PMAIP1 expression using quantitative real-time PCR (qRT-PCR) in vitro and in vivo. Furthermore, we manipulated PMAIP1 expression in chicken fibroblast cells (DF-1) and assessed its effects on ALV-J infection through qRT-PCR, immunofluorescence assay (IFA), and western blotting (WB). Our findings reveal a significant down-regulation of PMAIP1 in the spleen, lung, and kidney, coupled with an up-regulation in the bursa and liver of ALV-J infected chickens compared to uninfected ones. Additionally, DF-1 cells infected with ALV-J displayed a notable up-regulation of PMAIP1 at 6, 12, 24, 48, 74, and 108 h. Over-expression of PMAIP1 enhanced ALV-J replication, interferon expression, and proinflammatory factors. Conversely, interference led to contrasting results. Furthermore, we observed that PMAIP1 promotes virus replication by modulating mitochondrial function. In conclusion, the PMAIP1 gene facilitates virus replication by regulating mitochondrial function, thereby enriching our understanding of mitochondria-related genes and their involvement in ALV-J infection, offering valuable insights for avian leukosis disease resistance strategies.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Galinhas , Mitocôndrias , Doenças das Aves Domésticas , Replicação Viral , Animais , Vírus da Leucose Aviária/fisiologia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/genética , Mitocôndrias/metabolismo , Leucose Aviária/virologia , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismoRESUMO
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.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Animais , Embrião de Galinha , Vírus da Leucose Aviária/genética , Vírus da Leucose Aviária/metabolismo , Galinhas , Mamíferos , Provírus , Transdução de Sinais , Ubiquitinas , Fator de Transcrição STAT3/metabolismo , Fatores Reguladores de Interferon/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Lymphoid leukosis is a poultry neoplastic disease caused by avian leukosis virus (ALV) and is characterized by high morbidity and variable mortality rates in chicks. Currently, no effective treatment and vaccination is the only means to control it. This study exploited the immunoinformatics approaches to construct multi-epitope vaccine against ALV. ABCpred and IEDB servers were used to predict B and T lymphocytes epitopes from the viral proteins, respectively. Antigenicity, allergenicity and toxicity of the epitopes were assessed and used to construct the vaccine with suitable adjuvant and linkers. Secondary and tertiary structures of the vaccine were predicted, refined and validated. Structural errors, solubility, stability, immune simulation, dynamic simulation, docking and in silico cloning were also evaluated.The constructed vaccine was hydrophilic, antigenic and non-allergenic. Ramchandran plot showed most of the residues in the favored and additional allowed regions. ProsA server showed no errors in the vaccine structure. Immune simulation showed significant immunoglobulins and cytokines levels. Stability was enhanced by disulfide engineering and molecular dynamic simulation. Docking of the vaccine with chicken's TLR7 revealed competent binding energies.The vaccine was cloned in pET-30a(+) vector and efficiently expressed in Escherichia coli. This study provided a potent peptide vaccine that could assist in tailoring a rapid and cost-effective vaccine that helps to combat ALV. However, experimental validation is required to assess the vaccine efficiency.
Assuntos
Vírus da Leucose Aviária , Animais , Simulação de Acoplamento Molecular , Vacinas de Subunidades Proteicas , Imunoinformática , Galinhas , Epitopos de Linfócito T , Simulação de Dinâmica Molecular , Epitopos de Linfócito B , Vacinas de Subunidades Antigênicas , Biologia ComputacionalRESUMO
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.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Coinfecção , Neoplasias , Doenças das Aves Domésticas , Animais , Galinhas , Coinfecção/veterinária , Virulência , Viremia/veterinária , Leucose Aviária/epidemiologia , Neoplasias/veterinária , Peso Corporal , Doenças das Aves Domésticas/epidemiologiaRESUMO
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.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Doenças das Aves Domésticas , Animais , Vírus da Leucose Aviária/genética , Vírus da Leucose Aviária/química , Mutação , Galinhas , Isoformas de Proteínas/genética , Proteínas do Envelope Viral/genéticaRESUMO
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.