Specific and Sensitive Visual Proviral DNA Detection of Major Pathogenic Avian Leukosis Virus Subgroups Using CRISPR-Associated Nuclease Cas13a.

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.

Viral miRNA delivered by exosomes from Marek's disease virus-transformed lymphoma cell line exerts regulatory function in internalized primary chicken embryo fibroblast cells.

In the past decade, research has demonstrated that viral miRNAs encoded by a number of viral genomes, particularly by most of the herpesvirus including Marek's disease virus (MDV), play important regulatory roles in viral infection, replication, and regulation of tumorigenesis. As macrovesicles in cells, exosomes can deliver viral miRNAs and exert gene regulatory functions. Whether the exosomes play a role in the replication, pathogenesis/tumorigenesis of avian herpesviruses such as oncogenic Marek's disease virus (MDV) remains unclear. Herein we extracted and identified the exosomes from MDV-transformed T cell line MSB-1 and demonstrated high abundance of MDV-1 miRNA expression. Using dual luciferase-based reporter assay, we also demonstrated that the exosomes derived from MSB-1 can deliver functional miRNA successfully into primary chicken embryo fibroblasts. These findings provide new insights into the role of exosomes and the mechanisms of how virus-encoded miRNA function in MDV latency/activation switching, viral replication, pathogenesis and/or tumorigenesis.

Immune escape of avian oncogenic Marek's disease herpesvirus and antagonistic host immune responses.

Marek's disease virus (MDV) is a highly pathogenic and oncogenic alpha herpesvirus that causes Marek's disease (MD), which is one of the most important immunosuppressive and rapid-onset neoplastic diseases in poultry. The onset of MD lymphomas and other clinical diseases can be efficiently prevented by vaccination; these vaccines are heralded as the first demonstration of a successful vaccination strategy against a cancer. However, the persistent evolution of epidemic MDV strains towards greater virulence has recently resulted in frequent outbreaks of MD in vaccinated chicken flocks worldwide. Herein, we provide an overall review focusing on the discovery and identification of the strategies by which MDV evades host immunity and attacks the immune system. We have also highlighted the decrease in the immune efficacy of current MD vaccines. The prospects, strategies and new techniques for the development of efficient MD vaccines, together with the possibilities of antiviral therapy in MD, are also discussed.

Regulation of Wnt/ß-catenin signaling by Marek's disease virus in vitro and in vivo.

Marek's disease virus (MDV) infection causes immunosuppression in the host, ultimately inducing tumor formation and causing significant economic losses to the poultry industry. While the abnormal activation of the Wnt/ß-catenin signaling pathway is closely associated with the occurrence and development of tumors. However, the relationship between MDV and the Wnt/ß-catenin pathway remains unclear. In this study, we found that the MDV RB1B strain, but not the MDV vaccine strain CVI988, activated the Wnt/ß-catenin signaling pathway by increasing the phosphorylation level of GSK-3ß in chicken embryo fibroblast (CEF). In vivo infection experiments in SPF chickens also confirmed that the RB1B strain activated the Wnt/ß-catenin signaling pathway, while the CVI988 strain did not lead to its activation. Moreover, unlike the Meq protein encoded by the CVI988 strain, the Meq protein encoded by the RB1B strain specifically activated the Wnt/ß-catenin signaling pathway in CEF cells. The findings from these studies extend our understanding of the regulation of Wnt/ß-catenin signaling by MDV, which make a new contribution to understanding the virus-host interactions of MDV.

Metabolomics analysis of CEF cells infected with avian leukosis virus subgroup J based on UHPLC-QE-MS.

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.

Dissection of key factors correlating with H5N1 avian influenza virus driven inflammatory lung injury of chicken identified by single-cell analysis.

Chicken lung is an important target organ of avian influenza virus (AIV) infection, and different pathogenic virus strains lead to opposite prognosis. Using a single-cell RNA sequencing (scRNA-seq) assay, we systematically and sequentially analyzed the transcriptome of 16 cell types (19 clusters) in the lung tissue of chickens infected with H5N1 highly pathogenic avian influenza virus (HPAIV) and H9N2 low pathogenic avian influenza virus (LPAIV), respectively. Notably, we developed a valuable catalog of marker genes for these cell types. Compared to H9N2 AIV infection, H5N1 AIV infection induced extensive virus replication and the immune reaction across most cell types simultaneously. More importantly, we propose that infiltrating inflammatory macrophages (clusters 0, 1, and 14) with massive viral replication, pro-inflammatory cytokines (IFN-ß, IL1ß, IL6 and IL8), and emerging interaction of various cell populations through CCL4, CCL19 and CXCL13, potentially contributed to the H5N1 AIV driven inflammatory lung injury. Our data revealed complex but distinct immune response landscapes in the lung tissue of chickens after H5N1 and H9N2 AIV infection, and deciphered the potential mechanisms underlying AIV-driven inflammatory reactions in chicken. Furthermore, this article provides a rich database for the molecular basis of different cell-type responses to AIV infection.

3'UTR of ALV-J can affect viral replication through promoting transcription and mRNA nuclear export.

IMPORTANCE: 3'UTRs can affect gene transcription and post-transcriptional regulation in multiple ways, further influencing the function of proteins in a unique manner. Recently, ALV-J has been mutating and evolving rapidly, especially the 3'UTR of viral genome. Meanwhile, clinical symptoms caused by ALV-J have changed significantly. In this study, we found that the ALV-J strains containing △-r-TM-type 3'UTR are the most abundant. By constructing ALV-J infectious clones and subgenomic vectors containing different 3'UTRs, we prove that 3'UTRs directly affect viral tissue preference and can promote virus replication as an enhancer. ALV-J strain containing 3'UTR of △-r-TM proliferated fastest in primary cells. All five forms of 3'UTRs can assist intron-containing viral mRNA nuclear export, with similar efficiency. ALV-J mRNA half-life is not influenced by different 3'UTRs. Our results dissect the roles of 3'UTR on regulating viral replication and pathogenicity, providing novel insights into potential anti-viral strategies.

Emerging Hypervirulent Marek's Disease Virus Variants Significantly Overcome Protection Conferred by Commercial Vaccines.

As one of the most important avian immunosuppressive and neoplastic diseases, Marek's disease (MD), caused by oncogenic Marek's disease virus (MDV), has caused huge economic losses worldwide over the past five decades. In recent years, MD outbreaks have occurred frequently in MD-vaccinated chicken flocks, but the key pathogenic determinants and influencing factors remain unclear. Herein, we analyzed the pathogenicity of seven newly isolated MDV strains from tumor-bearing chickens in China and found that all of them were pathogenic to chicken hosts, among which four MDV isolates, SDCW01, HNXZ05, HNSQ05 and HNSQ01, were considered to be hypervirulent MDV (HV-MDV) strains. At 73 days of the virus infection experiment, the cumulative incidences of MD were 100%, 93.3%, 90% and 100%, with mortalities of 83.3%, 73.3%, 60% and 86.7%, respectively, for the four viruses. The gross occurrences of tumors were 50%, 33.3%, 30% and 63.3%, respectively, accompanied by significant hepatosplenomegaly and serious atrophy of the immune organs. Furthermore, the immune protection effects of four commercial MD vaccines against SDCW01, CVI988, HVT, CVI988+HVT, and 814 were explored. Unexpectedly, during the 67 days of post-virus challenge, the protection indices (PIs) of these four MD vaccines were only 46.2%, 38.5%, 50%, and 28%, respectively, and the birds that received the monovalent CVI988 or HVT still developed tumors with cumulative incidences of 7.7% and 11.5%, respectively. To our knowledge, this is the first demonstration of the simultaneous comparison of the immune protection efficacy of multiple commercial MD vaccines with different vaccine strains. Our study revealed that the HV-MDV variants circulating in China could significantly break through the immune protection of the classical MD vaccines currently widely used. For future work, there is an urgent need to develop novel, more effective MD vaccines for tackling the new challenge of emerging HV-MDV strains or variants for the sustainable control of MD.

Efficient Cross-Screening and Characterization of Monoclonal Antibodies against Marek's Disease Specific Meq Oncoprotein Using CRISPR/Cas9-Gene-Edited Viruses.

Marek's disease (MD) caused by pathogenic Marek's disease virus type 1 (MDV-1) is one of the most important neoplastic diseases of poultry. MDV-1-encoded unique Meq protein is the major oncoprotein and the availability of Meq-specific monoclonal antibodies (mAbs) is crucial for revealing MDV pathogenesis/oncogenesis. Using synthesized polypeptides from conserved hydrophilic regions of the Meq protein as immunogens, together with hybridoma technology and primary screening by cross immunofluorescence assay (IFA) on Meq-deleted MDV-1 viruses generated by CRISPR/Cas9-gene editing, a total of five positive hybridomas were generated. Four of these hybridomas, namely 2A9, 5A7, 7F9 and 8G11, were further confirmed to secrete specific antibodies against Meq as confirmed by the IFA staining of 293T cells overexpressing Meq. Confocal microscopic analysis of cells stained with these antibodies confirmed the nuclear localization of Meq in MDV-infected CEF cells and MDV-transformed MSB-1 cells. Furthermore, two mAb hybridoma clones, 2A9-B12 and 8G11-B2 derived from 2A9 and 8G11, respectively, displayed high specificity for Meq proteins of MDV-1 strains with diverse virulence. Our data presented here, using synthesized polypeptide immunization combined with cross IFA staining on CRISPR/Cas9 gene-edited viruses, has provided a new efficient approach for future generation of specific mAbs against viral proteins.

Chicken Glycogen Synthase Kinase 3ß Suppresses Innate Immune Responses and Enhances Avian Leukosis Virus Replication in DF-1 Cells.

Glycogen synthase kinase 3ß (GSK3ß) is a widely distributed multifunctional serine/threonine kinase. In mammals, GSK3ß regulates important life activities such as proinflammatory response, anti-inflammatory response, immunity, and cancer development. However, the biological functions of chicken GSK3ß (chGSK3ß) are still unknown. In the present study, the full-length cDNA of chGSK3ß was first cloned and analyzed. Absolute quantification of chicken chGSK3ß in 1-day-old specific-pathogen-free birds has shown that it is widely expressed in all tissues, with the highest level in brain and the lowest level in pancreas. Overexpression of chGSK3ß in DF-1 cells significantly decreased the gene expression levels of interferon beta (IFN-ß), IFN regulatory factor 7 (IRF7), Toll-like receptor 3 (TLR3), melanoma differentiation-associated protein 5 (MDA5), MX-1, protein kinase R (PKR), and oligoadenylate synthase-like (OASL), while promoting the replication of avian leukosis virus subgroup J (ALV-J). Conversely, levels of most of the genes detected in this study were increased when chGSK3ß expression was knocked down using small interfering RNA (siRNA), which also inhibited the replication of ALV-J. These results suggest that chGSK3ß plays an important role in the antiviral innate immune response in DF-1 cells, and it will be valuable to carry out further studies on the biological functions of chGSK3ß. IMPORTANCE GSK3ß regulates many life activities in mammals. Recent studies revealed that chGSK3ß was involved in regulating antiviral innate immunity in DF-1 cells and also could positively regulate ALV-J replication. These results provide new insights into the biofunction of chGSK3ß and the virus-host interactions of ALV-J. In addition, this study provides a basis for further research on the function of GSK3 in poultry.

Critical roles of non-coding RNAs in lifecycle and biology of Marek's disease herpesvirus.

Over the past two decades, numerous non-coding RNAs (ncRNAs) have been identified in different biological systems including virology, especially in large DNA viruses such as herpesviruses. As a representative oncogenic alphaherpesvirus, Marek's disease virus (MDV) causes an important immunosuppressive and rapid-onset neoplastic disease of poultry, namely Marek's disease (MD). Vaccinations can efficiently prevent the onset of MD lymphomas and other clinical disease, often heralded as the first successful example of vaccination-based control of cancer. MDV infection is also an excellent model for research into virally-induced tumorigenesis. Recently, great progress has been made in understanding the functions of ncRNAs in MD biology. Herein, we give a review of the discovery and identification of MDV-encoded viral miRNAs, focusing on the genomics, expression profiles, and emerging critical roles of MDV-1 miRNAs as oncogenic miRNAs (oncomiRs) or tumor suppressor genes involved in the induction of MD lymphomas. We also described the involvements of host cellular miRNAs, lincRNAs, and circRNAs participating in MDV life cycle, pathogenesis, and/or tumorigenesis. The prospects, strategies, and new techniques such as the CRISPR/Cas9-based gene editing applicable for further investigation into the ncRNA-mediated regulatory mechanisms in MDV pathogenesis/oncogenesis were also discussed, together with the possibilities of future studies on antiviral therapy and the development of new efficient MD vaccines.

Infection phase-dependent dynamics of the viral and host N6-methyladenosine epitranscriptome in the lifecycle of an oncogenic virus in vivo.

Dynamic alteration of the epitranscriptome exerts regulatory effects on the lifecycle of oncogenic viruses in vitro. However, little is known about these effects in vivo because of the general lack of suitable animal infection models of these viruses. Using a model of rapid-onset Marek's disease lymphoma in chickens, we investigated changes in viral and host messenger RNA (mRNA) N6-methyladenosine (m6 A) modification during Marek's disease virus (MDV) infection in vivo. We found that the expression of major epitranscriptomic proteins varies among viral infection phases, reprogramming both the viral and the host epitranscriptomes. Specifically, the methyltransferase-like 3 (METTL3)/14 complex was suppressed during the lytic and reactivation phases of the MDV lifecycle, whereas its expression was increased during the latent phase and in MDV-induced tumors. METTL3/14 overexpression inhibits, whereas METTL3/14 knockdown enhances, MDV gene expression and replication. These findings reveal the dynamic features of the mRNA m6 A modification program during viral replication in vivo, especially in relation to key pathways involved in tumorigenesis.

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.

Fully Attenuated meq and pp38 Double Gene Deletion Mutant Virus Confers Superior Immunological Protection against Highly Virulent Marek's Disease Virus Infection.

Marek's disease virus (MDV) induces immunosuppression and neoplastic disease in chickens. The virus is controllable via an attenuated meq deletion mutant virus, which has the disadvantage of retaining the ability to induce lymphoid organ atrophy. To overcome this deficiency and produce more vaccine candidates, a recombinant MDV was generated from the highly virulent Md5BAC strain, in which both meq and a cytolytic replication-related gene, pp38, were deleted. Replication of the double deletion virus, Md5BAC ΔmeqΔpp38, was comparable with that of the parental virus in vitro. The double deletion virus was shown to be fully attenuated and to reduce lymphoid organ atrophy in vivo. Crucially, Md5BAC ΔmeqΔpp38 confers superior protection against highly virulent virus compared with a commercial vaccine strain, CVI988/Rispens. Transcriptomic profiling indicated that Md5BAC ΔmeqΔpp38 induced a different host immune response from CVI988/Rispens. In summary, a novel, effective, and safe vaccine candidate for prevention and control of MD caused by highly virulent MDV is reported. IMPORTANCE MDV is a highly contagious immunosuppressive and neoplastic pathogen. The virus can be controlled through vaccination via an attenuated meq deletion mutant virus that retains the ability to induce lymphoid organ atrophy. In this study, we overcame the deficiency by generating meq and pp38 double deletion mutant virus. Indeed, the successfully generated meq and pp38 double deletion mutant virus had significantly reduced replication capacity in vivo but not in vitro. It was fully attenuated and conferred superior protection efficacy against very virulent MDV challenge. In addition, the possible immunological protective mechanism of the double deletion mutant virus was shown to be different from that of the gold standard MDV vaccine, CVI988/Rispens. Overall, we successfully generated an attenuated meq deletion mutant virus and widened the range of potential vaccine candidates. Importantly, this study provides for the first time the theoretical basis of vaccination induced by fully attenuated gene-deletion mutant virus.

Proviral ALV-LTR Sequence Is Essential for Continued Proliferation of the ALV-Transformed B Cell Line.

Avian leukosis virus (ALV) induces B-cell lymphomas and other malignancies in chickens through insertional activation of oncogenes, and c-myc activation has been commonly identified in ALV-induced tumors. Using ALV-transformed B-lymphoma-derived HP45 cell line, we applied in situ CRISPR-Cas9 editing of integrated proviral long terminal repeat (LTR) to examine the effects on gene expression and cell proliferation. Targeted deletion of LTR resulted in significant reduction in expression of a number of LTR-regulated genes including c-myc. LTR deletion also induced apoptosis of HP45 cells, affecting their proliferation, demonstrating the significance of LTR-mediated regulation of critical genes. Compared to the global effects on expression and functions of multiple genes in LTR-deleted cells, deletion of c-myc had a major effect on the HP45 cells proliferation with the phenotype similar to the LTR deletion, demonstrating the significance of c-myc expression in ALV-induced lymphomagenesis. Overall, our studies have not only shown the potential of targeted editing of the LTR for the global inhibition of retrovirus-induced transformation, but also have provided insights into the roles of LTR-regulated genes in ALV-induced neoplastic transformation.

A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology.

Marek's disease virus (MDV) is an important oncogenic α-herpesvirus that induces Marek's disease (MD), characterized by severe immunosuppression and rapid-onset T-cell lymphomas in its natural chicken hosts. Historically, MD is regarded as an ideal biomedical model for studying virally induced cancers. Monoclonal antibodies (mAbs) against viral or host antigenic epitopes are crucial for virology research, especially in the exploration of gene functions, clinical therapy, and the development of diagnostic reagents. Utilizing the CRISPR/Cas9-based gene-editing technology, we produced a pp38-deleted MDV-1 mutant-GX0101Δpp38-and used it for the rapid screening and identification of pp38-specific mAbs from a pool of MDV-specific antibodies from 34 hybridomas. The cross-staining of parental and mutated MDV plaques with hybridoma supernatants was first performed by immunofluorescence assay (IFA). Four monoclonal hybridomas-namely, 4F9, 31G7, 34F2, and 35G9-were demonstrated to secrete specific antibodies against MDV-1's pp38 protein, which was further confirmed by IFA staining and confocal analysis. Further experiments using Western blotting, immunoprecipitation (IP), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and immunohistochemistry (IHC) analysis demonstrated that the pp38-specific mAb 31G7 has high specificity and wide application potential for further research in MD biology. To the best of our knowledge, this is the first demonstration of the use of CRISPR/Cas9-based gene-editing technology for efficient screening and identification of mAbs against a specific viral protein, and provides a meaningful reference for the future production of antibodies against other viruses-especially for large DNA viruses such as herpesviruses.

Pathogenicity and Pathotype Analysis of Henan Isolates of Marek's Disease Virus Reveal Long-Term Circulation of Highly Virulent MDV Variant in China.

In recent years, outbreaks of Marek's disease (MD) have been frequently reported in vaccinated chicken flocks in China. Herein, we have demonstrated that four Marek's disease virus (MDV) isolates, HN502, HN302, HN304, and HN101, are all pathogenic and oncogenic to hosts. Outstandingly, the HN302 strain induced 100% MD incidence, 54.84% mortality, and 87.10% tumor incidence, together with extensive atrophy of immune organs. Pathotyping of HN302 was performed in comparison to a standard very virulent (vv) MDV strain Md5. We found that both CVI988 and HVT vaccines significantly reduced morbidity and mortality induced by HN302 or Md5 strains, but the protection indices (PIs) provided by these two vaccines against HN302 were significantly lower (27.03%) or lower (33.33%) than that against Md5, which showed PIs of 59.89% and 54.29%, respectively. These data suggested that HN302 possesses a significant higher virulence than Md5 and at least could be designated as a vvMDV strain. Together with our previous phylogenetic analysis on MDV-1 meq genes, we have presently suggested HN302 to be a typical highly virulent MDV variant belonging to an independent Chinese branch. To our knowledge, this is the first report to provide convincible evidence to identify a pathogenic MDV variant strain with a higher virulence than Md5 in China, which may have emerged and circulating in poultry farms in China for a long time and involved in the recent MD outbreaks.

Establishment and Application of a Real-Time Recombinase Polymerase Amplification Assay for the Detection of Avian Leukosis Virus Subgroup J.

Avian leukosis caused by avian leukosis virus (ALV), belonging to the genus Alpharetrovirus of the family Retroviridae, is associated with benign and malignant tumors in hemopoietic cells in poultry. Although several methods have been developed for ALV detection, most of them are not suitable for rapid on-site testing due to instrument limitations, professional operators, or the low sensitivity of the method. Herein, we described the real-time recombinase polymerase amplification (RPA) assay for rapid detection of ALV subgroup J (ALV-J). The major viral structural glycoprotein gp85, highly specific for the subgroup, was used as the molecular target for the real-time RPA assay. The results were obtained at 38°C within 20 min, with the detection sensitivity of 10 copies/µl of standard plasmid pMD18-T-gp85 as the template per reaction. Real-time RPA was capable of ALV-J-specific detection without cross-reaction with other non-targeted avian pathogens. Of the 62 clinical samples tested, the ALV-positive rates of real-time RPA, PCR, and real-time PCR were 66.13% (41/62), 59.68% (37/62), and 67.74% (42/62), respectively. The diagnostic agreement between real-time RPA and real-time PCR was 98.39% (61/62), and the kappa value was 0.9636. The developed real-time ALV-J assay seems promising for rapid and sensitive detection of ALV-J in diagnostic laboratories. It is suitable for on-site detection, especially in a poor resource environment, thus facilitating the prevention and control of ALV-J.

TMT-based proteomic analysis reveals integrins involved in the synergistic infection of reticuloendotheliosis virus and avian leukosis virus subgroup J.

BACKGROUND: Co-infection with the avian leukosis virus subgroup J (ALV-J) and the reticuloendotheliosis virus (REV) increases mutual viral replication, causing a more serious pathogenic effect by accelerating the progression of neoplasia and extending the tumor spectrum. However, the molecular mechanism underlying the synergistic replication of ALV-J and REV remains unclear. RESULTS: Here, we performed this study to compare the differentially expressed proteins among CEF cells infected with ALV-J, REV or both at the optimal synergistic infection time using TMT-based quantitative proteomics. We identified a total of 719 (292 upregulated and 427 downregulated) and 64 (35 upregulated and 29 downregulated) proteins by comparing co-infecting both viruses with monoinfecting ALV-J and REV, respectively. GO annotation and KEGG pathway analysis showed the differentially expressed proteins participated in virus-vector interaction, biological adhesion and immune response pathways in the synergistic actions of ALV-J and REV at the protein levels. Among the differentially expressed proteins, a large number of integrins were inhibited or increased in the co-infection group. Further, eight integrins, including ITGα1, ITGα3, ITGα5, ITGα6, ITGα8, ITGα9, ITGα11 and ITGß3, were validated in CEF cells by qRT-PCR or western blot. CONCLUSIONS: These findings proved that integrins may be key regulators in the mechanism of synergistic infection of REV and ALV-J, which will provide more insight into the pathogenesis of synergism of REV and ALV-J at protein level.

Targeted Deletion of Glycoprotein B Gene by CRISPR/Cas9 Nuclease Inhibits Gallid herpesvirus Type 3 in Dually Infected Marek's Disease Virus-Transformed Lymphoblastoid Cell Line MSB-1.

Marek's disease virus (MDV) is a member of the genus Mardivirus in the subfamily Alphaherpesvirinae. There are three different serotypes of MDV designated as MDV-1 (Gallid herpesvirus type 2), MDV-2 (Gallid herpesvirus type 3), and MDV-3 (Meleagrid herpesvirus 1, herpesvirus of turkeys, HVT). MDV-1 is the only serotype that induces Marek's disease (MD), a lymphoproliferative disorder resulting in aggressive T-cell lymphomas and paralytic symptoms. In the lymphomas and lymphoblastoid cell lines (LCL) derived from them, MDV establishes latent infection with limited viral gene expression. The latent viral genome in LCL can be activated by co-cultivation with chicken embryo fibroblast (CEF) monolayers. MSB-1, one of the first MDV-transformed LCL established from the splenic lymphoma, is distinct in harboring both the oncogenic MDV-1 and non-oncogenic MDV-2 viruses. Following the successful application of CRISPR/Cas9 editing approach for precise knockdown of the MDV-1 genes in LCL, we describe here the targeted deletion of MDV-2 glycoprotein B (gB) in MSB-1 cells. Due to the essential nature of gB for infectivity, the production of MDV-2 plaques on CEF was completely abolished in the MDV-2-gB-deleted MSB-1 cells. Our study has demonstrated that the CRISPR/Cas9 system can be used for targeted inactivation of the co-infecting MDV-2 without affecting the MDV-1 in the MSB-1 cell line. Successful inactivation of MDV-2 demonstrated here also points toward the possibility of using targeted gene editing as an antiviral strategy against pathogenic MDV-1 and other viruses infecting chickens. IMPORTANCE Marek's disease (MD) is a lymphoproliferative disease of chickens characterized by rapid-onset lymphomas in multiple organs and by infiltration into peripheral nerves, causing paralysis. Lymphoblastoid cell lines (LCL) derived from MD lymphomas have served as valuable resources to improve understanding of distinct aspects of virus-host interactions in transformed cells including transformation, latency, and reactivation. MDV-transformed LCL MSB-1, derived from spleen lymphoma induced by the BC-1 strain of MDV, has a unique feature of harboring an additional non-pathogenic MDV-2 strain HPRS-24. By targeted deletion of essential gene glycoprotein B from the MDV-2 genome within the MSB-1 cells, we demonstrated the total inhibition of MDV-2 virus replication on co-cultivated CEF, with no effect on MDV-1 replication. The identified viral genes critical for reactivation/inhibition of viruses will be useful as targets for development of de novo disease resistance in chickens to avian pathogens.