Virulence evaluation of Israeli Marek's disease virus isolates from commercial poultry using their meq gene sequence.

Fifty-seven Gallid alphaherpesvirus 2 (GaHV-2) isolates, collected during a 30-year period (1990-2019) from commercial poultry flocks affected by Marek's disease (MD), were molecularly characterised. The GaHV-2 meq gene was amplified and sequenced to evaluate the virus virulence, based on the number of PPPPs within the proline-rich repeats (PRRs) of its transactivation domain. The present illustration of virus virulence evaluation on a large scale of field virus isolates by molecular analysis exemplifies the practical benefit and usefulness of the molecular marker in commercial GaVH-2 isolates. The alternative assay of GaVH-2 virulence pathotyping is the classical Gold Standard ADOL method, which is difficult and impossible to employ on a large scale using the Specific Pathogen Free (SPF) chicks of the ADOL strains kept in isolators for two months. The phylogenetic analysis performed in the present study showed that the meq gene amino acid sequences of the 57 Israeli strains divide into 16 phylogenetic branches. The virulence evaluation was performed in comparison with 36 GaHV-2 prototype strains, previously characterised by the in vivo Gold Standard ADOL assay. The results obtained revealed that the GaHV-2 strains circulating in Israel have evolved into a higher virulence potential during the years, as the four-proline stretches number in the meq gene decreased over the investigated period, typically of very virulent virus prototypes. The present study supports the meq gene molecular markers for the assessment of field GaVH-2 strains virulence.

Efficacy and tolerability of an mRNA vaccine expressing gB and pp38 antigens of Marek's disease virus in chickens.

Marek's disease is a contagious proliferative disease of chickens caused by an alphaherpesvirus called Marek's disease virus. A bivalent mRNA vaccine encoding MDV's glycoprotein-B and phosphoprotein-38 antigens was synthesized and encapsulated in lipid nanoparticles. Tumor incidence, lesion score, organ weight indices, MDV genome load and cytokine expression were used to evaluate protection and immunostimulatory effects of the tested mRNA vaccine after two challenge trials. Results from the first trial showed decreased tumor incidence and a reduction in average lesion scores in chickens that received the booster dose. The second trial demonstrated that vaccination with the higher dose of the vaccine (10 µg) significantly decreased tumor incidence, average lesion scores, bursal atrophy, and MDV load in feather tips when compared to the controls. Changes in expression of type I and II interferons suggested a possible role for these cytokines in initiation and maintenance of the vaccine-originated immune responses.

Ancient chicken remains reveal the origins of virulence in Marek's disease virus.

The pronounced growth in livestock populations since the 1950s has altered the epidemiological and evolutionary trajectory of their associated pathogens. For example, Marek's disease virus (MDV), which causes lymphoid tumors in chickens, has experienced a marked increase in virulence over the past century. Today, MDV infections kill >90% of unvaccinated birds, and controlling it costs more than US$1 billion annually. By sequencing MDV genomes derived from archeological chickens, we demonstrate that it has been circulating for at least 1000 years. We functionally tested the Meq oncogene, one of 49 viral genes positively selected in modern strains, demonstrating that ancient MDV was likely incapable of driving tumor formation. Our results demonstrate the power of ancient DNA approaches to trace the molecular basis of virulence in economically relevant pathogens.

Phenotypic Characterization of Recombinant Marek's Disease Virus in Live Birds Validates Polymorphisms Associated with Virulence.

Marek's disease (MD) is a highly infectious lymphoproliferative disease in chickens with a significant economic impact. Mardivirus gallidalpha 2, also known as Marek's disease virus (MDV), is the causative pathogen and has been categorized based on its virulence rank into four pathotypes: mild (m), virulent (v), very virulent (vv), and very virulent plus (vv+). A prior comparative genomics study suggested that several single-nucleotide polymorphisms (SNPs) and genes in the MDV genome are associated with virulence, including nonsynonymous (ns) SNPs in eight open reading frames (ORF): UL22, UL36, UL37, UL41, UL43, R-LORF8, R-LORF7, and ICP4. To validate the contribution of these nsSNPs to virulence, the vv+MDV strain 686 genome was modified by replacing nucleotides with those observed in the vMDV strains. Pathogenicity studies indicated that these substitutions reduced the MD incidence and increased the survival of challenged birds. Furthermore, using the best-fit pathotyping method to rank the virulence, the modified vv+MDV 686 viruses resulted in a pathotype similar to the vvMDV Md5 strain. Thus, these results support our hypothesis that SNPs in one or more of these ORFs are associated with virulence but, as a group, are not sufficient to result in a vMDV pathotype, suggesting that there are additional variants in the MDV genome associated with virulence, which is not surprising given this complex phenotype and our previous finding of additional variants and SNPs associated with virulence.

LORF9 of Marek's disease virus is involved in the early cytolytic replication of B lymphocytes and can act as a target for gene deletion vaccine development.

IMPORTANCE: Marek's disease virus (MDV) is a highly infectious and oncogenic virus that can induce severe T cell lymphomas in chickens. MDV encodes more than 100 genes, most of which have unknown functions. This work indicated that the LORF9 gene is necessary for MDV early cytolytic replication in B lymphocytes. In addition, we have found that the LORF9 deletion mutant has a comparative immunological protective effect with CVI988/Rispens vaccine strain against very virulent MDV challenge. This is a significant discovery that LORF9 can be exploited as a possible target for the development of an MDV gene deletion vaccine.

The novel lncRNA-9802/miR-1646 axis affects cell proliferation of DF-1 by regulating Bax/Bcl-2 signaling pathway.

Marek's disease (MD) is a severe infectious and immunosuppressive neoplastic condition that significantly impacts the global poultry industry. Investigating the role of non-coding RNA in pathogenic mechanisms of MD virus (MDV) offers valuable insights for the effective prevention and management of MD. A higher expression of the novel lncRNA-9802 can be found in spleen tissues of MDV-infected chickens from our prior research, and there is a potential association between lncRNA-9802 and cell proliferation. In this study, we further demonstrated that over-expression of lncRNA-9802 could promote the proliferation of DF-1 cells. It has been established that lncRNA-9802 mediated its effects by binding to miR-1646, and further modulated the expression of the Bax and Bcl-2 genes. Deciphering the role of the recently discovered MD-associated lncRNA-9802/miR-1646 axis provides valuable theoretical basis for decoding the molecular mechanisms underlying MDV pathogenesis.

The genome evolution of Marek's disease viruses in chickens and turkeys in China.

The virus that causes Marek's disease (MD) is globally ubiquitous in chickens, continuously evolving, and poses a significant threat to the poultry industry. Although vaccines are extensively used, MD still occurs frequently and the virus has evolved increased virulence in China. Here, we report an outbreak of MD in vaccinated chickens and unvaccinated turkeys in a backyard farm in Guangdong province, China, in 2018. Phylogenetic analysis revealed two lineages of MDVs at this farm, with one lineage, containing isolates from two turkeys and five chickens, clustering with virulent Chinese strains and displays a relatively high genetic divergence from the vaccine strains. These new isolates appear to have broken through vaccine immunity, yielding this outbreak of MD in chickens and turkeys. The second lineage included four chicken isolates that clustered with the CVI988 and 814 vaccine strains. The large diversity of MDVs in this single outbreak reveals a complex circulation of MDVs in China. Poor breeding conditions and the weak application of disease prevention and control measures make backyard farms a hotbed for the evolution of viruses that cause infectious diseases. This is especially important in MDV as the MD vaccines do not provide sterilizing immunity, which allows the replication and shedding of virulent field viruses by vaccinated individuals and supporting the continuous evolution of MDVs. Hence, constant monitoring of the evolution of MDVs is necessary to understand the evolution of these field viruses and potential expansions of their host range.

Transgenerational epigenetic inheritance and immunity in chickens that vary in Marek's disease resistance.

Marek's disease virus (MDV), a naturally oncogenic, highly contagious alpha herpesvirus, induces a T cell lymphoma in chickens that causes severe economic loss. Marek's disease (MD) outcome in an individual is attributed to genetic and environmental factors. Further investigation of the host-virus interaction mechanisms that impact MD resistance is needed to achieve greater MD control. This study analyzed genome-wide DNA methylation patterns in 2 highly inbred parental lines 63 and 72 and 5 recombinant congenic strains (RCS) C, L, M, N, and X strains from those parents. Lines 63 and 72, are MD resistant and susceptible, respectively, whereas the RCS have different combinations of 87.5% Line 63 and 12.5% Line 72. Our DNA methylation cluster showed a strong association with MD incidence. Differentially methylated regions (DMRs) between the parental lines and the 5 RCS were captured. MD-resistant and MD-susceptible markers of DNA methylation were identified as transgenerational epigenetic inheritable. In addition, the growth of v-src DNA tumors and antibody response against sheep red blood cells differed among the 2 parental lines and the RCS. Overall, our results provide very solid evidence that DNA methylation patterns are transgenerational epigenetic inheritance (TEI) in chickens and also play a vital role in MD tumorigenesis and other immune responses; the specific methylated regions may be important modulators of general immunity.

Characterization of integrated Marek's disease virus genomes supports a model of integration by homology-directed recombination and telomere-loop-driven excision.

IMPORTANCE: Marek's disease virus (MDV) is a ubiquitous chicken pathogen that inflicts a large economic burden on the poultry industry, despite worldwide vaccination programs. MDV is only partially controlled by available vaccines, and the virus retains the ability to replicate and spread between vaccinated birds. Following an initial infection, MDV enters a latent state and integrates into host telomeres and this may be a prerequisite for malignant transformation, which is usually fatal. To understand the mechanism that underlies the dynamic relationship between integrated-latent and reactivated MDV, we have characterized integrated MDV (iMDV) genomes and their associated telomeres. This revealed a single orientation among iMDV genomes and the loss of some terminal sequences that is consistent with integration by homology-directed recombination and excision via a telomere-loop-mediated process.

Molecular characterization and phylogenetic analysis of Marek's disease virus in chickens from Ogun State, Nigeria.

Marek's disease (MD) is caused by oncogenic MD virus serotype 1 (MDV1) and is characterized by lymphoproliferative lesions resulting in high morbidity and mortality in chickens. Despite being ubiquitous on poultry farms, there is a dearth of information on its molecular characteristics in Nigeria. This study aimed at characterizing three virulence genes (Meq, pp38, and vIL-8) of MDV1 from chickens in Ogun state, Nigeria. Blood, feather quill, and tumour samples of chickens from different commercial poultry farms in Ogun State were pooled, spotted on 107 FTA cards, and screened for MDV1 by polymerase chain reaction (PCR). Phylogenetic analysis was carried out to compare Nigerian MDV1 Meq, pp38, and vIL-8 genes sequences with the published references. Thirteen samples were MDV1-positive and the Meq, as well as pp38, and vIL-8 genes from the different samples were 100% identical. The Meq genes contained 339 amino acids (aa) with three PPPP motifs in the transactivation domain and two interruptions of the PPPP motifs due to proline-to-arginine substitutions at positions 176 and 217 resulting in a 20.88% proline composition. Phylogenetic analysis revealed that the Meq gene clustered with strains from Egypt and very virulent ATE2539 strain from Hungary. Mutations were observed in the pp38 protein (at positions 107 and 109) and vIL-8 protein (at positions 4 and 31). Based on the molecular analysis of the three genes, the results indicate the presence of MDV1 with virulence signatures; therefore, further studies on in vivo pathotyping of Nigerian MDV1 from all states should be performed.RESEARCH HIGHLIGHTS Meq, pp38 and vIL-8 genes were 100% identical between Nigerian MDV strains.Proline content in Nigerian meq gene was 20.88% with two PPPP motifs interruptions.Meq, pp38 and vIL-8 genes of Nigerian MDV were similar to Egyptian and Indian strains.

Effect of Pre-Treatment with a Recombinant Chicken Interleukin-17A on Vaccine Induced Immunity against a Very Virulent Marek's Disease Virus.

The host response to pathogenic microbes can lead to expression of interleukin (IL)-17, which has antimicrobial and anti-viral activity. However, relatively little is known about the basic biological role of chicken IL-17A against avian viruses, particularly against Marek's disease virus (MDV). We demonstrate that, following MDV infection, upregulation of IL-17A mRNA and an increase in the frequency of IL-17A+ T cells in the spleen occur compared to control chickens. To elaborate on the role of chIL-17A in MD, the full-length chIL-17A coding sequence was cloned into a pCDNA3.1-V5/HIS TOPO plasmid. The effect of treatment with pcDNA:chIL-17A plasmid in combination with a vaccine (HVT) and very virulent(vv)MDV challenge or vvMDV infection was assessed. In combination with HVT vaccination, chickens that were inoculated with the pcDNA:chIL-17A plasmid had reduced tumor incidence compared to chickens that received the empty vector control or that were vaccinated only (66.6% in the HVT + empty vector group and 73.33% in HVT group versus 53.3% in the HVT + pcDNA:chIL-17A). Further analysis demonstrated that the chickens that received the HVT vaccine and/or plasmid expressing IL-17A had lower MDV-Meq transcripts in the spleen. In conclusion, chIL-17A can influence the immunity conferred by HVT vaccination against MDV infection in chickens.

Newcastle disease virus (NDV) recombinant expressing Marek's disease virus (MDV) glycoprotein B significantly protects chickens against MDV and NDV challenges.

Marek's disease (MD) is a highly contagious viral neoplastic disease of chickens caused by Marek's disease virus (MDV), resulting in significant economic losses to the poultry industry worldwide. The commonly used live and/or vectored MDV vaccines are expensive to produce and difficult to handle due to the requirement of liquid nitrogen for manufacturing and delivering frozen infected cells that are viable. In this study, we aimed to develop a Newcastle disease virus (NDV) vectored MDV vaccine that can be lyophilized, stored, and transported at 4 °C. Four NDV LaSota (LS) vaccine strain-based recombinant viruses expressing MDV glycoproteins gB, gC, gE, or gI were generated using reverse genetics technology. The biological assessments showed that these recombinant viruses were slightly attenuated in vivo yet retained similar growth kinetics and virus titers in vitro compared to the parental LaSota virus. Vaccination of leghorn chickens (Lines 15I5x71 F1 cross) with these recombinant viruses via intranasal and intraocular routes conferred different levels of protection against virulent MDV challenge. The recombinant expressing the MDV gB protein, rLS/MDV-gB, protected vaccinated birds significantly against MDV-induced tumor formation when challenged at 14 days post-vaccination (DPV) but moderately at 5 DPV. Whereas the other three recombinants provided little protection against the MDV challenge. All four recombinants conferred complete protection against the velogenic NDV challenge. These results demonstrated that the rLS/MDV-gB virus is a safe and efficacious dual vaccine candidate that can be lyophilized and potentially mass-administered via aerosol or drinking water to large chicken populations at a meager cost.

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.

Ultrasonographic features of Marek's disease in a chicken.

A 4-year-old chicken was presented with a history of anorexia, depression, and blindness. An ultrasound examination of the coelomic cavity was performed that revealed splenomegaly, hepatic nodules, and hypoechoic thickening of the intestinal wall. Ultrasonography of the coelomic cavity was done and revealed splenomegaly, nodular hepatic changes, and hypoechoic thickening of the intestinal wall. A diagnosis of Marek's disease was made based on the history and extension of the abdominal organ changes and confirmed by histopathology. This study describes an ultrasonographic appearance of Marek's disease in a chicken and emphasizes the importance and benefits of ultrasonography in staging the progression of Marek's disease.

Viral proteogenomic and expression profiling during productive replication of a skin-tropic herpesvirus in the natural host.

Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission, mostly due to a lack of natural virus-host model systems. Marek's disease is a devastating herpesviral disease of chickens caused by Marek's disease virus (MDV) and an excellent natural model to study skin-tropic herpesviruses and transmission. Like varicella zoster virus that causes chicken pox in humans, the only site where infectious cell-free MD virions are efficiently produced is in epithelial skin cells, a requirement for host-to-host transmission. Here, we enriched for heavily infected feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined short- and long-read RNA sequencing and LC/MS-MS bottom-up proteomics. Enrichment produced a previously unseen breadth and depth of viral peptide sequencing. We confirmed protein translation for 84 viral genes at high confidence (1% FDR) and correlated relative protein abundance with RNA expression levels. Using a proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts and identified a novel, abundant isoform of the 14 kDa transcript family via IsoSeq transcripts, short-read intron-spanning sequencing reads, and a high-quality junction-spanning peptide identification. We identified peptides representing alternative start codon usage in several genes and putative novel microORFs at the 5' ends of two core herpesviral genes, pUL47 and ICP4, along with strong evidence of independent transcription and translation of the capsid scaffold protein pUL26.5. Using a natural animal host model system to examine viral gene expression provides a robust, efficient, and meaningful way of validating results gathered from cell culture systems.

The pUL51 Tegument Protein Is Essential for Marek's Disease Virus Growth In Vitro and Bears a Function That Is Critical for Pathogenesis In Vivo.

pUL51 is a minor tegument protein important for viral assembly and cell-to-cell spread (CCS) but dispensable for replication in cell culture of all Herpesviruses for which its role has been investigated. Here, we show that pUL51 is essential for the growth of Marek's disease virus, an oncogenic alphaherpesvirus of chickens that is strictly cell-associated in cell culture. MDV pUL51 localized to the Golgi apparatus of infected primary skin fibroblasts, as described for other Herpesviruses. However, the protein was also observed at the surface of lipid droplets in infected chicken keratinocytes, hinting at a possible role of this compartment for viral assembly in the unique cell type involved in MDV shedding in vivo. Deletion of the C-terminal half of pUL51 or fusion of GFP to either the N- or C-terminus were sufficient to disable the protein's essential function(s). However, a virus with a TAP domain fused at the C-terminus of pUL51 was capable of replication in cell culture, albeit with viral spread reduced by 35% and no localization to lipid droplets. In vivo, we observed that although the replication of this virus was moderately impacted, its pathogenesis was strongly impaired. This study describes for the first time the essential role of pUL51 in the biology of a herpesvirus, its association to lipid droplets in a relevant cell type, and its unsuspected role in the pathogenesis of a herpesvirus in its natural host. IMPORTANCE Viruses usually spread from cell to cell through two mechanisms: cell-released virus and/or cell-to-cell spread (CCS). The molecular determinants of CCS and their importance in the biology of viruses during infection of their natural host are unclear. Marek's disease virus (MDV) is a deadly and highly contagious herpesvirus of chickens that produces no cell-free particles in vitro, and therefore, spreads only through CCS in cell culture. Here, we show that viral protein pUL51, an important factor for CCS of Herpesviruses, is essential for MDV growth in vitro. We demonstrate that the fusion of a large tag at the C-terminus of the protein is sufficient to moderately impair viral replication in vivo and almost completely abolish pathogenesis while only slightly reducing viral growth in vitro. This study thus uncovers a role for pUL51 associated with virulence, linked to its C-terminal half, and possibly independent of its essential functions in CCS.

The immune cell landscape and response of Marek's disease resistant and susceptible chickens infected with Marek's disease virus.

Genetically resistant or susceptible chickens to Marek's disease (MD) have been widely used models to identify the molecular determinants of these phenotypes. However, these prior studies lacked the basic identification and understanding of immune cell types that could be translated toward improved MD control. To gain insights into specific immune cell types and their responses to Marek's disease virus (MDV) infection, we used single-cell RNA sequencing (scRNAseq) on splenic cells from MD resistant and susceptible birds. In total, 14,378 cells formed clusters that identified various immune cell types. Lymphocytes, specifically T cell subtypes, were the most abundant with significant proportional changes in some subtypes upon infection. The largest number of differentially expressed genes (DEG) response was seen in granulocytes, while macrophage DEGs differed in directionality by subtype and line. Among the most DEG in almost all immune cell types were granzyme and granulysin, both associated with cell-perforating processes. Protein interactive network analyses revealed multiple overlapping canonical pathways within both lymphoid and myeloid cell lineages. This initial estimation of the chicken immune cell type landscape and its accompanying response will greatly aid efforts in identifying specific cell types and improving our knowledge of host response to viral infection.

Integrated analysis of methylation profiles and transcriptome of Marek's disease virus-infected chicken spleens reveal hypomethylation of CD4 and HMGB1 genes might promote Marek's disease tumorigenesis.

Marek's disease (MD) is a lymphoproliferative neoplastic disease caused by Marek's disease virus (MDV). Previous studies have showed that DNA methylation was involved in MD development, but systematic studies are still lacking. Herein, we performed whole genome bisulfite sequencing (WGBS) and RNA-seq in MDV-infected tumorous spleens (IN), noninfected spleens (NoIN), and survivor (SUR) spleens of chickens to identify the genes playing important roles in MD tumor transformation. We generated the first genome-wide DNA methylation profile of MDV-infected, noninfected, and survivor chickens. Combined the WGBS and RNA-Seq, we found that the expression of 25% differential expression genes (DEGs) were significantly correlated with methylation of CpG sites in their gene bodies or promoters. Further, we focused on the DEGs with differentially methylated regions (DMRs) on genes' body and promoter, and it showed the expression of 60% DEGs were significantly correlated with methylation of CpG sites in DMRs. Finally, we identified 8 genes, including CD4, CTLA4, DTL, HMGB1, LGMN, NUP210, RAD52, and ZAP70, and their expression was negatively correlated with methylation of DMRs in their promoters in both IN vs. NoIN and IN vs. SUR. These 8 genes showed specifically high expression in IN groups and clustered in module turquoise analyzed by WGCNA. Out of 8 genes, CD4 and HMGB1 were drop in QTLs associated with MD resistance. Thus, we overexpressed the 2 genes to simulate their high expression in the IN group and found they significantly promoted MDCC-MSB-1 cell proliferation, which revealed they might play promoting roles in MD tumorigenesis in IN due to their high expression induced by hypomethylation.

Differences in Pathogenicity and Vaccine Resistance Discovered between Two Epidemic Strains of Marek's Disease Virus in China.

Despite highly effective vaccines, Marek's disease (MD) causes great economic loss to the poultry industry annually, largely due to the continuous emergence of new MD virus (MDV) strains. To explore the pathogenic characteristics of newly emerged MDV strains, we selected two strains (AH/1807 and DH/18) with clinically different pathotypes. We studied each strain's infection process and pathogenicity and observed differences in immunosuppression and vaccine resistance. Specific pathogen-free chickens, unvaccinated or vaccinated with CVI988, were challenged with AH/1807 or DH/18. Both infections induced MD damage; however, differences were observed in terms of mortality (AH/1807: 77.8%, DH/18: 50%) and tumor rates (AH/1807: 50%, DH/18: 33.3%). The immune protection indices of the vaccine also differed (AH/1807: 94.1, DH/18: 61.1). Additionally, while both strains caused interferon-ß and interferon-γ expression to decline, DH/18 infection caused stronger immunosuppression than AH/1807. This inhibition persisted even after vaccination, leading to increased replication of DH/18 that ultimately broke through vaccine immune protection. These results indicate that both strains have different characteristics, and that strains such as DH/18, which cause weaker pathogenic damage but can break through vaccine immune protection, require further attention. Our findings increase the understanding of the differences between epidemic strains and factors underlying MD vaccination failure in China.

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.