Development and evaluation of a chicken embryo fibroblast cell culture based live attenuated Indian strain duck plague vaccine.

Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 107.5 TCID50/mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.

mRNA Splicing of UL44 and Secretion of Alphaherpesvirinae Glycoprotein C (gC) Is Conserved among the Mardiviruses.

Marek's disease (MD), caused by gallid alphaherpesvirus 2 (GaAHV2) or Marek's disease herpesvirus (MDV), is a devastating disease in chickens characterized by the development of lymphomas throughout the body. Vaccine strains used against MD include gallid alphaherpesvirus 3 (GaAHV3), a non-oncogenic chicken alphaherpesvirus homologous to MDV, and homologous meleagrid alphaherpesvirus 1 (MeAHV1) or turkey herpesvirus (HVT). Previous work has shown most of the MDV gC produced during in vitro passage is secreted into the media of infected cells although the predicted protein contains a transmembrane domain. We formerly identified two alternatively spliced gC mRNAs that are secreted during MDV replication in vitro, termed gC104 and gC145 based on the size of the intron removed for each UL44 (gC) transcript. Since gC is conserved within the Alphaherpesvirinae subfamily, we hypothesized GaAHV3 (strain 301B/1) and HVT also secrete gC due to mRNA splicing. To address this, we collected media from 301B/1- and HVT-infected cell cultures and used Western blot analyses and determined that both 301B/1 and HVT produced secreted gC. Next, we extracted RNAs from 301B/1- and HVT-infected cell cultures and chicken feather follicle epithelial (FFE) skin cells. RT-PCR analyses confirmed one splicing variant for 301B/1 gC (gC104) and two variants for HVT gC (gC104 and gC145). Interestingly, the splicing between all three viruses was remarkably conserved. Further analysis of predicted and validated mRNA splicing donor, branch point (BP), and acceptor sites suggested single nucleotide polymorphisms (SNPs) within the 301B/1 UL44 transcript sequence resulted in no gC145 being produced. However, modification of the 301B/1 gC145 donor, BP, and acceptor sites to the MDV UL44 sequences did not result in gC145 mRNA splice variant, suggesting mRNA splicing is more complex than originally hypothesized. In all, our results show that mRNA splicing of avian herpesviruses is conserved and this information may be important in developing the next generation of MD vaccines or therapies to block transmission.

Molecular characterization of the meq oncogene of Marek's disease virus in vaccinated Brazilian poultry farms reveals selective pressure on prevalent strains.

Marek's disease virus (MDV) has become an increasingly virulent pathogen in the poultry industry despite vaccination efforts to control it. Brazil has experienced a significant rise of Marek's disease (MD) outbreaks in recent years. Our study aimed to analyze the complete meq gene sequences to understand the molecular epidemiological basis of MD outbreaks in Brazilian vaccinated layer farms. We detected a high incidence rate of visceral MD (67.74%) and multiple circulating MDV strains. The most prevalent and geographically widespread genotype presented several clinical and molecular characteristics of a highly virulent strain and evolving under positive selective pressure. Phylogenetic and phylogeographic analysis revealed a closer relationship with strains from the USA and Japan. This study sheds light on the circulation of MDV strains capable of infecting vaccinated birds. We emphasize the urgency of adopting preventive measures to manage MDV outbreaks threatening the poultry farming industry.

RNA-seq analysis of chlorogenic acid intervention in duck embryo fibroblasts infected with duck plague virus.

INTRODUCTION: Chlorogenic acid, the primary active component in Chinese medicines like honeysuckle, exhibits anti-inflammatory and antiviral effects. It has been demonstrated that chlorogenic acid effectively prevents and treats Duck enteritis virus (DEV) infection. This study aims to further elucidate the mechanism by which chlorogenic acid prevents DEV infection. METHODS: Duck embryo fibroblast (DEF) cells were pre-treated with chlorogenic acid before being infected with DEV. Cell samples were collected at different time points for transcriptomic sequencing, while qPCR was used to detect the proliferation of DEV. Additionally, 30-day-old ducks were treated with chlorogenic acid, and their lymphoid organs were harvested for histopathological sections to observe pathological damage. The proliferation of DEV in the lymphoid organs was also detected using qPCR Based on the transcriptomic sequencing results, NF-κB1 gene was silenced by RNAi technology to analyze the effect of NF-κB1 gene on DEV proliferation. RESULTS: Compared to the viral infection group, DEF cells in the chlorogenic acid intervention group exhibited significantly reduced DEV load (P < 0.05). Transcriptomic sequencing results suggested that chlorogenic acid inhibited DEV proliferation in DEF cells by regulating NF-κB signaling pathway. The results of RNAi silencing suggested that in the three treatment groups, compared with the DEV experimental group, there was no significant difference in the effect of pre-transfection after transfection on DEV proliferation, while both the pre-transfection after transfection and the simultaneous transfection group showed significant inhibition on DEV proliferation Furthermore, compared to the virus infection group, ducks in the chlorogenic acid intervention group showed significantly decreased DEV load in their lymphoid organs (P < 0.05), along with alleviated pathological damage such as nuclear pyretosis and nuclear fragmentation. CONCLUSIONS: Chlorogenic acid effectively inhibits DEV proliferation in DEF and duck lymphatic organs, mitigates viral-induced pathological damage, and provides a theoretical basis for screening targeted drugs against DEV.

Duck plague virus Us3 regulates the expression of pUL48.

Duck plague (DP) is one of the contagious diseases caused by Duck plague virus (DPV), which is a serious threat to the development of duck farming. Us3 is a PKA-like protein kinase in alphaherpesvirus, which can regulate the biological functions of many viral proteins, but whether Us3 regulates pUL48 protein has not been reported. In this paper, Western Blot, qRT-PCR, dual luciferase reporter system and Co-IP were used to investigate the relationship between pUL48 and Us3. The results showed that: 1) pUL48 interacted with Us3 at 138-256aa through its DBD region. 2) Us3 enhanced the protein expression of pUL48 in a dose-dependent manner. 3) Us3 promoted the mRNA level of pUL48 by activating its promoter activity. 4) Us3 inhibited the transcriptional activation function of pUL48. The results can provide scientific data for perfecting and supplementing the function of alpha herpesvirus Us3 and pUL48.

Tracing the origin of virulence.

Microbial genomes from ancient chickens uncover the drivers of pathogenicity.

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.

Duck plague virus tegument protein vp22 plays a key role in the secondary envelopment and cell-to-cell spread.

Duck plague virus (DPV) is one of the major infectious and fatal diseases of geese, ducks, and other wild waterfowl. The DPV UL49 gene product VP22 is one of the most abundant tegument proteins. However, the role of the DPV VP22 is enigmatic to be clarified. In this study, we found deletion of the UL49 gene resulted in reduced viral growth curve and smaller plaque size in duck embryo fibroblast (DEF) cells, confirming that DPV VP22 is required for efficient viral growth in vitro. In addition, deletion of the UL49 gene inhibited the secondary envelopment of the virus, the release of viral particles, and the spread of viruses between cells. Our study signified the importance of VP22 for DPV secondary envelopment, release, cell-to-cell spread, and accumulation of viral RNA. These findings provide a basis for further study of the function of VP22 in DPV or other herpesviruses.

N-Linked Glycosylation and Expression of Duck Plague Virus pUL10 Promoted by pUL49.5.

Duck plague virus (DPV) is a member of the alphaherpesvirus subfamily, and its genome encodes a conserved envelope protein, protein UL10 (pUL10). pUL10 plays complex roles in viral fusion, assembly, cell-to-cell spread, and immune evasion, which are closely related to its protein characteristics and partners. Few studies have been conducted on DPV pUL10. In this study, we identified the characteristics of pUL10, such as the type of glycosylation modification and subcellular localization. The characteristic differences in pUL10 in transfection and infection suggest that there are other viral proteins that participate in pUL10 modification and localization. Therefore, pUL49.5, the interaction partner of pUL10, was explored. We found that pUL10 interacts with pUL49.5 during transfection and infection. Their interaction entailed multiple interaction sites, including noncovalent forces in the pUL49.5 N-terminal domains and C-terminal domains and a covalent disulfide bond between two conserved cysteines. pUL49.5 promoted pUL10 expression and mature N-linked glycosylation modification. Moreover, deletion of UL49.5 in DPV caused the molecular mass of pUL10 to decrease by approximately3 to 10 kDa, which suggested that pUL49.5 was the main factor affecting the N-linked glycosylation of DPV pUL10 during infection. This study provides a basis for future exploration of the effect of pUL10 glycosylation on virus proliferation. IMPORTANCE Duck plague is a disease with high morbidity and mortality rates, and it causes great losses for the duck breeding industry. Duck plague virus (DPV) is the causative agent of duck plague, and DPV UL10 protein (pUL10) is a homolog of glycoprotein M (gM), which is conserved in herpesviruses. pUL10 plays complex roles in viral fusion, assembly, cell-to-cell spread, and immune evasion, which are closely related to its protein characteristics and partners. In this study, we systematically explored whether pUL49.5 (a partner of pUL10) plays roles in the localization, modification, and expression of pUL10.

Effects of US3 protein kinase activity on localization of UL31/UL34 protein and nucleocapsids egress of duck plague virus.

Duck plague virus (DPV) is a pathogen causing duck plague and has caused huge economic losses in poultry industry. In our previous report, US3 gene deletion from DPV genome seriously impaired virus replication. In this study, we constructed a US3 kinase-inactive mutant (US3K213A) to further explore the function of US3 protein (pUS3) in DPV. Our results showed that the loss of pUS3 kinase activity caused lower viral titers, smaller plaque sizes and a blockage of capsids nuclear egress including primary enveloped virion (PEV) accumulation compared to the parental virus infection. It indicates that the effects of DPV pUS3 on viral propagation depended on its kinase activity. In addition, we conducted electron microscopy analysis to show the outer nuclear membrane (ONM) evaginations and the nuclear envelope (NE) deep invagination in US3K213A-infected cells. Finally, an irregular distribution of pUL31/pUL34 in the NE in △US3- and US3K213A-infected cells and an interaction of pUS3 and pUL31 were found, which suggests that pUS3 potentially targets pUL31 and regulates the localization of pUL31/pUL34 to promote nucleocapsids egress through its kinase activity.

Deletion of Double Copies of the US1 Gene Reduces the Infectivity of Recombinant Duck Plague Virus In Vitro and In Vivo.

Duck plague caused by duck plague virus (DPV) is one of the main diseases that seriously endangers the production of waterfowl. DPV possesses a large genome consisting of 78 open reading frames (ORFs), and understanding the function and mechanism of each encoded protein in viral replication and pathogenesis is the key to controlling duck plague outbreaks. US1 is one of the two genes located in the repeat regions of the DPV genome, but the function of its encoded protein in DPV replication and pathogenesis remains unclear. Previous studies found that the US1 gene or its homologs exist in almost all alphaherpesviruses, but the loci, functions, and pathogenesis of their encoded proteins vary among different viruses. Here, we aimed to define the roles of US1 genes in DPV infection and pathogenesis by generating a double US1 gene deletion mutant and its revertant without any mini-F cassette retention. In vitro and in vivo studies found that deletion of both copies of the US1 gene significantly impaired the replication, gene expression, and virulence of DPV, which could represent a potential candidate vaccine strain for the prevention of duck plague. IMPORTANCE Duck plague virus contains nearly 80 genes, but the functions and mechanisms of most of the genes have not yet been elucidated, including those of the newly identified immediate early gene US1. Here, we found that US1 deletion reduces viral gene expression, replication, and virus production both in vitro and in vivo. This insight defines a fundamental role of the US1 gene in DPV infection and indicates its involvement in DPV transcription. These results provide clues for the study of the pathogenesis of the US1 gene and the development of attenuated vaccines targeting this gene.

Evaluation of safety and immunogenicity of duck-plague virus gC/gE double gene deletion.

Duck plague caused by duck plague virus (DPV) is a highly contagious disease that can cause serious morbidity and death in waterfowl such as ducks and geese, and bring huge economic losses to the duck industry. In this study, on the basis of the duck plague virus gC gene deletion strain CHv-ΔgC, based on the duck plague virus bacterial artificial chromosome (BAC) platform in our laboratory, the gE gene was knocked out using the traceless deletion technology to obtain gC/gE double gene deletion candidate vaccine strain CHv-ΔgC/gE. The double gene deletion strain (CHv-ΔgC/gE) constructed in this study has greatly weakened virulence, no pathogenicity to ducks, and stable genetic characteristics in vitro and in vivo. Ducks immunized with CHv-ΔgC/gE can produce neutralizing antibodies and ELISA antibody levels comparable to those of commercial duck plague attenuated vaccine immunization, and can resist 100 LD50 CHv challenge of ducks, with good immune protection effect. It has the potential to be further developed into duck plague gC/gE double gene deletion, marked attenuated vaccine.

Differential expression profile and in-silico functional analysis of long noncoding RNA and mRNA in duck embryo fibroblasts infected with duck plague virus.

BACKGROUND: Duck plague virus (DPV), belonging to herpesviruses, is a linear double-stranded DNA virus. There are many reports about the outbreak of the duck plague in a variety of countries, which caused huge economic losses. Recently, increasing reports revealed that multiple long non-coding RNAs (lncRNAs) can possess great potential in the regulation of host antiviral immune response. Furthermore, it remains to be determined which specific molecular mechanisms are responsible for the DPV-host interaction in host immunity. Here, lncRNAs and mRNAs in DPV infected duck embryonic fibroblast (DEF) cells were identified by high-throughput RNA-sequencing (RNA-seq). And we predicted target genes of differentially expressed genes (DEGs) and formed a complex regulatory network depending on in-silico analysis and prediction. RESULT: RNA-seq analysis results showed that 2921 lncRNAs were found at 30 h post-infection (hpi). In our study, 218 DE lncRNAs and 2840 DE mRNAs were obtained in DEF after DPV infection. Among these DEGs and target genes, some have been authenticated as immune-related molecules, such as a Macrophage mannose receptor (MR), Anas platyrhynchos toll-like receptor 2 (TLR2), leukocyte differentiation antigen, interleukin family, and their related regulatory factors. Furthermore, according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis, we found that the target genes may have important effects on biological development, biosynthesis, signal transduction, cell biological regulation, and cell process. Also, we obtained, the potential targeting relationship existing in DEF cells between host lncRNAs and DPV-encoded miRNAs by software. CONCLUSIONS: This study revealed not only expression changes, but also the possible biological regulatory relationship of lncRNAs and mRNAs in DPV infected DEF cells. Together, these data and analyses provide additional insight into the role of lncRNAs and mRNAs in the host's immune response to DPV infection.

Duck Plague Virus Negatively Regulates IFN Signaling to Promote Virus Proliferation via JNK Signaling Pathway.

Duck plague virus (DPV), a member of the alphaherpesvirus subfamily, can cause severe damage and immunosuppression in ducks and geese in China. Since lacking an available cell model, the antiviral signal transduction pathways induction and regulation mechanisms related to DPV infection in duck cells are still enigmatic. Our previous study developed a monocyte/macrophages cell model, which has been applied to study innate immunity with DPV. In the present study, we compared and analyzed transcriptome associated with the DPV infection of CHv (virulent strain) and CHa (avirulent strain) at 48hpi based on the duck monocyte/macrophages cell model and RNA-seq technology. Differentially expressed genes (DEGs) analysis showed 2,909 and 2,438 genes altered in CHv and CHa infected cells compared with control cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the DEGs were mainly involved in biological processes such as metabolic pathways, viral infectious diseases, immune system, and signal transduction. The CHv and CHa virus differentially regulated MAPK, NF-κB, and IFN signaling pathways based on transcriptome sequencing data and RT-qPCR results. The JNK inhibitor SP600125 enhanced the IFN signaling, but potentially reduced the VSV and DPV titers in the cell culture supernatant, indicating that JNK negatively regulates the IFN pathway and the inflammatory pathway to promote virus proliferation. The research results may provide promising information to understand the pathogenesis of DPV and provide a novel mechanism by which DPV modulates antiviral signaling and facilitate virus proliferation through hijacking the JNK pathway, which provides a new means for the prevention and control of DPV infection.

Evaluation of the Safety and Immunogenicity of Duck-Plague Virus gE Mutants.

Duck plague (DP) is an acute infectious disease in the duck industry. The duck plague virus (DPV) is the pathogen, a subfamily of alphaherpesvirinae. gE is a type I membrane protein that contains three parts: an extracellular domain, a transmembrane domain, and a cytoplasmic domain. gE is the major virulence determinant of α-herpesvirus. However, the functions of the gE extracellular and cytoplasmic domains have not been reported in DPV. In this study, a gE extracellular domain deletion mutant and a gE cytoplasmic domain deletion mutant were constructed from DPV. Virus replication kinetics showed that the growth titers of both the gE ectodomain-deleted mutant virus and the gE cytoplasmic domain-deleted virus in DEFs were lower than that of the parental virus CHv-50. DPV CHv-gEΔET and DPV CHv-gEΔCT were continuously passed to the 20th passage in DEFs and the 10th in ducklings. The mutant virus DNA after passage was extracted for identification. The results showed that the gE ectodomain and gE cytoplasmic domain deletion mutant viruses have good genetic stability. The ducklings in each group (n=10) were inoculated with the same titers of DPV CHv-gEΔET, DPV CHv-gEΔCT, DPV CHv-ΔgE, and parental CHv-50, respectively. Clinical symptoms and serum antibody levels were detected after inoculation. The results showed that the virulence of DPV CHv-gEΔCT to ducklings was reduced compared with parental CHv-50, while the virulence of DPV CHv-gEΔET to ducklings was significantly reduced. 105 TCID50 DPV CHv-gEΔET or DPV CHv-ΔgE can induce ducklings to produce DPV-specific antibodies, protect the ducklings from virulent CHv challenge. Therefore, DPV CHv-gEΔET may serve as a promising vaccine candidate to prevent and control duck plague.

The protein encoded by the duck plague virus UL14 gene regulates virion morphogenesis and affects viral replication.

To investigate the pivotal roles of the duck plague virus (DPV) tegument protein UL14 in viral replication, we generated 2 mutated viruses of DPV by using the bacterial artifcial chromosome system, the UL14-null mutant virus (CHv-BAC-ΔUL14) and the corresponding revertant virus (CHv-BAC-ΔUL14R). We found that the CHv-BAC-ΔUL14 viruses exhibited impaired virion morphogenesis in transmission electron microscopy (TEM) studies. Furthermore, CHv-BAC-ΔUL14 exhibited a plaque size reduction in duck embryo fibroblasts (DEFs). Finally, CHv-BAC-ΔUL14 exhibited a significant viral growth defect. Taken together, our findings suggest that DPV UL14 protein regulates viral morphogenesis for efficient viral replication.

V5 and GFP Tagging of Viral Gene pp38 of Marek's Disease Vaccine Strain CVI988 Using CRISPR/Cas9 Editing.

Marek's disease virus (MDV) is a member of alphaherpesviruses associated with Marek's disease, a highly contagious neoplastic disease in chickens. The availability of the complete sequence of the viral genome allowed for the identification of major genes associated with pathogenicity using different techniques, such as bacterial artificial chromosome (BAC) mutagenesis and the recent powerful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based editing system. Thus far, most studies on MDV genome editing using the CRISPR/Cas9 system have focused on gene deletion. However, analysis of the expression and interactions of the viral proteins during virus replication in infected cells and tumor cells is also important for studying its role in MDV pathogenesis. The unavailability of antibodies against most of the MDV proteins has hindered the progress in such studies. This prompted us to develop pipelines to tag MDV genes as an alternative method for this purpose. Here we describe the application of CRISPR/Cas9 gene-editing approaches to tag the phosphoprotein 38 (pp38) gene of the MDV vaccine strain CVI988 with both V5 and green fluorescent protein (GFP). This rapid and efficient viral-gene-tagging technique can overcome the shortage of specific antibodies and speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.

Exploration of Alternative Splicing (AS) Events in MDV-Infected Chicken Spleens.

Marek's disease (MD) was an immunosuppression disease induced by Marek's disease virus (MDV). MD caused huge economic loss to the global poultry industry, but it also provided an ideal model for studying diseases induced by the oncogenic virus. Alternative splicing (AS) simultaneously produced different isoform transcripts, which are involved in various diseases and individual development. To investigate AS events in MD, RNA-Seq was performed in tumorous spleens (TS), spleens from the survivors (SS) without any lesion after MDV infection, and non-infected chicken spleens (NS). In this study, 32,703 and 25,217 AS events were identified in TS and SS groups with NS group as the control group, and 1198, 1204, and 348 differently expressed (DE) AS events (p-value < 0.05 and FDR < 0.05) were identified in TS vs. NS, TS vs. SS, SS vs. NS, respectively. Additionally, Function enrichment analysis showed that ubiquitin-mediated proteolysis, p53 signaling pathway, and phosphatidylinositol signaling system were significantly enriched (p-value < 0.05). Small structural variations including SNP and indel were analyzed based on RNA-Seq data, and it showed that the TS group possessed more variants on the splice site region than those in SS and NS groups, which might cause more AS events in the TS group. Combined with previous circRNA data, we found that 287 genes could produce both circular and linear RNAs, which suggested these genes were more active in MD lymphoma transformation. This study has expanded the understanding of the MDV infection process and provided new insights for further analysis of resistance/susceptibility mechanisms.

Marek's disease virus prolongs survival of primary chicken B-cells by inducing a senescence-like phenotype.

Marek's disease virus (MDV) is an alphaherpesvirus that causes immunosuppression and deadly lymphoma in chickens. Lymphoid organs play a central role in MDV infection in animals. B-cells in the bursa of Fabricius facilitate high levels of MDV replication and contribute to dissemination at early stages of infection. Several studies investigated host responses in bursal tissue of MDV-infected chickens; however, the cellular responses specifically in bursal B-cells has never been investigated. We took advantage of our recently established in vitro infection system to decipher the cellular responses of bursal B-cells to infection with a very virulent MDV strain. Here, we demonstrate that MDV infection extends the survival of bursal B-cells in culture. Microarray analyses revealed that most cytokine/cytokine-receptor-, cell cycle- and apoptosis-associated genes are significantly down-regulated in these cells. Further functional assays validated these strong effects of MDV infections on cell cycle progression and thus, B-cell proliferation. In addition, we confirmed that MDV infections protect B-cells from apoptosis and trigger an accumulation of the autophagy marker Lc3-II. Taken together, our data indicate that MDV-infected bursal B-cells show hallmarks of a senescence-like phenotype, leading to a prolonged B-cell survival. This study provides an in-depth analysis of bursal B-cell responses to MDV infection and important insights into how the virus extends the survival of these cells.

Pathologic Characterization of Coinfection with Histomonas meleagridis, Marek's Disease Virus, and Subtype J Avian Leukosis Virus in Chickens.

Histomonas meleagridis is a trichomonad protozoan parasite that can cause an important poultry disease known as histomoniasis; Marek's disease virus (MDV) and subtype J avian leukosis virus (ALV-J) usually cause avian oncogenic diseases. Although these diseases have been reported in a single pathogen infection, information about their coinfection is scarce. This study reports a naturally occurring case of coinfection with H. meleagridis, MDV, and ALV-J in a local chicken flock at the age of 150 days. Necropsy revealed necrosis and swelling in the liver and spleen. Histologic analysis showed large areas of mild to severe necrosis of hepatocytes, with numerous intralesional trophozoites of H. meleagridis by H&E and periodic acid-Schiff staining; H&E staining showed pleomorphic and neoplastic lymphoid tumor cells in the liver and myeloid cells with eosinophilic cytoplasmic granules in the spleen. Coexpression of MDV and ALV-J antigens was detected in the liver by fluorescence multiplex immunohistochemistry staining. The 18S rRNA gene of H. meleagridis, meq gene of MDV, and gp85 gene of ALV-J were identified in mixed liver and spleen tissues by PCR and sequencing, respectively.

Reporte de caso­Caracterización patológica de la coinfección con Histomonas meleagridis, el virus de la enfermedad de Marek y el virus de la leucosis aviar subtipo J en pollos Histomonas meleagridis es un parásito protozoario tricomonial que puede causar una enfermedad avícola importante conocida como histomoniasis; El virus de la enfermedad de Marek (MDV) y el virus de la leucosis aviar subtipo J (ALV-J) suelen causar enfermedades oncogénicas aviares. Aunque estas enfermedades se han reportado como infecciones patógenas separadas, la información sobre coinfección es escasa. Este estudio reporta un caso natural de coinfección con H. meleagridis, el virus de la enfermedad de Marek y el virus de la leucosis aviar subtipo J en una parvada de pollos local a la edad de 150 días. La necropsia reveló necrosis e inflamación del hígado y el bazo. El análisis histológico mostró grandes áreas de necrosis de hepatocitos de leve a severa, con numerosos trofozoítos intralesionales de H. meleagridis por tinción de hematoxilina y eosina y por tinción de ácido periódico-Schiff. La tinción de hematoxilina y eosina mostró células linfoides neoplásicas y pleomórficas en el hígado y en el bazo presencia de células mieloides con gránulos citoplásmicos eosinofílicos. La coexpresión de antígenos del virus de Marek y de la leucosis aviar subtipo J se detectó en el hígado mediante tinción inmunohistoquímica de fluorescencia múltiple. El gene de ARNr 18S de H. meleagridis, el gene meq del virus de Marek y el gene gp85 del virus de la leucosis aviar subtipo J se identificaron en tejidos mixtos de hígado y bazo mediante PCR y secuenciación, respectivamente.