Recombinant Marek's Disease Virus Research Articles

Efficacy of Recombinant Marek's Disease Virus Vaccine 301B/1 Expressing Membrane-Anchored Chicken Interleukin-15.

Cytokines are co-administrated with vaccines or co-expressed in the vaccine virus genome to improve protective efficacy by stimulating immune responses. Using glycosylphosphatidylinositol (GPI) anchoring by attachment to the target cytokine, we constructed recombinant Marek's disease virus (MDV) vaccine strain 301B/1 (v301B/1-rtg-IL-15) that expresses chicken interleukin-15 (IL-15) as the membrane-bound form at the cell surface. We evaluated the vaccine efficacy of v301B/1-rtg-IL-15 given as a bivalent Marek's disease (MD) vaccine in combination with turkey herpesvirus (HVT) against a very virulent plus MDV strain 648A challenge. The efficacy was compared with that of conventional bivalent MD vaccine, as a mixture with HVT plus parental v301B/1 or v301B/1-IL-15, which expresses a natural form of IL-15. The membrane-bound IL-15 expression did not interfere with the virus growth of recombinant v301B/1-rtg-IL-15. However, the MD incidence in birds vaccinated with v301B/1-rtg-IL-15 was higher than that of birds given the conventional bivalent MD vaccine containing parental v301B/1 virus, although the v301B/1-rtg-IL-15 vaccinated group showed increased natural killer cell activation at day 5 postvaccination, the same day as challenge. Overall, the protection of v301B/1-rtg-IL-15 was not improved from that of v301B/1 against very virulent plus MDV challenge.

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.

Emerging natural recombinant Marek's disease virus between vaccine and virulence strains and their pathogenicity.

Marek's disease virus (MDV), an oncogenic virus belonging to the subfamily Alphaherpesvirinae, causes Marek's disease (MD). Vaccines can control MD but cannot block the viral infection; they are considered imperfect vaccines, which carry the risk of recombination. In this study, six natural recombinant MDV strains were isolated from infected chickens in commercial flocks in China. We sequenced and analysed the genetic characteristics of the isolates (HC/0803, CH/10, SY/1219, DH/1307, DH/1504 and Hrb/1504). We found that the six strains resulted from recombination between the vaccine CVI988/Rispens (CVI988) strain skeleton and the virulence strain's partial unique short region. Additionally, a pathogenicity study was performed on recombinant strains (HC/0803 and DH/1307) and reference strains (CVI988 and LHC2) to evaluate their virulence. LHC2 induced 84.6% mortality in infected chickens; however, no mortality was recorded in chickens inoculated with HC/0803, DH/1307 or CVI988. However, HC/0803 and DH/1307 induced a notable spleen enlargement, and mild thymus and bursa atrophy at 11-17 days post-challenge (dpc). The viral genome load in the HC/0803- and DH/1307-challenged chickens peaked at approximately 107 viral copies per million host cells at 17 dpc and was similar to that in LHC2-challenged chickens, but significantly higher than that of CVI988-challenged chickens. In summary, HC/0803 and DH/1307 displayed mild virulence with temporal damage to the immune organs of chicken and a higher reproduction capability than the vaccine strain CVI988. Our study provides direct evidence of the emergence of recombinant MDV strains between vaccine and virulence strains in nature. The emergence of natural recombinant strains suggests that live vaccines can act as genetic donors for genomic recombination, and recombination may be a safety concern when administering live vaccines. These findings demonstrate that recombination promotes genetic diversity and increases the complexity of disease diagnosis, prevention and control.

Deletion of the Viral Thymidine Kinase in a Meq-Deleted Recombinant Marek's Disease Virus Reduces Lymphoid Atrophy but Is Less Protective.

Marek’s disease (MD) is a ubiquitous disease of domesticated chickens and its etiologic agent is the Gallid alphaherpesvirus 2 (GaHV-2), also known as Marek’s disease virus (MDV). MD is currently controlled by vaccination using live attenuated strains of MDV (e.g., CVI988/Rispens), non-pathogenic serotypes of MDV (GaHV-3), or non-pathogenic strains of the related Melagrid alphaherpesvirus 1 (MeHV-1). One attractive strategy for the production of new vaccine strains is a recombinant MDV attenuated by the deletion of the major viral oncogene meq. However, meq-deleted variants of MDV cause atrophy of the bursa and thymus in maternal antibody-negative chickens, and the resulting immunosuppression makes them unsuitable. Herein we detail our attempt to mitigate the lymphoid atrophy caused by meq-deleted MDV by further attenuation of the virus through ablation of the viral thymidine kinase (tk) gene. We demonstrate that ablation of the viral tk from the meq-deleted virus rMd5B40/Δmeq resulted in a virus attenuated for replication in vitro and which spared chickens from atrophy of the lymphoid organs in vivo. When the rMd5B40/Δmeq/Δtk/GFP was used as a vaccine it was protective against challenge with the vv+MDV strain 686, but the protection was less than that provided by the CVI988/Rispens vaccine.

Research Note: Characterization of S-Meq containing the deletion in Meq protein's transactivation domain in a Marek's disease virus strain in Japan

Marek's disease virus (MDV) causes malignant lymphomas in chickens (Marek's disease; MD). Although MD has caused significant economic losses to the poultry industry, currently, its occurrence in the field is effectively controlled by vaccination. However, the genetic characteristics of MDV strains have changed, and the poultry industry has experienced MD outbreaks in vaccinated chickens because of enhanced virulence. Meq, an oncoprotein of MDV, is a key transcription factor correlated with the tumorigenesis in MD. Animal experiments using recombinant MDV revealed that distinct polymorphisms in Meq affect the virulence of MDV strains. Meq containing an insertion or deletion is present in some MDV strains. In the 2010s, field strains that encode Meq containing the deletion (S-Meq) were reported. In this study, we characterized the genetic features of S-Meq detected in a Japanese MDV strain and analyzed its transactivation activity to investigate S-Meq's protein function. S-Meq lacked 41 amino acids, and the deletion was at the same position as those observed in other countries. In addition, S-Meq exhibited higher transactivation activity than Meq from other MDV strains circulating in Japan. These results suggest that the deletion in the transactivation domain may enhance the Meq protein's function. Further investigation is needed to clarify whether the deletion in the transactivation domain of Meq affects MDV's virulence.

The role of Meq-vIL8 in regulating Marek's disease virus pathogenesis.

Marek's disease virus (MDV) is a highly cell-associated oncogenic alphaherpesvirus that causes T cell lymphoma in chickens. MDV-encoded Meq and vIL8 proteins play important roles in transformation and early cytolytic infection, respectively. Previous studies identified a spliced transcript, meq-vIL8, formed by alternative splicing of meq and vIL8 genes in MDV lymphoblastoid tumour cells. To determine the role of Meq-vIL8 in MDV pathogenesis, we generated a recombinant MDV (MDV-meqΔSD) by mutating the splice donor site in the meq gene to abrogate the expression of Meq-vIL8. As expected, our results show that MDV-meqΔSD virus grows similarly to the parental and revertant viruses in cell culture, suggesting that Meq-vIL8 is dispensable for MDV growth in vitro. We further characterized the pathogenic properties of MDV-meqΔSD virus in chickens. Our results show that lack of Meq-vIL8 did not affect virus replication during the early cytolytic phase, as determined by immunohistochemistry analysis and/or viral genome copy number, but significantly enhanced viral DNA load in the late phase of infection in the spleen and brain of infected chickens. In addition, we observed that abrogation of Meq-vIL8 expression reduced the mean death time and increased the prevalence of persistent neurological disease, common features of highly virulent strains of MDV, in inoculated chickens. In conclusion, our study shows that Meq-vIL8 is an important virulence factor of MDV.

Role of Marek's Disease Virus (MDV)-Encoded US3 Serine/Threonine Protein Kinase in Regulating MDV Meq and Cellular CREB Phosphorylation.

Marek's disease (MD) is a neoplastic disease of chickens caused by Marek's disease virus (MDV), a member of the subfamily Alphaherpesvirinae Like other alphaherpesviruses, MDV encodes a serine/threonine protein kinase, US3. The functions of US3 have been extensively studied in other alphaherpesviruses; however, the biological functions of MDV US3 and its substrates have not been studied in detail. In this study, we investigated potential cellular pathways that are regulated by MDV US3 and identified chicken CREB (chCREB) as a substrate of MDV US3. We show that wild-type MDV US3, but not kinase-dead US3 (US3-K220A), increases CREB phosphorylation, leading to recruitment of phospho-CREB (pCREB) to the promoter of the CREB-responsive gene and activation of CREB target gene expression. Using US3 deletion and US3 kinase-dead recombinant MDV, we identified US3-responsive MDV genes during infection and found that the majority of US3-responsive genes were located in the MDV repeat regions. Chromatin immunoprecipitation sequencing (ChIP-seq) studies determined that some US3-regulated genes colocalized with Meq (an MDV-encoded oncoprotein) recruitment sites. Chromatin immunoprecipitation-PCR (ChIP-PCR) further confirmed Meq binding to the ICP4/LAT region, which is also regulated by US3. Furthermore, biochemical studies demonstrated that MDV US3 interacts with Meq in transfected cells and MDV-infected chicken embryonic fibroblasts in a phosphorylation-dependent manner. Finally, in vitro kinase studies revealed that Meq is a US3 substrate. MDV US3 thus acts as an upstream kinase of the CREB signaling pathway to regulate the transcription function of the CREB/Meq heterodimer, which targets cellular and viral gene expression.IMPORTANCE MDV is a potent oncogenic herpesvirus that induces T-cell lymphoma in infected chickens. Marek's disease continues to have a significant economic impact on the poultry industry worldwide. US3 encoded by alphaherpesviruses is a multifunctional kinase involved in the regulation of various cellular pathways. Using an MDV genome quantitative reverse transcriptase PCR (qRT-PCR) array and chromatin immunoprecipitation, we elucidated the role of MDV US3 in viral and cellular gene regulation. Our results provide insights into how viral kinase regulates host cell signaling pathways to activate both viral and host gene expression. This is an important step toward understanding host-pathogen interaction through activation of signaling cascades.

Safety and Efficacy Evaluation of Recombinant Marek's Disease Virus with REV-LTR.

Recently, chickens vaccinated with the CVI988/Rispens vaccine showed increased tumor incidence. Moreover, many strains of Marek’s disease virus (MDV) that were naturally integrated with the long terminal repeat (LTR) of the avian reticuloendotheliosis virus (REV) have been isolated, which means it is necessary to develop a new vaccine. In this study, two LTR sequences were inserted into Rispens to construct a recombinant MDV (rMDV). Then, the safety and efficacy of rMDV were evaluated separately in chickens. The growth rate curves showed that the insertion of REV-LTR into MDV enabled a faster replication in vitro than Rispens. Chickens immunized with high or repeated dose rMDV had no MD clinical signs. Further, no tumor, tissue lesions, or evident pathological changes were observed in the chicken organs. Polymerase chain reaction (PCR) and virus isolation revealed that rMDV had the ability to spread horizontally to non-immunized chickens and had no impact on the environment. After five passages in chickens, there were no obvious lesions, and the LTR insertion was stable. There were also no deletions or mutations, which indicates that rMDV is safe in chickens. In addition, rMDV has an advantage over Rispens against vvMDV Md5 at low doses. All results demonstrate that the transgenic strain of rMDV with REV-LTR can be used as a live attenuated vaccine candidate.

Attenuation of a recombinant Marek's disease virus lacking the meq oncogene and evaluation on its immune efficacy against Marek's disease virus

SC9-2 is a recombinant Marek's disease virus (MDV) strain lacking the meq oncogene. Previous study demonstrated that SC9-2 virus provides good protection against challenge with a very virulent MDV rMd5, but it induces immunosuppressive effects in specific pathogen-free (SPF) chickens. In the present study, SC9-2 was serially passaged on chicken embryo fibroblast (CEF) cell cultures. The pathogenicity and immune efficacy of SC9-2/10th and SC9-2/40th against rMd5 were evaluated. Animal experimental results showed that SC9-2/10th and SC9-2/40th showed no lethality or tumorigenicity in SPF chickens. Body weight of chickens inoculated with SC9-2/40th were significantly higher than that of the chickens inoculated with SC9-2/10th but lower than that of the uninoculated controls. The severity of bursa and thymus atrophy (BTA) and spleen enlargement in SC9-2/40th-inoculated chickens were also weaker than the SC9-2/10th-inoculated ones but stronger than the uninoculated controls. Chickens inoculated with SC9-2/40th and SC9-2/10th showed similar antibody levels induced by H9N2 subtype avian influenza virus/Newcastle disease virus inactivated vaccines, both of which were lower than the uninoculated controls. Replication of SC9-2/40th was significantly lower than SC9-2/10th in feather follicle epithelium (FFE) of infected chickens. The immune protection index of SC9-2/40th was also lower than that of SC9-2/10th, but the difference was not significantly, and both of which were significant higher than that of the commercial MDV vaccine CVI988/Rispens. The results of our studies demonstrated that SC9-2/40th showed weaker severity of BTA, spleen enlargement, and body weight loss and lower replication level in FFE than SC9-2/10th in SPF chickens. However, SC9-2/40th was able to confer better immune protection as compared with CVI988/Rispens vaccination in SPF chickens. In conclusion, serially attenuation of SC9-2 in CEFs reduced the lymphoid organ atrophy and replication in SPF chickens, and the immune protective efficacy of attenuated viruses was still superior than CVI988/Rispens.

Increased Horizontal Transmission of Recombinant Marek's Disease Virus Due to Reticuloendotheliosis Virus Long Terminal Repeat Is the Major Competitive Advantage of the Virus Being a Prevalent Strain.

GX0101 is the first field Marek’s disease virus (MDV) recombinant with an REV LTR insert isolated in China. We speculated that there was a selective advantage of GX0101 becoming the more prevalent field strain from a very low percentage of recombinant virus. In the study, dual fluorescence quantitative real-time PCR (DF-qPCR) that detects GX0101 and GX0101ΔLTR simultaneously was established based on the genomic difference of GX0101 and its LTR deletion strain GX0101ΔLTR. MDV natural transmission was simulated in specific-pathogen-free (SPF) chicks, and continuous tracking of GX0101 and GX0101ΔLTR in chicks was carried out. The results showed that GX0101 possessed high horizontal transmission capacity, which could infect SPF chicks by contact in a short time and became the predominant strain following contact infections in chicken flocks. GX0101 still had a more significant advantage of horizontal transmission than GX0101ΔLTR after continuous passage even if the initially infectious dose was significantly lower. There were 72 differentially expressed MDV genes between GX0101 and GX0101ΔLTR, with the genes and gene products mainly involved in virus replication, tegument protein, glycoprotein, nucleocapsid protein, immune evasion, tumor development and/or pathogenesis, and hypothetical protein. Sixteen genes related to virus replication and transmission were significantly up-regulated. This is the first study to illuminate that increased horizontal transmission of recombinant MDV due to REV LTR was the competitive advantage of the virus being a prevalent strain and define the differential transcription profile of viral genes between GX0101 and GX0101ΔLTR. This will be helpful for in-depth study on the molecular mechanism of increased horizontal transmission of MDV by REV LTR.

Differentially expressed genes during spontaneous lytic switch of Marek's disease virus in lymphoblastoid cell lines determined by global gene expression profiling.

Marek’s disease virus (MDV), an alphaherpesvirus of poultry, causes Marek’s disease and is characterized by visceral CD4+TCRαβ+ T-cell lymphomas in susceptible hosts. Immortal cell lines harbouring the viral genome have been generated from ex vivo cultures of MD tumours. As readily available sources of large numbers of cells, MDV-transformed lymphoblastoid cell lines (LCLs) are extremely valuable for studies of virus–host interaction. While the viral genome in most cells is held in a latent state, minor populations of cells display spontaneous reactivation identifiable by the expression of lytic viral genes. Spontaneous reactivation in these cells presents an opportunity to investigate the biological processes involved in the virus reactivation. For detailed characterization of the molecular events associated with reactivation, we used two lymphoblastoid cell lines derived from lymphomas induced by pRB1B-UL47eGFP, a recombinant MDV engineered to express enhanced green fluorescent protein (EGFP) fused with the UL47. We used fluorescence-activated cell sorting to purify the low-frequency EGFP-positive cells with a spontaneously activating viral genome from the majority EGFP-negative cells and analysed their gene expression profiles by RNA-seq using Illumina HiSeq2500. Ingenuity pathway analysis on more than 2000 differentially expressed genes between the lytically infected (EGFP-positive) and latently infected (EGFP-negative) cell populations identified the biological pathways involved in the reactivation. Virus-reactivating cells exhibited differential expression of a significant number of viral genes, with hierarchical differences in expression levels. Downregulation of a number of host genes including those directly involved in T-cell activation, such as CD3, CD28, ICOS and phospholipase C, was also noticed in the LCL undergoing lytic switch.

Protective efficacy of a novel recombinant Marek's disease virus vector vaccine against infectious bursal disease in chickens with or without maternal antibodies

Infectious bursal disease (IBD) causes significant clinical and economic losses to the poultry industry worldwide. Current vaccine programs using live attenuated and inactivated vaccines have numerous drawbacks. As an alternative solution to control IBD, a Marek's disease virus (MDV) vector vaccine (rMDV-VP2) expressing the VP2 gene of infectious bursal disease virus (IBDV) has been developed. In this study, the protective efficacy of rMDV-VP2 was evaluated in a dose-related experiment which showed that a single dose of 1000 PFU was sufficient to fully protect chickens against IBDV infection. Chickens inoculated with lower doses of rMDV-VP2 (250 or 500 PFU) conferred 80 and 90% protection against IBDV. Next, rMDV-VP2 vaccine provided 90% protection against IBDV in commercial layer chickens with maternal antibodies, which was higher than the protective efficacy using the B87 live vaccine of IBDV. Additionally, rMDV-VP2 conferred effective protection against very virulent MDV challenge in chickens (95% for chickens vaccinated with 250 or 500 PFU and 100% for chickens vaccinated with 1000 or 2000 PFU). These results demonstrated that rMDV-VP2 may be a novel bivalent vaccine against IBD and Marek's disease in chickens.

Influence of avian leukosis virus long terminal repeat on biological activities of Marek's disease virus

GX0101 was the first reported field strain of recombinant Marek's disease virus (MDV) that contained a long terminal repeat (LTR) from the reticuloendotheliosis virus (REV). It is a very virulent MDV strain, with relatively high horizontal transmission ability. The REV LTR in GX0101 genome was proved to decrease the pathogenicity but increase the potential for horizontal transmission of the virus. Here we constructed a recombinant MDV GX0101-ALV-LTR to study stability of avian leukosis virus (ALV) LTR at the REV LTR insertion site in GX0101 genome and its influence on biological activities of the recombinant virus. The results showed that GX0101-ALV-LTR was able to replicate stably both in vitro and in vivo. ALV LTR remained stable in chickens infected either by inoculation with the recombinant virus GX0101-ALV-LTR or by horizontal transmission, as well as in cell culture. The pathogenic properties of GX0101-ALV-LTR virus were evaluated in infected specific-pathogen-free chickens. The present study demonstrated that the GX0101-ALV-LTR virus had a weaker inhibitory effect on the growth rates of the infected chickens and induced weaker immunosuppressive effects. Horizontal transmission ability of the GX0101-ALV-LTR virus appeared to be similar with its parental virus GX0101. In short, ALV LTR was stable in GX0101 after replacing REV LTR, and the recombinant virus showed similar horizontal transmission ability but decreased pathogenicity.

Addition of a UL5 helicase-primase subunit point mutation eliminates bursal–thymic atrophy of Marek's disease virus ∆Meq recombinant virus but reduces vaccinal protection

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus and the causative agent of Marek's disease (MD), characterized by immunosuppression, paralysis, nerve enlargement and induction of T-cell lymphomas in chickens. Despite widespread usage of vaccines since the 1970s to control MD, more virulent field strains of MDV have emerged that overcome vaccinal protection, necessitating the development of new and more protective MD vaccines. The ∆Meq virus, a recombinant Md5 strain MDV lacking the viral oncogene Meq, is one candidate MD vaccine with great potential but unfortunately it also causes bursal–thymic atrophy (BTA) in maternal antibody negative chickens, raising concerns that impede commercial use as a vaccine. Previously, we identified a point mutation within UL5 that reduced in vivo replication in attenuated viruses. We proposed that introduction of the UL5 point mutation into the ∆Meq virus would reduce in vivo replication and eliminate BTA yet potentially retain high protective abilities. In birds, the ∆Meq+UL5 recombinant MDV had reduced replication compared to the original ∆Meq virus, while weights of lymphoid organs indicated that ∆Meq+UL5 did not induce BTA, supporting the hypothesis that reduction of in vivo replication would also abolish BTA. Vaccine trials of the ∆Meq+UL5 virus compared to other ∆Meq-based viruses and commercial vaccines show that, while the ∆Meq+UL5 does provide vaccinal protection, this protection was also reduced compared to the original ∆Meq virus. Therefore, it appears that a very delicate balance is required between levels of replication able to induce high vaccinal protection, yet not so high as to induce BTA.

Expression of fluorescent proteins within the repeat long region of the Marek's disease virus genome allows direct identification of infected cells while retaining full pathogenicity

Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus and causes Marek's disease (MD) in chickens. RLORF4 is an MDV-specific gene located in the repeat long (RL) regions of the genome and is directly involved in attenuation. In this report, we generated recombinant (r)MDVs in which eGFP or mRFP was inserted in-frame of the 3′ end of the RLORF4 gene. In vitro growth was unaffected and infected cells could be identified by using fluorescent microscopy. Interestingly, though inserted in-frame with RLORF4, eGFP and mRFP were expressed alone, confirming mRNA expression and splicing within the RL of MDV is complex. In vivo, rMDVs expressing mRFP or eGFP caused tumors similar to wild-type MDV. Fluorescent protein expression could be seen in spleen, tumor, and feather follicle epithelial cells. These results show that expression of fluorescent proteins within the RL region results in fluorescent rMDVs that still maintains full pathogenicity in the chicken.

Mutations within ICP4 acquired during in vitro attenuation do not alter virulence of recombinant Marek's disease viruses in vivo

Marek's disease (MD) is a T-cell lymphoma of chickens caused by the oncogenic Marek's disease virus (MDV). MD is primarily controlled by live-attenuated vaccines generated by repeated in vitro serial passage. Previous efforts to characterize attenuated MDVs identified numerous mutations, particularly a convergence of high-frequency mutations around amino acids 60–63 within ICP4 (RS1), therefore, ICP4 was considered a candidate gene deserving further characterization. Recombinant MDVs were generated containing a single Q63H mutation or double Q63H+S1630P mutations. Despite the repetitive nature of mutations within ICP4, neither recombinant virus decreased virulence, although one mutant reduced in vivo replication and failed to transmit horizontally. Our results indicate that these mutations are insufficient to reduce disease incidence in infected birds, and suggest that variants in ICP4 do not directly alter virulence, but rather may enhance MDV replication rates in vitro, offering an explanation for the widespread occurrence of ICP4 mutations in a variety of attenuated herpesviruses.

Transcriptional activity comparison of different sites in recombinant Marek's disease virus for the expression of the H9N2 avian influenza virus hemagglutinin gene

Over the last two decades, much attention has been paid to MDV-vectored recombinant vaccines. Many factors have influenced their protective efficacy, and insertion site has been among the main influential factors for the expression of foreign genes in recombinant Marek's disease virus (rMDV). To compare the transcriptional activity of different sites of rMDV, an H9N2 avian influenza virus hemagglutinin gene (AIV-H9N2-HA) expression cassette that used the bi-directional promoter of serotype 1 MDV (MDV1) in the 1.8kb RNA transcript direction (p1.8kb) as a promoter was inserted into 4 different regions of MDV using the bacterial artificial chromosome (BAC) vector and FLP/FRT recombination technique. The insertion regions included 3 of its own sites (US2, US10 and one of Meq genes) in the MDV genome and a foreign site (gpt gene) in the BAC vector. Quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to analyze and compare the H9N2-HA expression levels of these different rMDVs both at the mRNA level and at the protein level. The results indicated that among the four tested insertion regions, the HA expression cassette in the US2 region demonstrated the highest activity, followed by that in the Meq region, which was almost equal to that of US10. Further, the expression cassette had the lowest activity in the foreign region gpt gene. The above data could be useful for choosing proper recombinant insertion regions in the construction of rMDV to express different foreign genes, and it is a prerequisite for developing effective MDV-vectored recombinant vaccines.

Construction of recombinant Marek's disease virus (MDV) lacking the meq oncogene and co-expressing AIV-H9N2 HA and NA genes under control of exogenous promoters

To develop a recombinant Marek's disease virus (rMDV1) co-expressing the hemagglutinin gene (HA) and neuramidinase gene (NA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain and lacking the meq oncogene that shares homology with the Jun/Fos family of transcriptional factors, a wild strain of MDV GX0101 was used as parental virus, the HA and NA genes co-expression cassette under control of the CMV and SV40 early promoters was inserted at two meq sites of GX0101 to form a new meq knock-out mutant MDV (MZC12HA/NA) through homologous recombination. MZC12HA/NA was reconstituted by transfection of recombinant BAC–MDV DNA into the secondary chicken embryo fibroblast (CEF) cells. Highly purified MZC12HA/NA was obtained after four rounds of plaque purification and proliferation. In vitro growth properties of recombinant virus were also inspected and concluded that the MZC12HA/NA had the same growth kinetics in CEF cultures as its parental wild type virus GX0101. Southern blot indicated that co-expression cassette was successfully inserted at two copies sites of meq gene, so two meq genes were knocked-out completely. RT-qPCR showed transcription and expression levels of the HA and NA genes were both significantly higher than that of GX0101 own pp38 gene. Indirect fluorescence antibody (IFA) test, and Western blot analyses indicated that HA and NA genes were co-expressed simultaneously under control of the different promoters but meq genes were not. These results herald a new and effective recombinant meq-deleted MDV-based AIV-H9N2 vaccine may be useful in protecting chickens from very virulent MDV and H9N2 challenges.

Construction of Recombinant Marek's Disease Virus (rMDV) Co-Expressing AIV-H9N2-NA and NDV-F Genes under Control of MDV's Own Bi-Directional Promoter

To qualitatively analyze and evaluate a bi-directional promoter transcriptional function in both transient and transgenic systems, several different plasmids were constructed and recombinant MDV type 1 strain GX0101 was developed to co-express a Neuraminidase (NA) gene from Avian Influenza Virus H9N2 strain and a Fusion (F) gene from the Newcastle disease virus (NDV). The two foreign genes, NDV-F gene and AIV-NA gene, were inserted in the plasmid driven in each direction by the bi-directional promoter. To test whether the expression of pp38/pp24 heterodimers are the required activators for the expression of the foreign genes, the recombinant plasmid pPpp38-NA/1.8kb-F containing expression cassette for the two foreign genes was co-transfected with a pp38/pp24 expression plasmid, pBud-pp38-pp24, in chicken embryo fibroblast (CEF) cells. Alternatively, plasmid pPpp38-NA/1.8kb-F was transfected in GX0101-infected CEFs where the viral endogenous pp38/pp24 were expressed via virus infection. The expression of both foreign genes was activated by pp38/pp24 dimers either via virus infection, or co-expression. The CEFs transfected with pPpp38-NA/1.8kb-F alone had no expression. We chose to insert the expression cassette of Ppp38-NA/1.8kb-F in the non-essential region of GX0101ΔMeq US2 gene, and formed a new rMDV named MZC13NA/F through homologous recombination. Indirect fluorescence antibody (IFA) test, ELISA and Western blot analyses indicated that F and NA genes were expressed simultaneously under control of the bi-directional promoter, but in opposite directions. The data also indicated the activity of the promoter in the 1.8-kb mRNA transcript direction was higher than that in the direction for the pp38 gene. The expression of pp38/pp24 dimers either via co-tranfection of the pBud-pp38-pp24 plasmid, or by GX0101 virus infection were critical to activate the bi-directional promoter for expression of two foreign genes in both directions. Therefore, the confirmed function of the bi-directional promoter provides better feasibilities to insert multiple foreign genes in MDV genome based vectors.

Visualization of Marek’s disease virus in vitro using enhanced green fluorescent protein fused with US10

Marek's disease virus (MDV) is an highly cell-associated avian alphaherpesvirus. Although viral replication is supported in chicken embryo fibroblasts (CEF) or duck embryo fibroblasts, identification of MDV-infected cells is quite cumbersome especially during the early stages of virus replication when plaques can be difficult to recognize. To visualize MDV replication in infected cells and characterize MDV US10 in vitro, rMd5-US10-EGFP, a recombinant MDV, was generated that expresses enhanced green fluorescent protein (EGFP) as a tagged protein fused with US10 at the C-terminal end. The expression of US10-EGFP was detected in infected CEF using fluorescent microscopy and the expression intensity was quantified using flow cytometry analysis. In addition, confocal microscopic analysis provided information on subcellular localization of US10-EGFP in virus-infected cells. In conclusion, rMd5-US10-EGFP virus can be used to help monitor virus activity in vitro.