Dynamic Changes in Viral Loads during Co-Infection with a Recombinant Turkey Herpesvirus Vector Vaccine and Very Virulent Marek's Disease Virus In Vivo.

Marek's disease (MD), caused by the Marek's disease virus (MDV), is a common infectious tumor disease in chickens and was the first neoplastic disease preventable by vaccination. However, the vaccine cannot completely prevent virulent MDV infections, allowing both the vaccine and virulent MDV to coexist in the same chicken for extended periods. This study aims to investigate the changes in viral load of the very virulent strain Md5 and the rHVT-IBD vaccine in different chicken tissues using a real-time PCR assay. The results showed that the rHVT-IBD vaccine significantly reduced the viral load of MDV-Md5 in different organs, while the load of rHVT-IBD was significantly increased when co-infected with Md5. Additionally, co-infection with Md5 and rHVT-IBD in chickens not only changed the original viral load of both viruses but also affected the positive rate of Md5 at 14 days post-vaccination. The positive rate decreased from 100% to 14.29% (feather tips), 0% (skin), 33.33% (liver), 16.67% (spleen), 28.57% (thymus), 33.33% (bursa), and 66.67% (PBL), respectively. This study enhances our understanding of the interactions between HVT vector vaccines and very virulent MDV in chickens and provides valuable insights for the future development of MD vaccines.

A Marek's Disease Virus Messenger RNA-Based Vaccine Modulates Local and Systemic Immune Responses in Chickens.

Marek's disease (MD), caused by the Marek's disease virus, is a lymphoproliferative disease in chickens that can be controlled by vaccination. However, the current vaccines can limit tumor growth and death but not virus replication and transmission. The present study aimed to evaluate host responses following intramuscular injection of an mRNA vaccine encoding gB and pp38 proteins of the MDV within the first 36 h. The vaccine was injected in low and high doses using prime and prime-boost strategies. The expression of type I and II interferons (IFNs), a panel of interferon-stimulated genes, and two key antiviral cytokines, IL-1ß and IL-2, were measured in spleen and lungs after vaccination. The transcriptional analysis of the above genes showed significant increases in the expression of MDA5, Myd88, IFN-α, IFN-ß, IFN-γ, IRF7, OAS, Mx1, and IL-2 in both the spleen and lungs within the first 36 h of immunization. Secondary immunization increased expression of all the above genes in the lungs. In contrast, only IFN-γ, MDA5, MyD88, Mx1, and OAS showed significant upregulation in the spleen after the secondary immunization. This study shows that two doses of the MDV mRNA vaccine encoding gB and pp38 antigens activate innate and adaptive responses and induce an antiviral state in chickens.

Impact of viral telomeric repeat sequences on herpesvirus vector vaccine integration and persistence.

Marek's disease virus (MDV) vaccines were the first vaccines that protected against cancer. The avirulent turkey herpesvirus (HVT) was widely employed and protected billions of chickens from a deadly MDV infection. It is also among the most common vaccine vectors providing protection against a plethora of pathogens. HVT establishes latency in T-cells, allowing the vaccine virus to persist in the host for life. Intriguingly, the HVT genome contains telomeric repeat arrays (TMRs) at both ends; however, their role in the HVT life cycle remains elusive. We have previously shown that similar TMRs in the MDV genome facilitate its integration into host telomeres, which ensures efficient maintenance of the virus genome during latency and tumorigenesis. In this study, we investigated the role of the TMRs in HVT genome integration, latency, and reactivation in vitro and in vivo. Additionally, we examined HVT infection of feather follicles. We generated an HVT mutant lacking both TMRs (vΔTMR) that efficiently replicated in cell culture. We could demonstrate that wild type HVT integrates at the ends of chromosomes containing the telomeres in T-cells, while integration was severely impaired in the absence of the TMRs. To assess the role of TMRs in vivo, we infected one-day-old chickens with HVT or vΔTMR. vΔTMR loads were significantly reduced in the blood and hardly any virus was transported to the feather follicle epithelium where the virus is commonly shed. Strikingly, latency in the spleen and reactivation of the virus were severely impaired in the absence of the TMRs, indicating that the TMRs are crucial for the establishment of latency and reactivation of HVT. Our findings revealed that the TMRs facilitate integration of the HVT genome into host chromosomes, which ensures efficient persistence in the host, reactivation, and transport of the virus to the skin.

Circulating hypervirulent Marek's disease viruses in vaccinated chicken flocks in Taiwan by genetic analysis of meq oncogene.

Marek's disease (MD) is an important neoplastic disease caused by serotype 1 Marek's disease virus (MDV-1), which results in severe economic losses worldwide. Despite vaccination practices that have controlled the MD epidemic, current increasing MD-suspected cases indicate the persistent viral infections circulating among vaccinated chicken farms in many countries. However, the lack of available information about phylogeny and molecular characterization of circulating MDV-1 field strains in Taiwan reveals a potential risk in MD outbreaks. This study investigated the genetic characteristics of 18 MDV-1 strains obtained from 17 vaccinated chicken flocks in Taiwan between 2018 and 2020. Based on the sequences of the meq oncogene, the phylogenetic analysis demonstrated that the circulating Taiwanese MDV-1 field strains were predominantly in a single cluster that showed high similarity with strains from countries of the East Asian region. Because the strains were obtained from CVI988/Rispens vaccinated chicken flocks and the molecular characteristics of the Meq oncoprotein showed features like vvMDV and vv+MDV strains, the circulating Taiwanese MDV-1 field strains may have higher virulence compared with vvMDV pathotype. In conclusion, the data presented demonstrates the circulation of hypervirulent MDV-1 strains in Taiwan and highlights the importance of routine surveillance and precaution strategies in response to the emergence of enhanced virulent MDV-1.

Optimizing protocols for monitoring in vivo replication of a novel chimeric Marek's disease vaccine with an insertion of the long terminal repeat of reticuloendotheliosis virus in the CVI988 strain genome (CVI-LTR).

Monitoring Marek's disease (MD) vaccination is routinely done by evaluating the load of MD vaccine in the feather pulp (FP) between 7 and 10 days of age. However, attempts in our laboratory to detect a novel CVI-LTR vaccine in the FP samples from commercial flocks failed. The objective of this study was to evaluate the most suitable tissue and age to monitor CVI-LTR vaccination. We used two different commercial CVI988 vaccines as controls. One hundred and sixty 1-day-old commercial brown layers were vaccinated with either CVI-LTR, CVI988-A, CVI988-B or remained unvaccinated. Samples of the spleen, thymus, and bursa were collected at 3, 4, 5, and 6 days of age and samples of FP were collected at 7 and 21 days for DNA isolation. Our results showed that CVI-LTR replicated earlier than CVI988 vaccines in the lymphoid organs but was not detected in the FP at either 7 or at 21 days of age. We also confirmed that either the spleen or thymus collected at 4-6 days was a suitable sample to monitor CVI-LTR vaccination in commercial flocks. Finally, we evaluated the load of oncogenic MDV DNA in five commercial flocks that were vaccinated with either CVI-LTR + rHVT or CVI988-A + rHVT. The load of oncogenic MDV DNA was evaluated at 21 days in the FP in 20 chickens per group. Our results demonstrated that CVI-LTR was more successful in reducing oncogenic MDV DNA at 21 days of age than the CVI988-A strain.RESEARCH HIGHLIGHTSCVI-LTR replicates in the thymus and spleen earlier than CVI988.CVI-LTR replicates in lymphoid organs but it cannot be detected in feather pulp.CVI-LTR reduced the load of oncogenic MDV DNA more efficiently than CVI988.

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.

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.

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.

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.

Early Challenge with Oncogenic Marek's Disease Virus Does Not Interfere with Load of Marek's Disease Vaccines DNA in the Feather Pulp at 7 Days of Age.

In the last decade, monitoring Marek's disease (MD) vaccination by real-time PCR has become a common practice. Evaluating in vivo replication of MD vaccines in the feather pulp (FP) at 7 days of age provides information on how well a flock has been vaccinated. Factors such as vaccine dose, combination with other vaccines, age and route of vaccination, and the origin of the vaccine can influence the results and need to be taken into consideration. Early infection with oncogenic MD virus (MDV) could also affect how vaccines replicate in the first week and therefore might influence the results. The objective of this study was to evaluate if coinfection with oncogenic MDV could affect MD vaccine DNA viral load in the FP at 7 days of age. A retrospective study was done using data from nine animal experiments (46 treatment groups) in which chickens were vaccinated against MD either in ovo or at 1 day of age and challenged with various oncogenic strains at 1 day of age by contact. In each experiment, vaccinated but not challenged groups were used as controls. Replication of MD vaccine was evaluated in samples of FP collected at 7 days of age by real-time PCR, and percentage of positives and vaccine load were analyzed. Our results show that CVI-988 (13 treatment groups), SB-1 (six treatment groups), and in most cases turkey herpesvirus (HVT; 24 out of 27 treatment groups) replication was not affected by early infection with oncogenic MDV. There were three treatment groups in which HVT replication differed between challenged and unchallenged chickens, however the effect was not clear; replication of HVT in nonchallenged chickens was higher (one treatment group) or lower (two treatment groups) than in challenged chickens and factors other than coinfection with MDV might have contributed to such differences.

Nota de investigación­El desafío temprano con un virus oncogénico de la enfermedad de Marek no interfiere con la carga de ADN de las vacunas contra la enfermedad de Marek en la pulpa de la pluma a los siete días de edad. En la última década, el seguimiento de la vacunación contra la enfermedad de Marek (EM) mediante PCR en tiempo real se ha convertido en una práctica común. La evaluación de la replicación in vivo de las vacunas de Marek en la pulpa de la pluma (FP) a los siete días de edad proporciona información sobre qué tan bien se ha vacunado una parvada. Factores como la dosis de la vacuna, la combinación con otras vacunas, la edad, la vía de vacunación y el origen de la vacuna pueden influir en los resultados y deben tenerse en cuenta. La infección temprana con un virus de Marek oncogénico (MDV) también podría afectar la forma en que las vacunas se replican en la primera semana y por lo tanto, podría influir en los resultados. El objetivo de este estudio fue evaluar si la coinfección con un virus de Marek oncogénico podría afectar la carga viral del ADN de la vacuna de Marek en la pulpa de la pluma a los siete días de edad. Se realizó un estudio retrospectivo utilizando datos de nueve experimentos con animales (46 grupos de tratamientos) en los que se vacunaron pollos contra la enfermedad de Marek ya sea in ovo o al día de edad y se desafiaron con varias cepas oncogénicas al día de edad por contacto. En cada experimento, se utilizaron como controles los grupos vacunados, pero no desafiados. Se evaluó la replicación de la vacuna de Marek en muestras de pulpa de la pluma recolectadas a los siete días de edad por PCR en tiempo real, y se analizó el porcentaje de positivos y la carga vacunal. Los resultados de este estudio muestran que la replicación de la cepa CVI-988 (13 grupos de tratamiento), la cepa SB-1 (seis grupos de tratamiento) y en la mayoría de los casos, del herpesvirus de pavo (HVT; 24 de 27 grupos de tratamiento) no se vio afectada por la infección temprana con un virus de Marek oncogénico. Hubo tres grupos de tratamiento en los que la replicación de la vacuna HVT difería entre pollos desafiados y no desafiados, sin embargo, el efecto no fue claro; la replicación de la vacuna HVT en pollos no desafiados fue mayor (un grupo de tratamiento) o menor (dos grupos de tratamiento) que en los pollos desafiados y otros factores distintos a la coinfección con el virus de la enfermedad de Marek podrían haber contribuido a tales diferencias.

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.

Differential Replication and Cytokine Response between Vaccine and Very Virulent Marek's Disease Viruses in Spleens and Bursas during Latency and Reactivation.

Marek's disease virus (MDV) infection results in Marek's disease (MD) in chickens, a lymphoproliferative and oncogenic deadly disease, leading to severe economic losses. The spleen and bursa are the most important lymphoid and major target organs for MDV replication. The immune response elicited by MDV replication in the spleen and bursa is critical for the formation of latent MDV infection and reactivation. However, the mechanism of the host immune response induced by MDV in these key lymphoid organs during the latent and reactivation infection phases is not well understood. In the study, we focused on the replication dynamics of a vaccine MDV strain MDV/CVI988 and a very virulent MDV strain MDV/RB1B in the spleen and bursa in the latent and reactivation infection phases (7-28 days post-inoculation [dpi]), as well as the expression of some previously characterized immune-related molecules. The results showed that the replication ability of MDV/RB1B was significantly stronger than that of MDV/CVI988 within 28 days post-infection, and the replication levels of both MDV strains in the spleen were significantly higher than those in the bursa. During the latent and reactivation phase of MDV infection (7-28 dpi), the transcriptional upregulation of chicken IL-1ß, IL6, IL-8L1 IFN-γ and PML in the spleen and bursa induced by MDV/RB1B infection was overall stronger than that of MDV/CVI988. However, compared to MDV/RB1Binfection, MDV/CVI988 infection resulted in a more effective transcriptional activation of CCL4 in the latent infection phase (7-14 dpi), which may be a characteristic distinguishing MDV vaccine strain from the very virulent strain.

Characterization of Md5-BAC-REV-LTR virus as Marek's disease vaccine in commercial meat-type chickens: protection and immunosuppression.

Md5-BAC-REV-LTR is a recombinant Marek's disease virus (MDV), with an insertion of the long terminal repeat (LTR) of reticuloendotheliosis virus (REV) into the genome of the highly virulent MDV strain rMd5. It has been shown that Md5-BAC-REV-LTR does not induce tumours and confers high protection against challenge with MDV in 15 × 7 chickens. The objective of the present study was to evaluate the protection and safety (in terms of oncogenicity and immunosuppression) of Md5-BAC-REV-LTR in commercial meat-type chickens bearing maternal antibodies against MDV. Our results show that sub-cutaneous administration of Md5-BAC-REV-LTR at 1 day of age conferred high protection (protection index PI = 84.2) against an early challenge (1 day) by contact exposure to shedder birds infected with the vv+ MDV 648A strain. In such stringent challenge conditions, Md5-BAC-REV-LTR was more protective than a commercial CVI988 (PI = 12.4) and similar to the experimental vaccine Md5-BACΔmeq (PI = 92.4). Furthermore, Md5-BAC-REV-LTR did not induce either tumours or immunosuppression in this study. Immunosuppression was evaluated by the relative lymphoid organ weights and also by the ability of the vaccine to induce late-MDV-induced immunosuppression associated with reactivation of the virus. This study shows that Md5-BAC-REV-LTR has the potential to be used as a MD vaccine and is highly protective against early challenge with vv+ MDV.RESEARCH HIGHLIGHTSMd5-BAC-REV-LTR is highly protective against early challenge with vv+ MDV in commercial meat-type chickens.Md5-BAC-REV-LTR does not cause early immunosuppression.Md5-BAC-REV-LTR does not cause late immunosuppression.Unlike other serotype 1 vaccines, Md5-BAC-REV-LTR is not detected in feather pulp at 7 days post vaccination.

Distinct polymorphisms in a single herpesvirus gene are capable of enhancing virulence and mediating vaccinal resistance.

Modified-live herpesvirus vaccines are widely used in humans and animals, but field strains can emerge that have a higher virulence and break vaccinal protection. Since the introduction of the first vaccine in the 1970s, Marek's disease virus overcame the vaccine barrier by the acquisition of numerous genomic mutations. However, the evolutionary adaptations in the herpesvirus genome responsible for the vaccine breaks have remained elusive. Here, we demonstrate that point mutations in the multifunctional meq gene acquired during evolution can significantly alter virulence. Defined mutations found in highly virulent strains also allowed the virus to overcome innate cellular responses and vaccinal protection. Concomitantly, the adaptations in meq enhanced virus shedding into the environment, likely providing a selective advantage for the virus. Our study provides the first experimental evidence that few point mutations in a single herpesviral gene result in drastically increased virulence, enhanced shedding, and escape from vaccinal protection.

Codon Deoptimization of UL54 in meq-Deleted Marek's Disease Vaccine Candidate Eliminates Lymphoid Atrophy but Reduces Vaccinal Protection.

Marek's disease (MD) is an oncogenic, lymphoproliferative, and highly contagious disease of chickens. Its etiologic agent is the alphaherpesvirus Marek's disease virus (MDV, Gallid alphaherpesvirus 2), and it is a chronic and ubiquitous problem for the poultry industry with significant economic impact in the United States and worldwide. We have previously demonstrated that MDV attenuated by dicodon deoptimization of the UL54 gene results in reduced gene product accumulation in vitro, with reduced viral genome copy number upon infection and reduced atrophy of bursa and thymus in vivo as well. In this report we detail our attempts to use the same attenuation strategy on a meq-deleted MDV mutant, rMd5B40ΔMeq. Unlike the wild-type rMd5B40 virus the rMd5B40ΔMeq is no longer oncogenic, but infected birds experience an unacceptable amount of bursa and thymus atrophy (BTA). We produced two meq-deleted MDV recombinants with a dicodon-deoptimized UL54 (rMd5B40ΔMeq/UL54deop1 and -deop2) and tested their tendency to cause BTA and to serve as a protective vaccine. We found that, although dicodon deoptimization of the UL54 gene results in a virus that spares the infected animal from atrophy of the bursa and thymus, the meq-deleted UL54-deoptimized recombinant is also less protective than the meq-deleted virus without UL54 deoptimization, the HVT + SB1 combination vaccine, or the Rispens (CVI988) vaccine.

The Emergence of a vv + MDV Can Break through the Protections Provided by the Current Vaccines.

Marek's disease (MD) is an infectious malignant T-cell lymphoma proliferative disease caused by Marek's disease virus (MDV). In recent years, the emergence of very virulent (vv) and/or very virulent plus (vv +) strains of MDV in the field has been suggested as one of the causes of vaccination failure. The pathogenicity of the MDV strain GX18NNM4, isolated from a clinical outbreak in a broiler breeder flock that was vaccinated with CVI988/Rispens, was investigated. In the vaccination-challenge test, GX18NNM4 was able to break through the protections provided by the vaccines CVI988 and 814. It also significantly reduced body weight gain and caused marked gross lesions and a large area of infiltration of neoplastic lymphocyte cells in the heart, liver, pancreas, etc. of the infected birds. In addition, the expressions of programmed death 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1), in the spleens and cecal tonsils (CTs) of the unvaccinated challenged birds were significantly increased compared to those in the vaccinated challenged birds, indicating that the PD-1/PD-L1 pathway is related to immune evasion mechanisms. The results showed that the GX18NNM4 strain could cause severe immunosuppression and significantly decrease the protections provided by the current commercial vaccines, thus showing GX18NNM4 to be a vv + MDV strain.

Marek's disease virus oncogene Meq expression in infected cells in vaccinated and unvaccinated hosts.

Marek's disease (MD) vaccines are unique in their capability to prevent MD lymphomas as early as a few days after vaccination, despite the fact that they do not eliminate virulent viruses from the host. To help understand the mechanism behind this unique MD vaccine effect, we compared the expression of MDV oncoprotein Meq among CD4+ T cells between vaccinated and unvaccinated birds. Chickens were vaccinated by an MD vaccine, herpesvirus of turkeys, and then challenged by a recombinant virulent MDV that expresses green fluorescent protein simultaneously with Meq. We found significantly fewer Meq-expressing CD4+ T cells appeared in peripheral blood mononuclear cells (PBMC) of the vaccinated birds compared to the unvaccinated birds as early as one week after the virulent virus challenge. In contrast, the quantity of virulent MDV genome remained similar in Meq- PBMC in both vaccinated and unvaccinated birds. Our results suggest that MD vaccination affects the dynamics of Meq-expressing, possibly transformed, cells while impact on the overall infection in the Meq- cells was not significant.

Abrogation of Marek's disease virus replication using CRISPR/Cas9.

Marek's disease virus (MDV) is a highly cell-associated alphaherpesvirus that causes deadly lymphomas in chickens. While vaccination protects against clinical symptoms, MDV field strains can still circulate in vaccinated flocks and continuously evolve towards greater virulence. MDV vaccines do not provide sterilizing immunity, allowing the virus to overcome vaccine protection, and has increased the need for more potent vaccines or alternative interventions. In this study, we addressed if the CRISPR/Cas9 system can protect cells from MDV replication. We first screened a number of guide RNAs (gRNAs) targeting essential MDV genes for their ability to prevent virus replication. Single gRNAs significantly inhibited virus replication, but could result in the emergence of escape mutants. Strikingly, combining two or more gRNAs completely abrogated virus replication and no escape mutants were observed upon serial passaging. Our study provides the first proof-of-concept, demonstrating that the CRISPR/Cas9 system can be efficiently used to block MDV replication. The presented findings lay the foundation for future research to completely protect chickens from this deadly pathogen.

Vertical transmission of ALV from ALV-J positive parents caused severe immunosuppression and significantly reduced marek's disease vaccine efficacy in three-yellow chickens.

In order to evaluate the influence of the vertical transmission of avian leukosis virus (ALV) from J subgroup (ALV-J) positive parents on the vaccine efficacy of Marek's disease virus (MDV), ALV-J positive male breeders × female breeders of Three-yellow chickens and the ALV negative male breeder × the negative female breeders were used respectively for crossbreeding to produce eggs and the hatching offspring. The commercial CVI988/Rispens vaccine was used to vaccinate the crossbred offspring at 1-day-old. At 7-days-old, the birds were inoculated with the inactivated oil-emulsion vaccines (OEVs) AIV-H5 monovalent and NDV + AIV-H9 bivalent, respectively. Then the birds were challenged with a Chinese very virulent (vv) MDV field strain GXY2 at 14-day-old. The results showed that the viral load of the challenged GXY2 in the offspring from the ALV-J positive breeders was significantly higher than that from the ALV-negative breeders' (P < 0.05), and the mortality and tumor incidence of offspring from the ALV-J positive breeders were higher than those of the ALV-negative breeders. Also the offspring of the ALV-J positive breeders exhibited a significant negative effect on the development of the immune organs (P < 0.05) and lower antibody responses to the vaccinations with the commercial OEVs (P<0.05). The MD vaccine protective index in the offspring from the ALV-J positive breeders was lower than that from the ALV-negative breeders. The results of the study demonstrated that the vertical transmission of ALV from the ALV-J positive parents caused severe immunosuppression and significantly reduced the Marek's disease vaccine efficacy in Three-yellow chickens.