Marek's Disease Virus Field Strains Research Articles

Genetic evolution of Marek's disease virus in vaccinated poultry farms.

Background and Aim:The Marek’s disease virus (MDV) is a neoplastic disease causing serious economic losses in poultry production. This study aimed to investigate MDV occurrence in poultry flocks in the Lower Egypt during the 2020 breakout and genetically characterized Meq, gL, and ICP4 genes in field strains of MDV.Materials and Methods:Forty samples were collected from different breeds from eight Egyptian governorates in 2020. All flocks had received a bivalent vaccine (herpesvirus of turkey FC-126 + Rispens CVI988). However, weight loss, emaciation, reduced egg production, paralysis, and rough/raised feather follicles occurred. Samples were collected from feather follicles, liver, spleen, and nerve tissue for diagnosis by polymerase chain reaction. MDV genetic characterization was then performed by sequencing the Meq, gL, and ICP4 genes of five positive samples representing different governorates and breeds.Results:A total of 28 samples were positive for MDV field strains, while two were related to MDV vaccinal strains. All samples tested negative for ALV (A, B, C, D, and J) and REV. Phylogenetic analysis of the Meq gene of sequenced samples revealed that all MDVs were related to the highly virulent European viruses (Gallid herpesvirus 2 ATE and PC12/30) with high amino acid (A.A.) identity 99.2-100%. Alternatively, there was low A.A. identity with the vaccine strains CVI988 and 3004 (up to 82.5%). These results indicate that further investigation of the efficacy of current Egyptian vaccines is required. The Egyptian strains also harbor a specific mutation, allowing clustering into two subgroups (A and B). By mutation analysis of the Meq gene, the Egyptian viruses in our study had R101K, P217A, and E263D mutations present in all Egyptian viruses. Furthermore, R176A and T180A mutations specific to our strains contributed to the high virulence of highly virulent strains. There were no mutations of the gL or ICP4 genes.Conclusion:Further studies should evaluate the protection contributed by current vaccines used in Egypt.

Positive Selection Drives Rapid Evolution of the meq Oncogene of Marek’s Disease Virus

Marek’s disease (MD), caused by Marek’s disease virus (MDV), a poultry-borne alphaherpesvirus, is a devastating disease of poultry causing an estimated annual loss of one billion dollars to poultry producers, worldwide. Despite decades of control through vaccination, MDV field strains continue to emerge having increased virulence. The evolutionary mechanism driving the emergence of this continuum of strains to increased MDV virulence, however, remains largely enigmatic. Increase in MDV virulence has been associated with specific amino acid changes within the C-terminus domain of Mareks’s EcoRI-Q (meq)-encoded oncoprotein. In this study, we sought to determine whether the meq gene has evolved adaptively and whether past vaccination efforts have had any significant effect on the reduction or increase of MDV diversity over time. Our analysis suggests that meq is estimated to be evolving at a much faster rate than most dsDNA viruses, and is comparable with the evolutionary rate of RNA viruses. Interestingly, most of the polymorphisms in meq gene appear to have evolved under positive selection and the time of divergence at the meq locus coincides with the period during which the poultry industry had undergone transitions in management practices including the introduction and widespread use of live attenuated vaccines. Our study has revealed that the decades-long use of vaccines did not reduce MDV diversity, but rather had a stimulating effect on the emergence of field strains with increased genetic diversity until the early 2000s. During the years 2004–2005, there was an abrupt decline in the genetic diversity of field isolates followed by a recovery from this bottleneck in the year 2010. Collectively, these data suggest that vaccination seems to not have had any effect on MDV eradication, but rather had a stimulating effect on MDV emergence through adaptation.

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.

Molecular evolution of Marek's disease virus (MDV) field strains in a 40-year time period.

Marek's disease (MD) presents a serious threat in poultry production. The disease has been limited for over 40 yr by protective vaccination. The widely applied vaccination against MD is also one of the factors causing evolutionary pressure onto field Marek's disease virus (MDV) virulent strains. Molecular evolution of MDV genes involved in oncogenesis may increase the pathogenicity of MDV virulent strains. The goal of the presented study was to sum up the molecular evolution of MDV field strains isolated in the last 40 yr in Poland. In total, 85 field MDV strains collected between 1974 and 2012 were propagated in chicken embryo fibroblasts. After DNA extraction, three sets of primers were designed for PCR complementary to the MDV076 (RLORF7) region encoding the meq oncogene as well to the MDV077 (23 kDa protein binding alpha-enolase) and MDV077.5 (RLORF6) genes. The obtained 85 MDV076, 60 MDV077, and 58 MDV077.5 cloned fragments were sequenced and aligned with the sequences of reference MDV strains showing different pathogenicity levels. The retrieved nucleotide (nt) and deduced amino acid sequences of RLORF7, 23 kDa protein, and LORF6 of Polish field strains showed several mutations and substitutions homologous to those observed in reference strains with a determined pathogenicity. The observed changes indicated the continuous evolution of field MDV strains. The RLORF7 nt sequence of analyzed MDV isolates showed similarity to virulent and very virulent MDV reference strains. The obtained 23 kDa and LORF6 nt sequences provided more important data and were more similar to mildly pathogenic strains than to virulent and very virulent MDV. The specific nt motifs in all three genes may indicate an increase of MDV virulence and were found in strains starting from 2006. According to the obtained results, the strains isolated in 2012 are similar to the very virulent plus MDV group. The study showed that RLORF7, 23 kDa protein, and RLORF6 fragments harbor sequence motifs that may have some association with MDV pathogenicity level. However, the exact role of the investigated regions in pathogenicity should be further examined by knock-out MDV strains. Also, the true MDV pathotype may only be determined by traditional in vivo experiments.

A Deletion in the Glycoprotein L (gL) Gene of U.S. Marek's Disease Virus (MDV) Field Strains Is Insufficient to Confer Increased Pathogenicity to the Bacterial Artificial Chromosome (BAC)–Based Strain, RB-1B

Marek's disease (MD) is a highly transmissible, herpesvirus-associated malignancy of chickens and turkeys caused by Marek's disease virus (MDV). MD is currently controlled through the use of nonsterilizing vaccines composed of antigenically related, apathogenic herpesviruses Mardivirus 2 (MDV-2), Meleagrid herpesvirus 1 (herpesvirus of turkeys, HVT), or attenuated MDV-1 strain CVI988 (Rispens). Since the mid-1960s, field strains of MDV have increased in virulence, due, in part, to the widespread use of vaccines since the early 1970s. One mutation that we have identified common to very virulent field strains (vv and vv+MDVs) since the 1990s has been a mutation in the UL1 gene, encoding glycoprotein L (gL). This mutation, a 12-nucleotide (nt) deletion in the signal peptide of gL, has been associated with increased virulence and decreased vaccine protection in the context of challenge with a vv+MDV, strain TK. To determine whether this mutation alone was sufficient to confer increased virulence, we introduced this mutation into the transmission-competent pRB-1B bacterial artificial chromosome (BAC) using two-step, Red-mediated recombination. The resulting mutant, pRB-1BgLdelta, was tested for changes in replication in cell culture using multistep growth curves, plaque size analysis, viral burst analysis, and the ability to compete with the parental virus when co-transfected at different ratios and sequentially passaged. In addition, we examined this mutant for changes in pathogenicity in inoculated and contact-exposed unvaccinated and vaccinated chickens. Our data show minor differences in plaque sizes in cell culture, but no discernible changes in the infection of specific-pathogen-free (SPF) leghorn chickens. We therefore conclude that although this mutation is indeed common to MDV field strains isolated in the eastern United States, it is insufficient to confer increased virulence or loss of vaccine protection previously observed for a vv+MDV strain having this mutation.

Differentiation between pathogenic serotype 1 isolates of Marek's disease virus and the Rispens CVI988 vaccine in Australia using real-time PCR and high resolution melt curve analysis

Two real-time PCR assays were developed which enable quantitation and differentiation between pathogenic Australian isolates of Marek's disease virus (MDV) serotype 1 and the serotype 1 vaccine strain Rispens CVI988. The assays are based on a DNA sequence variation in the meq gene between pathogenic and vaccinal MDV1 which has been confirmed by sequencing of 20 Australian field strains of MDV. Complete specificity has been demonstrated in samples containing pathogenic MDV (n=20), Rispens (3 commercial vaccine strains), or both. The limit of detection of both the Rispens-specific and the pathogenic MDV1-specific assays was 10 viral copies/reaction. The tests successfully differentiated and quantified MDV in mixtures of pathogenic and vaccinal Rispens virus. A high resolution melt curve analysis targeting the same SNP used for the real-time PCR assays was also developed which successfully detected sequence variation between Md5, six Australian MDV1 isolates and the three Rispens vaccines. However it was ineffective at differentiating mixtures of pathogenic and vaccinal MDV1. The real-time PCR assays have both diagnostic and epidemiological applications as they enable differentiation and quantitation of Rispens CVI988 and pathogenic MDV1 in co-infected chickens in Australia.

Deletion of 1.8-kb mRNA of Marek's disease virus decreases its replication ability but not oncogenicity

BackgroundThe 1.8-kb mRNA was reported as one of the oncogenesis-related genes of Marek's disease virus (MDV). In this study, the bacterial artificial chromosome (BAC) clone of a MDV field strain GX0101 was used as the platform to generate mutant MDV to examine the functional roles of 1.8-kb mRNA.ResultsBased on the BAC clone of GX0101, the 1.8-kb mRNA deletion mutant GX0101Δ(A+C) was constructed. The present experiments indicated that GX0101Δ(A+C) retained a low level of oncogenicity, and it showed a decreased replication capacity in vitro and in vivo when compared with its parent virus, GX0101. Further studies in vitro demonstrated that deletion of 1.8-kb mRNA significantly decreased the transcriptional activity of the bi-directional promoter between 1.8-kb mRNA and pp38 genes of MDV.ConclusionThese results suggested that the 1.8-kb mRNA did not directly influence the oncogenesis but related to the replication ability of MDV.

Molecular and biological characterization of a Marek’s disease virus field strain with reticuloendotheliosis virus LTR insert

A Marek's disease virus (MDV) field strain designated GX0101 was isolated from a layer flock and confirmed to be a recombinant virus with an insert of a long terminal repeat (LTR) from the reticuloendotheliosis virus (REV). A chimeric molecule containing an REV-LTR insert of 539 bp and its flanking sequences from MDV was amplified and sequenced. An REV-LTR downstream from the Internal Repeat Short (IRS) region has 77.4-98.6% homology to seven REV field strains isolated from different avian species in different parts of the world. The insertion site is located downstream of SORF 1 and upstream of SORF2 in the IRS region near the junction with the Unique Short (US) region in the MDV serotype 1 genome. Chicken experiments were conducted to determine the oncogenicity of the recombinant GX0101 virus and its transmissibility to contact chickens. Dot blot hybridization was used to detect the presence of the pp38 gene in feather tips from GX0101 or Md5 infected and contact birds. The pp38 was detected in GX0101 contact birds about 1-2 weeks earlier than in Md5 birds when both groups were vaccinated with HVT vaccine. Long term pathogenicity tests in specific pathogen free (SPF) chickens reveal that the recombinant GX0101 has a higher virulence than GA, but less virulence than Md5, the very virulent pathotype of MDV. This is the first report on an oncogenic serotype 1 MDV field strain with LTR insert and its pathogenicity.

A Deletion Within Glycoprotein L of Marek's Disease Virus (MDV) Field Isolates Correlates with a Decrease in Bivalent MDV Vaccine Efficacy in Contact-Exposed Chickens

We examined the functional role of a naturally occurring deletion within the glycoprotein L (gL) gene of Marek's disease virus (MDV) field isolates. We previously showed that this mutation incrementally increased the virulence of an MDV in contact-exposed SPF leghorn chickens, when chickens shedding this virus were co-infected with herpesvirus of turkeys (HVT). In our present study, we examined this mutation using two stocks of the very virulent plus (vv+)MDV strain TK, one of which harbored this deletion (TK1a) while the other did not (TK2a). We report that TK1a replicating in vaccinated chickens overcame bivalent (HVT/SB1) vaccine protection in contact-exposed chickens. Treatment groups exposed to vaccinated chickens inoculated with a 1:1 mix of TK1a and TK2a showed decreased bivalent vaccine efficacy, and this decrease correlated with the prevalence of the gL deletion indicative of TK1a. These results were also found using quadruplicate treatment groups and bivalently vaccinated chickens obtained from a commercial hatchery. As this deletion was found in 25 out of 25 recent field isolates from Delaware, Maryland, North Carolina, Pennsylvania, and Virginia, we concluded that there is a strong selection for this mutation, which appears to have evolved in HVT or bivalently vaccinated chickens. This is the first report of a mutation in a vv+MDV field strain for which a putative biological phenotype has been discerned. Moreover, this mutation in gL has apparently been selected in MDV field isolates through Marek's disease vaccination.

Isolation of recombinant field strains of Marek’s disease virus integrated with reticuloendotheliosis virus genome fragments

Two Marek's disease virus (MDV) field strains were isolated from chickens with tumors independently from Guangdong and Guangxi provinces, and it was confirmed that there were no co-infections with reticuloendotheliosis viruses (REV) in chicken embryo fibroblast cells (CEF) in indirect fluorescence antibody test (IFA) with REV-specific monoclonal antibodies. By dot blot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated that LTR fragments of REV genome were integrated into genome of these two MDV field strains. To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primers from REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hot points were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDV for each pair) were used in PCR while using the genomic DNA of both strains as the templates. The sequence data demonstrated that both recombinant field strains contained the same REV LTR inserted into MDV at the identical sites in US fragment of the genomes. From the above, it was speculated that both recombinant field MDVs were originated from a same recombinant virus and spread among chicken flocks in two provinces.