BACKGROUND: Marek\'s disease (MD) is a lymphoproliferative disease caused by Gallid alphaherpesvirus 2 (GaHV-2, MDV-1), which primarily affects chickens. However, the virus is also able to induce tumors and polyneuritis in turkeys, albeit less frequently than in chickens.
RESULTS: This is the first study in Turkey reporting the molecular characterization of a MDV-1 strain detected in a flock of backyard turkeys exhibiting visceral lymphoma. Here, MEQ, vIL-8, pp38 and 132-bp tandem repeat regions, which are frequently preferred in the pathotyping of MDV-1, were examined. It was determined that the MEQ gene of MDV-1/TR-21/turkey strain obtained in the present study encoded 339 amino acids (1020 nt) and had four proline-rich repeat regions (PPPP). Based on the nucleotide sequence of the MEQ gene of the MDV-1/TR-21/turkey strain, a phylogenetic tree was created using the MEGA-X software with the Maximum Likelihood Method (in 1000 replicates). Our strain was highly identical (> 99.8) to the Italian/Ck/625/16, Polish (Polen5) and some Turkish (Layer-GaHV-2-02-TR-2017, Tr/MDV-1/19) MDV-1 strains. Also, nt and aa sequences of the MEQ gene of our strain were 99.1 and 99.41% identical to another Turkish strain (MDV/Tur/2019) originated from chickens. Sequence analysis of pp38 and vIL-8 genes also supported the above finding. The identity ratios of nucleotide and amino acid sequences of vIL-8 and pp38 genes of MDV-1/TR-21/turkey strain were 99.64-100% and 99.79-100%, respectively, when compared with those of the Polish strain. According to 132-bp tandem repeat PCR results, the MDV-1/TR-21/turkey strain had five copies.
CONCLUSIONS: These results suggested that the MDV-1/TR-21/turkey strain obtained from backyard turkeys can be either very virulent or very virulent plus pathotype, though experimental inoculation is required for precise pathotyping.

Several recombinant turkey herpesviruses (rHVTs) have been developed within the past decades, and they are now used commercially worldwide. In broiler chickens, rHVTs are usually administered alone, but in long-living birds they are used in combination with Marek\'s disease (MD) vaccines of other serotypes (i.e., CVI988). The objectives of this work were to 1) evaluate protection against MD conferred by HVT and two rHVTs when combined with CVI988 and 2) optimize the use of rHVT in combination with CVI988 to maximize replication of rHVT without compromising MD protection. Various vaccine protocols, all using rHVT or HVT at the recommended dose (RD), were evaluated. Protocols evaluated included in ovo vaccination with HVT+CVI988 or rHVT+CVI988 (using either the double dose [DD] or the RD of CVI988), day of age vaccination of rHVT+CVI988 at DD, and revaccination protocols using rHVT in ovo followed by CVI988 at DD at day of age. Our results show that, when combined with CVI988, HVT and rHVTs confer a similar level of protection against MD (>90%) regardless of whether CVI988 was used at RD or at DD. However, the combination of rHVT with CVI988 at DD resulted in reduced replication rates of rHVT (60%-76% vs. 95%-100%). Our results show that such a negative effect could be avoided without jeopardizing MD protection by administering CVI988 at RD (if combined in ovo with rHVT) or administered rHVT first in ovo followed by CVI988 at DD at day of age.
Estudio de la eficacia y replicación de vacunas con vectores recombinantes mediante el uso del virus del herpes del pavo combinado con otras vacunas contra la enfermedad de Marek. Varios herpesvirus de pavo recombinantes (rHVT) se han desarrollado en las últimas décadas y ahora se utilizan comercialmente en todo el mundo. En pollos de engorde, los rHVT generalmente se administran solos, pero en aves de vida larga se usan en combinación con vacunas contra la enfermedad de Marek (MD) de otros serotipos (especialmente, CVI988). Los objetivos de este trabajo fueron 1) evaluar la protección contra la enfermedad de Marek conferida por herpesvirus de pavo (HVT9 y por dos rHVT cuando se combinan con la cepa CVI988 y 2) optimizar el uso de rHVT en combinación con la cepa CVI988 para maximizar la replicación de rHVT sin comprometer la protección contra la enfermedad de Marek. Se evaluaron varios protocolos de vacunas, todos con rHVT o con HVT a la dosis recomendada (RD). Los protocolos evaluados incluyeron la vacunación in ovo con HVT + CVI988 o rHVT + CVI988 (usando la dosis doble o la dosis recomendada de la cepa CVI988), la vacunación al día de la edad con rHVT + CVI988 con dosis doble, y los protocolos de revacunación usando rHVT seguido por la cepa CVI988 con dosis doble al día de edad. Los resultados muestran que cuando se combinan con CVI988, HVT y rHVT confieren un nivel de protección similar contra la enfermedad de Marek (> 90%) independientemente de que la cepa CVI988 se haya usado a la dosis recomendada o con dosis doble. Sin embargo, la combinación de rHVT con la cepa CVI988 con doble dosis produjo una reducción en las tasas de replicación de rHVT (60% –76% vs. 95% –100%). Estos resultados muestran que dicho efecto negativo podría evitarse sin poner en peligro la protección contra la enfermedad de Marek administrando la cepa CVI988 a la dosis recomendada (si se combina in ovo con rHVT) o administrando rHVT primero in ovo, seguido de CVI988 con dosis doble al día de la edad.

Marek\'s disease virus (MDV) is a member of alphaherpesviruses associated with Marek\'s disease, a highly contagious neoplastic disease in chickens. Complete sequencing of the viral genome and recombineering techniques using infectious bacterial artificial chromosome (BAC) clones of Marek\'s disease virus genome have identified major genes that are associated with pathogenicity. Recent advances in CRISPR/Cas9-based gene editing have given opportunities for precise editing of the viral genome for identifying pathogenic determinants. Here we describe the application of CRISPR/Cas9 gene editing approaches to delete the Meq and pp38 genes from the CVI988 vaccine strain of MDV. This powerful technology will speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.

Marek\'s disease (MD) is a major cause of mortality in backyard chickens. The diagnosis of MD is complex, however, and knowledge of Marek\'s disease virus (MDV) in spontaneous field cases such as in backyard chickens is largely unknown. In this study, 40 backyard chickens with a presumptive MD diagnosis based on histologic lymphoid infiltrations in peripheral nerves with and without lymphomas were investigated. Twenty-eight of the birds were submitted to the diagnostic laboratory for disease explorations, and 12 chickens were from a flock in which some members demonstrated anisocoria and pupil irregularities compatible with ocular MD. Histologic scores were established for brain, peripheral nerves, heart, lung, liver, kidney, and gonad sections, ranging from mild (+) to severe (+++) lymphoid infiltrations. Twelve chickens had gross lymphomas, and all but two chickens had mild to severe peripheral nerve lymphoid infiltrates. There were no age or breed predispositions in the study group. Quantification of serotypes MDV-1, -2, and -3 performed with real-time PCR demonstrated high correlation (R2 = 0.94) between fresh and fixed spleen specimens, as well as between histopathology scores and MDV-1 viral loads. MDV-2 DNA was detected in a portion of the chickens, likely consistent with naturally occurring virus, whereas the vaccine strain MDV-3 was rarely detected. Significant differences in MDV-1 viral loads between tumorous and nontumorous chickens were observed, in which a ratio of MDV-1 glycoprotein B/glyceraldehyde-3-phosphate dehydrogenase ≥ 0.5 was suggestive of gross tumors in this study. We propose that real-time PCR may be a good tool for MD diagnosis in backyard chickens.

Marek\'s disease virus (MDV) is a herpesvirus that induces lymphomas and immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is divided into two phases: early-MDV-IS occurring mainly in chickens lacking maternal antibodies (MAb) against MDV and associated with lymphoid organ atrophy; and late-MDV-IS occurring once MDV enters latency and during tumour development. Our objectives were to document the impact of late-MDV-IS on commercial poultry (meat-type chickens bearing MAb against MDV and that were vaccinated or unvaccinated against MD) and to optimize a model to study late-MDV-IS under laboratory conditions. The impact of late-MDV-IS was evaluated by assessing the effect of early infection (day of age) with a very virulent plus MDV (vv+MDV) on the efficacy of chicken-embryo-origin (CEO) infectious laryngotracheitis (ILT) virus vaccine against ILT challenge. The CEO ILT vaccine was administered in water at 14 days of age and ILT virus (ILTV) challenge was done intratracheally at 30 days of age. Development of ILT was monitored by daily evaluation of clinical signs, development of gross and histological lesions in trachea, and quantification of ILTV transcripts in trachea. Infection with vv+MDV strain 648A resulted in total abrogation of protection conferred by the CEO vaccine against ILTV challenge even in chickens vaccinated at 1 day of age with either HVT, HVT+SB-1, or CVI988. Chickens exposed to vv+MDV prior to vaccination with CEO ILTV vaccine had similar (P < 0.05) clinical scores, gross lesions, histopathologic lesion scores, and load of ILTV transcripts in trachea after ILTV challenge, as chickens that were not vaccinated with CEO ILTV vaccine.

Marek\'s disease virus (MDV) is an alpha-herpesvirus causing Marek\'s disease in chickens, mostly associated with T-cell lymphoma. VP22 is a tegument protein abundantly expressed in cells during the lytic cycle, which is essential for MDV spread in culture. Our aim was to generate a pathogenic MDV expressing a green fluorescent protein (EGFP) fused to the N-terminus of VP22 to better decipher the role of VP22 in vivo and monitor MDV morphogenesis in tumors cells. In culture, rRB-1B EGFP22 led to 1.6-fold smaller plaques than the parental virus. In chickens, the rRB-1B EGFP22 virus was impaired in its ability to induce lymphoma and to spread in contact birds. The MDV genome copy number in blood and feathers during the time course of infection indicated that rRB-1B EGFP22 reached its two major target cells, but had a growth defect in these two tissues. Therefore, the integrity of VP22 is critical for an efficient replication in vivo, for tumor formation and horizontal transmission. An examination of EGFP fluorescence in rRB-1B EGFP22-induced tumors showed that about 0.1% of the cells were in lytic phase. EGFP-positive tumor cells were selected by cytometry and analyzed for MDV morphogenesis by transmission electron microscopy. Only few particles were present per cell, and all types of virions (except mature enveloped virions) were detected unequivocally inside tumor lymphoid cells. These results indicate that MDV morphogenesis in tumor cells is more similar to the morphorgenesis in fibroblastic cells in culture, albeit poorly efficient, than in feather follicle epithelial cells.

Marek\'s disease virus (MDV), a poultry pathogen, has been increasing in virulence since the mid twentieth century. Since multiple vaccines have been developed and widely implemented, losses due to MDV have decreased. However, vaccine failure has occurred in the past and vaccine breakthroughs remain a problem. Failure of disease control with current vaccines would have significant economic and welfare consequences. Nevertheless, the epidemiology of the disease during a farm outbreak is not well understood. Here we present a mathematical model to predict the effectiveness of vaccines to reduce the outbreak probability and disease burden within a barn. We find that the chance of an outbreak within a barn increases with the virulence of an MDV strain, and is significantly reduced when the flock is vaccinated, especially when there the contaminant strain is of low virulence. With low quantities of contaminated dust, there is nearly a 100% effectiveness of vaccines to reduce MDV outbreaks. However, the vaccine effectiveness drops to zero with an increased amount of contamination with a middle virulence MDV strain. We predict that the larger the barn, and the more virulent the MDV strain is, the more virus is produced by the time the flock is slaughtered. With the low-to-moderate virulence of the strains studied here, the number of deaths due to MDV is very low compared to all-cause mortality regardless of the vaccination status of the birds. However, the cumulative MD incidence can reach 100% for unvaccinated cohorts, and 35% for vaccinated cohorts. These results suggest that death due to MDV is an insufficient metric to assess the prevalence of MDV broiler barns regardless of vaccine status, such that active surveillance is required to successfully assess the probability of MDV outbreaks, and to limit transmission of MDV between successive cohorts of broiler chickens.

A genome-wide association study (GWAS) using Bayesian variable selection was performed to determine genomic regions associated with mortality due to Marek\'s disease virus (MDV) infection in layers. Mortality (%) under experimental disease challenge (500 plaque-forming units of a very virulent plus MDV strain) was recorded for progeny groups (average 15.5 birds; range 3 to 30) of 253 genotyped sires from four generations of a brown-egg layer line. An additional generation of 43 sires with progeny data was used to validate results. Sires were genotyped with a 42K Illumina single-nucleotide polymorphism (SNP) chip. Methods BayesB (pi = 0.995) and BayesCpi, with or without weighting residuals by the size of progeny groups were applied. The proportion of genetic variance contributed by SNPs within each 1-megabase (Mb) genomic region was quantified. Average mortality was 33% but differed significantly between generations. Genetic markers explained about 11% of phenotypic variation in mortality. Correlations between genomic estimated breeding values and percentage of progeny mortality for the validation generation (sons of individuals in training) were 0.12, 0.17, 0.02, and 0.16 for BayesB, weighted BayesB, BayesCpi, and weighted BayesCpi, respectively, when using the whole genome, and 0.03, 0.20, -0.06, and 0.14, when using only SNP from the 10, 1-Mb regions, explaining the largest proportion of genetic variance according to each method. Results suggest that regions on chromosomes 2, 3, 4, 9, 15, 18, and 21 are associated with Marek\'s disease resistance and can be used for selection and that accounting for the size of progeny groups has a large impact on correct localization of such genomic regions.

Transcription of cytokine genes was evaluated in the lung and spleen of chickens vaccinated with various serotype 1 Marek\'s disease (MD) vaccines. Three vaccine pairs/series, each consisting of one or two high protective (HP) and one low protective (LP) vaccine, were used. Vaccinated chickens had increased transcripts of IFN-γ in the lung and spleen at 3, 5, and 10 days post vaccination (dpv) compared to unvaccinated control chickens. In addition, transient increase of cytokine transcripts (iNOS, IL-1β, IL-18, IL-8, and IL-6 in the lung and iNOS, IL-18, and IL-6 in the spleen) was detected. Compared to chickens vaccinated with LP vaccines, HP vaccinated chickens had increased transcripts of iNOS at 5 dpv but decreased transcripts of IL-6, IL-8, and IL-18 at 10 dpv in the lung. HP vaccinated chickens had increased IFN-γ in the spleen at 3 and at 10 dpv. This study demonstrated that MD vaccines administered subcutaneously elicit a pulmonary immune response and identified differences in the cytokine gene expression between HP and LP vaccinated chickens.

Chicken anaemia virus (CAV) is the most important confounding pathogen in Marek\'s disease virus (MDV) infection. The effect of CAV co-infection at 4 weeks of age after inoculation of virulent MDV (vMDV, KS strain) or very virulent MDV (vvMDV, Md/5 strain) in 1-day-old chicks was investigated by pathological and immunohistochemical studies. CAV increased the mortality rates induced by vMDV or vvMDV. The packed cell volume was reduced significantly in vMDV-CAV infection; however, no reduction or non-significant reduction was observed in vMDV infection. Bone marrow hypoplasia was related to CAV co-infection and none of the birds inoculated with vMDV or vvMDV had hypoplasia. Severe atrophy of the thymus and bursa of Fabricius was observed in the vvMDV-CAV and vvMDV groups. Complete regeneration of the thymus cortex and bursa of Fabricius in the vMDV group was noted and was in contrast to sequential lymphoid depletion after CAV inoculation in the vMDV-CAV group. The spleen was either regenerated, lymphoid depleted or had lymphoproliferative lesions. Lymphoid depletion in the spleen was not detected in the vMDV group; however, it was prominent in the vMDV-CAV and vvMDV-CAV groups during the first 2 weeks after CAV inoculation. CAV inclusions and antigens were detected in the thymus cortex and spleen of vMDV-CAV and vvMDV-CAV groups during the experiment. Severe depletion of CD8(+) T cells was observed in depleted spleen and thymus. The neoplastic foci appeared around splenic arterioles and venules, and stained mainly by CD4 antibody; however, CD8(+) T cells were singly dispersed or were present in clusters. It could be concluded that CAV was responsible for bone marrow hypoplasia, severe anaemia and hindrance of lymphoid organ regeneration in MDV-CAV co-infection.