Marek's disease virus encoded miR-M6 and miR-M10 are dispensable for virus replication and pathogenesis in chickens.

MicroRNAs (miRNAs) are a class of approximately 22 nucleotides long non-coding RNAs, and virus-encoded miRNAs play an important role in pathogenesis. Marek's disease virus (MDV) is an oncogenic avian alphaherpesvirus that causes immunosuppression and tumors in its natural host, chicken. In the MDV genome, 14 miRNA precursors and 26 mature miRNAs were identified, thus MDV has been used as a model to study the function of viral miRNAs in vivo. Recently, a cluster of miRNAs encoded by MDV, Cluster 3 miRNAs (miR-M8-M10), has been shown to restrict early cytolytic replication and pathogenesis of MDV. In this study, we further analyzed the role of miR-M6 and miR-M10, members of cluster miR-M8-M10, in MDV replication and pathogenicity. We found that, compared to parental MDV, deletion of miR-M6-5p significantly enhanced the replication of MDV in cell culture, but not in chickens. The replication of miR-M6-5p deletion MDV was restored once the deleted sequences were re-inserted. Our results also showed that deletion of miR-M10-5p did not affect the replication of MDV in vitro and in vivo. In addition, our animal study results showed that deletion of miR-M6-5p or miR-M10-5p did not alter the pathogenesis of MDV. In conclusion, our study shows that both miR-M6 and miR-M10 are dispensable for MDV replication and pathogenesis in chickens, while also suggests a repressive role of miR-M6 in MDV replication in cell culture.

Adiponectin and its receptor genes' expression in response to Marek's disease virus infection of White Leghorns.

Marek's disease virus (MDV) causes T-cell lymphoma in susceptible chicken and is also related to an imbalance of the lipid metabolism. Adiponectin is a circulatory cytokine secreted from adipose tissue and exerts critical metabolic functions. Although the associations between adiponectin and diseases, including lipid disorder and noncardiac vascular diseases, have been reported, little is known about the relationship between MDV infection and adiponectin. Here, we challenged white Leghorns from Marek's disease (MD)-susceptible and MD-resistant lines with MDV at 7 D of age and then explored the body weight and plasma lipoprotein levels at 21 D after MDV infection. Meanwhile, adiponectin and the expression of its receptors were detected using quantitative real-time PCR and Western blot. The results showed that MDV infection induced body weight loss in all the experimental birds. Meanwhile, the concentrations of total cholesterol and high-density lipoprotein were lower after the infection, although there was no significant difference (P > 0.05). However, the infection did not affect adiponectin circulating levels in plasma. MD-susceptible birds had much lower plasma adiponectin than MD-resistant birds (P < 0.01). In abdominal fat, there was no significant difference in adiponectin mRNA level. Still, we observed a significant decrease in adiponectin protein concentration, as well as adipoR1 and adipoR2, at both mRNA and protein levels in the infected compared with the noninfected MD-susceptible chickens. In the spleen, MDV infection significantly reduced the adiponectin mRNA expression but increased the protein in MD-susceptible chickens, which decreased both adipoR1 mRNA expression and protein levels. Also interestingly, the adipoR1 mRNA expression level was significantly increased in MD-susceptible chickens in the liver after MDV infection. All findings in the present study provided interesting insights into adiponectin metabolism in chickens after MDV infection, which helps to advance the understanding of lipid metabolism in response to herpesvirus infection.

Neurolymphomatosis in non-Hodgkin lymphoma with cranial multineuritis: A case report.

RATIONALE: Neurolymphomatosis (NL) is a rare syndrome of lymphoma and leukemic infiltration of cranial or peripheral nerves. PATIENT CONCERNS: We report a case of non-Hodgkin Lymphoma (NHL) in a 24-year-old man presented with difficulty in swallowing, hypersalivation, hoarseness, ptosis, facial paralysis, and facial hypoesthesia associated with NL. DIAGNOSIS: NL was diagnosed based upon cranial magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) examination. INTERVENTIONS: The patient was treated with intrathecal methotreaxate (12.5 mg) and cytosine arabinoside (70 mg), systemic high-dose methotrexate therapy, and cranial radiotherapy. OUTCOME: Due to the deterioration of general condition of the patient, he was admitted to intensive care unit, but died 22 days after the onset of symptoms in spite of aggressive treatment. LESSONS: In this case, we present a patient with T cell lymphoma and multineuritis of NL diagnosed by MRI and as far as we know, this is the first reported case in which so many cranial nerves (3, 5, 7, 8, 9, and 10 th) were involved. Briefly, in a patient with hematologic malignancy and neurological complaints, NL should be considered. Early and effective use of imaging modalities such as positron emission tomography (PET-CT), MRI, and aggressive therapies are important for prolonged survival.

Atrophy of primary lymphoid organs induced by Marek's disease virus during early infection is associated with increased apoptosis, inhibition of cell proliferation and a severe B-lymphopenia.

Marek's disease is a multi-faceted highly contagious disease affecting chickens caused by the Marek's disease alphaherpesvirus (MDV). MDV early infection induces a transient immunosuppression, which is associated with thymus and bursa of Fabricius atrophy. Little is known about the cellular processes involved in primary lymphoid organ atrophy. Here, by in situ TUNEL assay, we demonstrate that MDV infection results in a high level of apoptosis in the thymus and bursa of Fabricius, which is concomitant to the MDV lytic cycle. Interestingly, we observed that in the thymus most of the MDV infected cells at 6 days post-infection (dpi) were apoptotic, whereas in the bursa of Fabricius most of the apoptotic cells were uninfected suggesting that MDV triggers apoptosis by two different modes in these two primary lymphoid organs. In addition, a high decrease of cell proliferation was observed from 6 to 14 dpi in the bursa of Fabricius follicles, and not in the thymus. Finally, with an adapted absolute blood lymphocyte count, we demonstrate a major B-lymphopenia during the two 1st weeks of infection, and propose this method as a potent non-invasive tool to diagnose MDV bursa of Fabricius infection and atrophy. Our results demonstrate that the thymus and bursa of Fabricius atrophies are related to different cell mechanisms, with different temporalities, that affect infected and uninfected cells.

Patient With 2 Hematologic Malignancies Presenting as Neurolymphomatosis.

Peripheral nervous system damage from hematologic malignancies is related to neoplastic cells infiltration of peripheral nerves or to monoclonal antibody production cross-reacting with peripheral nerves' antigens. Neurolymphomatosis (NL), a rare manifestation of hematologic malignancies, occurs when malignant cells invade the peripheral nerves leading to various manifestations. Here, we report a case of NL with 2 hematologic malignancies in a 79-year-old woman presenting with lower extremity pain/weakness. Investigation revealed anemia, IgM kappa monoclonal gammopathy, and elevated anti-MAG titer. Electrodiagnostic studies were consistent with mononeuropathy multiplex while imaging suggested malignancy in her ovaries and right S1 nerve root. Bone marrow and ovarian biopsies revealed chronic myelomonocytic leukemia, Waldenstrom macroglobulinemia, and diffuse large B-cell lymphoma. She received standard chemotherapy resulting in radiographic resolution of disease and symptomatic relief. NL can precede the diagnosis of hematologic malignancy but its symptoms are not easily identifiable, whereas management depends on the treatment of the underlying tumor.

Induction of DNA Damages upon Marek's Disease Virus Infection: Implication in Viral Replication and Pathogenesis.

Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that infects chickens and causes a deadly neoplastic disease. We previously demonstrated that MDV infection arrests cells in S phase and that the tegument protein VP22 plays a major role in this process. In addition, expression of VP22 induces double-strand breaks (DSBs) in the cellular DNA, suggesting that DNA damage and the associated cellular response might be favorable for the MDV life cycle. Here, we addressed the role of DNA damage in MDV replication and pathogenesis. We demonstrated that MDV induces DSBs during lytic infection in vitro and in the peripheral blood mononuclear cells of infected animals. Intriguingly, we did not observe DNA damage in latently infected MDV-induced lymphoblastoid cells, while MDV reactivation resulted in the onset of DNA lesions, suggesting that DNA damage and/or the resulting DNA damage response might be required for efficient MDV replication and reactivation. In addition, reactivation was significantly enhanced by the induction of DNA damage using a number of chemicals. Finally, we used recombinant viruses to show that VP22 is required for the induction of DNA damage in vivo and that this likely contributes to viral oncogenesis.IMPORTANCE Marek's disease virus is an oncogenic alphaherpesvirus that causes fatal T-cell lymphomas in chickens. MDV causes substantial losses in the poultry industry and is also used in small-animal models for virus-induced tumor formation. DNA damage not only is implicated in tumor development but also aids in the life cycle of several viruses; however, its role in MDV replication, latency, and reactivation remains elusive. Here, we demonstrate that MDV induces DNA lesions during lytic replication in vitro and in vivo DNA damage was not observed in latently infected cells; however, it was reinitiated during reactivation. Reactivation was significantly enhanced by the induction of DNA damage. Recombinant viruses that lacked the ability to induce DNA damage were defective in their ability to induce tumors, suggesting that DNA damage might also contribute to cellular transformation processes leading to MDV lymphomagenesis.

In vivo characterisation of two Australian isolates of Marek's disease virus including pathology, viral load and neuropathotyping based on clinical signs.

OBJECTIVE: To evaluate the pathogenicity of Australian Marek's disease virus (MDV) isolate MPF23 (1985) against the reference strain MPF57 based on pathology, viral load and neuropathotyping on the basis of clinical signs. PROCEDURE: Two MDV challenge isolates (MPF57 or MPF23) were administered to unvaccinated specific-pathogen free (SPF) layer chicks on day 5 after hatch at three challenge doses (500, 2000 or 8000 plaque-forming units (pfu)/chick). Mortality, body weight, immune organ weights, MDV load in peripheral blood lymphocytes (PBL) and clinical signs were measured to 56 days post challenge (dpc). RESULTS: MPF23 was the more pathogenic of the two viruses, inducing higher mortality (81% vs 62%) and incidence of MD lesions (100% vs 76%). MPF23 induced earlier, more sustained and more severe neurological signs in the period 26-56 dpc. However, there were few differences during the 0-23 dpc used in the neuropathotyping classification under test. The observed pattern during this earlier period classified both viruses as neuropathotype B, consistent with a very virulent pathotype. MDV load in PBL at 7 and 44 dpc did not differ between virus isolates, but the load at 7 dpc was significantly and negatively associated with time to euthanasia or death. CONCLUSION: MPF23 appears to be as, or more, virulent than the MDV strains isolated over the subsequent two decades. The neuropathotyping system developed in the USA did not clearly differentiate between the two isolates under test; however, extension of the period of assessment of clinical signs beyond 26 dpc did reveal clear differences.

Temporal expression and DNA hypomethylation profile of CD30 in Marek's disease virus-infected chicken spleens.

Marek's disease (MD) is a viral neoplastic disease of chickens caused by Marek's disease virus (MDV), which is serious threat to worldwide poultry industry. Our previous studies showed that the CD30 gene was hypomethylated in MD lymphoma. In this study, we further analyzed differential expression patterns and methylation levels of the CD30 gene between MDV-infected and noninfected spleens at 4, 7, 14, 21, and 28 d postinfection (dpi). The results showed that the expression of CD30 in MDV-infected spleens was significantly lower than that in noninfected spleens at 4 dpi. The expression of CD30 did not present significant difference between MDV-infected and noninfected spleens at 7 and 14 dpi. However, an increased expression of CD30 was presented in MDV-infected spleens at both 21 and 28 dpi. Simultaneously, CD30 showed a lower DNA methylation level in MDV-infected spleens at 14, 21, and 28 dpi. The results indicated that CD30 gene was involved in the whole process of MD tumorigenesis and upregulated expression of CD30 in MDV-infected spleens might be attributed to the hypomethylation of promoter of CD30 gene.

Correlation between Marek's disease virus pathotype and replication.

Marek's disease (MD) virus (MDV) is an alphaherpesvirus that causes MD, a lymphoproliferative disease in chickens. Pathotyping has become an increasingly important assay for monitoring shifts in virulence of field strains; however, it is time-consuming and expensive, and alternatives are needed to provide fast answers in the face of current outbreaks. The purpose of this study was to determine whether differences in virus replication between pathotypes that have been reported using a small number of virulent (v) and very virulent plus (vv+) MDV strains could be confirmed with a large collection of MD viruses. Based on pilot study data, bursa, brain, and lung samples were collected at 9 and 11 days postinoculation (dpi) from birds challenged with 1 of 15 MDV strains. The correlation between virus replication and virulence was confirmed between vMDV strains and higher virulent strains, but in most cases, there was no significant difference between very virulent (vv) and vv+MDV groups. At both 9 and 11 dpi, chickens infected with vv and vv+MDV had significantly lower body weights and relative thymus and bursa weights compared with chickens challenged with vMDV. However, similar to virus quantity, there was no significant difference between weights in birds challenged with vv or vv+MDV. The significant differences observed in maternal antibody negative (ab-) chickens were not significant in maternal antibody positive (ab+) chickens, demonstrating the requirement of ab- birds for this type of comparison. These data do not support the use of virus replication or organ weights as an alternative to pathotyping for discrimination between all three virulent MDV pathotypes but may be useful for determining a virus replication threshold to choose which field strains meet a minimum virulence to be pathotyped by traditional methods.

gga-miR-26a targets NEK6 and suppresses Marek's disease lymphoma cell proliferation.

MicroRNA (miRNA) are a class of highly conserved, small noncoding RNA that emerge as key posttranscriptional regulators in various neoplastic transformations. Our previous study profiling the miRNA transcriptome in Marek's disease virus (MDV)-induced lymphoma revealed many novel and differentially expressed miRNA, including gga-miR-26a, which was downregulated in MDV-infected spleens of chickens. In this study, differential expression of gga-miR-26a between MDV-infected and noninfected spleens at 4, 7, 14, 21, and 28 d postinfection was analyzed by real-time PCR. The results showed gga-miR-26a were downregulated in MDV-infected spleens at cytolytic infection, latency, and tumor transformation phases. Subsequent cell proliferation assay revealed cell viability was lower in gga-miR-26a mimic transfection group than that in negative controls. Target genes of gga-miR-26a were identified by luciferase reporter gene assay. The results showed significant interaction between gga-miR-26a and Never In Mitosis Gene A (NIMA)-related kinase 6 (NEK6) gene. Subsequent gain of function experiment and Western blot assay showed that mRNA and protein levels of NEK6 were downregulated after gga-miR-26 mimic was transfected into MDV-transformed lymphoid cell line (MSB-1), indicating that NEK6 was modulated by gga-miR-26a. The expression of NEK6 showed a higher trend in MDV-infected samples including tumorous spleen and MD lymphoma from liver than that in noninfected controls. The results suggested that gga-miR-26a inhibited MSB-1 cell proliferation. Gga-miR-26a and its direct target, NEK6, might play important roles in MDV infection.

Marek's disease virus and skin interactions.

Marek's disease virus (MDV) is a highly contagious herpesvirus which induces T-cell lymphoma in the chicken. This virus is still spreading in flocks despite forty years of vaccination, with important economical losses worldwide. The feather follicles, which anchor feathers into the skin and allow their morphogenesis, are considered as the unique source of MDV excretion, causing environmental contamination and disease transmission. Epithelial cells from the feather follicles are the only known cells in which high levels of infectious mature virions have been observed by transmission electron microscopy and from which cell-free infectious virions have been purified. Finally, feathers harvested on animals and dust are today considered excellent materials to monitor vaccination, spread of pathogenic viruses, and environmental contamination. This article reviews the current knowledge on MDV-skin interactions and discusses new approaches that could solve important issues in the future.

Very virulent plus strains of MDV induce an acute form of transient paralysis in both susceptible and resistant chicken lines.

Marek's disease (MD) is a lymphoproliferative disease of domestic chickens caused by a highly cell-associated alpha herpesvirus, Marek's disease virus (MDV). Clinical signs of MD include depression, crippling, weight loss, and transient paralysis (TP). TP is a disease of the central nervous system that affects MD-susceptible chickens 8-11 days post-infection (dpi), normally resulting in recovery 1-3 d after the onset of clinical signs. In this study we inoculated chickens from lines 7(2) (MD-susceptible) and 6(3) (MD-resistant) with a very virulent plus strain of MDV at 2 wk of age, and collected brain samples from birds with and without TP at 5, 11, and 21 dpi for gene expression profiling and histological analysis. Data revealed that chickens inoculated with MDV had higher levels of IL-6, IL-10, IL-18, IFN-α, IFN-ß, IFN-γ, MHC I, and CD18 in their brains at 11 dpi compared to the uninfected control birds. In addition, the expression levels of IL-6, IL-10, IFN-α, IFN-ß, and IFN-γ were significantly higher in the brains of the birds showing clinical signs of TP than in asymptomatic chickens. Comparative analysis between the two chicken lines showed that the expression levels of IL-6, IL-10, IFN-ß, IFN-γ, IL-18, CD18, and MHC I were significantly higher in the brains of the birds from line 6(3) with TP than those of line 7(2) exhibiting neurological disorders. A differential expression pattern was observed for some of the tested genes at different time points post-inoculation. Histological analysis showed lymphocytic meningitis, perivascular cuffing, and neuronal degeneration within the brains of birds from both susceptible and resistant lines exhibiting TP at 11 dpi. Vaccination prevented development of TP and other MD-associated clinical symptoms. These observations are suggestive of an underlying immunological mechanism for viral-induced neurological dysfunction, and the differential responses of the two chicken lines to MDV infection.

Detection of virulent Marek's disease virus in poultry in India.

Marek's disease virus (MDV) isolated from poultry flocks in three states of India was monitored for the virus occurrence in the field. The MDV genome was isolated from feather follicles, spleen, and liver of the chicken (173 samples). Twenty two samples were positive for MDV genome in PCR and belonged to the serotype 1. The sequencing of MEQ gene of 11 samples revealed that nucleotide sequences of the isolate Ind-KA-01-06 was similar to the very virulent MDV, strain RB-1B. In situ hybridization studies also confirmed a presence of MDV serotype 1 in the infected liver tissues. Furthermore, the ability of the virus to induce apoptosis detected by flow cytometry showed that the virulent MDV induced apoptosis more efficiently than Turkey herpesvirus (HVT) vaccine virus. The present study showed the presence of virulent/very virulent MDV strains in the Indian poultry flocks.

Marek's disease virus: from miasma to model.

Marek's disease virus (MDV) is an oncogenic herpesvirus that causes various clinical syndromes in its natural host, the chicken. MDV has long been of interest as a model organism, particularly with respect to the pathogenesis and immune control of virus-induced lymphoma in an easily accessible small-animal system. Recent advances in MDV genetics and the determination of the chicken genome sequence, aided by functional genomics, have begun to dramatically increase our understanding not only of lytic MDV replication, but also of the factors and mechanisms leading to latency and tumour formation. This new information is helping to elucidate cellular signalling pathways that have undergone convergent evolution and are perturbed by different viruses, and emphasizes the value of MDV as a comparative biomedical model. Furthermore, the door is now open for rational and efficient engineering of new vaccines against one of the most important and widespread infectious diseases in chickens.

Marek's disease virus-encoded vIL-8 gene is involved in early cytolytic infection but dispensable for establishment of latency.

Marek's disease, a lymphoproliferative disease of chickens, is caused by an alphaherpesvirus, Marek's disease virus (MDV). This virus encodes a virokine, vIL-8, with general homology to cellular CXC chemokines such as interleukin-8 (IL-8) and Gro-alpha. To study the function of vIL-8 gene, we deleted both copies of vIL-8 residing in the terminal repeat long and internal repeat long region of the viral genome and generated a mutant virus with vIL-8 deleted, rMd5/DeltavIL-8. Growth kinetics study showed that vIL-8 gene is dispensable for virus replication in cell culture. In vivo, the vIL-8 gene is involved in early cytolytic infections in lymphoid organs, as evidenced by limited viral antigen expression of rMd5/DeltavIL-8. However, the rMd5/DeltavIL-8 virus is unimpaired in virus replication in the feather follicle epithelium. vIL-8 does not appear to be important for establishment of latency, since rMd5/DeltavIL-8 and the wild-type virus have similar viremia titers at 14 days postinfection, a period when the virus titer comes primarily from reactivated latent genomes. Nevertheless, because of the impaired cytolytic infections, the overall transformation efficiency of the virus with vIL-8 deleted is much lower, as reflected by the reduced number of transformed cells at 5 weeks postinoculation and the presence of fewer gross tumors. Importantly, the revertant virus that restored the expression of vIL-8 gene also restored the wild-type phenotype, indicating the deficient phenotypes are results of vIL-8 deletion. One of the interesting differences between the MDV vIL-8 gene and its cellular counterpart is the presence of a DKR (Asp-Lys-Arg) motif instead of ELR (Glu-Leu-Arg) preceding the invariable CXC motif. To study the significance of this variation, we generated recombinant MDV, rMd5/vIL-8-ELR, carrying the ELR motif. Both in vitro and in vivo studies revealed that the DKR motif is as competent as ELR in pathogenesis of MDV.

Expansion of a unique region in the Marek's disease virus genome occurs concomitantly with attenuation but is not sufficient to cause attenuation.

Pathogenic Marek's disease viruses (MDVs) have two head-to-tail copies of a 132-bp repeat. As MDV is serially passaged in cell culture, the virus becomes attenuated and the number of copies of the 132-bp repeat increases from 2 to often more than 20 copies. To determine the role of the repeats in attenuation, we used five overlapping cosmid clones that spanned the MDV genome to reconstitute infectious virus (rMd5). By mutating the appropriate cosmids, we generated clones of infectious MDVs that contained zero copies of the 132-bp repeats, rMd5(Delta132); nine copies of the 132-bp repeats, rMd5(9-132); and nine copies of the 132-bp repeats inserted in the reverse orientation, rMd5(rev9-132). After two passages in cell culture, wild-type Md5, rMd5, and rMd5(Delta132) were stable. However, rMd5(9-132) and rMd5(rev9-132) contained a population of viruses that contained from 3 to over 20 copies of the repeats. A major 1.8-kb mRNA, containing two copies of the 132-bp repeat, was present in wild-type Md5 and rMd5 but was not present in rMd5(Delta132), rMd5(9-132), rMd5(rev9-132), or an attenuated MDV. Instead, the RNAs transcribed from the 132-bp repeat region in rMd5(9-132) and rMd5(rev9-132) closely resembled the pattern of RNAs transcribed in attenuated MDVs. When inoculated into susceptible day-old chicks, all viruses produced various lesions. Thus, expansion of the number of copies of 132-bp repeats, which accompanies attenuation, is not sufficient in itself to attenuate pathogenic MDVs.

An acute form of transient paralysis induced by highly virulent strains of Marek's disease virus.

A novel syndrome was observed after inoculation of 3-wk-old chickens with highly virulent Marek's disease virus (MDV) strains. This syndrome was characterized by the acute onset of neurologic signs including flaccid paralysis of neck and limbs 9-10 days postinoculation, typically resulting in death 1-3 days after the onset of clinical signs. Most affected birds died, and spontaneous recovery was rare. Few if any gross tissue changes were found. Histologic brain lesions included acute vasculitis with vasogenic edema and perivascular cuffing. The syndrome was influenced by the virus strain and dose and by chicken strain and B haplotype and was prevented by vaccination with turkey herpesvirus. Chickens up to 18 wk of age were susceptible. On the basis of clinical signs and histopathology, the syndrome was determined to be an acute form of transient paralysis (TP); its more acute nature and virtual lack of spontaneous recovery differentiated this syndrome from classical TP. Affected birds were viremic, and brains were positive for viral DNA by polymerase chain reaction assays, but these tests were also positive in inoculated chickens without clinical signs and may have limited value for diagnosis. Although acute TP should occur only rarely in Marek's disease-vaccinated commercial flocks, this syndrome may be important in laboratory studies, where it could interfere with pathogenesis trials. Finally, acute TP appears to be one component in the pathogenesis of the early mortality syndrome, a previously described immunodepressive disease induced by inoculation of 1-day-old chicks with highly virulent MDV.