Pathogen transmission from vaccinated hosts can cause dose-dependent reduction in virulence.

Many livestock and human vaccines are leaky because they block symptoms but do not prevent infection or onward transmission. This leakiness is concerning because it increases vaccination coverage required to prevent disease spread and can promote evolution of increased pathogen virulence. Despite leakiness, vaccination may reduce pathogen load, affecting disease transmission dynamics. However, the impacts on post-transmission disease development and infectiousness in contact individuals are unknown. Here, we use transmission experiments involving Marek disease virus (MDV) in chickens to show that vaccination with a leaky vaccine substantially reduces viral load in both vaccinated individuals and unvaccinated contact individuals they infect. Consequently, contact birds are less likely to develop disease symptoms or die, show less severe symptoms, and shed less infectious virus themselves, when infected by vaccinated birds. These results highlight that even partial vaccination with a leaky vaccine can have unforeseen positive consequences in controlling the spread and symptoms of disease.

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.

Plexus and peripheral nerve metastasis.

Cancer in the form of solid tumors, leukemia, and lymphoma can infiltrate and metastasize to the peripheral nervous system, including the cranial nerves, nerve roots, cervical, brachial and lumbosacral plexuses, and, rarely, the peripheral nerves. This review discusses the presentation, diagnostic evaluation, and treatment options for metastatic lesions to these components of the peripheral nervous system and is organized based on the anatomic distribution. As skull base metastases (also discussed in Chapter 14) result in cranial neuropathies, these will be covered in detail, as well as cancers that directly infiltrate the cranial nerves. Particular emphasis is placed on the clinical, imaging, and electrodiagnostic features that differentiate neoplastic plexopathies from radiation-induced plexopathies. Neurolymphomatosis, in which malignant lymphocytes invade the cranial nerves, nerve roots, brachial and lumbosacral plexuses, and peripheral nerves, is a rare manifestation of lymphoma and leukemia. Diagnoses of neurolymphomatosis are often missed or delayed given its varied presentations, resulting in poorer outcomes. Thus this disease will also be discussed in depth.

Long intergenic non-coding RNA GALMD3 in chicken Marek's disease.

Long intergenic non-coding RNAs (lincRNAs) are transcribed from non-coding DNA sequences. Studies have revealed that aberrant expressions of lincRNAs are associated with various types of cancers and neurological disorders. Marek's disease (MD) is a highly contagious T-cell lymphoid neoplasia of chicken induced by Marek's disease virus (MDV). In this study, we first identified and validated linc-GALMD3 highly expressed in MDV-infected CD4+ T cells by RNA-Seq and qRT-PCR. By RNA-Seq analysis in MDCC-MSB1 cells after loss of function of linc-GALMD3 by shRNA, we found that linc-GALMD3 could positively cis-regulate its downstream gga-miR-223 gene expression. In contrast, it could trans-regulate the 748 differentially expressed genes (FDR < 0.01) that were mainly enriched into mitochondrial structure and cell cycle processes using GO analysis. Of these, the most significantly expressed gene EPYC might cause iris lesion in MD. The other eight genes, NDUFA4, NDUFB6, NDUFV1, NDUFS8, SDHB, UQCRC1, UQCRC2, and COX7A2, actively participated in oxidative phosphorylation in mitochondrial dysfunction and cell death. Most importantly, we found that the MDV replication was repressed when linc-GALMD3 was knocked down in CEF cells. Our results suggested that linc-GALMD3 might be a critical regulator in chicken MD and could be used as a candidate-promising mark for MD prevention, diagnosis, and treatment.

Splenic marginal zone lymphoma: An indolent malignancy leading to the development of neurolymphomatosis.

INTRODUCTION: Acute neuropathic pain and weakness with a sensory level in a patient with a history of lymphoma has a broad differential diagnosis. Evaluation of such a presentation often includes MRI, neurophysiologic studies, and cerebrospinal fluid evaluation. We report a patient with splenic marginal zone lymphoma who developed acute weakness, sensory loss, and neuropathic pain due to neurolymphomatosis. METHODS: Clinical evaluation, MRI of the lumbar spine, cerebrospinal fluid evaluation, electrodiagnostic (EDx) studies, and biopsy of a dorsal nerve root were undertaken. RESULTS: EDx studies were consistent with an acute, acquired demyelinating sensorimotor polyradiculoneuropathy. Treatment with intravenous immunoglobulin and plasma exchange did not lead to clinical improvement. Ultimately, biopsy of a dorsal nerve root was performed and revealed neurolymphomatosis. CONCLUSION: This case emphasizes that, when it can be performed safely, biopsy for suspected neurolymphomatosis is imperative for appropriate diagnosis and treatment. Muscle Nerve 55: 440-444, 2017.

Chicken gga-miR-103-3p Targets CCNE1 and TFDP2 and Inhibits MDCC-MSB1 Cell Migration.

Marek's disease (MD) is a highly contagious viral neoplastic disease caused by Marek's disease virus (MDV), which can lead to huge economic losses in the poultry industry. Recently, microRNAs (miRNAs) have been found in various cancers and tumors. In recent years, 994 mature miRNAs have been identified through deep sequencing in chickens, but only a few miRNAs have been investigated further in terms of their function. Previously, gga-miR-103-3p was found downregulated in MDV-infected samples by using Solexa deep sequencing. In this study, we further verified the expression of gga-miR-103-3p among MDV-infected spleen, MD lymphoma from liver, noninfected spleen, and noninfected liver, by qPCR. The results showed that the expression of gga-miR-103-3p was decreased in MDV-infected tissues, which was consistent with our previous study. Furthermore, two target genes of gga-miR-103-3p, cyclin E1 (CCNE1) and transcription factor Dp-2 (E2F dimerization partner 2) (TFDP2), were predicted and validated by luciferase reporter assay, qPCR, and western blot analysis. The results suggested that CCNE1 and TFDP2 are direct targets of gga-miR-103-3p in chickens. Subsequent cell proliferation and migration assay showed that gga-miR-103-3p suppressed MDCC-MSB1 migration, but did not obviously modulate MDCC-MSB1 cell proliferation. In conclusion, gga-miR-103-3p targets the CCNE1 and TFDP2 genes, and suppresses cell migration, which indicates that it might play an important role in MD tumor transformation.

Growth inhibitive effect of betulinic acid combined with tripterine on MSB-1 cells and its mechanism.

Marek's disease (MD), a highly infectious lymphoproliferative disease in chickens, is caused by a cell-associated oncogenic herpesvirus, Marek's disease virus (MDV). MSB-1 is a MD-derived lymphoblastoid cell line and can induce tumors when inoculated into susceptible chickens. Betulinic acid, which is present as one of the major effective components in many traditional Chinese medicines, has recently been reported to inhibit growth of cancer cells and employed as a potential anticancer agent. Tripterine, a major active compound extracted from the Chinese herb Tripterygium wilfordii Hook F, has now also shown anti-tumor activities in various cancers. The aim of this study was to investigate the synergistic growth-inhibitive effect of betulinic acid combined with tripterine on MSB-1 cells and its mechanism. Viability of MSB-1 cells was assessed by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) method. Cell apoptotic analysis was performed by fluorescence detection. NF-κB transcription activity was detected by measuring luciferase activity. Western blotting was used to analyze the expression of p65, IκB and Meq. Our results showed that the proliferation in the combination group was significantly decreased as compared with that of monotherapy using betulinic acid or tripterine, accompanied by an induction of apoptosis, inhibition of NF-κB transcriptional activity and its targeting oncogenic gene Meq. The results suggest that the combination of betulinic acid and tripterine at lower concentration may produce a synergistic inhibitive effect on MSB-1 cells that warrants further investigation for its potential clinical applications.

Critical role of the virus-encoded microRNA-155 ortholog in the induction of Marek's disease lymphomas.

Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.

High frequency of neurolymphomatosis as a relapse disease of intravascular large B-cell lymphoma.

BACKGROUND: Intravascular large B-cell lymphoma (IVL) is characterized by lymphoma cell proliferation in the lumina of small vessels in various organs. A high incidence of neurologic symptoms associated with the central nervous system has been reported, but peripheral nerve involvement (neurolymphomatosis [NL]) rarely has been described. METHODS: The medical records from patients who were diagnosed with IVL over the past 4 years were reviewed. A diagnosis of NL was made based on the combination of neurologic symptoms and their correspondence with imaging studies, such as magnetic resonance imaging (MRI), (18) F-fluoro-2-deoxy-D-glucose (FDG)-positron emission tomography/computed tomography (PET/CT), and/or the histologic confirmation of lymphoma cells within the peripheral nerves, nerve root/plexuses, or cranial nerves. RESULTS: Four patients with NL were identified among 11 patients who had IVL. All cases of NL occurred as relapsed disease during or shortly after the completion of chemotherapy. Although MRI studies of the brains and whole spines revealed nerve infiltration by gadolinium enhancement in 2 patients, the technology was not sensitive enough to detect such infiltration in the remaining 2 patients. In contrast, FDG-PET/CT studies successfully revealed cranial or peripheral nerve lesions in all 4 patients and was useful for evaluating therapeutic response. Patients received treatment with high-dose methotrexate with or without other systemic chemotherapy, which achieved varied success. Further studies will be needed to determine the optimal treatment. CONCLUSIONS: Considering the rarity of IVL and NL, the current observations suggested that IVL may have a predilection not only for the vessels but also for both the central and peripheral nervous systems.

vLIP, a viral lipase homologue, is a virulence factor of Marek's disease virus.

The genome of Marek's disease virus (MDV) has been predicted to encode a secreted glycoprotein, vLIP, which bears significant homology to the alpha/beta hydrolase fold of pancreatic lipases. Here it is demonstrated that MDV vLIP mRNA is produced via splicing and that vLIP is a late gene, due to its sensitivity to inhibition of DNA replication. While vLIP was found to conserve several residues essential to hydrolase activity, an unfavorable asparagine substitution is present at the lipase catalytic triad acid position. Consistent with structural predictions, purified recombinant vLIP did not show detectable activity on traditional phospholipid or triacylglyceride substrates. Two different vLIP mutant viruses, one bearing a 173-amino-acid deletion in the lipase homologous domain, the other having an alanine point mutant at the serine nucleophile position, caused a significantly lower incidence of Marek's disease in chickens and resulted in enhanced survival relative to two independently produced vLIP revertants or parental virus. These data provide the first evidence that vLIP enhances the replication and pathogenic potential of MDV. Furthermore, while vLIP may not serve as a traditional lipase enzyme, the data indicate that the serine nucleophile position is nonetheless essential in vivo for the viral functions of vLIP. Therefore, it is suggested that this particular example of lipase homology may represent the repurposing of an alpha/beta hydrolase fold toward a nonenzymatic role, possibly in lipid bonding.

Oncogenicity of virulent Marek's disease virus cloned as bacterial artificial chromosomes.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that induces T-cell lymphomas in poultry. We report the construction of bacterial artificial chromosome (BAC) clones of the highly oncogenic RB-1B strain by inserting mini-F vector sequences into the U(S)2 locus. MDV reconstituted from two BAC clones induced rapid-onset lymphomas similar to those induced by the wild-type virus. Virus reconstituted from another BAC clone that showed a 7.7-kbp deletion in the internal and terminal unique long repeat regions was nononcogenic, suggesting that the deleted region may be associated with oncogenicity. The generation of the oncogenic BAC clones of MDV is a significant step in unraveling the oncogenic determinants of this virus.

Marek's disease virus-encoded Meq gene is involved in transformation of lymphocytes but is dispensable for replication.

Marek's disease virus (MDV) causes an acute lymphoproliferative disease in chickens, resulting in T cell lymphomas in visceral organs and peripheral nerves. Earlier studies have determined that the repeat regions of oncogenic serotype 1 MDV encode a basic leucine zipper protein, Meq, which structurally resembles the Jun/Fos family of transcriptional activators. Meq is consistently expressed in MDV-induced tumor cells and has been suggested as the MDV-associated oncogene. To study the function of Meq, we have generated an rMd5DeltaMeq virus by deleting both copies of the meq gene from the genome of a very virulent strain of MDV. Growth curves in cultured fibroblasts indicated that Meq is dispensable for in vitro virus replication. In vivo replication in lymphoid organs and feather follicular epithelium was also not impaired, suggesting that Meq is dispensable for lytic infection in chickens. Reactivation of the rMd5DeltaMeq virus from peripheral blood lymphocytes was reduced, suggesting that Meq is involved but not essential for latency. Pathogenesis experiments showed that the rMd5DeltaMeq virus was fully attenuated in chickens because none of the infected chickens developed Marek's disease-associated lymphomas, suggesting that Meq is involved in lymphocyte transformation. A revertant virus that restored the expression of the meq gene, showed properties similar to those of the parental virus, confirming that Meq is involved in transformation but not in lytic replication in chickens.

Pathogenesis of a Marek's disease virus mutant lacking vIL-8 in resistant and susceptible chickens.

A homologue of interleukin-8, viral interleukin-8 (vIL-8) has been identified in the genome of Marek's disease virus (MDV). This protein attracts peripheral blood mononuclear cells in vitro although its role in the pathogenesis of Marek's disease (MD) is not known. P chickens, genetically susceptible to MD, and N chickens, genetically resistant to the disease, were inoculated with either RB1B MDVor RB1BvIL-8smGFP, a vIL8 knockout RB1B MDV, to assess the role of vIL8 in the pathogenesis of MD. The tumor incidence was highest in the P birds given the RBIB virus, where the incidence was 100%. Tumor incidence in N birds given RB1B was 41.5%. Thirty-one percent of the P birds given RB1BvIL-8smGFP developed tumors, and no N bird given RB1BvIL-8smGFP developed tumors. Histologically, the tumors from RB1B-inoculated birds were larger and more invasive and had a more homogeneous cellular composition than those from RB1BvIL-8smGFP-inoculated birds, which were best described as microtumors. These microtumors did not obliterate the normal architecture of the tissues, and in contrast to the RBIB tumors, moderate numbers of heterophils were admixed with the proliferating lymphocytes. Susceptible birds receiving RB1B had the highest viral titers throughout the study, followed by the resistant birds inoculated with RB1B. P and N birds receiving RB1BvIL-8smGFP virus had consistently lower levels of viremia than their RB1B-inoculated counterparts although virus could be recovered from the birds during all stages of MD. In addition, the RB1BvIL-8smGFP virus was detected in birds held in contact with the inoculated group, although no tumors developed in contact control birds. This result indicates that RB1BvIL-8smGFP replicates in vivo but not as well as RB1B and that vIL8 is not essential for the completion of the pathogenesis of MD.

Risk factors for Marek's disease and mortality in white Leghorns in Norway.

A prospective longitudinal field study was conducted in the period from January 1994 to January 1996 to analyse the relationship between some selected risk factors in the growing and laying periods and (1) the flock-level occurrence of Marek's disease (MD) during the period from 16 to 32 weeks of age and (2) the cumulative mortality during the same period. A total of 171 layer flocks in 102 egg-production farms were included in the statistical analyses.A logistic regression (with strain of layer and vaccination program against MD as fixed effects) of flock-level MD-status during the first 16weeks of the laying period was conducted. Of the risk factors investigated, "multi-age management" and "housing system" were significantly associated at the rearing farm, and "number of hens in each cage" at the egg-production farm. Flocks kept in single-age facilities had a lower risk of MD than flocks housed in farms with multi-age management. The odds of MD were larger for flocks housed on a litter floor in the rearing farms compared to flocks housed in battery cages. At the egg-production stage, flocks kept in battery cages housing more than three hens were at greater risk of MD than those held in cages for three hens or less.A weighted least-squares regression (with strain of layer and flock-level MD-status as fixed effects) of cumulative mortality during the period from 16 to 32weeks of age was also run. The same risk factors (with the same directions of effects) and "size of the rearing farm" were included in the final model of mortality. Chicks reared in medium-sized farms were at higher risk of dying than those coming from either small or large rearing farms. Our results confirm the importance of preventing chicks from being exposed to MD-virus during the rearing period, to reduce the risk of MD-outbreaks (and thereby, mortality losses) during the early stage of the egg-laying period.

Open reading frame L1 of Marek's disease herpesvirus is not essential for in vitro and in vivo virus replication and establishment of latency.

Two mutant CV1988 Marek's disease virus (MDV) strains were developed in which a part of ORF L1 was replaced by lacZ with the SV40 early promoter. These mutant strains, CVIL1LacZ-A and -B, were inoculated into chickens to test the hypothesis that ORF L1 is involved in the induction and/or maintenance of latency. Mutant virus could be reisolated from lymphocytes obtained from chickens during both the lytic and latent phase of infection, indicating that ORF L1 is not essential for the induction and/or maintenance of latency or the reactivation from latency. Beta-galactosidase-positive lymphocytes were detected during the latent infection demonstrating that the SV40 early promoter can be active in recombinant MDV strains during latent infection. Although the insertion of lacZ was stable in cell culture, recombination within lacZ and the BamHI-L fragment was observed during in vivo infection.

Transforming potential of the herpesvirus oncoprotein MEQ: morphological transformation, serum-independent growth, and inhibition of apoptosis.

Marek's disease virus (MDV) induces the rapid development of overwhelming T-cell lymphomas in chickens. One of its candidate oncogenes, meq (MDV Eco Q) which encodes a bZIP protein, has been biochemically characterized as a transcription factor. Interestingly, MEQ proteins are expressed not only in the nucleoplasm but also in the coiled bodies and the nucleolus. Its novel subcellular localization suggests that MEQ may be involved in other functions beyond its transcriptional potential. In this report we show that MEQ proteins are expressed ubiquitously and abundantly in MDV tumor cell lines. Overexpression of MEQ results in transformation of a rodent fibroblast cell line, Rat-2. The criteria of transformation are based on morphological transfiguration, anchorage-independent growth, and serum-independent growth. Furthermore, MEQ is able to distend the transforming capacity of MEQ-transformed Rat-2 cells through inhibition of apoptosis. Specifically, MEQ can efficiently protect Rat-2 cells from cell death induced by multiple modes including tumor necrosis factor alpha, C2-ceramide, UV irradiation, and serum deprivation. Its antiapoptotic function requires new protein synthesis, as treatment with a protein synthesis inhibitor, cycloheximide, partially reversed MEQ's antiapoptotic effect. Coincidentally, transcriptional induction of bcl-2 and suppression of bax are also observed in MEQ-transformed Rat-2 cells. Taken together, our results suggest that MEQ antagonizes apoptosis through regulation of its downstream target genes involved in apoptotic and/or antiapoptotic pathways.

Chicken anaemia virus influences the pathogenesis of Marek's disease in experimental infections, depending on the dose of Marek's disease virus.

Eight groups of 1-day-old chickens were inoculated with 0, 250, 5000, or 100,000 white blood cells of chickens infected with Marek's disease virus strain K (MDV-WBC). Four of these groups were additionally infected with 10(5) TCID50 chicken anaemia virus (CAV). At day 14 after inoculation, chickens infected with CAV had reduced haematocrit levels, reduced body weights, and depletion of the thymic cortex and bone marrow. Semi-quantitative immunohistochemical examination of nerves and visceral organs was performed at day 28 by immunoperoxidase staining in which a monoclonal antibody specific for leucocytes was used. CAV significantly enhanced the number of lymphoproliferative lesions induced by 5000 MDV-WBC. In contrast, CAV significantly reduced the number of lymphoproliferative lesions induced by 100,000 MDV-WBC. Comparable results were found at day 61 after macroscopic examination of nerves and visceral organs. These findings show that the pathogenesis of MD in experimental infections appears to be enhanced or inhibited by CAV, depending on the dose of MDV.

Evaluation of pathogenicity and protective efficacy of serotype 2 Marek's disease virus from birds belonging to genus Gallus in Japan.

The new cloned serotype 2 Marek's disease viruses (MDV) of ML-6, ML-9, and ML-22 strains were inoculated in specific-pathogen-free (SPF) chicks to evaluate the pathogenicity and protective efficacy. Chicks inoculated or contact-infected with ML strains showed no gross and histological lesions in lymphoid organs, sciatic plexuses and other visceral organs during 10 weeks of observation periods, indicating that the viruses were non-pathogenic. Moreover, the viruses were found to be spread horizontally among chicks by demonstrating the presence of viremia in contacted chicks at 2 weeks-old. Chicks vaccinated with ML-6 at one day-old were protected against subsequent challenge by inoculation with virulent MDV strain of Md/5 at 4 or 7 days old or by contact infection at 7 days old with chickens previously inoculated with the same strain.