A rapid and accurate method for herpesviral gnome editing / 生物工程学报

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.

Marek's disease vaccine activates chicken macrophages

To provide insights into the role of innate immune responses in vaccine-mediated protection, we investigated the effect of Marek's disease (MD) vaccine, CVI988/Rispens, on the expression patterns of selected genes associated with activation of macrophages in MD-resistant and MD-susceptible chicken lines. Upregulation of interferon γ, interleukin (IL)-1β, IL-8, and IL-12 at different days post-inoculation (dpi) revealed activation of macrophages in both chicken lines. A strong immune response was induced in cecal tonsils of the susceptible line at 5 dpi. The highest transcriptional activities were observed in spleen tissues of the resistant line at 3 dpi. No increase in the population of CD3³ T cells was observed in duodenum of vaccinated birds at 5 dpi indicating a lack of involvement of the adaptive immune system in the transcriptional profiling of the tested genes. There was, however, an increase in the number of macrophages in the duodenum of vaccinated birds. The CVI988/Rispens antigen was detected in the duodenum and cecal tonsils of the susceptible line at 5 dpi but not in the resistant line. This study sheds light on the role of macrophages in vaccine-mediated protection against MD and on the possible development of new recombinant vaccines with enhanced innate immune system activation properties.

Expression and polyclonal antibody preparation of the tegument protein UL48 encoded by MDV / 中国兽医学报

UL48 plays essential role in replication of MDV genome and interacts with UL36 as well as other MDV tegument proteins.To investigate the interaction between UL48 and UL36 during MDV oncogenisis,antibody against UL48 was prepared and characterized in current study.UL48 gene was amplified from MDV-Ⅰ genome and then subcloned into pTYB1 and pGEX-4T3 vectors for UL48 expression with induction of IPTG in BL21(DE3) E..coli cells.Chitin-sepharose and Glutathion-sepharose were,respectively,used to purify fusion protein intein-UL48 and GST-UL48.Four subcutaneous injections of intein-UL48 fusion protein were done on the lower back and the thigh of rabbit and then other three injections with an interval 10 days.The titer of antibody was measured by the sandwich ELISA with UL48 protein isolated from GST-UL48 after cleavage of thrombin.Western blot was carried out for specificity analysis of antibody against UL48 protein.The results suggested that UL48 antibody was succesfully prepared,and its titer was 1 ∶ 512 000.

Isolation and identification of three Marek's disease virus strains and sequence comparison of genes involved in pathogenesis / 中国兽医学报

To understand epidemiological characteristics of Marek's disease virus (MDV) prevalent in china currently,3 Marek's disease (MD) strains were isolated and identified from white feather meat chickens vaccined with MDV CVI988 or 814 through necropsy,histopathological observation,virus isolation and IFA detection,named SDAU1501,SDAU1502 and SDAU1503,respectively.vIL8,pp38,MEQ gene of the three strains of MDV were amplified using PCR,and compared with reference strains.The homology between SDAU1501 and SDAU1502 and virulent strains was above 97％,suggesting some features of virulent strains;while meq gene of SDAU1503 lost P amino acid at the 194 th site as that in CVI988,But the distinctive 177 nucleotide insertion mutations was not existed,predicting that it may be a attenuated vaccine strain.New variations of MDV continued and different types of variants emerged,therefore,prevalence and genetic monitoring of MD should be proceeded;meanwhile,more attentions should be given to MDV vaccine development.

Marek Disease Virus: molecular approach to the virus and host immune response / Virus de la Enfermedad de Marek: aproximación molecular al virus y respuesta inmune del hospedero / Vírus da doença de Marek: abordagem molecular do vírus e a resposta imune do hospedeiro

Marek's disease virus affects dramatically the production of broiler chicken, hens breeding and commercial due to lost causes for carcass condemnation, presence of tumors and high mortality. Give them us derivatives by the VEM they affect significant economic losses in the poultry industry worldwide and impact the comprehensive management of poultry health and health in general. The genetic and molecular characteristics of the VEM highlight the diversity of the genome in each of the 3 serotypes of the virus; genes involved in pathogenicity, evasion of the immune response and replication strategies are consistent with the difficulty of their infection control. Viral latency and the pump of the immune response of the host, particularly the control of type I interferons, are the mechanism to help the perpetuation in the poultry and thus hamper their effective environmental control. All those conditions have allowed that the virus evolves to forms more virulent that with the use of the vaccines current does not provide a protection adequate against these; for this reason, it is necessary to reconsider current vaccination plans to improve the immune response of active type, particularly involving cell type, to control his evasion and control on the immune system.

El virus de la enfermedad de Marek afecta dramaticamente la producción de pollo de engorde, gallinas comerciales y reproductoras debido a las perdidas causas por decomisos en mataderos, presencia de tumores y alta mortalidad. Las daños derivados por el VEM repercuten en pérdidas económicas significativas en la industria avícola mundial e impactan el manejo integral de la sanidad y salud avícola en general. Las características genéticas y moleculares del VEM resaltan la diversidad genómica en cada uno de los tres serotipos del virus; genes involucrados en sus estrategias de replicación, patogenicidad y evasión de la respuesta inmune son consistentes con la dificultad del control de su infección. La latencia viral y la evación de la respuesta inmune del hospedero, particularmente el control de interferones tipo I, son los mecanismo que ayudan a la perpetuacion en las granjas avicolas y por ende dificultan su efectivo control medio ambiental. Todas esas condiciones han permitido que el virus evolucione a formas más virulentas que con el uso de las vacunas actuales no proporcionen una protección adecuada contra estas; por eso, se hace necesario replantear los planes actuales de vacunación para mejorar la respuesta inmune de tipo activo, involucrando particularmente la de tipo celular, para controlar su evasion y control sobre el sistema inmune.

O vírus da doença de Marek afeta drasticamente a produçã o de frango de engorda, galinhas poedeiras comerciais e reproduçã o devido a eles causas perdidas por convulsões em matadouros, presença de tumores e alta mortalidade. Dar-lhes nos derivados pelo VEM que afectem perdas económicas significativas na indústria avícola em todo o mundo e afetar o gerenciamento abrangente de saúde das aves e saúde em geral. As características genéticas e moleculares do VEM destacam a diversidade do genoma em cada um dos 3 sorotipos do vírus; genes envolvidos em suas estratégias de replicaçã o, patogenicidade e evasã o da resposta imune sã o consistentes com a dificuldade do controle de sua infecçã o. Latência viral e a bomba da resposta imune do hospedeiro, especialmente o controle de interferons do tipo I, sã o o mecanismo para ajudar a perpetuaçã o em aves de capoeira e, assim, dificultar o seu controle ambiental eficaz. Todas essas condições permitiram que o vírus evolui para formas mais virulentas que com o uso das vacinas atuais nã o fornece uma proteçã o adequada contra estes; por isso, é necessário faz-los repensar corrente de planos de vacinaçã o para melhorar o imune resposta do tipo ativo, envolvendo particularmente o de célula do tipo, para controlar sua evasã o e controle sobre o imunológico do sistema.

Visceral Marek's disease in white-peafowl (Pavo cristatus) / Doença de Marek visceral em pavão-branco (Pavo cristatus)

Marek's disease (MD) is a lymphoproliferative disorder caused by Gallid herpesvirus 2 (MDV) that infects mainly domestic gallinaceous birds although wild birds may occasionally be affected. The current report describes the anatomopathological and molecular findings of a case of MD in a white-peafowl (Pavo cristatus). The signs included apathy, hyporexia, and diarrhea. Grossly, 0.5 to 1.5cm in diameter, yellow, soft nodules were observed in the skeletal muscle, lung, kidney, air sacs, small intestine, heart, ovary, ventriculus, and proventriculus. Microscopically, numerous atypical round neoplastic cells were noted. The molecular detection of MDV DNA was implemented to amplify part of the meq gene and products were sequenced for the phylogenetic analysis. Template DNA was obtained from tissues of the affected bird and from blood of all the gallinaceous birds of the Zoo. The expected amplicon for the partial amplification of MDV meq gene was obtained and the amplicons were sequenced. Sequences obtained enabled grouping the strain (accession no. KT768121) with MDV serotype 1 strains from the GenBank. Based on the anatomopathological and molecular findings, the diagnosis of MD in a white-peafowl was reached, and to the authors' knowledge, no previous report regarding MD was published in Pavo cristatus.(AU)

Doença de Marek (MD) é uma desordem linfoproliferativa causada pelo Gallid herpesvirus 2 (MDV), que infecta principalmente galináceos domésticos, porém aves silvestres podem ser ocasionalmente afetadas. O presente relato descreve os achados anatomopatológicos e moleculares de um caso de MD em um pavão-branco (Pavo cristatus). Os sinais clínicos incluíram apatia, hiporexia e diarreia. Macroscopicamente, foram observados nódulos macios, de 0,5 a 1,5cm de diâmetro, no músculo esquelético, no pulmão, nos rins, nos sacos aéreos, no intestino delgado, no coração, no ovário, no ventrículo e no proventrículo. Microscopicamente, numerosas células redondas neoplásicas atípicas foram notadas. A detecção molecular do DNA do MDV foi implementada para amplificar parte do gene meq, e os produtos foram sequenciados para análise filogenética. DNA foi obtido de tecidos de aves afetadas e do sangue de todos os galináceos do zoológico. A esperada amplificação de parte do gene meq de MDV amplificado foi ampliada e sequenciada. As sequências obtidas permitiram o agrupamento da cepa (acesso KT768121) com cepas do sorotipo 1 de MDV do GenBank.. O diagnóstico de MD em pavão-branco foi obtido com base nos achados anatomopatológicos e moleculares e, pelo conhecimento dos autores, não há relatos anteriores publicados de MD em Pavo cristatus.(AU)

Imaging of neurolymphomatosis with 18F-FDG PET/CT: A case report and review of literature / 解放军医学杂志

Objective To explore the value of FDG PET-CT in the diagnosis of neurolymphomatosis (NL). Methods The clinical manifestation and FDG PET/CT imaging results in a patient with diffuse large B cell lymphoma accompanying peripheral neuropathy which was confirmed by pathological examination, were introduced. The images as shown by PET/CT were compared with the findings of traditional imaging including MRI and CT. Relevant literature was reviewed. Results A 38-year female patient complaining of left chest-back pain for 2 months came to hospital for treatment. An enhanced MRI of thoracic vertebrae showed osseous destruction on the left side of 4th thoracic vertebra and left posterior segment of 5th rib, and it was primarily diagnosed as a tumor. FDG PET/CT revealed a massively increased radioactive uptake in intervertebral foramen of left 4th, 5th thoracic vertebrae. The lesion was shown as an increase in uptake of radio-active substance along the left 5th intercostal nerve in the form of bundle or threads. A round-like nodule with increased radioactive uptake was observed in the left parasternal 2nd intercostal space. A CT-guided percutaneous needle biopsy of the nodule revealed a diffuse large B-cell lymphoma (A type). The lesion was shown to involve 4th, 5th thoracic vertebrae and left 5th intercostal nerve. It was diagnosed as NL. Repeated FDG PET imaging after chemotherapy showed normal radioactive distribution in the site of primary lesion area. Conclusions PET/CT is effective and sensitive in the diagnosis of NL, especially in patient with a history of malignant hematologic disease with clinical symptoms concerning peripheral nerve, accompanied by negative results with other examinations. Comparing with MRI, PET/CT can reveal involvement of peripheral nerve earlier, better reflect the degree of pathological condition, and reveal the number of nerves involved, as well as size and morphology of the lesion. It can reveal the active lesions of NL, and provide the target for needle biopsy.

Downregulation of cellular prion protein inhibited the proliferation and invasion and induced apoptosis of Marek's disease virus-transformed avian T cells

Cellular prion protein (PrP(C)) is ubiquitously expressed in the cytomembrane of a considerable number of eukaryotic cells. Although several studies have investigated the functions of PrP(C) in cell proliferation, cell apoptosis, and tumorigenesis of mammals, the correlated functions of chicken PrP(C) (chPrP(C)) remain unknown. In this study, stable chPrP(C)-downregulated Marek's disease (MD) virus-transformed avian T cells (MSB1-SiRNA-3) were established by introducing short interfering RNA (SiRNA) targeting chicken prion protein genes. We found that downregulation of chPrP(C) inhibits proliferation, invasion, and migration, and induces G1 cell cycle phase arrest and apoptosis of MSB1-SiRNA-3 cells compared with Marek's disease virus-transformed avian T cells (MSB1) and negative control cells. To the best of our knowledge, the present study provides the first evidence supporting the positive correlation between the expression level of chPrP(C) and the proliferation, migration, and invasion ability of MSB1 cells, but appears to protect MSB1 cells from apoptosis, which suggests it functions in the formation and development of MD tumors. This evidence may contribute to future research into the specific molecular mechanisms of chPrP(C) in the formation and development of MD tumors.

Construction of Transferring Vector of Marek’s Disease Virus Expressing GFP Gene and Its Primary Application / 中国生物工程杂志

The expressing cassette, LoxP-CMV-gpt-IRES-LoxP( about 2.9kb), was amplified by PCR from a plasmid, pIRES-gpt, by use of the primers , which contained the loxP sites in 5' terminals, respectively. The loxP sites were designed into primers by the software of Primer primer 5.0. Then the cassette was cloned into the site of BalI in pBUS10 to obtain pUS-gptIRES(L). The sequencing analysis for pUS-gptIRES(L) indicated that two loxP sites with the same direction were correctly inserted into pUS-gptIRES(L).The gpt gene in pUS-gptIRES(L) was replaced by a fragment including the full length GFP gene as well as SV40 poly A sequence to get pUS-GFPIRES(L). pUS-GFPIRES(L) was transiently transfected into CHO cell lines, and then the green fluorescence could be seen, the results showed that GFP gene could be expressed correctly. Moreover, pUS-GFPIRES(L) was transfected into the CEF infected MDV CVI988 strain and recombinant virus was selected by the green fluorescence. The growth curve of virus showed the characteristic of recombinant virus was the same as that of CVI988 in vitro. These results give the basis for further studying the characteristic of MDV in vivo and the application of the Cre/LoxP system to MDV genome.

Prokaryotic Expression of the Partial gB Gene of the Marek’s Disease Virus / 中国生物工程杂志

The partial segment of Marek′s disease virus (MDV) glycoprotein B (gB) gene was amplified by PCR. The segment was cloned into pET-28a vector to obtain the recombinant pET-gB plasmid. The recombinant plasmid was transformed into E.coli BL21,and expressed in very high level as inclusion body after induced with 1.0mmol/L IPTG. The inclusion body was solubilized in urea (8mol/L) . The purified protein was obtained by use of His?Bind affinity chromatography. Mice were immunized i.p. by the purified protein to make the polyclonal antibody. The titer of the antibody by indirect ELISA was 1?10~ -5 . Moreover, the analysis by western blot proved that antibody was specific to the recombinant protein. These works lay a favorable foundation for the study of the immune response by MDV gB.

Neurolymphomatosis, a case report / 北京大学学报(医学版)

Neurolymphomatosis(NL) is characterized by lymphomatous infiltration of the peripheral nervous system. We report a case of neurolymphomatosis(NL) which was confirmed by sural nerve biopsy. Sural nerve specimen from a 49-year-old female patient with weakness of limbs was examined with routine histochemical and immunohistochemistry staining, in which the first antibodies against CD3, CD20, CD45, CD45RO and CD68 were used. Numerous T-lymphoma cells invaded in the adipose tissue of epineurium of sural nerve. The nerve biopsy showed marked axonal degeneration of myelinated fibers. The clinical and histopathologic findings confirmed the diagnosis of neurolymphomatosis.