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.

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)

Marek's disease virus can infect chicken brain microglia and promote the transcription of toll-like receptor 15 and 1LB genes / 病毒学报

Microglial cells were purified from a mixed neuroglia culture prepared from the neonatal chicken brain in vitro, and were infected with the vvMDV YL040920 isolate and an attenuated MDV vaccine strain CVI988/Rispens, respectively. The presence of cytopathic effect (CPE) was examined daily, and the MEQ expression in MDV-infected microglia was detected by immunohistochemistry assay. DNA replication of the MDV meq gene and transcription of the gB gene were determined by real-time quantitative PCR (qPCR) and qRT-PCR, respectively. The transcripts of Toll-like receptor (TLR) mRNA in microglia post MDV infection were quantified by qRT-PCR. The results of this study showed that both vvMDV YL040920 and attenuated vaccine strain CVI988/Rispens could infect microglia and produce characteristic CPE with plaque formation. The plaques were formed due to cells shedding at multi-sites, then quickly expanded and integrated. Furthermore, the MEQ protein was detected in nuclei of YL040920 and CVI988/ Rispens-infected microglia, and MDV meq DNA replication and gB gene transcription in MDV-infected microglia were also confirmed. Although both MDV DNA copies and gB transcripts were increased in the virus-infected microglia, the higher viral DNA load and gB transcript were observed for CVI988/Rispens than for YL040920 in vitro (P < or = 0.05/0.001). The transcriptions of TLR15 and TLR1LB gene were found to be up-regulated in microglia following MDV infection in vitro. Purified microglia infected with YL040920 was observed increased TLR15 and TLR1LB transcripts as early as 1 day post infection (dpi), and reached its peak level at 3 dpi, then decreased mildly at 5 dpi. For CVI988/Rispens, it induced an increase of TLR15 transcript as early as 1 dpi, and rose rapidly at 3 dpi, and then decreased slightly at 5 dpi. At the same time, CVI988/Rispens induced the increase of chTLR1LB transcript at 3 dpi and decreased at 5 dpi. By comparing the TLRs transcription between YL040920 and CVI988/Rispens-infected microglia, it was suggested that vvMDV YL040920 might induce more TLR15 transcript than the attenuated vaccine strain CVI988/Rispens (P < or = 0.01/0.001), while CVI988/Rispens induced more TLR1LB transcript than YL040920 (P < or = 0.001).

Cloning and sequence analysis of the Meq gene of 4 Marek's disease virus isolates from China / 病毒学报

Earlier studies have determined that the repeat regions of oncogenic serotype 1 MDV (Marek's disease virus) encode a basic leucine zipper protein, Meq, which structurally resembles the Jun/Fos family of transcriptional activators. Meq has been suggested as the MDV-associated oncogene. In this paper, based on the published sequence of Meq gene of GA strain of MDV, a pair of primers were designed and synthesized. Meq gene ORF (Open reading frame) of the four Chinese local MDV isolates, the reference strain J-1 and the vaccine strain 814 were amplified by using polymerase chain reaction(PCR). Then the PCR products were cloned and sequenced respectively. The results of sequence comparison indicated that the sequences of Meq gene in different strains are relatively conserved and homology of the amino acid sequences is 96.5%-99.7%. The proline-rich repeats of Meq gene of four MDV isolates have site mutations, and it is related to MDV's virulence. Two unique site mutations appear in Meq gene of Chinese local MDV isolates, but they aren't present in Meq gene of the published MDV strains from abroad and the early domestic strains. It seems that some regularities exist between such mutations in four Chinese local MDV isolates and the virulence of MDV, but the regularities need further research.

Induction of cell mediated immune response by nonspecific stimulator of immunity against antigenically unrelated oncogenic virus (Marek's disease virus).

An enzyme treated preparation of Mycobacterium phlei (NSI), induced strong cell mediated immune response (CMIR) against specific as well as against nonspecific oncogenic Marek's disease (MDV) in birds, as evinced by Lymphocyte migration inhibition, (LMIT) lymphocyte transformation test (LT) and Lymphokine (Lymphocyte migration inhibition factor) LyIF assay. Maximum CMIR could be observed towards third week post inoculation. All the three tests exhibited a positive correlation. Such phenomenon of CMIR induction by NSI, nonspecifically to unrelated viral/cancerous diseases (MD) in birds generates hopes for immunoprevention of these maladies by utilizing such phenomenon.

Ocorrência de anticorpos para vírus aviários em frangos de corte em regiäo de intensa produçäo avícola / Occurrence of antibodies for avian viruses in broilers in a region of high density poultry production

Amostras de sangue foram obtidas ao abate de 60 frangos de corte, de 43 a 56 dias de idade, de cada uma de 60 granjas sem problemas sanitários aparentes localizadas em uma regiäo de alta densidade de produçäo avícola. As granjas correspondiam a 10% do número total que existe na regiäo e estavam localizadas em um raio de 50 km. Todos os frangos tinham sido vacinados contra a doença de Marek no primeiro dia de vida. Os soros foram avaliados no teste de imunodifusäo para anticorpos para reovírus aviário (RVA), vírus da doença infecciosa da bursa (VDIB), vírus da bouba aviária (VBA), vírus herpes de perus (HVT), adenovírus aviário do grupo 1 (AVA-1) e do grupo 2 (AVA-2). Os mesmos soros foram examinados no microteste de soroneutralizaçäo em placas para anticorpos para o vírus da bronquite infecciosa (VBI) e o vírus da laringotraqueíte infecciosa (VLTI), bem como anticorpos inibidores da hemoaglutinaçäo para o vírus da doença de Newcattle (VDN). Anticorpos para RVA, detectados em 2172 (63,6%) de 3418 soros, e para AVA-1, em 2701 (78,2%) de 3456 soros, foram demonstrados em todas as 60 granjas, enquanto que, anticorpos para o VDIB, em 3086 (89,4%) de 3452 soros, foram encontrados em somente 57 destas granjas. Estes resultados indicam que os três vírus säo ubíquos em frangos de corte da regiäo estudada. Anticorpos para o VBA em 14 (0,5%) de 2935 soros, para o VDN em seis (0,2%) de 3320 soros, foram encontrados em cinco de 59 e em uma de 60 granjas, respectivamente, indicando que esses dois vírus ocorrem apenas raramente. Anticorpos para HVT, em 315 (9,0%) de 3484 soros, foram detectados em 39 de 60 granjas testadas, sugerindo resposta sorológica pobre à vacinaçäo e baixa exposiçäo de campo ao vírus da doença de Marek. Anticorpos para o VBI, em 61 (2,1%) de 2925 soros, foram encontrados em 12 de 57 granjas testadas. Porém, em apenas uma granja a percentagem de reagentes era significativa (41%); nas outras 11 granjas a percentagem de frangos com anticorpos variava de 1,7 a 16,1%. Finalmente, näo foram detectados anticorpos em 3491 soros de 60 granjas testada para AVA-2, nem em 3035 soros de 59 granjas testadas para o VLTI, indicando que esses vírus näo existem na área geográfica estudada. O significado de todos os achados é discutido