Fatores associados à infecção pelo vírus da estomatite vesicular em equídeos no estado do Rio Grande do Norte, Brasil / Factors associated with infection by the vesicular stomatitis virus in equines in the state of Rio Grande do Norte, Brazil

Objetivou-se investigar a presença do Vírus da Estomatite Vesicular (VEV) e seus fatores de risco para ocorrência e disseminação da enfermidade em equídeos das mesorregiões Leste e Oeste Potiguar do estado do Rio Grande do Norte, Brasil. Foram analisadas pela técnica de virusneutralização, 809 amostras sanguíneas de equídeos provenientes de noventa propriedades de dezesseis municípios Potiguares durante os meses de julho de 2018 a fevereiro de 2019. Os fatores de riscos associados ao VEV foram avaliados por meio de questionário epidemiológico e os dados submetidos a análise estatística no programa IBM SPSS Statistics versão 21.0 com nível de confiança de 95%. Posteriormente, todas as variáveis estatisticamente significantes foram submetidas a análise de regressão de Poisson. A soroprevalência de anticorpos anti-VEV foi 24,6% (199/809), sendo 3,2% (13/402) de soropositivos na mesorregião Leste e 45,7% (186/407) na do Oeste Potiguar. Com relação aos sorotipos, observou-se uma prevalência de 3,8% (31/809) e 24,5% (198/809) para Indiana 2 e 3 respectivamente, com 15,1% (30/198) de coinfecção. Equídeos criados na mesorregião Oeste, em propriedades que não realizam quarentena e onde os animais enfermos são mantidos no rebanho, foram consideradas fatores predisponentes a infecção pelo VEV. Esses resultados demonstram a circulação do VEV em equídeos no Rio Grande do Norte, com destaque ao Oeste Potiguar, e sendo necessário a aplicação de medidas sanitárias que impeçam introdução e disseminação do vírus ente as espécies susceptíveis, principalmente em condições climáticas favoráveis para a sua manutenção, no ambiente de criação e pastagens.

This study aimed to investigate the presence of Vesicular stomatitis virus (VSV) and risk factors for its occurrence and dissemination in equines from the Eastern and Western mesoregions of the state of Rio Grande do Norte, Brazil. Blood samples were analyzed, by Serum Virus Neutralization Assay, from 809 animals belonging to 90 properties distributed in sixteen municipalities from July 2018 to February 2019. Risk factors were assessed using an epidemiological questionnaire. Data were submitted to statistical analysis using the software IBM SPSS Statistics, version 21.0 with a 95% confidence level. Also, all statistically significant variables were subjected to Poisson regression analysis. The occurrence of anti-VSV antibodies was 24.6% (199/809) with 3.2% (13/402) and 45.7% (186/407) of seropositivity in the Western and Eastern mesoregion, respectively. Regarding serotypes, there were an occurrence of 3.8% (31/809) and 24.5% (198/809) for Indiana 2 and 3, respectively, and 15.1% (30/198) of co-infection for both. Equines kept of the Western mesoregion, on properties that do not quarantine, and where sick animals are kept in the herd, were considered risk factors for LVV infection. These results demonstrate the presence of VSV in equines in Rio Grande do Norte, with emphasis on Oeste Potiguar, and that sanitary measures must be adopted to prevent the introduction and viral spreading among susceptible species, especially due to favorable climatic conditions for the maintenance of VSV in the breeding and pasture environment.

Fatores associados à infecção pelo vírus da estomatite vesicular em equídeos no estado do Rio Grande do Norte, Brasil / Factors associated with infection by the vesicular stomatitis virus in equines in the state of Rio Grande do Norte, Brazil

Objetivou-se investigar a presença do Vírus da Estomatite Vesicular (VEV) e seus fatores de risco para ocorrência e disseminação da enfermidade em equídeos das mesorregiões Leste e Oeste Potiguar do estado do Rio Grande do Norte, Brasil. Foram analisadas pela técnica de virusneutralização, 809 amostras sanguíneas de equídeos provenientes de noventa propriedades de dezesseis municípios Potiguares durante os meses de julho de 2018 a fevereiro de 2019. Os fatores de riscos associados ao VEV foram avaliados por meio de questionário epidemiológico e os dados submetidos a análise estatística no programa IBM SPSS Statistics versão 21.0 com nível de confiança de 95%. Posteriormente, todas as variáveis estatisticamente significantes foram submetidas a análise de regressão de Poisson. A soroprevalência de anticorpos anti-VEV foi 24,6% (199/809), sendo 3,2% (13/402) de soropositivos na mesorregião Leste e 45,7% (186/407) na do Oeste Potiguar. Com relação aos sorotipos, observou-se uma prevalência de 3,8% (31/809) e 24,5% (198/809) para Indiana 2 e 3 respectivamente, com 15,1% (30/198) de coinfecção. Equídeos criados na mesorregião Oeste, em propriedades que não realizam quarentena e onde os animais enfermos são mantidos no rebanho, foram consideradas fatores predisponentes a infecção pelo VEV. Esses resultados demonstram a circulação do VEV em equídeos no Rio Grande do Norte, com destaque ao Oeste Potiguar, e sendo necessário a aplicação de medidas sanitárias que impeçam introdução e disseminação do vírus ente as espécies susceptíveis, principalmente em condições climáticas favoráveis para a sua manutenção, no ambiente de criação e pastagens.

This study aimed to investigate the presence of Vesicular stomatitis virus (VSV) and risk factors for its occurrence and dissemination in equines from the Eastern and Western mesoregions of the state of Rio Grande do Norte, Brazil. Blood samples were analyzed, by Serum Virus Neutralization Assay, from 809 animals belonging to 90 properties distributed in sixteen municipalities from July 2018 to February 2019. Risk factors were assessed using an epidemiological questionnaire. Data were submitted to statistical analysis using the software IBM SPSS Statistics, version 21.0 with a 95% confidence level. Also, all statistically significant variables were subjected to Poisson regression analysis. The occurrence of anti-VSV antibodies was 24.6% (199/809) with 3.2% (13/402) and 45.7% (186/407) of seropositivity in the Western and Eastern mesoregion, respectively. Regarding serotypes, there were an occurrence of 3.8% (31/809) and 24.5% (198/809) for Indiana 2 and 3, respectively, and 15.1% (30/198) of co-infection for both. Equines kept of the Western mesoregion, on properties that do not quarantine, and where sick animals are kept in the herd, were considered risk factors for LVV infection. These results demonstrate the presence of VSV in equines in Rio Grande do Norte, with emphasis on Oeste Potiguar, and that sanitary measures must be adopted to prevent the introduction and viral spreading among susceptible species, especially due to favorable climatic conditions for the maintenance of VSV in the breeding and pasture environment.

Early and late neuropathological features of meningoencephalitis associated with Maraba virus infection

Maraba virus is a member of the genus Vesiculovirus of the Rhabdoviridae family that was isolated in 1983 from sandflies captured in the municipality of Maraba, state of Pará, Amazônia, Brazil. Despite 30 years having passed since its isolation, little is known about the neuropathology induced by the Maraba virus. Accordingly, in this study the histopathological features, inflammatory glial changes, cytokine concentrations, and nitric oxide activity in the encephalon of adult mice subjected to Maraba virus nostril infection were evaluated. The results showed that 6 days after intranasal inoculation, severe neuropathological-associated disease signs appeared, including edema, necrosis and pyknosis of neurons, generalized congestion of encephalic vessels, and intra- and perivascular meningeal lymphocytic infiltrates in several brain regions. Immunolabeling of viral antigens was observed in almost all central nervous system (CNS) areas and this was associated with intense microglial activation and astrogliosis. Compared to control animals, infected mice showed significant increases in interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon (INF)-γ, MCP-1, nitric oxide, and encephalic cytokine levels. We suggest that an exacerbated inflammatory response in several regions of the CNS of adult BALB/c mice might be responsible for their deaths.

Cell Death Mechanisms in Esophageal Squamous Cell Carcinoma Induced by Vesicular Stomatitis Virus Matrix Protein

OBJECTIVES: Vesicular stomatitis virus (VSV) is under development as an oncolytic virus due to its preferential replication in cancer cells and oncolytic activity, however the viral components responsible have not yet been determined. In this study the effects of VSV wild-type (wt) and M51R-mutant matrix proteins (M51R-mMP) on apoptosis, pyroptosis, necroptosis, and autophagy pathways, in an esophagus cancer cell line (KYSE-30) were investigated. METHODS: The KYSE-30 cells were transfected with pcDNA3.1 plasmids encoding wt or M51R-mMP, and apoptosis, pyroptosis, necroptosis, and autophagy were evaluated 48 and 72 hours after transfection. RESULTS: KYSE-30 cells transfected with VSV wt and M51R-mMPs significantly reduced cell viability to < 50% at 72 hours post-transfection. M51R-MP significantly increased the concentration of caspase-8 and caspase-9 at 48 and 72 hours post-transfection, respectively ( p < 0.05). In contrast, no significant changes were detected following transfection with the VSV wt plasmid. Moreover, VSV wt and M51R-mMP transfected cells did not change the expression of caspase-3. VSV wt and M51R-mMPs did not mMP change caspase-1 expression (a marker of pyroptosis) at 48 and 72 hours post-transfection. However, M51R-mMP and VSV wt transfected cells significantly increased RIP-1 (a marker of necroptosis) expression at 72 hours post-infection ( p < 0.05). Beclin-1, a biomarker of autophagy, was also induced by transfection with VSV wt or M51R-mMPs at 48 hours post-transfection. CONCLUSION: The results in this study indicated that VSV exerts oncolytic activity in KYSE-30 tumor cells through different cell death pathways, suggesting that M51R-mMP may potentially be used to enhance oncolysis.

Occurrence of viral diseases in donkeys (Equus asinus) in São Paulo State, Brazil / Ocorrência de enfermidades virais em asininos (Equus asinus) no estado de São Paulo, Brasil

Among the diseases that affect equines, viral diseases play an important role from a health and economic point of view, especially influenza, viral arteritis, herpes infections and vesicular stomatitis. In the Brazilian literature, there is little or no account of the occurrence of infectious diseases in donkeys. Given the importance of donkeys in different activities and the lack of information on infections that may occur in these animals, the aim of this study was to determine the frequency of anti-equine herpesvirus (EHV), anti-equine arteritis virus (EAV), anti-vesicular stomatitis, and anti-equine influenza (H3N8) antibodies in the serum of 85 donkeys bred in some regions of the state of São Paulo. We found the following antibody frequencies: 50.6% (43/85) antibodies against influenza virus subtype H3N8, 47% (40/85) anti-EHV, and 20% (17/85) anti-EAV. The donkeys were not seropositive for vesicular stomatitis. The results suggested that the agents EHV, EAV, and equine influenza subtype H3N8 circulate among donkeys in some regions of the state of São Paulo, Brazil, reinforcing the importance of establishing a routine diagnosis and epidemiological study of this species.(AU)

Dentre as doenças que acometem os equídeos, as enfermidades virais assumem um papel importante do ponto de vista sanitário e econômico, especialmente a influenza, arterite viral, as infecções herpéticas e a estomatite vesicular. Na literatura nacional, existe pouco ou nenhum relato sobre a ocorrência de enfermidades infecciosas nos asininos. Tendo em vista a importância dos asininos para diferentes atividades e a falta de informações sobre as doenças que acometem esses animais, este trabalho teve como objetivo estudar a frequência de anticorpos anti-EHV, antivírus da arterite equina, anti-estomatite vesicular e anti-influenza equina (H3N8) em 85 soros de jumentos criados no estado de São Paulo. Estimou-se que 50,6% apresentavam anticorpos contra o subtipo H3N8 do vírus da influenza; 47% (40/85) apresentavam anticorpos contra o EHV e 20% apresentavam anticorpos contra o vírus da arterite. Os jumentos não foram soro reagentes contra a estomatite vesicular. Os resultados obtidos sugerem que os agentes EHV, vírus da arterite equina e influenza equina subtipo H3N8, circulam entre os jumentos do estado de São Paulo, caracterizando a importância do estabelecimento de uma rotina diagnóstica e estudos epidemiológicos na espécie.(AU)

Species Specific Antiviral Activity of Porcine Interferon-α8 (IFNα8)

Interferons (IFNs) have been known as antiviral genes and they are classified by type 1, type 2, and type 3 IFN. The type 1 IFN consists of IFNα, IFNβ, IFNτ, and IFNω whereas the type 2 IFN consists of only IFNγ, which is a key cytokine driving T helper cell type 1 immunity. IFNλ belongs to the type 3 IFN, which is also known as IL-28 and IL-29 possessing antiviral activities. Type 1 IFN is produced by viral infection whereas type 2 IFN is induced by mitogenic or antigenic T-cell stimuli. The IFNτ of bovine was first discovered in an ungulate ruminant recognition hormone. IFNτ belongs to the type 1 IFN with the common feature of type 1 IFN such as antiviral activity. IFNs have been mostly studied for basic research and clinical usages therefore there was no effort to investigate IFNs in industrial animals. Here we cloned porcine IFNα8 from peripheral blood mononuclear cells of Korean domestic pig (Sus scrofa domestica). The newly cloned IFNα8 amino acid sequence from Korean domestic pig shares 98.4% identity with the known porcine IFNα8 in databank. The recombinant porcine IFNα8 showed potent antiviral activity and protected bovine Madin-Darby bovine kidney epithelial (MDBK) cells from the cytopathic effect of vesicular stomatitis virus, but it failed to protect human Wistar Institute Susan Hayflick (WISH) cells and canine Madin-Darby canine kidney epithelial-like (MDCK) cells. The present study demonstrates species specific antiviral activity of porcine IFNα8.

A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.

Investigação epidemiológica de estomatite vesicular por achados clínicos em bovinos e equinos no Estado do Maranhão / Stomatitis vesicular in cattle and horse in the Maranhão state

A Estomatite Vesicular (EV) é uma doença infecciosa que acomete equinos, bovinos, suínos, mamíferos silvestres e humanos. Por apresentar sinais clínicos semelhantes a outras doenças vesiculares, principalmente, febre aftosa, sua presença em determinadas regiões pode interferir no intercâmbio comercial internacional dos animais, seus produtos e subprodutos. Apesar de sua importância, a epidemiologia e a manutenção do vírus no ambiente não estão totalmente esclarecidas dificultando a aplicação de medidas de controle efetivas. A doença já foi diagnosticada em todas as regiões brasileiras. Bovinos com sialorréia, perda do epitélio lingual, lesões abertas com bordas amareladas nas gengivas, lábios, língua e mucosa oral e equinos com sialorréia e lesões abertas na mucosa oral e lábios foram observados e notificados ao Serviço Veterinário Oficial do Estado do Maranhão, Agência Estadual de Defesa Agropecuária do Maranhão (AGRD/MA). Amostras de soro de equinos e bovinos com sintomas de EV foram coletadas para investigação por ELISA e por neutralização viral, além do diagnóstico diferencial para Febre Aftosa (FA). Fragmentos epiteliais de bovinos com lesões na língua foram coletados para identificação molecular do agente. Todos os animais foram negativos para FA. Todos os bovinos e equinos foram reativos para EV nos testes sorológicos. A partir dos fragmentos epiteliais de bovinos enviados ao Instituto Biológico de São Paulo para PCR, foi possível caracterizar o agente como VesiculovirusIndiana III (Alagoas/VSAV).

Vesicular stomatitis (VS) is an infectious viral disease that affects bovines, equines, swine, wild animals and humans. As it is indistinguishable from other vesicular diseases, mainly Foot and Mouth Disease (FMD), it causes restrictions in commercial livestock trade at national and international levels and also significant economic losses. As the epidemiology and maintenance of VS virus in nature are not clearly understood it is difficult to take effective control measures. VS was diagnosed in some regions of Brazil, such as Minas Gerais, Santa Catarina, São Paulo and Alagoas. Cattle and horses with clinical symptoms of drooling, shedding of the lingual epithelium, presence of vesicles on the oral mucosa were observed and reported to the National Animal Health Office health of Maranhão State, Brazil. Samples of serum of these animals were collected and sent to Laboratório Nacional de Agropecuaria for ELISA and virus neutralization and differential diagnosis for Foot and Mouth Disease (FMD). The results of ELISA confirmed the VS. In the differential diagnosis, the results were negative for FMD. Samples of bovine epithelial tissue for VS by PCR confirmation of diagnosis were collected and sent to Biological Institute of São Paulo. Molecular results confirmed the VesiculovirusIndiana III (Alagoas/VSAV) infection.

[Cellular immune response following neonatal mice immunization with vesicular stomatitis virus]

Infectious microorganisms are major sources of illness and death worldwide, and the leading cause of death in neonates. Effective vaccination of this age group is of particular importance. The lack of a response and greater susceptibility to tolerance are two major features that limit the effectiveness of vaccines in neonates. In this study we compare the cellular immune response generated following antigen injections at different times of life in newborn mice to that of adult mice. Adult and different age neonate mice were vaccinated with vesicular stomatitis virus [VSV]. One week after the last injection, cellular immunity was assayed on spleen cells that targeted EL4 infected cells using lactate dehydrogenase cytotoxicity assay. Antigen injection induced a decreased immune response in newborn mice compared with mice that had been immunized with subsequent injections. In the adult group, due to the evolution of the immune system, we observed a stronger immune response. Immunization of newborn mice may induce a reduced response when compared to adult vaccinations. However this can be corrected by the administration of additional booster doses

Vesicular Stomatitis Virus G Glycoprotein and ATRA Enhanced Bystander Killing of Chemoresistant Leukemic Cells by Herpes Simplex Virus Thymidine Kinase/Ganciclovir

Refractoriness of acute myeloid leukemia (AML) cells to chemotherapeutics represents a major clinical barrier. Suicide gene therapy for cancer has been attractive but with limited clinical efficacy. In this study, we investigated the potential application of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) based system to inhibit chemoresistant AML cells. We first generated Ara-C resistant K562 cells and doxorubicin-resistant THP-1 cells. We found that the HSV-TK/GCV anticancer system suppressed drug resistant leukemic cells in culture. Chemoresistant AML cell lines displayed similar sensitivity to HSV-TK/GCV. Moreover, HSV-TK/GCV killing of leukemic cells was augmented to a mild but significant extent by all-trans retinoic acid (ATRA) with concomitant upregulation of Connexin 43, a major component of gap junctions. Interestingly, HSV-TK/GCV killing was enhanced by expression of vesicular stomatitis virus G glycoprotein (VSV-G), a fusogenic membrane protein, which also increased leukemic cell fusion. Co-culture resistant cells expressing HSV-TK and cells stably transduced with VSV-G showed that expression of VSV-G could promote the bystander killing effect of HSV-TK/GCV. Furthermore, combination of HSV-TK/GCV with VSV-G plus ATRA produced more pronounced antileukemia effect. These results suggest that the HSV-TK/GCV system in combination with fusogenic membrane proteins and/or ATRA could provide a strategy to mitigate the chemoresistance of AML.

Molecular cloning and expression of bone marrow stromal antigen-2 and detection of its biological activity / 病毒学报

To clone porcine bone marrow stromal antigen-2 (BST-2) gene, construct its recombinant eukaryotic expression plasmid and induce the expression of the fusion antiviral protein, we amplified BST-2 gene by RT-PCR from the total RNA extracted from PK15 cells. The recombinant expression plasmid pcDNA-BST-2 was constructed and then was transfected into HEK293T cells to expresse the BST-2 fusion protein. Western blot and indirect immunofluorescence assay (IFA) were performed, and the biological activity was detected. The results showed that the construction of recombinant plasmid pcDNA-BST-2 was confirmed by restriction enzyme digestion and sequencing. The expressed product had antiviral activity against Vesicular stomatitis virus (VSV), Avian influenza virus (AIV) and Porcine reproductive and respiratory syndrome virus (PRRSV). In conclusion, the research paves the way for further research on bioactivity assayand antiviral medication.

Ribosomal Protein L19 and L22 Modulate TLR3 Signaling

BACKGROUND: Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA) and induces inflammation. In this study we attempted to ascertain if there are endogenous host molecules controlling the production of cytokines and chemokines. Two candidates, ribosomal protein L19 and L22, were analyzed to determine if they influence cytokine production followed by TLR3 activation. In this study we report that L19 acts upon production of IP-10 or IL-8 differently in glioblastoma cells. METHODS: L19 or L22 was transfected into HEK293-TLR3, A549 or A172 cells. After treatment with several inhibitors of NF-kB, PI3K, p38 or ERK, production of IL-8 or IP-10 was measured by ELISA. siRNA was introduced to suppress expression of L19. After Vesicular stomatitis virus infection, viral multiplication was measured by western blot. RESULTS: L19 increased ERK activation to produce IL-8. In A172 cells, in which TLR3 is expressed at endosomes, L19 inhibited interferon regulatory factor 3 (IRF3) activation and IP-10 production to facilitate viral multiplication, whereas L19 inhibited viral multiplication in A549 cells bearing TLR3 on their cell membrane. CONCLUSION: Our results suggest that L19 regulates TLR3 signaling, which is cell type specific and may be involved in pathogenesis of autoimmune diseases and chronic inflammatory diseases.

Bioinformatics analysis and function prediction of the novel gene AY358935 / 南方医科大学学报

<p><b>OBJECTIVE</b>To obtain the functional information of AY358935 gene.</p><p><b>METHODS</b>The properties, subcellular location, and structure of AY358935 protein, and the expression profile of AY358935 gene were analyzed by bioinformatics software and the biological functions of the gene were predicted. AY358935 expression was detected by Western blot analysis in early virus infection.</p><p><b>RESULTS</b>AY358935 was evolutionally conserved. The human AY358935 protein had an amino acid similarity of 74%, 60%, 38% and 33% with its counterpart in horses, mice, zebrafish and Xenopus laevis, respectively. Bioinformatics analysis indicated that AY358935 protein was located likely in the mitochondria. There was a N-terminal signal peptide and single transmembrane structure in AY358935 protein, which contained several phosphorylation sites. The secondary structure mainly comprised of alpha helices and random coils. AY358935 was ubiquitously expressed in normal tissues and carcinomas and regulated by the expression of double-stranded RNA-dependent protein kinase. AY358935 protein expression was obviously upregulated in cells 2 h after infection by vesicular stomatitis virus.</p><p><b>CONCLUSION</b>As a predicted secretary protein with a small molecular weight, AY358935 might have important functions in cellular proliferation and anti-viral innate immune regulation.</p>

The Effect of Recombinant Baculovirus (BacG-CMV-P53) Mediated Gene Therapy in the HT-1376 Bladder Cancer Cell Line / 대한비뇨기과학회지

PURPOSE: In preliminary studies, it was found that mammalian cells can be infected by recombinant baculovirus in vitro. Therefore, the potential use of recombinant baculovirus(BacG-cytonegalovirus(CMV)-P53) for the bladder gene therapy was investigated. MATERIALS AND METHODS: The recombinant Baculovirus(BV) pseudotyped was developed with the vesicular stomatitis virus(VSV) G protein. The presence of the VSV-G protein in purified BV preparations was confirmed by Western blotting analysis. The bladder cancer cells of human(HT-1376) were infected with various multiplicity of infection(MOI) of the BV, and the percentage of apoptotic cells determined by methyl thiazolyl tetrazolium(MTT) assay. RESULTS: The suppression effect of the recombinant BV with a P53 insertion in human bladder cancer cells(HT-1376) increased as the MOI of the recombinant BV increased; 100% cell survival in the group with PBS, and 81.6+/-4.3, 52.0+/-5.6 and 39.8+/-3.7% at 1, 10 and 100 MOI, respectively (p<0.05). CONCLUSIONS: Significant growth suppression was observed following infection with BacG-CMV-P53 in a human bladder cancer cell line. This observation suggests that BacG-CMV-P53 may be a potentially effective agent to prevent recurrence for P53 mutated bladder cancer. Bladder gene therapy using recombinant baculovirus could be a safe and effective treatment of bladder cancer.

The Effect of Recombinant Baculovirus (BacG-CMV-P53) Mediated Gene Therapy in the HT-1376 Bladder Cancer Cell Line / 대한비뇨기과학회지

PURPOSE: In preliminary studies, it was found that mammalian cells can be infected by recombinant baculovirus in vitro. Therefore, the potential use of recombinant baculovirus(BacG-cytonegalovirus(CMV)-P53) for the bladder gene therapy was investigated. MATERIALS AND METHODS: The recombinant Baculovirus(BV) pseudotyped was developed with the vesicular stomatitis virus(VSV) G protein. The presence of the VSV-G protein in purified BV preparations was confirmed by Western blotting analysis. The bladder cancer cells of human(HT-1376) were infected with various multiplicity of infection(MOI) of the BV, and the percentage of apoptotic cells determined by methyl thiazolyl tetrazolium(MTT) assay. RESULTS: The suppression effect of the recombinant BV with a P53 insertion in human bladder cancer cells(HT-1376) increased as the MOI of the recombinant BV increased; 100% cell survival in the group with PBS, and 81.6+/-4.3, 52.0+/-5.6 and 39.8+/-3.7% at 1, 10 and 100 MOI, respectively (p<0.05). CONCLUSIONS: Significant growth suppression was observed following infection with BacG-CMV-P53 in a human bladder cancer cell line. This observation suggests that BacG-CMV-P53 may be a potentially effective agent to prevent recurrence for P53 mutated bladder cancer. Bladder gene therapy using recombinant baculovirus could be a safe and effective treatment of bladder cancer.

Vírus da estomatite vesicular (Sorotipo Indiana 2) como modelo experimental para o estudo de encefalite aguda: aspectos morfológicos / Vesicular stomatitis virus (Indiana 2 Serotype) as experimental model to study acute encephalitis: morphological features

O vírus da estomatite vesicular (VEV) é um vesiculovírus da família Rhabdoviridae que infecta mamíferos e causa lesões vesiculares semelhantes às observadas na febre aftosa. A encefalite experimental pode ser induzida em roedores e os sintomas são semelhantes aos observados na raiva; entretanto, as lesões observadas no encéfalo dos animais são diferentes. Corpúsculos de inclusão não são observados, há necrose especialmente da região do bulbo olfatório e em alguns casos, ventriculite. Observamos que o padrão temporal de disseminação do VEV e os aspectos morfológicos das lesões são similares aos descritos na literatura. O vírus parece se disseminar através dos ventrículos cerebrais, multiplicando-se em células do epêndima e em neurônios, além de utilizar o transporte retrógrado e anterógrado. Constatamos que devido à facilidade de manipulação do vírus, este modelo experimental tem sido utilizado em inúmeros trabalhos de pesquisa em diversas áreas. Se por um lado, os relatos sobre a patogenia da infecção são numerosos, por outro lado, ainda existem muitas lacunas que envolvem, por exemplo, aspectos sobre a transmissão do vírus, a recuperação dos animais infectados e a participação de células gliais durante a fase aguda e a fase de recuperação dos animais

Interferon consensus sequence binding protein: Not essential for interferon alpha-mediated antiviral response to vesicular-stomatitis virus infection in HL-60 cells

BACKGROUND: The role of the interferon consensus sequence binding protein (ICSBP), a member of interferon regulatory factor family, in protecting against a vesicular stomatitis virus (VSV) infection has not been firmly elucidated. Thus, it was investigated utilizing the human promyelocytic leukemia HL-60 cells which do not express ICSBP. METHODS: HL-60 cells were stably transfected with plasmid containing cDNA for either ICSBP or DNA binding domain (DBD) and tested for their VSV-susceptibilities. The susceptibility of each transfectant group to a VSV infection was determined by a plaque assay at 1 h, 24 h, and 48 h post-infection in the presence (500 IU/ml) or absence of interferon alpha(IFN alpha). RESULTS: In the absence of IFN alpha, the three groups showed similar sensitivities to a VSV infection. However, when pre-treated with IFN, the viral titers in both the ICSBP and control clones steadily decreased over 48 h of incubation, indicating the existence of IFN alpha-mediated protection against VSV infection. The IFN alpha-treated ICSBP clones appeared to be more resistant to infection compared with the control clones, although the difference was not great . On the contrary, the viral titers in the IFN alpha-treated DBD clones increased at 24 h then decreased by 48 h. CONCLUSION: The expression of truncated ICSBP (DBD) does not appear to underlie the impaired protection against a VSV infection in the DBD clones, since even the control clones lacking ICSBP were protected from a VSV infection. This suggests that ICSBP does not play a critical role in the IFN alpha-mediated anti-VSV response of HL-60 cells, although it appears to confer some resistance to a VSV infection.

The Role of Noncoding Region in Hantaan Viral S Genome for Expression of Nucleocapsid Protein

The genome of Hantaan virus, the prototype of the hantavirus genus, is composed of three segmented, single stranded negative sense RNA genome. The 5' and 3' termini of the Hantaan virus RNA genome contain noncoding regions (NCRs) that are highly conserved and complementary to form panhandle stuctures. There are some reports that these NCRs seems to control gene expression and viral replication in influenza virus and vesicular stomatitis virus. In this study, we examined whether NCRs in Hantaan virus play a role in expression of the viral nucleocapsid protein (Np) and foreign (luciferase) gene. The 5' and/or 3' NCR-deleted mutants were constructed and analysed. The Np expression of 5' NCR-deleted clone, it showed 40% reduction. To investigate the role of NCR in foreign gene expression, the clones which are replaced ORF of Hantaan viral Np gene with that of luciferase gene were constructed. The results were similar to those of the experiments using Np gene. These results suggest that 3' NCR is more important than 5' NCR in protein expression. To find out a critical region of 3' NCR in more important than 5' NCR in protein expression. To find out a critical region of 3' NCR in protein expression, several clones with a deleted part of 3' NCR were constructed and analyzed. The deletion of the conserved region in 3' NCR showed 20~30% decrease in Np expression. However there were no change in luciferase activities between clones with or without non-conserved region of 3' NCR. These results suggest that the 3' NCR of Hantaan virus S genome, especially conserved region in 3' NCR, plays and important role in the expression of Hantaan viral Np and foreign genes.