Construction and verification of recombinant chimeric vesicular stomatitis virus expressing rabies virus G protein / 中国生物制品学杂志

@#Objective To construct recombinant chimeric vesicular stomatitis virus(VSV)expressing G protein of rabies virus(RV)using VSV as vector.MethodsTo rescue the recombinant virus,G gene of VSV antigenome was replaced with G gene of RV vaccine CTN-1 strain,and co-transfected into 293T cells with helper plasmids coding T7 RNA polymerase and proteins N,P and L of VSV. The expression of RV G gene and G protein was detected by RT-PCR,immunofluorescence assay and Western blot. The recombinant virus was subcultured in Vero cells,the virus titer of different generations was detected and the virus growth curve was drawn.ResultsThe recombinant virus VSV-RVG was successfully rescued. RTPCR results demonstrated that the RV G gene was successfully inserted into the genome of the recombinant virus,and the expression of RVG protein was detected by immunofluorescence assay and Western blot. The recombinant virus was continuously passaged for 5 generations,and the virus titer was stable within 7. 5 ～ 8. 5 lgTCID50/mL.ConclusionThe recombinant chimeric VSV expressing RV G protein was successfully constructed with good genetic stability,which lays a foundation of the construction of reverse genetics technology platform based on VSV vector.

Enfermedad boca mano pie. Presentación de un caso / Foot, hand and mouth disease. Case presentation

La enfermedad boca mano pie es una infección altamente contagiosa, causada por el virus Coxsackie A16 y el enterovirus 71. La transmisión ocurre por contacto directo con secreciones nasales, orales, materia fecal y gotas aerolizadas, en una ruta fecal-oral o ruta oral-oral, y a través de objetos contaminados. Se presenta el caso de un paciente de cuatro años de edad que acudió a la consulta de estomatología por la presencia de vesículas dolorosas en la mucosa bucal, las cuales dificultaban su alimentación. Además presentaba rash en manos y pies. Luego de indicársele tratamiento estomatológico, fue remitido al pediatra de su área de salud, quien concluyó el diagnostico de enfermedad boca mano pie. El componente bucal de esta entidad constituye, por lo general, el principal síntoma y el motivo de consulta, sin embargo, es poco conocida en el perfil estomatológico. En ello radica el interés de la presentación, ya que el conocimiento de la fisiopatología y el cuadro clínico de la afección, permite al estomatólogo realizar el diagnóstico diferencial y sospechar clínicamente la enfermedad. 

Foot, hand and mouth disease is a highly contagious disease, caused by the A16 Coxsackie virus and 71 enterovirus. The transmission occurs by the direct contact with nasal and oral secretions or fecal material and sprayed drops, in an oral fecal or fecal oral route and through contaminated objects. A case of a 4 year old patient came to the dental office due to the presence of painful blisters in the oral mucosa which made his feeding difficult. In addition he had a rash in hands and feet. After prescribing dental treatment he was referred to the pediatrician of his health area who conclude the diagnosis of foot, hand and mouth disease. The oral component is generally the main symptom and the chief complaint, however, its almost unknown in its oral profile. That is the reason for the interest of this presentation because knowing its physiopathology and the clinical characteristics of the presentation allows differential diagnosis and clinically suspect the disease.

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.

Clinical observation on Yinhuang decoction and external application of Xilei decoction in treating vesicular stomatitis / 国际中医中药杂志

Objective To evaluate the effects ofYinhuangdecoction and external application ofXilei decoction in treating vesicular stomatitis.Methods From January 2012 to October 2013, 126 enrolled cases were randomly divided into a treatment group and a control group with 63 in each. The treatment group, which was treated with acycloguanosine tablets as the control group did, was additionally given orallyYinhuang decoction and external application ofXilei decoction. The symptoms, clinical effects comparison were observed before and after the treatment.Results After 7-days treatment, the cure rate of the treatment group was 95.2%(60/63), and the control group was 73.0%(46/63). Their difference was conspicuous(χ2=17.407,P<0.05). The treatment group attained better curative effects(P<0.05) and got shorter symptomatic relief time of the duration of fever, time of tain disappeared, time of ulcer dissipated and cure time(The T-value is respectively 9.590, 6.983, 5.864, 5.814,P<0.01).ConclusionsYinhuang decoction and external application ofXilei decoction is effective for treating vesicular stomatitis.

Observation on effect of Livin on VSV-induced apoptosis of A549 cells / 肿瘤研究与临床

Objective To investigate the effect of Livin expression on VSV-induced apoptsis of A549 cells.Methods The expression of Livin of A549 cells was inhibited by RNA interference.VSV-induced apoptosis of A549 cells was observed by Tunel assay.Protein Level of livin was detected by Western blot.Caspase-3 activity was detected by the fluorescence-based quantitative method.Results Livin downregulation VSV-induced apoptosis of A549 cells.Inhibited the expression of Livin of A549 cells had increased Caspase-3 activity.Conclusion The effect of Livin on VSV-induced apoptotic of A549 cells could be increased by RNA interference.

Reviewing host proteins of Rhabdoviridae: Possible leads for lesser studied viruses.

Rhabdoviridae, characterized by bullet-shaped viruses, is known for its diverse host range, which includes plants, arthropods, fishes and humans. Understanding the viral–host interactions of this family can prove beneficial in developing effective therapeutic strategies. The host proteins interacting with animal rhabdoviruses have been reviewed in this report. Several important host proteins commonly interacting with animal rhabdoviruses are being reported, some of which, interestingly, have molecular features, which can serve as potential antiviral targets. This review not only provides the generalized importance of the functions of animal rhabdovirus-associated host proteins for the first time but also compares them among the two most studied viruses, i.e. Rabies virus (RV) and Vesicular Stomatitis virus (VSV). The comparative data can be used for studying emerging viruses such as Chandipura virus (CHPV) and the lesser studied viruses such as Piry virus (PIRYV) and Isfahan virus (ISFV) of the Rhabdoviridae family.

Mejoramiento de la producción de una vacuna oleosa contra estomatitis vesicular bivalente / Improvement of oil vesicular stomatitis bivalent vaccine production

El presente estudio calculó diferentes MI (Multiplicidad de Infección) para la producción de cultivos industriales de virus de Estomatitis Vesicular (EV) y evaluó el efecto de la cantidad de glicoproteína G en la inducción de respuesta de anticuerpos neutralizantes contra el virus de EV en cobayos inmunizados con una vacuna oleosa bivalente (Indiana (I) y New Jersey (NJ)). Al establecer el MI más eficiente se logró mejorar la cinética de infección de los cultivos industriales disminuyendo los tiempos de cultivo y mejorando los títulos infectantes. Adicionalmente se encontró que títulos de anticuerpos neutralizantes de cobayos inmunizados con vacuna de EV conteniendo aproximadamente 5 microgramos de glicoproteína G de cada serotipo fueron de 3.66 log10 para I y 4.06 log10 para NJ, los cuales se correlacionan con títulos de protección en bovinos. De este estudio se puede concluir que al seleccionar un mejor MI se puede hacer más eficiente el proceso de producción de cultivos virales industriales de EV y que la formulación de una vacuna contra estomatitis vesicular a partir de la cuantificación de la glicoproteína G puede ser una metodología de gran utilidad en la producción industrial de vacunas de buena calidad.

This experiment assess different MI for Vesicular Stomatitis VS virus industrial culture production and evaluated the effect of glycoprotein G concentration in relation to antibodies induction against VS on guinea pigs vaccinated with oil bivalent vaccine (Indiana I and New Jersey NJ). With efficient MI it was possible to get better kinetic of infection at industrial cultures, reducing time of culture and improving viral titers. In addition, it was found that neutralizing titers of guinea pigs immunized with an EV vaccine containing 5 micrograms of glycoprotein G, were 3.66 log10 for I and 4.06 log10 for NJ, which are correlated to protection titers in cattle. About this study can be concluded that selecting a superior MI, efficiency of industrial VE virus production can be improved; on the other hand, glycoprotein G quantification methodology can be useful for a good quality VS Vaccine industrial manufacture.

Retroviral Expression System and Its Application in Efficient Foreign Gene Expression / 中国生物工程杂志

Retroviral expression system which consists of retroviral vector,envelop protein vector and packaging cell line is an efficient expression system for recombinant protein.It has great potential in gene therapy and biopharmacy.Transcriptional active genome regions are the preferred targets for retrovirus integration.Furthermore,VSV-G protein enables this system a broader host range and makes virus integration more efficient.After infection of high-titer virus,high production clones can be selected through simple screening.So far,the research on retrovirus expression system has developed into application in bio-pharmacy industry.Here the composition of this system and the mechanism of virus transduction and summarize the application and prospect of retroviral expression system are introduced.

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.

Effect of proteolytic digestion on the function of vesicular stomatitis virus ribonucleoprotein complex.

The nucleocapsid protein (49 Kd) of vesicular stomatitis virus is tightly bound to the genome rendering the latter transcriptionally competent. Controlled digestion with chymotrypsin removed a 12 Kd peptide from the complex. The resulting complex failed to serve as template for genome transcription in vitro when the polymerase components L and NS proteins were added. A template-associated protein kinase activity was also lost upon chymotrypsin treatment. However, the cleaved nucleocapsid protein (37 Kd) was still capable of binding tightly with the genome template and retained the epitope recognized by a monoclonal antibody. These results suggest that the nucleocapsid protein possesses separate domains that mediate binding to polymerase complex and maintain the structural integrity of the template.