Construction and characterization of EGFP reporter gene labeled Sindbis virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct and characterize EGFP reporter gene labeled Sindbis virus (SINV).</p><p><b>METHODS</b>The reporter gene EGFP was inserted into the genome of infectious clone pBR-XJ160 by using multi-fusion long fragment PCR method. Then apply reverse genetic manipulation technique to rescue and obtain EGFP labeled SINV.</p><p><b>RESULTS</b>We successively obtained labeled SINV, which has good fluorescent expression characteristics and genetic stability.</p><p><b>CONCLUSION</b>The labeled virus can be seen in living cells and living body, and this serves as a good tool for cell and tissue tropism and biological function study of viruses. This study laid a foundation for further studying the cell tropism, biological functions and infection mechanism of SINV.</p>

Preparation and identification of a recombinant hepatitis C virus (HCV) based on sindbis virus vector / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct the recombinant virus-like particles containing HCV envelope glycoprotein E1E2 based on sindbis virus vector.</p><p><b>METHODS</b>The gene encoding HCV envelope glycoprotein E1E2 was cloned into sindbis virus vector to construct recombinant plasmids pBR-XJE1E2 and pVA-XJE1E2, and transfect them into BHK-21 cells to obtain recombinant virus-like particles. The expression of E1 and E2 protein were verified by Western Blot and indirect immunofluorescent assay (IFA).</p><p><b>RESULTS</b>The results of restriction enzyme digestion, PCR and sequencing analysis showed that the recombinant plasmids were constructed successfully. And the results of RT-PCR, Western blotting and IFA detection showed that the transfect cells could package HCV-like particles of expressing structural proteins E1E2.</p><p><b>CONCLUSION</b>The recombinant expression plasmids pBR-XJE1E2 and pVA-XJE1E2 based on sindbis virus vector could package HCV-like particles in eukaryotic cell, which provides a foundation for further study of its in vivo animal immune response.</p>

Effects of the site-directed mutagenesis at nsP2-726Pro on replicon vector derived from XJ-160 virus / 病毒学报

To investigate the effects of site-directed mutagenesis at nsP2-726Pro on the characteristics of replicon vector derived from XJ-160 virus, a Sindbis virus (SINV) isolated in China. The mutant vector pBRep-726L, pBRep-726S, pBRep-726V or pBRep-726A was constructed by introducing nsP2-726Pro --> Leu, nsP2-726Pro --> Ser, nsP2-726Pro --> Val or nsP2-726Pro --> Ala into XJ-160 viral replicon vector pBRepXJ respectively. To quantitatively and qualitatively determine the site-directed mutagenesis on the replicon, the recombinant plasmids expressing Neomycinr (Neo(r)), enhanced green fluorescent protein (EGFP) or Renilla luciferase (R. luc) were constructed by cloning report genes into pBRepXJ or mutant XJ-160 vector respectively. And in vitro-synthesized RNA from expression vectors were electroporated into BHK-21 cells. Compared with the wild-type replicon, the mutation nsP2-726Pro --> Val or nsP2-726Pro --> Ala accelerated the processing of CPE on BHK-21 cells and simultaneously enhanced its self-replicating capacity. The mutant vector pBRep-726L with Leu substitution exhibited similar packaging capacity to that of pBRepXJ. In contrast, pBRep-726S exhibited a medium phenotype, including the process of CPE and the activity of R. luc expression in BHK-21 cells. The site-directed mutagenesis at nsP2-726Pro not only regulates directly XJ-160 virus vector-host cell interactions, but also plays an important role in its packaging capacity. All of these results lay a basis for researching the relation between the structure and function of alphavirus genome and developing alphavirus vector system with Chinese intellectual property.

Molecular basis of one-way serological reaction between SINV and XJ-160 virus / 病毒学报

The purpose of this study is to elucidate the molecular mechanism of one-way serological reaction between XJ-160 virus and SINV by recombinant viruses which exchanged the glycoprotein genes individually or simultaneously. Three recombinant viruses were obtained based on the whole-length infectious cDNA clone of XJ-160 virus. The infectivity and pathogenesis to BHK-21 cells and animals were studied and the gene which controlled this one-way serological reaction phenomenon was searched by MCPENT. The results showed that the E2 glycoprotein was the main factor which influenced the growth rate, plaque morphology and pathogenicity of BHK-21 cells and suckling mice. The results of MCPENT showed that the E2 glycoprotein of SINV played a major role in this one-way serological reaction phenomenon. Our study identified the SINE2 gene was the determined gene for one way serological reaction between XJ-160 virus and SINV, and this research laid the foundation for further analysis of the genomic structure and function of SINV.

Construction and identification of replicon vector derived from an infectious full-length cDNA clone of a Sindbis virus / 病毒学报

To construct vector system of XJ-160 virus, a Sindbis virus isolated in China, recombinant vector pBRepXJ together with its helper plasmid pBR-H were derived from XJ-160 viral infectious clone pBR-XJ160 by overlap-PCR. To quantitatively and qualitatively verify the function of the replicon system, recombinant plasmids pSinRep-EGFP, pBRepXJ-EGFP, pSinRep-R and pBRepXJ-R were constructed by cloning report genes of enhanced green fluorescent protein (EGFP) or Renilla luciferase (R. luc) into pBRepXJ or pSinRep5, a commercial Sindbis vector. And in Vitro-synthesized RNA from expression vectors were electroporated into BHK-21 cells. The results indicated that the replicon vector system was capable of self-replicating in host cell, and the expression efficiency of heterologous genes corresponded with that of the commercial Sindbis vector (pSinRep5). Our study laid the basis for developing alphavirus vector system with Chinese intellectual property.

Using real-time PCR to evaluate the effect of viral inactivation by Methylene Blue with visible light / 病毒学报

To investigate the feasibility of using Real-Time PCR to evaluate the effectiveness of Sindbis virus inactivation by Methylene Blue with visible light. Sindbis virus was treated by Methylene Blue with different intensity of visible light and the transcribed cDNA was quantified by Real-Time PCR. Residual infectivity of treated virus was tested by cell infection method as parallel control at the same time. The residual infectivity of virus decreased from 6.50 lgTCID50/mL to under the limit of detection as light intensity increased. Meanwhile, the quantity of virus cDNA decreased significantly (P < 0.05), which correlated to the decline of virus infectivity (R2 > 0.98). Methylene Blue with visible light could cause lesion to nucleic acid of Sindbis virus, the extent of which was light intensity-dependent and correlated to the decrease of virus infectivity. The results demonstrated that Real-Time PCR can be a useful tool for evaluating effect of virus inactivation after Methylene Blue treatment with light.

Prostaglandin A1 inhibits the replication of Sindbis virus in monkey kidney and mosquito cells

The present study reports the effect of prostaglandin A1 (PGA1) on the replication of Sindbis virus in monkey kidney and mosquito cells. In PGA1 treated cells we observed a severe reduction of virus yield. In both cells lines the highest nontoxic concentration of PGA1 (10 ìg/mL) decreased virus replication, dose dependently, by more than 90 por cento. SDS-PAGE analysis of [35S] methionine labeled proteins showed that viral proteins (E1/E2 and C) were normally synthesized in PGA1 treated Vero cells, and induction of stress proteins (HSP70 and HSP90 ) was detected in uninfected and infected cells. In Vero cells the inhibition of virus replication was accompanied by a decrease in [3H] glucosamine incorporation into the virus glycoproteins.

Intracellular Localization of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

Porcine reproductive and respiratory syndrome virus (PRRSV) is a small enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. It causes the porcine reproductive and respiratory syndrome in swine. The virus has 7 structural proteins: Of the seven, the N protein is the nucleocapsid that comprises a core of the virus particle. We have expressed the N protein of PRRSV PL97-1/LP1 strain using a heterologous gene expression vector derived from Sindbis virus, called pSinrep5. Immunofluorescence analysis showed that the N proteins were mainly found in the cytoplasm as well as in the nucleus of BHK-21 cells transfected with pSinrep5-N-derived RNA. Moreover, expression of the N protein did not change the incompetence of RNA replication of Mutant/nt14900 that lacks a 3' cis-acting replication element and the efficiency of RNA replication of Mutant/nt14800 that has a low level of RNA replication. Overall, our findings are consistent with previous results and help to understand a role of the N protein in PRRSV biology.

Detection of Sindbis virus-specific nucleic acid with SYBR GREEN I real time PCR assay / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To develop a rapid, specific and sensitive method for detecting Sindbis virus (SINV) with SYBR GREEN I real time PCR.</p><p><b>METHODS</b>Total RNA of strains of Sindbis virus and a related virus were extracted and reverse transcribed to cDNAs. With the cDNAs as template, the SYBR GREEN I real time PCR assay was developed and optimized on ABI 7300 apparatus for specific-detection of Sindbis virus, and the sensitivity, specificity and reproducibility were evaluated.</p><p><b>RESULTS</b>For the PCR, 55 degrees C was chosen as the optimal anneal temperature and 0.5 micromol/L as the optimal primer concentration. Using this method, all the selected SINV were detected as positive, while the results of control arboviruses such as Geta virus, Japanese encephalitis virus (JEV), Batai virus, Seadornavirus Orbiviruses and synthesized WEEV cDNA were negative. With this system, 0.1 PFU/ml SINV cDNA could be detected; The sensitivity of this assay was about 100 times higher than standard RT-PCR. All the results were reproducible within two compatible tests, and the stability of the detection system was very good. The test results of simulated infection human serum samples showed that human serum had no obvious interference with this system. With this system, 6 of 151 clinical samples with unknown fever or encephalitis were determined as positive.</p><p><b>CONCLUSION</b>The developed SYBR GREEN I real time PCR assay for detecting Sindbis virus was highly sensitive, specific and showed a good reproducibility and stability. It is our belief that the present method can be further used in clinic sample to verify its stringency.</p>

Expression of the Structural Proteins of Japanese Encephalitis Virus

Japanese encephalitis virus (JEV) is one of the most important human pathogens, which causes the permanent neuropsychiatric sequelae and even fatal diseases with high mortality and morbidity, especially among children. In this study, we expressed the structural proteins (C, prM, and E) of JEV using a Sindbis virus-based heterologous gene expression vector, the pSinRep5. We designed two expression vectors (pSinRep5/JEV C-E and pSinRep5/JEV C-NS1), which encode the precise coding sequence of JEV C-E and JEV C-NS1 proteins, respectively. These cloned JEV structural protein genes were designed to express under the Sindbis virus subgenomic promoter. Upon the transfection and expression of the pSinRep5/JEV C-E or pSinRep5/JEV C-NS1 plasmid, the transfected cells expressed approximately 55 kDa JEV E prtiens. As designed, the JEV NS1 proteins were expressed only in the SinRep5/JEV C-NS1 RNAtransfected cells. In addition, we found in the pSinRep5/JEV C-NS1-transfected cells that the viral proteins were predominantly localized around the perinuclear membranes. On the other hand, cytoplasmic staining was mainly observed in the pSinRep5/JEV C-E RNA-transfected cells in the absence of NS1 protein. Thus, our system will provide a useful tool to dissect intracellular membrane localization signals located in the JEV structural proteins without handling the infectious JEV viral particles and to characterize viral morphogenesis of this pathogen.

Susceptibility of Aedes krombeini cell line to some arboviruses.

The susceptibility of the newly established Ae. krombeini cell line (NIVI-AK-453) to six arboviruses, belonging to four different families, was studied. Sindbis (SIND), Vesicular stomatitis (VSV) Chandipura (CHP) and African horse sickness (AHS) viruses multiplied in these cultures. A four-to-five-fold increase in the virus titres was observed. The maximum titre of SIND, VSV, CHP and AHS viruses were observed on 1st, 4th, 3rd and 10th post infection days, respectively. A steady and significant increase in the titre of AHS was observed over a period of ten days. The sandfly fever virus (SFV) and the tick-borne, Kaisodi virus did not multiply in the cultures.

Preservaçäo de interferon de membrana amniótica humana / Preservation of the human amniotic membrane interferon

Foram realizadas investigaçöessobre a presença do interferon de membrana amniótica humana (IFN-AM) a fim de permitir pesquisas posteriores a respeito de sua purificaçäo, propriedades químicas e biológicas e ensaios clínicos.Testes de inativaçäo rápida foram realizados para ensejar resultados imediatos.IFN-AM foi preparado pela infecçäo de âmnios pelo vírus da doença de Newcastle.A atividade anti-vírica do IFN-AM foi determinada em um sistema de células Vero-vírus da encefalomiocardite de camundongo ou vírus Sindbis.Para os testes de inativaçäo, preparaçöes de IFN-AM contendo 0;1 e 2 por cento de soro de carneiro foram aquecidas em temperaturas de 35ºC a 100ºC em tempos variáveis e foi medida a atividade antivírica residual.Destes dados, foi calculada a constante de Arrhenius de queda de atividade pelo calor, que foi representada graficamente contra a temperatura absoluta.Os dados de temperaturas altas(45ºC e acima) foram extrapolados para temperaturas baixas(37ºC e abaixo).Os resultados mostraram que o IFN-AM preservou-se melhor em pH 2 em presença de soro.Os dados obtidos nas temperaturas de 45º, 55º e 65ºC permitiram o cálculo de constante de Arrhenius de queda para baixas temperaturas por extrapolaçäo.A -4, 2ºC näo deverá ocorrer perda de ativadade do IFN-AM.

Japanese encephalitis virus surveillance in the Taipei area, Taiwan in 1978.

Attempts were made to isolate Japanese encephalitis virus from mosquitoes collected with three light traps operated twice a week at three sentinel pigpens in the Taipei area from May to Oct. 1978. A total of 6,549 mosquitoes trapped alive were processed in 283 pools. Suspensions were inoculated into Aedes albopictus cell cultures and into suckling mice. A total of 19 JE virus isolates were made; 12 from pools of Cx. tritaeniorhynchus, 6 from Cx. annulus and one from Cx. quinquefaciatus. Sindbis virus was isolated for the first time in Taiwan from a pool of Cx. tritaeniorhynchus. More isolates were made from mosquito cell cultures (20) than by mouse inoculation (6). Positive isolations of JE virus by mouse inoculation were all from the mosquitoes obtained during an 11 day period from 20 to 30 June 1978 when all six sentinel pigs were viremic. This study shows the decided advantage of the use of mosquito-cell cultures over that of mice.

Alphaviruses in Peninsular Malaysia: II. Serological evidence of human infection.

A serum survey of several characteristic groups of humans in urban, rural, and forested areas of Peninsular Malaysia for evidence of infection with three alphaviruses (Sindbis, getah, and chikungunya) was made on 4384 specimens collected between 1965 and 1969. Analysis of the serological results indicated that 1) persons residing in predominantly rural and forested areas have higher frequencies of specific alphavirus antibody of all three viruses than persons residing in urban areas, 2) human infection with chikungunya virus appears to be at a low level of activity but is widespread, although more common and recent in the northern part of the country, and 3) Sindbis and getah viruses probably do not represent a threat to the public health, but chikungunya virus remains a potential menance and may be responsible for future epidemics transmitted by A. aegypti and A. albopictus mosquitoes.

Alphaviruses in Peninusular Malaysia: I. Virus isolations and animal serology.

A survey of the activity of three alphaviruses (Sindbis, getah and chikungunya) in Peninsular Malaysia was conducted between 1962 and 1970. Serum samples were examined from 3,917 vertebrates representing a wide variety of wild and domestic animals throughout the peninsula for hemagglutination-inhibiting and neutralizing antibodies. A total of 548,939 mosquitoes were collected from different habitats, including jungle, rural, suburban and urban areas, and the majority of the females taken were examined for the presence of virus. Two strains of Sindbis virus and one strain of getah virus were isolated from pools of Culex mosquitoes collected in and around domestic animal shelters. Analysis of the serological results indicated that, 1) getah virus is associated principally with large domestic animals, particularly swine, 2) Sindbis virus is associated with large domestic animals and birds, especially domestic ducks, and 3) chikungunya virus, which has not yet been isolated in Malaysia, appeared to be present at a very low level of activity, probably with wild monkeys as the vertebrate hosts.