ABSTRACT
Japanese encephalitis virus (JEV), a member of mosquito-borne flaviviruses, is the leading cause of viral encephalitis in a large geographic area of Southeast Asia and Australia. JEV contains a single-stranded positive-sense RNA genome, which encodes its own RNA-dependent RNA polymerase (NS5) that is required for genomic RNA replication. In this study, we have described a pair of mouse antisera specific to the N- or C-terminal region of the NS5. Initially, two hydrophilic regions corresponding to the N-terminus and C-terminus of the NS5 protein were individually amplified by reverse transcription-PCR from the genomic RNA of JEV K87P39 strain. The amplified DNA fragments were cloned into a prokaryotic expression vector, pGEX-4T-1; the resulting constructs were used for the expression of GST fusion proteins, designated GST/NS5N and GST/NS5C, in E. coli BL-21 strain. Following immunization of three BALB/c mice with each of the purified GST/NS5N and GST/NS5C, we obtained two pools of the antisera, specifically recognizing the ~103-kDa NS5 and several smaller NS5-related proteins in BHK-21 and Vero cells infected with JEV K87P39 strain. Overall, we have successfully expressed the N- and C-terminal regions of JEV NS5 fused to the C-terminus of GST and generated the mouse antisera capable of recognizing the NS5 and its related proteins in JEV-infected cells. This would provide a valuable reagent for the study of JEV NS5 in the viral life cycle.
Subject(s)
Animals , Humans , Mice , Antibody Formation , Asia, Southeastern , Asian People , Australia , Clone Cells , DNA , Encephalitis Virus, Japanese , Encephalitis, Japanese , Encephalitis, Viral , Flavivirus , Genome , Immune Sera , Immunization , Proteins , RNA , RNA-Dependent RNA Polymerase , Sprains and Strains , Vero CellsABSTRACT
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.
Subject(s)
Humans , Arteriviridae , Cytoplasm , Fluorescent Antibody Technique , Gene Expression , Nucleocapsid , Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Proteins , RNA , RNA Viruses , Sindbis Virus , Sprains and Strains , Swine , Virion , VirusesABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the genus Arterivirus in the family Arteriviridae, is the most important viral pathogens in swine industry worldwide. Here, we have investigated 5' and 3' cis-acting RNA elements required for PRRSV genome replication. Using the infectious PRRSV cDNA, we have manipulated the genomic RNA to generate mutant genomic RNAs, transfected these mutants into susceptible MARC-145 cells, and examined the competence of RNA replication. We found three genetic factors that were essential for viral replication. First, the cap structure present at the 5'-end of the genome was absolutely required for RNA replication. Secondly, polyadenylation of the genomic RNA at the 3'-end was also essential for RNA replication. Thirdly, approximately 100-nucleotide region just upstream of the N protein-coding region was crucial for genomic RNA replication. Taken together, our findings indicate that replication of PRRSV genomic RNA requires three important cis-acting RNA elements: 5' cap structure, 3' poly(A) motif, and an internal sequence of about 100 nucleotides. Further investigation is needed to elucidate the molecular mechanism(s) of how these elements act on PRRSV genome replication.
Subject(s)
Humans , Arteriviridae , Arterivirus , DNA, Complementary , Genome , Mental Competency , Nucleotides , Polyadenylation , Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , RNA , SwineABSTRACT
Japanese encephalitis virus (JEV) is a member of mosquito-borne flaviviruses. To investigate whether there is a cis-acting genetic element in the coding region of the JEV C protein, which is required for viral replication, we generated four mutants by introducing a various size of deletions in each structural protein-coding region, designated as pJEV/Rep/deltaCC/LUC, pJEV/Rep/deltaC/LUC, pJEV/Rep/deltaprM/LUC, and pJEV/Rep/deltaE/LUC, of these, all replicons except for pJEV/Rep/deltaCC/LUC were competent in replication. Since pJEV/Rep/deltaCC/LUC is the same as pJEV/Rep/ deltaC/LUC except for an additional 5' deletion (nt 132~201) in the coding region of the C protein, this region appeared to be essential for RNA replication. This is consistent with the proposed cyclization sequence motif in the 5' region of the C gene, which has been recently shown to be required for replication in other mosquito-borne flaviviruses such as DV, YFV, KUN, and WNV. Thus, our results suggest that a cis-acting genetic element in the coding region of the JEV C protein may play an important role in RNA replication. This study will facilitate the current understanding of JEV RNA replication.
Subject(s)
Humans , Asian People , Capsid Proteins , Clinical Coding , Cyclization , Encephalitis Virus, Japanese , Encephalitis, Japanese , Flavivirus , Replicon , RNAABSTRACT
Japanese encephalitis virus (JEV) is a member of Flaviviruses, transmitted by mosquitoes. The core of JEV is composed of the capsid (C) proteins. In order to produce the recombinant viral C protein and the antiserum specifically recognizing the JEV C protein, we have expressed and purified the JEV C protein as a Glutathion-S-Transferase (GST) fusion protein in E. coli. The JEV C protein-coding region was PCR-amplified using the infectious cDNA of a JEV Korean isolate, and the amplicons were cloned into the pGEX4T-1 E. coli expression vector. GST-C fusion proteins were purified using a glutathione sepharose column. Subsequently, the GST-C fusion proteins were used for immunization of rabbits, and the antisera were obtained from those immunized animals. Western blot analysis using the JEV-infected BHK21 cell lysates showed that these antisera specifically reacted with the JEV C proteins. This study will provide a useful reagent for the diagnosis and understanding of the viral morphogenesis in the JEV-infected cells.
Subject(s)
Animals , Humans , Rabbits , Asian People , Blotting, Western , Capsid Proteins , Capsid , Clone Cells , Culicidae , Diagnosis , DNA, Complementary , Encephalitis Virus, Japanese , Encephalitis, Japanese , Flavivirus , Glutathione , Immune Sera , Immunization , Morphogenesis , Sepharose , Staphylococcal Protein AABSTRACT
Sera of patients visited at the Kangnam St. Mary Hospital in Seoul were collected randomly at the Department of Clinicopathology from January, 1998 to December, 2002. Specimens were collected two twice a month, in a 15-day interval, and 100 specimens were collected at a time. Specimens test in duplicate, and/or displaying antinuclear antibody reaction were excluded from the seroepidemiological analyses. Detection of antibodies to Hantaan virus, an etiologic agent of hemorrhagic fever with renal syndrome (HFRS), was done by indirect immunofluorescent antibody (IFA) technique. Out of 11,361 sera tested, 445 cases (3.9%) showed specific antibody to Hantaan virus. Sexual difference was not noted. Annual incidence of HFRS cases showed a 3 year-periodicity. In the monthly incidence analysis, two peaks of incidence were appeared in the male cases, the first peak in March and the second in August. Female cases showed a single peak in October. The age distribution showed that 64.9% of the sero-positive cases were from 40 to 69 years of age. Peak age-group was in the 6th decade. Each decade of age-group showed diverse patterns of annual and monthly incidences. These results suggest the incidence of HFRS shows a periodicity and a unique pattern in each age group.