ABSTRACT
Hantaviruses are negative-strand RNA viruses that contain three segmented (L/M/S) genome and belong to the genus hantavirus of the family Bunyaviridae. Due to such an unique structure of segmented RNA genome, hantaviruses have a possibility to produce reassortants that containing genomic sets mixed with different segments originated from both parental viruses during the genetic interaction. To investigate whether this phenomenon occurs in vitro, Hantaan (HTN) and Seoul (SEO) viruses were co-infected into Vero-E6 cells and virulent Maaji (MAA) virus was superinfected into avirulent Prospect Hill (PH) virus-infected Vero-E6 cells, respectively. To select only reassortants among progeny viruses, well separated plaque clones were analyzed by multiplex RT-PCR. The putative reassortant viruses detected by 1st multiplex RT-PCR were plaque-purified three times and confirmed by 2nd multiplex RT-PCR. Only 3 reassortants like HTN/HTN/SEO, SEO/HTN/HTN and SEO/HTN/SEO and only 2 reassortants like PH/MAA/MAA, MAA/MAA/PH as designated in order of L/M/S of genomic segments have been identified so far. These results indicate that genetic reassortment can be induced by mixed-infection of two more distantly related serotypes of hantavirus. Interestingly, reassortant SEO/HTN/SEO containing HTN viral M RNA segment is isolated more frequently. This implies that preferential selection of M genome segments occurred when RNA genomes were packaged into virion and also the process of packaging of RNA segments into virion is not random phenomenon. These reassortants would be helpful to know whether genetic reassortment is dependent on genetic distance between hantaviruses and which viral RNA segment plays an important role in coding for virulence marker. Therefore, genetic reassortment can be useful genetic tool to understand genetical, and biological function of hantavirus.
Subject(s)
Humans , Bunyaviridae , Clinical Coding , Clone Cells , Genome , Orthohantavirus , Parents , Product Packaging , Reassortant Viruses , RNA , RNA Viruses , RNA, Viral , Seoul , Virion , VirulenceABSTRACT
A system for the expression of synthetic hantavirus-like luciferase RNA was developed using Hantaan (HTN) virus as a helper virus. The hantavirus-like luciferase RNA was constructed by the deletion of the coding region in HTN virus (76~118) S genome and by replacement of a luciferase gene. PCR was performed using primers designed to amplify the whole region of hantavirus-like foreign gene. The resulting PCR product was placed under the control of the T7 RNA polymerase promoter for in vitro transcription. The produced hantavirus-like luciferase RNA was transfected into Vero-E6 cell infected with HTN virus using lipofectin. The level of expression was measured using a luminometer. The hantavirus-like luciferase RNA was allowed to amplify and express. And also, the hantavirus-like luciferase RNA was packaged into HTN virions. The 5' terminal and 3' terminal conserved sequences of HTN virus genome were sufficient to provide the signals for RNA amplification and packaging. This suggests that the RNA promoter region for hantavirus RNA synthesis is located in 3' terminal region. The luciferase activity was analyzed from the progeny virus-infected cells in order to examine if the 5' and 3' terminal sequences play a role in regulating a packaging pathway while genome of HTN virus is packaged. The luciferase activity was detectable in every cell passage. However, the activity of luciferase was decreased gradually after each passage. The fact that the hantavirus-like luciferase RNA can be packaged into progeny virus suggests that the 5' and 3' terminal sequences of HTN virus genome play an important role in regulating a packaging pathway.
Subject(s)
Clinical Coding , Conserved Sequence , DNA-Directed RNA Polymerases , Genome , Orthohantavirus , Helper Viruses , Luciferases , Polymerase Chain Reaction , Product Packaging , Promoter Regions, Genetic , RNA , VirionABSTRACT
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.
Subject(s)
Animals , Clone Cells , Ecthyma, Contagious , Gene Expression , Genome , Hantaan virus , Orthohantavirus , Luciferases , Nucleocapsid Proteins , Nucleocapsid , Orthomyxoviridae , RNA , Vesicular StomatitisABSTRACT
No abstract available.
ABSTRACT
Sero-epidemiologic survey has been carried out to establish serologically the presence of hantavirus in areas of South Africa. The survey was oriented to search natural infection in both of humans and wild rodents and involvement of human disease. The normal human sera were collected from the residents in urban and rural areas of Western Cape, and rural area of Eastern Cape province. The rodent sera came from various species of rodents trapped in Northern Cape and Western Free provinces. The patient sera were selected from the patients of renal failure, pulmonary syndrome and pyrexia of unknown origin (PVO) according to diagnostic chart among the patients hospitalized in major hospitals of Cape Town area. The sera were screened and titrated by IFA test using antigens of Hantaan (HTN), Seoul (SEO), Puumala (PUU), and Prospect Hill (PH) viruses primarily. Positive cases were subjected to differential IFA test using HTN, PUU and PH antigens and plaque reduction neutralization test for further confirmation. Anti-hantavirus antibodies were detected from 2 of 352 rural, 1 of 172 urban residents of E. Cape, and 5 of 118 rural, 5 of 368 urban residents of W. Cape. The antibody was also demonstrated from 5 of 221 wild rodents, and it was appeared that 2 different species, Aethomys namaquensis and Tatera leucogaster, are involved. Among 318 patients tested, 3 who were diagnosed as chronic renal failure, acute respiratory distress syndrome (ARDS) and glomerulonephritis were proved to be positive. The reaction patterns obtained from all of these positive sera were distinct from hantaviral sero-patterns ever established. This result suggests that new viruses may exist in this area and play an possible etiologic role in human disease. The feature of serologic survey on anti-hantavirus antibody demonstrable newly from African wild rodents which are different from reservoir species in other continents elicits a conjecture that the virus may be different from known hantaviruses ever found. This fact also suggests that an expanded role in etiologic involvement with other unknown human diseases by newly emerging hantaviruses may be possible in this areas.
Subject(s)
Humans , Antibodies , Fever , Glomerulonephritis , Orthohantavirus , Hydrogen-Ion Concentration , Kidney Failure, Chronic , Neutralization Tests , Renal Insufficiency , Respiratory Distress Syndrome , Rodentia , Seoul , South AfricaABSTRACT
Hantaviruses are negative-strand RNA viruses that contain three segmented genome and belong to the family Bunyaviridae. Due to such an unique structure of segmented RNA genome, these viruses have a possibility to produce reassortant that have genomic sets mixed with different segments originated from both parental virus during the genetic interaction. To investigate whether this phenomenon occurs actually, Hantaan virus (HTN) could be coinfected with Seoul virus (SEO) to Vero-E6 cell. And also, HTN could be coinfected with Prospect hill virus (PH) to investigate the rate of genetic reassortment according to the genetic distance among the HTN, SEO and PH viruses. As a available method to differentiate the reassortant, we used the multiplex RT-PCR that detect and differentiate the serotype of hantaviruses in mixed infection. Each progeny clone could be screened by multiplex RT-PCR. So, we have constructed the multiplex RT-PCR system that separated amplifications for each segment of Hantaviruses. Our multiplex RT-PCR system provide sensitive, specific and simplified tools for the rapid differentiation of hantaviruses serotypes and also the diagnosis of Hantavirus infections.
Subject(s)
Humans , Bunyaviridae , Clone Cells , Coinfection , Diagnosis , Genome , Hantaan virus , Hantavirus Infections , Orthohantavirus , Hydrogen-Ion Concentration , Parents , RNA , RNA Viruses , Seoul virus , SerotypingABSTRACT
Hantaan virus is the causative agent of rather severe form of hemorrhagic fever with renal syndrome which occurs widely in north-eastern Asia including Korea, China and far eastcrn part of Russia. Although several types of vaccine for this disease have been developed, the therapeutic agent has not been developed yet. Therefore, we launched the construction of ribozyme to be used as the therapeutic purpose of the disease. Ribozyme which cleaves RNA as an enzyme is a RNA oligonucleotide specific to target RNA. We constructed a ribozyme oligonucleotide aimed at S genomic RNA segment of Hantaan virus (strain 76-118) containing T7 promoter region cornplementary to promoter primer oligonucleotide. Then two oligonucleotides were annealed to prepare double stranded transcription template, and transcription was performed in vitro. Thus, we could prepare the clone of whole S segment of the virus by RT-PCR, and then BamHI/HinCII fragment of the S genome segment was subcloned to pT7T319U vector containing T7 promoter in genome sense. The substrate transcript was made by run-off transcription. These substrate and ribozyme transcripts were used to detect cleavage activity of the ribozyme to the target RNA substrate prior to its application to cultured cell. The cleavage reaction showed that the ribozyme cleaves the target RNA which is S segment of Hantaan virus. To know whether the ribozyme works in cell infected with Hantaan virus as well, the ribozyme was transfected to Vero-E6 cell by lipofectin after inoculation of the virus. The transfected ribozyme was detectable in the cell by RT-PCR utilizing ribozyme specific primers. On 7 days after inoculation, the culture media were harvested and used to determinate viral titers by immunoenzyme plaque assay. In contrast to the mock transfected negative control, the viral titers of the cultures transfected at 1, 2 and 3 days after the virus inoculation were lowered to 1/100 level. This result suggests that the ribozyme inhibits the multiplication of Hantaan virus in cultured cell successfully in early stage of infection, and ribozyme is a possible new anti-viral drug against the virus infection.
Subject(s)
Asia , Cells, Cultured , China , Clone Cells , Culture Media , Genome , Hantaan virus , Hemorrhagic Fever with Renal Syndrome , Korea , Oligonucleotides , Promoter Regions, Genetic , RNA , RussiaABSTRACT
Prospect Hill virus (PHV) is related antigenically but distinct to Hantaan virus. As a member of genus Hantavirus, PHV has three segmented RNA genome. Among these segments, Small segment(S) encodes nucleocapsid protein (NP) as structural protein and also may do functional nonstructural protein(NSs). We performed in vitro transcription to produce vRNA of PHV S genome. For the first step of in vitro transcription of S genome of PHV, the S RNA segrnent which is 1,675 nucleotides long was amplified by RT-PCR using PCR primers built according to cDNA sequence of PHV S genome. Next, a new PCR primer appended above downstream primer to T7 phage promoter sequence was reconstructed to obtain PCR product containing T7 promoter. Then another PCR was performed. Using this PCR product as the template, in vitro run-off transcription without cloning by transcriptional vector was performed to obtain viral- sense RNA transcript. Thereafter, the size of transcript was assessed by running on formaldehyde agarose gels. Since the transcription reactants contain a-S UTP, the transcript is detectable by autoradiography. The transcript was also detectable by northern hybridization with a-P dCTP- labelled PHV amplicon probe (319 bp) and the initiation start point of run-off transcription was also determined by primer extention analysis. Our data indicate that the in vitro transcript could be produced from the PCR product amplified by PCR primer containing T7 phage promoter without cloning into a phage transcription vector.