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      Development of a Chimeric DNA-RNA Hammerhead Ribozyme Targeting SARS Virus

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          Abstract

          Objective: Severe acute respiratory syndrome (SARS) is a severe pulmonary infectious disease caused by a novel coronavirus. To develop an effective and specific medicine targeting the SARS-coronavirus (CoV), a chimeric DNA-RNA hammerhead ribozyme was designed and synthesized using a sequence homologous with the mouse hepatitis virus (MHV). Method: Chimeric DNA-RNA hammerhead ribozyme targeting MHV and SARS-CoV were designed and synthesized.To confirm its activity, in vitro cleavage reactions were performed with the synthesized ribozyme. Effects of the chimeric ribozyme were evaluated on multiplication of MHV. Effects of the chimeric ribozyme on expression of SARS-CoV were evaluated in cultured 3T3 cells. Result: The synthetic ribozyme cleaved the synthetic target MHV and SARS-CoV RNA into fragments of predicted length. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited multiplication of MHV in DBT cells by about 60%. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited the expression of SARS-CoV RNA in 3T3 cells transfected with the recombinant plasmid. The chimeric DNA-RNA ribozyme targeting SARS-CoV significantly inhibited MHV viral activity and expression of recombinant SARS RNA in vitro. Conclusion: These findings indicate that the synthetic chimeric DNA-RNA ribozyme could provide a feasible treatment for SARS.

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          Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome

          Christian Drosten, Stephan Günther, Wolfgang Preiser … (2003)
          The severe acute respiratory syndrome (SARS) has recently been identified as a new clinical entity. SARS is thought to be caused by an unknown infectious agent. Clinical specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and molecular techniques. A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a polymerase-chain-reaction (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60 percent of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clinical specimens from patients with SARS but not in controls. High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum. Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. The novel coronavirus might have a role in causing SARS. Copyright 2003 Massachusetts Medical Society
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            Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.

            J. Milligan, D R Groebe, G Witherell … (1987)
            A method is described to synthesize small RNAs of defined length and sequence using T7 RNA polymerase and templates of synthetic DNA which contain the T7 promoter. Partially single stranded templates which are base paired only in the -17 to +1 promoter region are just as active in transcription as linear plasmid DNA. Runoff transcripts initiate at a unique, predictable position, but may have one nucleotide more or less on the 3' terminus. In addition to the full length products, the reactions also yield a large amount of smaller oligoribonucleotides in the range from 2 to 6 nucleotides which appear to be the result of abortive initiation events. Variants in the +1 to +6 region of the promoter are transcribed with reduced efficiency but increase the variety of RNAs which can be made. Transcription reaction conditions have been optimized to allow the synthesis of milligram amounts of virtually any RNA from 12 to 35 nucleotides in length.
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              The epidemiology of severe acute respiratory syndrome in the 2003 Hong Kong epidemic: an analysis of all 1755 patients.

              Gabriel Leung, Anthony J. Hedley, Lai-Ming Ho … (2004)
              As yet, no one has written a comprehensive epidemiologic account of a severe acute respiratory syndrome (SARS) outbreak from an affected country. To provide a comprehensive epidemiologic account of a SARS outbreak from an affected territory. Epidemiologic analysis. The 2003 Hong Kong SARS outbreak. All 1755 cases and 302 deaths. Sociodemographic characteristics; infection clusters by time, occupation, setting, and workplace; and geospatial relationships were determined. The mean and variance in the time from infection to onset (incubation period) were estimated in a small group of patients with known exposure. The mean and variance in time from onset to admission, from admission to discharge, or from admission to death were calculated. Logistic regression was used to identify important predictors of case fatality. 49.3% of patients were infected in clinics, hospitals, or elderly or nursing homes, and the Amoy Gardens cluster accounted for 18.8% of cases. The ratio of women to men among infected individuals was 5:4. Health care workers accounted for 23.1% of all reported cases. The estimated mean incubation period was 4.6 days (95% CI, 3.8 to 5.8 days). Mean time from symptom onset to hospitalization varied between 2 and 8 days, decreasing over the course of the epidemic. Mean time from onset to death was 23.7 days (CI, 22.0 to 25.3 days), and mean time from onset to discharge was 26.5 days (CI, 25.8 to 27.2 days). Increasing age, male sex, atypical presenting symptoms, presence of comorbid conditions, and high lactate dehydrogenase level on admission were associated with a greater risk for death. Estimates of the incubation period relied on statistical assumptions because few patients had known exposure times. Temporal changes in case management as the epidemic progressed, unavailable treatment information, and several potentially important factors that could not be thoroughly analyzed because of the limited sample size complicate interpretation of factors related to case fatality. This analysis of the complete data on the 2003 SARS epidemic in Hong Kong has revealed key epidemiologic features of the epidemic as it evolved.
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                Author and article information

                Journal
                Journal ID (publisher-id): INT
                Journal ID (nlm-ta): Intervirology
                Journal ID (doi): 10.1159/issn.0300-5526
                Title: Intervirology
                Publisher: S. Karger AG
                ISSN (Print): 0300-5526
                ISSN (Electronic): 1423-0100
                Publication date Subscription-year: 2009
                Publication date (Print): May 2009
                Publication date (Electronic): 07 May 2009
                Volume: 52
                Issue: 2
                Pages: 92-99
                Affiliations
                aDivision of Gastroenterology and Hepatology, Department of Medicine, Nihon University School of Medicine, bAdvanced Research Institute of the Science and Humanities, Nihon University Graduate School, cDivision of Nephrology, Hypertension and Endocrinology, Department of Medicine, Nihon University School of Medicine, dDepartment of Virology III, National Institute of Infectious Disease, and eDepartment of Immunology and Microbiology, Nihon University School of Medicine, Tokyo, Japan
                Article
                Publisher ID: 215946 Other ID: Intervirology 2009;52:92–99
                DOI: 10.1159/000215946
                PubMed ID: 19420961
                SO-VID: 2eb37544-7785-4a19-bc9d-50add9c2522a
                Copyright © © 2009 S. Karger AG, Basel
                License:

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                History
                Date received : 10 November 2008
                Date accepted : 22 February 2009
                Page count
                Figures: 5, References: 33, Pages: 8
                Categories
                Subject: Original Paper

                ScienceOpen disciplines: Internal medicine,Respiratory medicine,Clinical Psychology & Psychiatry,Microbiology & Virology,Infectious disease & Microbiology
                Keywords: Antiviral,SARS virus,Ribozyme,Mouse hepatitis virus
                Data availability:
                ScienceOpen disciplines: Internal medicine, Respiratory medicine, Clinical Psychology & Psychiatry, Microbiology & Virology, Infectious disease & Microbiology
                Keywords: Antiviral, SARS virus, Ribozyme, Mouse hepatitis virus

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