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      Detección del virus Guanarito mediante la técnica de RT-PCR en un solo paso Translated title: Detection of Guanarito virus by one step RT-PCR


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          Se estandarizó una RT-PCR en un solo paso para la detección del virus Guanarito (VGTO), con el propósito de sustituir la técnica de cultivo celular, empleada para el diagnóstico de la fiebre hemorrágica venezolana, y minimizar el riesgo biológico por exposición a este virus. Se diseñaron cebadores específicos para la detección del segmento S del gen de la nucleocápside de VGTO y se estandarizó un protocolo donde se analizaron 44 muestras: 18 sueros agudos, 11 macerados de tejidos y 15 sobrenadantes de cultivos en células Vero-E6 como controles positivos. Los resultados obtenidos por la RT-PCR fueron comparados con la técnica de cultivo celular, encontrándose un 100% de sensibilidad y especificidad, con valores predictivos positivos y negativos de 97,7% y 100%, respectivamente. El coeficiente de correlación de Pearson entre ambas pruebas fue de 99%. Este es el primer reporte del uso de una metodología para el diagnóstico molecular específico del VGTO en Venezuela.

          Translated abstract

          A one-step RT-PCR was standardized for the detection of Guanarito virus (VGTO), with the purpose of replacing the cell culture technique, used for the diagnosis of Venezuelan hemorrhagic fever, and to minimize the biological risk from exposure to this virus. Specific primers were designed for the detection of the S segment of the nucleocapsid gene of VGTO. A protocol was standardized where 44 samples were analyzed: 18 acute sera, 11 macerated tissues and 15 culture supernatants in Vero-E6 cells as positive controls. The results obtained by the RT-PCR were compared with the cell culture technique, finding 100% sensitivity and specificity, with positive and negative predictive values of 97.7% and 100%, respectively. The Pearson correlation coefficient between both tests was 99%. This is the first report of the use of a methodology for the molecular diagnosis specific to VGTO in Venezuela.

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          Most cited references12

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          The phylogeny of New World (Tacaribe complex) arenaviruses.

          Several New World (Tacaribe complex) arenaviruses (Arenaviridae) are known to cause severe hemorrhagic disease in humans. Phylogenetic reconstruction of the Tacaribe complex arenaviruses previously has been limited by the relative scarcity of sequence data for arenavirus genomes. In the present study, oligonucleotide primers were designed based on conserved regions of the nucleocapsid (N) protein gene and then used to amplify, by reverse transcription--polymerase chain reaction, a 613-to 649-nucleotide region of the N gene of all known Tacaribe complex arenaviruses. This has allowed completion of the first detailed genetic characterization and phylogenetic analysis of all known members of the Tacaribe complex. These viruses formed three lineages. Lineage A contained Flexal, Parana, Pichinde, and Tamiami viruses; lineage B contained Amapari, Guanarito (GUA), Junin (JUN), Machupo (MAC), Sabia (SAB), and Tacaribe viruses. Latino and Oliveros viruses occupied lineage C. The highly pathogenic Tacaribe complex arenaviruses (GUA, JUN, MAC, SAB) were all members of lineage B, suggesting the possibility that the highly pathogenic phenotype is the result of evolutionary radiation from a common ancestor. The approach described here provides a rapid method for characterization of novel Tacaribe complex arenaviruses and may provide clues as to their potential public health importance.
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            Description of Guanarito virus (Arenaviridae: Arenavirus), the etiologic agent of Venezuelan hemorrhagic fever.

            This paper characterizes Guanarito virus, the etiologic agent of Venezuelan hemorrhagic fever. Based on its morphology and antigenic properties, Guanarito virus appears to be a new member of the Tacaribe complex of the genus Arenavirus, family Arenaviridae. Complement fixation and indirect fluorescent antibody tests showed that Guanarito virus and its antiserum are broadly cross-reactive with other members of the Tacaribe complex, but it can be differentiated from other members of the complex by neutralization test. Guanarito virus causes mortality in suckling mice and adult guinea pigs, but not in adult mice. Inoculated rhesus monkeys developed viremia and became ill; however, they subsequently recovered and responded with production of antibody. To date, all isolates of Guanarito virus have come from sick persons or wild rodents living within a single geographic focus in the central plains of Venezuela.
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              Phylogeny of New World arenaviruses based on the complete coding sequences of the small genomic segment identified an evolutionary lineage produced by intrasegmental recombination.

              Previous studies suggested that the small genomic segments (S-RNA) of the South American arenaviruses (SA-AVs) represent three phylogenetic lineages (designated A, B, and C) and indicated that the S-RNA of Whitewater Arroyo virus (WWAV) (a North American arenavirus [NA-AV]) is a product of genetic recombination between a lineage A and lineage B virus. The purpose of this study was to extend our knowledge on the phylogenetic relationships between WWAV, the two other NA-AVs (Tamiami and bear canyon), and the 15 SA-AVs. Therefore, we determined the complete sequence of the S-RNA of nine arenaviruses previously uncharacterized or sequenced only partially. Phylogenetic analyses of the two complete coding regions indicated that the S-RNA of the three NA-AVs have descended from a single ancestral virus, which was the product of recombination between a lineage A and lineage B arenavirus. No such evidence for genetic recombination was found in cupixi virus (a novel arenavirus isolated from a wild rodent captured in Northeastern Brazil) or the 14 other SA-AVs. The recombinant nature of the S-RNA of NA-AVs distinguishes them from the SA-AVs, and thus, indicates that the NA-AVs represent a fourth phylogenetic lineage in the Tacaribe serocomplex.

                Author and article information

                Revista de la Sociedad Venezolana de Microbiología
                Rev. Soc. Ven. Microbiol.
                Organo Oficial de la Sociedad Venezolana de Microbiología. (Caracas, DF, Venezuela )
                December 2017
                : 37
                : 2
                : 82-86
                [01] Caracas orgnameInstituto Nacional de Higiene Rafael Rangel orgdiv1Gerencia Sectorial de Diagnóstico y Vigilancia Epidemiológica Venezuela
                S1315-25562017000200011 S1315-2556(17)03700200011


                : 10 October 2017
                : 02 February 2017
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 12, Pages: 5

                SciELO Venezuela

                Comunicación Corta

                fiebre hemorrágica venezolana,Guanarito virus,RT-PCR,Venezuelan hemorrhagic fever,Virus Guanarito


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