Asunto(s)
Aciclovir/farmacología , Farmacorresistencia Viral/genética , Encefalitis por Herpes Simple/genética , Encefalitis por Herpes Simple/inmunología , Huésped Inmunocomprometido/genética , Aciclovir/uso terapéutico , Anciano , Antivirales/farmacología , Antivirales/uso terapéutico , Encefalitis por Herpes Simple/tratamiento farmacológico , Femenino , Humanos , Timidina Quinasa/genéticaRESUMEN
Here, we describe a novel reliable method to assess the significance of individual mutations within the thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) to nucleoside analogue resistance. Eleven defined single nucleotide polymorphisms that occur in the TK gene of clinical HSV-1 isolates and a fluorescence reporter were introduced into the HSV-1 strain 17(+) that had been cloned into a bacterial artificial chromosome. The susceptibility of these different strains to aciclovir, penciclovir, brivudin, and foscarnet was determined with a modified cytopathic effect reduction assay. The strains were also tested for their aciclovir susceptibility by measuring the relative fluorescence intensity as an indicator for HSV-1 replication and by quantifying the virus yield. Our data indicate that the amino acid substitutions R41H, R106H, A118V, L139V, K219T, S276R, L298R, S345P, and V348I represent natural polymorphisms of the TK protein, whereas G61A and P84L mediate broad cross-resistance against aciclovir, penciclovir, brivudin, and susceptibility to foscarnet. This method allows the definition of the resistance genotype of otherwise unclear mutations in the TK gene of HSV-1. Thus, it provides a scientific basis for antiviral testing in clinical isolates of patients suffering from serious diseases and will facilitate testing of new antivirals against HSV-1.
Asunto(s)
Aciclovir/farmacología , Farmacorresistencia Viral/genética , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/genética , Mutación/genética , Recombinación Genética/genética , Animales , Antivirales/farmacología , Chlorocebus aethiops , Farmacorresistencia Viral/efectos de los fármacos , Herpesvirus Humano 1/enzimología , Cinética , Reacción en Cadena de la Polimerasa , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Transfección , Células Vero , Carga Viral/genética , Replicación Viral/efectos de los fármacosRESUMEN
Serological methods are used widely for the determination of herpes simplex virus (HSV) IgG and IgM antibodies in virological laboratories. The present study evaluates the automated performance of the Virion\Serion (Würzburg, Germany) and Orgentec (Mainz, Germany) enzyme-linked immunosorbent assays (ELISA) for the determination of HSV type-common and type-specific IgG and IgM antibodies. Two hundred sixty-three sera from HSV-negative children, healthy blood donors as well as patients without and with acute HSV infections were included. The Serion ELISAs classic HSV 1+2, HSV 1 and HSV 2 IgG showed sensitivities between 89.1% and 98.0% and specificities from 82.8% to 100%. Sensitivities of the Orgentec ELISAs Anti-HSV-1 and Anti-HSV-2 IgG were calculated as 91.0-96.0% and 88.5-95.4% accompanied by specificities between 93.1% and 100%. The HSV type-common Serion IgM ELISA revealed also a high sensitivity and specificity. However, the single-type HSV-1 and HSV-2 IgM ELISAs from both companies did not detect reliably HSV-1- and HSV-2-specific IgM antibodies. In conclusion, the automated performance of Serion ELISAs classic HSV 1+2, HSV 1 and HSV 2 IgG as well Orgentec ELISAs Anti-HSV-1 and Anti-HSV-2 IgG provide highly dependable results for identifying HSV-1 and HSV-2 IgG-positive or -negative individuals. While HSV type-common IgM ELISAs can be useful to confirm acute newly acquired HSV infections, the use of single-type IgM ELISAs on the basis of whole-virus antigen is dispensable.
Asunto(s)
Anticuerpos Antivirales/sangre , Técnicas de Laboratorio Clínico/métodos , Herpes Simple/diagnóstico , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 2/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Adolescente , Adulto , Anciano , Automatización de Laboratorios/métodos , Preescolar , Pruebas Diagnósticas de Rutina/métodos , Femenino , Humanos , Técnicas para Inmunoenzimas/métodos , Lactante , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Adulto JovenRESUMEN
The analysis of the viral thymidine kinase (TK) genotype is of rising significance for testing resistance of herpes simplex virus (HSV) to antivirals especially acyclovir. However, numerous of the described amino acid (aa) substitutions are diagnostically less conclusive because of the pronounced natural polymorphism of this gene. In this study, several aa substitutions in the TK sequence of HSV-1 with unclear significance for resistance were analyzed by expression of recombinant TK proteins and determination of enzymatic activity on the basis of an enzyme linked immunosorbent assay using bromodeoxyuridine (BrdU) as TK substrate. The recombinant TK wild-type protein resulted in high TK activity and TK mutant with stop of translation showed negative results. The recombinant TK proteins containing the aa substitutions R41H or V348I had high phosphorylation activities suggesting most likely natural gene polymorphisms. By contrast, the aa changes Y53H, L139V, R163H, L298A and L315S were accompanied by negative or weakly positive TK activities indicating resistance association. In conclusion, the combination of methods described here represents a useful tool to evaluate the significance of aa substitutions for resistance of clinical HSV-1 strains.