RESUMEN
A quantitative real-time PCR (qPCR) assay using SYBR Green dye was established in order to detect and quantify the proviral DNA of HTLV-1 in peripheral blood mononuclear cells (PBMCs). Primers were designed, and the assay was standardized to amplify a novel, conserved HTLV-1 tax region. Proviral load was normalized to the amount of cellular DNA by quantitation of the human albumin gene. Firstly, the qPCR was assessed determining the specificity, sensitivity, dynamic range and intra- and inter-assay reproducibility of the technique. The limit of detection as determined by PROBIT analysis using dilutions of the standard was 2.97 copies. The assay had an excellent dynamic range from 105 to 10¹ copies per reaction and good intra- and inter-assay reproducibility, CVs less than 2%. Secondly, the performance of the qPCR was tested on 40 HTLV-1 seropositive individuals. Proviral load for HTLV-1 carriers ranged from 2.2×10² to more than 8.3×104 copies/106 PBMCs. The high sensitivity and wide dynamic range allowed the determination of a broad range of HTLV-1 proviral loads in infected individuals. This assay is a valuable alternative diagnostic tool when current available serological assays are insufficient. In addition, it will facilitate the study of the relationship between proviral load and pathogenesis.
Asunto(s)
Genes pX , Infecciones por HTLV-I/diagnóstico , Infecciones por HTLV-I/virología , Virus Linfotrópico T Tipo 1 Humano/aislamiento & purificación , Provirus/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Carga Viral/métodos , Cartilla de ADN/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Provirus/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Carga Viral/normasRESUMEN
The province of San Salvador de Jujuy, located in the northwest of Argentina, is a highly endemic area for HTLV-1 infection and a foci of tropical spastic paraparesis/HTLV-1-associated myelopathy (HAM/TSP). Therefore, to better understand this, we carried out a genetic characterization of a large set of HTLV-1 strains (n = 65) of descendants of Amerindians from this region. The LTR and env regions were analyzed. The genetic analysis showed that all of these new HTLV-1 isolates from Argentina belong to the Transcontinental subgroup A of the HTLV-1a Cosmopolitan subtype, with the exception of three isolates that cluster within the Japanese subgroup B. Interestingly, the majority of the sequences from Jujuy province belonged to a distinct cluster within the Latin America Transcontinental subgroup, referred to here as the Jujuy subcluster, and were characterized by specific signatures in the LTR. Given that the samples analyzed in this study belong to the Amerindian population and the high prevalence of HTLV-1 in Jujuy in contrast to the low prevalence of this virus in the country, it could be that HTLV-1aA was spread in Argentina from the Amerindians to the cosmopolitan population. Moreover, this is the first report of an HTLV-1aB or Japanese subgroup in descendants of non-Japanese people in South America.
Asunto(s)
Infecciones por HTLV-I/virología , Virus Linfotrópico T Tipo 1 Humano/clasificación , Virus Linfotrópico T Tipo 1 Humano/genética , ARN Viral/genética , Argentina , Análisis por Conglomerados , Enfermedades Endémicas , Productos del Gen env/genética , Genotipo , Infecciones por HTLV-I/epidemiología , Virus Linfotrópico T Tipo 1 Humano/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Filogenia , Mutación Puntual , Grupos de Población , Análisis de Secuencia de ADN , Homología de Secuencia , Secuencias Repetidas TerminalesRESUMEN
Serum samples (n: 110) from blood donors and high risk individuals from Cordoba, Argentina with indeterminate HIV-1 and HTLV-I/II Wb profiles were studied for specific antibodies to HTLV-I/II and HIV-1 by indirect immunofluorescence assay (IFA) and for the presence or absence of HIV-1 and HTLV-I/II specific bands by Wb. This study was carried out in order to characterize their putative reactions with HIV-1 and HTLV-I/II proteins and to resolve the retrovirus infection status of these individuals. Results indicated that blood donors sera displaying indeterminate HIV-1 or HTLV-I/II Wb patterns were not immunoreactive to HTLV-I/II and HIV-1 on IFA. However, a high rate of indeterminate HIV-1 and HTLV-I/II Wb samples from high risk individuals had positive HTLV-I/II and HIV-1 IFA results respectively. Our study supports the growing evidence that HTLV-HIV indeterminate seroreactivity in low risk population is due to a cross reaction against nonviral antigens, and in high risk populations the indeterminate samples show serological cross-recognition between HIV-1 proteins and HTLV-I/II proteins on Wb. These results point out the necessity to investigate the HTLV-I/II reactivity in indeterminate HIV-1 samples and viceversa in order to confirm the diagnosis. Finally, this study shows the potential usefulness of IFA in elucidating the status of HIV-1 and HTLV-I/II infection of individuals with indeterminate Wb profiles, thus enabling resolution of retrovirus infection status