Browsing by Author "Pinilla, Milagros"
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Publication Identification of new splice sites used for generation of rev transcripts in human immunodeficiency virus type 1 subtype C primary isolates(Public Library of Science (PLOS), 2012-02) Delgado, Elena; Carrera, Cristina; Nebreda, Paloma; Fernandez-Garcia, Aurora; Pinilla, Milagros; Garcia, Valentina; Perez-Alvarez, Lucia; Thomson, Michael M; Ministerio de Ciencia e Innovación (España)The HIV-1 primary transcript undergoes a complex splicing process by which more than 40 different spliced RNAs are generated. One of the factors contributing to HIV-1 splicing complexity is the multiplicity of 3' splice sites (3'ss) used for generation of rev RNAs, with two 3'ss, A4a and A4b, being most commonly used, a third site, A4c, used less frequently, and two additional sites, A4d and A4e, reported in only two and one isolates, respectively. HIV-1 splicing has been analyzed mostly in subtype B isolates, and data on other group M clades are lacking. Here we examine splice site usage in three primary isolates of subtype C, the most prevalent clade in the HIV-1 pandemic, by using an in vitro infection assay of peripheral blood mononuclear cells. Viral spliced RNAs were identified by RT-PCR amplification using a fluorescently-labeled primer and software analyses and by cloning and sequencing the amplified products. The results revealed that splice site usage for generation of rev transcripts in subtype C differs from that reported for subtype B, with most rev RNAs using two previously unreported 3'ss, one located 7 nucleotides upstream of 3'ss A4a, designated A4f, preferentially used by two isolates, and another located 14 nucleotides upstream of 3'ss A4c, designated A4g, preferentially used by the third isolate. A new 5' splice site, designated D2a, was also identified in one virus. Usage of the newly identified splice sites is consistent with sequence features commonly found in subtype C viruses. These results show that splice site usage may differ between HIV-1 subtypes.Publication Incidence of non-B subtypes of HIV-1 in Galicia, Spain: high frequency and diversity of HIV-1 among men who have sex with men(European Centre for Disease Prevention and Control (ECDC), 2009-11-26) Cuevas, Maria Teresa; Fernandez-Garcia, Aurora; Sanchez-Garcia, Ana M; Gonzalez-Galeano, Maria; Pinilla, Milagros; Sanchez-Martinez, Monica; Garcia, Valentina; Perez-Alvarez, Lucia; Instituto de Salud Carlos IIIAn increase in HIV transmission among men who have sex with men (MSM) has been reported in eight regions of Spain from 2003 to 2007. In order to study the incidence of HIV-1 genetic forms in Galicia, northwest of Spain, in particular the spread of HIV-1 variants among MSM, 93 newly diagnosed HIV-1 patients, including those with acute and recently acquired infections, were studied for a year from August 2008 to August 2009. Thirty eight (41%) were MSM. Of them, nine (24%) were infected by non-B viruses, including seven different genetic forms. The analysis of transmission clusters showed that 23 (60%) MSM grouped in different clusters and mostly in large clusters. Resistance mutations were detected in six (16%) MSM.