Study of the genetic variability of the HIV in Brazilian samples of blood givers aiming at the characterization of a biological panel: analysis of the subtypes and the resistance to the anti-retrovirais
The study of HIV/AIDS infection comprises among others, epidemiological, clinical and genetic aspects. In order to develop a biologic panel of plasma samples with molecular and serologic characterization for HIV-1 we have studied the genetic variability, the dynamics of viral subtypes and its potential implication on the development of new reagents, correlating serum-incidence in different geographic regions, time of infection and resistance to antiretroviral drugs. Seventy-four plasma samples from drug-naïve blood donors living in North, Northeast and Southeast Brazil were received and evaluated by two EIA and one IFA in order to confirm their HIV-1 reactivity. Those samples allow the preparation of biologic panels due to their volume amounts. The discrimination between long term and recent infection was estimated by using an HIV-1 BED Incidence CEIA. RNA was extracted from all samples, followed by reverse transcription to obtain cDNA to be used in nested PCR protocols using primers for the env (gp120 and gp41), RT and PRO regions. Automatic genomic sequencing was also conducted to define HIV-1 subtyping and to assess drug resistance based on env (gp41), RT and PRO genes. Phylogenetic analysis of gp120, gp41, PRO and RT was performed for the definition of viral subtypes using the Clustal X and Mega v3.0 softwares. Subtype B was found in 83,8% (62/74) of all samples while subtype F corresponded to 2,7% (2/74) of them. BF mosaics occurred in 11% (8/74) of the studied samples and different genetic profiles were evidenced for subtypes B and F: BPRBTRBgp120Bgp41, FPRFRTFgp120Fgp41, FPRFTRBgp120Bgp41, FPRBTRBgp120Bgp41 and BPRBTRFgp120Bgp41. Only one sample (1.4%) was characterized as a BC mosaic (BPRCTR ?gp120Cgp41) and, for the first time, the occurrence of an AGH mosaic (AGPRGTRHgp120Hgp41) was observed in the country. Sixteen out of the seventy-four analyzed samples (21.6%) were characterized as recent infections. With respect to anti-retroviral resistance, one sample (1,35%) presented M41L and T215S primary mutations, one sample (1.35%) showed a T69N mutation conferring a resistance profile to NRTI, two samples (2.7%) with the V82I polymorphism conferring resistance to PI and eight samples (12.9%) with a pattern of resistance to T20 previously unseen amongst drug-naïve HIV-1 infected individuals in Brazil. Due to the predominance of B subtype samples and the low number of non-B genotypes in the present study it was not possible to establish correlations between subtypes and mutations/ polymorphisms nor between subtypes and time of infection. However, the availability of subtyped plasma samples and HIV isolates will allow the development of biologic reagents and reference materials necessary to implement quality control programs, to evaluate the performance of diagnostic reagents and to develop and validate new products, such as, the Bio-Manguinhos HIV-1 Viral Load Quantification assay. Thus, it will be possible to improve and expand the diagnosis of HIV-1 infection, adjusting the tests so as to consider all variants circulating in the country.
Advisor:Amilcar Tanuri; Mariza Goncalves Morgado
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:HIV Blood Donors Drug Resistance, Viral Variation (Genetics) Anti-Retroviral Agents
Date of Publication:04/18/2006