Abstract

Molecular tools for the early detection and the epidemiological characterization of HIV-1 infections based on the strains circulating in Argentina

G.R. Perez^1,2, D. Gardiol^1,2, M.A. Taborda¹, A.A. Giri<sup>1,2

1</sup>Virology Area, School of Biochemistry and Pharmaceutical Sciences, Rosario National University, Microbiology, Rosario, Argentina, ²Institute of Molecular and Cell Biology of Rosario (CONICET), Rosario, Argentina

In the Americas, different patterns of HIV-1 subtypes and recombinants distribution have been found. In Argentina, approximately 80% isolates are BF recombinants, almost 20% are B subtype and scattered cases of A, C and F subtypes. This scenario represents a risk factor for spreading unidentified infections due to differences in the specificity/sensitivity of commercially available tests, and highlights the need for developing diagnostic tools tailored on the strains circulating in each region with affordable technology.
We developed the CIC_HIV-1/HIV-1 RT-PCR assay, a method that includes a competitive internal control (CIC_HIV-1) and combines RT-PCR, liquid hybridization and EIA. First, we performed a genomic analysis using 1,745 HIV-1 subtypes and inter-subtypes listed at NCBI. A gag-gene fragment presenting the lesser mismatches among strains circulating in Argentina and South America was selected to design generic primers and probes. Primers specificity and sensitivity were demonstrated with plasmids containing B or F subtype genomes. Once primers functionality was achieved, the CIC_HIV-1 was constructed. Briefly, sequences of the A1 region from Qβ coliphage were replaced by the selected HIV-1 primers, using a pBR322 derivative in which the Qβ phage genome cDNA was cloned downstream the T7 RNA polymerase promoter as template. The plasmid bearing the modified Qβ genome was used to transform E. coli. After induction, recombinant Qβ particles (CIC_HIV-1) were obtained. CIC_HIV-1 is RNase resistant, stable at 4°C, economical to produce and useful to verify the efficiency of the whole procedure, including RNA extraction.
The CIC_HIV-1/HIV-1 RT-PCR assay fulfills the requirements for diagnostic tests for HIV-1 early detection and/or molecular blood donor screening, that includes a CIC_HIV-1 consisting in a recombinant RNA packaged into capsids which hybridizes with the same primers. This approach can be adapted for qualitative/quantitative detection of other subtypes and inter-subtypes circulating in many developing regions, contributing to a more effective HIV-1 epidemiological control.
Funding: FIC-NIH(D43TW001037).