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Evaluation of Prototype gp41 Genotyping Reagents for Analysis of Diverse HIV-1 Strains
Natalia Marlowe*1, S Hudelson2, R Bruce1, T Lee3, M Owens1, J Hackett, Jr4, P Swanson4, S Devare4, and S Eshleman2
1Celera Diagnostics, Alameda, CA, US; 2Johns Hopkins Univ Sch of Med, Baltimore, MD, US; 3Cornell Univ, Ithaca, NY, US; and 4Abbott Diagnostics, Abbott Park, IL, US
Background: The HIV fusion inhibitor, enfuvirtide
(T-20), targets the HIV transmembrane domain, env gp41.
Mutations in gp41 are associated with T-20 resistance. We evaluated the
performance of a prototype HIV-1 gp41 genotyping reagent developed by Celera Diagnostics.
Methods: HIV-1-infected plasma samples were collected from 126
individuals in Cameroon (n = 61), Brazil
(n = 21), Uganda
(n = 17), South
Africa (n = 15),
Thailand (n = 9), and Argentina (n = 3) between 1993 and 2001. Samples were obtained from
prospective blood donors who were subsequently found to have HIV-1 infection.
HIV viral loads were determined using the LCx HIV RNA
Quantitative assay. Subtype determinations were based on phylogenetic
analysis of gag p24, pol integrase, and env gp41 genes.
Samples were amplified and sequenced using reagents and protocols provided by
Celera Diagnostics for HIV-1 gp41 genotyping. The 676-bp region spanning gp41
HR1 and HR2 was analyzed using ABI Prism SeqScape
v2.5 software. Sequences were examined for the presence of amino acid
polymorphisms in the HR1 domain (amino acids 30-70), including the enfuvirtide resistance domain (amino acids 36-45).
Results: The panel included the following subtypes and circulating
recombinant forms (CRFs): A/A2 (18), B (11), C (14), D (10), CRF01_AE
(9), F/F2 (9), G (7), CRF02_AG (33), and recombinant (15). Viral loads of the samples ranged from 2.92 to > 6 log10
RNA copies/mL. The mean viral load was 4.24 log10 RNA copies/mL.
Amplification of the gp41 region was successful for 124 (98.4%) of the 126
samples. Bi-directional gp41 sequencing (amino acids 1-180) was successful for
114 (91.9%) of the 124 samples that amplified. Overall, analysis was successful
for 119 (94.4%) of the 126 samples tested. Many samples contained polymorphisms
in the HR1 domain. In the T-20 resistance domain, the only polymorphism
detected was N42S, which is not associated with T-20 resistance. This
polymorphism was detected in the majority of samples with subtypes A, C, G,
CRF01_AE, CRF02_AG, and mosaic strains, and was also observed in nearly half of
the subtype F samples.
Conclusions: The gp41 HIV-1 research reagents developed by
Celera Diagnostics are useful tools for genotyping analysis of the gp41 region
in diverse HIV-1 strains.
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