title: Sensitive Next-Generation Sequencing Method Reveals Deep Genetic Diversity of HIV-1 in the Democratic Republic of the Congo.
abstract: As the epidemiological epicenter of the human immunodeficiency virus (HIV) pandemic, the Democratic Republic of the Congo (DRC) is a reservoir of circulating HIV strains exhibiting high levels of diversity and recombination. In this study, we characterized HIV specimens collected in two rural areas of the DRC between 2001 and 2003 to identify rare strains of HIV. Theenvgp41 region was sequenced and characterized for 172 HIV-positive specimens. Theenvsequences were predominantly subtype A (43.02%), but 7 other subtypes (33.14%), 20 circulating recombinant forms (CRFs; 11.63%), and 20 unclassified (11.63%) sequences were also found. Of the rare and unclassified subtypes, 18 specimens were selected for next-generation sequencing (NGS) by a modified HIV-switching mechanism at the 5' end of the RNA template (SMART) method to obtain full-genome sequences. NGS produced 14 new complete genomes, which included pure subtype C (n= 2), D (n= 1), F1 (n= 1), H (n= 3), and J (n= 1) genomes. The two subtype C genomes and one of the subtype H genomes branched basal to their respective subtype branches but had no evidence of recombination. The remaining 6 genomes were complex recombinants of 2 or more subtypes, including subtypes A1, F, G, H, J, and K and unclassified fragments, including one subtype CRF25 isolate, which branched basal to all CRF25 references. Notably, all recombinant subtype H fragments branched basal to the H clade. Spatial-geographical analysis indicated that the diverse sequences identified here did not expand globally. The full-genome and subgenomic sequences identified in our study population significantly increase the documented diversity of the strains involved in the continually evolving HIV-1 pandemic.IMPORTANCEVery little is known about the ancestral HIV-1 strains that founded the global pandemic, and very few complete genome sequences are available from patients in the Congo Basin, where HIV-1 expanded early in the global pandemic. By sequencing a subgenomic fragment of the HIV-1 envelope from study participants in the DRC, we identified rare variants for complete genome sequencing. The basal branching of some of the complete genome sequences that we recovered suggests that these strains are more closely related to ancestral HIV-1 strains than to previously reported strains and is evidence that the local diversification of HIV in the DRC continues to outpace the diversity of global strains decades after the emergence of the pandemic.
authors.affiliation: Infectious Disease Research, Abbott Diagnostics, Abbott Park, Illinois, USA mary.rodgers@abbott.com.
authors.forename: Mary A
authors.initials: MA
authors.lastname: Rodgers
authors.affiliation: Wellcome Trust-Africa Centre for Population Health, University of KwaZulu-Natal, Durban, Republic of South Africa.|College of Health Sciences, University of KwaZulu-Natal, Durban, Republic of South Africa.
authors.forename: Eduan
authors.initials: E
authors.lastname: Wilkinson
authors.affiliation: Infectious Disease Research, Abbott Diagnostics, Abbott Park, Illinois, USA.
authors.forename: Ana
authors.initials: A
authors.lastname: Vallari
authors.affiliation: School of Dentistry, University of Missouri-Kansas City, Kansas City, Missouri, USA.
authors.forename: Carole
authors.initials: C
authors.lastname: McArthur
authors.affiliation: Presbyterian Church (USA), Kinshasa, Democratic Republic of the Congo.
authors.forename: Larry
authors.initials: L
authors.lastname: Sthreshley
authors.affiliation: Infectious Disease Research, Abbott Diagnostics, Abbott Park, Illinois, USA.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Brennan
authors.affiliation: Infectious Disease Research, Abbott Diagnostics, Abbott Park, Illinois, USA.
authors.forename: Gavin
authors.initials: G
authors.lastname: Cloherty
authors.affiliation: Wellcome Trust-Africa Centre for Population Health, University of KwaZulu-Natal, Durban, Republic of South Africa.|College of Health Sciences, University of KwaZulu-Natal, Durban, Republic of South Africa.|Research Department of Infection, University College London, London, United Kingdom.
authors.forename: Tulio
authors.initials: T
authors.lastname: de Oliveira
mesh_terms: D016000:Cluster Analysis; D015023:Democratic Republic of the Congo / Q000453:epidemiology; D014644:Genetic Variation; D016679:Genome, Viral; D005838:Genotype; D015700:HIV Envelope Protein gp41 / Q000235:genetics; D015658:HIV Infections / Q000453:epidemiology / Q000821:virology*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D017720:Molecular Epidemiology; D012424:Rural Population; D017385:Sequence Homology
chemical_list: D015700:HIV Envelope Protein gp41; C514787:gp41 protein, Human immunodeficiency virus 1
keywords: HIV-1 surveillance; full-length genome; genetic diversity; next-generation sequencing; phylogenetic analysis; recombination