Utility of Metagenomic Next-Generation Sequencing for Characterization of HIV and Human Pegivirus Diversity

Abstract

Given the dynamic changes in HIV-1 complexity and diversity, next-generation sequencing (NGS) has the potential to revolutionize strategies for effective HIV global surveillance. In this study, we explore the utility of metagenomic NGS to characterize divergent strains of HIV-1 and to simultaneously screen for other co-infecting viruses. Thirty-five HIV-1-infected Cameroonian blood donor specimens with viral loads of >4.4 log10 copies/ml were selected to include a diverse representation of group M strains. Random-primed NGS libraries, prepared from plasma specimens, resulted in greater than 90% genome coverage for 88% of specimens. Correct subtype designations based on NGS were concordant with sub-region PCR data in 31 of 35 (89%) cases. Complete genomes were assembled for 25 strains, including circulating recombinant forms with relatively limited data available (7 CRF11_cpx, 2 CRF13_cpx, 1 CRF18_cpx, and 1 CRF37_cpx), as well as 9 unique recombinant forms. HPgV (formerly designated GBV-C) co-infection was detected in 9 of 35 (25%) specimens, of which eight specimens yielded complete genomes. The recovered HPgV genomes formed a diverse cluster with genotype 1 sequences previously reported from Ghana, Uganda, and Japan. The extensive genome coverage obtained by NGS improved accuracy and confidence in phylogenetic classification of the HIV-1 strains present in the study population relative to conventional sub-region PCR. In addition, these data demonstrate the potential for metagenomic analysis to be used for routine characterization of HIV-1 and identification of other viral co-infections.

Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

“…Linkeramplified shotgun library (LASL) was applied for virus sequencing from marine water samples [8]. The anchored random PCR approach, as used in our study, has been frequently used and described in detail in previous studies [16,[36][37][38][39][40][41][42].…”

A Pan-HIV Strategy for Complete Genome Sequencing

“…As to whether the HIV-SMART approach represents an improvement over random priming for direct sequencing from patient samples, a comprehensive side-by-side evaluation has not been attempted. However, libraries made from comparable non-subtype B Cameroonian specimens with viral loads of Ն4.5 log 10 prepared by RdA/RdB random priming (37-39) yielded a median of 0.14% Ϯ 0.8% HIV-1 reads, whereas those prepared by HIV-SMART virus-specific priming yielded a median of 2.48% Ϯ 2.33% HIV-1 reads, for a Ͼ17-fold increase in viral reads (15). The HIV-SMART approach is also an improvement relative to published methods; one reported Յ311 viral reads for numerous high-titer (5 to 6 log 10 copies/ml) HIV specimens, and another which started with a larger volume of specimen (4 to 8 ml of plasma) yielded incomplete coverage and 0.007 to 1.44% viral reads (24,40).…”

“…However, full genomes are also of interest for detection of quasispecies and tracking immune evasion through variation in CD8 T lymphocyte epitopes with linkage elsewhere in the genome (13,14). We recently implemented a randomly primed NGS approach to conduct HIV surveillance and characterize clinical specimens for coinfecting viruses (15). For specimens with viral loads of Ͼ4.8 log 10 copies/ml, full genomes were obtained 70% of the time; however, the percentage of HIV reads varied significantly among specimens (0.002 to 4.11%).…”

Human pegivirus isolates characterized by deep sequencing from hepatitis C virus‐RNA and human immunodeficiency virus‐RNA–positive blood donations, France

“…Exploration of diversity, distribution, and potential hosts of pegiviruses is in perpetual re‐evaluation . In the case of human infections, at least six genotypes of the virus have been described, typically classified by phylogenetic comparison of 5′‐untranslated region (5′‐UTR), E2, or complete genomic sequences: genotype 1 is retrieved in West Africa, genotypes 1 and 2 in Europe and North America, genotype 3 in parts of Asia, genotype 4 in South East Asia, genotype 5 in South Africa, and genotype 6 in Indonesia; recombination events have also been described …”