The application of deep sequencing in analyzing drug resistance and characterizing HIV-1 quasispecies in families
Objective To utilize deep sequencing to investigate HIV-1 quasispecies among family members,and to analyze their transmission dynamics,evolutionary relationships,and drug resistance profiles.Methods Blood samples were collected from a couple and their newborns,all testing positive for HIV-1 at delivery,48 h post-delivery,42 days post-delivery,and at 2 years old.Plasma viral loads were determined in maternal plasma and neonatal dried blood spots.HIV-1 DNA was extracted from dried blood spots,and gag,pol,and env gene regions were amplified by nested PCR for subtype identification,drug resistance analysis,and HiSeq high-throughput sequencing.Genetic dispersion rate within and between samples were calculated,and phylogenetic trees of quasispecies were constructed using the maximum likelihood method.Results Maternal and infant viral loads were 5 600 copies/mL and 1 000 copies/mL,respectively.All family members were infected with the CRF07_BC subtype.The proportion of the K65R minority drug-resistant variant in the infant at 2 years old was higher than at birth.The genetic dispersion rate of infected viruses between the mother and infant was smaller than between the father and infant.The phylogenetic tree of the pol region indicated virus transmission in the order of"father-mother-infant".Genetic dispersion rate of infant samples at different timepoints varied between 0.47％to 0.68％,and 0.49％to 0.82％compared to baseline samples.Phylogenetic trees of the pol and env regions showed that dominant quasispecies in the infant evolved from baseline quasispecies.Conclusions Deep sequencing allows for determination of transmission patterns,genetic characteristics,dynamic changes,and drug resistance profiles of HIV-1 quasispecies over time,informing follow-up combination antiretroviral therapy(cART).
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