Comparisons of Human Immunodeficiency Virus Type 1 Envelope Variants in Blood and Genital Fluids near the Time of Male-to-Female Transmission.

To better understand the transmission of human immunodeficiency virus type 1 (HIV-1), the genetic characteristics of blood and genital viruses from males were compared to those of the imputed founding virus population in their female partners. Initially serodiscordant heterosexual African couples with sequence-confirmed male-to-female HIV-1 transmission and blood and genital specimens collected near the time of transmission were studied. Single viral templates from blood plasma and genital tract RNA and DNA were sequenced across HIV-1 env gp160. Eight of 29 couples examined yielded viral sequences from both tissues. Analysis of these couples' sequences demonstrated, with one exception, that the women's founding viral populations arose from a single viral variant and were CCR5 tropic, even though CXCR4 variants were detected within four males. The median genetic distance of the imputed most recent common ancestor of the women's founder viruses showed that they were closer to the semen viruses than to the blood viruses of their transmitting male partner, but this finding was biased by detection of a greater number of viral clades in the blood. Using multiple assays, the blood and genital viruses were consistently found to be compartmentalized in only two of eight men. No distinct amino acid signatures in the men's viruses were found to link to the women's founders, nor did the women's env sequences have shorter variable loops or fewer N-linked glycosylation sites. The lack of selective factors, except for coreceptor tropism, is consistent with others' findings in male-to-female and high-risk transmissions. The infrequent compartmentalization between the transmitters' blood and semen viruses suggests that cell-free blood virus likely includes HIV-1 sequences representative of those of viruses in semen.IMPORTANCE Mucosal transmissions account for the majority of HIV-1 infections. Identification of the viral characteristics associated with transmission would facilitate vaccine design. This study of HIV strains from transmitting males and their seroconverting female partners found that the males' genital tract viruses were rarely distinct from the blood variants. The imputed founder viruses in women were genetically similar to both the blood and genital tract variants of their male partners, indicating a lack of evidence for genital tract-specific lineages. These findings suggest that targeting vaccine responses to variants found in blood are likely to also protect from genital tract variants.

Transmission of HIV-1 drug resistance mutations within partner-pairs: A cross-sectional study of a primary HIV infection cohort.

BACKGROUND: Transmission of human immunodeficiency virus type 1 (HIV-1) drug resistance mutations, particularly that of minority drug-resistant variants, remains poorly understood. Population-based studies suggest that drug-resistant HIV-1 is less transmissible than drug-susceptible viruses. We compared HIV-1 drug-resistant genotypes among partner-pairs in order to assess the likelihood of transmission of drug resistance mutations and investigate the role of minority variants in HIV transmission. METHODS AND FINDINGS: From 1992-2010, 340 persons with primary HIV-1 infection and their partners were enrolled into observational research studies at the University of Washington Primary Infection Clinic (UWPIC). Out of 50 partner-pairs enrolled, 36 (72%) transmission relationships were confirmed by phylogenetic distance analysis of HIV-1 envelope (env) sequences, and 31 partner-pairs enrolled after 1995 met criteria for this study. Drug resistance mutations in the region of the HIV-1 polymerase gene (pol) that encodes protease and reverse transcriptase were assessed by 454-pyrosequencing. In 25 partner-pairs where the transmission direction could be determined, 12 (48%) transmitters had 1-4 drug resistance mutations (23 total) detected in their HIV-1 populations at a median frequency of 6.0% (IQR 1.5%-98.7%, range 1.0%-99.6%). Of 10 major mutations detected in five transmitters at a frequency >95%, 100% (95% CI 69.2%-100%) were detected in recipients. All of these transmitters were antiretroviral (ARV)-naïve at the time of specimen collection. Fourteen mutations (eight major mutations and six accessory mutations) were detected in nine transmitters at low frequencies (1.0%-11.8%); four of these transmitters had previously received ARV therapy. Two (14% [95% CI 1.8%-42.8%]) G73S accessory mutations were detected in both transmitter and recipient. This number is not significantly different from the number expected based on the observed frequencies of drug-resistant viruses in transmitting partners. Limitations of this study include the small sample size and uncertainties in determining the timing of virus transmission and mutation history. CONCLUSIONS: Drug-resistant majority variants appeared to be commonly transmitted by ARV-naïve participants in our analysis and may contribute significantly to transmitted drug resistance on a population level. When present at low frequency, no major mutation was observed to be shared between partner-pairs; identification of accessory mutations shared within a pair could be due to transmission, laboratory artifact, or apolipoprotein B mRNA-editing enzyme, catalytic polypeptides (APOBECs), and warrants further study.

Validation of an oligonucleotide ligation assay for quantification of human immunodeficiency virus type 1 drug-resistant mutants by use of massively parallel sequencing.

Global HIV treatment programs need sensitive and affordable tests to monitor HIV drug resistance. We compared mutant detection by the oligonucleotide ligation assay (OLA), an economical and simple test, to massively parallel sequencing. Nonnucleoside reverse transcriptase inhibitor (K103N, V106M, Y181C, and G190A) and lamivudine (M184V) resistance mutations were quantified in blood-derived plasma RNA and cell DNA specimens by OLA and 454 pyrosequencing. A median of 1,000 HIV DNA or RNA templates (range, 163 to 1,874 templates) from blood specimens collected in Mozambique (n = 60) and Kenya (n = 51) were analyzed at 4 codons in each sample (n = 441 codons assessed). Mutations were detected at 75 (17%) codons by OLA sensitive to 2.0%, at 71 codons (16%; P = 0.78) by pyrosequencing using a cutoff value of ≥ 2.0%, and at 125 codons (28%; P < 0.0001) by pyrosequencing sensitive to 0.1%. Discrepancies between the assays included 15 codons with mutant concentrations of ∼2%, one at 8.8% by pyrosequencing and not detected by OLA, and one at 69% by OLA and not detected by pyrosequencing. The latter two cases were associated with genetic polymorphisms in the regions critical for ligation of the OLA probes and pyrosequencing primers, respectively. Overall, mutant concentrations quantified by the two methods correlated well across the codons tested (R(2) > 0.8). Repeat pyrosequencing of 13 specimens showed reproducible detection of 5/24 mutations at <2% and 6/6 at ≥ 2%. In conclusion, the OLA and pyrosequencing performed similarly in the quantification of nonnucleoside reverse transcriptase inhibitor and lamivudine mutations present at >2% of the viral population in clinical specimens. While pyrosequencing was more sensitive, detection of mutants below 2% was not reproducible.

Development and validation of an oligonucleotide ligation assay to detect lamivudine resistance in hepatitis B virus.

Treatment of chronic hepatitis B virus (HBV) infection with lamivudine-monotherapy rapidly selects mutant variants in a high proportion of individuals. Monitoring lamivudine resistance by consensus sequencing is costly and insensitive for detection of minority variants. An oligonucleotide ligation assay (OLA) for HBV lamivudine-resistance was developed and compared to consensus sequencing. Both assays detected drug resistance mutations in 35/64 (54.7%) specimens evaluated, and OLA detected minority mutants in an additional six (9.4%). OLA may offer a sensitive and inexpensive alternative to consensus sequencing for detection of HBV drug resistance in resource-limited settings.

Nevirapine-Resistant HIV-1 DNA in Breast Milk After Single-Dose Nevirapine With or Without Zidovudine for Prevention of Mother-to-Child Transmission.

Among 30 human immunodeficiency virus type 1 (HIV-1)-infected women who received single-dose nevirapine (NVP), 17 (57%) had NVP-resistant HIV-1 detected in breast milk. NVP resistance in breast milk persisted for at least 8 months postpartum and was apparently transmitted to at least 1 infant. NVP resistance was detected less often in women who also received zidovudine.