Risk of HIV-1 transmission by breastfeeding among mothers infected with recombinant and non-recombinant HIV-1 genotypes

BACKGROUND: Viral genotype and intersubtype recombination may influence the rate and/or timing of mother-to-child HIV-1 transmission. METHODS: We determined the HIV-1 subtype of the C2-C5 env and 5'LTR regions from milk and blood samples of 61 Tanzanian mothers who transmitted the virus through breastfeeding and their HIV-1 positive non-transmitting controls. Cases and controls were matched on infant's age at sample collection. All mothers resided in Dar es Salaam, Tanzania. RESULTS: Most infections among cases were due to recombinant viruses (41.0%), followed by HIV-1 subtype A (26.2%), subtype D (19.7%), and subtype C (13.1%). In multivariate analysis including maternal CD4+ cell counts, HIV disease stage, and proviral load in breast milk, the odds of breast milk transmission were 7.2 times higher if the mother carried an intersubtype recombinant genome in comparison to a subtype C virus (p=0.02). Viruses with recombinant LTRs were 4.9 times more likely to be transmitted through breastfeeding than viruses with non-recombinant LTRs of subtype A, C or D combined (p=0.01). CONCLUSIONS: This suggested that intersubtype recombinant genomes, and especially recombination within the LTR, might render HIV-1 more fit for transmission via breast milk in comparison with non-recombinant subtypes A, C, and D.     

Detection of intersubtype recombinants with respect to env and nef genes of HIV-1 among female sex workers in Calcutta, India

HIV-1 detected among female sex workers in Calcutta, India was characterized in respect to env and nef genes. A total of 39 HIV-1 seropositive samples were used in the study. Phylogenetic analysis of the nucleotide sequences of respective regions showed that 22 out of 39 samples (56.4%) were infected with subtype C with respect to both env and nef genes; however, 17 samples (43.6%) showed distinct subtype discordance. Simplot analysis of these samples showed the presence of intersubtype recombination between subtypes C and B. Both env C/nef B and env B/nef C recombinants were found to be present; 16 samples were found to be env C/nef B and 1 sample was detected as env B/nef C. This result indicates the emergence of intersubtype recombinants of HIV-1 for the first time in this eastern part of India.     

Determination of gag and env subtypes of HIV-1 detected among injecting drug users (IDUs) in Manipur, India: evidence for intersubtype recombination

The majority of HIV-1 transmission in Manipur, one of the northeastern states of India, is through the sharing of needles and syringes among the injecting drug users (IDUs). A total of 28 HIV seropositive samples were used to determine the HIV-1 subtypes with respect to both gag and envelope genes. The specific regions within gag and envelope genes were amplified from PBMC DNA by nested PCR using appropriate primers. These amplicons were used in heteroduplex mobility assay followed by DNA sequencing. Phylogenetic analysis of the nucleotide sequences of respective regions showed that 89% of samples (25/28) were infected with subtype C with respect to both gag and envelope genes; however, 11% of the samples (3/28) showed subtype discordance with respect to the envelope (C2-V3) and gag (p24-p7) genomic regions. Simplot analysis of the discordant samples showed the presence of intersubtype recombination between subtype C and Thai B; two samples were found to be subtype C in envelope but Thai B in gag, whereas, one sample was found to be subtype Thai B in envelope and 'C' in gag region.     

The first B/G intersubtype recombinant form of human immunodeficiency virus type 1 (HIV-1) identified in Germany was undetected or underquantitated by some commercial viral load assays

The high level of genetic diversity of human immunodeficiency virus type 1 (HIV-1) and the continual emergence of recombinant forms have important implications not only for the global evolution of HIV but also for diagnosis, monitoring, and treatment strategies. The present study reports the first intersubtype B/G recombinant strain of HIV-1 in Germany. This strain is notable from a clinical perspective, since it was undetectable in the NucliSens HIV-1 QT assay (Organon Tecknika/bioMérieux) and was significantly underquantitated in the Monitor v1.5 test (Roche Molecular Systems) relative to the LCx HIV RNA Quantitative assay (Abbott Laboratories). Gag-encoded p24 (gag p24), pol-encoded integrase (pol IN), and env-encoded gp41 (env gp41) immunodominant region (IDR) sequences were characterized to establish group and subtype designation and to evaluate the degree of genetic diversity at primer and probe binding sites of the viral load assays. Phylogenetic analysis revealed that this virus is an intersubtype B/G recombinant strain. The gag p24 region is subtype G, env gp41 IDR is subtype B, and pol IN is a B/G chimera. Nucleotide mismatches within primer and probe-binding sites provided the molecular basis for differences in quantitation observed between viral load assays. Genetic diversity of HIV-1 continues to challenge the reliability of detection and quantitation by viral load assays.     

Phylogenetic structure in African HIV-1 subtype C revealed by selective sequential pruning

Subtype C is the most prevalent clade in the HIV-1 pandemic. Previous studies suggested that African HIV-1 subtype C lacks a well-defined phylogenetic structure. Here we show that, by sequential pruning of ambiguously positioned taxa, a well-defined intrasubtype C phylogenetic structure becomes apparent, with 52% African HIV-1 subtype C isolates analyzed in envelope sequences branching within 11 clusters, also supported in a tree of full-length genomes, and all with geographical associations. Among 46 viruses recently transmitted in South Africa, 70% branched within 7 clusters (41% in the largest one) and 15% additional isolates were intercluster recombinants. Choice of the outgroup sequence and inclusion of intrasubtype recombinant viruses in the analyses could greatly affect support of clusters. The identification of clusters comprising a large proportion of African HIV-1 subtype C viruses may have implications for the design of vaccines intended for use in areas where subtype C is prevalent.     

Identification of a newly characterized HIV-1 BG intersubtype circulating recombinant form in Galicia, Spain, which exhibits a pseudotype-like virion structure

We recently reported the finding of phylogenetically related HIV-1 BG intersubtype recombinant and G subtype nonrecombinant viruses circulating among injecting drug users in the region of Galicia in northwestern Spain. Here, we report the characterization of near full-length genome sequences of nine of these viruses (seven BG recombinant and two of nonrecombinant G subtype), obtained from epidemiologically unlinked individuals. Bootscan analysis reveals that six recombinant viruses share an identical mosaic structure, with two intersubtype breakpoints delimiting a B subtype segment comprising most of Env gp120 and the external portion of Env gp41, with the remaining portions of the genome being of subtype G, thus mimicking a pseudotype virion structure. The seventh BG recombinant virus exhibits breakpoints in env coincident with the other BG viruses but contains additional B subtype segments in gag and pol. In phylogenetic trees of complete genomes and of the B subtype segment of env, all seven BG viruses group in a monophyletic cluster. G subtype portions of the BG viruses group uniformly with the newly derived nonrecombinant G subtype viruses of Galicia in bootscan analysis, which points to the locally circulating G subtype strain as parental of the recombinants. These results allow us to define a new HIV-1 circulating recombinant form (CRF14_BG), the first reported to originate in Western Europe.     

One-tube real-time isothermal amplification assay to identify and distinguish human immunodeficiency virus type 1 subtypes A, B, and C and circulating recombinant forms AE and AG

To halt the human immunodeficiency virus type 1 (HIV-1) epidemic requires interventions that can prevent transmission of numerous HIV-1 subtypes. The most frequently transmitted viruses belong to the subtypes A, B, and C and the circulating recombinant forms (CRFs) AE and AG. A fast one-tube assay that identifies and distinguishes among subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The assay amplifies a part of the gag gene sequence of the genome of all currently known HIV-1 subtypes and can identify and distinguish among the targeted subtypes as the reaction proceeds, because of the addition of subtype-specific molecular beacons with multiple fluorophores. The combination of isothermal nucleic acid sequence-based amplification and molecular beacons is a new approach in the design of real-time assays. To obtain a sufficiently specific assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype A and CRF AG isolates reacted with the same molecular beacon. We tested the specificity and sensitivity of the assay on a panel of the culture supernatant of 34 viruses encompassing all HIV-1 subtypes: subtypes A through G, CRF AE and AG, a group O isolate, and a group N isolate. Assay sensitivity on this panel was 92%, with 89% correct subtype identification relative to sequence analysis. A linear relationship was found between the amount of input RNA in the reaction mixture and the time that the reaction became positive. The lower detection level of the assay was approximately 10(3) copies of HIV-1 RNA per reaction. In 38% of 50 serum samples from HIV-1-infected individuals with a detectable amount of virus, we could identify subtype sequences with a specificity of 94% by using sequencing and phylogenetic analysis as the "gold standard." In conclusion, we showed the feasibility of the approach of using multiple molecular beacons labeled with different fluorophores in combination with isothermal amplification to identify and distinguish subtypes A, B, and C and CRFs AE and AG of HIV-1. Because of the low sensitivity, the assay in this format would not be suited for clinical use but can possibly be used for epidemiological monitoring as well as vaccine research studies.     

Near full-length genomic characterization of a HIV type 1 BC recombinant strain from Manipur, India

Genetic complexity of HIV-1 is brought about by recombination between HIV-1 subtypes which leads to the development of epidemiologically significant founder strains. In the present study, the near full-length genome sequence of an HIV-1 isolate from an injecting drug user of Manipur (India) was determined, which evidenced the presence of a novel HIV-1 BC recombinant strain. Near full-length genome was amplified by polymerase chain reaction using primer walking approach. The recombination break points were detected using bootscan and simplot analyses. This isolate exhibited a mosaic structure consisting of subtype C backbone with subtype B insertions at the upstream of pol gene (3026-3259) and the downstream of env gene which spanned till the nef gene (8183-8961). Phylogenetic relationships determined with neighbor-joining trees, revealed that the subtype C sequences clustered with sequences from Indian subtype C HIV-1 strains, and the subtype B sequences clustered with HIV-1 subtype B strains from Thailand. This finding may create a complex scenario of HIV-1 epidemic among the injecting drug users of Manipur in near future.     

Differences in perinatal transmission among human immunodeficiency virus type 1 genotypes

OBJECTIVE: To determine whether genotypes from human immunodeficiency virus type 1 (HIV-1) subtypes A, C, or D or intersubtype recombinants have the same probability of being transmitted from mother to child. METHODS: We determined the HIV-1 genetic subtype and maternal risk factors of 51 matched transmitting and nontransmitting mothers from Tanzania. The HIV-1 gag (p24-p7) and env (C2-C5) nucleotide sequences were used for genotype classification, and matched logistic regression analysis was used to assess differences among genotypes. RESULTS: Mothers infected with HIV-1 subtype A (odds ratio, 3.8; 95% CI, 0.8-24.7%), HIV-1 subtype C (odds ratio, 5.1; 95% CI, 1.3-30.8%), or HIV-1 intersubtype recombinant viruses (odds ratio, 5.3; 95% CI, 1.2-33.4%) were more likely to transmit HIV-1 to their infants than mothers infected with HIV-1 subtype D. Lower CD4 cell counts at enrollment were associated with transmission, but CD4 cell counts within each genotype did not explain differences in transmission among HIV-1 genotypes. CONCLUSION: We have shown that HIV-1 genotypes might be associated with differential risk for vertical transmission. These findings provide the first evidence that HIV-1 genetic subtypes may play a role in rates of vertical transmission in an African setting.     

Determination of human immunodeficiency virus type 1 subtypes in Taiwan by vpu gene analysis

The genetic diversity of human immunodeficiency virus (HIV) type 1 (HIV-1) has been characterized mainly by analysis of the env and gag genes. Information on the vpu genes in the HIV sequence database is very limited. In the present study, the nucleotide sequences of the vpu genes were analyzed, and the genetic subtypes determined by analysis of the vpu gene were compared with those previously determined by analysis of the gag and env genes. The vpu genes were amplified by nested PCR of proviral DNA extracted from 363 HIV-1-infected individuals and were sequenced directly by use of the PCR products. HIV-1 subtypes were determined by sequence alignment and phylogenetic analysis with reference strains. The strains in all except one of the samples analyzed could be classified as subtype A, B, C, E, or G. The vpu subtype of one strain could not be determined. Of the strains analyzed, genetic subtypes of 247 (68.0%) were also determined by analysis of the env or gag gene. The genetic subtypes determined by vpu gene analysis were, in general, consistent with those determined by gag and/or env gene analysis except for those for two AG recombinant strains. All the strains that clustered with a Thailand subtype E strain in the vpu phylogenetic analyses were subtype E by env gene analysis and subtype A by gag gene analysis. In summary, our genetic typing revealed that subtype B strains, which constituted 73.8% of all strains analyzed, were most prevalent in Taiwan. While subtype E strains constituted about one-quarter of the viruses, they were prevalent at a higher proportion in the group infected by heterosexual transmission. Genetic analysis of vpu may provide an alternate method for determination of HIV-1 subtypes for most of the strains, excluding those in which intersubtype recombination has occurred.     

HIV-1 LTR subtype and perinatal transmission

Multiple subtypes of HIV-1 have been identified; however, there is little data on the relative transmissibility of viruses belonging to different subtypes. A matched case-control study addressed whether viruses with different long terminal repeat (LTR) subtypes were transmitted equally from mother to infant. The LTR subtype was determined for 45 matched cases and controls who participated in a clinical trial in Tanzania. HIV-1 subtypes A, C, and D and intersubtype recombinant sequences were identified. Exact matched logistic regression analysis showed that viruses containing subtype A or intersubtype recombinant LTRs were 3.2 and 4.8 times more likely to be transmitted from mother to infant than viruses with subtype D LTRs. Viruses containing subtype C LTRs were 6.1 times more likely to be transmitted than those with subtype D LTRs. These differences in transmission were independent of maternal CD4 at enrollment. Thus, it appears that HIV-1 subtype may be associated with differing rates of perinatal transmission in Tanzania.     

Identification of HIV superinfection in seroconcordant couples in Rakai, Uganda, by use of next-generation deep sequencing

HIV superinfection, which occurs when a previously infected individual acquires a new distinct HIV strain, has been described in a number of populations. Previous methods to detect superinfection have involved a combination of labor-intensive assays with various rates of success. We designed and tested a next-generation sequencing (NGS) protocol to identify HIV superinfection by targeting two regions of the HIV viral genome, p24 and gp41. The method was validated by mixing control samples infected with HIV subtype A or D at different ratios to determine the inter- and intrasubtype sensitivity by NGS. This amplicon-based NGS protocol was able to consistently identify distinct intersubtype strains at ratios of 1% and intrasubtype variants at ratios of 5%. By using stored samples from the Rakai Community Cohort Study (RCCS) in Uganda, 11 individuals who were HIV seroconcordant but virally unlinked from their spouses were then tested by this method to detect superinfection between 2002 and 2005. Two female cases of HIV intersubtype superinfection (18.2%) were identified. These results are consistent with other African studies and support the hypothesis that HIV superinfection occurs at a relatively high rate. Our results indicate that NGS can be used for detection of HIV superinfection within large cohorts, which could assist in determining the incidence and the epidemiologic, virologic, and immunological correlates of this phenomenon.     

Generation of intersubtype human immunodeficiency virus type 1 recombinants in env gene in vitro: influences in the biological behavior and in the establishment of productive infections

The occurrence of human immunodeficiency virus type 1 (HIV-1) recombinant genomes belonging to different subtypes is a common event in regions where more than two subtypes cocirculate. Although there are accumulating data toward an increase in the number of intersubtype recombinants, little has been addressed about the biological behavior of such mosaic genomes. This work reports the biological characterization of engineered in vitro HIV-1 intersubtype recombinants in the gp120 region. The recombinants possess the entire gp120 of B or F Brazilian isolates in the Z6 (subtype D) backbone. Here we show that this type of recombinant structure results in profound impairment to the establishment of productive infections in CD4-positive cells. The characterization of biological properties of those recombinant viruses demonstrated viral production occurring only during a transient peak early on infection and that they are not able to down-regulate the expression of CD4 receptor on the cell surface. We also report the phenotype reversion of one recombinant virus studied here, after 62 days in culture. Two amino acid substitutions in highly constant gp120 regions (C1 and C4) were identified in the revertant virus. The mutation occurring in the C4 region is localized near two amino acid residues critical for gp120/CD4 interaction. Based on these data, we suggest that failure in CD4 down-modulation by recombinant viruses can be due to a structural dysfunction of gp160 protein unable to block CD4 at the endoplasmic reticule. The possibilities that the establishment of latent infections can be directly related to the continuous expression of CD4 on the infected cell surface and that the occurrence of mutations in amino acid nearby residues critical for gp120/CD4 interaction can restore the fully productive infectious process are discussed.     

人免疫缺陷病毒1型CRF07_BC毒株准种间的重组

目的 通过研究人类免疫缺陷病毒1型（human immunodeficiency virus1，HIV-1）感染者个体内准种间的差异，探讨HIV的系统进化的发生模式。方法 从HIV-1感染者血浆中提取总RNA，通过逆转录多聚酶链反应（RT-PCR）获得HIV．1gp120全长基因，纯化后装入T载体，转化至TOP10大肠埃希菌内增殖，通过蓝白斑筛选获得阳性克隆，对所获得的目的克隆测序并分析。结果 获得同一患者的16个克隆的gp120全长基因序列，通过系统进化树分析，克隆序列均为CRF07_BC亚型，但在系统进化树上16个克隆可明显分为A、B两群，其中13个克隆属于A群，2个克隆属于B群，1个克隆（编号XPD7）位于A、B群之间，通过simplot软件的重组分析，发现XPD7克隆为A群和B群的重组株。结论 发现了我国广泛流行的HIV-1CRF07-BC毒株准种间的重组现象，准种间的重组作为HIV进化的一种有效手段将导致HIV毒株的快速进化的发生，可能更易逃脱宿主的免疫监控。     

我国西南西北地区吸毒人群重组人类免疫缺陷病毒1？…

目的 寻找人类免疫缺陷病毒１型在中国可能的重组。方法从流行２种以上ＨＩＶ－１亚型的地区收集ＨＩＶ感染者的血样。从ＰＭＣｓ中应用套式聚合酶链反应方法,对ＨＩＶ病毒的ｔａｔ和ｅｎｖ基因进行扩增,ＰＣＲ产物直接测序并进行序列分析。结果 对中国Ｂ’亚型和Ｃ亚型浒区域收集的１４个ＨＩＶ－１毒株进行序列分析,对ｅｎｖ基因进行测序后没有发现重组毒株的证据。     

北京市男男性接触HIV-1感染者膜蛋白基因序列测定及亚型分析

目的 了解北京市男男性接触人群(MSM)中HIV-1的最新流行趋势及膜蛋白V3环序列特征.方法 巢式聚合酶链式反应(n-PER)扩增2007年提取的北京市男男性接触HIV感染者基因组DNA样品,对膜蛋白基因C2-V3区测序,进行病毒亚型及V3环序列特点分析.结果 11例样本中,4例是欧美B亚型,5例是AE重组亚型,1例是BC重组亚型,1例是01B重组亚型.V3环顶端四肽以GPGQ和GPGR为主.结论 北京市男男性接触HIV-1感染者中重组亚型呈蔓延流行趋势.     

Diversity of the HIV-1 long terminal repeat following mother-to-child transmission

A study of the human immunodeficiency virus Type 1 (HIV-1) 5' long terminal repeat (LTR) was performed to determine the extent of variation found within the LTR from 19 mother-infant pairs in Tanzania and to assess whether the LTR is useful in distinguishing maternal sequences that were transmitted to infants. HIV-1 subtypes A, C, and D as well as intersubtype recombinant LTR sequences were detected in mothers and infants. The LTR subtype was 100% concordant between mothers and their infants. Diversity calculations showed a significant reduction in LTR variation in infants compared to their mothers. However, the overall magnitude of LTR variation was less than that found in the env gene from the same individuals. These data suggest a selective constraint active upon the 5' long terminal repeat that is distinct from immune selective pressure(s) directed against HIV-1 structural genes. Detection of maternal LTR variants that were transmitted to infants may yield important information concerning nonstructural determinants of HIV-1 transmission from mother to infant.     

Identification of 3 phylogenetically related HIV-1 BG intersubtype circulating recombinant forms in Cuba

BG intersubtype recombinants represented 11.6% of HIV-1 isolates in a recent survey in Cuba based on pol sequences, most of them forming a single clade further subdivided into 3 subclades. Here, we analyze 8 near full-length genomes and 1 gag-pol sequence from epidemiologically unlinked Cuban BG recombinants from these 3 subclades (3 from each). Near full-length sequences were also obtained from 3 subtype G and 2 subtype B Cuban viruses. Phylogenetic relationships were estimated via maximum likelihood, and mosaic structures of the recombinants were inferred with the bootscanning, MaxChi, Genconv, and GARD methods. For the near full-length genomes, all recombinants formed a strongly supported clade further subdivided into the same subclades previously defined in pol. Mosaic structures were identical within each subclade and different among subclades, although 5 breakpoints were coincident among all recombinants. Individual phylogenetic trees for nonrecombinant fragments (concatenated B and G subtype segments) indicated a common ancestry for the parental viruses and their relationships to local subtype B and G strains. These results allow us to identify 3 new BG intersubtype circulating recombinant forms in Cuba derived from a common recombinant ancestor, which originated from B and G subtype parental strains circulating in Cuba.