HIV type 1 subtypes among blood donors in the Mbeya region of southwest Tanzania

HIV-1 is endemic in Tanzania where three different subtypes, A, C, and D, have been identified. Information on HIV-1 genetic diversity is crucial to define requirements for an effective vaccine, in regions where HIV-1 vaccine trials are planned. To define the subtype distribution of HIV-1 in the Mbeya region of southwest Tanzania, peripheral blood mononuclear cells (PBMC) and plasma were obtained from 36 discarded HIV seropositive blood units. Multiregion hybridization assay (MHA) was performed on both PBMC DNA and plasma RNA to determine the subtype distribution. Twenty virtually full-length HIV-1 sequences were amplified from the extracted DNA, sequenced, and phylogenetically analyzed. Subtype distribution determined by all three assays was comparable. More than 50% of the samples analyzed were subtype C, followed by a high proportion of subtype C-containing intersubtype recombinants. Based on this work, subtype C appears to be the prevalent subtype in southwest Tanzania, followed by a high proportion of intersubtype recombinants.     

Isolates from four different HIV type 1 clades circulating in Cuba identified by DNA sequence of the C2-V3 region

According to the epidemiology of human immunodeficiency virus infection in Cuba, the main sources of infection have been persons coming from foreign countries, mainly from Africa, and individuals who have had sexual contacts with foreigners in Cuba. However, the first Cuban HIV-1 isolates sequenced have been all classified as subtype B. In this note we report the sequence of the gp120 C2/V3 region from 11 HIV-1 isolates from Cuban patients. DNA was isolated either directly from blood PBMC or from primary isolates, PCR amplified and sequenced. Six isolates were classified as subtype B and three of them had the atypical sequences GRGR, GWGR, and TPGR on the tip of the V3 loop. Besides, two other sequences were classified as subtype A, two as subtype H, and one as subtype C. These results confirm that although subtype B seems to be predominant, HIV-1 isolates from various subtypes do circulate in Cuba.     

Sequence features downstream of the primer-binding site of HIV type 1 subtype E shared by subtype G and a subset of subtype A

Two distinct sequence features downstream of the primer-binding site (PBS) were identified in a full-length HIV-1 subtype E clone amplified in this study. Both features are frequently found in HIV-1 subtypes A and G and in more than half of the full-length intersubtype recombinant clones. One of these is the absence of a trinucleotide sequence, which is located 14 nucleotides downstream of the PBS and found only in subtypes B, C, D, F, and H. The other is an insertion of 24 nucleotides immediately downstream of the PBS, which was previously reported as a sequence feature shared by subtypes A, E, and G. The analysis conducted here revealed that this 24-nucleotide insertion contained two sequence motifs duplicated in adjacent regions and was not found in all HIV-1 subtype A clones. Furthermore, our finding suggests that the PBS region of all known full-length subtype E clones, which are A/E intersubtype recombinants, is derived from the group of HIV-1 subtype A, which contains a similar insertion.     

Full-length HIV type 1 proviral sequencing of 10 highly exposed women from Nairobi, Kenya reveals a high proportion of intersubtype recombinants

Phylogenetic analysis has revealed that the current HIV/AIDS pandemic consists of a multitude of different viral clades and recombinant viruses. The predominant circulating HIV-1 clade in Kenya is A1; however, Kenya borders countries where different subtypes are prominent, making Kenya a likely location for recombination. Previous studies have reported significant differences in the proportions of sequences in Kenya that are intersubtype recombinants. Studies that performed sequence-based typing on multiple HIV-1 genomic regions or full-length sequences found higher rates of recombination than those that examined a single gene or gene fragment. In this study, we describe full-length HIV-1 proviral sequence-based genotyping after limited peripheral blood mononuclear cell (PBMC) coculture. Ten subjects from a highly exposed cohort located in Nairobi, Kenya were examined. Pairwise comparison found minimal difference between sequences generated directly from patient PBMC DNA compared to sequences from cocultured PBMC DNA. Of the 10 full-length HIV-1 sequences examined, five were nonrecombinant clade A1, while the other five were unique intersubtype recombinants. Although this frequency of recombination is higher than previously described in Kenya, this finding is in agreement with previous full-length sequence data. Interestingly, although all the nonrecombinant sequences were clade A1, not all the recombinant sequences contained a clade A1 sequence.     

Identification of a major restriction in HIV-1 intersubtype recombination

Genetic recombination increases diversity in HIV-1 populations, thereby allowing variants to escape from host immunity or antiviral therapies. In addition to the currently described nine subtypes of HIV-1, many of the circulating strains are intersubtype recombinants. In this study, we determined the recombination rate between two HIV-1 subtype C viruses and between a subtype B virus and a subtype C virus during a single round of virus replication. Although HIV-1 subtype C recombines at a high rate, similar to that of HIV-1 subtype B, the recombination rate between a subtype B virus and a subtype C virus is much lower than the intrasubtype recombination rate. A 3-nt sequence difference in the dimerization initiation signal (DIS) region between HIV-1 subtypes B and C accounts for most of the reduction of intersubtype recombination. By matching the DIS sequences, the B/C intersub-type recombination rate was elevated 4-fold; by introducing mismatches in the 3-nt sequences, the B/B intrasubtype recombination rate was reduced 4-fold. Further analyses showed that the intermolecular template-switching frequency was unaffected by the sequence identity of the DIS region. These results support the hypothesis that mismatched sequences in the DIS region alter the formation of heterozygous virions, thereby lowering the observable recombination rate. Here, we present the discovery of a major restriction in HIV-1 intersubtype recombination. These results have important implications for virus evolution, the mechanism of HIV-1 RNA packaging, high negative interference in recombination, and the generation of circulating intersubtype recombinants within the infected population.     

Sequence variability of the integrase protein from a diverse collection of HIV type 1 isolates representing several subtypes

HIV-1 recombinants between viruses from different subtypes appear to be surprisingly common in several regions of the world. To detect such intersubtype recombinants that contain mosaic genomes, we have analyzed sequences from the integrase (IN)-coding region of the polymerase (pol) gene from 23 viruses of known envelope (env) subtype from South America and Africa. As defined by env sequences, these viral genomes included nine subtype A, four subtype B, three subtype C, and four subtype D viruses from group M, and three viruses from group O HIV-1. Mosaic genomes were common, with 7 mosaic genomes among the 20 group M isolates analyzed. Two of these isolates had mosaic IN-coding regions that were distinct, but that had recombination breakpoints at the same location, in the highly conserved polypurine track. Mosaic genomes were particularly common in the viruses from Kenya (five of nine), consistent with our previous prediction that there was a high frequency of intersubtype recombinants circulating in this country. The IN amino acid sequence was highly conserved among the several represented subtypes, including group O. Group M IN sequences shared 94% or greater amino acid sequence identity within a subtype and 91% or greater identity between subtypes. The most divergent M and O variant amino acid sequences differed by only 19%, and the known functional domains were conserved among all of the isolates. The high degree of genetic homogeneity among the virus isolates representing several subtypes indicates that a single drug targeted against IN might be effective for all HIV-1 infections.     

Construction of an infectious HIV type 1 molecular clone from an African patient with a subtype D/C Recombinant Virus

The majority of HIV-1 infections worldwide occur in Africa, where subtype B viruses are rare and intersubtype recombinants are common. Pathogenesis and vaccine studies need to focus on viruses derived from African patients, and infectious HIV-1 molecular clones can be useful tools. To clone non-B subtypes and recombinant viruses from patients, we cultivated HIV-1 from the plasma of a Kenyan long-term survivor. Viral DNA was cloned into a plasmid, which was transfected into COS cells; progeny virus was propagated in PBMCs. Sequence analyses revealed that both the patient's plasma HIV-1 RNA and the cloned DNA genomes were recombinants between subtypes D and C; subtype C sequences comprised the nef and LTR regions. The cloned virus used the CCR5 coreceptor and did not form syncytia in vitro. This infectious HIV-1 subtype D/C recombinant molecular clone obtained from a Kenyan long-term survivor promises to be useful to study pathogenesis and vaccine design.     

Full-length genomic sequencing and analysis of four HIV type 1 subtype B isolates circulating in the territory of Russia

We describe four full-length genomic sequences of HIV-1 subtype B isolates from Russia. These full-length HIV-1 genomes were amplified by nested polymerase chain reaction and sequenced. The sequences obtained were subjected to neighbor-joining phylogenetic analysis using a Kimura two-parameter model and to detailed sequence analysis. Comparison of the sequences obtained with 68 near full-length HIV-1 subtype B sequences from different geographic regions of the world revealed that isolates from Russia did not cluster significantly with other subtype B genomes. Sequences AY819715 and AY751407 significantly formed one cluster, which indicates their close similarity. The HIV-1 genomes obtained from Russia did not form a distinct homogeneous group inside subtype B. Their genetic diversity probably reflects the result of multiple introduction events of different subtype B strains to Russia. Sequences AY819715 and AY751406 possess some features of viruses from long-term survivors, such as specific extensions of the Env V2 region.     

Identification and characterization of HIV type 1 subtypes present in the Kingdom of Saudi Arabia: high level of genetic diversity found

Saudi Arabia has a very low prevalence of HIV infections and nothing is known about HIV strains present in the population. Here specimens were collected from 62 HIV-1-infected patients at the King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia. Viral sequences were PCR amplified using primers for HIV-1 group M in gag p24, pol integrase, and env gp41 and genetic subtype was determined by phylogenetic analysis. HIV-1 viral sequences were amplified from 56 of the 62 specimens. Based on phylogenetic analysis of viral sequences, subtype C was the most common subtype present and accounted for 39.3% of the infections followed by subtype G (25%), subtype B (17.9%), subtype D (3.6%), and subtypes A and CRF02_AG (1.8% each). In addition, for six specimens subtype classifications were discordant between gag, pol, and/or env; these intersubtype recombinant viruses account for 10.7% of the infections and consisted of recombinants of subtypes A/CRF01, A/CRF02, A/G, B/G, and D/CRF02. The high HIV-1 strain diversity suggests that there have been multiple introductions of HIV-1 into Saudi Arabia from several sources. Within the study population, there were five husband/wife pairs. For each pair, the viral sequences obtained were closely related to each other showing that heterosexual transmission occurred.     

Identification of new CRF43_02G and CRF25_cpx in Saudi Arabia based on full genome sequence analysis of six HIV type 1 isolates

Recently, we reported a high level of HIV-1 strain diversity in patients at the King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia. Based on phylogenetic analysis of gag p24, pol integrase, and env gp41 sequences, subtypes A, B, C, D, and G, and CRF02_AG, as well as unique recombinant forms were identified. Subtype G accounted for 25% of the infections in the Saudi population and this high prevalence was unexpected. Although subtype G is found in west central Africa, pure subtype G strains are uncommon. To further characterize the subtype G infections in Saudi Arabia, six strains that appeared to be pure subtype G were selected for full genome sequencing. Near full-length genomes were obtained using RT-PCR amplification to generate overlapping fragments from viral RNA extracted from plasma. The six strains are not subtype G throughout their entire genome. Four isolates have a recombinant structure composed of CRF02_AG and subtype G and share three identical breakpoints. This recombinant form defines a new CRF designated CRF43_02G. The remaining two isolates are CRF25_cpx, a circulating recombinant form identified in Cameroon composed of subtypes A and G and unclassified segments. Reanalysis of the previously reported Saudi HIV-1 partial genome sequences revealed additional isolates classified as CRF43_02G and CRF25_cpx and one isolate was reclassified to CRF22_01A. Identification of CRF43_02G in Saudi Arabia could indicate a transmission network within the country. Alternatively, the new CRF could have been introduced from an external source where this CRF is not yet recognized.     

Sequence analysis of env C2/V3, gag p17/p24, and pol protease regions of 25 HIV type 1 isolates from Ho Chi Minh City, Vietnam

Env C2/V3, gag p17/p24, pol protease, and RT regions of HIV-1 isolates recently obtained from 25 HIV-1 seropositive individuals from Ho Chi Minh City (Vietnam) were studied, and genes subtypes were determined by DNA sequence analyses. Twenty-three isolates out of 25 were identified as belonging to subtype E, now recognized as circulating recombinant form 1 (CRF01_AE). The motif at the top of the V3 loop (generally GPGQ) was then preceded by an isoleucine or a methionine (M) residue; the M residue might be a local signature of Vietnamese E isolates compared to Thai E viruses. Two isolates (8%) were shown to be intersubtype recombinants: one E/B and one CRF02_AG(IBNG)/D. The polymorphism of pol protease was considered only for CRF01_AE isolates and is clearly different from that recorded for B viruses with substitutions at positions 13, 35, 36, 41, 69, and 89.     

Assay of plasma samples representing different HIV-1 genetic subtypes: an evaluation of new versions of the amplicor HIV-1 monitor assay.

Quantification of plasma HIV-1 RNA levels has rapidly become the main tool for monitoring disease progression and treatment. However, some first-generation assays do not accurately quantify all HIV-1 subtypes. This study compares the first-generation and two newer prototype Amplicor HIV-1 Monitor assays (Roche Diagnostic Systems) in terms of their performance in quantifying HIV-1 RNA in stored plasma samples from 101 individuals infected with various genetic subtypes of HIV-1 (28 subtype A, 18 B, 26 C, 20 D, 2 E, 3 G, 2 H, and 2 J). The HIV-1 subtype had previously been determined by direct sequencing of the V3 domain of the env gene. The results from the three assays agreed for subtypes B and C, as well as for most subtype D samples. In contrast, the first-generation assay reported significantly lower plasma HIV-1 RNA levels than did the two newer versions of the assay for most subtype A, E, G, and H samples. There were no differences in mean plasma HIV-1 RNA levels between individuals infected with subtypes A, B, C, and D if the results from the two newer test versions were used, and if an adjustment was made between subtypes for differences in CD4 count. Thus, this study confirms that the first-generation assay does not accurately quantify HIV-1 RNA levels in many individuals infected with subtype A, E, G, and H. These problems appeared to have been greatly reduced in the two new prototype versions.     

Emerging recombinant human immunodeficiency viruses: uneven representation of the envelope V3 region.

OBJECTIVE: To determine whether the envelope V3 region from HIV-1 subtypes A, C or D had the same probability of being present in intersubtype recombinant genomes. MATERIALS AND METHODS: The envelope C2-C5 and the gag p24-p7 regions from one hundred infants infected perinatally in Tanzania were compared using phylogenetic and recombination analysis. Exact binomial and Fisher's exact tests were used to assess if various genomic regions were more likely to be overrepresented in intersubtype recombinants. RESULTS: Of one hundred HIV-1 positive infants analyzed, twenty-two (22%) showed exclusively subtype A sequence in gag and env. Subtype C accounted for twenty-two infants (22%) whereas nineteen infants (19%) were infected by HIV-1 subtype D. Intersubtype recombinant genomes accounted for thirty-seven infections (37%). The V3 region from subtype A was found in all fifteen A-D recombinants (P = 0.00003) and the V3 region from subtype C was found in all twelve C-D recombinants (P = 0.0002). Conversely, subtype D gag sequences were preferentially represented in the gag of A-D recombinants (P = 0.0003) as well as C-D recombinants (P = 0.002). In A-D recombinants, the V3 region of subtype A was generally surrounded by subtype A C3-C5 sequences. In contrast, the V3 region from subtype C was surrounded by subtype D C3-C5 sequences in C-D recombinants. Significant differences were not found in the number of subtype A or subtype C sequences in A-C recombinants. CONCLUSION: We have shown that several recombinant HIV-1 viruses have been generated and efficiently transmitted to infants in Tanzania. The recombination patterns showed that the V3 region of subtypes A or C was always selected in A-D and C-D recombinants. This selection suggests that the fitness of subtype D-V3 in perinatal transmission may be reduced with respect to V3 from subtype A and/or subtype C. The elevated number of recombinants transmitted perinatally suggests that co-infection or super-infection by two HIV-1 subtypes is not uncommon in this population.     

Identification of a new HIV-2 subtype based on phylogenetic analysis of full-length genomic sequence

Human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus from sooty mangabey (SIV(SM) form one of the six primate lentivirus lineages. The close phylogenetic relationship and geographic coincidence indicate that HIV-2 originated from cross-species transmission of SIV(SM) to humans. HIV-2 exhibits considerable genetic diversity, with subtypes A-F identified. Previously, we reported the partial gag and env sequences of an unusual HIV-2 isolate, Abt96. Abt96 was collected in Ivory Coast from an asymptomatic blood donor. Here we describe the near full-length genomic sequence of Abt96. The genome was assembled from overlapping PCR fragments amplified from viral RNA isolated from plasma. Phylogenetic analysis of sequences derived from segments of the Abt96 genome demonstrate that the Abt96 isolate branches independently of all other characterized HIV-2 isolates. On the basis of the phylogenetic data being presented, we propose that Abt96 is a new HIV-2 subtype and designate it subtype G.     

Full-length genome analysis of HIV-1 subtype C utilizing CXCR4 and intersubtype recombinants isolated in South Africa

The HIV-1 epidemic in South Africa is largely due to subtype C viruses, which preferentially use CCR5 as a coreceptor for infection. We describe full-length genome sequences of two CXCR4-utilizing HIV-1 subtype C viruses and two intersubtype recombinants from South Africa. Three of the viruses (99ZACM4, 99ZACM9, and 99ZASW7) were isolated in 1999 from AIDS patients in Johannesburg, and a fourth virus (98ZADu178) was isolated in Durban in 1998 from an asymptomatic female sex worker. Isolates 99ZASW7 and 99ZACM9 from Johannesburg were subtype C throughout the genome, 99ZASW7 used the CXCR4 coreceptor, and 99ZACM9 used both CCR5 and CXCR4. Isolate 98ZADu178 from Durban was a novel recombinant between subsubtype A2 and subtype C. The third isolate from Johannesburg, 99ZACM4, was a complex, novel recombinant with multiple breakpoints and contained segments of subtypes A, C, D, G, and K. These results establish the presence of intersubtype recombinants in South Africa, indicating that ongoing surveillance for other subtypes and recombinants is necessary.     

Genetic subtypes of HIV type 1 in Hungary

We examined the diversity of HIV-1 subtypes in 11 adults from Hungary, using the heteroduplex mobility assay (HMA) and DNA sequencing. HMA results showed that HIV-1 gp120 sequences from 10 patients were of subtype B, whereas 1 patient, infected in Africa, carried a subtype C strain. DNA sequencing confirmed the HMA results and revealed a high intrasubtype diversity in the C2V3 region of env in different clade B isolates, which suggests multiple introduction of subtype B to Hungary. This study shows that subtype B is the predominant HIV-1 clade in Hungary.     

一例HIV-1急性感染者奠基病毒的gp160基因序列特征及其假病毒构建

目的观察急性感染期艾滋病病毒I型（HIV-1）gp160的全长基因序列及感染特征。方法从一例处于Feibig I期HIV-1感染者血浆中提取RNA,扩增gp160全长基因并测序,分析其生物学信息;将gp160全长基因与pcDNA3.1His/V5真核表达载体连接,构建Env-pcDNA3.1真核表达质粒,与骨架质粒pNL4-3.Luc.R-E-共转染293细胞,包装出假病毒。用包装的假病毒感染ghost细胞,测定感染细胞的荧光素酶活性（RLU）,鉴定假病毒的感染活性。结果成功扩增出gp160全长基因,嗜性预测为CCR5,N-糖基化位点数与标准株HXB2相同,但gp120糖基化程度更高,氨基酸变异主要集中在V1-V5区。假病毒感染试验显示,RLU值达到7log。结论获得了处于急性感染期的HIV-1gp160基因序列和高感染活性的假病毒。     

Near full-length clones and reference sequences for subtype C isolates of HIV type 1 from three different continents

Among the major circulating HIV-1 subtypes, subtype C is the most prevalent. To generate full-length subtype C clones and sequences, we selected 13 primary (PBMC-derived) isolates from Zambia, India, Tanzania, South Africa, Brazil, and China, which were identified as subtype C by partial sequence analysis. Near full-length viral genomes were amplified by using a long PCR technique, sequenced in their entirety, and phylogenetically analyzed. Amino acid sequence analysis revealed 10.2, 6.3, and 17.3% diversity in predicted Gag, Pol, and Env protein sequences. Ten of 13 viruses were nonmosaic subtype C genomes, while all three isolates from China represented B/C recombinants. One of them was composed primarily of subtype C sequences with three small subtype B portions in gag, pol, and nef genes. Two others exhibited these same mosaic regions, but contained two additional subtype B portions at the gag/pol overlap and in the accessory gene region, suggesting ongoing B/C recombination in China. All subtype C genomes contained a prematurely truncated second exon of rev, but other previously proposed subtype C signatures, including three potential NF-kappa B-binding sites in the viral promoter-enhancer regions, were found in only a subset of these genomes.