Determinants of late HIV diagnosis among different transmission groups in Finland from 1985 to 2005.

OBJECTIVES: To study determinants of late HIV diagnosis in a low-HIV-prevalence (<0.1%) country where HIV spread among men who have sex with men (MSM) and heterosexuals in the 1980s, and among injecting drug users (IDUs) in the late 1990s. METHODS: Newly diagnosed HIV cases referred to the Helsinki University Central Hospital between 1985 and 2005 were reviewed to identify determinants of late HIV diagnosis, defined as diagnosis when the first CD4 count was <200 cells/microL, or when AIDS occurred within 3 months of HIV diagnosis. Determinants of late diagnosis were analysed using multivariate logistic regression. RESULTS: Among 934 HIV cases, 211 (23%) were diagnosed late. In the first 4-year interval of each sub-epidemic (1985-1989 for MSM and heterosexuals, 1998-2001 for IDUs), rates of late HIV diagnosis were 13%, 18% and 6%, respectively, but increased thereafter to 29%, 27% and 37%. Late diagnosis was associated with non-Finnish ethnicity, older age, male gender, lack of earlier HIV testing, diagnosis at health care settings and later stage of the sub-epidemic. CONCLUSIONS: The lower rate of late diagnosis in the first 4-year interval of each HIV sub-epidemic suggests that the early stages of the HIV epidemic in Finland were detected early. This factor may have contributed to the low prevalence of HIV infection in Finland. The stage and age of the epidemic should be taken into account when interpreting the data on late HIV diagnosis, especially in cross-country comparisons.

Construction and biological characterization of infectious molecular clones of HIV-1 subtypes B and E (CRF01_AE) generated by the polymerase chain reaction.

We previously described the use of extended polymerase chain reaction (PCR) to amplify contiguous 9.2-kilobase (kb) single-long terminal repeat (LTR) proviral sequences from HIV-1 genetic subtypes A through G. We now extend these findings by describing a novel vector system to recover infectious molecular clones from long PCR amplicons. Directional ligation of 9.2-kb proviral amplicons into a recovery vector reconstitutes missing LTR sequences, providing candidate molecular clones for infectivity screening. We show that a previously characterized infectious molecular clone of HIV-1 retains its biological properties upon recovery with this strategy. Three additional infectious molecular clones generated, from primary isolates of subtype B (HIV-1(WR27)) and circulating recombinant form 01_AE (subtype E) (HIV-1(CM235)) by subtype-specific LTR reconstitution, displayed biological properties reflecting their cognate parental isolates. This represents the first report of infectious molecular clones from circulating recombinant form 01_AE (subtype E).

Analysis of HIV-1 genetic subtypes in Finland reveals good correlation between molecular and epidemiological data.

The molecular epidemiology of HIV-1 genetic subtypes was studied in a cross-sectional sample collected from HIV-infected individuals living in Finland between 1988 and 1994 and compared with independently collected epidemiological data. Subtypes were determined by sequencing and phylogenetic analysis of the gag NCp7 and the env coding regions of PBMC provirus. Finnish viruses belonging to 7 subtypes were found. Two thirds (n = 70) of the sequences could be classified as subtype B, while others belonged to subtypes A, C, D, F and G and the circulating recombinant form AE(CM240) (n = 25). There were significant differences in gender distribution and mode-of-transmission between B-type infections and infections with the other subtypes. Most subtype B strains in Finland were associated with homosexual transmission and about half of these were acquired in Finland, while most individuals harbouring non-B infections indicated heterosexual transmission and direct or indirect contact with Africa or Southeast Asia. The heterogeneity of genetic subtypes in the country was in good agreement with the epidemiological data suggesting that a significant proportion of infections were imported. HIV-1 subtype determination may prove to be a valuable tool for providing objective epidemiological data.

HIV-1 subtype H near-full length genome reference strains and analysis of subtype-H-containing inter-subtype recombinants.

OBJECTIVE: To characterize near-full-length genomes of two HIV-1 subtype H strains. To extend sequence data to include full env and gag, and analyse and redefine, previously documented subtype H strains. DESIGN: Near-full-length genomes of HIV-1 env subtype H strains VI991 and VI997 were amplified, cloned, sequenced, phylogenetically analysed and compared with a panel of 23 HIV-1 group M reference isolates. The mosaic nature of previously published subtype H strains VI557 and CA13 was reanalysed. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) from individuals harbouring strains VI991 and VI997 were co-cultivated with PHA stimulated donor PBMC. Near-full-length genomes of VI991 and VI997, and gag and env genes of CA13 and VI557, were amplified by polymerase chain reaction, cloned and sequenced. Intersubtype recombination analyses were performed by similarity plot, bootscanning and phylogenetic analysis. RESULTS: Near-full-length clones of HIV-1 VI991 and VI997 are representative of subtype H. They form a phylogenetic cluster with the only previously described subtype H representative HIV-1 90CF056.1, regardless of the genome region analysed. VI557 is redefined as a gag and env subtype H mosaic virus containing unclassified fragments. CA13 is a complex intersubtype recombinant between subtypes A, H and unclassified strains CONCLUSION: Near-full-length genome analysis identified HIV-1 VI991 and VI997 as two new subtype H representatives. These reagents will allow defining and classifying non-recombinant as well as recombinant HIV-1, eventually helping to solve the puzzle of HIV-1 subtypes.

Near full-length genome analysis of HIV type 1 CRF02.AG subtype C and CRF02.AG subtype G recombinants.

HIV-1 CRF02.AG strains are prevalent in west and west-central Africa, suggesting a longstanding presence of these subtype A/G recombinants in the global epidemic. Cocirculation of CRF02.AG strains with other group M subtypes may give rise to HIV-1 recombinants constituting a mosaic genome comprising fragments of three different subtypes. We report on the genetic analysis of the near-full-length genomes of such recombinants (VI1035 and VI1197) as well as CRF02.AG strains in Belgian individuals. VI1035 and VI1197 may be the result of successful "second-generation" recombinations of HIV-1 strains CRF02.AG with, respectively, subtype C (VI1035) and G (VI1197) strains in a dually infected individual.

Virtually full-length subtype F and F/D recombinant HIV-1 from Africa and South America.

For reliable classification of HIV-1 strains appropriate reference sequences are needed. The HIV-1 genetic subtype F has a wide geographic spread, causing significant epidemics in South America, Africa, and some regions of Europe. Previously only two full-length sequences of each of the HIV-1 subtype F subclusters F1 and F2 have been described. To extend the knowledge of subtype F variation on a complete genome level, three new virtually full-length F1 sequences were cloned and sequenced, two from Africa and one from South America. Comparison of the new and previously described sequences showed that monophyletic clustering of the subcluster F1 of subtype F is consistent and highly supported in all genome regions. Two additional full-length strains were shown to be mosaics of subtypes F and D. These epidemiologically unrelated F/D sequences showed similar chimeric structure, suggesting that they may represent a previously undescribed circulating recombinant form (CRF). This was supported by partial sequences from three additional unlinked F/D recombinants. Genetic distances in the phylogenetic trees suggest that the recombination event leading to the putative CRF occurred relatively long ago, close to the divergence of the F1 and F2 subclusters. Furthermore, all five F/D recombinants are linked to the Democratic Republic of Congo, suggesting that the original recombination event took place in central Africa.

Characterization of subtype A HIV-1 from Africa by full genome sequencing.

OBJECTIVE: To improve our understanding of the genetic complexity of HIV-1 subtype A by increasing the number of subtype A isolates that have been sequenced in their entirety. METHODS: Nine HIV-1-seropositive patients from Africa living in Sweden contributed peripheral blood mononuclear cells (PBMC) for this study. Sequencing of the C2-V3 region of env had shown them to be subtype A. DNA from virus cultures was used for the amplification of virtually full-length proviral sequences, and the resulting fragment was sequenced. RESULTS: Six of the nine viral isolates were subtype A throughout the genome, or non-recombinant, and all of these were from east Africa. One virus from the Ivory Coast had the AG(IbNG) genetic form, a recombinant form common in west Africa. Two of the isolates were novel recombinants: one was an A/C recombinant and the other was A/D. Analysis of gag reveals three subclusters within the A subtype: one containing the AG(IbNG) subtype viruses, one containing the AE(CM240) viruses and one containing the non-recombinant A viruses. These genetic clusters have different geographical distributions in Africa. CONCLUSION: The prevailing view of HIV-1 subtype A forming a uniform band across the center of sub-Saharan Africa needs revision. In all probability, the most common subtype in west Africa and west central Africa is the AG recombinant, AG(IbNG), whereas in east central Africa it is the non-recombinant subtype A.

An isolate of human immunodeficiency virus type 1 originally classified as subtype I represents a complex mosaic comprising three different group M subtypes (A, G, and I).

Full-length reference clones and sequences are currently available for eight human immunodeficiency virus type 1 (HIV-1) group M subtypes (A through H), but none have been reported for subtypes I and J, which have only been identified in a few individuals. Phylogenetic information for subtype I, in particular, is limited since only about 400 bp of env gene sequences have been determined for just two epidemiologically linked viruses infecting a couple who were heterosexual intravenous drug users from Cyprus. To characterize subtype I in greater detail, we employed long-range PCR to clone a full-length provirus (94CY032.3) from an isolate obtained from one of the individuals originally reported to be infected with this subtype. Phylogenetic analysis of C2-V3 env gene sequences confirmed that 94CY032.3 was closely related to sequences previously classified as subtype I. However, analysis of the remainder of its genome revealed various regions in which 94CY032.3 was significantly clustered with either subtype A or subtype G. Only sequences located in vpr and nef, as well as the middle portions of pol and env, formed independent lineages roughly equidistant from all other known subtypes. Since these latter regions most likely have a common origin, we classify them all as subtype I. These results thus indicate that the originally reported prototypic subtype I isolate 94CY032 represents a triple recombinant (A/G/I) with at least 11 points of recombination crossover. We also screened HIV-1 recombinants with regions of uncertain subtype assignment for the presence of subtype I sequences. This analysis revealed that two of the earliest mosaics from Africa, Z321B (A/G/?) and MAL (A/D/?), contain short segments of sequence which clustered closely with the subtype I domains of 94CY032.3. Since Z321 was isolated in 1976, subtype I as well as subtypes A and G must have existed in Central Africa prior to that date. The discovery of subtype I in HIV-1 hybrids from widely distant geographic locations also suggests a more widespread distribution of this virus subtype, or at least segments of it, than previously recognized.

HIV-1 genetic subtype A/B recombinant strain causing an explosive epidemic in injecting drug users in Kaliningrad.

OBJECTIVES: To investigate the molecular epidemiology and genetic structure of the virus strain(s) causing an outbreak of HIV-1 infection in the Kaliningrad province of the Russian Federation and to investigate the relationship of this outbreak to some other emerging HIV-1 epidemics in the countries of the former Soviet Union. DESIGN: A molecular epidemiological investigation was conducted in the city of Kaliningrad amongst individuals recently diagnosed as HIV-1-positive. Samples were also collected from neighbouring Lithuania and from the Ukraine. METHODS: Incident and population data was collected from official health statistics in Kaliningrad. A standardized questionnaire was administered to newly diagnosed individuals to assess risk factors for HIV-1 infection. For genotyping, two regions of the virus (env C2-V3 and gag NCp7) were directly sequenced. RESULTS: The number of newly diagnosed individuals testing seropositive for HIV-1 infection in Kaliningrad rose from less than one per month to more than 100 per month during the period of July-October 1996. A total of 1335 new infections were identified between 1 July 1996 and 30 June 1997. The main reported risk factor for HIV-1 infection (80%) was injecting drug use, in particular with a locally produced opiate. Sequence analysis of patient viruses in Kaliningrad (n = 50) showed that the epidemic was caused by a highly homogenous HIV-1 strain, recombinant between the genetic subtypes A and B. Comparison with subtype A strains prevalent amongst injecting drug users (IDU) in the Ukraine showed that one of these strains was the direct subtype A parent of the epidemic A/B recombinant strain in Kaliningrad. CONCLUSIONS: The HIV-1 epidemic in Kaliningrad probably started from a single source, with rapid spread of the virus through the IDU population. The origin of the epidemic strain is a recombination event occurring between the subtype A strain virus prevalent among IDU in some southern CIS countries, and a subtype B strain of unknown origin.

Full genome sequences of human immunodeficiency virus type 1 subtypes G and A/G intersubtype recombinants.

Multiple genetic subtypes and intersubtype recombinant strains have been identified among isolates of HIV-1. The greatest diversity of strains has been recovered from Central Africa, where mixtures of subtypes and recombinant forms have been recovered. However, many of the HIV-1 subtypes and recombinants have been characterized by partial rather than full-length genome sequencing. Here we report the first two virtually full-length genome sequences from HIV-1 subtype G, isolated in Sweden and Finland but originating in Congo and Kenya, and from two Djibouti isolates sharing the A/G recombinant structure of Nigerian isolate, IbNG. By comparison with reference sequences of other subtypes, it appears that the subtype G strains are largely nonrecombinant, while the Djibouti strains show alternating segments from subtypes A and G. In the cytoplasmic domain of the gp41 protein of the Djibouti viruses the E, G, and IbNG strains form a single cluster, separate from subtype A, clouding the subtype origin of these particular segments. Within the resolution of current technology, the structure of the Djibouti strains is identical to that of IbNG, establishing for the first time the geographic spread of this recombinant in Africa. The geographic spread of the IbNG-like strains suggests that, like the subtype E recombinants, these should be given a specific name to facilitate future identification and tracking; the name "IbNG subtype" is proposed.

Infectious molecular clones with the nonhomologous dimer initiation sequences found in different subtypes of human immunodeficiency virus type 1 can recombine and initiate a spreading infection in vitro.

Recombinant forms of human immunodeficiency virus type 1 (HIV-1) have been shown to be of major importance in the global AIDS pandemic. Viral RNA dimer formation mediated by the dimerization initiation sequence (DIS) is believed to be essential for viral genomic RNA packaging and therefore for RNA recombination. Here, we demonstrate that HIV-1 recombination and replication are not restricted by variant DIS loop sequences. Three DIS loop forms found among HIV-1 isolates, DIS (CG), DIS (TA), and DIS (TG), when introduced into deletion mutants of HIV-1 recombined efficiently, and the progeny virions replicated with comparable kinetics. A fourth DIS loop form, containing an artificial AAAAAA sequence disrupting the putative DIS loop-loop interactions [DIS (A6)], supported efficient recombination with DIS loop variants; however, DIS (A6) progeny virions exhibited a modest replication disadvantage in mixed cultures. Our studies indicate that the nonhomologous DIS sequences found in different HIV-1 subtypes are not a primary obstacle to intersubtype recombination.

Diversity of the human immunodeficiency virus type 1 envelope glycoprotein in San Francisco Men's Health Study participants.

Multiple genetic subtypes of HIV-1, differing by up to 30% of nucleotides in their envelope coding sequences, have been identified in the global epidemic. In the United States, where HIV-1 infection with subtype B predominates, the interisolate diversity in envelope is 15% or more. It is recognized that geographic, temporal, and demographic variables can affect the genetic diversity of HIV-1 strains, but there have been few opportunities to evaluate these factors by population-based sampling. We have evaluated HIV-1 envelope diversity among participants in the San Francisco Men's Health Study (SFMHS), which represents a geographically, temporally, and demographically defined subset of HIV-1 infections in the United States. DNA was extracted from primary PBMCs obtained within 6 months of seroconversion and from individuals whose HIV-1 infection occurred between 1985 and 1989. The full-length envelope gene was PCR amplified, cloned, and sequenced from 17 different individuals. The sequences were compared within the cohort and with reference sequences from the United States and overseas, and their relationship to vaccine prototype strains LAI, MN, and SF2 was evaluated. SFMHS participants harbored HIV-1 subtype B infections with limited interpatient variation and a higher proportion of atypical V3 loop crown sequences than reference sequences of this subtype. Throughout gp160, the MN strain was less representative than LAI or SF2 among the patients examined. The geographic component of variation was apparently more substantial than the temporal, emphasizing the need for widely distributed geographic sampling in estimations of HIV diversity.

Evolution and probable transmission of intersubtype recombinant human immunodeficiency virus type 1 in a Zambian couple.

The extraordinary genetic diversity of human immunodeficiency virus type 1 (HIV-1) results from the introduction of mutations by an error-prone reverse transcriptase and from recombination of the two RNA genomes packaged in the virion during the synthesis of proviral DNA. The occurrence of multiple, genetically distant HIV-1 subtypes and their geographic intermixing set up conditions for dramatic, rather than gradual, changes in genotype whenever genomes from different subtypes are copackaged in virions. Here we describe, for the first time, the sequential generation of multiple different, but related, intersubtype HIV-1 recombinants within an infected individual. Full-length gag and env genes were recovered directly from peripheral blood mononuclear cells or from primary virus cultures, using serial blood samples from a Zambian woman and a sample from her spouse. DNA sequencing and phylogenetic analysis established that two different A/C recombinant forms of HIV-1 predominated at two time points in the woman. A related but distinct recombinant HIV-1 was recovered from her spouse. Intersubtype recombination apparently played a central role in the evolution of HIV-1 in this couple and may contribute substantially to the rapid emergence of HIV-1 variants whenever mixed-subtype HIV-1 infections occur.

HIV-1 genetic diversity.

BACKGROUND: HIV-1 evolves by rapid mutation and by recombination, both processes actively contributing to its genetic diversity. Most of the multiple genetic subtypes and intersubtype recombinations of HIV-1 that comprise the global pandemic have not been characterized by full genome sequencing. METHODS: DNA from primary virus cultures on donor peripheral blood mononuclear cells was used as template for long polymerase chain reaction amplification, molecular cloning, and automated sequencing of virtually full-length HIV-1 genomes from subtypes A, C, E, G and A/D recombinant forms. Standard phylogenetic analysis methods were employed, and some were modified for the detection and mapping of recombinant breakpoints. RESULTS: Subtypes A, B, C and D are largely, if not entirely, distinguishable throughout the genome and show no clear evidence of intersubtype recombination. In contrast, all available sequences of subtypes E and G are recombinant with subtype A. Full-length sequences of subtypes F, H, I and J are still unavailable. Subtype E and G, and some A/D recombinant HIV, have retained the cytoplasmic domain of gp41 from subtype A. Some recombinants possess the matrix and core of one subtype and the outer envelope of another, resembling pseudotypes. Certain pairs of subtypes may have recombined more often than others. CONCLUSION: Recombinant HIV-1 have already established a global reservoir and are largely responsible for the rapidly expanding subtype E epidemic in Southeast Asia. Recombination may have played a key role in the evolution of HIV-1 and the geographic intermixing of subtypes, which is increasing, may foster the emergence of a even greater variety of recombinant strains.

Detection of HIV type 1 env subtypes A, B, C, and E in Asia using dried blood spots: a new surveillance tool for molecular epidemiology.

Global surveillance of HIV-1 subtypes for genetic characterization is hampered by the biohazard of processing and the difficulties of shipping whole blood or cells from many developing country regions. We developed a technique for the direct automated sequencing of viral DNA from dried blood spot (DBS) specimens collected on absorbent paper, which can be mailed unrefrigerated in sturdy paper envelopes with low biohazard risk. DBS were collected nonrandomly from HIV-1-infected, mostly asymptomatic, patients in five Asian countries in 1991, and shipped via airmail or hand carried without refrigeration to Bangkok, and then transshipped to North America for processing. After more than 2 years of storage, including 6 months at ambient temperatures, proviral DNA in the DBS was amplified by nested PCR, and a 389-nucleotide segment of the C2-V3 env gene region was sequenced, from which 287 base pairs were aligned and subtyped by phylogenetic analysis with neighbor-joining and other methods. From southern India, there were 25 infections with subtype C and 2 with subtype A. From Myanmar (Burma), we identified the first subtype E infection, as well as six subtype BB, a distinct cluster within subtype B that was first discovered in Thailand and that has now appeared in China, Malaysia, and Japan. From southwest China, one BB was identified, while a "classical" B typical of North American and European strains was found in Indonesia. From Thailand, five DBS of ambiguous serotype were identified as three B, one BB, and one E. A blinded control serotype E specimen was correctly identified, but a serotype BB control was not tested. Most HIV-1 in southern India appears to be env subtype C, with rare A, as others have reported in western and northern India. The subtypes BB and E in Myanmar, and the BB in China, suggest epidemiological linkage with these subtypes in neighboring Thailand. DBS are a practical, economical technique for conducting large-scale molecular epidemiological surveillance to track the global distribution and spread of HIV-1 variants.

PIP: The global surveillance of HIV-1 subtypes for genetic characterization is frustrated by the danger of processing and the difficulties of shipping whole blood or cells from many developing country regions. The authors therefore developed a technique for the direct automated sequencing of viral DNA from dried blood spot (DBS) specimens collected on absorbent paper. Such specimens can be mailed unrefrigerated in paper envelopes with low biohazard risk. 51 DBS specimens were collected nonrandomly from HIV-1-infected, mostly asymptomatic individuals in India, Myanmar, China, Indonesia, and Thailand in 1991, then shipped via airmail or hand carried without refrigeration to Bangkok from where they were forwarded to North America for processing. After more than 2 years in storage, including 6 months at ambient temperatures, proviral DNA in 42 of the DBS was successfully amplified by nested polymerase chain reaction, and a 389-nucleotide segment of the C2-V3 env gene region was sequenced, from which 287 base pairs were aligned and subtyped by phylogenetic analysis with neighbor-joining and other methods. From southern India, there were 25 infections with subtype C and two with subtype A; the first subtype E infection was identified from Myanmar, as well as six subtype B(B); one B(B) was identified from southwest China; subtype B was identified from Indonesia; and five DBS of ambiguous serotype classified as three B, one B(B), and one E were identified from Thailand. DBS can be used as a practical, cost-effective way of tracking the global distribution and spread of HIV-1 variants.

Full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from Thailand.

Human immunodeficiency virus type 1 isolates of envelope genotype E are contributing substantially to the global pandemic. These strains appear to be mosaics, with the gag gene from clade A and the envelope from clade E; the parental clade E strain has not been found. Here we report the first full genomic sequence of one such mosaic virus, isolate CM240 from Thailand. Multiple breakpoints between the two parental genotypes have been found in a CM240 virus. The entire gag-pol region and most, if not all, of the accessory genes vif, vpr, tat, rev, and vpu appear to derive from clade A. The genotype switches to E shortly after the signal peptide of the envelope and back to clade A near the middle of gp41; thus, the portion of the envelope that lies on the cytoplasmic side of the membrane appears to be principally derived not from clade E, as previously thought, but from clade A. Another small segment not belonging to any recognized clade and presumably also contributed by the parental E strain has been found in the long terminal repeat. It may be significant that the implied virion structure resembles a pseudotype virus with the matrix and core from one clade and the outer envelope from another. In the long terminal repeat, differences were observed between CM240 and other clades in the number of NF-kappa B binding sites, the sequence of the TATA box, and the putative secondary structure of the transactivation response region stem-loop. The mosaic structure of a CM240 virion is suggestive of phenotypic differences which might have contributed to the emergence of this variant.

PIP: A new variant of human immunodeficiency virus (HIV)-1 with a mosaic genomic structure was identified in Thailand in 1992. This variant, termed genotype E, was characterized by an envelope gene sequence equidistant from genotypes A through D. The gag gene, encoding the matrix and core virus proteins, grouped with genotype A rather than forming a new clade. More than 500,000 Thais are estimated to be infected with the envelope clade E virus and its type 1 isolates, previously assumed to be rare outliers, are contributing substantially to the global acquired immunodeficiency syndrome pandemic. Reported here is the first complete genomic analysis of one such mosaic virus, isolate CM240 from Thailand. The entire gag-pol region and most of the accessory genes vif, vpr, tat, rev, and vpu appear to derive from clade A. The genotype switches to E shortly after the signal peptide of the envelope and back to clade A near the middle of gp41. Thus, the portion of the envelope that lies on the cytoplasmic side of the membrane appears to be derived from clade A. Another small segment presumably contributed by the parental E strain has been found in the long terminal repeat. The multiple crossover points detected in CM240 may reflect a common mechanism of frequent strand switching by reverse transcriptase. Full genomic analyses of other mosaic HIV-1 genomes are recommended to determine whether the breakpoints found in CM240 are recurrent and to identify the functional implications of the virion alterations.