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Background Occult or latent hepatitis B virus (HBV) infection is defined as infection with detectable HBV DNA and undetectable surface antigen (HBsAg) in patients' blood. The cause of an overt HBV infection becoming an occult one is unknown. To gain insight into the mechanism of the development of occult infection, we compared the full-length HBV genome from a blood donor carrying an occult infection (d4) with global genotype D genomes. Results The phylogenetic analysis of polymerase, core and X protein sequences did not distinguish d4 from other genotype D strains. Yet, d4 surface protein formed the evolutionary outgroup relative to all other genotype D strains. Its evolutionary branch was the only one where accumulation of substitutions suggests positive selection (dN/dS = 1.3787). Many of these substitutiions accumulated specifically in regions encoding the core/surface protein interface, as revealed in a 3D-modeled protein complex. We identified a novel RNA splicing event (deleting nucleotides 2986-202) that abolishes surface protein gene expression without affecting polymerase, core and X-protein related functions. Genotype D strains differ in their ability to perform this 2986-202 splicing. Strains prone to 2986-202 splicing constitute a separate clade in a phylogenetic tree of genotype D HBVs. A single substitution (G173T) that is associated with clade membership alters the local RNA secondary structure and is proposed to affect splicing efficiency at the 202 acceptor site. Conclusion We propose an evolutionary scenario for occult HBV infection, in which 2986-202 splicing generates intracellular virus particles devoid of surface protein, which subsequently accumulates mutations due to relaxation of coding constraints. Such viruses are deficient of autonomous propagation and cannot leave the host cell until it is lysed.

Surface protein and polymerase of hepatitis B virus provide a striking example of gene overlap. Inclusion of more coding constraints in the phylogenetic analysis forces the tree toward accepted topology. Three-dimensional protein modeling demonstrates that participation in local protein function underlies the observed mosaic patterns of amino acid conservation and variability. Conserved amino acid residues of polymerase were typically clustered at the catalytic core marked by the YMDD motif. The proposed tertiary structure of surface protein displayed the expected transmembrane helices in a 2-domain constellation. Conserved amino acids like, for instance, cysteine residues are involved in the spatial orientation of the two domains, the exposed location of the a-determinant and the dimer formation of surface protein. By means of computational alanine replacement scanning, we demonstrated that the interfaces between domains in monomeric surface protein, between the monomers in dimeric surface protein and in a capsid–surface protein complex mainly consist of relatively well-conserved amino acid residues.

We identified an HIV-1 variant that belongs to the M group, with limited similarity of short genetic regions (100-200 nt) to subtype K, but the remainder of the genome is unrelated to any established HIV-1 subtype. The isolate was obtained from an HIV-1-positive male, living in the Netherlands, who encountered the virus before 1989, most probably via heterosexual contact in Africa. We describe the full-length genome sequence of four biological clones that were obtained from two samples collected 5 years apart. At both time points all open reading frames were intact. Within the 5-year interval, the person received antiretroviral therapy with zalcitabine and zidovudine for almost 4 years. Evolution of drug-resistant variants is likely given the increase in viral RNA load to +/-10,000 copies/ml during the last year of treatment. Surprisingly, the only regular RT mutation acquired during this period was K70R, which suggests that the genetic background of this variant is perhaps not suitable for the generation of the standard 41L, 67N, and 215Y/F mutations that typically arise during prolonged, nonsuccessful, zidovudine treatment. Awaiting the discovery of at least two additional, epidemiologically unrelated patients with a phylogenetically related HIV-1 variant, we can designate this variant a new HIV-1 subtype, or a distinct branch of subtype K.

The rate of processes accompanying the transition of the HIV-1 epidemic from nascent stage to concentrated one in the Former Soviet Union (FSU) during intravenous drug user (IDU)-associated HIV infection outbreaks in 1994-1999 has not been analyzed. To define the rates, we studied susceptible populations and circulating viruses before, during, and after the outbreaks. Our findings included the following: (1) the pattern of high HIV-1 genetic diversity characteristic of the nascent epidemic changed to a concentrated one within 1 year in St. Petersburg and in Moscow; (2) different FSU regions were at different stages of the HIV-1 epidemic in 1994-1996; (3) the change of serotypic patterns characteristic of different stages of the HIV/AIDS epidemic for the non-IDU risk group occurred within 1 year in Moscow, suggesting an extremely high rate of IDU-associated epidemic pattern distributions in regions and susceptible populations in the FSU.

Background Complete genome sequences of the Astroviridae include human, non-human mammalian and avian species. A consensus topology of astroviruses has been derived from nucleotide substitutions in the full-length genomes and from non-synonymous nucleotide substitutions in each of the three ORFs. Analyses of synonymous substitutions displayed a loss of tree structure, suggesting either saturation of the substitution model or a deviant pattern of synonymous substitutions in certain virus species. Results We analyzed the complete Astroviridae family for the inference of adaptive molecular evolution at sites and in branches. High rates of synonymous mutations are observed among the non-human virus species. Deviant patterns of synonymous substitutions are found in the capsid structural genes. Purifying selection is a dominant force among all astrovirus genes and only few codon sites showed values for the dN/dS ratio that may indicate site-specific molecular adaptation during virus evolution. One of these sites is the glycine residue of a RGD motif in ORF2 of human astrovirus serotype 1. RGD or similar integrin recognition motifs are present in nearly all astrovirus species. Conclusion Phylogenetic analysis directed by maximum likelihood approximation allows the inclusion of significantly more evolutionary history and thereby, improves the estimation of dN and dS. Sites with enhanced values for dN/dS are prominent at domains in charge of environmental communication (f.i. VP27 and domain 4 in ORF1a) more than at domains dedicated to intrinsic virus functions (f.i. VP34 and ORF1b (the virus polymerase)). Integrin recognition may play a key role in astrovirus to target cell attachment.

The presence of the novel parvovirus PARV4 and a related variant, PARV5, was recently demonstrated in pooled plasma used in the manufacture of blood and plasma-derived medicinal products. DNA sequence analysis of nearly full-length genomes of four PARV4 and two PARV5 strains from manufacturing plasma pools is now presented. Like PARV4, PARV5 encodes two non-overlapping open reading frames (ORF1 and ORF2), homologous to the non-structural and capsid proteins of other parvoviruses, respectively. A highly conserved region in ORF2 contains phospholipase A2 motifs involved in parvovirus infectivity. Hybridization of strand-specific probes to DNA extracted from high-titre, PARV4-positive plasma revealed that the positive and negative strands are packaged into PARV4 virions in similar quantities. This extended analysis of nearly full-length PARV4 and PARV5 sequences suggests that they are closely related genotypes and the use of a single virus name, PARV4, comprising genotypes 1 and 2 (previously termed PARV5) is proposed.

To study the molecular epidemiology of HIV-1 in Belarus, where the rapid spread of HIV-1 has been registered since 1996, we obtained HIV-1 sequences from 30 individuals living in five cities in both the main geographic areas of the epidemic (Gomel and Minsk regions) and territories where spreading of the epidemic remains limited (Grodno region). Analysis of env V3 and gag p17/p24 sequences demonstrated that infections in all 12 injecting drug users and 14 of 18 individuals infected through sexual contacts were caused by subtype A viruses that are specific for the epidemic in the former Soviet Union (IDU-A viruses), while the remaining four infections were caused by phylogenetically unrelated to each other subtype B viruses. Extrapolation of these results to the total population of HIV-1-infected individuals in Belarus allowed us to estimate that IDU-A viruses account for nearly 95% of HIV-1 infections in Belarus.

Retroviral RNA genomes are known to have a biased nucleotide composition. For instance, the plus-strand RNA of human immunodeficiency virus (HIV) is A-rich, and the genome of human T cell leukemia virus (HTLV) is C-rich, and other retroviruses have a U-rich or G.-rich genome. The biased composition of these genomes is most likely caused by directional mutational pressure of the respective reverse transcriptase enzymes. Using a set of retroviral genomes with a distinct nucleotide composition, we performed skew analyses of the nucleotide bias along the complete viral genome. Distinct nucleotide signatures were apparent, and these typical patterns were generally conserved across the viral genome. Furthermore, it is demonstrated that this typical nucleotide bias, combined with a profound discrimination against the CpG dinucleotide sequence, strongly influences the codon usage of the retroviruses in a direct manner, and their amino acid usage in an indirect manner. The fact that both codon usage and amino acid usage are so closely entwined with the genome composition has important practical implications. For instance, the typical trends in nucleotide usage could influence the molecular phylogenetic reconstruction of the family Retroviridae

To study the evolutionary relationships among astroviruses, all available sequences for members of the family Astroviridae were collected. Phylogenetic analysis distinguished two deep-rooted groups: one comprising mammalian astroviruses, with ovine astrovirus being an outlier, and the other comprising avian astroviruses. All virus species as well as serotypes of human astroviruses represented individual lineages within the tree. All human viruses clustered together and separately from non-human viruses, which argue for their common evolutionary origin and against ongoing animal-to-human transmissions. The branching order of mammalian astroviruses was exactly the opposite of that of their host species, suggesting at least two cross-species transmissions involving pigs, cats and humans, possibly through intermediate hosts. Analysis of synonymous (Ds) versus non-synonymous (Da) distances revealed that negative selection is dominating in the evolution of astroviruses, with the Ds:Da ratios being up to 46 for the comparisons of the most closely related viruses. Phylogenetic analyses of all open reading frames (ORFs) based on Ds resulted in the loss of tree structures, with virus species – and in ORF2, even serotypes of human astroviruses – branching out from virtually a single node, suggesting their ancient separation. The strong selection against non-synonymous substitutions, the low number of which is, therefore, not proof of a recent separation between lineages, together with the position of the oldest available human astrovirus strain (1971) far from the common node of its serotype 4, suggest that intraserotype diversification originates from an earlier date.

Objective: To examine the epidemiological factors influencing the distribution and spread of HIV-1 subtypes among heterosexuals in the Netherlands. Method: A nationwide serosurveillance in 21 HIV/AIDS centres from 1997 to 1999 involved 200 individuals for whom the mode of HIV transmission was heterosexual contact or unknown. HIV-1 subtypes were determined by phylogenetic analysis of env V3 sequences and correlated with sociodemographic characteristics of the subjects and their sexual partners. Results: HIV-1 subtype B infection occurred in 121 subjects (60%). Non-B subtypes were identified in 31 (A), 24 (C), 10 (D), six (E), four (F) and three (G) individuals; one had an unclassified subtype. The proportion of subtype B was about 60% in four of the six regions of the Netherlands, but in the Northwest and Southwest regions these proportions were 76% and 46%, respectively. The Surinamese and Antilleans, large immigrant groups, were all infected with subtype B, as were almost all individuals with an unknown source. The proportions of non-B viruses did not change significantly over time in Amsterdam, where subtyping was available from 1988 onward, but a shift in the various subtype B strains was observed, suggesting introductions of new subtype B strains in Amsterdam. Conclusion: To date, HIV-1 non-B subtypes in the Netherlands are still found predominantly among heterosexuals with an epidemiological link with sub-Saharan Africa. Despite continuing introductions of non-B subtypes, the B/non-B distribution has been stable over time, most likely as a result of introductions of subtype B strains from Caribbean and South American countries.

The current classification of parvoviruses is based on virus host range and helper virus dependence, while little data on evolutionary relationships among viruses are available. We identified and analyzed 472 sequences of parvoviruses, among which there were (virtually) full-length genomes of all 41 viruses currently recognized as individual species within the family Parvoviridae . Our phylogenetic analysis of full-length genomes as well as open reading frames distinguished three evolutionary groups of parvoviruses from vertebrates: (i) the human helper-dependent adeno-associated virus (AAV) serotypes 1 to 6 and the autonomous avian parvoviruses; (ii) the bovine, chipmunk, and autonomous primate parvoviruses, including human viruses B19 and V9; and (iii) the parvoviruses from rodents (except for chipmunks), carnivores, and pigs. Each of these three evolutionary groups could be further subdivided, reflecting both virus-host coevolution and multiple cross-species transmissions in the evolutionary history of parvoviruses. No parvoviruses from invertebrates clustered with vertebrate parvoviruses. Our analysis provided evidence for negative selection among parvoviruses, the independent evolution of their genes, and recombination among parvoviruses from rodents. The topology of the phylogenetic tree of autonomous human and simian parvoviruses matched exactly the topology of the primate family tree, as based on the analysis of primate mitochondrial DNA. Viruses belonging to the AAV group were not evolutionarily linked to other primate parvoviruses but were linked to the parvoviruses of birds. The two lineages of human parvoviruses may have resulted from independent ancient zoonotic infections. Our results provide an argument for reclassification of Parvovirinae based on evolutionary relationships among viruses.

We studied the intrahost evolution and dynamics of a multidrug-resistant HIV-1, which contains an insertion of two amino acids (aa) and several aa changes within the reverse transcriptase (RT) gene. From an individual receiving intermittent therapy, sequences of 231 full-length molecular clones of HIV-1 RT were obtained from serum-derived viruses at 12 consecutive time points over a period of 6 years, 17 to 20 clones per time point. In the 3.5-year period prior to the first course of therapy, only wild-type (wt) viruses were found. As soon as 6 months after the start of zidovudine (AZT) monotherapy, all viruses contained an insertion of two aa between positions 68 and 69 of the RT and aa changes at positions 67 and 215, a combination conferring resistance to multiple nucleoside analogs. After termination of therapy, the insertion mutants were rapidly and completely replaced by the wt viruses. In turn, the insertion mutants replaced the wt viruses after initiation of therapy with 3TC, d4T, and saquinavir. After termination of triple therapy, the wt viruses completely replaced the mutants within 1 month, which is markedly faster than has been observed earlier for the replacement of AZT-resistant viruses. Fast replacements of the mutant virus populations after termination of therapy indicate gross competitive disadvantage of the insertion mutant in the absence of therapy, which we estimated by using several models. The insertion mutants attained high virus loads, demonstrating that virus load cannot be used as a direct measure of virus fitness.

We studied sequence differences in regulatory elements of the long terminal repeat (LTR) and primer-binding site (PBS) among various human immunodeficiency virus type 1 (HIV-1) subtypes. Phylogenetic sequence analysis of a fragment of 729 base pairs (bp) covering the Gag-coding region for half of p24 and all of p17 revealed the gag subtype of all 60 viruses included in the study: A (n = 20), B (n = 12), C (n = 7), D (n = 10), E (n = 3), F (n = 4), G (n = 3), and H (n = 1). The subtype was also determined by analysis of a 689-bp fragment comprising the LTR and the PBS motif. Comparison of the LTR versus gag sequences showed a mosaic genome for seven isolates. After analysis of all sequences, we could describe subtype-specific differences in sequences encompassing the regulatory elements of the LTR and the PBS motif.

The human genome contains a large number of sequences that belong to the HERV-K family of human endogenous retroviruses. Most of these elements are likely remnants of ancient infections by ancestral exogenous retroviruses. To obtain further insight into the evolutionary history and molecular mechanisms responsible for the diversity of the human HERV-K elements, we analyzed several aspects of their genome structure. The nucleotide composition of the HERV-K genome was found to be highly biased and asymmetric, with an abundance of the A nucleotide in the viral (+) strand. A similar trend has been reported for the genomes of several exogenous retroviruses, with different nucleotides as the preferred building block. Other genome characteristics that were reported previously for actively replicating retroviruses are also apparent for the endogenous HERV-K virus. In particular, we observed suppression of the dinucleotide CpG, which represents potential methylation sites, and a strong preference for synonymous substitutions within the open reading frame of the reverse transcriptase (RT) enzyme. Furthermore, the mutational spectrum of the HERV-K RT enzyme was evaluated by nucleotide sequence comparison of 34 available elements. Interestingly, this analysis revealed a striking similarity with the mutational pattern of the HIV-1 RT enzyme, with a preference for G-to-A and C-to-T transitions. It is proposed that the mutational bias of the HERV-K RT enzyme played a role in the shaping of this retroviral genome, which was actively replicating more than 30 million years ago. This effect can still be observed in the contemporary endogenous HERV-K elements.

The moment in history when subtypes of the human immunodeficiency virus HIV-1 became distinguishable is hotly debated1,2. Zhu et al.3 have provided a unique HIV-1 sequence from 1959, known as ZR59. Based on the position of ZR59 in phylogenetic trees and its distance from the common node of the viral B/D/F-subtype lineages, they suggest that subtypes B and D have evolved from a single introduction “not long before 1959”, and that HIV-1 group M viruses probably shared a common ancestor “in the 1940s or the early 1950s”. Here we caution against such precise dating of these evolutionary events.

To gain insight into the natural history of hepatitis C virus (HCV), 13 human immunodeficiency virus (HIV)-seronegative injecting drug users were studied who seroconverted for HCV as determined by third-generation enzyme-linked immunosorbent assay, showed an ensuing antibody response to HCV, and were not treated with any antiviral drugs during follow-up. Subjects included 13 untreated HIV-negative individuals, of whom 5 (38.5%) apparently cleared HCV and were polymerase chain reaction (PCR)-negative in at least 3 consecutive samples, 3 (23.1%) showed transient viremia and were PCR-negative in 1 sample during the study period, and the other 5 (38.5%) showed persistent viremia. Viremia was determined longitudinally by reverse-transcription PCR (RT-PCR) and quantified by branched DNA (bDNA). HCV genotypes were determined on serial samples during follow-up. Quantitative antibody levels to core, NS3, NS4, and NS5 were determined using the Chiron RIBA HCV-titering Strip Immunoblot Assay, which is based on HCV genotype 1. The antibody responses to core, NS3, NS4, and NS5 were erratic. In individuals infected with HCV genotype 1, significantly higher median antibody responses to core (P =.02) and to NS4 (P =.04) were found as compared with those infected with other genotypes, showing a significant impact of HCV genotype specificity of the assay. In groups infected with HCV genotype 1, significantly higher median NS3 antibody titers (2.61 relative intensity [RI] vs. 0.38 RI; P =.003) were found in the individuals with persistent viremia than in those with apparent resolution of HCV RNA in blood. In groups infected with genotypes other than genotype 1, significantly higher median NS3 antibody titers (0.89 RI vs. 0.03 RI; P =.0004) and NS5 antibody titers (1.86 RI vs. 0.01 RI; P =.006) were found in the individuals with persistent viremia than in those with apparent resolution of HCV RNA in blood. Individuals with viral persistence had higher HCV-RNA loads with higher antibody responses as compared with individuals with apparent viral clearance from blood. Apparent viral clearance from blood was observed in an unexpectedly high percentage (38.5%), associated with a significant decrease of antibodies to NS3, independent of HCV genotype, as compared with individuals with persistent viremia (P

Objective: To identify genotypic drug resistance patterns of HIV-1 in patients who were extensively pretreated with anti-HIV drugs and not responding to their current antiretroviral combination therapy. Methods: Drug susceptibility of the viruses was tested by a phenotypic recombinant virus assay. Genotypic analysis of HIV resistance was performed by sequencing of the amino-terminal part of the corresponding reverse transcriptase (RT) gene (amino acids 1-280) for serum-derived and recombinant viruses. Results: Among viruses from 92 patients studied, three (3%) viruses contained a T215Y amino-acid change as well as a previously unseen combination of an aminoacid change at codon 67 (N→E/S) and a two amino-acid insertion between codons 68 and 69 of the RT gene of HIV-1. Phenotypic resistance analysis showed high levels of resistance to zidovudine, lamivudine and stavudine (in all patients) and moderate levels of resistance to didanosine and zalcitabine (in two patients), whereas neither serum-derived nor recombinant viruses contained previously known amino-acid changes conferring resistance to didanosine, zalcitabine, lamivudine and stavudine. However, all recombinant viruses contained an insertion of two amino acids between codons 68 and 69 of RT as well as an amino-acid change at codon 67, as was seen in the serum-derived viruses. Conclusions: Antiretroviral therapy including zidovudine may yield replicating viruses with a two amino-acid insertion in RT in combination with amino-acid changes at codons 67 and 215, which are highly resistant to lamivudine and stavudine on top of zidovudine and have unpredictable susceptibility to didanosine and zalcitabine despite lack of previously reported corresponding resistance-associated amino-acid changes. It is currently unknown what regimens can induce the emergence of this type of multidrug-resistant viruses. This will only be elucidated when resistance assays are capable of detecting these mutants.

In the present study, the RIBA HCV serotyping SIA was evaluated with a cohort of injecting drug users. Serotyping may be a rapid and cost-effective method of determining genotypes in cohort studies. In this study, hepatitis C virus (HCV) antibody-positive sera from a cohort of 331 chronically infected injecting drug users, of which 167 were coinfected with human immunodeficiency virus (HIV), were serotyped by the RIBA HCV Serotyping SIA. Among the 331 specimens, serotype-specific antibodies were detected in 250 (sensitivity, 75.5%), in which serotype 1 was predominant (57.2%), followed by serotype 3 (26.8%). Among the 331 specimens, 164 were HIV negative, and serotype-specific antibodies were detected in 151 (sensitivity, 92.1%), in which serotype 1 was predominant (59.6%), followed by serotype 3 (33.8%). For a subset of 58 samples taken from 19 chronically infected HCV seroconverters with a mean follow-up of 5 years, serotypes were compared with genotypes, which were determined by a line probe assay (HCV LiPa) and by direct sequencing of the products obtained by nested PCR of the 5′ untranslated region. Among the 58 samples with known genotypes, serotype-specific antibodies were detected in 38 (total sensitivity, 65.5%), with a specificity of 78.9%. Thirty of these serotypeable samples revealed a serotype that corresponded to the genotype in the 58 samples (total positive predictive value, 51.7%). Of the 58 samples, 23 were coinfected with HIV, and when these were excluded, the total sensitivity increased to 76.5%, with a total specificity of 80.8% and a total positive predictive value of 61.8%. The serotyping assay showed a high total sensitivity (96.3%) for samples positive by HCV RIBA, version 3.0, with four bands. We conclude that the sensitivity of the RIBA HCV serotyping SIA is limited by the immunocompetence of the HCV-infected host. In general, samples from HIV-negative individuals containing genotype 1a had higher sensitivity, specificity, and concordance in the serotyping assay compared with genotyping, whereas samples containing genotype 3a were found to be more cross-reactive and untypeable. Therefore, the prevalence of genotypes other than genotype 1 could be underestimated if they are determined by serotyping, and improvements in specificity are recommended.

Objective: To compare the viral suppression of two antiretroviral regimens using three drugs or five drugs. Design: Two open-label studies using a three-drug (zidovudine, lamivudine and ritonavir) and a five-drug regimen (zidovudine, lamivudine, abacavir, indinavir and nevirapine) in study-drug-naive patients, except for one in the five-drug study. Methods: Participants with ≤ 10,000 HIV-1 RNA copies/ml in plasma at baseline were compared by means of Kaplan-Meier curves for time to < 50 copies/ml, as well as linear regression analysis for the first phase of decline using log-transformed copy numbers. Results: The elimination rate constants For HIV-1 RNA in 15 participants of the three-drug study were compared with nine participants of the five-drug study. The level of < 50 copies/ml was reached earlier when using the five-drug than when using the three-drug regimen (P log rank = 0.0005): median time to reach this level was 4 weeks and 12 weeks, respectively. No differences were found in HIV-1 RNA elimination rate constants in the first 2 weeks after the initiation of therapy. When the viral load declines were calculated from day 2 onwards, adjusting for differences in pharmacological delay of the drugs used, again no differences in early viral load decline were found between the two regimens. Conclusion: With the five drugs used in this study, the median time to reach < 50 HIV-1 RNA copies/ml was 8 weeks shorter than with the three-drug regimen. This finding shows that suppression of viral load in HIV-infection by standard triple-drug therapy can be improved upon.

The prototype endogenous retrovirus HERV-K10 was identified in the human genome by its homology to the exogenous mouse mammary tumour virus. By analysis of a short 244 bp segment of the reverse transcriptase (RT) gene of other HERV-K10-like sequences, it has become clear that these elements represent an extended family consisting of multiple groups (the HML-1 to HML-6 subgroups). Some of these elements are transcriptionally active and contain an intact open reading frame for the RT protein, raising the possibility that this family is still expanding through retrotransposition. To better define the relationship of these endogenous retroviruses, we identified ten new members of the HML-2 subgroup. PCR was used to amplify reverse-transcribed RNA of a 595 bp region of the RT gene in a variety of human cell samples, including normal and leukaemic bone marrow and peripheral blood, placenta cells and a transformed T cell line. We provide an extensive phylogenetic analysis of the relationships for this cluster of HERV-K-related endogenous retroviral elements. Nucleotide diversity values for nonsynonymous versus synonymous codon positions indicate that moderately strong selection is or was operating on these retroviral RT gene segments. The evolution of this class of endogenous retroelements is discussed.

To study human immunodeficiency virus type 1 (HIV-1) compartmentalization between intestine and blood, paired faecal and serum samples were collected from 204 HIV-1-infected persons. Direct sequencing of the gp120 V3 region obtained from 33 persons showed that faecal and serum sequences could be nearly homologous (0.3% different) or very dissimilar (11.3% different). Individual clones were obtained and sequenced from the faecal and serum samples of 13 persons. In 6 persons the HIV-1 subpopulations in faeces and serum were similar, whereas in 7 persons, distribution of V3 genotypes showed a marked difference. Genetic characterization of the HIV-1 subpopulations showed less heterogeneity in faecal subpopulations than in serum subpopulations in 5 of the 7 subjects. Furthermore, faecal and serum subpopulations differed predominantly by nonsynonymous nucleotide substitutions (in 6 of 7 persons). Comparison of the HIV-1 subpopulations in faeces and serum of these 7 persons, using resampling techniques, revealed a significant difference between faecal and serum subpopulations at an N-linked glycosylation site, C-terminal of the V3 loop (amino acids 331-333). Sequences from faecal subpopulations of all 7 persons contained a glycosylation site at amino acid position 331-333. Four of these 7 harboured serum variants lacking a glycosylation site at this position. The faecal subpopulations in these 4 persons showed limited nonsynonymous substitutions compared to synonymous substitutions, indicating that purifying selection is operational on these subpopulations.

Naturally occurring mutations in the polymerase gene of human immunodeficiency virus type 1 (HIV-1) have important implications for therapy and the outcome of clinical studies. Using 42 virus isolates obtained from the UNAIDS sample collection, we analyzed the protease (99 amino acids [aa]) and the first 297 aa of reverse transcriptase (RT) coding regions. Based on the V3 sequence analysis, the collection includes subtype A (n = 5), subtype B (n = 12), subtype C (n = 1), subtype D (n = 11), and subtype E (n = 13) viruses. Of the 42 protease genes, 37 contained naturally occurring mutations at positions in the gene that contribute to resistance to protease inhibitors (indinavir, saquinavir, ritonavir, and nelfinavir) in clade B isolates. The phenotypic effect of these substitutions in non-B isolates is unclear. The The 5'half RT coding region of the 42 isolates was found to be less variable, although 19 of the 42 RT sequences contained amino acid substitutions known to contribute to nucleoside and/or nonnucleoside drug resistance. Since the virus isolates were obtained in 1992, it is unlikely that the infected subjects received protease inhibitors, but we found evidence that one subject acquired a zidovudine (AZT)-resistant HIV-1 strain from a contact who had received AZT. Phylogenetic analysis identified five subtype pol clusters: A, B, C, D, and A'. Comparison of env and pol sequences of the same viruses showed no more recombination events than were already identified on the basis of gag/env comparison (M. Cornelissen, G. Kampinga, F. Zorgdrager, J. Goudsmit, and the UNAIDS Network for HIV Isolation and Characterization, J. Virol. 70:8209-8212, 1996). In one of the known recombinants, a crossover site between subtypes A and C could be identified, and in another, a crossover site could not be identified due to lack of a reference subtype F pol sequence. We analyzed the ds/da ratio of gag, pol, and env sequences of 35 isolates, excluding the recombinants. Our analysis showed that gag and pol are subjected to purifying selection with an average ds/da ratio above 1, independent of the subtype and in contrast with V3 (ds/da approximately 1). Based on the low ds/da ratio of the intergroup analysis of A/E and B/D gag and pol sequences, we analyzed the evolutionary relation between subtypes B and D in more detail by constructing separate phylogenetic trees for synonymous and nonsynonymous substitutions. Our analysis suggests a common ancestry for subtypes B and D that is distinct from that of subtypes A and E.

We and others have shown that in individual human immunodeficiency virus type 1 (HIV-1) infection, the adaptive evolution of HIV-1 is influenced by host immune competence. In this study, we tested the hypothesis that in addition to selective forces operating within the host, transmission bottlenecks have an impact on HIV-1 intrahost evolution. Therefore, we studied the intrahost evolution of the V3 region of the external glycoprotein gp120 of HIV-1 during the 3- and 5-year periods following seroconversion after parenteral versus sexual (male-to-male) transmission in 41 participants of the Amsterdam prospective cohorts of homosexual men (n = 31) and intravenous drug users (IVDUs; n = 10) who were AIDS free and had comparable numbers of CD4+ cells. We observed that HIV-1 strains in homosexual men accumulated over 5 years more nonsynonymous substitutions within the V3 loop than HIV-1 strains in IVDUs as a result of lower rates of nonsynonymous evolution in both the initial 3-year period from seroconversion and the following 2-year period as well as a larger proportion of nonsynonymous back substitutions in IVDUs. The mean numbers of synonymous substitutions did not differ between the two risk groups. Since HIV-1 strains in IVDUs could be distinguished from the viruses of homosexual men based on several nucleotide substitutions of which the most conserved is a synonymous substitution at the tip of the V3 loop (GGC pattern), we studied whether the founder virus population itself has an impact on the intrahost evolution of HIV-1. The mean number of nonsynonymous substitutions accumulated over 5 years within the V3 loop was lower in 10 IVDUs infected by the HIV-1 strains with the GGC signature than in 4 IVDUs infected by HIV-1 strains lacking this pattern, while the mean numbers of synonymous substitutions were similar in the two groups.

To establish an epidemiological link between HIV-1 epidemics in U.S. and European homosexual men and intravenous drug users (IVDUs) we analyzed the HIV-1 gp120 V3 sequences in both risk groups. Signature pattern analysis revealed that the V3 sequences of viruses from IVDUs in Northern Europe are distinguishable from those of homosexual men on the basis of one amino acid and two synonymous nucleotide substitutions, which the most conserved was a synonymous nucleotide substitution in the second glycine codon at the tip of the gp120 V3 loop (GGC). This substitution was seen in 17 of 20 (85%) viruses of IVDUs in Northern Europe, in none of 41 homosexual men in either Europe or the United States, and in 5 of 11 (45%) U.S. IVDUs sequences analyzed. Subsequent phylogenetic and multivariate principal coordinate (PCOORD) analyses showed that 16 of 20 (80%) of the Northern European IVDU sequences clustered together with the 5 U.S. IVDU sequences carrying the GGC substitution and away from the sequences of homosexual men from either Europe or the United States. Taken together with the higher level of heterogeneity of U.S. IVDU sequences compared to the Dutch IVDU sequences taken at the same time, these data present suggestive evidence for a U.S. instead of a European origin of the AIDS epidemic among Northern European IVDUs.

Objective : To study the development of the V3 region of the HIV-1 envelope over time, both within subjects and population-wide. Methods : Direct V3 sequences were obtained from viral RNA from seroconversion samples of 138 individuals [32 intravenous drug users (IVDU), 106 homosexual men], as well as from 5-year follow-up samples of 45 of these individuals (11 IVDU, 34 homosexual men). Results : The population-wide variation of the V3 region in both the seroconversion samples and the 5-year samples steadily increased over consecutive years and were of similar magnitude in each calendar year. The variation in the IVDU group was slightly lower (presumably lagging behind) than in the homosexual group, but also increased over time. The consensus sequence, representing the centre of the swarm of variants, remained almost stationary in 10 years of evolution. The V3 sequences from virions in serum collected 5 years after seroconversion still resembled those from the seroconversion sample, either in overall similarity or in specific (signature) amino acids. Seroconversion and late sequences from a donor-recipient pair were also very similar. Conclusions : The variation in V3 sequences from seroconversion samples is as large as that in 5-year follow-up samples from the same calendar year, suggesting that there is no strong selection for a particular V3 genotype at transmission. The HIV-1 subtype B quasispecies in a naive population appears to evolve through unbiased expansion around a stationary consensus sequence. Despite its large variability, the V3 region retains many of its individual characteristics after 5 years of infection. This indicates that the sampling moment (relative to the seroconversion data) will not greatly influence the results of phylogenetic analyses.

The antigenic diversity threshold theory predicts that antigenic sites of human immunodeficiency virus type 1, such as the V3 region of the external glycoprotein gp120, evolve more rapidly during the symptom-free period in individuals progressing to AIDS than in those who remain asymptomatic for a long time. To test this hypothesis, genomic RNA sequences were obtained from the sera of 44 individuals at seroconversion and 5 years later. The mean number of nonsynonymous nucleotide substitutions in the V3 region of the viruses circulating in 31 nonprogressors (1.1 x 10(-2) +/- 0.1 x 10(-2) per site per year) was higher than the corresponding value for 13 progressors (0.66 x 10(-2) +/- 0.1 x 10(-2) per site per year) (P < 0.01), while no difference between the mean numbers of synonymous substitutions in the two groups was seen (0.37 x 10(-2) +/- 0.1 x 10(-2) and 0.51 x 10(-2) +/- 0.2 x 10(-2) per site per year for nonprogressors and progressors, respectively; P > 0.1). The mean ratios of synonymous nucleotide p distance to nonsynonymous p distance were 0.35 for nonprogressors and 0.62 for progressors. The number of nonsynonymous substitutions was not associated with virus load or virus phenotype, which are established predictors of disease progression, but correlated strongly with the duration of the immunocompetent period (r2 = 0.41; P = 0.001). This indicates that there is no causative relationship between intrahost evolution and CD4+ cell decline. Our data suggest that intrahost evolution in human immunodeficiency virus type 1 infection is driven by selective forces, the strength of which is related to the duration of the immunocompetent period.

Objective : To address the question of whether T-cell-line adaptation of the original LAI and MN (NM) HIV-1 populations biased the interpretation of the intraindividual and population-wide virus distributions. Patients and methods : HIV-1 genomic RNA coding for the gp120 C2V3 region was obtained from serum samples of patients LAI and MN and compared to the proviral DNA derived from simultaneously sampled peripheral blood mononuclear cells (PBMC) as well as B- and T-cell lines. Results : Two (10%) of 20 clones of HIV-1 LAI RNA and none of 16 clones of the HIV-1 MN RNA carried syncytium-inducing (SI)-determining amino-acid changes. HIV-1 LAI RNA formed one SI and two non-SI (NSI) phylogenetic clusters. The HIV-1 LAI DNA in PBMC included both SI and NSI clones but lacked one NSI cluster and contributed NSI clones to the SI cluster as well as an SI clone to the NSI cluster, indicating the existence of an intermediate SI/NSI genotype. In vitro culture using either primary cells or B-cell lines yielded only SI clones, distributed over two Sl/NSI mixed clusters. Long-term propagation in T-cell lines further restricted the clonality and yielded SI clones belonging to only one cluster. On the population level, HIV-1 LAI, MN and BRU sequences all clustered according to the individual host and apart from each other and separate from the epidemiological controls without notable influence of Sl/NSI distinction or cell-culture adaptation. Conclusion : Our data demonstrate a selection bias during the cell-line adaptation of HIV-1 strains LAI and MN with more impact on phenotypic than on genotypic characteristics.

To investigate the mechanism of action of the 22-amino-acid HIV fusion peptide on HIV infection, we studied its influence on virus adsorption and HIV-induced syncytium formation. The effect of the peptide preparations on the synthesis of viral antigens in HIV-infected cell cultures was determined by antigen capture assay, and the inhibition of proviral DNA synthesis was detected by hybridization with a HIV-specific oligonucleotide probe after PCR amplification. Fusion peptides inhibited HIV-induced syncytium formation and antigen production in lytic infected cells, and this effect was increased in conjugation with bovine serum albumin or with synthetic net-charged polymer by its C-terminus. The association of peptide with carrier by N-terminus, or with positive-charged polymer or gelatin completely abolished its effect on HIV infection. No peptide preparations influenced HIV-1 chronically infected cells. Because peptide preparations blocked the HIV-specific DNA synthesis 2 hr after infection without influencing virus adsorption and reverse transcription, we concluded that the block of infection occurred during the penetration of virions through the cell membrane. On the basis of results obtained we propose that our peptide preparations could be used for anti-HIV chemotherapy. The possibility of the existence of receptors for gp41 N-terminal region on target cell membrane is discussed.