Re-analysis of unassigned hepatitis C virus (HCV) strain CYHCV025: Evidence of a highly divergent lineage within genotype 1.

HCV global sequences have been classified into 7 genotypes, several subtypes and a number of unassigned sequences. Our aim was to perform an in depth investigation of the taxonomic relationships of the unclassified CYHCV025 strain by means of phylogenetic analysis. Phylogenetic tree reconstructions were performed using ML methods on full-length genomic and partial HCV alignments. Phylogenetic analysis of full-length sequences revealed that CYHCV025 clustered close to the root node of genotype 1, showing distant genetic relationships to all previously classified subtypes and unclassified sequences. Single section analysis using the SSE showed that the distances between the query and all subtypes were much higher than distances within and between subtypes of genotype 1 (p<0.05). Recombination analysis revealed no evidence for intersubtype or genetic mixing between the query and the references from different genotype 1 subtypes. Different analyses revealed that CYHCV025 is the most genetically divergent within genotype 1, showing no high- or low-level clustering with any of the previous subtypes or unclassified sequences. Identification of a single lineage within a genotype without any late branching can be explained by "genetic isolation" until the late stage of HCV epidemic spread.

Primary resistance to integrase strand-transfer inhibitors in Europe.

OBJECTIVES: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. METHODS: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score ≥ 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing. RESULTS: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score ≥ 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score ≥ 10 were found in 8 (14.3%) individuals. CONCLUSIONS: No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.

A chemokine receptor CCR2 allele delays HIV-1 disease progression and is associated with a CCR5 promoter mutation.

Viral and host factors influence the rate of HIV-1 disease progression. For HIV-1 to fuse, a CD4+ cell must express a co-receptor that the virus can use. The chemokine receptors CCR5 and CXCR4 are used by R5 and X4 viruses, respectively. Most new infections involve transmission of R5 viruses, but variants can arise later that also use CXCR4 (R5-X4 or X4 viruses). This is associated with an increased rate of CD4+ T-cell loss and poor prognosis. The ability of host cells to support HIV-1 entry also influences progression. The absence of CCR5 in approximately 1% of the Caucasian population, due to homozygosity for a 32-nucleotide deletion in the coding region (delta32-CCR5 allele), very strongly protects against HIV-1 transmission. Heterozygosity for the delta32-CCR5 allele delays progression typically by 2 years. A recent study showed that a conservative substitution (V64I) in the coding region of CCR2 also has a significant impact on disease progression, but not on HIV-1 transmission. This was unexpected, since CCR2 is rarely used as a co-receptor in vitro and the V64I change is in a transmembrane region. Because a subsequent study did not confirm this effect on progression to disease, we analyzed CCR2-V64I using subjects in the Chicago MACS. We show that CCR2-V64I is indeed protective against disease progression and go on to show that the CCR2-V64I allele is in complete linkage disequilibrium with a point mutation in the CCR5 regulatory region.

Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy.

Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of 'covert' virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1-infected individuals and for the development of virus eradication strategies.

Effects of in vivo CD8(+) T cell depletion on virus replication in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine.

The role of CD8(+) T lymphocytes in controlling replication of live, attenuated simian immunodeficiency virus (SIV) was investigated as part of a vaccine study to examine the correlates of protection in the SIV/rhesus macaque model. Rhesus macaques immunized for >2 yr with nef-deleted SIV (SIVmac239Deltanef) and protected from challenge with pathogenic SIVmac251 were treated with anti-CD8 antibody (OKT8F) to deplete CD8(+) T cells in vivo. The effects of CD8 depletion on viral load were measured using a novel quantitative assay based on real-time polymerase chain reaction using molecular beacons. This assay allows simultaneous detection of both the vaccine strain and the challenge virus in the same sample, enabling direct quantification of changes in each viral population. Our results show that CD8(+) T cells were depleted within 1 h after administration of OKT8F, and were reduced by as much as 99% in the peripheral blood. CD8(+) T cell depletion was associated with a 1-2 log increase in SIVmac239Deltanef plasma viremia. Control of SIVmac239Deltanef replication was temporally associated with the recovery of CD8(+) T cells between days 8 and 10. The challenge virus, SIVmac251, was not detectable in either the plasma or lymph nodes after depletion of CD8(+) T cells. Overall, our results indicate that CD8(+) T cells play an important role in controlling replication of live, attenuated SIV in vivo.

Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques.

To determine the role of CD8(+) T cells in controlling simian immunodeficiency virus (SIV) replication in vivo, we examined the effect of depleting this cell population using an anti-CD8 monoclonal antibody, OKT8F. There was on average a 99.9% reduction of CD8 cells in peripheral blood in six infected Macaca mulatta treated with OKT8F. The apparent CD8 depletion started 1 h after antibody administration, and low CD8 levels were maintained until day 8. An increase in plasma viremia of one to three orders of magnitude was observed in five of the six macaques. The injection of a control antibody to an infected macaque did not induce a sustained viral load increase, nor did it significantly reduce the number of CD8(+) T cells. These results demonstrate that CD8 cells play a crucial role in suppressing SIV replication in vivo.

Measuring recent thymic emigrants in blood of normal and HIV-1-infected individuals before and after effective therapy.

The role of the thymus in HIV-1 pathogenesis remains unclear. We developed an assay to quantify the number of recent thymic emigrants in blood based on the detection of a major excisional DNA byproduct (termed alpha1 circle) of T cell receptor rearrangement. By studying 532 normal individuals, we found that alpha1 circle numbers in blood remain high for the first 10-15 yr of life, a sharp drop is seen in the late teen years, and a gradual decline occurs thereafter. Compared with age-matched uninfected control individuals, alpha1 circle numbers in HIV-1-infected adults were significantly reduced; however, there were many individuals with normal alpha1 circle numbers. In 74 individuals receiving highly active antiretroviral therapy, we found no appreciable effect on alpha1 circle numbers in those whose baseline values were already within the normal range, but significant increases were observed in those with a preexisting impairment. The increases in alpha1 circle numbers were, however, numerically insufficient to account for the rise in levels of naive T lymphocytes. Overall, it is difficult to invoke thymic regenerative failure as a generalized mechanism for CD4 lymphocyte depletion in HIV-1 infection, as alpha1 circle numbers are normal in a substantial subset of HIV-1-infected individuals.

T cell receptor excision circles and HIV-1 2-LTR episomal DNA to predict AIDS in patients not receiving effective therapy.

OBJECTIVE: To determine whether improved prediction of AIDS-free survival following HIV-1 seroconversion is achieved by measuring HIV-1 2-LTR episomal DNA (2-LTR) circles and T cell receptor rearrangement excision circles (TREC), reflecting HIV replication and lymphocyte emigration from the thymus, respectively. DESIGN: Subanalysis of a cohort of 154 patients with hemophilia who became HIV positive between 1978 and 1985 and were followed prospectively. METHODS: Relative hazards (RH) of AIDS, in the absence of highly effective anti-HIV therapy, were estimated for age, HIV-1 viral load, CD4 lymphocyte count and levels of HIV-1 2-LTR circles and TREC [per 106 peripheral blood mononuclear cells (PBMC)]. RESULTS: TREC correlated significantly with CD4 cell counts (r = 0.30) and age (r = -0.60). 2-LTR circles correlated significantly with HIV-1 viral load (r = 0.35). If viral load, CD4 lymphocytes and age were included in a proportional hazards model, the risk of AIDS during a median of 11.6 years of follow-up was increased significantly with fewer TREC (adjusted RH, 2.0 per log10 copies/106 PBMC) and more 2-LTR circles (RH, 1.7 per log10 copies/106 PBMC). AIDS prediction with TREC and 2-LTR circles held for most subgroups defined by median viral load, CD4 lymphocytes and age. CONCLUSIONS: PBMC that have high levels of HIV-1 replication and low levels of recent thymic emigrants are associated with a substantially increased risk of AIDS. It is not known if measurement of either TREC or 2-LTR circles will complement HIV-1 viral load as an estimation of the risk of AIDS for patients who are receiving highly effective anti-HIV therapy.

Global distribution of the CCR2-64I/CCR5-59653T HIV-1 disease-protective haplotype.

OBJECTIVES: Several natural polymorphisms in the genes for the human CC-chemokine receptors CCR5 and CCR2 are associated with HIV-1 disease. The CCR2-64I genetic variant [a G to A substitution resulting in a valine (V) to isoleucine (I) change at position 64] is in strong linkage disequilibrium with a mutation within the CCR5 regulatory region (CCR5-59653T). Individuals with two CCR2-64I alleles are not resistant to sexual transmission of HIV-1, but progress significantly more slowly to HIV-1 disease. It is therefore important to determine the global distributions of CCR2-64I and CCR5-59653T genetic variants and define the degree of linkage between them. DESIGN AND METHODS: We have developed molecular beacon-based, real-time PCR allele discrimination assays for all three chemokine receptor mutations, and used these spectral genotyping assays to genotype 3923 individuals from a globally distributed set of 53 populations. RESULTS: CCR2-64I and CCR5-59653T genetic variants are found in almost all populations studied: their allele frequencies are greatest (approximately 35%) in Africa and Asia but decrease in Northern Europe. We confirm that CCR2-64I is in strong linkage disequilibrium with CCR5-59653T (96.92% of individuals had the same genotype for both CCR2-64I and CCR5-59653T polymorphisms). CONCLUSIONS: The greater geographical distribution of the CCR2-64I/CCR5-59653T haplotype compared with that of CCR5-delta32 suggests that it is a much older mutation whose origin predates the dispersal of modern humans.

Genetic analysis of human immunodeficiency virus type 1 strains in Kenya: a comparison using phylogenetic analysis and a combinatorial melting assay.

We surveyed human immunodeficiency virus (HIV) subtype distribution from peripheral blood mononuclear cells (PBMCs) collected in 1995 from 24 HIV-1-infected Kenyan residents (specimens from predominantly male truck drivers and female sex workers near Mombasa and Nairobi). Processed lysates from the PBMC samples were used for env amplification, directly sequenced, and analyzed by phylogenetic analysis. Envelope amplification products were also used for analysis in a polymerase chain reaction (PCR)-based assay, called the combinatorial melting assay (COMA). Results of the two tests were compared for assignment of subtype for this Kenyan cohort. The COMA, a PCR capture technique with colorimetric signal detection, was used with HIV reference subtype strains as well as regional (East Africa) HIV strains for subtype identification. Performance of the COMA was at 100% concordance (24 of 24) as compared with DNA sequencing analysis. Phylogenetic analysis showed 17 isolates to be subtype A, 3 subtype D, and 4 subtype C viruses. This may represent an increase in subtype C presence in Kenya compared with previously documented reports. The COMA can offer advantages for rapid HIV-1 subtype screening of large populations, with the use of previously identified regional strains to enhance the identification of local strains. When more detailed genetic information is desired, DNA sequencing and analysis may be required.

Evidence of two distinct subsubtypes within the HIV-1 subtype A radiation.

Members of HIV-1 group M are responsible for the vast majority of AIDS cases worldwide and have been classified on the basis of their phylogenetic relationships into nine roughly equidistant clades, termed subtypes. Although there are no known phenotypic correlates for these genotypes, the disproportionate spread of certain of these lineages has been taken to indicate that subtype-specific biological differences may exist. The subtype nomenclature thus remains an important molecular epidemiological tool with which to track the course of the group M pandemic. In this study, we have characterized HIV-1 strains described previously as unusual subtype A variants on the basis of partial sequence analysis. Six such strains from Cyprus (CY), South Korea (KR), and the Democratic Republic of Congo (CD) were PCR amplified from infected cell culture or patient PBMC DNA, cloned, and sequences in their entirety (94CY017, 97KR004, 97CDKTB48, and 97CDKP58) or as half genomes (97CDKS10 and 97CDKFE4). Distance and phylogenetic analyses showed that four of these viruses (94CY017, 97CDKTB48, 97CDKFE4, and 97CDKS10) were closely related to each other, but quite divergent from all other HIV-1 strains, except for subtype A viruses, which represented their closest relatives. In phylogenetic trees from gag, pol, env, and nef regions, the four newly characterized HIV-1 strains formed a distinct sister clade to subtype A, which was as closely related to subtype A as subsubtypes F1 and F2 are to each other. According to current nomenclature rules, this defines a subsubtype, which we have tentatively termed A2. The two other viruses, 97KR004 and 97CDKP58, as well as a full-length HIV-1 sequence from the sequence database (ZAM184), were found to represent complex A2/D, A2/G, and A2/C recombinants, respectively. These results indicate that HIV-1 subtype A is composed of two subsubtypes (A1 and A2), both of which appear to have a widespread geographic distribution. The A2 viruses described here represent the first reference reagents for this new group M lineage.

HIV-specific cytotoxic T lymphocytes, HLA-A11, and chemokine-related factors may act synergistically to determine HIV resistance in CCR5 delta32-negative female sex workers in Chiang Rai, northern Thailand.

Understanding how highly HIV-exposed individuals remain HIV uninfected may be useful for HIV vaccine design and development of new HIV prevention strategies. To elucidate mechanisms associated with resistance to HIV infection, immunologic and genetic factors were examined in 14 HIV-exposed but persistently seronegative (HEPS) female sex workers from Chiang Rai, northern Thailand and in ethnically matched, HIV-positive (n = 9) and HIV-negative women (n = 9). The HEPS women were identified in a study of commercial sex workers who had an HIV-1 incidence of 20.3 per 100 person-years. A high frequency of HLA-A11 was observed in HEPS women (86%) compared with northern Thai controls (56%). HIV-specific cytotoxic T lymphocyte (CTL) lytic responses were detected in cryopreserved peripheral blood mononuclear cells (PBMCs), using HLA-A-matched subtype E HIV-1 peptides in four of seven (57%) HEPS women, eight of eight HIV-positive women, and zero of nine HIV-negative unexposed controls (p = 0.019 HEPS women vs. HIV-negative controls). CTL lysis levels were low, but responses were detected to peptides from Nef, Pol, Gag, and Env. Nef responses predominated in HEPS women. Compared with controls, HEPS women tended to have higher frequencies of CCR5 promotor 59402GG and SDF-1 3'UTR 801A genotypes known to influence HIV transmission or course of disease. HEPS women also had higher levels of spontaneous RANTES production by PBMCs than other groups. Each of these factors could potentially contribute to HIV resistance. As most HEPS women had one or more of these factors, they may prevent HIV infection synergistically by blocking HIV cell entry, delaying its dissemination, or killing HIV-infected cells.

Ultraviolet absorbance and circular dichroism of Pf1 virus: nucleotide/subunit ratio of unity, hyperchromic tyrosines and DNA bases, and high helicity in the subunits.

Data have been obtained for the Pf1 virion that establish its stoichiometry and conformational features of its DNA and its protein. The absorbance spectrum of the dissociated virus under alkaline denaturing conditions is fit exactly by spectra for DNA and protein at a mole ratio of one nucleotide per protein subunit. This result, together with three previous values by independent methods, establishes that the nucleotide/subunit ratio (n/s) of Pf1 is unity. The absorbance spectrum of DNA in the intact native virus is assigned as the spectrum for heat denatured Pf1 DNA, with epsilon (P) = 8400 M-1 cm-1 at 259 nm. The absorbance spectrum assigned to protein (two tyrosines) in the intact virus has = 2500 M-1 cm-1 per tyrosine at lambda max of 281.5 nm; this is the most red-shifted and hyperchromic tyrosine spectrum known. The CD spectrum of the intact virus from 250 to 320 nm has no apparent DNA contribution, but has a strong contribution from the red-shifted tyrosine(s). The CD spectrum from 185 to 250 nm has the shape of alpha-helical CD reference spectra, but is perceptibly blue-shifted, with a crossover from negative to positive ellipticity at 199.7 nm, and it has very high amplitudes (e.g. [theta 207.5nm] = -44,000 deg cm2 dmol-1). This spectrum indicates completely helical protein in the virus, with a predominance of alpha-helix and perhaps some 3(10)-helix. The unit n/s ratio, the high absorbance and negligible near-UV CD for the DNA bases, and the high amplitudes for the helical protein are critical input data for the determination of Pf1 virus structure.

Genotyping HIV-1 and HCV strains by a combinatorial DNA melting assay (COMA).

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) strains can be genetically classified into genetic lineages known as genetic types or subtypes according to phylogenetic analyses of complete or partial nucleotide sequences of their genomes. The genetic classification of HIV-1 and HCV strains has important implications for the development of globally effective vaccines and for the management of patients. MATERIALS AND METHODS: A new method, termed combinatorial DNA melting assay (COMA), allows rapid accessing of comparative genetic information between related DNA sequences, making it possible to rapidly and accurately genotype unknown HIV-1 and HCV strains. COMA is mainly based on the differential melting properties of long DNA heteroduplexes. Combinatorial arrays of DNA heteroduplexes are formed when captured PCR-amplified reference DNA with known nucleotide sequences are combined with solution-phase complementary and antigenically labeled DNA with unknown sequences. Genetic divergence between the known and the unknown sequences is inferred as the experimentally derived melting curves of the two strands of the DNA heteroduplexes increasingly diverge. RESULTS: COMA was successfully applied to the genetic classification of HIV-1 and HCV strains into phylogenetic lineages or subtypes. CONCLUSIONS: Use of this assay should accelerate current efforts to understand the global molecular epidemiology of HIV-1 and HCV and may extend to the genetic characterization of other genetically diverse infectious pathogens associated with numerous diseases.

Re-analysis of human immunodeficiency virus type 1 isolates from Cyprus and Greece, initially designated 'subtype I', reveals a unique complex A/G/H/K/? mosaic pattern.

Human immunodeficiency virus type 1 (HIV-1) has been classified into three main groups and 11 distinct subtypes. Moreover, several circulating recombinant forms (CRFs) of HIV-1 have been recently documented to have spread widely causing extensive HIV-1 epidemics. A subtype, initially designated I (CRF04_cpx), was documented in Cyprus and Greece and was found to comprise regions of sequence derived from subtypes A and G as well as regions of unclassified sequence. Re-analysis of the three full-length CRF04_cpx sequences that were available revealed a mosaic genomic organization of unique complexity comprising regions of sequence from at least five distinct subtypes, A, G, H, K and unclassified regions. These strains account for approximately 2% of the total HIV-1-infected population in Greece, thus providing evidence of the great capability of HIV-1 to recombine and produce highly divergent strains which can be spread successfully through different infection routes.

CXCR4 mediates entry and productive infection of syncytia-inducing (X4) HIV-1 strains in primary macrophages.

CCR5 and CXCR4 are the main coreceptors for non-syncytia-inducing (NSI) and syncytia-inducing (SI) HIV-1 strains, respectively. NSI HIV-1 isolates do not infect either human lymphoid or monocytoid cell lines, and this inability correlates with the absence of CCR5 expression in these cell types. The ability of SI HIV-1 isolates to infect human primary macrophages has been disputed. Here, we report that CXCR4 is expressed in primary blood-derived human mononuclear phagocytes at all stages of differentiation, although the maturation process correlates with downregulation of CXCR4 mRNA. Infection experiments with the SI molecular clone NL4-3 tagged with a mutant of the green fluorescent protein established that both monocytes and attached macrophages are susceptible to infection with CXCR4-restricted HIV-1 strains. NL4-3 entry into primary macrophages could be blocked by SDF-1alpha in a dose-dependent manner, or by the anti-CXCR4 monoclonal antibody 12G5. HIV-1 entry led to productive infection. No evidence of postentry defects or nuclear import delay for CXCR4-restricted HIV-1 strains was detected using a quantitative real-time PCR assay measuring HIV-1 DNA entry into the nucleus. Macrophages infected by HIV-1 and expressing virus were maintained in culture for long periods of time (up to 5 months). These results demonstrate that CXCR4 is the main HIV-1 SI coreceptor in human primary macrophages and underline the importance of the macrophage as a long-living viral reservoir for HIV-1.

Genetic analysis of human immunodeficiency virus type 1 strains from patients in Cyprus: identification of a new subtype designated subtype I.

DNA sequences encoding the C2 to V3 region of envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) were amplified by PCR from uncultured peripheral blood mononuclear cells obtained from 24 of 25 HIV-1-seropositive patients from Cyprus. By using a heteroduplex mobility assay (HMA), all amplified products were studied genetically and compared with 16 previously characterized HIV-1 strains belonging to subtypes A through F. HMA results revealed that HIV-1 gp120 sequences from 15 of our patients were of subtype B of HIV-1, whereas one isolate was of subtype C. However, gp120 sequences from eight patients had no obvious similarities to the known subtypes as defined by HMA. DNA sequencing and phylogenetic analyses of molecular clones confirmed the HMA results and placed the eight undefined HIV-1 isolates into three distinct genetic clusters. On the basis of branch topology and lengths of the phylogenetic tree, we conclude that one group consisting of three clones from two patients represents a new HIV-1 env subtype, which we have termed subtype I. The remaining two sequence clusters, consisting of five sequences from four patients and two sequences from two other patients, are distally related to subtypes A and F. These data demonstrate the extensive heterogeneity of HIV-1 in Cyprus, including the presence of new subtype.