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Today, many technologies for the deposition of various functional coatings using volatile compounds are united under the general name chemical vapor deposition processes from the gas phase (CDV, MOCVD, ALD, CVI, PECVD, etc [...]

Model studies of the subtype B and non-subtype B integrases are still required to compare their susceptibility to antiretroviral drugs, evaluate the significance of resistance mutations and identify the impact of natural polymorphisms on the level of enzymatic reactivity. We have therefore designed the consensus integrase of the HIV-1 subtype A strain circulating in the former Soviet Union territory (FSU-A) and two of its variants with mutations of resistance to the strand transfer inhibitor raltegravir. Their genes were synthesized, and expressed in E coli; corresponding His-tagged proteins were purified using the affinity chromatography. The enzymatic properties of the consensus integrases and their sensitivity to raltegravir were examined in a series of standard in vitro reactions and compared to the properties of the integrase of HIV-1 subtype B strain HXB2. The consensus enzyme demonstrated similar DNA-binding properties, but was significantly more active than HXB-2 integrase in the reactions of DNA cleavage and integration. All integrases were equally susceptible to inhibition by raltegravir and elvitegravir, indicating that the sporadic polymorphisms inherent to the HXB-2 enzyme have little effect on its susceptibility to drugs. Insensitivity of the mutated enzymes to the inhibitors of strand transfer occurred at a cost of a 30-90% loss of the efficacies of both 3'-processing and strand transfer. This is the first study to describe the enzymatic properties of the consensus integrase of HIV-1 clade A and the effects of the resistance mutations when the complex actions of sporadic sequence polymorphisms are excluded.

The results of the experimental and numerical study of the influence of obstacle geometry on the conditions of flame stabilisation in the turbulent boundary layer with fuel injection near a porous surface are presented. The features of the detachment zones formation in the turbulent reacting flow behind the obstacle are studied. The obtained results are compared with the data on flame-off conditions when flowing over a flat surface.

To analyze HIV-1 genotypes in Lithuania and the transmission of drug-resistant viruses, HIV-1 sequences were obtained from 138 individuals, who were diagnosed as HIV-1 infected in 1990–2008 and represented all major risk groups. Subtype A strains, dominating in the former Soviet Union (90% of cases), were found in 60% of individuals, followed by subtype B (22%) and CRF03_AB (12%) strains. The remaining 7% of the strains included variants belonging to subtype C, CRF01_AE, CRF02_AG, more complex recombinant forms, and strains that could not be reliably genotyped. Analysis of virus genotypes per risk group revealed the circulation of distinct HIV-1 strains in different risk groups: subtype A viruses were present in 82% of injecting drug users (IDUs), but less than a half of heterosexually infected individuals and cases with unknown transmission route, and none of men having sex with men (MSM). We observed no mutations causing drug resistance among 27 newly diagnosed HIV-1 cases.

BackgroundThe concept of demand characteristics, which involves research participants being aware of what the researcher is investigating, is well known and widely used within psychology, particularly in laboratory-based studies. Studies of this phenomenon may make a useful contribution to broader consideration of the effects of taking part in research on participant behaviour. This systematic review seeks to summarise data from studies of the effects of demand characteristics on participant behaviours in non-laboratory settings.Methodology/Principal FindingsElectronic databases were searched to identify eligible studies. These had to be purposely designed to evaluate possible effects of demand characteristics on at least one behavioural outcome under the autonomous control of the participants and use longitudinal study designs. Only 7 studies were included, 6 providing observational data and 1 experimental study, with 5 studies involving examination of possible effects on health behaviours. Although studies provided some evidence of effects of demand characteristics on participant behaviour, heterogeneous operationalisation of the construct, the limited number of studies and poor quality of study designs made synthesis and interpretation of study findings challenging.Conclusions/SignificanceAlthough widely accepted as important in psychology, there have been few dedicated studies of the effects of demand characteristics on research participant behaviours outside laboratory settings. This body of literature does not currently contribute to the wider study of research participation effects. A systematic review of data from laboratory-based studies is needed, as are high-quality primary studies in non-laboratory settings. We suggest that unqualified use of the term demand characteristics should be abandoned.

To study the molecular epidemiology of HIV-1 in Krasnoyarsk region, Russia, where HIV-1 has spread rapidly since 2000, we obtained pol sequences from individuals living in this region (n = 67) as well as in the geographically closely related Altay region (n = 13). In both regions, subtype A viruses specific for the former Soviet Union (IDU-A strains) were dominant (92.5%). Virus sequences clustered according to the geographic origin of the infected individuals rather than to their risk group, demonstrating the role of geographically defined epidemiological networks in the propagation of the HIV-1 epidemic in the region. Six viruses belonged to subtype B. Three of them were phylogenetically (and therefore epidemiologically) closely related to each other, demonstrating that even though IDU-A viruses dominate the epidemic, the spread of other virus strains does occur. Most viruses (75%) had an A62V mutation in reverse transcriptase, specific for HIV-1 strains in Russia. Remarkably, 26 of 47 (55%) patients under HAART with detectable virus loads did not have any known drug-resistant mutation, indicating the need to increase compliance to therapy.

The effectiveness of highly active antiretroviral therapy (HAART), the standard of care for the treatment of human immunodeficiency virus type 1 (HIV-1) infection, is assessed by measuring the viral RNA load in plasma. A patient is considered to be successfully treated when the HIV-1 load in plasma stays below the detection limit of commercial assays. However, virus replication and evolution do continue in patients under HAART, which may eventually result in the development of drug-resistant HIV-1 strains and therapy failure. To monitor this low-level virus replication in peripheral blood mononuclear cells (PBMC), sensitive methods are required to measure HIV-1 molecular markers. We report the development of highly sensitive methods for the quantitation of unspliced and multiply spliced HIV-1 RNA and proviral DNA in PBMC. The methods are based on innovative seminested real-time reverse transcription-PCR (RT-PCR) that combines the accuracy and precision of real-time PCR and the sensitivity of nested PCR. We show that the newly developed methods are superior to the conventional single-step real-time RT-PCR in their sensitivity, accuracy, dynamic range, and the power of quantitative detection of HIV-1 RNA and DNA in clinical samples. These easy-to-perform methods can be widely used in research, including clinical studies, to monitor intracellular processes of virus replication.

The evolutionary history of the Astroviridae comprises the ancient separation between avian and mammalian astrovirus lineages followed by diversification among mammalian astroviruses. The latter process included several cross-species transmissions. We found that the recent, but not the ancient, evolution of astroviruses was associated with a switch in nucleotide composition and codon usage among non-human mammalian versus human/avian astroviruses. Virus and hosts phylogenies based on codon usage agreed with each other and matched the hosts' evolutionary emergence order. This recent switch in driving forces acting at the synonymous level points to the adaptation of codon usage by viruses to that of their hosts after cross-species transmissions. This is the first demonstration of nucleotide composition and codon usage being active driving forces during the recent evolutionary history of a virus group in the host–parasite system.

The genome of hepatitis B virus (HBV) provides a striking example of gene overlapping. In particular, the surface protein gene S is overlapped completely by the polymerase gene P. Evolutionary constraints in overlapping genes have been demonstrated for many viruses, with one of the two overlapping genes being subjected to positive selection (adaptive evolution), while the other one is subjected to purifying selection. Yet, for HBV to persist successfully, adaptive evolution of both the P and S genes is essential. We propose that HBV employs a mechanism that allows the independent adaptive evolution of both genes. We hypothesize that (i) the adaptive evolution of P occurs via p1/s3 non-synonymous substitutions, which are synonymous in S, (ii) the adaptive evolution of S occurs via p3/s2 non-synonymous substitutions, which are synonymous in P, and (iii) p2/s1 substitutions are rare. Analysis of 450 HBV sequences demonstrated that this mechanism is operational in HBV evolution both within and among genotypes. Positions were identified in both genes where adaptive evolution is operational. Whilst significant parts of the P and S genes were subjected to positive selection, with the K a/K s ratio for either the P or the S gene being >1, there were only a few regions where the K a/K s ratios in both genes were >1. This mechanism of independent evolution of the overlapping regions could also apply to other viruses, taking into account the increased frequency of amino acids with a high level of degeneracy in the proteins encoded by overlapping genes of viruses.

Fever of unknown origin (FUO) is a serious problem in the United States. An unidentified agent was cultured from the stool of an infant who presented with FUO. This virus showed growth in HFDK cells and suckling mice. Using DNase sequence-independent single-primer amplification, we identified several nucleotide sequences with a high homology to Theiler's murine encephalomyelitis virus. Nearly full-length viral genome sequencing and phylogenetic analysis demonstrate that this virus is a member of the Cardiovirus genus of the Picornaviridae family.

Recombination between (human) enteroviruses is a common event in nature. Recently, it has been recognised that this feature has a major impact on the use of the live-attenuated polio vaccine during the end stage of polio eradication. The constraints for successful recombination between (vaccine-derived) polioviruses and human enteroviruses are, however, largely unknown. Here, we describe the identification and characterisation of a HEV-C field strain, isolated from the stool of a 2-year-old Dutch boy. Serotyping indicated that strain 89490 is a variant of strain CAV20a, which is already a variant of the prototype CAV20 strain. Amino acid sequence identity of 94.1% for the entire P1-region, and 92.4% for the major structural protein (VP1) indicates that this strain is indeed related to CAV20. However, virus neutralisation and Western blot analysis failed to show antigenic homology between the prototype CAV20 strain and our field strain. Furthermore, the 89490 field strain, just like the sub-prototype CAV20a, is able to replicate on RD-cells, while the prototype CAV20 and another sub-prototype CAV20b are not. On the basis of the phylogenetic analysis of the P2 and P3 region we expect that strain 89490 can act as recombination partner for the attenuated poliovirus strains of the Oral Polio Vaccine (OPV).

A sequence-independent PCR amplification method was used to identify viral nucleic acids in the plasma samples of 25 individuals presenting with symptoms of acute viral infection following high-risk behavior for human immunodeficiency virus type 1 transmission. GB virus C/hepatitis G virus was identified in three individuals and hepatitis B virus in one individual. Three previously undescribed DNA viruses were also detected, a parvovirus and two viruses related to TT virus (TTV). Nucleic acids in human plasma that were distantly related to bacterial sequences or with no detectable similarities to known sequences were also found. Nearly complete viral genome sequencing and phylogenetic analysis confirmed the presence of a new parvovirus distinct from known human and animal parvoviruses and of two related TTV-like viruses highly divergent from both the TTV and TTV-like minivirus groups. The detection of two previously undescribed viral species in a small group of individuals presenting acute viral syndrome with unknown etiology indicates that a rich yield of new human viruses may be readily identifiable using simple methods of sequence-independent nucleic acid amplification and limited sequencing.

Control of viremia in natural human immunodeficiency virus type 1 (HIV-1) infection in humans is associated with a virus-specific T-cell response. However, still much is unknown with regard to the extent of CD8+cytotoxic T-lymphocyte (CTL) responses required to successfully control HIV-1 infection and to what extent CTL epitope escape can account for rises in viral load and ultimate progression to disease. In this study, we chose to monitor through full-length genome sequence of replication-competent biological clones the modifications that occurred within predicted CTL epitopes and to identify whether the alterations resulted in epitope escape from CTL recognition. From an extensive analysis of 59 biological HIV-1 clones generated over a period of 4 years from a single individual in whom the viral load was observed to rise, we identified the locations in the genome of five CD8+CTL epitopes. Fixed mutations were identified within the p17, gp120, gp41, Nef, and reverse transcriptase genes. Using a gamma interferon ELIspot assay, we identified for four of the five epitopes with fixed mutations a complete loss of T-cell reactivity against the wild-type epitope and a partial loss of reactivity against the mutant epitope. These results demonstrate the sequential accumulation of CTL escape in a patient during disease progression, indicating that multiple combinations of T-cell epitopes are required to control viremia.

The vast majority of HIV-1 strains from the epidemic in the former Soviet Union (FSU) belong to subtype A (IDU-A) and CRF03_AB (IDU-A/B), for which IDU-A is one of parental strains; no epidemic by another parental virus, belonging to subtype B (IDU-B), has yet been identified. To characterize viruses present during the early stage of the epidemic in southern Ukraine, where the first outbreaks in the FSU were registered, we obtained partial env and pol sequences from IDUs from Odessa and Nikolaev and compared them with viruses from other outbreaks. All viruses from Odessa belonged to the IDU-A type, which is in accord with previous studies. At the same time, we found that the outbreak in Nikolaev was caused by IDU-B viruses, indicating that this outbreak is the result of an independent virus introduction. Phylogenetic analysis of viruses from the FSU supported the epidemiological data suggesting that the HIV-1 epidemic in the FSU started in southern Ukraine.

We obtained and studied HIV-1 sequences with a known sampling year from three outbreaks of the HIV-1 epidemic: 141 env V3 (270 nt) sampled between 1984 and 1992 and 117 pol prot/RT (804 nt) sequences sampled between 1986 and 1999 from Dutch homosexual men and injecting drug users (IDUs), as well as 77 env V3 sequences sampled between 1983 and 1994 in the United States. Since retrospective serological and/or epidemiological data on these populations are available, providing estimates of the dates of the onset of the HIV-1 epidemics, we had the opportunity to test different phylogenetic models for their accuracy in deriving the recent evolutionary history of HIV-1 subtype B and the onset date of the HIV-1 epidemic. We observed that, in any given year, individual sequences vary widely in their distances to the common ancestor, and sequences close to the ancestors were found decades after the onset of the epidemic. Nevertheless, the mean evolutionary distances of virus strains to ancestors were increasing significantly during the course of the studied epidemics, which indicates that the molecular clock is operational in the recent evolution of HIV-1. When the relationship between the sampling years of sequences and their nucleotide distances to the common ancestor was extrapolated to the past, analysis of pol sequences provided accurate estimates of the onset years of the epidemics, whereas analysis of V3 sequences by the maximum-likelihood or neighbor-joining methods led to an overestimation of the age of the epidemics. Separate analysis of nonsynonymous and synonymous distances revealed that this overestimation results from nonsynonymous substitutions, whose numbers were not increasing significantly in all three virus populations over the observation period. In contrast, analysis of synonymous env V3 distances provided accurate estimates of the onset years for the outbreaks we studied.

Viruses circulating in Ethiopia during the 1990s cluster with main subtype C, but a significant subcluster, C', was noted in multiple analyses. This subcluster of subtype C(C') was in a fifty-fifty equilibrium with the main subtype C (Abebe et al., AIDS Res Hum Retroviruses 2000;16:1909-1914). To analyze genetic diversification within the subcluster of HIV-1 subtype C designated C' in the course of the epidemic in Ethiopia, we analyzed 165 env gp120 V3 sequences obtained between 1988 and 1999. We observed a highly significant positive correlation between sampling years of individual sequences and their synonymous distances to the reconstructed common ancestor of the HIV-1 subtype C' subcluster. The extrapolation of the regression line of synonymous distances back to the date when no synonymous heterogeneity was present among the Ethiopian HIV-1 C' population allowed us to estimate 1982 (95% CI, 1980-1983) as the year of the onset of HIV-1 C' genetic diversification and expansion in Ethiopia. These results are in agreement with retrospective epidemiological and serological data, which demonstrated the absence of an HIV-1 epidemic in the Ethiopian population before the 1980s.

Objective To reconstruct the epidemiological relationships of the HIV epidemics among injecting drug users (IDU) in western Europe. Methods HIV env V3 sequences of and epidemiological data were obtained from 145 IDU who seroconverted in three sequential periods: 1984-1988, 1989-1992 and 1993-1997. The sequences were phylogenetically analysed and examined for signature patterns characteristic of northern European IDU, including the conserved GGC codon in the V3 loop. Results Subpopulations of genetically related HIV strains were observed in Italy, France, Scotland and Spain, in contrast to the Netherlands, Austria and Switzerland. This difference between the two groups of countries suggests that the HIV epidemics amongst IDU in the latter group was caused by multiple virus introductions. In Edinburgh and the surrounding area, most IDU were infected with the same GGC strain over the 12-year study period. The epidemic among IDU in north-western Europe started with GGC viruses, whereas in south-western Europe non-GGC viruses predominated. This geographical separation has faded during the course of the epidemic, most likely because of virus exchange among IDU populations.

OBJECTIVE: To trace the introduction of HIV-1 subtype C into Ethiopia based on virus diversification during the epidemic. DESIGN: A set of 474 serum samples obtained in Ethiopia in 1982-1985 was tested for HIV-1. HIV-1 env gp120 V3 and gag or pol regions were sequenced and analysed together with sequences from later stages of the epidemic. RESULTS: None of 98 samples from 1982-1983, one of 193 samples from 1984, and one of 183 samples from 1985 were HIV-1 positive. Phylogenetic analysis of virus sequences from positive samples revealed that they belong to the Ethiopian C, and not the C', cluster. Analysis of 81 Ethiopian C V3 sequences from 1984-1997 revealed that the consensus sequence of the Ethiopian epidemic has been stable over time. Both the 1984 and 1985 V3 sequences, in contrast with three out of 27 (11%) of the 1988 and none out of 51 of the 1992-1997 sequences, had no synonymous substitutions compared to the reconstructed common ancestor of the Ethiopian C viruses. A highly significant correlation between sampling years of the V3 sequences and their synonymous distances to the common ancestor was demonstrated. CONCLUSIONS: The increasing genetic heterogeneity together with stable consensus sequence of the Ethiopian HIV-1 C population demonstrates that evolution of the virus population is characterized by an unbiased expansion around a stationary consensus. Based on the rate of synonymous diversification of HIV-1 strains within the Ethiopian population, we were able to estimate 1983 (95% confidence interval, 1980-1984) as the year of HIV-1 C introduction into Ethiopia

Broad use of antiretroviral drugs is becoming a factor that is important to consider for understanding the HIV-1 epidemiology. Since 1993, we observe that a proportion of new infections within major risk groups in Amsterdam is caused by azidothymidine (AZT)-resistant viruses. After the introduction of combination therapy in The Netherlands in 1997, new infections with drug-resistant viruses have not been documented. Large-scale monitoring of anti-HIV-1 therapy failures revealed that antiretroviral drugs may yield previously undescribed resistant viruses, which contain a two amino acid insertion (68SS/V69) within their reverse transcriptase genes in combination with mutations at codons 67 and 215. These viruses are highly resistant to AZT, 3TC, and d4T, and moderately resistant to ddI and ddC.

New independent states of the former Soviet Union are facing a rapidly growing epidemic of HIV-1 among injecting drug users (IDUs). This epidemic is caused by three HIV-1 populations, one belonging to HIV-1 subtype A (IDU-A), another to subtype B (IDU-B), and the third being a recombinant of the IDU-A and IDU-B viruses (IDU-A/B, gagA/envB). Each of these populations is characterized by a high level of genetic homogeneity. We identified a unique synonymous nucleotide substitution in the first isoleucine codon at the IHIGPGR motif (ATT), which was observed in the env subtype B V3 sequences derived from IDUs in Russia and the Ukraine. This substitution was observed in none of 179 sequences obtained from IDUs in western Europe, northern America, and Asia. Molecular epidemiological analysis of HIV-1 strains based on this sequence pattern could be useful for tracing the origin and spread of the IDU-B viruses to other countries and risk groups.

The aim of this study was to analyze the association of hepatocellular carcinoma (HCC) with hepatitis C virus (HCV) in Egypt, using hepatitis B virus (HBV) and hepatitis E virus (HEV) as virus controls. In addition, the association of HCC with HCV RNA levels among persons seropositive for HCV was analyzed. We compared 131 patients with proven HCC, 247 with bladder cancer, and 466 healthy hospital employees. Age, sex, and place of residence were recorded to study confounding factors. Among the healthy controls, 16% were seropositive for HCV, 21% for HBV, and 31% for HEV. When healthy controls were age-matched with HCC patients, the latter were significantly (P < 0.001) more often HCV seropositive (67%) than were the controls (30%). The seropositivity for HBV and HEV did not differ significantly in frequency between the two groups. The seropositivity for HCV was also significantly (P < 0.001) more often found in HCC patients (76%) than in BC patients (47%), with seroprevalences for HBV and HEV not differing significantly in these age-matched groups. In HBV-negative HCC and bladder cancer patients, seroprevalence for HCV was significantly (P = 0.002) higher in HCC patients (68%) than in bladder cancer patients (36%). This difference was even more pronounced (P < 0.001) in HBV-positive HCC and bladder cancer patients (78% versus 52%, respectively). Of HCV-seropositive individuals, 49% were HCV RNA positive by branched DNA assay, and of these, 96% were infected by HCV genotype 4. No correlation between HCV RNA load and seropositivity of HBV or age or disease state was found. Infection with HCV and HCV-HBV double infection, but not HBV or HEV infection alone, is strongly correlated with HCC in Egypt.

In addition to development or selection of resistance, failure to continuously suppress HIV-1 production while still using initially effective combination antiretroviral therapy (cART) may result from super-infection with a drug-resistant strain. Both transmission of drug resistant HIV and super-infection have been demonstrated. We analysed HIV pol genes obtained before start of initially successful cART and during failure while still on cART in 101 patients. Difference in precART and cART failure sequences were explained by evolution and not by super-infection.

Countries of the former Soviet Union are experiencing an emerging HIV-1 epidemic due to a rapid expansion of HIV-1 among injecting drug users (IDUs). To study the molecular epidemiology of HIV-1 among IDUs in St. Petersburg, Russia, virus sequences were obtained from 22 individuals. Phylogenetic analysis of the env and gag regions revealed circulation of two major HIV-1 populations, one belonging to HIV-1 subtype A, and another being a recombinant of subtype A and B viruses (gagA/envB). Both virus populations were highly homogeneous, with a mean pairwise genetic distance of

During six blood passages of simian immunodeficiency virus SIVsm in rhesus macaques, the asymptomatic period shortened from 18 months to 1 month. To study SIVsm envelope gene ( env ) evolution during passage in rhesus macaques, the C1 to CD4 binding regions of multiple clones were sequenced at seroconversion and again at death. The env variation found during adaptation was almost completely confined to the variable regions. Intrasample sequence variation among clones at seroconversion was lower than the variation among clones at death. Intrasample variation among clones from a single time point as well as intersample variation decreased during the passage. In the variable regions, the mean number of intrasample nonsynonymous nucleotide substitutions decreased from the first passage (5.26 × 10 −2 ± 0.6 × 10 −2 per site) to the fifth passage (2.24 × 10 −2 ± 0.4 × 10 −2 per site), whereas in the constant regions, the mean number of intrasample nonsynonymous nucleotide substitutions differed less between the first and fifth passages (1.14 × 10 −2 ± 0.27 × 10 −2 and 0.80 × 10 −2 ± 0.24 × 10 −2 per site). Shortening of the asymptomatic period coincided with a rise in the Ks/Ka ratio (ratio between the number of synonymous [ Ks ] and the number of nonsynonymous [ Ka ] substitutions) from 1.080 in passage one to 1.428 in passage five and mimicked the difference seen in the intrahost evolution between asymptomatic and fast-progressing individuals infected with human immunodeficiency virus type 1. The distribution of nonsynonymous substitutions was biphasic, with most of the adaptation of env variable regions occurring in the first three passages. This phase, in which the symptom-free period fell to 4 months, was followed by a plateau phase of apparently reduced adaptation. Analysis of codon usage revealed decreased codon redundancy in the variable regions. Overall, the results suggested a biphasic pattern of adaptation and evolution, with extremely rapid selection in the first three passages followed by an equilibrium or stabilization of the variation between env clones at different time points in passages four to six.

The objective of this retrospective cohort study is to assess the mechanism by which human immunodeficiency virus type 1 (HIV) influences hepatitis C virus (HCV) replication in injecting drug users. Virological (HCV and HIV RNA levels) and immunological (CD4+, CD8+ cell counts, and anti-CD3 reactivity) parameters were determined in 19 HCV seroconverters in sequential samples over a period of 1 to 9 years. Among these subjects, 10 were HIV-seronegative (HIVneg), 4 were HIV-seropositive (HIVpos), and 5 seroconverted for HIV (HIVsc) during the observation period. HCV RNA levels were higher in HIVpos subjects than in HIVneg subjects. In subjects seroconverting for HIV, HCV, RNA levels increased significantly immediately after HIV seroconversion (P 500 cells/microliter (P = 0.001), but not in the population with CD4+ cell counts

Human immunodeficiency virus type 1 (HIV-1) strains can be separated into genetic subtypes based on phylogenetic analysis of the envelope gene. Once it had been shown that population-wide intrasubtype genetic variation of HIV-1 strains increases in the course of the AIDS epidemic, it remained uncertain whether HIV-1 subtypes are phenotypic entities spreading as distinct virus populations. To examine this, we applied Eigen's concepts of sequence geometry and fitness topography to the analysis of intrasubtype evolution of the gp120 V3 domain of HIV-1 subtypes A, B, C, and D in the course of the global AIDS epidemic. We observed that despite the high evolution rate of HIV-1, the nonsynonymous distances to the subtype consensus of sequences obtained early in the epidemic are similar to those obtained more than 10 years later, in contrast to the synonymous distances, which increased steadily over time. For HIV-1 subtype B, we observed that the evolution rate of the individual sequences is independent of their distance from the subtype B consensus, but for the individual sequences most distant from the consensus evolution away from the consensus is constrained. As a result, individual HIV-1 genomes fluctuate within a sequence space with fixed distance to the subtype consensus. Our findings suggest that the evolution of the V3 domain of HIV-1 subtypes A, B, C, and D is confined to an area in sequence space within a fixed distance to the consensus of a respective subtype. This in turn indicates that each HIV-1 subtype is a distinct viral quasispecies that is well adapted to the present environment, able to maintain its identity in the V3 region over time, and unlikely to merge during progression of the AIDS epidemic.

Materials and methods Autologous CD4 T lymphocytes from HIV-infected subjects were genetically modified ex vivo with the vector, expanded, and 10-80 billion vector-modified cells were reinfused into patients. Longitudinal effects of the therapy on HIV-1 env evolution were analyzed in 17 subjects sampled both pre-infusion and monthly postinfusion for 6 to 12 months. Plasma-derived viral RNA from 144 samples was amplified, cloned, and the fulllength gp120 coding region was sequenced in 8-10 clones for each sample.

In most HIV-infected individuals adherent to modern antiretroviral therapy (ART), plasma viremia stays undetectable by clinical assays and therefore, additional virological markers for monitoring and predicting therapy responses and for measuring the degree of HIV persistence in patients on ART should be identified. For the above purposes, quantitation of cell-associated HIV biomarkers could provide a useful alternative to measurements of viral RNA in plasma. This review concentrates on cell-associated (CA) HIV RNA with the emphasis on its use as a virological biomarker. We discuss the significance of CA HIV RNA as a prognostic marker of disease progression in untreated patients and as an indicator of residual virus replication and the size of the dynamic viral reservoir in ART-treated patients. Potential value of this biomarker for monitoring the response to ART and to novel HIV eradication therapies is highlighted.

The paper presents the results of experimental investigations of convective heat and mass transfer by sublimation of a single particle of metallorganic compounds mixture in an argon flow. The gas temperature is 180-290 ºС, the flow velocity is up to 2 m/s. The influence of the Zr(dpm)4 and Y(dpm)3 proportions on the characteristics of the transport processes was considered. An increase in the fraction of the more fusible component reduces the temperature of the particle during the sublimation process.

Our objective is to create gene immunogens targeted against drug-resistant HIV-1, focusing on HIV-1 enzymes as critical components in viral replication and drug resistance. Consensus-based gene vaccines are specifically fit for variable pathogens such as HIV-1 and have many advantages over viral genes and their expression-optimized variants. With this in mind, we designed the consensus integrase (IN) of the HIV-1 clade A strain predominant in the territory of the former Soviet Union and its inactivated derivative with and without mutations conferring resistance to elvitegravir. Humanized IN gene was synthesized; and inactivated derivatives (with 64D in the active site mutated to V) with and without elvitegravir-resistance mutations were generated by site-mutagenesis. Activity tests of IN variants expressed in E coli showed the consensus IN to be active, while both D64V-variants were devoid of specific activities. IN genes cloned in the DNA-immunization vector pVax1 (pVaxIN plasmids) were highly expressed in human and murine cell lines (>0.7 ng/cell). Injection of BALB/c mice with pVaxIN plasmids followed by electroporation generated potent IFN-γ and IL-2 responses registered in PBMC by day 15 and in splenocytes by day 23 after immunization. Multiparametric FACS demonstrated that CD8+ and CD4+ T cells of gene-immunized mice stimulated with IN-derived peptides secreted IFN-γ, IL-2, and TNF-α. The multi-cytokine responses of CD8+ and CD4+ T-cells correlated with the loss of in vivo activity of the luciferase reporter gene co-delivered with pVaxIN plasmids. This indicated the capacity of IN-specific CD4+ and CD8+ T-cells to clear IN/reporter co-expressing cells from the injection sites. Thus, the synthetic HIV-1 clade A integrase genes acted as potent immunogens generating polyfunctional Th1-type CD4+ and CD8+ T cells. Generation of such response is highly desirable for an effective HIV-1 vaccine as it offers a possibility to attack virus-infected cells via both MHC class I and II pathways.

Objectives To characterize HIV-1 epidemiological networks of men having sex with men (MSM) and drug users (DUs) in the Netherlands for >30 years. Design and methods Previously, we demonstrated different origin of the HIV-1 epidemics in Dutch MSM and DUs. To achieve the study objectives, risk group-specific genetic markers in the pol gene were examined in 315 participants of the Amsterdam Cohort Studies on HIV/AIDS who were registered as HIV-1 infected in 1981-2011. Results Phylogenetic analysis demonstrated circulation of distinct virus strains in the 2 networks, with 98% of viruses of MSM clustering together and apart from strains of 73% DUs. Nine genetic markers that significantly distinguished virus strains specific for DUs were identified, of which 3 were ≥90% conserved. Over the total observation period, only 6% of viruses (4 of MSM and 14 of DUs) clustered with those of the other risk group. Among these sequences, the 3 most conserved genetic markers of that other risk group were 87% conserved.All 4 cases of DU-specific viruses among MSM occurred in 1980s-early 1990s. Viruses nonspecific for DUs were causing new infections among DUs at the rate of 20% till 2002 and replaced DU-specific strains among new infections thereafter, coinciding with switching of DUs to low-harm drug practices. Conclusions Dutch MSM and DUs have remained separate epidemiological networks for decades, despite their geographical and behavioral overlap. Switching to low-harm drug practices among DUs resulted in new infections caused by HIV-1 strains originating from other risk groups.

The history of the HIV-1 B epidemic is the subject of a continuing debate. Did the epidemic start in the 1970s, as it was established based on the epidemiological data, or decades earlier, as it was suggested based on the analysis of nucleotide distances in the env gene? Our study [Lukashov and Goudsmit,J Mol Evol (2002) 54: 680-691] found that the overestimation of the age of the epidemic in the analysis of env sequences was a bias resulting from the non-clock-like evolution at nonsynonymous sites, while the estimates based on synonymous substitutions agreed with the results of epidemiological studies. Besides the principal difference between the evolution of synonymous and nonsynonymous sites, several issues have to be addressed: (i) the onset of the HIV/AIDS epidemic, and not the circulation of the preepidemic viruses, should be taken as the gold standard for the timeline of HIV diversification; (ii) the circulation of ancient, preepidemic, viruses, whether long- or short-term, does not influence the increase of HIV divergence during the epidemic; and (iii) application of the same random latency probability for all viruses, irrespective to their age and distance from the common ancestor, biases the estimation of the age of the epidemic.

Positively charged amino acid substitutions at positions 11 and 25 within the loop of the third variable region (V3) of HIV-1 subtype B envelope have been shown to be associated with the syncytium-inducing (SI) phenotype of the virus. The present study was designed to examine SI and NSI-associated V3 mutations in HIV-1 subtypes other than B. HIV-1 RNA was isolated from 53 virus stocks and 26 homologous plasma samples from 53 recently infected individuals from Brazil, Rwanda, Thailand, and Uganda. The C2-V3 region of the viral envelope was converted to cDNA, amplified, and sequenced. Of 53 primary virus stock samples 49 were biologically phenotyped through measurement of the syncytium-inducing capacity in MT-2 cells (to differentiate between SI and NSI phenotypes). In addition, after passage of primary isolates through PHA stimulated donor PBMC, the replication capacity was determined in U937-2, CEM, MT-2, and Jurkat-tat cell lines (to differentiate rapid/high and slow/low phenotypes). According to the seq...

Background Modern antiretroviral therapy (ART) is assumed to allow a certain degree of nonadherence while still maintaining complete suppression of viral replication ("forgiveness”), as virological suppression, measured by the commercial plasma viral load assays, is common at adherence levels >55-70% [1]. Yet, it is unknown whether HIV-1 replication is completely suppressed at these levels of adherence. [2]Here we investigated whether modest nonadherence to ART influences levels of HIV-1 RNA and DNA in peripheral blood mononuclear cells (PBMC).

Orthohepadnavirus (mammalian hosts) and avihepadnavirus (avian hosts) constitute the family of Hepadnaviridae and differ by their capability and inability for expression of protein X, respectively. Origin and functions of X are unclear. The evolutionary analysis at issue of X indicates that present strains of orthohepadnavirus started to diverge about 25,000 years ago, simultaneously with the onset of avihepadnavirus diversification. These evolutionary events were preceded by a much longer period during which orthohepadnavirus developed a functional protein X while avihepadnavirus evolved without X. An in silico generated 3D-model of orthohepadnaviral X protein displayed considerable similarity to the tertiary structure of DNA glycosylases (key enzymes of base excision DNA repair pathways). Similarity is confined to the central domain of MUG proteins with the typical DNA-binding facilities but without the capability of DNA glycosylase enzymatic activity. The hypothetical translation product of a vestigial X reading frame in the genome of duck hepadnavirus could also been folded into a DNA glycosylase-like 3D-structure. In conclusion, the most recent common ancestor of ortho- and avihepadnavirus carried an X sequence with orthology to the central domain of DNA glycosylase.

Objective: To compare the dynamics of HIV-1 molecular markers in peripheral blood mononuclear cells (PBMCs) and in plasma during the asymptomatic phase of untreated HIV-1 infection. Design and methods: Using seminested real-time PCR assays, we measured the levels of HIV-1 proviral (pr) DNA, unspliced (us) RNA, and multiply spliced RNA in the PBMCs of 10 untreated HIV-1-infected men at multiple time points during the asymptomatic phase of infection and compared the longitudinal trends of these markers with those of viral RNA in plasma. Results: Whereas plasma RNA levels did not significantly change in any of the individuals, levels of usRNA significantly increased with time in six out of 10 persons, and levels of prDNA in four. Slopes, changes, and time-weighted changes from baseline of usRNA, prDNA, and CD4(+) cell count, but not of plasma RNA, were significantly different from zero (P

Results The HIV/AIDS epidemic in Belarus can be divided into two stages. The first stage (1986-1996) was characterized by low prevalence and incidence of HIV-1. The total number of HIV-1-infected individuals in this period was 117, of whom 43 were foreigners who have arrived for study or work in Belarus. Since July 1996, the epidemiological situation in Belarus has changed. An HIV-1 outbreak in Svetlogorsk and Zhlobin in 1996-1997 has caused 1021 new HIV-1 infections within a six-month period. This first stage of the epidemic was characterized by: 1. Young men being mainly affected, with >60% of new infections being registered among individuals of 1424 years old; 2. Parenteral route of infection (92% of all transmissions); 3. Epidemiological process being largely limited to the cities of Svetlogorsk and Zhlobin; 4. The outbreak was caused by a subtype A HIV-1 strain. For October, first, 2009 10428 cases of a HIV-infection or 107,8 cases per 100 thousand population are officially registered in Belarus. HIV/AIDS Cases are registered more, than in 190 administrative territories. The current stage of the epidemic is characterized by: 1. The epidemic still affecting young population (individuals of 19-29 years old around 66% of new infections); 2. Heterosexual route of transmission becoming dominant (2009 77% of new infections); 3. Geographical expansion the epidemic is registered in 198 administrative territories with the highest prevalence in Svetlogorsk (3, 100 cases per 100,000 inhabitants); 4. About 95% of new HIV-1 cases are caused by the “Svetlogorsk” subtype A strain. With two-three new HIV-1 cases being registered daily in Belarus, mostly among the young generation. Such situation leads to that the quantity of children born to HIV-infected mothers annually increases. Despite of spent preventive therapy, 11% of such children are HIV-infected.

An experimental and numerical study of a laminar boundary layer with combustion has been carried out at hydrogen and nitrogen fuel mixture blow through a porous plate. At that main flow velocity ranged from 2 to 4 m/sec and the mass fraction of hydrogen in the fuel from 1 to 11%. The lower limit of stable combustion depending on the blow intensity and hydrogen content in the fuel mixture was obtained experimentally. Data on the temperature distribution in the boundary layer have been obtained and analyzed. The simulation results show that in this range of parameters combustion occurs in the kinetic mode.Copyright © 2010 by ASME

HIV researchers have to consider themselves lucky with their selection of the object of their studies: besides the importance of studying HIV as a human pathogen, this virus is in many ways a biological gold mine. Its recent origin and high level of variability make analysis of HIV attractive to evolutionary biologists. The error-prone mechanism of virus replication, its short generation time, and distinct selection mechanisms are resulting in HIV evolution rates being orders of magnitude higher than evolution rates of many other viruses and cellular organisms. This gives the researchers a unique opportunity to study ongoing evolutionary processes and to address fundamental evolutionary issues in real time.Recent developments in the area of sequencing methods and equipment have made it possible to obtain huge amounts of genetic information—currently, the GenBank contains partial or complete sequence information for more than 30,000 HIV strains. The boost given by HIV research to the whole field of molecular evolution and phylogeny, can be recognized in The Evolution of HIV, edited by Keith A. Crandall. The book represents a beautiful collection of 14 essays that are written by authors for whom HIV is the primary object of research as well as those for whom HIV is a tool in addressing general evolutionary issues. Chapters 3–8 and 10 provide us with a comprehensive overview of how phylogenetic and statistical analyses help in studying the epidemiology and evolution of HIV, and, on the other hand, how the validity of phylogenetic methods and evolutionary assumptions can be tested by using HIV sequence information. Particular attention in the book is given to the use of sequence data for the confirmation of instances of HIV transmission, both at theoretical and practical levels (chapters 7 and 10).Genetic variation of HIV is usually considered at several levels: virus heterogeneity within individual hosts (intrahost evolution), heterogeneity among infected hosts within a human population (interhost evolution), and heterogeneity among human populations (global variation). Let us follow this increasing scale of complexity, as the book edited by Keith A. Crandall does so nicely.Over the last few years, the relationship between the intrahost heterogeneity of HIV and disease progression has been widely debated. The original thoughts that the increase of virus heterogeneity above a certain level is required for AIDS progression were found to be inconsistent with multiple observations of faster nonsynonymous evolution and more heterogeneous virus populations in immunocompetent HIV-infected individuals (nonprogressors) compared to immunocompromised host (progressors). At the same time, patterns of synonymous intrahost evolution are similar in progressors and nonprogressors. The extent of nonsynonymous intrahost evolution among a group of patients over years of observations appeared to be proportional to the length of a period during which each patient has remained immunocompetent. These observations demonstrated that faster AIDS progression is not related to a higher variability of the HIV populations. Virus variability (ability to produce mutants) is the same in progressors and nonprogressors—it is virus variation (the probability of new mutants to be fixed in virus population) that is different. As a result of these observations, the role of virus heterogeneity and evolution in AIDS pathogenesis, has been revised. Intrahost variation of HIV by itself is no longer considered a driving force of AIDS progression, but rather a consequence of the host's immune competence. This concept and experimental findings supporting it are broadly discussed in the book (chapters 9, 11, and 13). For a more mathematical view on the population dynamics of HIV as well as other viruses, we strongly recommend another recently published book—Origin and Evolution of Viruses, edited by Esteban Domingo, Robert G. Webster, and John J. Holland (Academic Press, London, 1999, ISBN 0-12-220360-7).Previous genetic analysis revealed that HIV populations in newly infected individuals are relatively homogeneous and often represent a minor virus donor subpopulation. These observations have inspired a continuing discussion whether specific virus strains are more transmissible or more prone to be selected at the port of entry. To explain limitation of HIV genetic heterogeneity during or after virus transmission, several alternative hypotheses have been put forward. Although the relative impact of random and nonrandom selective processes remains to be established, the preference is currently given to the hypothesis that explains relative virus homogeneity soon after the infection by random transmission-associated population bottlenecks (discussed in chapters 9 and 12).The discussion of limits of virus heterogeneity at the time of transmission overlaps with a debate on the issue of biological significance of HIV subtypes, which is reviewed in chapter 13. In the U.S. and Western Europe, where HIV-1 subtype B is prevalent, most HIV cases occur among homosexual men and injecting drug users, while in Africa, where non-B subtypes are circulating, the HIV epidemic is mainly due to heterosexual transmissions. This uneven global distribution of HIV subtypes has led to a hypothesis that particular HIV subtypes may be adapted to specific risk groups or routes of transmission. However, many studies have not substantiated differences in in vivo virulence among HIV subtypes. We currently witness rapidly increasing numbers of non-B infections among traditionally subtype B risk groups, including recent non-B epidemics among injecting drug users in the former Soviet Union and China. This negates the idea that HIV subtypes are associated with a transmission route or risk behavior. Since we cannot prove that the existence of HIV subtypes is a consequence of adaptation of the original ancestral virus to different conditions, could we instead suggest that subtype diversity is a result of different ways of adapting to the same environment? This point of view goes along with the classical concept of adaptive sequence landscapes. Each and every HIV strain, belonging to a given HIV subtype, is rapidly evolving at the mean rate of about 1% per year for the most variable envelope gene. But does this mean that this HIV subtype is actually evolving? Are HIV-1 strains circulating today in, say, the U.S. 20% different from viruses that have been present in this country at the start of the epidemic 20 years ago? Our own analysis of genetic distances among HIV strains belonging to various HIV subtypes revealed that the mean intrasubtype nonsynonymous heterogeneity of the env gene is not increasing over time, in contrast to growing synonymous heterogeneity (Lukashov and Goudsmit, J. Virol. 71, 6332–6338, 1997). HIV subtypes appeared to be stable in the course of the epidemic, in spite of rapid genetic changes observed for each and every individual HIV strain. This phenomenon could be compared to Brownian movements, in which seemingly chaotic traffic of a body's atoms does not lead to desintegration of the body. Evolution is a vector, and besides its length (the evolution rate), its direction has to be considered. The high evolution rate of HIV can be combined with the stability of HIV subtypes only if virus evolution is constrained to the limited sequence space around the subtype consensus sequence.In the area of molecular epidemiology of HIV, enormous international efforts have resulted in several thousands of HIV samples from virtually all countries and risk groups being collected and sequenced. These data, which are summarized in chapter 2, emphasize the pivotal role in shaping HIV molecular epidemiology of multiple founder effects during HIV migration among host populations at risk. The nature of HIV variation allows for efficacious ways to pose and answer epidemiological questions, like those concerning the spread of viruses by different transmission routes, within different genetically defined risk groups, and in particular ecological niches.For such an excellent book, we miss a section on the evolutionary history of HIV. The continuing discussion on the origin of HIV and HIV subtypes has been heated up recently by the discovery of several HIV strains that are unique in terms of their age and/or position in the HIV phylogenetic tree. Although there seems to be no firm consensus on when exactly this virus was introduced into humans, most researchers agree that the human virus is relatively “young.” The timescale in the midst of this debate is unprecedented—for the first time, when talking about the evolutionary origin of a virus, we are fighting about decades, not millions of years. Researchers are generally optimists, and HIV scientists are no exception: based on longitudinal observation of a growing plant, we should be able to estimate its growth rate and to trace back in time quite precisely the moment when the seed started to grow. However, what we cannot easily estimate is the age of this seed and how much time it spent in the soil after being put there by the farmer.For the AIDS researchers less versed in HIV molecular biology, the Retroviruses textbook, edited by John M. Coffin, Stephen H. Hughes, and Harold E. Varmus (Cold Spring Harbor Laboratory Press, Plainview, NY, 1997, ISBN 0–87969–497–1 cloth, ISBN 0–87969–497–1 paper), should be kept at hand. All in all, for every researcher who is interested in evolution and molecular epidemiology of HIV, The Evolution of HIV is the book to read.

Background: Combination antiretroviral therapy (cART), the standard of care for HIV-1 infection, is considered to be successful when plasma viremia remains below the detection limit of commercial assays. Yet, cART fails in a substantial proportion of patients after the apparent success. No laboratory markers are known that are predictive of cART outcome in initial responders during the period of undetectable plasma viremia. Methodology/Principal Findings: Here, we report the results of a retrospective longitudinal study of twenty-six HIV-infected individuals who initially responded to cART by having plasma viremia suppressed to