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We report here the sequences of two reference strains of parvovirus B19 (B19V) used for quantitation of B19V DNA. One reference strain has been established by the World Health Organization (WHO) and the other by the European Pharmacopeia (Ph. Eur.) and belong to B19V genotype 1a1 and 1a2, respectively.

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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.

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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.

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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.

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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.

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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.

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Parvovirus B19 (B19V) can cause infection in humans. To date, three genotypes of B19V, with subtypes, are known, of which genotype 1a is the most prevalent genotype in the Western world. We sequenced the genome of B19V strains of 65 asymptomatic, recently infected Dutch blood donors, to investigate the spatio-temporal distribution of B19V strains, in the years 2003-2009. The sequences were compared to B19V sequences from Dutch patients with fifth disease, and to global B19V sequences as available from GenBank. All Dutch B19V strains belonged to genotype 1a. Phylogenetic analysis of the strains from Dutch blood donors showed that two groups of genotype 1a co-exist. A clear-cut division into the two groups was also found among the B19V strains from Dutch patients, and among the B19V sequences in GenBank. The two groups of genotype 1a co-exist around the world and do not appear to differ in their ability to cause disease. Strikingly, the two groups of B19V predominantly differ in synonymous mutations, distributed throughout the entire genome of B19V. We propose to call the two groups of B19V genotype 1a respectively subtype 1a1 and 1a2.

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BACKGROUND: Modern antiretroviral therapy (ART) regimens are widely assumed to forgive modest nonadherence, because virological suppression in plasma is common at adherence levels of >70%. Yet, it is unknown whether human immunodeficiency virus type 1 (HIV-1) replication is completely suppressed at these levels of adherence. METHODS: We longitudinally quantified levels of cell-associated HIV-1 RNA and DNA in 40 patients (median duration of successful ART before study initiation, 46 months), whose 1-week adherence to therapy prior to the sampling moments was measured electronically. RESULTS: Patients were constantly 100% adherent (the optimal-adherence group), demonstrated improving adherence over time (the improving-adherence group), or neither of the above (the poor-adherence group). Adherence never decreased to <70% in any patient, and no rebound in plasma virological levels was observed. Nevertheless, poor adherence but not optimal or improving adherence caused a significant longitudinal increase in cell-associated HIV RNA levels (P = .006). Time-weighted changes and regression slopes of viral RNA load for the poor-adherence group were significantly higher than those for the optimal-adherence group (P < .01). CONCLUSIONS: Because ART only blocks infection of new cells but not viral RNA transcription in cells infected before therapy initiation, the observed effects strongly suggest that modest nonadherence can cause new cycles of HIV-1 replication that are undetectable by commercial plasma viral load assays.

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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.

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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 < 0.01). No significant longitudinal trend of plasma RNA was observed in the study group using linear mixed models, whereas the trends of usRNA, prDNA, and CD4 cell count were highly significant (P < 0.001). usRNA levels increased significantly faster than those of plasma RNA or prDNA, suggesting a temporal increase in viral replication rates in PBMCs. Finally, CD4 cell count inversely correlated with levels of usRNA and prDNA, but not with plasma RNA level. CONCLUSION: During the asymptomatic phase of untreated HIV-1 infection, when virion production and clearance are balanced, resulting in stable plasma viremia, viral load in PBMCs steadily increases and is a sensitive and direct longitudinal virological marker of infection progression.

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OBJECTIVE: To obtain insight in the HIV-1 transmission networks among men having sex with men (MSM) in the Netherlands. DESIGN: A phylogenetic tree was constructed from polymerase sequences isolated from 2877 HIV-1 subtype B-infected patients monitored as part of the AIDS Therapy Evaluation in the Netherlands (ATHENA) nationwide observational cohort. METHODS: For MSM with a known date of infection, the most similar sequences were selected as potential transmission pairs when they clustered with bootstrap value of at least 99%. Time from infection to onward transmission was estimated as the median time between dates of infection for each transmission pair. The source of infections with a resistant strain was traced using the entire phylogenetic tree. RESULTS: Of sequences from 403 MSM with a known date of infection between 1987 and 2007, 175 (43%) formed 63 clusters. Median time to onward transmission was 1.4 years (interquartile range 0.6-2.7). Twenty-four (6%) MSM carried a virus with resistance-related mutations, 13 of these were in eight clusters together with sequences from 28 other patients in the entire phylogenetic tree. Six clusters contained sequences obtained from 29 men all presenting the same resistance-related mutations. CONCLUSION: From our selection of likely transmission pairs, we conclude that onward transmission of HIV-1 from infected MSM in the Netherlands happens both during and after primary infection. Transmission of resistant strains from the antiretroviral therapy-treated population is limited, but strains with resistance-related mutations have formed subepidemics.

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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.

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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 <50 copies/ml. Eleven of these patients remained virologically suppressed, whereas fifteen experienced subsequent cART failure. Using sensitive methods based on seminested real-time PCR, we measured the levels of HIV-1 proviral (pr) DNA, unspliced (us) RNA, and multiply spliced RNA in the peripheral blood mononuclear cells (PBMC) of these patients at multiple time points during the period of undetectable plasma viremia on cART. Median under-therapy level of usRNA was significantly higher (0.43 log(10) difference, P = 0.0015) in patients who experienced subsequent cART failure than in successfully treated patients. In multivariate analysis, adjusted for baseline CD4(+) counts, prior ART experience, and particular cART regimens, the maximal usRNA level under therapy was the best independent predictor of subsequent therapy failure (adjusted odds ratio [95% CI], 24.4 [1.5-389.5], P = 0.024). The only other factor significantly associated with cART failure was prior ART experience (adjusted odds ratio [95% CI], 12.3 [1.1-138.4], P = 0.042). Levels of usRNA under cART inversely correlated with baseline CD4(+) counts (P = 0.0003), but did not correlate with either baseline usRNA levels or levels of prDNA under therapy. CONCLUSION: Our data demonstrate that the level of HIV-1 usRNA in PBMC, measured in cART-treated patients with undetectable plasma viremia, is a strong predictive marker for the outcome of therapy.

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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 substitutions 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.

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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.

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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.

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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.

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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.

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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.

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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.

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