Silent codon positions in the A-rich HIV RNA genome that do not easily become A: Restrictions imposed by the RNA sequence and structure.

There is a strong evolutionary tendency of the human immunodeficiency virus (HIV) to accumulate A nucleotides in its RNA genome, resulting in a mere 40 per cent A count. This A bias is especially dominant for the so-called silent codon positions where any nucleotide can be present without changing the encoded protein. However, particular silent codon positions in HIV RNA refrain from becoming A, which became apparent upon genome analysis of many virus isolates. We analyzed these 'noA' genome positions to reveal the underlying reason for their inability to facilitate the A nucleotide. We propose that local RNA structure requirements can explain the absence of A at these sites. Thus, noA sites may be prominently involved in the correct folding of the viral RNA. Turning things around, the presence of multiple clustered noA sites may reveal the presence of important sequence and/or structural elements in the HIV RNA genome.

Euclidean Distance Analysis Enables Nucleotide Skew Analysis in Viral Genomes.

Nucleotide skew analysis is a versatile method to study the nucleotide composition of RNA/DNA molecules, in particular to reveal characteristic sequence signatures. For instance, skew analysis of the nucleotide bias of several viral RNA genomes indicated that it is enriched in the unpaired, single-stranded genome regions, thus creating an even more striking virus-specific signature. The comparison of skew graphs for many virus isolates or families is difficult, time-consuming, and nonquantitative. Here, we present a procedure for a more simple identification of similarities and dissimilarities between nucleotide skew data of coronavirus, flavivirus, picornavirus, and HIV-1 RNA genomes. Window and step sizes were normalized to correct for differences in length of the viral genome. Cumulative skew data are converted into pairwise Euclidean distance matrices, which can be presented as neighbor-joining trees. We present skew value trees for the four virus families and show that closely related viruses are placed in small clusters. Importantly, the skew value trees are similar to the trees constructed by a "classical" model of evolutionary nucleotide substitution. Thus, we conclude that the simple calculation of Euclidean distances between nucleotide skew data allows an easy and quantitative comparison of characteristic sequence signatures of virus genomes. These results indicate that the Euclidean distance analysis of nucleotide skew data forms a nice addition to the virology toolbox.

HIV-1 tolerates changes in A-count in a small segment of the pol gene.

BACKGROUND: The HIV-1 RNA genome has a biased nucleotide composition with a surplus of As. Several hypotheses have been put forward to explain this striking phenomenon, but the A-count of the HIV-1 genome has thus far not been systematically manipulated. The reason for this reservation is the likelihood that known and unknown sequence motifs will be affected by such a massive mutational approach, thus resulting in replication-impaired virus mutants. We present the first attempt to increase and decrease the A-count in a relatively small polymerase (pol) gene segment of HIV-1 RNA. RESULTS: To minimize the mutational impact, a new mutational approach was developed that is inspired by natural sequence variation as present in HIV-1 isolates. This phylogeny-instructed mutagenesis allowed us to create replication-competent HIV-1 mutants with a significantly increased or decreased local A-count. The local A-count of the wild-type (wt) virus (40.2%) was further increased to 46.9% or reduced to 31.7 and 26.3%. These HIV-1 variants replicate efficiently in vitro, despite the fact that the pol changes cause a quite profound move in HIV-SIV sequence space. CONCLUSIONS: Extrapolating these results to the complete 9 kb RNA genome, we may cautiously suggest that the A-rich signature does not have to be maintained. This survey also provided clues that silent codon changes, in particular from G-to-A, determine the subtype-specific sequence signatures.

A Novel Astrovirus-Like RNA Virus Detected in Human Stool.

Several novel clades of astroviruses have recently been identified in human faecal samples. Here, we describe a novel astrovirus-like RNA virus detected in human stools, which we have tentatively named bastrovirus. The genome of this novel virus consists of 6,300 nucleotides organized in three open reading frames. Several sequence divergent strains were detected sharing 67-93 per cent nucleotide identity. Bastrovirus encodes a putative structural protein that is homologous to the capsid protein found in members of the Astroviridae family (45% amino acid identity). The virus also encodes a putative non-structural protein that is genetically distant from astroviruses but shares some homology to the non-structural protein encoded by members of the Hepeviridae family (28% amino acid identity). This novel bastrovirus is present in 8.7 per cent (35/400) of faecal samples collected from 300 HIV-1-positive and 100 HIV-1-negative individuals suggesting common occurrence of the virus. However, whether the source of the virus is infected human cells or other, for example, dietary, remains to be determined.

Impact of the biased nucleotide composition of viral RNA genomes on RNA structure and codon usage.

We are interested in the influence of nucleotide composition on the fundamental characteristics of the virus RNA genome. Most RNA viruses have genomes with a distinct nucleotide composition, e.g. ranging from minimally 12.9 % to maximally 40.3 % (C- and U-count, respectively, in coronavirus HKU). We present a global analysis of diverse virus types, including plus-strand, minus-strand and double-strand RNA viruses, for the impact of this nucleotide preference on the predicted structure of the RNA genome that is packaged in virion particles and on the codon usage in the viral open reading frames. Several virus-specific features will be described, but also some general conclusions were drawn. Without exception, the virus-specific nucleotide bias was enriched in the unpaired, single-stranded regions of the RNA genome, thus creating an even more striking virus-specific signature. We present a simple mechanism that is based on elementary aspects of RNA structure folding to explain this general trend. In general, the nucleotide bias was the major determinant of the virus-specific codon usages, thus limiting a role for codon selection and translational control. We will discuss molecular and evolutionary scenarios that may be responsible for the diverse nucleotide biases of RNA viruses.

Nucleotide composition of the Zika virus RNA genome and its codon usage.

BACKGROUND: RNA viruses have genomes with a distinct nucleotide composition and codon usage. We present the global characteristics of the RNA genome of Zika virus (ZIKV), an emerging pathogen within the Flavivirus genus. ZIKV was first isolated in 1947 in Uganda, caused a widespread epidemic in South and Central America and the Caribbean in 2015 and has recently been associated with microcephaly in newborns. METHODS: The nearly 11 kb positive-stranded RNA genome of ZIKV was analyzed for its nucleotide composition, also in the context of the folded RNA molecule. Nucleotide trends were investigated along the genome length by skew analyses and we analyzed the codons used for translation of the ZIKV proteins. RESULTS: ZIKV RNA has a biased nucleotide composition in being purine-rich and pyrimidine-poor. This preference for purines is a general characteristic of the mosquito-borne and tick-borne flaviviruses. The virus-specific nucleotide bias is further enriched in the unpaired, single-stranded regions of the structured ZIKV RNA genome, thus further imposing this ZIKV-specific signature. The codons used for translation of the ZIKV proteins is also unusual, but we show that it is the underlying bias in nucleotide composition of the viral RNA that largely dictates these codon preferences. CONCLUSIONS: The ZIKV RNA genome has a biased nucleotide composition that dictates the codon usage of this flavivirus. We discuss the evolutionary scenarios and molecular mechanisms that may be responsible for these distinctive ZIKV RNA genome features.

In silico prediction of ebolavirus RNA polymerase inhibition by specific combinations of approved nucleotide analogues.

BACKGROUND AND OBJECTIVE: The urgency of ebolavirus drug development is obvious in light of the current local epidemic in Western Africa with high morbidity and a risk of wider spread. We present an in silico study as a first step to identify inhibitors of ebolavirus polymerase activity based on approved antiviral nucleotide analogues. STUDY DESIGN: Since a structure model of the ebolavirus polymerase is lacking, we performed combined homology and ab initio modeling and report a similarity to known polymerases of human enterovirus, bovine diarrhea virus and foot-and-mouth disease virus. This facilitated the localization of a nucleotide binding domain in the ebolavirus polymerase. We next performed molecular docking studies with nucleotides (ATP, CTP, GTP and UTP) and nucleotide analogues, including a variety of approved antiviral drugs. RESULTS AND CONCLUSIONS: Specific combinations of nucleotide analogues significantly reduce the ligand-protein interaction energies of the ebolavirus polymerase for natural nucleotides. Any nucleotide analogue on its own did not reduce ligand-protein interaction energies. This prediction encourages specific drug testing efforts and guides future strategies to inhibit ebolavirus replication.

On the biased nucleotide composition of the human coronavirus RNA genome.

We investigated the nucleotide composition of the RNA genome of the six human coronaviruses. Some general coronavirus characteristics were apparent (e.g. high U, low C count), but we also detected species-specific signatures. Most strikingly, the high U and low C proportions are quite variable and act like communicating vessels, C goes down when U goes up and vice versa. U ranges among virus isolates from 30.7% to 40.3%, and C makes the opposite movement from 20.0% to 12.9%, respectively. The nucleotide biases are more pronounced in the unpaired regions of the structured RNA genome, which may suggest a certain biological function for these distinctive sequence signatures. Coronaviruses have an atypical codon usage that has been linked to mutational events operating on the viral RNA genome on an evolutionary time scale. We suggest that the atypical nucleotide bias may serve a distinct biological function and that it is the direct cause of the characteristic codon usage in these viruses. The relevance for evolution of the novel human pathogens MERS and SARS is discussed.

Specific mutations in the C-terminus domain of HBV surface antigen significantly correlate with low level of serum HBV-DNA in patients with chronic HBV infection.

BACKGROUND: To define HBsAg-mutations correlated with different serum HBV-DNA levels in HBV chronically-infected drug-naive patients. METHODS: This study included 187 patients stratified into the following ranges of serum HBV-DNA:12-2000 IU/ml, 2000-100,000 IU/ml, and >100,000 IU/ml. HBsAg-mutations were associated with HBV-DNA levels by applying a Bayesian-Partitional-Model and Fisher-exact test. Mutant and wild-type HBV genotype-D genomes were expressed in Huh7 cells and HBsAg-production was determined in cell-supernatants at 3 days-post-transfection. RESULTS: Specific HBsAg-mutations (M197T,-S204N-Y206C/H-F220L) were significantly correlated with serum HBV-DNA <2000 IU/ml (posterior-probability>90%, P < 0.05). The presence of Y206C/H and/or F220L was also associated with lower median (IQR) HBsAg-levels and lower median (IQR) transaminases (for HBsAg:250[115-840] IU/ml for Y206C/H and/or F220L versus 4300[640-11,838] IU/ml for wild-type, P = 0.023; for ALT:28[21-40] IU/ml versus 53[34-90] IU/ml, P < 0.001). These mutations were localized in the HBsAg C-terminus, known to be involved in virion and/or HBsAg secretion. The co-occurrence of Y206C + F220L was found significant by cluster-analysis, (P = 0.02). In addition, in an in-vitro model Y206C + F220L determined a 2.8-3.3 fold-reduction of HBsAg-amount released in supernatants compared to single mutants and wt (Y206C + F220L = 5,679 IU/ml; Y206H = 16,305 IU/ml; F220L = 18,368 IU/ml; Y206C = 18,680 IU/ml; wt = 14,280 IU/ml, P < 0.05). CONCLUSIONS: Specific HBsAg-mutations (compartmentalized in the HBsAg C-terminus) correlated with low-serum HBV-DNA and HBsAg-levels. These findings can be important to understand mechanisms underlying low HBV replicative potential including the inactive-carrier state.

Differential binding of tenofovir and adefovir to reverse transcriptase of hepatitis B virus.

INTRODUCTION: Resistance of the reverse transcriptase (RT) of hepatitis B virus (HBV) to the tenofovir nucleotide drug has not been observed since its introduction for treatment of hepatitis B virus (HBV) infection in 2008. In contrast, frequent viral breakthrough and resistance has been documented for adefovir. Our computational study addresses an inventory of the structural differences between these two nucleotide analogues and their binding sites and affinities to wildtype (wt) and mutant RT enzyme structures based on in silico modeling, in comparison with the natural nucleotide substrates. RESULTS: Tenofovir and adefovir only differ by an extra CH3-moiety in tenofovir, introducing a center of chirality at the carbon atom linking the purine group with the phosphates. (R)-Tenofovir (and not (S)-tenofovir) binds significantly better to HBV-RT than adefovir. "Single hit" mutations in HBV-RT associated with adefovir resistance may affect the affinity for tenofovir, but to a level that is insufficient for tenofovir resistance. The RT-Surface protein gene overlap in the HBV genome provides an additional genetic constraint that limits the mutational freedom required to generate drug-resistance. Different pockets near the nucleotide binding motif (YMDD) in HBV-RT can bind nucleotides and nucleotide analogues with different affinities and specificities. CONCLUSION: The difference in binding affinity of tenofovir (more than two orders of magnitude in terms of local concentration), a 30x higher dosage of the (R)-tenofovir enantiomer as compared to conformational isomeric or rotameric adefovir, and the constrained mutational space due to gene overlap in HBV may explain the absence of resistance mutations after 6 years of tenofovir monotherapy. In addition, the computational methodology applied here may guide the development of antiviral drugs with better resistance profiles.

On the nucleotide composition and structure of retroviral RNA genomes.

Retroviral RNA genomes display a rich variety in their nucleotide composition. For instance, the single-stranded RNA genome of human T cell leukemia virus (HTLV-1) is C-rich and G-poor and that of the human immunodeficiency virus (HIV-1) is A-rich and C-poor. Animal retroviruses add further variation to this unexplained, but many times remarkable virus-specific property. We previously described that the nucleotide bias is even more extreme in the unpaired regions of the structured HIV-1 RNA genome, which has been probed by SHAPE technology. We now document that the same trend is apparent for the MFold-predicted RNA structure of HIV-1 RNA and subsequently investigated the predicted structures of the RNA genomes of other retroviruses. We conclude that all virus-specific signatures are enhanced for the unpaired nucleotides in the RNA genome. Consequently, the differences in nucleotide count between the diverse human and animal retroviruses are further exposed in the single stranded genome regions. We used a skew analysis to visualize these striking differences in nucleotide usage. Evolutionary events responsible for these nucleotide signatures will be discussed.

Unexplained diarrhoea in HIV-1 infected individuals.

BACKGROUND: Gastrointestinal symptoms, in particular diarrhoea, are common in non-treated HIV-1 infected individuals. Although various enteric pathogens have been implicated, the aetiology of diarrhoea remains unexplained in a large proportion of HIV-1 infected patients. Our aim is to identify the cause of diarrhoea for patients that remain negative in routine diagnostics. METHODS: In this study stool samples of 196 HIV-1 infected persons, including 29 persons with diarrhoea, were examined for enteropathogens and HIV-1. A search for unknown and unexpected viruses was performed using virus discovery cDNA-AFLP combined with Roche-454 sequencing (VIDISCA-454). RESULTS: HIV-1 RNA was detected in stool of 19 patients with diarrhoea (66%) compared to 75 patients (45%) without diarrhoea. In 19 of the 29 diarrhoea cases a known enteropathogen could be identified (66%). Next to these known causative agents, a range of recently identified viruses was identified via VIDISCA-454: cosavirus, Aichi virus, human gyrovirus, and non-A non-B hepatitis virus. Moreover, a novel virus was detected which was named immunodeficiency-associated stool virus (IASvirus). However, PCR based screening for these viruses showed that none of these novel viruses was associated with diarrhoea. Notably, among the 34% enteropathogen-negative cases, HIV-1 RNA shedding in stool was more frequently observed (80%) compared to enteropathogen-positive cases (47%), indicating that HIV-1 itself is the most likely candidate to be involved in diarrhoea. CONCLUSION: Unexplained diarrhoea in HIV-1 infected patients is probably not caused by recently described or previously unknown pathogens, but it is more likely that HIV-1 itself plays a role in intestinal mucosal abnormalities which leads to diarrhoea.

Identification of a new genotype of Torque Teno Mini virus.

BACKGROUND: Although human torque teno viruses (TTVs) were first discovered in 1997, still many associated aspects are not clarified yet. The viruses reveal a remarkable heterogeneity and it is possible that some genotypes are more pathogenic than others. The identification of all genotypes is essential to confirm previous pathogenicity data, and an unbiased search for novel viruses is needed to identify TTVs that might be related to disease. METHOD: The virus discovery technique VIDISCA-454 was used to screen serum of 55 HIV-1 positive injecting drug users, from the Amsterdam Cohort Studies, in search for novel blood-blood transmittable viruses which are undetectable via normal diagnostics or panvirus-primer PCRs. RESULTS: A novel torque teno mini virus (TTMV) was identified in two patients and the sequence of the full genomes were determined. The virus is significantly different from the known TTMVs (< 40% amino acid identity in ORF1), yet it contains conserved characteristics that are also present in other TTMVs. The virus is chronically present in both patients, and these patients both suffered from a pneumococcal pneumonia during follow up and had extremely low B-cells counts. CONCLUSION: We describe a novel TTMV which we tentatively named TTMV-13. Further research is needed to address the epidemiology and pathogenicity of this novel virus.

Anti-HBV treatment induces novel reverse transcriptase mutations with reflective effect on HBV S antigen.

INTRODUCTION: The identification of novel reverse-transcriptase (RT) drug-resistance mutations is critical in predicting the probability of success to anti-HBV treatment. Furthermore, due to HBV-RT/HBsAg gene-overlap, they can have an impact on HBsAg-detection and quantification. METHODS: 356 full-length HBV-RT sequences from 197 drug-naive patients and 159 patients experiencing virological-breakthrough to nucleoside/nucleotide-analogs (NUCs) were analyzed. Mutants and wild-type HBs-antigens were expressed in HuH7-hepatocytes and quantified in cell-supernatants and cell-lysates by Architect HBsAg-assay. RESULTS: Ten novel RT-mutations (rtN53T-rtS78T-rtS85F-rtS135T-rtA181I-rtA200V-rtK212Q-rtL229V/F-rtM309K) correlated with specific NUC-treatments and classical drug-resistance mutations on divergent evolutionary pathways. Some of them reduced RT-binding affinity for anti-HBV drugs and altered S-antigen structure. Indeed, rtS78T (prevalence: 1.1% in drug-naïve and 12.2% in adefovir-failing patients) decreased the RT-affinity for adefovir more than the classical adefovir-resistance mutations rtA181 T/V (WT:-9.63 kcal/mol, rtA181T:-9.30 kcal/mol, rtA181V:-7.96 kcal/mol, rtS78T:-7.37 kcal/mol). Moreover, rtS78T introduced a stop-codon at HBsAg-position 69, and completely abrogated HBsAg-quantification in both supernatants and cell-lysates, indicating an impaired HBsAg-secretion/production. Furthermore, the HBsAg-mutation sP217L, silent in RT, significantly correlated with M204V/I-related virological-breakthrough and increased HBsAg-quantification in cell-lysate. CONCLUSIONS: Mutations beyond those classically known can affect drug-binding affinity of mutated HBV-RT, and may have potential effects on HBsAg. Their cumulative effect on resistance and HBV-pathogenicity indicates the importance of preventing therapeutic failures.

The A-nucleotide preference of HIV-1 in the context of its structured RNA genome.

A bipartition of HIV-1 RNA genome sequences into single- and double-stranded nucleotides is possible based on the secondary structure model of a complete 9 kb genome. Subsequent analysis revealed that the well-known lentiviral property of A-accumulation is profoundly present in single-stranded domains, yet absent in double-stranded domains. Mutational rate analysis by means of an unrestricted model of nucleotide substitution suggests the presence of an evolutionary equilibrium to preserve this biased nucleotide distribution.

The hepatitis B virus x protein inhibits thymine DNA glycosylase initiated base excision repair.

The hepatitis B virus (HBV) genome encodes the X protein (HBx), a ubiquitous transactivator that is required for HBV replication. Expression of the HBx protein has been associated with the development of HBV infection-related hepatocellular carcinoma (HCC). Previously, we generated a 3D structure of HBx by combined homology and ab initio in silico modelling. This structure showed a striking similarity to the human thymine DNA glycosylase (TDG), a key enzyme in the base excision repair (BER) pathway. To further explore this finding, we investigated whether both proteins interfere with or complement each other's functions. Here we show that TDG does not affect HBV replication, but that HBx strongly inhibits TDG-initiated base excision repair (BER), a major DNA repair pathway. Inhibition of the BER pathway may contribute substantially to the oncogenic effect of HBV infection.

Novel HBsAg markers tightly correlate with occult HBV infection and strongly affect HBsAg detection.

Occult HBV infection (OBI) is a threat for the safety of blood-supply, and has been associated with the onset of HBV-related hepatocellular carcinoma and lymphomagenesis. Nevertheless, genetic markers in HBsAg (particularly in D-genotype, the most common in Europe) significantly associated with OBI in vivo are missing. Thus, the goal of this study is to define: (i) prevalence and clinical profile of OBI among blood-donors; (ii) HBsAg-mutations associated with OBI; (iii) their impact on HBsAg-detection. OBI was searched among 422,278 blood-donors screened by Nucleic-Acid-Testing. Following Taormina-OBI-definition, 26 (0.006%) OBI-patients were identified. Despite viremia <50IU/ml, HBsAg-sequences were obtained for 25/26 patients (24/25 genotype-D). OBI-associated mutations were identified by comparing OBI-HBsAg with that of 82 chronically-infected (genotype-D) patients as control. Twenty HBsAg-mutations significantly correlated for the first time with OBI. By structural analysis, they localized in the major HBV B-cell-epitope, and in HBsAg-capsid interaction region. 14/24 OBI-patients (58.8%) carried in median 3 such mutations (IQR:2.0-6.0) against 0 in chronically-infected patients. By co-variation analysis, correlations were observed for R122P+S167L (phi=0.68, P=0.01), T116N+S143L (phi=0.53, P=0.03), and Y100S+S143L (phi=0.67, p<0.001). Mutants (obtained by site-directed mutagenesis) carrying T116N, T116N+S143L, R122P, R122P+Q101R, or R122P+S167L strongly decreased HBsAg-reactivity (54.9±22.6S/CO, 31.2±12.0S/CO, 6.1±2.4S/CO, 3.0±1.0S/CO and 3.9±1.3S/CO, respectively) compared to wild-type (306.8±64.1S/CO). Even more, Y100S and Y100S+S143L supernatants show no detectable-HBsAg (experiments in quadruplicate). In conclusions, unique HBsAg-mutations in genotype-D, different than those described in genotypes B/C (rarely found in western countries), tightly correlate with OBI, and strongly affect HBsAg-detection. By altering HBV-antigenicity and/or viral-particle maturation, they may affect full-reliability of universal diagnostic-assays for HBsAg-detection.

Protein X of hepatitis B virus: origin and structure similarity with the central domain of DNA glycosylase.

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.

Rooting human parechovirus evolution in time.

BACKGROUND: The Picornaviridae family contains a number of important pathogenic viruses, among which the recently reclassified human parechoviruses (HPeVs). These viruses are widespread and can be grouped in several types. Understanding the evolutionary history of HPeV could answer questions such as how long the circulating lineages last shared a common ancestor and how the evolution of this viral species is shaped by its population dynamics. Using both strict and relaxed clock Bayesian phylogenetics we investigated 1) the substitutions rates of the structural P1 and capsid VP1 regions and 2) evolutionary timescale of currently circulating HPeV lineages. RESULTS: Our estimates reveal that human parechoviruses exhibit high substitution rates for both structural P1 and capsid VP1 regions, respectively 2.21 x 10(-3) (0.48 - 4.21 x 10(-3)) and 2.79 x 10(-3) (2.05 - 3.66 x 10(-3)) substitutions per site per year. These are within the range estimated for other picornaviruses. By employing a constant population size coalescent prior, the date of the most recent common ancestor was estimated to be at around 1600 (1427-1733). In addition, by looking at the frequency of synonymous and non-synonymous substitutions within the VP1 gene we show that purifying selection constitutes the dominating evolutionary force leading to strong amino acid conservation. CONCLUSION: In conclusion, our estimates provide a timescale for the evolution of HPeVs and suggest that genetic diversity of current circulating HPeV types has arisen about 400 years ago.

Occult hepatitis B infection: an evolutionary scenario.

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.