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The 1918 influenza pandemic was the deadliest respiratory pandemic of the 20th century and determined the genomic make-up of subsequent human influenza A viruses (IAV). Here, we analyze both the first 1918 IAV genomes from Europe and the first from samples prior to the autumn peak. 1918 IAV genomic diversity is consistent with a combination of local transmission and long-distance dispersal events. Comparison of genomes before and during the pandemic peak shows variation at two sites in the nucleoprotein gene associated with resistance to host antiviral response, pointing at a possible adaptation of 1918 IAV to humans. Finally, local molecular clock modeling suggests a pure pandemic descent of seasonal H1N1 IAV as an alternative to the hypothesis of origination through an intrasubtype reassortment. ispartof: NATURE COMMUNICATIONS vol:13 issue:1 ispartof: location:England status: published

The HIV-1 reservoir is the major hurdle to curing HIV-1. However, the impact of the viral genome on the HIV-1 reservoir, i.e. its heritability, remains unknown. We investigate the heritability of the HIV-1 reservoir size and its long-term decay by analyzing the distribution of those traits on viral phylogenies from both partial-pol and viral near full-length genome sequences. We use a unique nationwide cohort of 610 well-characterized HIV-1 subtype-B infected individuals on suppressive ART for a median of 5.4 years. We find that a moderate but significant fraction of the HIV-1 reservoir size 1.5 years after the initiation of ART is explained by genetic factors. At the same time, we find more tentative evidence for the heritability of the long-term HIV-1 reservoir decay. Our findings indicate that viral genetic factors contribute to the HIV-1 reservoir size and hence the infecting HIV-1 strain may affect individual patients’ hurdle towards a cure., The HIV reservoir is a major hurdle for a cure of HIV, but the factors determining its size and dynamics remain unclear. Here the authors show in a large cohort of 610 HIV-1 infected individuals, who are on suppressive ART for a median of 5.4 years, that viral genetic factors contribute substantially to the HIV-1 reservoir size.

Respiratory syncytial virus (RSV) infection is a leading cause of hospitalization and mortality in young children. Protective therapy options are limited. Currently, palivizumab, a monoclonal IgG1 antibody, is the only licensed drug for RSV prophylaxis, although other IgG antibody candidates are being evaluated. However, at the respiratory mucosa, IgA antibodies are most abundant and act as the first line of defense against invading pathogens. Therefore, it would be logical to explore the potential of recombinant human IgA antibodies to protect against viral respiratory infection, but very little research on the topic has been published. Moreover, it is unknown whether human antibodies of the IgA isotype are better suited than those of the IgG isotype as antiviral drugs to combat respiratory infections. To address this, we generated various human IgA antibody formats of palivizumab and motavizumab, two well-characterized human IgG1 anti-RSV antibodies. We evaluated their efficacy to prevent RSV infection in vitro and in vivo and found similar, but somewhat decreased efficacy for different IgA subclasses and formats. Thus, reformatting palivizumab or motavizumab into IgA reduces the antiviral potency of either antibody. Moreover, our results indicate that the efficacy of intranasal IgA prophylaxis against RSV infection in human FcαRI transgenic mice is independent of Fc receptor expression.

PURPOSE: To determine the incidence of elevated intraocular pressure (TOP) and secondary glaucoma in herpetic anterior uveitis (AU), owing to either herpes simplex or varicella zoster virus, by using the Standardization of Uveitis Nomenclature (SUN) criteria, and to identify risk factors for the development of glaucoma.DESIGN: Retrospective observational cohort study.METHODS: Patients with herpetic AU presenting themselves between 2001 and 2013 at the ophthalmology department of the University Medical Center Groningen were included. Main outcome measures were the incidence of elevated KW and glaucoma and risk factors for the development of glaucoma.RESULTS: Seventy-three herpetic AU patients were included. Ocular complications most commonly seen during follow-up for uveitis were elevated IOP (75%), keratitis (59%), dry eyes (34%), posterior synechiae (34%), cataract (32%), and glaucoma (15%). Glaucoma patients, in comparison to non-glaucoma patients, had a higher number of IOP peaks during their follow-up for uveitis (P CONCLUSIONS: Using the SUN criteria, our study confirmed that elevated IOP and secondary glaucoma are major complications in herpetic AU. If an elevated IOP occurred, it was usually already present at the start of a uveitis episode. A risk factor for the development of glaucoma was the number of endured IOP peaks. Future studies are needed to evaluate whether early and prolonged use of antiviral and IOP-lowering medication may prevent glaucoma.

Background: HIV Gag mutations have been reported to confer PI drug resistance. However, clinical implications are still controversial and most current genotyping algorithms consider solely the protease gene for assessing PI resistance. Objectives: Our goal was to describe for HIV infections in Switzerland the potential role of the C-terminus of Gag (NC–p6) in PI resistance. We aimed to characterize resistance-relevant mutational patterns in Gag and protease and their possible interactions. Methods: Resistance information on plasma samples from 2004–12 was collected for patients treated by two diagnostic centres of the Swiss HIV Cohort Study. Sequence information on protease and the C-terminal Gag region was paired with the corresponding patient treatment history. The prevalence of Gag and protease mutations was analysed for PI treatment-experienced patients versus PI treatment-naive patients. In addition, we modelled multiple paths of an assumed ordered accumulation of genetic changes using random tree mixture models. Results: More than half of all PI treatment-experienced patients in our sample set carried HIV variants with at least one of the known Gag mutations, and 17.9% (66/369) carried at least one Gag mutation for which a phenotypic proof of PI resistance by in vitro mutagenesis has been reported. We were able to identify several novel Gag mutations that are associated with PI exposure and therapy failure. Conclusions: Our analysis confirmed the association of Gag mutations, well known and new, with PI exposure. This could have clinical implications, since the level of potential PI drug resistance might be underestimated.

Kaposi sarcoma-associated herpesvirus (KSHV) ORF57, also known as Mta (mRNA transcript accumulation), enhances viral intron-less transcript accumulation and promotes splicing of intron-containing viral RNA transcripts. In this study, we identified KSHV PAN, a long non-coding polyadenylated nuclear RNA as a main target of ORF57 by a genome-wide CLIP (cross-linking and immunoprecipitation) approach. KSHV genome lacking ORF57 expresses only a minimal amount of PAN. In cotransfection experiments, ORF57 alone increased PAN expression by 20-30-fold when compared to vector control. This accumulation function of ORF57 was dependent on a structured RNA element in the 5' PAN, named MRE (Mta responsive element), but not much so on an ENE (expression and nuclear retention element) in the 3' PAN previously reported by other studies. We showed that the major function of the 5' PAN MRE is increasing the RNA half-life of PAN in the presence of ORF57. Further mutational analyses revealed a core motif consisting of 9 nucleotides in the MRE-II , which is responsible for ORF57 interaction and function. The 9-nt core in the MRE-II also binds cellular PABPC1, but not the E1B-AP5 which binds another region of the MRE-II. In addition, we found that PAN RNA is partially exportable in the presence of ORF57. Together, our data provide compelling evidence as to how ORF57 functions to accumulate a non-coding viral RNA in the course of virus lytic infection.

A large panel of drug-resistant mutants of human immunodeficiency virus type 1 reverse transcriptase (RT) was used to study the mechanisms of resistance to 1-(β- d -dioxolane)thymine triphosphate (DOT-TP) and other nucleotide analogs. RT containing thymidine analog-associated mutations (TAM) or RT with a T69S-SG insertion in combination with TAM removed 3′-azido-3′-deoxythymidine-5′-monophosphate or tenofovir more efficiently than DOT-monophosphate from chain-terminated DNA primer/template through ATP-mediated pyrophosphorolysis. For non-ATP-dependent discrimination toward DOT-TP, high levels of resistance were found for RT bearing the Q151M mutation with family mutations, while RT bearing only the M184V or the Y115F mutation conferred no resistance to DOT-TP. A lower degree of resistance to DOT-TP than to tenofovir diphosphate or carbovir-TP was found for RT containing the K65R mutation. In the present studies, 1-(β- d -dioxolane)guanine triphosphate, another nucleotide with a dioxolane sugar moiety, showed a resistance profile similar to that of DOT-TP. The results suggest that DOT, compared with other approved nucleoside analogs, is overall more resilient to mutations such as TAM, M184V, and K65R, which are commonly found in viruses derived from subjects failing multinucleoside therapy.

Supplementary Data; International audience; An HIV-infected patient presenting an unexpected viral escape under combined antiretroviral treatment is described. The virus isolated from plasma contained a large deletion in the HIV-1 integrase gene but no known resistance mutation. Nested polymerase chain reactions (PCRs) with patient virus integrase-specific primers and probes were developed and used to detect the mutant from plasma, blood, rectal biopsies, and sperm. The variant progressively emerged during a period of therapy-induced virosuppression, and persisted at a low but detectable level for at least 5 years. Surprisingly, proviral DNA from lymphocytes, rectal cells, and sperm cells was, and remained, mainly wild type. Cellular HIV RNA with the deletion was detected only once from the rectum. The origin and mechanisms underlying this so far not described production at a detectable level are largely hypothetical. This observation raised concern about the ability of defective viruses to spread.

International audience; BACKGROUND:Human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia/lymphoma, shows intrapatient genetic variability. Although HTLV-1 can replicate via the reverse transcription of virion RNA to a double-stranded DNA provirus (the conventional manner for retroviruses), its predominant mode of replication is via the clonal expansion (mitosis) of the infected cell. This expansion is achieved by the viral oncoprotein Tax, which keeps the infected CD4 T lymphocyte cycling. Because Tax also interferes with cellular DNA repair pathways, we investigated whether somatic mutations of the provirus that occur during the division of infected cells could account for HTLV-1 genetic variability.METHODS:An inverse polymerase chain reaction strategy was designed to distinguish somatic mutations from reverse transcription-associated substitutions. This strategy allows the proviral sequences to be isolated together with flanking cellular sequences. Using this method, we sequenced 208 HTLV-1 provirus 3' segments, together with their integration sites, belonging to 29 distinct circulating cellular clones from infected individuals.RESULTS:For 60% of the clones, 8%-80% of infected cells harbored a mutated HTLV-1 provirus, without evidence of reverse transcription-associated mutations. Mutations within flanking cellular sequences were also identified at a frequency of 2.8 x 10(-4) substitution per base pair. Some of these clones carried multiple discrete substitutions or deletions, indicating progressive accumulation of mutations during clonal expansion. The overall frequency of somatic mutations increased with the degree of proliferation of infected T cells.CONCLUSIONS:These data indicate that, in vivo, HTLV-1 variation results mainly from postintegration events that consist of somatic mutations of the proviral sequence occurring during clonal expansion. The finding of substitutions in flanking sequences suggests that somatic mutations occurring after integration, presumably coupled with selection, help move the cellular clones toward a transformed phenotype, of which adult T-cell leukemia/lymphoma is the end point.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69 also interact with RBM15 and OTT3, EBV EB2, which also promotes ORF59 expression, does not function like KSHV ORF57 to efficiently prevent RBM15-mediated nuclear accumulation of ORF59 RNA and RBM15's association with polyadenylated RNAs. Collectively, our data provide novel insight elucidating a molecular mechanism by which ORF57 promotes the expression of viral intronless genes.

OBJECTIVES: Estimating the prevalence of accumulated HIV drug resistance in patients receiving antiretroviral therapy (ART) is difficult due to lack of resistance testing at all occasions of virological failure and in patients with undetectable viral load. A method to estimate this for 6498 EuroSIDA patients who were under follow-up on ART at 1 July 2008 was therefore developed by imputing data on patients with no prior resistance test results, based on the probability of detecting resistance in tested patients with similar profiles.METHODS: Using all resistance test results available, predicted intermediate/high-level resistance to specific drug classes [nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs)] was derived using the Stanford algorithm v5.1.2. Logistic regression models were then employed to estimate predicted probability of resistance to each drug class for given values of current viral load, history of virological failure and previous virological suppression. Based on these predicted probabilities and patients' covariate profiles, estimates of prevalence in 5355 patients with no prior test results were obtained. Overall prevalence of resistance was estimated by pooling these data with those observed in the remaining 1143 tested patients.RESULTS: Prevalence of NRTI, NNRTI and PI resistance was estimated as 43% (95% confidence interval: 39%-46%), 15% (13%-18%) and 25% (22%-28%), respectively.CONCLUSIONS: This method provides estimates for the proportion of treated patients in a cohort who harbour resistance on a given date, which are less likely to be affected by selection bias due to missing resistance data and will allow us to estimate prevalence of resistance to different drug classes at specific timepoints in HIV-infected populations on ART.

The fingers subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a hotspot for nucleoside analogue resistance mutations. Some multi-nucleoside analogue-resistant variants contain a T69S substitution along with dipeptide insertions between residues 69 and 70. This set of mutations usually co-exists with classic zidovudine-resistance mutations (e.g. M41L and T215Y) or an A62V mutation and confers resistance to multiple nucleoside analogue inhibitors. As insertions lie in the vicinity of the dNTP-binding pocket, their influence on RT fidelity was investigated. Commonly occurring insertion mutations were selected, i.e. T69S-AG, T69S-SG and T69S-SS alone, in combination with 3′-azido-2′,3′-deoxythymidine-resistance mutations M41L, L210W, R211K, L214F, T215Y (LAGAZand LSGAZ) or with an alternate set where A62V substitution replaces M41L (VAGAZ, VSGAZand VSSAZ). Using alacZαgapped duplex substrate, the forward mutation frequencies of recombinant wild-type and mutant RTs bearing each of the above sets of mutations were measured. All of the mutants displayed significant decreases in mutation frequencies. Whereas the dipeptide insertions alone showed the least decrease (4·0- to 7·5-fold), the VAG series showed an intermediate reduction (5·0- to 11·4-fold) and the LAG set showed the largest reduction in mutation frequencies (15·3- and 16·3-fold for LAGAZand LSGAZ, respectively). Single dNTP exclusion assays for mutants LSGAZand LAGAZconfirmed their large reduction in misincorporation efficiencies. The increasedin vitrofidelity was not due to excision of the incorrect nucleotide via ATP-dependent removal. There was also no direct correlation between increased fidelity and template–primer affinity, suggesting a change in the active site that is conducive to better discrimination during dNTP insertion.

As the replication pattern of leukemogenic PTLVs possesses a strong pathogenic impact, we investigated HTLV-2 replication in vivo in asymptomatic carriers belonging into 2 distinct populations infected by the same HTLV-2b subtype. They include epidemically infected American blood donors, in whom HTLV-2b has been present for only 30 years, and endemically infected Bakola Pygmies from Cameroon, characterized by a long viral endemicity (at least few generations). In blood donors, both the circulating proviral loads and the degree of infected cell proliferation were largely lower than those characterizing asymptomatic carriers infected with leukemogenic PTLVs (HTLV-1, STLV-1). This might contribute to explain the lack of known link between HTLV-2b infection and the development of malignancies in this population. In contrast, endemically infected individuals displayed high proviral loads resulting from the extensive proliferation of infected cells. The route and/or the duration of infection, viral genetic drift, host immune response, genetic background, co-infections or a combination thereof might have contributed to these differences between endemically and epidemically infected subjects. As the clonality pattern observed in endemically infected individuals is very reminiscent of that of leukemogenic PTLVs at the pre-leukemic stage, our results highlight the possible oncogenic effect of HTLV-2b infection in such population.

Although Enteroviruses are mainly described as responsible for acute diseases, their role in severe chronic pathology has been also established. Echovirus 6-like sequences have been detected by PCR analysis in central nervous system specimens from patients presenting with Amyotrophic Lateral Sclerosis. These findings suggested a persistent infection with viruses that underwent, genetic changes precluding viral particle release. To support this hypothesis, we developed a model system of Echovirus 6 chronic infection in precursors of glial cells. The nucleotide sequences of the 5'non-translated region (5'NTR), 2A and 3C regions of the virus developing persistent genome were analysed during establishment of the chronic phenotype. This study revealed that at day 160 of chronic infection, several mutations were observed: one mutation at nucleotide 108 upstream the domain II of the internal ribosome entry site (IRES) structure, one mutation at nucleotide 30 in the cloverleaf, and two mutations in the 2A region (translated in His48 to Tyr, and Ile 123 to Met). No mutations were detected in the 3C region. The impact of these mutations on viral replication have been analysed in a rabbit reticulocyte lysate (RRL) translation assay supplemented with HeLa cell lysate, and by plaque assay. Viruses with these mutations displayed a phenotype with a significant reduction of replication, while in vitro translation was not affected by the nucleotide 108 mutation. This model allowed the description of molecular changes observed in the genome of Echovirus 6 during the establishment of a chronic infection phenotype, and may be helpful for the understanding of the mechanisms leading Enteroviruses to develop chronic infections in man.

1742-464X (Print) Journal Article; The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coli beta-galactosidase conferred by the single-chain Fv (scFv) fragment 13R4 equipped with nuclear import/export signals. Here, by appending to scFvs the proteolytic PEST signal sequence (a protein region rich in proline, glutamic acid, serine and threonine) of mouse ornithine decarboxylase, we tested whether short-lived or destabilized intrabodies could affect the steady-state level of target by redirecting it to the proteasomes. In the absence of antigen, the half-life of the modified scFv 13R4, relative to untagged molecules, was considerably reduced in vivo. However, after coexpression with either cytoplasmic or nuclear antigen, the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with beta-galactosidase. Analysis of destabilized site-directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of beta-galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.

Herpes simplex virus (HSV) infections are efficiently treated with antiviral drugs such as acyclovir (ACV). However, resistance has been reported, mainly among immunocompromised patients (prevalence around 5%) and particularly allogeneic bone marrow transplant patients (prevalence reaching 30%). Resistance to ACV is associated with mutations on one of the two viral enzymes involved in the ACV mechanism of action: thymidine kinase (TK) and DNA polymerase. In 95% of the cases, ACV resistance is associated with a mutation in the TK gene as this enzyme is not essential for viral replication, unlike viral DNA polymerase, which is rarely involved in resistance. Strains resistant to ACV are almost always cross-resistant to other TK-dependent drugs such as penciclovir and famciclovir. Resistant infections can be managed by foscarnet or cidofovir but both are more toxic than ACV. These drugs also inhibit viral DNA polymerase but they are active on most ACV-resistant HSV as they do not depend on TK; nevertheless virus resistant to ACV because of a mutation in the DNA polymerase may be cross-resistant to these molecules. Published data on genetic characterization of resistant clinical isolates point out hot spots in viral TK and DNA polymerase genes. TK mutations associated with resistance are either insertion or deletion (codons 92 and 146 of TK gene) or substitution (codon 176-177, 336 of TK gene). DNA polymerase mutations are mainly located in conserved sites of the enzyme. A high level of gene polymorphism has also been reported for these genes, especially for TK. These results are useful for the development of rapid genotypic assays for the detection of mutations associated with resistance.

Human pathogenic retroviruses do not have common loci of integration. However, many factors, such as chromatin structure, transcriptional activity, DNA-protein interaction, CpG methylation, and nucleotide composition of the target sequence, may influence integration site selection. These features have been investigated by in vitro integration reactions or by infection of cell lines with recombinant retroviruses. Less is known about target choice for integration in vivo. The present study was conducted in order to assess the characteristics of cellular sequences targeted for human T-cell leukemia virus type 1 (HTLV-1) integration in vivo. Sequencing integration sites from ≥200 proviruses (19 kb of sequence) isolated from 29 infected individuals revealed that HTLV-1 integration is not random at the level of the nucleotide sequence. The virus was found to integrate in A/T-rich regions with a weak consensus sequence at positions within and without of the hexameric repeat generated during integration. These features were not associated with a preference for integration near active regions or repeat elements of the host chromosomes. Most or all of the regions of the genome appear to be accessible to HTLV-1 integration. As with integration in vitro, integration specificity in vivo seems to be determined by local features rather than by the accessibility of specific regions.