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International audience; Currently, turkey coronaviruses (TCoV) are isolated from homogenized intestines of experimentally infected embryos to ensure a maximum recovery of viral particles from all components of the intestines. However, the process of homogenization also ensures release of a significant amount of cellular RNAs into the sample that hinders downstream viral genome sequencing. This is especially the case for next generation sequencing (NGS) which sequences molecules at random. This characteristic means that the heavily abundant cellular RNA in the sample drowns out the minority viral RNA during the sequencing process and, consequently, very little to no viral genome data are obtained. To address this problem, a method was developed, in which 10 descendent isolates of the European strain of TCoV were recovered uniquely from the intestinal lumen without homogenization of the tissue. For nine out of 10 samples, NGS produced viral RNA reads with good coverage depth over the entire TCoV genomes. This is a much-needed new, simple and cost effective method of isolating TCoV that facilitates downstream NGS of viral RNA and should be considered as an alternative method for isolating other avian enteric coronaviruses in the interest of obtaining full-length genome sequences.

Throughout the COVID-19 pandemic nasopharyngeal or nose and/or throat swabs (NTS) have been the primary approach for collecting patient samples for the subsequent detection of viral RNA. However, this procedure, if undertaken correctly, can be unpleasant and therefore deters individuals from providing high quality samples. To overcome these limitations other modes of sample collection have been explored. In a cohort of frontline health care workers we have compared saliva and gargle samples to gold-standard NTS. 93% of individuals preferred providing saliva or gargle samples, with little sex-dependent variation. Viral titers collected in samples were analyzed using standard methods and showed that gargle and saliva were similarly comparable for identifying COVID-19 positive individuals compared to NTS (92% sensitivity; 98% specificity). We suggest that gargle and saliva collection are viable alternatives to NTS swabs and may encourage testing to provide better disease diagnosis and population surveillance.

International audience; Since the beginning of the pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) the gastrointestinal (GI) tract has emerged as an important organ influencing the propensity to and potentially the severity of the related COVID-19 disease. However, the contribution of the SARS-CoV-2 intestinal infection on COVID-19 pathogenesis remains to be clarified. In this exploratory study, we highlighted a possible link between alterations in the composition of the gut microbiota and the levels of SARS-CoV-2 RNA in the gastrointestinal tract, which could be more important than the presence of SARS-CoV-2 in the respiratory tract, COVID-19 severity and GI symptoms. As established by metaproteomics, altered molecular functions in the microbiota profiles of high SARS-CoV-2 RNA level faeces highlight mechanisms such as inflammation-induced enterocyte damage, increased intestinal permeability and activation of immune response that may contribute to vicious cycles. Uncovering the role of this gut microbiota dysbiosis could drive the investigation of alternative therapeutic strategies to favour the clearance of the virus and potentially mitigate the effect of the SARS-CoV-2 infection.

The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identify an LNA ASO binding to the 5′ leader sequence of SARS-CoV-2 that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the COVID-19 mouse model potently suppresses viral replication (>80-fold) in the lungs of infected mice. We find that the LNA ASO is efficacious in countering all SARS-CoV-2 “variants of concern” tested both in vitro and in vivo. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce or prevent transmission and decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences and could be stockpiled for future coronavirus pandemics.

Background Children with cancer are frequently immunocompromised. While children are generally thought to be at less risk of severe SARS-CoV-2 infection than adults, comprehensive population-based evidence for the risk in children with cancer is unavailable. We aimed to produce evidence of the incidence and outcomes from SARS-CoV-2 in children with cancer attending all hospitals treating this population across the UK. Methods Retrospective and prospective observational study of all children in the UK under 16 diagnosed with cancer through data collection from all hospitals providing cancer care to this population. Eligible patients tested positive for SARS-CoV-2 on reverse transcription polymerase chain reaction (RT-PCR). The primary end-point was death, discharge or end of active care for COVID-19 for those remaining in hospital. Results Between 12 March 2020 and 31 July 2020, 54 cases were identified: 15 (28%) were asymptomatic, 34 (63%) had mild infections and 5 (10%) moderate, severe or critical infections. No patients died and only three patients required intensive care support due to COVID-19. Estimated incidence of hospital identified SARS-CoV-2 infection in children with cancer under 16 was 3%. Conclusions Children with cancer with SARS-CoV-2 infection do not appear at increased risk of severe infection compared to the general paediatric population. This is reassuring and supports the continued delivery of standard treatment.

In this recommendation, we update our 2016 table of reference sequences of subtypes of hepatitis E virus (HEV; species Orthohepevirus A, family Hepeviridae) for which complete genome sequences are available (Smith et al., 2016). This takes into account subsequent publications describing novel viruses and additional proposals for subtype names; there are now eight genotypes and 36 subtypes. Although it remains difficult to define strict criteria for distinguishing between virus subtypes, and is not within the remit of the International Committee on Taxonomy of Viruses (ICTV), the use of agreed reference sequences will bring clarity and stability to researchers, epidemiologists and clinicians working with HEV.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people globally since its first detection in late 2019. Besides humans, cats and, to some extent, dogs were shown to be susceptible to SARS-CoV-2, highlighting the need for surveillance in a One Health context. Seven veterinary clinics from regions with high incidences of coronavirus disease (COVID-19) were recruited during the early pandemic (March to July 2020) for the screening of patients. A total of 2257 oropharyngeal and nasal swab specimen from 877 dogs and 260 cats (including 18 animals from COVID-19-affected households and 92 animals with signs of respiratory disease) were analyzed for the presence of SARS-CoV-2 RNA using reverse transcriptase real-time polymerase chain reaction (RT-qPCR) targeting the viral envelope (E) and RNA dependent RNA polymerase (RdRp) genes. One oropharyngeal swab from an Italian cat, living in a COVID-19-affected household in Piedmont, tested positive in RT-qPCR (1/260; 0.38%, 95% CI: 0.01–2.1%), and SARS-CoV-2 infection of the animal was serologically confirmed six months later. One oropharyngeal swab from a dog was potentially positive (1/877; 0.1%, 95% CI: 0.002–0.63%), but the result was not confirmed in a reference laboratory. Analyses of convenience sera from 118 animals identified one dog (1/94; 1.1%; 95% CI: 0.02–5.7%) from Lombardy, but no cats (0/24), as positive for anti-SARS-CoV-2 receptor binding domain (RBD) antibodies and neutralizing activity. These findings support the hypothesis that the prevalence of SARS-CoV-2 infection in pet cat and dog populations, and hence, the risk of zoonotic transmission to veterinary staff, was low during the first wave of the pandemic, even in hotspot areas.

CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing., The Class 2 family of CRISPR nucleases named Cas12j, which shares only low sequence identity with other CRISPR nucleases was recently identified in the biggiephage clade of phages. Here, the authors present the cryo-EM structure of a functional Cas12j3−crRNA complex in the post-catalytic state and discuss Cas12j3 PAM recognition, hybrid stabilisation and the activation mechanism.

Background Testing is crucial for COVID-19 response and management, however, WHO’s preparedness index omits estimations of actual testing capabilities, which influence the ability to contain, mitigate and clinically manage infectious diseases. With one of the highest excess death rates globally, Ecuador had a comparatively low number of confirmed COVID-19 cases, which may have been influenced by limited availability of data for decision-making due to low laboratory capacity. Methods We examine de-identified data on 55,063 individuals with suspected COVID-19 between February 27 and April 30, 2020 included in the RT-PCR testing database collected by the Ministry of Health. Processing times and rates per province, and the number of pending tests, were tallied cumulatively. We assessed the relationship between sample shipping, laboratory capacity and case completion using a negative binomial generalized linear model. Results The national average time for case completion was 3 days; 12.1% of samples took ≥10 days to complete; the national average daily backlog was 29.1 tests per 100,000 people. Only 8 out of 24 provinces had authorized COVID-19 processing laboratories but not all processed samples. There was an association between samples coming from outside the processing laboratory province, the number of other samples present at the laboratory during processing, and the amount of time needed to process a sample. Samples from another province took 1.29 times as long to process, on average. The percentage of pending results on April 30 was 67.1%. Conclusion A centralized RT-PCR testing system contributes to critical delays in processing, which may mask a case burden higher than reported, impeding timely awareness, and adequate clinical care and vaccination strategies and subsequent monitoring. Although Ecuador adapted or authorized existing facilities to address limitations in laboratory capacity for COVID-19, this study highlights the need to estimate and augment laboratory capabilities for improved decision making and policies on diagnostic guidelines and availability. Support is needed to procure the necessary human and physical resources at all phases of diagnostic testing, including transportation of samples and supplies, and information management. Strengthening emergency preparedness enables a clear understanding of COVID-19 disparities within and across the country.

The detection of antisense RNA is hampered by reverse transcription (RT) non-specific priming, due to the ability of RNA secondary structures to prime RT in the absence of specific primers. The detection of antisense RNA by conventional RT-PCR does not allow assessment of the polarity of the initial RNA template, causing the amplification of non-specific cDNAs. In this study we have developed a modified protocol for the detection of human immunodeficiency virus type 1 (HIV-1) antisense protein (ASP) RNA. Using this approach, we have identified ASP transcripts in CD4+ T cells isolated from five HIV-infected individuals, either untreated or under suppressive therapy. We show that ASP RNA can be detected in stimulated CD4+ T cells from both groups of patients, but not in unstimulated cells. We also show that in untreated patients, the patterns of expression of ASP and env are very similar, with the levels of ASP RNA being markedly lower than those of env. Treatment of cells from one viraemic patient with α-amanitin greatly reduces the rate of ASP RNA synthesis, suggesting that it is associated with RNA polymerase II, the central enzyme in the transcription of protein-coding genes. Our data represent the first nucleotide sequences obtained in patients for ASP, demonstrating that its transcription indeed occurs in those HIV-1 lineages in which the ASP open reading frame is present.

Infectious Bronchitis virus (IBV) continues to cause significant economic losses for the chicken industry despite the use of many live IBV vaccines around the world. Several authors have suggested that vaccine-induced partial protection may contribute to the emergence of new IBV strains. In order to study this hypothesis, three passages of a challenge IBV were made in SPF chickens sham inoculated or vaccinated at day of age using a live vaccine heterologous to the challenge virus. All birds that were challenged with vaccine heterologous virus were positive for viral RNA. NGS analysis of viral RNA in the unvaccinated group showed a rapid selection of seven genetic variants, finally modifying the consensus genome of the viral population. Among them, five were non-synonymous, modifying one position in NSP 8, one in NSP 13, and three in the Spike protein. In the vaccinated group, one genetic variant was selected over the three passages. This synonymous modification was absent from the unvaccinated group. Under these conditions, the genome population of an IBV challenge virus evolved rapidly in both heterologous vaccinated and non-vaccinated birds, while the genetic changes that were selected and the locations of these were very different between the two groups.

Shifting frames to make more proteins Severe acute respiratory syndrome coronavirus 2 critically depends on the ribosomal frameshifting that occurs between two large open reading frames in its genomic RNA for expression of viral replicase. Programmed frameshifting occurs during translation, when the ribosome encounters a stimulatory pseudoknot RNA fold. Using a combination of cryo–electron microscopy and biochemistry, Bhatt et al. revealed that the pseudoknot resists unfolding as it lodges at the entry of the ribosomal messenger RNA channel. This causes back slippage of the viral RNA, resulting in a minus-1 shift of the reading frame of translation. A partially folded nascent viral polyprotein forms specific interactions inside the ribosomal tunnel that can influence the efficiency of frameshifting. Science , abf3546, this issue p. 1306

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic levels. Sufficient testing for SARS-CoV-2 has remained essential for tracking and containing the virus. SARS-CoV-2 testing capabilities are still limited in many countries. Here, we explore the use of conventional RNA purification as an alternative to automated systems for detection of SARS-CoV-2 by RT-qPCR. 87 clinical swab specimens were extracted by conventional phenol-chloroform RNA purification and compared to commercial platforms for RNA extraction and the fully integrated Cobas®6800 diagnostic system. Our results show that the conventional RNA extraction is fully comparable to modern automated systems regarding analytical sensitivity and specificity with respect to detection of SARS-CoV-2 as evaluated by RT-qPCR. Moreover, the method is easily scalable and implemented in conventional laboratories as a low cost and suitable alternative to automated systems for the detection of SARS-CoV-2. The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic levels. Sufficient testing for SARS-CoV-2 has remained essential for tracking and containing the virus. SARS-CoV-2 testing capabilities are still limited in many countries. Here, we explore the use of conventional RNA purification as an alternative to automated systems for detection of SARS-CoV-2 by RT-qPCR. 87 clinical swab specimens were extracted by conventional phenol-chloroform RNA purification and compared to commercial platforms for RNA extraction and the fully integrated Cobas®6800 diagnostic system. Our results show that the conventional RNA extraction is fully comparable to modern automated systems regarding analytical sensitivity and specificity with respect to detection of SARS-CoV-2 as evaluated by RT-qPCR. Moreover, the method is easily scalable and implemented in conventional laboratories as a low cost and suitable alternative to automated systems for the detection of SARS-CoV-2.

Objectives: The prevalence of active, chronic, and former hepatitis E virus (HEV) infections was investigated in a cohort of immunocompromised patients. The association with transfusion transmitted HEV was evaluated, and the HEV seroprevalence was compared with that in healthy blood donors. Study design and methods: Serum samples from 4023 immunocompromised patients at Rigshospitalet, Denmark were retrospectively tested for HEV RNA and anti-HEV IgG. HEV RNA-positive patients were followed up by HEV testing, clinical symptoms, and transfusion history. Factors associated with anti-HEV were explored by multivariable logistic regression analysis. Samples from 1226 blood donors were retrospectively tested for anti-HEV IgG. Results: HEV RNA was detected in six patients (0.15%) with no indications of chronic HEV infection. HEV RNA prevalence rates among recipients of allogeneic haematopoietic stem cell transplantation (allo-HSCT) and solid organ transplantation (SOT) were 0.58% and 0.21%, respectively. Transfusion transmitted infections were refuted, and transfusion history was not associated with anti-HEV positivity. The difference in HEV seroprevalence between patients (22.0%) and blood donors (10.9%) decreased when adjusting for age and sex (odds ratio 1.20, 95% confidence interval 0.97–1.48). Conclusions: HEV viremia among allo-HSCT and SOT recipients suggests that clinicians should be aware of this diagnosis. The lack of association of blood transfusion with anti-HEV positivity supports food-borne transmission as the main transmission route of HEV common to both patients and blood donors. Keywords: Hepatitis E, Epidemiology, Immunocompromised, Transfusion transmission, HEV RNA, Anti-HEV IgG, Chronic HEV infection

International audience; In this recommendation, we update our 2016 table of reference sequences of subtypes of hepatitis E virus (HEV; species Orthohepevirus A, family Hepeviridae) for which complete genome sequences are available (Smith et al., 2016). This takes into account subsequent publications describing novel viruses and additional proposals for subtype names; there are now eight genotypes and 36 subtypes. Although it remains difficult to define strict criteria for distinguishing between virus subtypes, and is not within the remit of the International Committee on Taxonomy of Viruses (ICTV), the use of agreed reference sequences will bring clarity and stability to researchers, epidemiologists and clinicians working with HEV

International audience; Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. The systematic use of improved tools for diagnosing and genotyping has completely changed our understanding of the epidemiology and clinical consequences of HEV infection. Most cases of HEV in Europe arise from infected animals such as pigs, wild boar, deer and rabbits. Zoonotic HEV genotypes (HEV genotypes 3-8) are mainly food-borne or transmitted by direct contact, but recent data suggest that infection can also be water-borne or even iatrogenic throught contamined blood products. HEV-3 is the most prevalent genotype in Europe but the geographic distributions of the 3 major clades and subgenotypes (HEV-3abjkchi, HEV-3efg, and HEV-3ra) differ. Most HEV-3 infections are asymptomatic but they can result in severe acute hepatitis in patients with chronic liver disease, chronic hepatitis in immunocompromised patients, and to extra-hepatic manifestations. Despite more frequent reports of symptomatic hepatitis E cases across Europe, systems for monitoring HEV infections vary greatly. Severe HEV-associated illnesses, hospitalizations and deaths are probably underestimated. The seroprevalence and incidence of locally acquired hepatitis E varies between and within European countries and over time. The precise origin of these variations is uncertain but may be linked to environmental factors or the degree to which HEV contaminates the human food chain. Collaborative initiatives such as the establishment of the One Health platform for HEV sequences (HEVnet database) will be very useful for a better understanding of the epidemiology of HEV in Europe and the development of effective prevention strategies.

ObjectiveHepatitis E virus (HEV), one of the most common agent of acute hepatitis worldwide, is mainly transmitted enterically, via contaminated water for HEV genotypes 1 (HEV1) and HEV2, or by eating raw or undercooked infected meat for HEV genotype 3 (HEV3) and HEV4. However, little is known about how the ingested HEV reaches the liver or its ability to replicate in intestinal cells.DesignWe developed human primary cultures of small intestine epithelial cells and intestinal explants obtained from small bowel resections. The epithelial cells were also polarised on transwells. Cells were infected with Kernow-p6 strain or clinically derived virions.ResultsPrimary intestinal cells supported the growth of Kernow-p6 strain and HEV1 and HEV3 clinically derived virions. Polarised enterocytes infected with HEV1 and HEV3 strains released HEV particles vectorially: mostly into the apical compartment with a little basally. Iodixanol density gradient centrifugation of enterocyte-derived HEV virions gave bands at a density of 1.06–1.08 g/cm3, corresponding to that of quasi-enveloped HEV particles. Ribavirin therapy inhibited HEV excretion from the basal surface but not from the apical side of infected human enterocytes. HEV virions also infected intestinal tissue explants. Lastly, HEV RNA and antigen were detected in the intestinal crypts of a chronically infected patient.ConclusionHEV can replicate in intestinal cells and reaches the liver as quasi-enveloped virions.

Contains fulltext : 208598.pdf (Publisher’s version ) (Closed access) This article reports the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in February 2019. Of note, in addition to seven new virus families, the ICTV has approved, by an absolute majority, the creation of the realm Riboviria, a likely monophyletic group encompassing all viruses with positive-strand, negative-strand and double-strand genomic RNA that use cognate RNA-directed RNA polymerases for replication.

Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.

International audience; While Tunisia is endemic for hepatitis B virus (HBV), a recent large-scale retrospective study, revealed a very low prevalence (2%) of hepatitis Delta virus (HDV) (Yacoubi et al. in J Clin Virol 72:126-132, 2015). All strains were classified within the genotype 1 (HDV-1) as assessed by nucleotide sequencing of the so-called 'R0' region of the genome described previously. In this study, we aimed to determine the full-length genome sequence of HDV isolates in order to fully characterize the HDV strains spreading in Tunisia. Eleven HDV antibody and RNA positive samples were obtained from the 1615 clinical samples previously studied. The whole genome sequence was obtained for 5 strains by sequencing and realignment of four overlapping regions covering the entire genome, followed by extensive phylogenetic analyses. Tunisian sequences segregated together with Turkish and African sequences and showed 60% GC content. Alignment with an HDV-1 consensus sequence revealed that they exhibited several point mutations in different functional domains of the delta proteins that, according to previous studies, might possibly affect their properties. In conclusion, the first full-length genome sequences of Tunisian HDV isolates are provided, isolates which are closely related to Turkish and Sub-Saharan Africa strains, supporting the hypothesis for the spread of HDV-1-strains from Africa via Tunisia to Turkey, before spread to the rest of the world.

Single-cell analyses allow uncovering cellular heterogeneity, not only per se, but also in response to viral infection. Similarly, single cell transcriptome analyses (scRNA-Seq) can highlight specific signatures, identifying cell subsets with particular phenotypes, which are relevant in the understanding of virus-host interactions.

International audience; Background: Clinical studies support biologically independent roles of cell-free HIV particles and HIV-infected cells in disease progression. The associations between the level of infected cells and immune markers have been poorly studied, particularly in perinatally infected children. Objective: We tested the hypothesis that independent roles of cell-free virus and infected cells in HIV pathogenesis should be revealed by different associations between each of them and specific immune markers. Methods: Levels of HIV RNA and DNA, HIV-specific CD8 T lymphocytes, activated and naïve/memory T lymphocytes were determined in 44 untreated HIV-1-infected children. Pearson’s partial correlation coefficients were used to assess associations between the variables. Results: Here we provide new information, by showing a direct correlation between the percentages of CD4+HLA-DR+ lymphocytes and HIV DNA levels. Furthermore, higher HIV-specific CD8 T lymphocyte frequencies were associated with lower HIV DNA levels. In contrast, CD8+38+ lymphocytes and memory CD4 lymphocytes were correlated only to the HIV RNA level. All correlations were independent of age and CD4 depletion.Conclusion: Several immune markers were correlated to either the HIV RNA or the HIV DNA level, but never to both of them, supporting the concept that cell-free virus and infected cells play different roles in HIV-1 immunopathogenesis.

International audience; Introduction: A lack of access to hepatitis C virus (HCV) diagnostics is a significant barrier to achieving the World Health Organization 2030 global elimination goal. HCV core antigen (HCVcAg) quantification and dried blood spot (DBS) are appealing alternatives to conventional HCV serology and nucleic acid testing (NAT) for resource-constraint settings, particularly in difficult-to-reach populations. We assessed the accuracy of serum and DBS HCVcAg testing in people who inject drugs in Tanzania using HCV NAT as a reference.Method: Between May and July 2015, consecutive HCV-seropositive patients enrolled in the local opioid substitution treatment centre were invited to participate in the study. All had HCV RNA detection (Roche Molecular Systems, Pleasanton, CA, USA), genotyping (NS5B gene phylogenetic analysis) and HCVcAg on blood samples and DBS (Architect assay; Abbott Diagnostics, Chicago, IL, USA).Results: Out of 153 HCV-seropositive individuals, 65 (42.5%) and 15 (9.8%) were co-infected with HIV (41 (63%) were on anti-retroviral therapy (ARVs)) and hepatitis B respectively. In total, 116 were viraemic, median viral load of 5.7 (Interquartile range (IQR); 4.0-6.3) log iU/ml (75 (68.2%) were genotype 1a, 35 (31.8%) genotype 4a). The median alanine transaminase (ALT) (iU/l), aspartate transaminase (AST) (iU/l) and gamma-glutamyl transferase (GGT) (iU/l) were 35 (IQR; 23-51), 46 (32-57) and 69 (35-151) respectively. For the quantification of HCV RNA, serum HCVcAg had a sensitivity at 99.1% and a specificity at 94.1%, with an area under the receiver operating curve (AUROC) at 0.99 (95% CI 0.98-1.00). DBS HCVcAg had a sensitivity of 76.1% and a specificity of 97.3%, with an AUROC of 0.87 (95% CI 0.83-0.92). HCVcAg performance did not differ by HIV co-infection or HCV genotype. Conclusions Our study suggests that HCVcAg testing in serum is an excellent alternative to HCV polymerase chain reaction in Africa. Although HCVcAg detection and quantification in DBS has a reduced sensitivity, its specificity and accuracy are good and it could therefore be used for scaling up HCV testing and care in resource-limited African settings.

Endogenous viral elements derived from non-retroviral RNA viruses were described in various animal genomes. Whether they have a biological function such as host immune protection against related viruses is a field of intense study. Here, we investigated the repertoire of endogenous flaviviral elements (EFVEs) inAedesmosquitoes, the vectors of arboviruses such as dengue and chikungunya viruses. Previous studies identified three EFVEs fromAe. albopictusand one fromAe. aegypticell lines. However, in-depth characterization of EFVEs in wild-type mosquito populations and individualsin vivohas not been performed. We detected the full-length DNA sequence of the previously described EFVEs and their respective transcripts in severalAe. albopictusandAe. aegyptipopulations from geographically distinct areas. However, EFVE-derived proteins were not detected by mass spectrometry. Using deep sequencing, we detected the production of piRNA-like small RNAs in antisense orientation, targeting the EFVEs and their flanking regionsin vivo. The EFVEs were integrated in repetitive regions of the mosquito genomes, and their flanking sequences varied among mosquito populations from different geographical regions. We bioinformatically predicted several new EFVEs from a VietnameseAe. albopictuspopulation and observed variation in the occurrence of those elements among mosquito populations. Phylogenetic analysis of anAe. aegyptiEFVE suggested that it integrated prior to the global expansion of the species and subsequently diverged among and within populations. Together, this study revealed substantial structural and nucleotide diversity of flaviviral integrations inAedesgenomes. Unraveling this diversity will help to elucidate the potential biological function of these EFVEs.ImportanceEndogenous viral elements (EVEs) are whole or partial viral sequences integrated in host genomes. Interestingly, some EVEs have important functions for host fitness and antiviral defense. Because mosquitoes also have EVEs in their genomes, we decided to thoroughly characterized them to lay the foundation of the potential use of these EVEs to manipulate the mosquito antiviral response. Here, we focused on EVEs related to theFlavivirusgenus, to which dengue and Zika viruses belong, inAedesmosquito individuals from geographically distinct areas. We showed the existencein vivoof flaviviral EVEs previously identified in mosquito cell lines and we detected new ones. We showed that EVEs have evolved differently in each mosquito population. They produced transcripts and small RNAs, but not proteins, suggesting a function at the RNA level. Our study uncovers the diverse repertoire of flaviviral EVEs inAedesmosquito populations and suggests a role in the host antiviral system.

PURPOSE: To describe the clinical presentation, diagnosis and treatment of periorbital extranodal natural killer/T-cell lymphoma, nasal type. METHODS: Case series of three patients with periorbital involvement of extranodal natural killer/T-cell lymphoma, nasal type, of whom clinical data, orbital imaging and immunohistochemical analysis were collected. For the purpose of this study, all histopathological and immunohistochemical slides were re-examined. RESULTS: All patients presented with painless eyelid swelling and a history of sinonasal disease, of whom one with bilateral panuveitis, not responding to systemic antibiotics. Extraocular muscle involvement was present in 2 cases upon presentation and in 1 case later on. Initial paranasal and orbital biopsies were negative in 2 patients, with only the second orbital biopsy leading to the diagnosis. Natural killer/T-cell and cytotoxic markers were present in all cases, as well as Epstein-Barr virus encoded RNA in situ hybridization. The patients died respectively 5, 9 and 35 months from diagnosis despite treatment with chemotherapy and radiotherapy. CONCLUSION: Extranodal natural killer/T-cell lymphoma, nasal type, should be suspected in a painless periorbital cellulitis with chronic sinusitis, not responding to conventional therapy. A high index of suspicion is necessary in biopsies showing angiodestruction and necrosis. Epstein-Barr virus encoded RNA in situ hybridization and expert hematopathologist consultation is necessary to decrease the delay in diagnosis.

HIV-1 replication is rapid and highly error-prone. Transmission of a drug-resistant HIV-1 strain is possible and occurs within the HIV-1-infected population. In this study, we aimed to determine the prevalence of transmitted drug resistance mutations (TDRMs) in 1,306 newly diagnosed untreated HIV-1-infected patients from 21 cities across six regions of Turkey between 2010 and 2015. TDRMs were identified according to the criteria provided by the World Health Organization's 2009 list of surveillance drug resistance mutations. The HIV-1 TDRM prevalence was 10.1% (133/1,306) in Turkey. Primary drug resistance mutations (K65R, M184V) and thymidine analogue-associated mutations (TAMs) were evaluated together as nucleos(t)ide reverse transcriptase inhibitor (NRTI) mutations. NRTI TDRMs were found in 8.1% (107/1,306) of patients. However, TAMs were divided into three categories and M41L, L210W, and T215Y mutations were found for TAM1 in 97 (7.4%) patients, D67N, K70R, K219E/Q/N/R, T215F, and T215C/D/S mutations were detected for TAM2 in 52 (3.9%) patients, and M41L + K219N and M41L + T215C/D/S mutations were detected for the TAM1 + TAM2 profile in 22 (1.7%) patients, respectively. Nonnucleoside reverse transcriptase inhibitor-associated TDRMs were detected in 3.3% (44/1,306) of patients (L100I, K101E/P, K103N/S, V179F, Y188H/L/M, Y181I/C, and G190A/E/S) and TDRMs to protease inhibitors were detected in 2.3% (30/1,306) of patients (M46L, I50V, I54V, Q58E, L76V, V82A/C/L/T, N83D, I84V, and L90M). In conclusion, long-term and large-scale monitoring of regional levels of HIV-1 TDRMs informs treatment guidelines and provides feedback on the success of HIV-1 prevention and treatment efforts. © Copyright 2016, Mary Ann Liebert, Inc. 2016.

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.

Migratory water birds are a natural reservoir for influenza A viruses. Viruses replicate in the intestines of ducks and are shed with the fecal materials. Virus isolation from collected fecal materials, therefore, is an integral part of the surveillance of avian influenza in water birds. In the present study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was assessed for its usefulness in detecting the RNA of influenza A viruses in fecal materials. It was found that, RT-LAMP specifically and sensitively detects the matrix gene of influenza A viruses. Influenza A viruses were isolated from the fecal materials in which viral RNA were detected by RT-LAMP in 35 min. The present findings indicate that RT-LAMP is useful as a high throughput screening method for field samples prior to virus isolation, allowing the processing of hundreds of samples per day.

In this study, the immunity level of the southern Greek population in the 1-10-year, 11-20-year, 21-30-year and 31-40-year age groups with regard to Sabin vaccine strains and a collection of 11 recombinant and three non-recombinant poliovirus vaccine strains was determined. The results showed the lowest neutralization titre in the 21-30-year-age group against poliovirus type 3. Moreover, the capsid coding region of OPV (oral poliovirus vaccine) derivatives was sequenced in order to identify mutations that might lead to antigenic changes. In Sabin-1 derivatives a tendency of accumulation of mutations was observed in or near antigenic sites while in Sabin-2 and Sabin-3 derivatives in sites known to be involved in restoring neurovirulence or eliminating their temperature-sensitive phenotype. It was concluded that the combination of mutations in the capsid coding region and not the number of specific mutations in antigenic sites determines the antigenic properties of OPV derivatives and their reactivity with human sera. Vaccine

Rotavirus is the leading cause of acute gastroenteritis among young children worldwide. A prospective multi-center study was conducted (2007-2008) in five Pediatric Hospitals to determine the prevalence, the clinical characteristics, and genotype distribution of rotavirus infection in Greece. Faecal samples were examined for the presence of group A rotavirus antigen by immunochromatography. Rotavirus strains were subjected to G and P genotyping by reverse-transcriptase polymerase chain reaction (PCR) and sequencing. A total of 393 children (216 boys) of median age 23 months, participated in the study. Rotavirus was the cause of acute gastroenteritis in 166 children, 42.3% (CI 95%, 37.4-47.1%) of non-hospitalized and 47.8% (CI 95%, 41.7-53.9%) of hospitalized patients. Rotavirus gastroenteritis occurred between December and April in 78.6% of the cases. Most children with RVG (77.8%) were between 3 months and 3 years old. The mean value of Clark severity score was 12.9 +/- 5.1 for RVG and 10.5 +/- 4.9 for non-RVG (P < 0.01). Genotypes were determined in 117 strains and their distribution was as following: G1P[8], 49%; G2P[4], 31%; G4P[8], 10%; G9P[8], 9%; and G8P[14], 1%. In conclusion, rotavirus is a frequent cause of acute gastroenteritis in Greece. The genotypes circulating are similar with those of other European countries. J Med Virol

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37 degrees C and 40 degrees C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37 degrees C and 40 degrees C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives. Eur J Clin Microbiol Infect Dis

Several novel astroviruses have been recently discovered in humans and in other animals. Here, we report results from our surveillance of astroviruses in human and rodent faecal samples in Hong Kong. Classical human astroviruses (n=9) and a human MLB1 astrovirus were detected in human faecal samples (n=622). Novel astroviruses were detected from 1.6% of the faecal samples of urban brown rat (Rattus norvegicus) (n=441), indicating the prevalence of astrovirus infection in rats might be much lower than that recently observed in bats. These rat astroviruses were phylogenetically related to recently discovered human astroviruses MLB1 and MLB2, suggesting that the MLB viruses and these novel rat astroviruses may share a common ancestor. © 2010 SGM., published_or_final_version

In the post-eradication era of wild polioviruses, the only remaining sources of poliovirus infection worldwide would be the vaccine-derived polioviruses (VDPVs). As the preponderance of countries certified to be polio-free has switched from OPV (oral poliovirus vaccine) to IPV (inactivated poliovirus vaccine), importation of recombinant evolved derivatives of vaccinal strains would have serious implication for public health. To test the robustness of the proposed RT-PCR screening analysis, eleven recombinant vaccine-derived polioviruses that were characterized previously by sequencing by our group, in addition to three recently identified recombinant environmental isolates were assayed. Although the most definitive characterization of VDPVs is by genomic sequencing, in this study we describe a new, inexpensive and broadly applicable RT-PCR assay for the identification of the predominant recombination types S3/Sx in 2C and S2/Sx in 3D genomic regions respectively of VDPVs, that can be readily implemented in laboratories lacking sequencing facilities as a first approach for the early detection of vaccine-derived poliovirus (VDPVs). Mol Cell Probes

Two enteroviruses from river water and four from sewage treatment plant were isolated in Larissa, Greece, that all shared the same sequence. A full genome analysis was conducted in an attempt to reveal the evolutionary pathways of one of the isolated strains (LR11F7). VP1 nucleotide and phylogenetic analysis revealed that the isolated strain had 78% homology with the echovirus 7 prototype strain Wallace. Full genome analysis revealed that LR11F7 P1 region is related to echoviruses 7 and that P2 and P3 regions are originating from contemporary enteroviruses isolated in South Asia. Two recombination events were shown to be involved into the evolutionary history of LR11F7, the one event concerning 3A, 3B, and 2C, and the other concerning 3D genomic region, both with new types of HEV-B. The contribution of recombination to enterovirus evolution is substantial, giving rise to new genetic lineages with unknown properties. Virus Genes

Human rhinoviruses (HRV), and to a lesser extent human enteroviruses (HEV), are important respiratory pathogens. Like other RNA viruses, these picornaviruses have an intrinsic propensity to variability. This results in a large number of different serotypes as well as the incessant discovery of new genotypes. This large and growing diversity not only complicates the design of real-time PCR assays but also renders immunofluorescence unfeasible for broad HRV and HEV detection or quantification in cells. In this study, we used the 5′ untranslated region, the most conserved part of the genome, as a target for the development of both a real-time PCR assay (Panenterhino/Ge/08) and a peptide nucleic acid-based hybridization oligoprobe (Panenterhino/Ge/08 PNA probe) designed to detect all HRV and HEV species members according to publicly available sequences. The reverse transcription-PCR assay has been validated, using not only plasmid and viral stocks but also quantified RNA transcripts and around 1,000 clinical specimens. These new generic detection PCR assays overcame the variability of circulating strains and lowered the risk of missing emerging and divergent HRV and HEV. An additional real-time PCR assay (Entero/Ge/08) was also designed specifically to provide sensitive and targeted detection of HEV in cerebrospinal fluid. In addition to the generic probe, we developed specific probes for the detection of HRV-A and HRV-B in cells. This investigation provides a comprehensive toolbox for accurate molecular identification of the different HEV and HRV circulating in humans.

Background. Defective hepatitis B virus (HBV) particles, generated from singly spliced HBV RNA, have been detected in chronic carriers of HBV. The present study was designed to quantify the expression of defective HBV (dHBV) and wild-type HBV (wtHBV) genomes in the serum of patients with HBV infection and its relation to the severity of liver disease. Methods. HBV and dHBV loads were determined by quantitative polymerase chain reaction in the serum of 89 untreatedHBV-infectedpatients(31coinfectedwithhumanimmunodeficiencyvirus[HIV]type1)withliverdisease of different stages. The ratio of dHBV DNA to total (wtHBV plus dHBV) HBV DNA (dHBV/HBV ratio) was used to express data independently of the level of viral replication. Results. DespiteaglobalcorrelationbetweendHBVandwtHBVload,thedHBV/HBVratiorangedfrom0.001% to 69%. The variation in dHBV/HBV ratio was independent of HIV coinfection, HBV genotype, and precore mutations. The mean dHBV/HBV ratio was higher in patients with severe liver necrosis and fibrosis. Conclusions. OurdataindicatethatanelevateddHBV/HBVratioisassociatedwithlivernecroinflammationand fibrosis disease, suggesting a regulation of dHBV expression according to the severity of the liver disease. The dHBV/ HBV ratio may help to better define liver disease stage during HBV infection. Persistent human hepatitis B virus (HBV) infection is a major public health problem. The major complications of chronic HBV infection are the development of liver cirrhosis and hepatocellular carcinoma (HCC) [1‐3]. Evidence indicating a direct involvement of HBV in this process is now available [3]; indeed, recent data have demonstrated that serum HBV DNA levels are strong predictorsoftheriskofHCC,independentofhepatitisB e antigen (HBeAg) expression, serum alanine amino

Clostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites., IMPORTANCE Clostridium difficile is the major cause of nosocomial infections associated with antibiotic therapy worldwide. To survive in bacteriophage-rich gut communities, enteropathogens must develop efficient systems for defense against foreign DNA elements. CRISPR-Cas systems have recently taken center stage among various anti-invader bacterial defense systems. We provide experimental evidence for the function of the C. difficile CRISPR system against plasmid DNA and bacteriophages. These data demonstrate the original features of active C. difficile CRISPR system and bring important insights into the interactions of this major enteropathogen with foreign DNA invaders during its infection cycle.

Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. In concert with other nonstructural proteins, it induces a specific membrane rearrangement, designated as membranous web, which serves as a scaffold for the HCV replicase. The N-terminal part of NS4B comprises a predicted and a structurally resolved amphipathic α-helix, designated as AH1 and AH2, respectively. Here, we report a detailed structure-function analysis of NS4B AH1. Circular dichroism and nuclear magnetic resonance structural analyses revealed that AH1 folds into an amphipathic α-helix extending from NS4B amino acid 4 to 32, with positively charged residues flanking the helix. These residues are conserved among hepaciviruses. Mutagenesis and selection of pseudorevertants revealed an important role of these residues in RNA replication by affecting the biogenesis of double-membrane vesicles making up the membranous web. Moreover, alanine substitution of conserved acidic residues on the hydrophilic side of the helix reduced infectivity without significantly affecting RNA replication, indicating that AH1 is also involved in virus production. Selective membrane permeabilization and immunofluorescence microscopy analyses of a functional replicon harboring an epitope tag between NS4B AH1 and AH2 revealed a dual membrane topology of the N-terminal part of NS4B during HCV RNA replication. Luminal translocation was unaffected by the mutations introduced into AH1, but was abrogated by mutations introduced into AH2. In conclusion, our study reports the three-dimensional structure of AH1 from HCV NS4B, and highlights the importance of positively charged amino acid residues flanking this amphipathic α-helix in membranous web formation and RNA replication. In addition, we demonstrate that AH1 possesses a dual role in RNA replication and virus production, potentially governed by different topologies of the N-terminal part of NS4B., Author Summary With an estimated 180 million chronically infected individuals, hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. HCV is a positive-strand RNA virus that builds its replication complex on rearranged intracellular membranes, designated as membranous web. HCV nonstructural protein 4B (NS4B) is a key organizer of HCV membranous web and replication complex formation. Here, we provide a detailed structure-function analysis of an N-terminal amphipathic α-helix of NS4B, named AH1, and demonstrate that it plays key roles in shaping the membranous web as well as in virus production. We also show that the N-terminal part of NS4B adopts a dual membrane topology in a replicative context, possibly reflecting the different roles of this protein in the viral life cycle.

Avian H9N2 influenza A virus has caused repeated human infections in Asia since 1998. Here we report that an H9N2 influenza virus infected a 5-year-old child in Hong Kong in 2003. To identify the possible source of the infection, the human isolate and other H9N2 influenza viruses isolated from Hong Kong poultry markets from January to October 2003 were genetically and antigenically characterized. The findings of this study show that the human H9N2 influenza virus, A/Hong Kong/2108/03, is of purely avian origin and is closely related to some viruses circulating in poultry in the markets of Hong Kong. The continued presence of H9N2 influenza viruses in poultry markets in southern China increases the likelihood of avian-to-human interspecies transmission. Copyright © 2005, American Society for Microbiology. All Rights Reserved., published_or_final_version

Background: The Epstein–Barr virus (EBV) encodes two anti-apoptotic cellular Bcl2 homologs, BALF1 and BHRF1. BHRF1 has an antiapoptotic activity but is rarely expressed in nasopharyngeal carcinoma (NPC). However, BALF1 is not yet well characterized. Objectives: The objective of the study was to characterize BALF1gene. First, the search of its transcriptional expression in EBV-positive B cell lines, EBV-positive Burkitt’s lymphoma’s cell lines and nasopharyngeal carcinoma’s biopsies. Second, the examination of its anti-apoptotic activity in serum dependent assays. Study design: We first analysed the transcriptional expression of BALF1 by reverse transcriptase DNA polymerase chain reaction (RT-PCR) method. For the analysis of its anti-apoptotic activity, we transfected NIH3T3 cells with pBABE-BALF1 expression plasmid and studied serum dependence of these transfectants. Results: BALF1expression was detected in the latent stage and increased more significantly during the lytic phase in IgG-treated AKATA and TPA-SB-treated P3HR1-TK negative cell lines. As its expression was not affected by the inhibitor of viral DNA synthesis, this gene does not belong to late gene family. When analysed its transcription in Burkitt’s lymphoma (BL)-derived cell lines and NPC biopsies, all BL-derived cell lines and more than 80% of NPC biopsies transcribed this gene. The study of serum dependence of BALF1-transfected NIH3T3 cells showed: with 10% of serum, BALF1 transfectants grew significantly more higher cell density than vector alone transfected NIH3T3 cell lines and with 1% of serum, BALF1 transfectants were capable of growing, but with about 40% reduced rate in comparison with those with 10% serum, while vector alone transfected NIH3T3 cells could not almost grow. Conclusion: BALF1gene was transcribed in EBV-associated tumor cells. BALF1 could render cells to serum independent. These results suggest that BALF1 gene could play its role in EBV oncogenesis. © 2005 Elsevier B.V. All rights reserved.

Classical swine fever virus (CSFV) is a member of the pestivirus family, which shares many features in common with hepatitis C virus (HCV). It is shown here that CSFV has an exceptionally efficient cis-acting internal ribosome entry segment (IRES), which, like that of HCV, is strongly influenced by the sequences immediately downstream of the initiation codon, and is optimal with viral coding sequences in this position. Constructs that retained 17 or more codons of viral coding sequence exhibited full IRES activity, but with only 12 codons, activity was ∼66% of maximum in vitro (though close to maximum in transfected BHK cells), whereas with just 3 codons or fewer, the activity was only ∼15% of maximum. The minimal coding region elements required for high activity were exchanged between HCV and CSFV. Although maximum activity was observed in each case with the homologous combination of coding region and 5′ UTR, the heterologous combinations were sufficiently active to rule out a highly specific functional interplay between the 5′ UTR and coding sequences. On the other hand, inversion of the coding sequences resulted in low IRES activity, particularly with the HCV coding sequences. RNA structure probing showed that the efficiency of internal initiation of these chimeric constructs correlated most closely with the degree of single-strandedness of the region around and immediately downstream of the initiation codon. The low activity IRESs could not be rescued by addition of supplementary eIF4A (the initiation factor with ATP-dependent RNA helicase activity). The extreme sensitivity to secondary structure around the initiation codon is likely to be due to the fact that the eIF4F complex (which has eIF4A as one of its subunits) is not required for and does not participate in initiation on these IRESs.

The first 11 nt at the 5' end of influenza virus genomic RNA were shown to be both necessary and sufficient for specific binding by the influenza virus polymerase. A novel in vitro transcription assay, in which the polymerase was bound to paramagnetic beads via a biotinylated 5'-vRNA oligonucleotide, was used to study the activities of different forms of the polymerase. Complexes composed of co-expressed PB1/PB2/PA proteins and a sub-complex composed of PB1/PA bound to the 5'-vRNA oligonucleotide, whereas PB1 expressed alone did not. The enriched 5'-vRNA/PB1/PB2/PA complex was highly active for ApG and globin mRNA primed transcription on a model 3'-vRNA template. RNA synthesis in the absence of added primers produced products with 5'-terminal tri- or diphosphate groups, indicating that genuine unprimed initiation of transcription also occurred. No transcriptase activity was detected for the PB1/PA complex. These results demonstrate a role for PA in the enhancement of 5' end binding activity of PB1, a role for PB2 in the assembly of a polymerase complex able to perform both cap-dependent and -independent synthesis and that NP is not required for the initiation of replicative transcription.

International audience; Like most arthropod-borne viruses (arboviruses), chikungunya virus (CHIKV) is a RNA virus maintained in nature in an alternating cycle of replication between invertebrate and vertebrate hosts. It has been assumed that host alternation restricts arbovirus genome evolution and imposes fitness trade-offs. Despite their slower rates of evolution, arboviruses still have the capacity to produce variants capable to exploit new environments. To test whether the evolution of the newly emerged epidemic variant of CHIKV (E1-226V) is constrained by host alternation, the virus was alternately-passaged in hamster-derived BHK-21 cells and Aedes aegypti-derived Aag-2 cells. It was also serially-passaged in BHK-21 or Aag-2 cells to promote adaptation to one cell type and presumably, fitness cost in the bypassed cell type. After 30 passages, obtained CHIKV strains were genetically and phenotypically characterized using in vitro and in vivo systems. Serially- and alternately-passaged strains can be distinguished by amino-acid substitutions in the E2 glycoprotein, responsible for receptor binding. Two substitutions at positions E2-64 and E2-208 only lower the dissemination of the variant E1-226V in Ae. aegypti. These amino-acid changes in the E2 glycoprotein might affect viral infectivity by altering the interaction between CHIKV E1-226V and the cellular receptor on the midgut epithelial cells in Ae. aegypti but not in Aedesalbopictus.

International audience; A molecular screening of wild-caught rodents was conducted in French Guiana, South America to identify hosts of the hantavirus Maripa described in 2008 in a hantavirus pulmonary syndrome (HPS) case. Over a 9-year period, 418 echimyids and murids were captured. Viral RNA was detected in two sigmodontine rodents, Oligoryzomys fulvescens and Zygodontomys brevicauda, trapped close to the house of a second HPS case that occurred in 2009 and an O. fulvescens close to the fourth HPS case identified in 2013. Sequences from the rodents had 96% and 97% nucleotide identity (fragment of S and M segments, respectively) with the sequence of the first human HPS case. Phylogenetic reconstruc-tions based on the complete sequence of the S segment show that Maripa virus is closely related to Rio Mamore hantavirus. Using environmental descriptors of trapping sites, including vegetation, landscape units, rain, and human disturbance, a maximal entropy-based species distribution model allowed for identification of areas of higher predicted occurrence of the two rodents, where emergence risks of Maripa virus are expected to be higher. Hantaviruses (Bunyaviridae) are distributed worldwide.

Objectives The genotypic analysis of human metapneumo-(HMPV) and boca-(HBoV) viruses circulating in Greece and their comparison to reference and other clinical strains. Design Genetic analysis of representative strains over three consecutive winter seasons of the years 2005–2008. Setting Representative positive specimens for HMPV and HBoV from paediatric patients of healthcare units and hospitals in Southern Greece with influenza-like illness or other respiratory tract infections. Sample Seven to ten positive specimens for either HMPV or HBoV from each winter period. In total, 24 specimens positive for HMPV and 26 for HBoV, respectively. Main outcome measures Sequence diversity of HMPV and HBoV strains by sequencing the complete G and VP1/VP2 genes, respectively. Results In total, 24 HMPV strains were found to have a 92–100% nucleotide and a 85.9–100% amino acid identity. Phylogenetic analysis based on the number of amino acid differences, revealed circulation of 4 different subclusters belonging to genetic lineage B2. Similarly, analysis of 26 HBoV strains indicated that 22 clustered within genotype St2, 2 into genotype St1 and the remaining 2 formed a third cluster derived from potential recombination between different St1 genotype strains. St2 HBoV genotype was observed throughout the whole observation period whereas St1 only during the second and the third winter period. Higher levels of heterogeneity were observed between HMPV compared to HBoV strains. Conclusions Phylogenetic analysis revealed circulation of one single lineage (B2) for HMPV viruses and predominance of St2 genotype for HBoV viruses. A possible recombination between St1 genotype strains of HBoV was observed.

Background/Aims: Patients infected with the hepatitis C virus (HCV) often have liver steatosis, suggesting the possibility of a viral cytopathic effect. The aim of this study was to correlate the occurrence and severity of liver steatosis with HCV RNA type, level and sequence of the core-encoding region. Methods: We scored the liver steatosis in 101 HCV-infected individuals carefully selected to exclude other risk factors of a fatty liver. Results were compared with HCV RNA genotype and level in serum and liver. In selected patients, we assessed the effect of antiviral therapy on steatosis and the relationship between nucleocapsid sequence heterogeneity and fat infiltration. Results: Steatosis was found in 41 (40.6%) patients, irrespective of sex, age or route of infection. HCV genotype 3 was associated with higher steatosis scores than other genotypes. A significant correlation between steatosis score and titer of intrahepatic HCV RNA was found in patients infected with genotype 3, but not in those infected with genotype 1. In selected patients, response to alpha-interferon was associated with the disappearance of steatosis. Analysis of the nucleocapsid of 14 HCV isolates failed to identify a sequence specifically associated with the development of steatosis. Conclusions: We provide virological and clinical evidence that the steatosis of the liver is the morphological expression of a viral cytopathic effect in patients infected with HCV genotype 3. At variance with published evidence from experimental models, the HCV nucleocapsid protein does not seem to fully explain the lipid accumulation in these patients.

We assessed the performance of the Ceeram and Altona assays, the first two commercially available hepatitis E virus (HEV) RNA assays, using serial dilutions of 4 HEV-positive reference samples (genotypes 3a, 3c, 3e, and 3f). Both assays provided good analytical sensitivity and high reproducibility for detecting genotype 3 HEV RNA.

From the cDNAs of two defective RNAs naturally exhibiting a large difference in replication efficiency, a series of Sendai virus RNA chimeras were constructed by reciprocal exchanges of their 3′ end primary sequences. Using a reverse genetics system, the ability of these RNAs to replicate when expressed from cDNAs in the context of the viral proteins N, P, and L, also expressed from plasmids, was analyzed. First the extent of potential RNA 3′/5′ end complementarity was tested by disrupting and restoring the terminal 110-nucleotide complementarity of a copy-back RNA. Alternatively, this base pairing potential was gradually increased from 12 to 57 or to 98 nucleotides by continuous substitutions. In all cases, the restoration or the creation of more extended base pairing potential had no effect on RNA replication. Reciprocal exchanges were then made in order to identifycis-acting sequences that could induce high replication efficiency. It was found that nucleotides 1–31 of the antigenome 3′ end were sufficient to confer a high replication property (more than a 10-fold increase), regardless of the sequence adjacent to these terminal nucleotides. It is concluded that one of the most important features that modulate replication efficiency is contained in the promoter end primary sequence and that this feature is likely to operate independently of the ability to form a potential terminal base pairing.