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Find video protocols related to scientific articles indexed in Pubmed.
Presence of sandfly-borne phleboviruses of two antigenic complexes ( Sandfly fever Naples virus and Sandfly fever Sicilian virus ) in two different bio-geographical regions of Tunisia demonstrated by a microneutralisation-based seroprevalence study in dogs.
Parasit Vectors
PUBLISHED: 09-24-2014
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BackgroundSandfly-borne phleboviruses are present in North Africa where they can infect humans in regions where Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis in the Western Mediterranean basin is present affecting both humans and dogs. We investigated the capacity of dogs to be used as sentinels for sandfly-borne phleboviruses as previously shown for leishmaniasis.FindingsA total of 312 sera were collected from guard dogs in two different bioclimatic regions (governorates of Kairouan and Bizerte) of Tunisia where zoonotic visceral leishmaniasis has been reported. These sera were tested for the presence of neutralising antibodies against 3 phleboviruses: Toscana virus, Punique virus and Sicilian virus. In the governorate of Kairouan, seroprevalence rates of 7.5%, 43.5%, and 38.1% were observed for Toscana, Punique and Sicilian virus, respectively. A high proportion of sera from the governorate of Bizerte were hemolyzed and showed high cytotoxicity for the cells and subsequently precluded detailed interpretation of this batch. However, validated results for 27 sera were in agreement with data observed in the governorate of Kairouan.ConclusionsToscana virus is present in the governorate of Kairouan but at a lower rate compared to Punique and Sicilian viruses. These three sandfly-borne phleboviruses can infect dogs. Direct detection and isolation of the viruses are now to be attempted in animals as well as in humans. Our findings showed that guard dogs are good sentinels for virus transmitted by sandflies and strongly suggested that the high seroprevalence rates observed in dogs merit further attention.
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Novel flaviviruses from mosquitoes: mosquito-specific evolutionary lineages within the phylogenetic group of mosquito-borne flaviviruses.
Virology
PUBLISHED: 08-09-2014
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Novel flaviviruses that are genetically related to pathogenic mosquito-borne flaviviruses (MBFV) have been isolated from mosquitoes in various geographical locations, including Finland. We isolated and characterized another novel virus of this group from Finnish mosquitoes collected in 2007, designated as Ilomantsi virus (ILOV). Unlike the MBFV that infect both vertebrates and mosquitoes, the MBFV-related viruses appear to be specific to mosquitoes similar to the insect-specific flaviviruses (ISFs). In this overview of MBFV-related viruses we conclude that they differ from the ISFs genetically and antigenically. Phylogenetic analyses separated the MBFV-related viruses isolated in Africa, the Middle East and South America from those isolated in Europe and Asia. Serological cross-reactions of MBFV-related viruses with other flaviviruses and their potential for vector-borne transmission require further characterization. The divergent MBFV-related viruses are probably significantly under sampled to date and provide new information on the variety, properties and evolution of vector-borne flaviviruses.
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Co-ordinating the clinical management of imported human cases suspected of being infected with a highly pathogenic virus such as Ebola.
Clin. Microbiol. Infect.
PUBLISHED: 08-08-2014
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In recent years, there have been increasing numbers of alerts for possible imported cases of infection by highly pathogenic viruses (HiPaV). Striking examples include severe respiratory pathogens (e.g., the SARS or MERS coronavirus)(6), or haemorrhagic fever viruses (e.g., Ebola virus) (3). Many countries have already implemented disease control measures that include the establishment of specialised facilities such as negative-air pressure isolation rooms for the containment of patients and reference diagnostic laboratories where potentially infectious samples can be safely analysed to provide diagnosis and virus characterisation. This article is protected by copyright. All rights reserved.
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Single-stranded positive-sense RNA viruses generated in days using infectious subgenomic amplicons.
J. Gen. Virol.
PUBLISHED: 07-22-2014
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Reverse genetics is a key methodology for producing genetically modified RNA viruses and deciphering cellular and viral biological properties, but methods based on the preparation of plasmid-based complete viral genomes are laborious and unpredictable. Here, both wild-type and genetically modified infectious RNA viruses were generated in days using the newly described ISA (infectious-subgenomic-amplicons) method. This new versatile and simple procedure may enhance our capacity to obtain infectious RNA viruses from PCR-amplified genetic material.
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Mutations in the chikungunya virus non-structural proteins cause resistance to favipiravir (T-705), a broad-spectrum antiviral.
J. Antimicrob. Chemother.
PUBLISHED: 06-20-2014
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T-705, also known as favipiravir, is a small-molecule inhibitor that is currently in clinical development for the treatment of influenza virus infections. This molecule also inhibits the replication of a broad spectrum of other RNA viruses. The objective of this study was to investigate the antiviral effect of favipiravir on chikungunya virus (CHIKV) replication and to contribute to unravelling the molecular mechanism of action against this virus.
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Complete coding sequence of zika virus from a French polynesia outbreak in 2013.
Genome Announc
PUBLISHED: 06-07-2014
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Zika virus is an arthropod-borne Flavivirus member of the Spondweni serocomplex, transmitted by Aedes mosquitoes. We report here the complete coding sequence of a Zika virus strain belonging to the Asian lineage, isolated from an infected patient returning from French Polynesia, an epidemic area in 2013/2014.
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Highly diverse morbillivirus-related paramyxoviruses in wild fauna of the southwestern Indian Ocean Islands: evidence of exchange between introduced and endemic small mammals.
J. Virol.
PUBLISHED: 05-14-2014
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The Paramyxoviridae form an increasingly diverse viral family, infecting a wide variety of different hosts. In recent years, they have been linked to disease emergence in many different animal populations and in humans. Bats and rodents have been identified as major animal populations capable of harboring paramyxoviruses, and host shifting between these animals is likely to be an important driving factor in the underlying evolutionary processes that eventually lead to disease emergence. Here, we have studied paramyxovirus circulation within populations of endemic and introduced wild small mammals of the southwestern Indian Ocean region and belonging to four taxonomic orders: Rodentia, Afrosoricida, Soricomorpha, and Chiroptera. We report elevated infection levels as well as widespread paramyxovirus dispersal and frequent host exchange of a newly emerging genus of the Paramyxoviridae, currently referred to as the unclassified morbillivirus-related viruses (UMRVs). In contrast to other genera of the Paramyxoviridae, where bats have been shown to be a key host species, we show that rodents (and, in particular, Rattus rattus) are significant spreaders of UMRVs. We predict that the ecological particularities of the southwestern Indian Ocean, where small mammal species often live in densely packed, multispecies communities, in combination with the increasing invasion of R. rattus and perturbations of endemic animal communities by active anthropological development, will have a major influence on the dynamics of UMRV infection.
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Evaluation of antiviral efficacy of ribavirin, arbidol, and T-705 (favipiravir) in a mouse model for Crimean-Congo hemorrhagic fever.
PLoS Negl Trop Dis
PUBLISHED: 05-01-2014
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Mice lacking the type I interferon receptor (IFNAR-/- mice) reproduce relevant aspects of Crimean-Congo hemorrhagic fever (CCHF) in humans, including liver damage. We aimed at characterizing the liver pathology in CCHF virus-infected IFNAR-/- mice by immunohistochemistry and employed the model to evaluate the antiviral efficacy of ribavirin, arbidol, and T-705 against CCHF virus.
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Xpert Flu for point-of-care diagnosis of human influenza in industrialized countries.
Expert Rev. Mol. Diagn.
PUBLISHED: 04-08-2014
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Respiratory infections, particularly those caused by influenza viruses, represent the third-most important cause of death in the world due to infectious diseases. Nevertheless, despite the enormous publicity attracted by epidemics due to these viruses, laboratory diagnosis, documentation and recording of respiratory diseases is still unsatisfactory. Available diagnostic tests capable of providing results rapidly are either limited and insufficiently sensitive or highly sensitive and specific but insufficiently rapid. Considerable investment and research efforts have been made towards the development of new diagnostics for influenza A and B viruses and the Xpert(®) Flu assay (Cepheid(®), CA, USA) has emerged as one of the most promising. In this article, we review current knowledge of the Xpert Flu test, discuss its potential value as a point-of-care test and outline the potential leads for future development.
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Widespread circulation of a new echovirus 30 variant causing aseptic meningitis and non-specific viral illness, South-East France, 2013.
J. Clin. Virol.
PUBLISHED: 03-28-2014
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Human enteroviruses (HEVs) are major cause of aseptic meningitis. A new outbreak of E-30 occurred between April and September 2013 in Marseille, South-East France.
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Development of generic Taqman PCR and RT-PCR assays for the detection of DNA and mRNA of ?-actin-encoding sequences in a wide range of animal species.
J. Virol. Methods
PUBLISHED: 02-19-2014
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As a member of the European Virus Archive (EVA) consortium, our laboratory is developing and maintaining a large collection of viruses. This collection implies the use of a panel of cell lines originating from various animal species. In order to make easier the handling of such a large panel of cell lines, wide spectrum real-time PCR and RT-PCR assays were developed to allow the detection and the quantification of DNA and mRNA of ?-actin, one of the most commonly used eukaryotic housekeeping genes. By using two degenerated primers and a unique probe, these two assays were shown to detect nucleic acids of a panel of vertebrate and invertebrate cell lines commonly used in animal virology. This panel included human, monkey, rodent, dog, pig, fish, batrachian, mosquito and tick cell lines. Additionally, the two assays amplified successfully ?-actin-encoding sequences of sandflies. Sensitivity evaluation performed on synthetic DNA and RNA sequences showed that the two assays were very sensitive and suitable for accurate quantification. The two assays constitute together a convenient method suitable for multiple purposes. They can be used for instance to estimate the amount of contaminating cellular genetic material prior to sequence-independent amplification of viral genomes achieved before high-throughput sequencing, to evaluate the efficiency of DNase and/or RNase treatments performed on cellular extract and to check nucleic acid extraction by using ?-actin-encoding sequences as endogenous control. This assay will constitute a precious tool for virologists working with multiple cell lines or animal models.
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Molecular detection of human rhinoviruses in respiratory samples: a comparison of Taqman probe-, SYBR green I- and BOXTO-based real-time PCR assays.
Virol. J.
PUBLISHED: 02-11-2014
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Human Rhinoviruses (HRV) are major causative agents of acute respiratory tract infections in all age group and important contributing factors of childhood morbidity and mortality. Clinical presentation is poorly specific and the great antigenic and genetic variability of HRVs renders the biological diagnosis complex. Here, we have evaluated several molecular diagnostic protocols, including Taqman probe-based and intercalating agent-based RT-PCR assays.
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Influenza C virus high seroprevalence rates observed in 3 different population groups.
J. Infect.
PUBLISHED: 02-10-2014
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The epidemiology of Influenza C virus (FLUCV) infections remains poorly characterised. Here, we have examined the age- and location-specific seroprevalence of antibodies against FLUCV in 1441 sera from metropolitan continental France (Marseille), South-West Indian Ocean French territories (Reunion Island) and United-Kingdom (Edinburgh) using a combination of haemagglutination inhibition, virus neutralisation and ELISA assays. Our results show that immunity to FLUCV is common in all locations studied (global seroprevalence values >50%) and that the first immunising contacts generally occur early in life (i.e., in the 0-4 year-old age group). The latter item is further supported by the detection of FLUCV RNA by RT-PCR in naso-pharyngeal samples collected in patient attending the Emergency Room of the Public hospitals of Marseille, France with a large majority of children under 10 years-old: 17 (60.7%) in children ?3 yo, 10 (35.7%) in the 4-10 yo age group and 1 (3.6%) in an adult (49yo). The temporal distribution of cases was atypical with regard to influenza (a large proportion of cases occurred in spring and summer) and the clinical presentation was diverse, including but being not limited to classical Influenza-like-Ilnesses. Altogether, our results indicate an intense circulation of FLUCV in the different study areas and an early occurrence of infection in human life. Flu C appears to be a widely under-diagnosed and under-studied human paediatric disease that obviously deserves further clinical and epidemiological characterisation.
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Viral aetiology influenza like illnesses in Santa Cruz, Bolivia (2010-2012).
Virol. J.
PUBLISHED: 02-10-2014
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Acute respiratory infections represent a serious public health issue worldwide but virological aetiologies of Influenza Like Illnesses (ILIs) remain largely unknown in developing countries. This study represents the first attempt to characterise viral aetiologies of ILIs in Bolivia.
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Determinants of individuals' risks to 2009 pandemic influenza virus infection at household level amongst Djibouti city residents--a CoPanFlu cross-sectional study.
Virol. J.
PUBLISHED: 01-20-2014
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Following the 2009 swine flu pandemic, a cohort for pandemic influenza (CoPanFlu) study was established in Djibouti, the Horn of Africa, to investigate its case prevalence and risk predictors' at household level.
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SYBR green real-time PCR for the detection of all enterovirus-A71 genogroups.
PLoS ONE
PUBLISHED: 01-01-2014
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Enterovirus A71 (EV-A71) has recently become an important public health threat, especially in South-East Asia, where it has caused massive outbreaks of Hand, Foot and Mouth disease every year, resulting in significant mortality. Rapid detection of EV-A71 early in outbreaks would facilitate implementation of prevention and control measures to limit spread. Real-time RT-PCR is the technique of choice for the rapid diagnosis of EV-A71 infection and several systems have been developed to detect circulating strains. Although eight genogroups have been described globally, none of these PCR techniques detect all eight. We describe, for the first time, a SYBR Green real-time RT-PCR system validated to detect all 8 EV-A71 genogroups. This tool could permit the early detection and shift in genogroup circulation and the standardization of HFMD virological diagnosis, facilitating networking of laboratories working on EV-A71 in different regions.
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Molecular comparison and evolutionary analyses of VP1 nucleotide sequences of new African human enterovirus 71 isolates reveal a wide genetic diversity.
PLoS ONE
PUBLISHED: 01-01-2014
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Most circulating strains of Human enterovirus 71 (EV-A71) have been classified primarily into three genogroups (A to C) on the basis of genetic divergence between the 1D gene, which encodes the VP1 capsid protein. The aim of the present study was to provide further insights into the diversity of the EV-A71 genogroups following the recent description of highly divergent isolates, in particular those from African countries, including Madagascar. We classified recent EV-A71 isolates by a large comparison of 3,346 VP1 nucleotidic sequences collected from GenBank. Analysis of genetic distances and phylogenetic investigations indicated that some recently-reported isolates did not fall into the genogroups A-C and clustered into three additional genogroups, including one Indian genogroup (genogroup D) and 2 African ones (E and F). Our Bayesian phylogenetic analysis provided consistent data showing that the genogroup D isolates share a recent common ancestor with the members of genogroup E, while the isolates of genogroup F evolved from a recent common ancestor shared with the members of the genogroup B. Our results reveal the wide diversity that exists among EV-A71 isolates and suggest that the number of circulating genogroups is probably underestimated, particularly in developing countries where EV-A71 epidemiology has been poorly studied.
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Estimating the burden of Japanese encephalitis virus and other encephalitides in countries of the mekong region.
PLoS Negl Trop Dis
PUBLISHED: 01-01-2014
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Diverse aetiologies of viral and bacterial encephalitis are widely recognized as significant yet neglected public health issues in the Mekong region. A robust analysis of the corresponding health burden is lacking. We retrieved 75 articles on encephalitis in the region published in English or in French from 1965 through 2011. Review of available data demonstrated that they are sparse and often derived from hospital-based studies with significant recruitment bias. Almost half (35 of 75) of articles were on Japanese encephalitis virus (JEV) alone or associated with dengue. In the Western Pacific region the WHO reported 30,000-50,000 annual JEV cases (15,000 deaths) between 1966 and 1996 and 4,633 cases (200 deaths) in 2008, a decline likely related to the introduction of JEV vaccination in China, Vietnam, or Thailand since the 1980s. Data on dengue, scrub typhus and rabies encephalitis, among other aetiologies, are also reviewed and discussed. Countries of the Mekong region are undergoing profound demographic, economic and ecological change. As the epidemiological aspects of Japanese encephalitis (JE) are transformed by vaccination in some countries, highly integrated expert collaborative research and objective data are needed to identify and prioritize the human health, animal health and economic burden due to JE and other pathogens associated with encephalitides.
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Co-circulation of Toscana virus and Punique virus in northern Tunisia: a microneutralisation-based seroprevalence study.
PLoS Negl Trop Dis
PUBLISHED: 09-01-2013
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In northern Tunisia, the co-circulation of two related sand fly-borne phleboviruses, Toscana virus (TOSV) and Punique virus (PUNV) was previously demonstrated. In contrast to TOSV, a prominent human pathogen, there is no data supporting that PUNV is capable to infect and cause disease to humans. We studied the respective involvement of TOSV and PUNV in human infections in northern Tunisia through a seroprevalence study.
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An epidemic of dengue-1 in a remote village in rural Laos.
PLoS Negl Trop Dis
PUBLISHED: 08-01-2013
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In the Lao PDR (Laos), urban dengue is an increasingly recognised public health problem. We describe a dengue-1 virus outbreak in a rural northwestern Lao forest village during the cool season of 2008. The isolated strain was genotypically "endemic" and not "sylvatic," belonging to the genotype 1, Asia 3 clade. Phylogenetic analyses of 37 other dengue-1 sequences from diverse areas of Laos between 2007 and 2010 showed that the geographic distribution of some strains remained focal overtime while others were dispersed throughout the country. Evidence that dengue viruses have broad circulation in the region, crossing country borders, was also obtained. Whether the outbreak arose from dengue importation from an urban centre into a dengue-naïve community or crossed into the village from a forest cycle is unknown. More epidemiological and entomological investigations are required to understand dengue epidemiology and the importance of rural and forest dengue dynamics in Laos.
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Genetic drift of influenza A(H3N2) viruses during two consecutive seasons in 2011-2013 in Corsica, France.
J. Med. Virol.
PUBLISHED: 07-16-2013
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The 2011-2012 and 2012-2013 post-pandemic influenza outbreaks were characterized by variability in the A(H3N2) influenza viruses, resulting in low to moderate vaccine effectiveness (VE). The aim of this study was to investigate the molecular evolution and vaccine strain match of the A(H3N2) influenza viruses, having been circulated throughout the population of the French Corsica Island in 2011-2012 and again in 2012-2013. Clinical samples from 31 patients with confirmed A(H3N2) influenza viruses were collected by general practitioners (GPs) over these two consecutive seasons. An analysis of genetic distance and antigenic drift was conducted. Based on a hemagglutinin (HA) aminoacid sequence analysis, the Corsican A(H3N2) viruses fell into the A/Victoria/208/2009 genetic clade, group 3. All influenza viruses were characterized by at least four fixed amino acid mutations which were: N145S (epitope A); Q156H and V186G (epitope B) Y219S (epitope D), with respect to the A/Perth/16/2009 (reference vaccine strain for the 2011-2012) and the A/Victoria/361/2011 (reference vaccine strain for the 2012-2013). Using the pepitope model, the percentages of the perfect match VE estimated against circulated strains declined within and between seasons, with estimations of <50%. Overall, these results seem to indicate an antigenic drift of the A(H3N2) influenza viruses which were circulated in Corsica. These findings highlight the importance of the continuous and careful surveillance of genetic changes in the HA domain during seasonal influenza epidemics, in order to provide information on newly emerging genetic variants. J. Med. Virol. © 2013 Wiley Periodicals, Inc.
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Isolation of Toscana virus from the cerebrospinal fluid of a man with meningitis in Marseille, France, 2010.
Vector Borne Zoonotic Dis.
PUBLISHED: 06-29-2013
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Toscana virus (TOSV; Bunyaviridae, Phlebovirus) is an emerging arthropod-borne virus transmitted by phlebotomine sandflies. TOSV is a frequent cause of central nervous system infection during the warm season in several countries bordering the Mediterranean Sea. Here, we report a case of TOSV aseptic meningitis diagnosed in 2012 in Marseille, France. The virus strain was recovered in cell culture from the cerebrospinal fluid. New-generation sequencing based on Ion Torrent technology was used to determine its complete genome sequence. Phylogenetic analysis based on the partial L segment revealed that this isolate belongs to the lineage B together with other French, Spanish, and Moroccan strains. Although several cases of TOSV meningitis are reported in the literature, few of them are diagnosed by RT-PCR combined with virus isolation and further sequence characterization. This case report supports that virus isolation should be attempted whenever possible because this remains the gold standard technique for diagnosis of arthropod-borne viral infections.
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Sheep-to-human transmission of Orf virus during Eid al-Adha religious practices, France.
Emerging Infect. Dis.
PUBLISHED: 06-05-2013
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Five persons in France were infected with Orf virus after skin wounds were exposed to infected sheep tissues during Eid al-Adha, the Muslim Feast of Sacrifice. Infections were confirmed by electron microscopy, PCR, and sequence analysis. Prevention and control of this underdiagnosed disease can be achieved by educating physicians, slaughterhouse workers, and persons participating in Eid al-Adha.
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Sandfly-borne phleboviruses of Eurasia and Africa: Epidemiology, genetic diversity, geographic range, control measures.
Antiviral Res.
PUBLISHED: 05-28-2013
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Sandfly-borne phleboviruses may cause a transient febrile illness (sandfly fever) or more severe neuroinvasive disease. In the Old World, they are vectored by phlebotomine flies, which are widely distributed in the Mediterranean basin, North Africa, the Indian subcontinent, the Middle East and central Asia. High seroprevalence rates have been recorded in humans and domestic animals in areas where sandflies are present. Most published studies have focused on phlebovirus infections of travelers and of soldiers stationed in endemic areas, but the health impact on local populations should not be underestimated, as seroprevalence studies indicate massive circulation of these viruses, even if disease is seldom documented. Except for Toscana virus, which shows a marked neurotropism and is a leading cause of aseptic meningitis in endemic regions, phlebovirus infections are inadequately considered by physicians and are generally underestimated. However, several properties of these viruses suggest that they will extend their geographic range. First, changes in the areas occupied by sandflies as a result of climate change have a direct impact on the epidemiology of associated human and animal diseases. Second, phleboviruses exhibit a high mutation rate, and their tri-segmented genome is prone to reassortment and recombination. Third, distinct virus strains can be transmitted by the same arthropod species. Recent studies have documented the distribution of sandfly-borne phleboviruses in Western Europe, but data for Eastern Europe, the Middle East and Africa are very limited. With the goal of filling knowledge gaps and planning new research programs, we have examined available information and present it as a comprehensive review, with a specific focus on understudied regions. We also discuss the need to conduct studies aimed at developing new antiviral drugs and vaccines.
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"Megavirales", a proposed new order for eukaryotic nucleocytoplasmic large DNA viruses.
Arch. Virol.
PUBLISHED: 05-07-2013
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The nucleocytoplasmic large DNA viruses (NCLDVs) comprise a monophyletic group of viruses that infect animals and diverse unicellular eukaryotes. The NCLDV group includes the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Mimiviridae and the proposed family "Marseilleviridae". The family Mimiviridae includes the largest known viruses, with genomes in excess of one megabase, whereas the genome size in the other NCLDV families varies from 100 to 400 kilobase pairs. Most of the NCLDVs replicate in the cytoplasm of infected cells, within so-called virus factories. The NCLDVs share a common ancient origin, as demonstrated by evolutionary reconstructions that trace approximately 50 genes encoding key proteins involved in viral replication and virion formation to the last common ancestor of all these viruses. Taken together, these characteristics lead us to propose assigning an official taxonomic rank to the NCLDVs as the order "Megavirales", in reference to the large size of the virions and genomes of these viruses.
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Chikungunya fever: epidemiology, clinical syndrome, pathogenesis and therapy.
Antiviral Res.
PUBLISHED: 04-07-2013
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Chikungunya virus (CHIKV) is the aetiological agent of the mosquito-borne disease chikungunya fever, a debilitating arthritic disease that, during the past 7years, has caused immeasurable morbidity and some mortality in humans, including newborn babies, following its emergence and dispersal out of Africa to the Indian Ocean islands and Asia. Since the first reports of its existence in Africa in the 1950s, more than 1500 scientific publications on the different aspects of the disease and its causative agent have been produced. Analysis of these publications shows that, following a number of studies in the 1960s and 1970s, and in the absence of autochthonous cases in developed countries, the interest of the scientific community remained low. However, in 2005 chikungunya fever unexpectedly re-emerged in the form of devastating epidemics in and around the Indian Ocean. These outbreaks were associated with mutations in the viral genome that facilitated the replication of the virus in Aedes albopictus mosquitoes. Since then, nearly 1000 publications on chikungunya fever have been referenced in the PubMed database. This article provides a comprehensive review of chikungunya fever and CHIKV, including clinical data, epidemiological reports, therapeutic aspects and data relating to animal models for in vivo laboratory studies. It includes Supplementary Tables of all WHO outbreak bulletins, ProMED Mail alerts, viral sequences available on GenBank, and PubMed reports of clinical cases and seroprevalence studies.
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Next generation sequencing of viral RNA genomes.
BMC Genomics
PUBLISHED: 03-26-2013
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With the advent of Next Generation Sequencing (NGS) technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field. Most RNA viruses have relatively small genomes in comparison to other organisms and as such, would appear to be an obvious success story for the use of NGS technologies. However, due to the relatively low abundance of viral RNA in relation to host RNA, RNA viruses have proved relatively difficult to sequence using NGS technologies. Here we detail a simple, robust methodology, without the use of ultra-centrifugation, filtration or viral enrichment protocols, to prepare RNA from diagnostic clinical tissue samples, cell monolayers and tissue culture supernatant, for subsequent sequencing on the Roche 454 platform.
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Complete Genome of a Genotype I Japanese Encephalitis Virus Isolated from a Patient with Encephalitis in Vientiane, Lao PDR.
Genome Announc
PUBLISHED: 02-21-2013
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Japanese encephalitis virus (JEV) (Flaviviridae, Flavivirus) is an arthropod-borne flavivirus transmitted by Culex species mosquitoes. We report here the complete genome of the JEV genotype I strain JEV_CNS769_Laos_2009 isolated from an infected patient in Vientiane, Lao Peoples Democratic Republic (PDR) (Laos).
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Random codon re-encoding induces stable reduction of replicative fitness of Chikungunya virus in primate and mosquito cells.
PLoS Pathog.
PUBLISHED: 02-21-2013
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Large-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (<0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine.
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Comparative analysis of rodent tissue preservation methods and nucleic acid extraction techniques for virus screening purposes.
J. Virol. Methods
PUBLISHED: 01-28-2013
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The polymerase chain reaction (PCR) has become an essential method for the detection of viruses in tissue specimens. However, it is well known that the presence of PCR inhibitors in tissue samples may cause false-negative results. Hence the identification of PCR inhibitors and evaluation and optimization of nucleic acid extraction and preservation methods is of prime concern in virus discovery programs dealing with animal tissues. Accordingly, to monitor and remove inhibitors we have performed comparative analyses of two commonly used tissue storage methods and five RNA purification techniques using a variety of animal tissues, containing quantified levels of added MS2 bacteriophages as the indicator of inhibition. The results showed (i) no significant difference between the two methods of sample preservation, viz. direct storage at -80°C or 4°C in RNAlater, (ii) lung rodent tissues contained lower levels of inhibitor than liver, kidney and spleen, (iii) RNA extraction using the EZ1+PK RNA kit was the most effective procedure for removal of RT-PCR inhibitors.
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Chikungunya fever: a clinical and virological investigation of outpatients on Reunion Island, South-West Indian Ocean.
PLoS Negl Trop Dis
PUBLISHED: 01-17-2013
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Chikungunya virus (CHIKV) is responsible for acute febrile polyarthralgia and, in a proportion of cases, severe complications including chronic arthritis. CHIKV has spread recently in East Africa, South-West Indian Ocean, South-Asia and autochthonous cases have been reported in Europe. Although almost all patients are outpatients, medical investigations mainly focused on hospitalised patients.
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Novel virus discovery and genome reconstruction from field RNA samples reveals highly divergent viruses in dipteran hosts.
PLoS ONE
PUBLISHED: 01-01-2013
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We investigated whether small RNA (sRNA) sequenced from field-collected mosquitoes and chironomids (Diptera) can be used as a proxy signature of viral prevalence within a range of species and viral groups, using sRNAs sequenced from wild-caught specimens, to inform total RNA deep sequencing of samples of particular interest. Using this strategy, we sequenced from adult Anopheles maculipennis s.l. mosquitoes the apparently nearly complete genome of one previously undescribed virus related to chronic bee paralysis virus, and, from a pool of Ochlerotatus caspius and Oc. detritus mosquitoes, a nearly complete entomobirnavirus genome. We also reconstructed long sequences (1503-6557 nt) related to at least nine other viruses. Crucially, several of the sequences detected were reconstructed from host organisms highly divergent from those in which related viruses have been previously isolated or discovered. It is clear that viral transmission and maintenance cycles in nature are likely to be significantly more complex and taxonomically diverse than previously expected.
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Factors associated with post-seasonal serological titer and risk factors for infection with the pandemic A/H1N1 virus in the French general population.
PLoS ONE
PUBLISHED: 01-01-2013
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The CoPanFlu-France cohort of households was set up in 2009 to study the risk factors for infection by the pandemic influenza virus (H1N1pdm) in the French general population. The authors developed an integrative data-driven approach to identify individual, collective and environmental factors associated with the post-seasonal serological H1N1pdm geometric mean titer, and derived a nested case-control analysis to identify risk factors for infection during the first season. This analysis included 1377 subjects (601 households). The GMT for the general population was 47.1 (95% confidence interval (CI): 45.1, 49.2). According to a multivariable analysis, pandemic vaccination, seasonal vaccination in 2009, recent history of influenza-like illness, asthma, chronic obstructive pulmonary disease, social contacts at school and use of public transports by the local population were associated with a higher GMT, whereas history of smoking was associated with a lower GMT. Additionally, young age at inclusion and risk perception of exposure to the virus at work were identified as possible risk factors, whereas presence of an air humidifier in the living room was a possible protective factor. These findings will be interpreted in light of the longitudinal analyses of this ongoing cohort.
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Molecular evolution of the insect-specific flaviviruses.
J. Gen. Virol.
PUBLISHED: 10-19-2011
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There has been an explosion in the discovery of insect-specific flaviviruses and/or their related sequences in natural mosquito populations. Herein we review all insect-specific flavivirus sequences currently available and conduct phylogenetic analyses of both the insect-specific flaviviruses and available sequences of the entire genus Flavivirus. We show that there is no statistical support for virus-mosquito co-divergence, suggesting that the insect-specific flaviviruses may have undergone multiple introductions with frequent host switching. We discuss potential implications for the evolution of vectoring within the family Flaviviridae. We also provide preliminary evidence for potential recombination events in the history of cell fusing agent virus. Finally, we consider priorities and guidelines for future research on insect-specific flaviviruses, including the vast potential that exists for the study of biodiversity within a range of potential hosts and vectors, and its effect on the emergence and maintenance of the flaviviruses.
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Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen.
BMC Microbiol.
PUBLISHED: 05-31-2011
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The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle.
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Epidemiologic relationship between Toscana virus infection and Leishmania infantum due to common exposure to Phlebotomus perniciosus sandfly vector.
PLoS Negl Trop Dis
PUBLISHED: 05-06-2011
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Sand flies are recognised vectors of parasites in the genus Leishmania and a number of arthropod-borne viruses, in particular viruses within the genus Phlebovirus, family Bunyaviridae. In southern France, Toscana phlebovirus (TOSV) is recognized as a prominent cause of summer meningitis. Since Leishmania and TOSV have a common vector (Phlebotomus perniciosus), an epidemiologic link has been assumed for a long time. However, there is no scientific evidence of such a link between human leishmaniosis and phleboviral infections. To identify a possible link, we investigated the presence and distribution of antibodies against these two microorganisms (i) in individuals and (ii) at a spatial level in the city of Marseille (south-eastern France). Five hundred sera were selected randomly in the biobank of the Department of Parasitology of the Public Hospitals of Marseille. All sera were previously tested for IgG against Leishmania by Western Blotting, and TOSV IgG were detected by indirect immunofluorescence. The seropositivity rates were 21.4% for TOSV and 28% for Leishmania. Statistical analysis demonstrated that seropositivity for one pathogen was significantly associated with seropositivity to the other pathogen. This result provided the first robust evidence for the existence of an epidemiological relationship between Leishmania infantum and TOSV. Addresses of tested patients were geolocalized and integrated into Geographical Information System software, in order to test spatial relationship between the two pathogens. Spatial analysis did not allow to identify (i) specific patterns for the spatial distribution of positive serological results for TOSV or Leishmania, and (ii) a spatial relationship between Leishmania and TOSV positive serological results. This may reflect the fact that the sample studied was not powerful enough to demonstrate either a spatial clustering or co-location, i.e. that the actual risk exposure area is smaller than the mean of distance between patients in our study (245 m).
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Revolutionizing clinical microbiology laboratory organization in hospitals with in situ point-of-care.
PLoS ONE
PUBLISHED: 03-22-2011
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Clinical microbiology may direct decisions regarding hospitalization, isolation and anti-infective therapy, but it is not effective at the time of early care. Point-of-care (POC) tests have been developed for this purpose.
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Nationwide surveillance of 18 respiratory viruses in patients with influenza-like illnesses: a pilot feasibility study in the French Sentinel Network.
J. Med. Virol.
PUBLISHED: 03-10-2011
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The aim of the present study was to test the feasibility of integrating the diagnosis of 18 respiratory viruses into clinical surveillance of influenza-like illness using a PCR-DNA microarray detection assay. The study took place in the French Sentinel Network, a nationwide surveillance network of General Practitioners (GPs) representative of French GPs in terms age, location, and type of practice (urban/rural). Three virological laboratories also participated in the study. The study was planned for 5 weeks from January 25, 2010 to February 27, 2010. A subset of 150 Sentinel GPs, located in mainland France, was enrolled to collect clinical data and nasopharyngeal samples from every first patient of the week having a medical visit for influenza-like illness defined as a sudden fever of 39°C or more with respiratory symptoms and myalgia. Sixty-three GPs (42%) collected 103 samples while 87 GPs (58%) did not. GPs did not differ with respect to their age, gender, urban/rural distribution, or years of inscription in the Sentinel Network. Patients included were of a similar age and had similar vaccination characteristics, but were more frequently men than influenza-like illness patients reported to the network during the study period. Sixty-one viruses were detected from 56 of 96 (58%) interpretable samples. The respiratory viruses detected most frequently were metapneumovirus and respiratory syncytial virus. This study showed that virological diagnosis of 18 respiratory viruses can be combined with surveillance of clinical influenza-like illness in general practice. Although feasibility has not been demonstrated yet, it will be evaluated over the winter of 2010-2011.
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A retrospective overview of enterovirus infection diagnosis and molecular epidemiology in the public hospitals of Marseille, France (1985-2005).
PLoS ONE
PUBLISHED: 02-22-2011
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Human enteroviruses (HEV) are frequent human pathogens and, associated in particular with large outbreaks of aseptic meningitis. Here, we have compiled a database of clinical HEV isolates from the Public Hospitals of Marseille, from 1985 to 2005. Amongst 654 isolates that could be characterized by complete sequencing of the VP1 gene, 98% belonged to species HEV-B; the most frequently isolated serotypes were Echovirus E30, E11, E7, E6 and E4. The high incidence of E30 and the recent emergence of E13 are consistent with reports worldwide and peak HEV isolation occurred mostly in the late spring and summer months. The proportion of echoviruses has decreased across the years, while that of coxsackieviruses has increased. Stool (the most frequent sample type) allowed detection of all identified serotypes. MRC5 (Human lung fibroblasts) cell line was the most conducive cell line for HEV isolation (84.9% of 10 most common serotype isolates, 96.3% in association with BGM (Buffalo green monkey kidney cells)). Previous seroneutralization-based serotype identification demonstrated 55.4% accuracy when compared with molecular VP1 analysis. Our analysis of a large number of clinical strains over 20 years reinforced the validity of VP1 serotyping and showed that comparative p-distance scores can be coupled with phylogenetic analysis to provide non-ambiguous serotype identification. Phylogenetic analysis in the VP1, 2C and 3D regions also provided evidence for recombination events amongst clinical isolates. In particular, it identified isolates with dissimilar VP1 but almost identical nonstructural regions.
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In vitro antiviral activity of arbidol against Chikungunya virus and characteristics of a selected resistant mutant.
Antiviral Res.
PUBLISHED: 02-10-2011
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Arbidol (ARB) is an antiviral drug originally licensed in Russia for use against influenza and other respiratory viral infections. Although a broad-spectrum antiviral activity has been reported for this drug, there is until now no data regarding its effects against alphavirus infection. Here, the in vitro antiviral effect of ARB on Chikungunya virus (CHIKV) replication was investigated and this compound was found to present potent inhibitory activity against the virus propagated onto immortalized Vero cells or primary human fibroblasts (MRC-5 lung cells) (IC(50)<10?g/ml). A CHIKV resistant mutant was then selected and adapted to growth in the presence of 30?g/ml ARB in MRC5 cells; its complete sequence analysis revealed a single amino acid substitution (G407R) localized in the E2 envelope protein. To confirm the G407R role in the molecular mechanism of ARB resistance, a CHIKV infectious clone harboring the same substitution was engineered, tested, and was found to display a similar level of resistance. Finally, our results demonstrated the effective in vitro antiviral activity of ARB against CHIKV and gave some tracks to understand the molecular basis of ARB activity.
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RNA and DNA bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than 45,000 routine PCR tests.
PLoS ONE
PUBLISHED: 02-09-2011
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Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs). In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i) to design specific ECs for both DNA and RNA viruses and (ii) to use T4 (DNA) or MS2 (RNA) phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids.
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Pandemic influenza due to pH1N1/2009 virus: estimation of infection burden in Reunion Island through a prospective serosurvey, austral winter 2009.
PLoS ONE
PUBLISHED: 01-18-2011
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To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season.
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Picornavirus non-structural proteins as targets for new anti-virals with broad activity.
Antiviral Res.
PUBLISHED: 01-12-2011
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Picornaviridae is one of the largest viral families and is composed of 14 genera, six of which include human pathogens. The best known picornaviruses are enteroviruses (including polio, PV, and rhinoviruses), foot-and-mouth disease virus (FMDV), and hepatitis A virus (HAV). Although infections often are mild, certain strains may cause pandemic outbreaks accompanied with meningitis and/or paralysis. Vaccines are available for PV, HAV and FMDV. When the oral vaccines are given to immunocompromised individuals, they may be chronically infected, and remain secretors of vaccine-derived variants of virus for years. There is no effective prophylaxis available for these or other picornaviruses. So far, only the 3C protease from viruses in three genera has been fully characterized as an anti-viral target, whereas the mode of action of compounds targeting other non-structural proteins have remained largely unaddressed. Within the EU-supported FP6 project-VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication), the non-structural proteins were studied to identify conserved binding sites for broadly reactive anti-virals. The putative 2C helicase from echovirus-30 was shown to form ring-shaped hexamers typical for DNA-encoded SF3 helicases, and to possess ATPase activity. Hexamer formation of 2C from enterovirus 76 was in vitro shown to be dependent on the 44 N-terminal residues. Crystal structures of three enterovirus 3C proteases were solved and shown to be similar to those of other picornaviruses. A new binding site of VPg to the bottom of the thumb domain of CV-B3 3D polymerase was identified as a potential target. Broad anti-enterovirus compounds against 2C and 3A proteins were also identified, including thiazolobenzimidazoles (active against 2C) and TTP-8307 (targeting 3A). There is a need for more potent inhibitors against PV and other picornaviruses, which are potential silent reservoirs for re-emerging PV-like disease.
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Molecular epidemiology of yellow fever in Bolivia from 1999 to 2008.
Vector Borne Zoonotic Dis.
PUBLISHED: 10-06-2010
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Yellow fever (YF) is a serious public health problem in Bolivia since at least the 19th century. Surprisingly, very limited information has been made available to date regarding the genetic characterisation and epidemiology of Bolivian YF virus (YFV) strains. Here, we conducted the genetic characterization of 12 human isolates of YFV collected in Bolivia between 1999 and 2008, by sequencing and analysis of two regions of the viral genome: a fragment encoding structural proteins "PrM" (premembrane and envelope) and a distal region "EMF," spanning the end of the virus genome. Our study reveals a high genetic diversity of YFV strains circulating in Bolivia during the last decade: we identified not only "Peruvian-like" genotype II viruses (related to previously characterized Bolivian strains), but also, for the fist time, "Brazilian-like" genotype I viruses. During the complete period of the study, only cases of "jungle" YF were detected (i.e., circulation of YFV via a sylvatic cycle) with no cluster of urban cases. However, the very significant spread of the Aedes aegypti mosquito across Bolivian cities threatens the country with the reappearance of an urban YFV transmission cycle and thus is required a sustained epidemiological surveillance.
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A derivate of the antibiotic doxorubicin is a selective inhibitor of dengue and yellow fever virus replication in vitro.
Antimicrob. Agents Chemother.
PUBLISHED: 09-13-2010
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A doxorubicin derivate, SA-17, that carries a squaric acid amide ester moiety at the carbohydrate (?-l-daunosaminyl) group was identified as a selective inhibitor of in vitro dengue virus (DENV) serotype 2 replication (50% effective concentration [EC(50)] = 0.34 ± 0.20 ?g/ml [0.52 ± 0.31 ?M]). SA-17 is markedly less cytostatic than the parent compound, resulting in a selectivity index value of ?100. SA-17 also inhibits yellow fever virus 17D (YFV-17D) replication (EC(50) = 3.1 ± 1.0 ?g/ml [4.8 ± 1.5 ?M]), although less efficiently than DENV replication, but proved inactive against a variety of enveloped and nonenveloped viruses. SA-17 inhibits in vitro flavivirus replication in a dose-dependent manner, as was assessed by virus yield reduction assays and quantification of viral RNA by means of real-time quantitative reverse transcriptase PCR (RT-qPCR) (?2 to 3 log reduction). The anti-DENV activity was confirmed using a Renilla luciferase-expressing dengue reporter virus. Time-of-drug-addition studies revealed that SA-17 acts at the very early stages of the viral replication cycle (i.e., virus attachment and/or virus entry). This observation was corroborated by the observation that SA-17, unlike the nucleoside analogue ribavirin, does not inhibit the replication of DENV subgenomic replicons. Preincubation of high-titer stocks of DENV or YFV-17D with ?5 ?g/ml SA-17 resulted in 100% inhibition of viral infectivity (?3 log reduction). SA-17, however, did not prove virucidal.
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Molecular and serological evidence for the presence of novel phleboviruses in sandflies from northern algeria.
Open Virol J
PUBLISHED: 04-22-2010
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During summer 2007, a total of 785 phlebotomine flies were trapped in northern Algeria, identified morphologically, organised as monospecific pools and tested for the presence of phlebovirus RNA using degenerate primers. Three pools were positive, and the corresponding PCR products were cloned and sequenced. Viral sequences corresponding to two phleboviruses distinct from each other were detected in sandflies circulating in two close locations (140 km apart) in Northern Algeria. The 3 sequences were aligned with homologous polymerase sequences retrieved from the Genbank database, in order to examine their phylogenetic relationships. One viral sequence (from Phlebotomus papatasi) was closely related to but distinct from a sequence obtained from Phlebotomus ariasi sandflies trapped in Algeria in 2006. The two other viral sequences (from Phlebotomus longicuspis) were genetically distantly related to sequences corresponding to virus members of the Sandfly fever Naples virus species and although falling within the same group, this clearly represents a second distinct novel lineage. These results are indicative of a high genetic heterogeneity within sandflies trapped in a relatively small geographic area. Seroprevalence studies conducted on sera from populations living in the same areas indicated that humans can be infected by these viruses.
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The N-terminal domain of the arenavirus L protein is an RNA endonuclease essential in mRNA transcription.
PLoS Pathog.
PUBLISHED: 04-07-2010
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Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a cap-snatching mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease ?/? architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.
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Detection of extensive cross-neutralization between pandemic and seasonal A/H1N1 Influenza Viruses using a pseudotype neutralization assay.
PLoS ONE
PUBLISHED: 03-15-2010
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Cross-immunity between seasonal and pandemic A/H1N1 influenza viruses remains uncertain. In particular, the extent that previous infection or vaccination by seasonal A/H1N1 viruses can elicit protective immunity against pandemic A/H1N1 is unclear.
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The 2C putative helicase of echovirus 30 adopts a hexameric ring-shaped structure.
Acta Crystallogr. D Biol. Crystallogr.
PUBLISHED: 03-03-2010
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The 2C protein, which is an essential ATPase and one of the most conserved proteins across the Picornaviridae family, is an emerging antiviral target for which structural and functional characterization remain elusive. Based on a distant relationship to helicases of small DNA viruses, piconavirus 2C proteins have been predicted to unwind double-stranded RNAs. Here, a terminally extended variant of the 2C protein from echovirus 30 has been studied by means of enzymatic activity assays, transmission electron microscopy, atomic force microscopy and dynamic light scattering. The transmission electron-microscopy technique showed the existence of ring-shaped particles with ?12 nm external diameter. Image analysis revealed that these particles were hexameric and resembled those formed by superfamily 3 DNA virus helicases.
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Likely correlation between sources of information and acceptability of A/H1N1 swine-origin influenza virus vaccine in Marseille, France.
PLoS ONE
PUBLISHED: 02-25-2010
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In France, there was a reluctance to accept vaccination against the A/H1N1 pandemic influenza virus despite government recommendation and investment in the vaccine programme.
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Role of host cell factors in flavivirus infection: Implications for pathogenesis and development of antiviral drugs.
Antiviral Res.
PUBLISHED: 02-22-2010
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The genus Flavivirus contains approximately 70 arthropod-borne enveloped RNA viruses many of which cause severe human and in some cases, animal disease. They include dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and tick-borne encephalitis virus. Hundreds of thousands of deaths due to flavivirus infections occur each year, many of which are unpreventable due to lack of availability of appropriate vaccines and/or antiviral drugs. Flaviviruses exploit the cytoplasmic cellular machinery to facilitate propagation of infectious progeny virions. They engage in dynamic and antagonistic interactions with host cell membranes and biochemical processes. Following infection, the cells initiate various antiviral strategies to counteract viral invasion. In its defense, the virus has alternative strategies to suppress these host responses to infection. The fine balance between these interactions determines the outcome of the viral infection and disease progression. Published studies have revealed specific effects of flaviviruses on cellular processes, but the underlying mechanisms that determine the specific cytopathogenetic changes induced by different flaviviruses have not, as yet, been elucidated. Independently of the suppression of the type I IFN response which has been described in detail elsewhere, this review focuses on recent discoveries relating to alterations of host metabolism following viral infection. Such studies may contribute to new approaches to antiviral drug development. The role of host cellular factors will be examined in the context of protection and/or pathogenesis resulting from flavivirus infection, with particular emphasis on West Nile virus and dengue virus.
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Evolution of dengue virus in Mexico is characterized by frequent lineage replacement.
Arch. Virol.
PUBLISHED: 02-12-2010
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Both dengue fever and its more serious clinical manifestation, dengue hemorrhagic fever, represent major public health concerns in the Americas. To understand the patterns and dynamics of virus transmission in Mexico, a country characterized by a marked increase in dengue incidence in recent years, we undertook a molecular evolutionary analysis of the largest sample of Mexican strains of dengue virus compiled to date. Our E gene data set comprises sequences sampled over a period of 27 years and representing all of the Mexican states that are endemic for dengue. Our phylogenetic analysis reveals that, for each of the four dengue viruses (DENV-1 to DENV-4), there have been multiple introductions of viral lineages in Mexico, with viruses similar to those observed throughout the Americas, but there has been strikingly little co-circulation. Rather, dengue virus evolution in Mexico is typified by frequent lineage replacement, such that only a single viral lineage dominates in a specific serotype at a specific time point. Most lineage replacement events involve members of the same viral genotype, although a replacement event involving different genotypes was observed with DENV-2, and viral lineages that are new to Mexico are described for DENV-1, DENV-3 and DENV-4.
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The 1932 Macau epidemic of cerebrospinal meningitis: a historical perspective and critical review of the data.
Infect. Genet. Evol.
PUBLISHED: 02-05-2010
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Since the first clinical description by Vieusseux (1805) of the epidemic form of meningitis known today as cerebrospinal meningitis, numerous epidemic outbreaks of the disease were reported globally during the nineteenth and early twentieth century. Historical medical data confirmed that clinical disease may occur either sporadically or in an epidemic form. Moreover, it may afflict children, young military recruits and/or populations living under crowded conditions. In 1932, an epidemic of meningitis occurred in Macau. The disease was sufficiently unusual to justify the publication of a special report by the Portuguese physician in charge of the control services of the epidemic. Here we present a critical review of the Macau epidemic data.
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Point of care strategy for rapid diagnosis of novel A/H1N1 influenza virus.
PLoS ONE
PUBLISHED: 01-25-2010
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Within months of the emergence of the novel A/H1N1 pandemic influenza virus (nA/H1N1v), systematic screening for the surveillance of the pandemic was abandoned in France and in some other countries. At the end of June 2009, we implemented, for the public hospitals of Marseille, a Point Of Care (POC) strategy for rapid diagnosis of the novel A/H1N1 influenza virus, in order to maintain local surveillance and to evaluate locally the kinetics of the pandemic.
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Novel virus influenza A (H1N1sw) in South-Eastern France, April-August 2009.
PLoS ONE
PUBLISHED: 01-25-2010
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In April 2009, the first cases of pandemic (H1N1)-2009 influenza [H1N1sw] virus were detected in France. Virological surveillance was undertaken in reference laboratories of the seven French Defence Zones.
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Nonstructural NS1 proteins of several mosquito-borne Flavivirus do not inhibit TLR3 signaling.
Virology
PUBLISHED: 01-22-2010
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Flaviviruses are single-stranded positive RNA viruses that replicate through double stranded RNA (dsRNA) intermediates. These dsRNA may be recognized as pathogen-associated molecular patterns by cellular receptors including membrane-bound Toll-like receptor 3 (TLR3) and cytosolic helicases RIG-I and MDA5. dsRNA stimulation results in signaling cascades converging to activation of interferon (IFN) regulatory factor 3 (IRF3) and to transcriptional activation of several interferon stimulated genes, including IFNss and inflammatory cytokines. There are conflicting reports concerning the ability of West Nile virus to counteract TLR3 signaling. In our analyses, transiently or stably expressed NS1 proteins from two West Nile viruses, two dengue 2 viruses and a yellow fever virus failed to inhibit TLR3 signaling in two different mammalian cell lines. Moreover, using siRNA inhibiting the helicase signalization pathway, we show that viral infection did not impede TLR3 responses to poly(I:C). We conclude that NS1 proteins from distinct mosquito-borne flaviviruses do not inhibit TLR3 signaling.
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High prevalence of both humoral and cellular immunity to Zaire ebolavirus among rural populations in Gabon.
PLoS ONE
PUBLISHED: 01-20-2010
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To better understand Zaire ebolavirus (ZEBOV) circulation and transmission to humans, we conducted a large serological survey of rural populations in Gabon, a country characterized by both epidemic and non epidemic regions. The survey lasted three years and covered 4,349 individuals from 220 randomly selected villages, representing 10.7% of all villages in Gabon. Using a sensitive and specific ELISA method, we found a ZEBOV-specific IgG seroprevalence of 15.3% overall, the highest ever reported. The seroprevalence rate was significantly higher in forested areas (19.4%) than in other ecosystems, namely grassland (12.4%), savannah (10.5%), and lakeland (2.7%). No other risk factors for seropositivity were found. The specificity of anti-ZEBOV IgG was confirmed by Western blot in 138 individuals, and CD8 T cells from seven IgG+ individuals were shown to produce IFN-gamma after ZEBOV stimulation. Together, these findings show that a large fraction of the human population living in forested areas of Gabon has both humoral and cellular immunity to ZEBOV. In the absence of identified risk factors, the high prevalence of "immune" persons suggests a common source of human exposure such as fruits contaminated by bat saliva. These findings provide significant new insights into ZEBOV circulation and human exposure, and raise important questions as to the human pathogenicity of ZEBOV and the existence of natural protective immunization.
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Punique virus, a novel phlebovirus, related to sandfly fever Naples virus, isolated from sandflies collected in Tunisia.
J. Gen. Virol.
PUBLISHED: 01-20-2010
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Sandflies are widely distributed around the Mediterranean Basin. Therefore, human populations in this area are potentially exposed to sandfly-transmitted diseases, including those caused by phleboviruses. Whilst there are substantial data in countries located in the northern part of the Mediterranean basin, few data are available for North Africa. In this study, a total of 1489 sandflies were collected in 2008 in Tunisia from two sites, bioclimatically distinct, located 235 km apart, and identified morphologically. Sandfly species comprised Phlebotomus perniciosus (52.2%), Phlebotomus longicuspis (30.1%), Phlebotomus papatasi (12.0%), Phlebotomus perfiliewi (4.6%), Phlebotomus langeroni (0.4%) and Sergentomyia minuta (0.5%). PCR screening, using generic primers for the genus Phlebovirus, resulted in the detection of ten positive pools. Sequence analysis revealed that two pools contained viral RNA corresponding to a novel virus closely related to sandfly fever Naples virus. Virus isolation in Vero cells was achieved from one pool. Genetic and phylogenetic characterization based on sequences in the three genomic segments showed that it was a novel virus distinct from other recognized members of the species. This novel virus was provisionally named Punique virus. Viral sequences in the polymerase gene corresponding to another phlebovirus closely related to but distinct from sandfly fever Sicilian virus were obtained from the eight remaining positive pools.
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Chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages.
J. Clin. Invest.
PUBLISHED: 01-06-2010
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Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that induces in humans a disease characterized by fever, rash, and pain in muscles and joints. The recent emergence or reemergence of CHIKV in the Indian Ocean Islands and India has stressed the need to better understand the pathogenesis of this disease. Previous CHIKV disease models have used young or immunodeficient mice, but these do not recapitulate human disease patterns and are unsuitable for testing immune-based therapies. Herein, we describe what we believe to be a new model for CHIKV infection in adult, immunocompetent cynomolgus macaques. CHIKV infection in these animals recapitulated the viral, clinical, and pathological features observed in human disease. In the macaques, long-term CHIKV infection was observed in joints, muscles, lymphoid organs, and liver, which could explain the long-lasting CHIKV disease symptoms observed in humans. In addition, the study identified macrophages as the main cellular reservoirs during the late stages of CHIKV infection in vivo. This model of CHIKV physiopathology should allow the development of new therapeutic and/or prophylactic strategies.
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Symptomatic infections less frequent with H1N1pdm than with seasonal strains: Antoine Flahault, Xavier de Lamballerie, Camille Pelat, Nicolas Salez, Thomas Hanslik.
PLoS Curr
PUBLISHED: 12-27-2009
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A serosurvey conducted in a sample of first quarter pregnant women in France at week 48-49 of 2009 exhibit a seroprevalence level of 10.6%. It has been extrapolated in male and female population living in France mainland, aged 20-39 yr, that 1,712,000, 95%CI (1,112,700 - 2,311,300) people were recently infected by H1N1pdm (recently vaccinated women were excluded from analysis). From week 36 to 46-47 of 2009, 336,288, 95%CI (207,303-421,299) patients visited their general practitioners with clinical influenza in France, mainland. We then extrapolated the proportion of symptomatic H1N1pdm influenza in both males and females aged 20-39 yr who visited their GP to be 19.6%.
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Improving the diagnostic efficiency of H1N1 2009 pandemic flu: analysis of predictive clinical signs through a prospective cohort.
PLoS Curr
PUBLISHED: 10-25-2009
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In late June 2009, we set up a dedicated flu-like illness outpatient consultation in the Infectious Diseases and Tropical Medicine department of Marseille university hospital to detect the new A/H1N1 pandemic influenza and to contain efficiently the A/H1N1 infected patients. For 3 months, we compiled data corresponding to a total of 307 patients who presented with a flu-like syndrome. 31 of them were positive for H1N1 pandemic flu through real-time RT-PCR (rRT-PCR); among them, 19 were positive for a rapid influenza detection test (RIDT). We report here the significant clinical characteristics of A/H1N1 pandemic flu patients compared with other flu-like illnesses, which were used to improve the predictive value of the diagnosis in the current epidemiological situation. We found that regardless of the prevalence of A/H1N1 positive cases in the suspected patients, the absence of cough rejects the diagnosis of A/H1N1 infection in 100% of cases. Among patients referred for flu-like illness, those with cough should be tested for A/H1N1 by RIDT. In the current situation, the PPV and NPV of RIDT for H1N1 reached 90.5% and 95.8 %, respectively. It is important to notice that the 2 RIDT-positive that were negative for H1N1 were seasonal H3N2 influenza indicating that specificity and PPV of RIDT for all influenza was 100%. Therefore, positive RIDT does not require rRT-PCR confirmatory test. Only negative RIDT should be tested with rRT-PCR assay. Respecting this algorithm would have saved up to 70,000 Euros ( 100.000 USD) for the 307 patients and would have resulted in a significant gain of time to transmit the laboratory results to the clinical ward.
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Point of care strategy for rapid diagnosis of novel A/H1N1 influenza virus.
PLoS Curr
PUBLISHED: 09-22-2009
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In late June 2009, we implemented for public hospitals of Marseille Point Of Care strategy for rapid diagnosis of novel A/H1N1 influenza virus. During two months, we have tested more than 900 specimens in both Point Of Care laboratories. We believe that implementation of Point of Care strategy for the largest number of suspects cases may improve quality of patients care and our knowledge of the epidemiology of the pandemic.
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Genomics and evolution of Aedes-borne flaviviruses.
J. Gen. Virol.
PUBLISHED: 09-09-2009
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We analysed the complete coding sequences of all recognized species of Aedes-borne flavivirus, including previously uncharacterized viruses within the yellow fever virus (YFV), Spondweni virus (SPOV) and dengue virus (DENV) groups. Two major phylogenetic lineages were revealed: one included the YFV and Entebbe bat virus groups, and the other included the DENV, SPOV and Culex-borne flavivirus groups. This analysis supported previous evidence that Culex-borne flaviviruses have evolved from ancestral Aedes-borne viruses. However, the topology at the junction between these lineages remains complex and may be refined by the discovery of viruses related to the Kedougou virus. Additionally, viral evolution was found to be associated with the appearance of new biological characteristics; mutations that may modify the envelope protein structure were identified for seven viruses within the YFV group, and an expansion of host-vector range was identified in the two major evolutionary lineages, which in turn may facilitate the emergence of mosquito-borne flaviviruses.
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Isolation of a novel species of flavivirus and a new strain of Culex flavivirus (Flaviviridae) from a natural mosquito population in Uganda.
J. Gen. Virol.
PUBLISHED: 08-05-2009
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The genus Flavivirus, which contains approximately 70 single-stranded, positive-sense RNA viruses, represents a unique model for studying the evolution of vector-borne disease, as it includes viruses that are mosquito-borne, tick-borne or have no known vector. Both theoretical work and field studies suggest the existence of a large number of undiscovered flaviviruses. Recently, the first isolation of cell fusing agent virus (CFAV) was reported from a natural mosquito population in Puerto Rico, and sequences related to CFAV have been discovered in mosquitoes from Thailand. CFAV had previously been isolated from a mosquito cell line in 1975 and represented the only known insect-only flavivirus, appearing to replicate in insect cells alone. A second member of the insect-only group, Kamiti River virus (KRV), was isolated from Kenyan mosquitoes in 2003. A third tentative member of the insect-only group, Culex flavivirus (CxFV), was first isolated in 2007 from Japan and further strains have subsequently been reported from the Americas. We report the discovery, isolation and characterization of two novel insect-only flaviviruses from Entebbe, Uganda: a novel lineage tentatively designated Nakiwogo virus (NAKV) and a new strain of CxFV. The individual mosquitoes from which these strains were isolated, identified retrospectively by using a reference molecular phylogeny generated using voucher specimens from the region, were Mansonia africana nigerrima and Culex quinquefasciatus, respectively. This represents the first isolation, to our knowledge, of a novel insect-only flavivirus from a Mansonia species and the first isolation of a strain of CxFV from Africa.
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Structure and functionality in flavivirus NS-proteins: perspectives for drug design.
Antiviral Res.
PUBLISHED: 07-02-2009
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Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.
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Molecular epidemiological analysis of dengue fever in Bolivia from 1998 to 2008.
Vector Borne Zoonotic Dis.
PUBLISHED: 06-10-2009
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Dengue fever was first recognized in Bolivia in 1931. However, very limited information was available to date regarding the genetic characterization and epidemiology of Bolivian dengue virus strains. Here, we performed genetic characterization of the full-length envelope gene of 64 Bolivian isolates from 1998 to 2008 and investigated their origin and evolution to determine whether strains circulated simultaneously or alternatively, and whether or not multiple introductions of distinct viral variants had occurred during the period studied. We determined that, during the last decade, closely related viruses circulated during several consecutive years (5, 6, and 6 years for DENV-1, DENV-2, and DENV-3, respectively) and the co-circulation of two or even three serotypes was observed. Emergence of new variants (distinct from those identified during the previous episodes) was identified in the case of DENV-1 (2007 outbreak) and DENV-2 (2001 outbreak). In all cases, it is likely that the viruses originated from neighboring countries.
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The crystal structures of Chikungunya and Venezuelan equine encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket.
J. Virol.
PUBLISHED: 04-22-2009
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Macro domains (also called "X domains") constitute a protein module family present in all kingdoms of life, including viruses of the Coronaviridae and Togaviridae families. Crystal structures of the macro domain from the Chikungunya virus (an "Old World" alphavirus) and the Venezuelan equine encephalitis virus (a "New World" alphavirus) were determined at resolutions of 1.65 and 2.30 A, respectively. These domains are active as adenosine di-phosphoribose 1-phosphate phosphatases. Both the Chikungunya and the Venezuelan equine encephalitis virus macro domains are ADP-ribose binding modules, as revealed by structural and functional analysis. A single aspartic acid conserved through all macro domains is responsible for the specific binding of the adenine base. Sequence-unspecific binding to long, negatively charged polymers such as poly(ADP-ribose), DNA, and RNA is observed and attributed to positively charged patches outside of the active site pocket, as judged by mutagenesis and binding studies. The crystal structure of the Chikungunya virus macro domain with an RNA trimer shows a binding mode utilizing the same adenine-binding pocket as ADP-ribose, but avoiding the ADP-ribose 1-phosphate phosphatase active site. This leaves the AMP binding site as the sole common feature in all macro domains.
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Concurrent chikungunya and dengue virus infections during simultaneous outbreaks, Gabon, 2007.
Emerging Infect. Dis.
PUBLISHED: 04-01-2009
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An outbreak of febrile illness occurred in Gabon in 2007, with 20,000 suspected cases. Chikungunya or dengue-2 virus infections were identified in 321 patients; 8 patients had documented co-infections. Aedes albopictus was identified as the principal vector for the transmission of both viruses.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.