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1.  Complete Genomic Sequence of European Bat Lyssavirus 1, Isolated from Eptesicus isabellinus in Spain 
Genome Announcements  2015;3(1):e01518-14.
All members of the lyssavirus genus cause the disease rabies. European bat lyssavirus 1 (EBLV-1) viruses are divided genetically into three groups according to geographic location and host reservoir. We report here the first genome sequence for an EBLV-1 isolated from Eptesiscus isabellinus in the Iberian Peninsula, Spain.
doi:10.1128/genomeA.01518-14
PMCID: PMC4333652  PMID: 25676752
2.  Highly Diverse Morbillivirus-Related Paramyxoviruses in Wild Fauna of the Southwestern Indian Ocean Islands: Evidence of Exchange between Introduced and Endemic Small Mammals 
Journal of Virology  2014;88(15):8268-8277.
ABSTRACT
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.
IMPORTANCE Identification of the infectious agents that circulate within wild animal reservoirs is essential for several reasons: (i) infectious disease outbreaks often originate from wild fauna; (ii) anthropological expansion increases the risk of contact between human and animal populations and, as a result, the risk of disease emergence; (iii) evaluation of pathogen reservoirs helps in elaborating preventive measures to limit the risk of disease emergence. Many paramyxoviruses for which bats and rodents serve as major reservoirs have demonstrated their potential to cause disease in humans and animals. In the context of the biodiversity hot spot of southwestern Indian Ocean islands and their rich endemic fauna, we show that highly diverse UMRVs exchange between various endemic animal species, and their dissemination likely is facilitated by the introduced Rattus rattus. Hence, many members of the Paramyxoviridae appear well adapted for the study of the viral phylodynamics that may be associated with disease emergence.
doi:10.1128/JVI.01211-14
PMCID: PMC4135957  PMID: 24829336
3.  Risk factors of pandemic influenza A/H1N1 in a prospective household cohort in the general population: results from the CoPanFlu-France cohort 
Background
The CoPanFlu-France household cohort was set up in 2009 to identify risk factors of infection by the pandemic A/H1N1 (H1N1pdm09) virus in the general population.
Objectives
To investigate the determinants of infection during the 2010–2011 season, the first complete influenza season of study follow-up for this cohort.
Patients/Methods
Pre- and post-epidemic blood samples were collected for all subjects, and nasal swabs were obtained in all subjects from households where an influenza-like illness was reported. Cases were defined as either a fourfold increase in the serological titer or a laboratory-confirmed H1N1pdm09 on a nasal swab, with either RT-PCR or multiplex PCR. Risk factors for H1N1pdm09 infections were explored, without any pre-specified hypothesis, among 167 individual, collective and environmental covariates via generalized estimating equations modeling. We adopted a multimodel selection procedure to control for model selection uncertainty.
Results
This analysis is based on a sample size of 1121 subjects. The final multivariable model identified one risk factor (history of asthma, OR = 2·17; 95% CI: 1·02–4·62) and three protective factors: pre-epidemic serological titer (OR = 0·51 per doubling of the titer; 95% CI: 0·39–0·67), green tea consumption a minimum of two times a week (OR = 0·39; 95% CI: 0·18–0·84), and proportion of subjects in the household always covering their mouth while coughing/sneezing (OR = 0·93 per 10% increase; 95% CI: 0·86–1·00).
Conclusion
This exploratory study provides further support of previously reported risk factors and highlights the importance of collective protective behaviors in the household. Further analyses will be conducted to explore these findings.
doi:10.1111/irv.12294
PMCID: PMC4280818  PMID: 25495468
Cohort studies; France; influenza A virus, H1N1 subtype; risk factor
4.  First Reported Chikungunya Fever Outbreak in the Republic of Congo, 2011 
PLoS ONE  2014;9(12):e115938.
Background
Chikungunya is an Aedes -borne disease characterised by febrile arthralgia and responsible for massive outbreaks. We present a prospective clinical cohort study and a retrospective serological study relating to a CHIK outbreak, in the Republic of Congo in 2011.
Methodology and Findings
We analysed 317 suspected cases, of which 308 (97.2%) lived in the city of Brazzaville (66.6% in the South area). Amongst them, 37 (11.7%) were CHIKV+ve patients (i.e., biologically confirmed by a real-time RT-PCR assay), of whom 36 (97.3%) had fever, 22 (66.7%) myalgia and 32 (86.5%) arthralgia. All tested negative for dengue. The distribution of incident cases within Brazzaville districts was compared with CHIKV seroprevalence before the outbreak (34.4% in 517 blood donors), providing evidence for previous circulation of CHIKV. We applied a CHIK clinical score to 126 patients recruited within the two first day of illness (including 28 CHIKV+ves (22.2%)) with sensitivity (78.6%) and specificity (72.4%) values comparing with those of the referent study in Reunion Island. The negative predictive value was high (92%), but the positive predictive value (45%) indicate poor potential contribution to medical practice to identify CHIKV+ve patients in low prevalence outbreaks. However, the score allowed a slightly more accurate follow-up of the evolution of the outbreak than the criterion "fever+arthralgia". The complete sequencing of a Congolase isolate (Brazza_MRS1) demonstrated belonging to the East/Central/South African lineage and was further used for producing a robust genome-scale CHIKV phylogenetic analysis.
Conclusions/Significance
We describe the first Chikungunya outbreak declared in the Republic of Congo. The seroprevalence study conducted amongst blood donors before outbreak provided evidence for previous CHIKV circulation. We suggest that a more systematic survey of the entomological situation and of arbovirus circulation is necessary in Central Africa for better understanding the environmental, microbiological and sociological determinants of emergence.
doi:10.1371/journal.pone.0115938
PMCID: PMC4277398  PMID: 25541718
5.  A Sero-epidemiological Study of Arboviral Fevers in Djibouti, Horn of Africa 
Arboviral infections have repeatedly been reported in the republic of Djibouti, consistent with the fact that essential vectors for arboviral diseases are endemic in the region. However, there is a limited recent information regarding arbovirus circulation, and the associated risk predictors to human exposure are largely unknown. We performed, from November 2010 to February 2011 in the Djibouti city general population, a cross-sectional ELISA and sero-neutralisation-based sero-epidemiological analysis nested in a household cohort, which investigated the arboviral infection prevalence and risk factors, stratified by their vectors of transmission. Antibodies to dengue virus (21.8%) were the most frequent. Determinants of infection identified by multivariate analysis pointed to sociological and environmental exposure to the bite of Aedes mosquitoes. The population was broadly naïve against Chikungunya (2.6%) with risk factors mostly shared with dengue. The detection of limited virus circulation was followed by a significant Chikungunya outbreak a few months after our study. Antibodies to West Nile virus were infrequent (0.6%), but the distribution of cases faithfully followed previous mapping of infected Culex mosquitoes. The seroprevalence of Rift valley fever virus was 2.2%, and non-arboviral transmission was suggested. Finally, the study indicated the circulation of Toscana-related viruses (3.7%), and a limited number of cases suggested infection by tick-borne encephalitis or Alkhumra related viruses, which deserve further investigations to identify the viruses and vectors implicated. Overall, most of the arboviral cases' predictors were statistically best described by the individuals' housing space and neighborhood environmental characteristics, which correlated with the ecological actors of their respective transmission vectors' survival in the local niche. This study has demonstrated autochthonous arboviral circulations in the republic of Djibouti, and provides an epidemiological inventory, with useful findings for risk mapping and future prevention and control programs.
Author Summary
The arboviruses are a group of viruses transmitted by arthropods such as mosquitoes, ticks, or sandflies. These pathogens have complex life cycles and depend on both arthropods and vertebrate hosts for survival and transmission. Recent global increase in cases confirms that they are of great public health concern. In this study, conducted in the winter of 2010, the seroprevalence and determinants of infections were investigated in the republic of Djibouti, Horn of Africa. The highest seroprevalence values were observed for mosquito-borne diseases, in particular dengue (transmitted by Aedes mosquitoes); antibodies to dengue virus were found in a fifth of the sampled population. Most Djiboutians were initially unexposed to Chikungunya virus (also transmitted by Aedes mosquitoes), but a few months later, many got infected, resulting in an outbreak. Of the few West Nile virus seropositive cases detected, the majority were in places where WNV had been previously identified in Culex mosquitoes. In addition, seropositive cases of Toscana-related viruses (transmitted by sandflies), and tick-borne encephalitis virus or Alkhumra-related viruses (transmitted by ticks) were also observed. In this study, the risk of arboviral infections was mostly associated with environmental and behavioural risk factors, with highest risk prevailing in the city centre (District 1). Overall, the results suggest a likely exposure to the local circulation of arboviruses, rather than infections acquired outside the study area. This knowledge, therefore, confirms the impact of arbovirus infections in Djibouti, and is essential for prevention and control programs.
doi:10.1371/journal.pntd.0003299
PMCID: PMC4263616  PMID: 25502692
6.  Single-stranded positive-sense RNA viruses generated in days using infectious subgenomic amplicons 
The Journal of General Virology  2014;95(Pt 11):2462-2467.
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.
doi:10.1099/vir.0.068023-0
PMCID: PMC4202267  PMID: 25053561
7.  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 
Parasites & Vectors  2014;7(1):476.
Background
Sandfly-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.
Findings
A 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.
Conclusions
Toscana 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.
doi:10.1186/s13071-014-0476-8
PMCID: PMC4197250  PMID: 25306250
Phleboviruses; Toscana virus; Sand flies; Dogs; Sentinels; Emerging; Mediterranean basin; Bunyaviridae
8.  Novel flaviviruses from mosquitoes: Mosquito-specific evolutionary lineages within the phylogenetic group of mosquito-borne flaviviruses 
Virology  2014;464-465:320-329.
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.
Highlights
•Mosquito-borne flavivirus-related viruses were isolated from Finnish mosquitoes.•Isolates were reactive with flavivirus antibodies but appeared mosquito-specific.•Sequence analysis identified related viruses from different parts of the world.•These viruses represent unique properties among the mosquito-borne flavivirus group.
doi:10.1016/j.virol.2014.07.015
PMCID: PMC4170750  PMID: 25108382
Flavivirus; Isolate; Mosquito; Complete genome; Phylogenetic analysis
9.  “Megavirales”, a proposed new order for eukaryotic nucleocytoplasmic large DNA viruses 
Archives of virology  2013;158(12):2517-2521.
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.
doi:10.1007/s00705-013-1768-6
PMCID: PMC4066373  PMID: 23812617
10.  Complete Coding Sequence of Zika Virus from a French Polynesia Outbreak in 2013 
Genome Announcements  2014;2(3):e00500-14.
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.
doi:10.1128/genomeA.00500-14
PMCID: PMC4047448  PMID: 24903869
11.  Evaluation of Antiviral Efficacy of Ribavirin, Arbidol, and T-705 (Favipiravir) in a Mouse Model for Crimean-Congo Hemorrhagic Fever 
Background
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.
Methodology/Principal Findings
CCHF virus-infected IFNAR−/− mice died 2–6 days post infection with elevated aminotransferase levels and high virus titers in blood and organs. Main pathological alteration was acute hepatitis with extensive bridging necrosis, reactive hepatocyte proliferation, and mild to moderate inflammatory response with monocyte/macrophage activation. Virus-infected and apoptotic hepatocytes clustered in the necrotic areas. Ribavirin, arbidol, and T-705 suppressed virus replication in vitro by ≥3 log units (IC50 0.6–2.8 µg/ml; IC90 1.2–4.7 µg/ml). Ribavirin [100 mg/(kg×d)] did not increase the survival rate of IFNAR−/− mice, but prolonged the time to death (p<0.001) and reduced the aminotransferase levels and the virus titers. Arbidol [150 mg/(kg×d)] had no efficacy in vivo. Animals treated with T-705 at 1 h [15, 30, and 300 mg/(kg×d)] or up to 2 days [300 mg/(kg×d)] post infection survived, showed no signs of disease, and had no virus in blood and organs. Co-administration of ribavirin and T-705 yielded beneficial rather than adverse effects.
Conclusions/Significance
Activated hepatic macrophages and monocyte-derived cells may play a role in the proinflammatory cytokine response in CCHF. Clustering of infected hepatocytes in necrotic areas without marked inflammation suggests viral cytopathic effects. T-705 is highly potent against CCHF virus in vitro and in vivo. Its in vivo efficacy exceeds that of the current standard drug for treatment of CCHF, ribavirin.
Author Summary
Crimean-Congo hemorrhagic fever (CCHF) is endemic in Africa, Asia, southeast Europe, and the Middle East. The case fatality rate is 30–50%. Studies on pathophysiology and treatment of CCHF have been hampered by the lack of an appropriate animal model. We have employed CCHF virus-infected transgenic mice, which are defective in the innate immune response, as a disease model. These mice die from the infection and show signs of disease similar to those found in humans. First, we studied the liver pathology in the animals, as hepatic necrosis is a prominent feature of human CCHF. Secondly, we used the model to test the efficacy of antiviral drugs that are in clinical use or in an advanced stage of clinical testing. Besides ribavirin, the standard drug for treatment of CCHF, we tested arbidol, a drug in clinical use against respiratory infections, and T-705, a new drug in clinical development for the treatment of influenza virus infection. While ribavirin and arbidol showed some or no beneficial effect, respectively, T-705 was highly efficacious in the animal model. These data hold promise for clinical efficacy of T-705 in human CCHF.
doi:10.1371/journal.pntd.0002804
PMCID: PMC4006714  PMID: 24786461
12.  SYBR Green Real-Time PCR for the Detection of All Enterovirus-A71 Genogroups 
PLoS ONE  2014;9(3):e89963.
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.
doi:10.1371/journal.pone.0089963
PMCID: PMC3961242  PMID: 24651608
13.  Molecular Comparison and Evolutionary Analyses of VP1 Nucleotide Sequences of New African Human Enterovirus 71 Isolates Reveal a Wide Genetic Diversity 
PLoS ONE  2014;9(3):e90624.
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.
doi:10.1371/journal.pone.0090624
PMCID: PMC3944068  PMID: 24598878
14.  Viral aetiology influenza like illnesses in Santa Cruz, Bolivia (2010–2012) 
Virology Journal  2014;11:35.
Background
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.
Methods
It was performed in Santa Cruz city from January 2010 to September 2012, based on 564 naso-pharyngeal swabs collected in a National Reference Laboratory and real-time PCR techniques, viral cultures and phylogenetic analyses.
Results
50.2% of samples were positive for at least one virus with influenza viruses (Flu A: ~15%; Flu B: ~9%), rhinoviruses (~8%), coronaviruses (~5%) and hRSV (~4%) being the most frequently identified. The pattern of viral infections varied according to age groups. The elucidation rate was the highest (>60%) amongst patients under 10 yo and the lowest (<40%) amongst patients ≥60 yo. Nearly 3% of samples showed dual viral infections. Epidemiological peaks were associated with a predominant virus but generally included 30-50% of infections by different viruses. Unexpectedly, the frequency of influenza in the 0–4 yo population was very low and a complete hRSV eclipse occurred in 2011. Genetic analyses indicated that distinct evolutionary lineages of Flu A(H1N1)pdm2009, Flu A/H3N2 and Flu B have co-circulated in Bolivia in the study period, originating from Central and North America, Europe, Asia and Australia.
Conclusion
Our results emphasise the requirement for a reinforced epidemiological and genetic follow-up of influenza and other ILIs in Bolivia to further inform the preparation of vaccines used in the region, guide vaccination campaigns and improve the medical management of patients.
doi:10.1186/1743-422X-11-35
PMCID: PMC4015617  PMID: 24564892
Epidemiology; Influenza like illness; Bolivia; South America; Influenza A(H1N1)pdm2009; Influenza phylogeny; Respiratory infections
15.  Molecular detection of human rhinoviruses in respiratory samples: a comparison of Taqman probe-, SYBR green I- and BOXTO-based real-time PCR assays 
Virology Journal  2014;11:31.
Background
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.
Methods
5,627 respiratory samples sent to the laboratory of Virology of the University Hospitals of Marseille, France, from March 2011 to February 2012, were tested using a real-time RT-PCR assay in the 5’NCR of the rhinoviral genome that associated a Taqman probe and the detection of DNA-BOXTO-dye complexes. A sample of 500 BOXTO-positive samples were further tested using the same probe assay (without BOXTO), and a SYBR Green assay (using the same amplification primers). The specific amplification of HRV sequences was assessed by NGS amplicon sequencing.
Results
The Taqman probe RT-PCR assay identified 696/5,627 samples (12,4%) as HRV-positive. BOXTO-positive samples included all probe-positive samples and 1,913 additional samples, of which only 24.3% were confirmed by sequencing. The SYBR Green assay was more specific (16/550 samples were probe-negative/SYBR Green-positive, all confirmed by 5′NCR sequencing), but 3/500 samples were probe-positive/SYBR Green-negative.
Conclusions
Our results highlight the difficulty in detecting HRVs in clinical samples using a single molecular detection system. Amongst the 3 systems tested, the best compromise was obtained with the SYBR Green assay, which, by comparison with our probe-based assay provided an improved sensitivity without altering the detection specificity. Interestingly, a majority of probe-negative/BOXTO- or SYBR Green-positive samples were not associated with mutations in the sequence targeted by the probe. Sequence-based modifications of the secondary structure of the HRV 5′NCR may be associated with a limited access to the probe hybridisation region. Further investigations may identify a test combining a probe based- and an intercalating agent-based detection, which will significantly improve the diagnosis of HRV infections.
doi:10.1186/1743-422X-11-31
PMCID: PMC3936951  PMID: 24548758
BOXTO; SYBR green; Taqman probe; Picornaviridae; Human rhinovirus; Respiratory infections; Molecular diagnosis
16.  Estimating the Burden of Japanese Encephalitis Virus and Other Encephalitides in Countries of the Mekong Region 
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.
doi:10.1371/journal.pntd.0002533
PMCID: PMC3907313  PMID: 24498443
17.  Determinants of individuals’ risks to 2009 pandemic influenza virus infection at household level amongst Djibouti city residents - A CoPanFlu cross-sectional study 
Virology Journal  2014;11:13.
Background
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.
Methods
From the four city administrative districts, 1,045 subjects from 324 households were included during a face-to-face encounter between 11th November 2010 and 15th February 2011. Socio-demographic details were collected and blood samples were analysed in haemagglutination inhibition (HI) assays. Risk assessments were performed in a generalised estimating equation model.
Results
In this study, the indicator of positive infection status was set at an HI titre of ≥ 80, which was a relevant surrogate to the seroconversion criterion. All positive cases were considered to be either recent infections or past contact with an antigenically closely related virus in humans older than 65 years. An overall sero-prevalence of 29.1% and a geometrical mean titre (GMT) of 39.5% among the residents was observed. Youths, ≤ 25 years and the elderly, ≥65 years had the highest titres, with values of 35.9% and 29.5%, respectively. Significantly, risk was high amongst youths ≤ 25 years, (OR 1.5-2.2), residents of District 4(OR 2.9), students (OR 1.4) and individuals living near to river banks (OR 2.5). Belonging to a large household (OR 0.6), being employed (OR 0.5) and working in open space-outdoor (OR 0.4) were significantly protective. Only 1.4% of the cohort had vaccination against the pandemic virus and none were immunised against seasonal influenza.
Conclusion
Despite the limited number of incident cases detected by the surveillance system, A(H1N1)pdm09 virus circulated broadly in Djibouti in 2010 and 2011. Age-group distribution of cases was similar to what has been reported elsewhere, with youths at the greatest risk of infection. Future respiratory infection control should therefore be tailored to reach specific and vulnerable individuals such as students and those working in groups indoors. It is concluded that the lack of robust data provided by surveillance systems in southern countries could be responsible for the underestimation of the epidemiological burden, although the main characteristics are essentially similar to what has been observed in developed countries.
doi:10.1186/1743-422X-11-13
PMCID: PMC3927658  PMID: 24468218
18.  Novel Virus Discovery and Genome Reconstruction from Field RNA Samples Reveals Highly Divergent Viruses in Dipteran Hosts 
PLoS ONE  2013;8(11):e80720.
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.
doi:10.1371/journal.pone.0080720
PMCID: PMC3832450  PMID: 24260463
19.  Co-Circulation of Toscana Virus and Punique Virus in Northern Tunisia: A Microneutralisation-Based Seroprevalence Study 
Background
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.
Methods
The presence of TOSV and PUNV neutralising antibodies (NT-Ab) was tested in human sera collected from 5 districts of the governorate of Bizerte, and the titres of NT-Ab were estimated by microneutralisation (MN) assay.
Principal Findings
A total of 1,273 sera were processed. TOSV and PUNV NT-Ab were detected in 522 (41%) and 111 sera (8.72%) respectively. TOSV seroprevalence varied from 17.2% to 59.4% depending on the district. Analysis of TOSV geometric mean titre values demonstrated a constant increase according to the age. The vast majority of sera containing NT-Ab were found to be more reactive toward TOSV than PUNV. Indeed, past infections with PUNV and TOSV were undisputable for 5 and 414 sera, respectively.
Conclusions
PUNV may be capable to infect humans but at a low rate. TOSV is responsible for the vast majority of human infections by sand fly-borne phleboviruses in northern Tunisia. TOSV must be considered by physician and tested in diagnostic laboratories for patients with meningitis and unexplained fever in northern Tunisia.
Author Summary
In northern Tunisia, two different pheboviruses are known to circulate in sand fly population, Toscana virus (TOSV) and Punique virus (PUNV). In contrast to TOSV, a prominent human pathogen, there is no data supporting that PUNV is capable to infect humans and to cause a disease. We studied the respective involvement of TOSV and PUNV in human infections in northern Tunisia through a seroprevalence study. Because TOSV and PUNV are antigenically and genetically closely related, it is difficult to distinguish between them by using broadly reactive serological tests, such as enzyme-linked immunosorbent assay (ELISA). Thus, we developed a method of microneutralisation assay using the two viruses in a comparative manner. A total of 1,273 sera were processed. We provide first evidence to support (i) that Punique virus may be capable to infect humans but at a low rate, (ii) that TOSV, the most prevalent arbovirus in Southern Europe, is responsible for the vast majority of human infections by sand fly-borne phleboviruses in northern Tunisia. Therefore, it is important to consider TOSV as an important pathogen that needs to be included in all virological diagnostic concerning patients with meningitis and unexplained febrile illness originated from Northern Tunisia.
doi:10.1371/journal.pntd.0002429
PMCID: PMC3772032  PMID: 24069484
20.  An Epidemic of Dengue-1 in a Remote Village in Rural Laos 
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.
Author Summary
Dengue disease is caused by a virus transmitted by mosquitoes. In Southeast Asia, where it is endemic, it represents a very important public health problem. Major outbreaks, including severe cases and death, occur every year. Two distinct transmission cycles have been described. Most common is the human-mosquito-human cycle observed throughout most tropical regions of the world, often associated with urban locations and always human habitations, often producing explosive outbreaks, whereas “sylvatic” dengue, genetically different, circulates in forest wild animals and has been reported to be able to infect humans. In the Lao PDR, a developing country where dengue is endemic, data on this disease are sparse. This study reports an unusual outbreak of dengue that occurred during the cold season in a village in a forested area. It also is the first extensive analysis of dengue virus nucleotide sequences, from 39 patients across the country, from Laos. Results suggest three patterns of dengue circulation in Laos: local transmission, transmission over the whole country, and transmission implicating bordering countries. The dengue virus isolated from patients in the forest village outbreak proved to be genetically similar to those found in urbanized areas throughout the country. More investigations are needed to understand the relationships between dengue in forested and urban areas.
doi:10.1371/journal.pntd.0002360
PMCID: PMC3738459  PMID: 23951379
21.  Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drug 
Objectives
Infection with yellow fever virus (YFV), the prototypic mosquito-borne flavivirus, causes severe febrile disease with haemorrhage, multi-organ failure and a high mortality. Moreover, in recent years the Flavivirus genus has gained further attention due to re-emergence and increasing incidence of West Nile, dengue and Japanese encephalitis viruses. Potent and safe antivirals are urgently needed.
Methods
Starting from the crystal structure of the NS3 helicase from Kunjin virus (an Australian variant of West Nile virus), we identified a novel, unexploited protein site that might be involved in the helicase catalytic cycle and could thus in principle be targeted for enzyme inhibition. In silico docking of a library of small molecules allowed us to identify a few selected compounds with high predicted affinity for the new site. Their activity against helicases from several flaviviruses was confirmed in in vitro helicase/enzymatic assays. The effect on the in vitro replication of flaviviruses was then evaluated.
Results
Ivermectin, a broadly used anti-helminthic drug, proved to be a highly potent inhibitor of YFV replication (EC50 values in the sub-nanomolar range). Moreover, ivermectin inhibited, although less efficiently, the replication of several other flaviviruses, i.e. dengue fever, Japanese encephalitis and tick-borne encephalitis viruses. Ivermectin exerts its effect at a timepoint that coincides with the onset of intracellular viral RNA synthesis, as expected for a molecule that specifically targets the viral helicase.
Conclusions
The well-tolerated drug ivermectin may hold great potential for treatment of YFV infections. Furthermore, structure-based optimization may result in analogues exerting potent activity against flaviviruses other than YFV.
doi:10.1093/jac/dks147
PMCID: PMC3888155  PMID: 22535622
antiviral drug discovery; flavivirus helicase inhibition; new use of existing drug; in silico docking; structure-based drug design
22.  Next generation sequencing of viral RNA genomes 
BMC Genomics  2013;14:444.
Background
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.
Results
As representative RNA viruses, full genome sequence was successfully obtained from known lyssaviruses belonging to recognized species and a novel lyssavirus species using these protocols and assembling the reads using de novo algorithms. Furthermore, genome sequences were generated from considerably less than 200 ng RNA, indicating that manufacturers’ minimum template guidance is conservative. In addition to obtaining genome consensus sequence, a high proportion of SNPs (Single Nucleotide Polymorphisms) were identified in the majority of samples analyzed.
Conclusions
The approaches reported clearly facilitate successful full genome lyssavirus sequencing and can be universally applied to discovering and obtaining consensus genome sequences of RNA viruses from a variety of sources.
doi:10.1186/1471-2164-14-444
PMCID: PMC3708773  PMID: 23822119
Next generation sequencing; Pyrosequencing; Lyssavirus; Genome; RNA; Virus
23.  Causes of non-malarial fever in Laos: a prospective study 
The Lancet Global Health  2013;1(3):e46-e54.
Summary
Background
Because of reductions in the incidence of Plasmodium falciparum malaria in Laos, identification of the causes of fever in people without malaria, and discussion of the best empirical treatment options, are urgently needed. We aimed to identify the causes of non-malarial acute fever in patients in rural Laos.
Methods
For this prospective study, we recruited 1938 febrile patients, between May, 2008, and December, 2010, at Luang Namtha provincial hospital in northwest Laos (n=1390), and between September, 2008, and December, 2010, at Salavan provincial hospital in southern Laos (n=548). Eligible participants were aged 5–49 years with fever (≥38°C) lasting 8 days or less and were eligible for malaria testing by national guidelines.
Findings
With conservative definitions of cause, we assigned 799 (41%) patients a diagnosis. With exclusion of influenza, the top five diagnoses when only one aetiological agent per patient was identified were dengue (156 [8%] of 1927 patients), scrub typhus (122 [7%] of 1871), Japanese encephalitis virus (112 [6%] of 1924), leptospirosis (109 [6%] of 1934), and bacteraemia (43 [2%] of 1938). 115 (32%) of 358 patients at Luang Namtha hospital tested influenza PCR-positive between June and December, 2010, of which influenza B was the most frequently detected strain (n=121 [87%]). Disease frequency differed significantly between the two sites: Japanese encephalitis virus infection (p=0·04), typhoid (p=0·006), and leptospirosis (p=0·001) were more common at Luang Namtha, whereas dengue and malaria were more common at Salavan (all p<0·0001). With use of evidence from southeast Asia when possible, we estimated that azithromycin, doxycycline, ceftriaxone, and ofloxacin would have had significant efficacy for 258 (13%), 240 (12%), 154 (8%), and 41 (2%) of patients, respectively.
Interpretation
Our findings suggest that a wide range of treatable or preventable pathogens are implicated in non-malarial febrile illness in Laos. Empirical treatment with doxycycline for patients with undifferentiated fever and negative rapid diagnostic tests for malaria and dengue could be an appropriate strategy for rural health workers in Laos.
Funding
Wellcome Trust, WHO–Western Pacific Region, Foundation for Innovative New Diagnostics, US Centers for Disease Control and Prevention.
doi:10.1016/S2214-109X(13)70008-1
PMCID: PMC3986032  PMID: 24748368
24.  2009 A(H1N1) Seroconversion Rates and Risk Factors among the General Population in Vientiane Capital, Laos 
PLoS ONE  2013;8(4):e61909.
Objective
To assess 2009 A(H1N1) seroconversion rates and their determinants within an unvaccinated population in Vientiane Capital, Laos.
Methods
CoPanFlu Laos, a general population cohort of 807 households and 4,072 participants was established in March 2010. Sociodemographic data, epidemiological data, and capillary blood samples were collected from all the household members in March, and again in October 2010, in order to assess the level of antibodies to 2009 A(H1N1) with the haemagglutination inhibition assay. 2009 A(H1N1) seroconversion was defined as a fourfold or greater increase in titre between inclusion and follow-up. Determinants for pandemic influenza infection were studied using the generalized estimating equations model, taking household clustering into account.
Results
Between March and November 2010, 3,524 paired sera were tested. Prior to the pandemic, our cohort was almost completely vaccine-naive for seasonal influenza. The overall seroconversion rate among nonvaccinated individuals (n = 2,810) was 14.3% (95%CI [13.0, 15.6]), with the highest rate for participants under 20 yo (19.8%, 95%CI [17.4, 22.4]) and the lowest rate for participants over 60 yo (6.5%, 95%CI [3.7, 10.4]). Participants with lower baseline titres had significantly higher infection rates, with a dose-effect relationship. Odds ratios (ORs) ranged from 76.5 (95%CI [27.1, 215.8]), for those with a titre at inclusion of 1∶10, to 8.1 (95%CI [3.3, 20.4]), for those with a titre of 1∶40. Having another household member with a titre ≥1∶80 was associated with a higher likelihood of immunity (OR = 3.3, 95%CI [2.8, 3.9]).
Conclusion
The determinants and age distribution for seroconversion within a vaccine-naive population were similar to those found in developed countries. This pandemic was characterized by strong epidemiological determinants, regardless of geographical zone and level of development. Moreover, we detected pre-existing cross-reacting antibodies in participants over 60 yo, which could not have originated from former multiple vaccination as has been suggested elsewhere.
doi:10.1371/journal.pone.0061909
PMCID: PMC3630132  PMID: 23637928
25.  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  2013;8(4):e60127.
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.
doi:10.1371/journal.pone.0060127
PMCID: PMC3629047  PMID: 23613718

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