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1.  Evaluation of Four Commercial Multiplex Molecular Tests for the Diagnosis of Acute Respiratory Infections 
PLoS ONE  2015;10(6):e0130378.
Acute Respiratory Infections (ARIs) are responsible for considerable morbidity and mortality worldwide. Documentation of respiratory specimens can help for an appropriate clinical management with a significant effect on the disease progress in patient, the antimicrobial therapy used and the risk of secondary spread of infection. Here, we compared the performances of four commercial multiplex kits used in French University Hospital diagnostic microbiology laboratories for the detection of ARI pathogens (i.e., the xTAG Respiratory Viral Panel Fast, RespiFinder SMART 22, CLART PneumoVir and Fast Track Diagnostics Respiratory Pathogen 33 kits). We used a standardised nucleic acids extraction protocol and a comprehensive comparative approach that mixed reference to well established real-time PCR detection techniques and analysis of convergent positive results. We tested 166 respiratory clinical samples and identified a global high degree of correlation for at least three of the techniques (xTAG, RespiFinder and FTD33). For these techniques, the highest Youden’s index (YI), positive predictive (PPV) and specificity (Sp) values were observed for Core tests (e.g., influenza A [YI:0.86–1.00; PPV:78.95–100.00; Sp:97.32–100.00] & B [YI:0.44–1.00; PPV:100.00; Sp:100.00], hRSV [YI:0.50–0.99; PPV:85.71–100.00; Sp:99.38–100.00], hMPV [YI:0.71–1.00; PPV:83.33–100.00; Sp:99.37–100.00], EV/hRV [YI:0.62–0.82; PPV:93.33–100.00; Sp:94.48–100.00], AdV [YI:1.00; PPV:100.00; Sp:100.00] and hBoV [YI:0.20–0.80; PPV:57.14–100.00; Sp:98.14–100.00]). The present study completed an overview of the multiplex techniques available for the diagnosis of acute respiratory infections.
PMCID: PMC4481272  PMID: 26107509
2.  Risk Factors Associated with Ebola and Marburg Viruses Seroprevalence in Blood Donors in the Republic of Congo 
PLoS Neglected Tropical Diseases  2015;9(6):e0003833.
Ebola and Marburg viruses (family Filoviridae, genera Ebolavirus and Marburgvirus) cause haemorrhagic fevers in humans, often associated with high mortality rates. The presence of antibodies to Ebola virus (EBOV) and Marburg virus (MARV) has been reported in some African countries in individuals without a history of haemorrhagic fever. In this study, we present a MARV and EBOV seroprevalence study conducted amongst blood donors in the Republic of Congo and the analysis of risk factors for contact with EBOV.
Methodology and Findings
In 2011, we conducted a MARV and EBOV seroprevalence study amongst 809 blood donors recruited in rural (75; 9.3%) and urban (734; 90.7%) areas of the Republic of Congo. Serum titres of IgG antibodies to MARV and EBOV were assessed by indirect double-immunofluorescence microscopy. MARV seroprevalence was 0.5% (4 in 809) without any identified risk factors. Prevalence of IgG to EBOV was 2.5%, peaking at 4% in rural areas and in Pointe Noire. Independent risk factors identified by multivariate analysis were contact with bats and exposure to birds.
This MARV and EBOV serological survey performed in the Republic of Congo identifies a probable role for environmental determinants of exposure to EBOV. It highlights the requirement for extending our understanding of the ecological and epidemiological risk of bats (previously identified as a potential ecological reservoir) and birds as vectors of EBOV to humans, and characterising the protection potentially afforded by EBOV-specific antibodies as detected in blood donors.
Author Summary
Ebola and Marburg viruses cause haemorrhagic fevers often fatal to humans. Here, we looked for antibodies to Ebola and Marburg viruses (i.e., markers of previous contact with these viruses) in Congolese blood donors with no previous history of haemorrhagic fever. We found serologic evidence for contact with Marburg and Ebola viruses in 0.5% and 2.5% of blood donors, respectively. The circulation of Marburg virus occurs at a very low rate without any identified risk factor. In contrast, prevalence to Ebola virus was peaking at 4% in rural areas and in Pointe Noire city. Importantly, we identified that contacts with bats and birds constituted two independent environmental determinants of exposure. This study confirms that contact with Ebola virus is not infrequent in Congo and can occur in the absence of haemorrhagic fever. It highlights the requirement for further investigating the role of bats and birds in the ecological cycle of Ebola, and for determining whether asymptomatic contact with Ebola virus can provide subsequent protection against severe forms of the Ebola disease.
PMCID: PMC4457487  PMID: 26047124
3.  Dengue Seroprevalence in the French West Indies: A Prospective Study in Adult Blood Donors 
Using an anti-dengue immunoglobulin G (IgG) indirect enzyme-linked immunosorbent assay, seroprevalence was determined among 783 adult blood donors in the French Caribbean islands of Guadeloupe and Martinique in 2011. Overall, 93.5% [91.5; 95.1] samples were positive for dengue antibodies, 90.7% (350 of 386) in Martinique and 96.2% (382 of 397) in Guadeloupe. Only 30% of these adults recalled having had dengue disease before. Serotype-specific neutralization assays applied to a subset of IgG-positive samples indicated that a majority (77 of 96; 80%) reacted to the four serotypes. These seroprevalence findings are the first reported for Guadeloupe and Martinique and are consistent with the dengue epidemiology in these territories.
PMCID: PMC4458816  PMID: 25846291
4.  Chikungunya Virus Transmission Potential by Local Aedes Mosquitoes in the Americas and Europe 
PLoS Neglected Tropical Diseases  2015;9(5):e0003780.
Chikungunya virus (CHIKV), mainly transmitted in urban areas by the mosquitoes Aedes aegypti and Aedes albopictus, constitutes a major public health problem. In late 2013, CHIKV emerged on Saint-Martin Island in the Caribbean and spread throughout the region reaching more than 40 countries. Thus far, Ae. aegypti mosquitoes have been implicated as the sole vector in the outbreaks, leading to the hypothesis that CHIKV spread could be limited only to regions where this mosquito species is dominant.
Methodology/Principal Findings
We determined the ability of local populations of Ae. aegypti and Ae. albopictus from the Americas and Europe to transmit the CHIKV strain of the Asian genotype isolated from Saint-Martin Island (CHIKV_SM) during the recent epidemic, and an East-Central-South African (ECSA) genotype CHIKV strain isolated from La Réunion Island (CHIKV_LR) as a well-characterized control virus. We also evaluated the effect of temperature on transmission of CHIKV_SM by European Ae. albopictus. We found that (i) Aedes aegypti from Saint-Martin Island transmit CHIKV_SM and CHIKV_LR with similar efficiency, (ii) Ae. aegypti from the Americas display similar transmission efficiency for CHIKV_SM, (iii) American and European populations of the alternative vector species Ae. albopictus were as competent as Ae. aegypti populations with respect to transmission of CHIKV_SM and (iv) exposure of European Ae. albopictus to low temperatures (20°C) significantly reduced the transmission potential for CHIKV_SM.
CHIKV strains belonging to the ECSA genotype could also have initiated local transmission in the new world. Additionally, the ongoing CHIKV outbreak in the Americas could potentially spread throughout Ae. aegypti- and Ae. albopictus-infested regions of the Americas with possible imported cases of CHIKV to Ae. albopictus-infested regions in Europe. Colder temperatures may decrease the local transmission of CHIKV_SM by European Ae. albopictus, potentially explaining the lack of autochthonous transmission of CHIKV_SM in Europe despite the hundreds of imported CHIKV cases returning from the Caribbean.
Author Summary
More than one million chikungunya cases have been reported in the Americas since October 2013, when the Asian genotype of chikungunya virus (CHIKV) was imported by a traveller returning from Asia. CHIKV is mainly transmitted in urban areas by the domestic mosquitoes Aedes aegypti and Aedes albopictus. In this study, we evaluate the potential for the CHIKV circulating in the Caribbean to initiate outbreaks in Aedes-infested regions of continental America and Europe by assessing the ability of local mosquitoes to experimentally transmit the virus. Mosquitoes were exposed to a blood-meal containing the virus which must overcome several barriers to infect various tissues in the vector before being secreted in the mosquito saliva when biting a host. We found that Ae. aegypti and Ae. albopictus transmitted similarly the virus. When exposing Ae. albopictus from Europe at a temperature of 20°C after infection, we detect a significant drop of CHIKV transmission potential. Our results suggest that the CHIKV outbreak in the Americas could potentially spread throughout Ae. aegypti- and Ae. albopictus-infested regions of the Americas however with a limited risk of spillovers in Ae. albopictus-infested regions in Europe. These data will be useful for adapting vector control strategies and epidemiological surveillance.
PMCID: PMC4439146  PMID: 25993633
5.  Caribbean and La Réunion Chikungunya Virus Isolates Differ in Their Capacity To Induce Proinflammatory Th1 and NK Cell Responses and Acute Joint Pathology 
Journal of Virology  2015;89(15):7955-7969.
Chikungunya virus (CHIKV) is a mosquito-borne arthralgic alphavirus that has garnered international attention as an important emerging pathogen since 2005. More recently, it invaded the Caribbean islands and the Western Hemisphere. Intriguingly, the current CHIKV outbreak in the Caribbean is caused by the Asian CHIKV genotype, which differs from the La Réunion LR2006 OPY1 isolate belonging to the Indian Ocean lineage. Here, we adopted a systematic and comparative approach against LR2006 OPY1 to characterize the pathogenicity of the Caribbean CNR20235 isolate and consequential host immune responses in mice. Ex vivo infection using primary mouse tail fibroblasts revealed a weaker replication efficiency by CNR20235 isolate. In the CHIKV mouse model, CNR20235 infection induced an enervated joint pathology characterized by moderate edema and swelling, independent of mononuclear cell infiltration. Based on systemic cytokine analysis, localized immunophenotyping, and gene expression profiles in the popliteal lymph node and inflamed joints, two pathogenic phases were defined for CHIKV infection: early acute (2 to 3 days postinfection [dpi]) and late acute (6 to 8 dpi). Reduced joint pathology during early acute phase of CNR20235 infection was associated with a weaker proinflammatory Th1 response and natural killer (NK) cell activity. The pathological role of NK cells was further demonstrated as depletion of NK cells reduced joint pathology in LR2006 OPY1. Taken together, this study provides evidence that the Caribbean CNR20235 isolate has an enfeebled replication and induces a less pathogenic response in the mammalian host.
IMPORTANCE The introduction of CHIKV in the Americas has heightened the risk of large-scale outbreaks due to the close proximity between the United States and the Caribbean. The immunopathogenicity of the circulating Caribbean CHIKV isolate was explored, where it was demonstrated to exhibit reduced infectivity resulting in a weakened joint pathology. Analysis of serum cytokine levels, localized immunophenotyping, and gene expression profiles in the organs revealed that a limited Th1 response and reduced NK cells activity could underlie the reduced pathology in the host. Interestingly, higher asymptomatic infections were observed in the Caribbean compared to the La Réunion outbreaks in 2005 and 2006. This is the first study that showed an association between key proinflammatory factors and pathology-mediating leukocytes with a less severe pathological outcome in Caribbean CHIKV infection. Given the limited information regarding the sequela of Caribbean CHIKV infection, our study is timely and will aid the understanding of this increasingly important disease.
PMCID: PMC4505608  PMID: 25995257
6.  Effect of Chemical Stabilizers on the Thermostability and Infectivity of a Representative Panel of Freeze Dried Viruses 
PLoS ONE  2015;10(4):e0118963.
As a partner of the European Virus Archive (EVA) FP7 project, our laboratory maintains a large collection of freeze-dried viruses. The distribution of these viruses to academic researchers, public health organizations and industry is one major aim of the EVA consortium. It is known that lyophilization requires appropriate stabilizers to prevent inactivation of the virus. However, few studies have investigated the influence of different stabilizers and lyophilization protocols on the thermostability of different viruses. In order to identify optimal lyophilization conditions that will deliver maximum retention of viral infectivity titre, different stabilizer formulations containing trehalose, sorbitol, sucrose or foetal bovine serum were evaluated for their efficacy in stabilizing a representative panel of freeze dried viruses at different storage temperatures (-20°C, +4°C and +20°C) for one week, the two latter mimicking suboptimal shipping conditions. The Tissue Culture Infectious Dose 50% (TCID50) assay was used to compare the titres of infectious virus. The results obtained using four relevant and model viruses (enveloped/non enveloped RNA/DNA viruses) still serve to improve the freeze drying conditions needed for the development and the distribution of a large virus collection.
PMCID: PMC4414529  PMID: 25923434
7.  Complete Genomic Sequence of Rabies Virus from an Ethiopian Wolf 
Genome Announcements  2015;3(2):e00157-15.
Ethiopian wolves are the rarest canid in the world, with only 500 found in the Ethiopian highlands. Rabies poses the most immediate threat to their survival, causing epizootic cycles of mass mortality. The complete genome sequence of a rabies virus (RABV) derived from an Ethiopian wolf during the most recent epizootic is reported here.
PMCID: PMC4384137  PMID: 25814597
8.  Attenuation of Tick-Borne Encephalitis Virus Using Large-Scale Random Codon Re-encoding 
PLoS Pathogens  2015;11(3):e1004738.
Large-scale codon re-encoding (i.e. introduction of a large number of synonymous mutations) is a novel method of generating attenuated viruses. Here, it was applied to the pathogenic flavivirus, tick-borne encephalitis virus (TBEV) which causes febrile illness and encephalitis in humans in forested regions of Europe and Asia. Using an infectious clone of the Oshima 5–10 strain ("wild-type virus"), a cassette of 1.4kb located in the NS5 coding region, was modified by randomly introducing 273 synonymous mutations ("re-encoded virus"). Whilst the in cellulo replicative fitness of the re-encoded virus was only slightly reduced, the re-encoded virus displayed an attenuated phenotype in a laboratory mouse model of non-lethal encephalitis. Following intra-peritoneal inoculation of either 2.105 or 2.106 TCID50 of virus, the frequency of viraemia, neurovirulence (measured using weight loss and appearance of symptoms) and neuroinvasiveness (detection of virus in the brain) were significantly decreased when compared with the wild-type virus. Mice infected by wild-type or re-encoded viruses produced comparable amounts of neutralising antibodies and results of challenge experiments demonstrated that mice previously infected with the re-encoded virus were protected against subsequent infection by the wild-type virus. This constitutes evidence that a mammalian species can be protected against infection by a virulent wild-type positive-stranded RNA virus following immunisation with a derived randomly re-encoded strain. Our results demonstrate that random codon re-encoding is potentially a simple and effective method of generating live-attenuated vaccine candidates against pathogenic flaviviruses.
Author Summary
The arbovirus Tick-borne encephalitis virus (TBEV; genus Flavivirus) is transmitted by ticks of the Ixodes genus. TBEV causes febrile illness and encephalitis in humans in forested regions of Europe and Asia. The incidence of TBE is increasing across Central and Eastern European countries despite the availability of several licensed inactivated vaccines and appropriate vaccination programmes. Large-scale codon re-encoding, a recently developed attenuation method that modifies viral RNA nucleotide composition of large coding regions without alteration of the encoded proteins, has been successfully applied to a variety of RNA viruses. In contrast with previous empirical methods of generating live attenuated vaccines, large-scale codon re-encoding facilitates rapid generation of vaccine candidates using reverse genetics methods, by direct control of the attenuation phenotype. Additional benefits include reduced costs and induction of long-term immunity. Here, we have applied the large-scale codon re-encoding method to the TBEV to demonstrate the principle of developing a live attenuated virus vaccine which protects mice against subsequent infection with the wild type virulent virus. This study therefore illustrates that codon re-encoding is potentially an easily derived and effective method of producing live attenuated vaccine candidates against positive-stranded RNA viruses.
PMCID: PMC4348424  PMID: 25734338
9.  Causal analysis of H1N1pdm09 influenza infection risk in a household cohort 
Obtaining a comprehensive quantitative figure of the determinants of influenza infection will help identify priority targets for future influenza mitigation interventions. We developed an original causal model integrating highly diverse factors and their dependencies, to identify the most critical determinants of pandemic influenza infection (H1N1pdm09) during the 2010–2011 influenza season.
We used data from 601 households (1450 participants) included in a dedicated cohort. Structural equations were used to model direct and indirect relationships between infection and risk perception, compliance with preventive behaviours, social contacts, indoor and outdoor environment, sociodemographic factors and pre-epidemic host susceptibility. Standardised estimates (βstd) were used to assess the strength of associations (ranging from −1 for a completely negative association to 1 for a completely positive association).
Host susceptibility to H1N1pdm09 and compliance with preventive behaviours were the only two factors directly associated with the infection risk (βstd=0.31 and βstd=−0.21). Compliance with preventive behaviours was influenced by risk perception and preventive measures perception (βstd=0.14 and βstd=0.27). The number and duration of social contacts were not associated with H1N1pdm09 infection.
Our findings suggest that influenza vaccination in addition to public health communication campaigns focusing on personal preventive measures should be prioritised as potentially efficient interventions to mitigate influenza epidemics.
PMCID: PMC4345517  PMID: 25416792
10.  New Insights into Flavivirus Evolution, Taxonomy and Biogeographic History, Extended by Analysis of Canonical and Alternative Coding Sequences 
PLoS ONE  2015;10(2):e0117849.
To generate the most diverse phylogenetic dataset for the flaviviruses to date, we determined the genomic sequences and phylogenetic relationships of 14 flaviviruses, of which 10 are primarily associated with Culex spp. mosquitoes. We analyze these data, in conjunction with a comprehensive collection of flavivirus genomes, to characterize flavivirus evolutionary and biogeographic history in unprecedented detail and breadth. Based on the presumed introduction of yellow fever virus into the Americas via the transatlantic slave trade, we extrapolated a timescale for a relevant subset of flaviviruses whose evolutionary history, shows that different Culex-spp. associated flaviviruses have been introduced from the Old World to the New World on at least five separate occasions, with 2 different sets of factors likely to have contributed to the dispersal of the different viruses. We also discuss the significance of programmed ribosomal frameshifting in a central region of the polyprotein open reading frame in some mosquito-associated flaviviruses.
PMCID: PMC4342338  PMID: 25719412
11.  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.
PMCID: PMC4333652  PMID: 25676752
12.  Isolation, Genetic Characterization, and Seroprevalence of Adana Virus, a Novel Phlebovirus Belonging to the Salehabad Virus Complex, in Turkey 
Journal of Virology  2015;89(8):4080-4091.
A new phlebovirus, Adana virus, was isolated from a pool of Phlebotomus spp. (Diptera; Psychodidae) in the province of Adana, in the Mediterranean region of Turkey. Genetic analysis based on complete coding of genomic sequences indicated that Adana virus belongs to the Salehabad virus species of the genus Phlebovirus in the family Bunyaviridae. Adana virus is the third virus of the Salehabad virus species for which the complete sequence has been determined. To understand the epidemiology of Adana virus, a seroprevalence study using microneutralization assay was performed to detect the presence of specific antibodies in human and domestic animal sera collected in Adana as well as Mersin province, located 147 km west of Adana. The results demonstrate that the virus is present in both provinces. High seroprevalence rates in goats, sheep, and dogs support intensive exposure to Adana virus in the region, which has not been previously reported for any virus included in the Salehabad serocomplex; however, low seroprevalence rates in humans suggest that Adana virus is not likely to constitute an important public health problem in exposed human populations, but this deserves further studies.
IMPORTANCE Until recently, in the genus Phlebovirus, the Salehabad virus species consisted of two viruses: Salehabad virus, isolated from sand flies in Iran, and Arbia virus, isolated from sand flies in Italy. Here we present the isolation and complete genome characterization of the Adana virus, which we propose to be included in the Salehabad virus species. To our knowledge, this is the first report of the isolation and complete genome characterization, from sand flies in Turkey, of a Salehabad virus-related phlebovirus with supporting seropositivity in the Mediterranean, Aegean, and Central Anatolia regions, where phleboviruses have been circulating and causing outbreaks. Salehabad species viruses have generally been considered to be a group of viruses with little medical or veterinary interest. This view deserves to be revisited according to our results, which indicate a high animal infection rate of Adana virus and recent evidence of human infection with Adria virus in Greece.
PMCID: PMC4442372  PMID: 25653443
13.  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.
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.
PMCID: PMC4135957  PMID: 24829336
14.  Risk factors of pandemic influenza A/H1N1 in a prospective household cohort in the general population: results from the CoPanFlu-France cohort 
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.
To investigate the determinants of infection during the 2010–2011 season, the first complete influenza season of study follow-up for this cohort.
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.
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).
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.
PMCID: PMC4280818  PMID: 25495468
Cohort studies; France; influenza A virus, H1N1 subtype; risk factor
15.  First Reported Chikungunya Fever Outbreak in the Republic of Congo, 2011 
PLoS ONE  2014;9(12):e115938.
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.
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.
PMCID: PMC4277398  PMID: 25541718
16.  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.
PMCID: PMC4263616  PMID: 25502692
17.  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.
PMCID: PMC4202267  PMID: 25053561
18.  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;7:476.
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.
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.
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.
PMCID: PMC4197250  PMID: 25306250
Phleboviruses; Toscana virus; Sand flies; Dogs; Sentinels; Emerging; Mediterranean basin; Bunyaviridae
19.  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.
•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.
PMCID: PMC4170750  PMID: 25108382
Flavivirus; Isolate; Mosquito; Complete genome; Phylogenetic analysis
20.  “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.
PMCID: PMC4066373  PMID: 23812617
21.  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.
PMCID: PMC4047448  PMID: 24903869
22.  Evaluation of Antiviral Efficacy of Ribavirin, Arbidol, and T-705 (Favipiravir) in a Mouse Model for Crimean-Congo Hemorrhagic Fever 
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.
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.
PMCID: PMC4006714  PMID: 24786461
23.  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.
PMCID: PMC3961242  PMID: 24651608
24.  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.
PMCID: PMC3944068  PMID: 24598878
25.  Viral aetiology influenza like illnesses in Santa Cruz, Bolivia (2010–2012) 
Virology Journal  2014;11:35.
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.
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.
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.
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.
PMCID: PMC4015617  PMID: 24564892
Epidemiology; Influenza like illness; Bolivia; South America; Influenza A(H1N1)pdm2009; Influenza phylogeny; Respiratory infections

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