Workshop on mobile laboratories deployed in the Ebola outbreak in West-Africa 2014-2015

First paragraph: Ebola virus disease (EVD) is a haemorrhagic fever caused by Ebola virus (EBOV) with high infectivity and mortality. EBOV is an enveloped, single-stranded, and negative-sense RNA virus belonging to the Filoviridae family. In contrast to the genus Marburgvirus which contains one single species, the genus Ebolavirus contains 5 species: Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SUDV), Taï Forest ebolavirus (TAFV), Bundibugyo ebolavirus (BDBV) which are pathogenic for humans, and Reston Ebolavirus (RESTV) which infects non human primates. EBOV was first discovered in 1976 in the Democratic Republic of Congo (DRC) and simultaneously in Sudan. Since 1976, EVD has appeared sporadically in DRC, Sudan, Gabon, Uganda, and Congo, with small to large outbreaks and lethality ranging from 50 to 100% with about 2500 cumulative cases until 2013

Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes

BACKGROUND Zika virus (ZIKV), a mosquito borne flavivirus is a pathogen affecting humans in Asia and Africa. ZIKV infection diagnosis relies on serology-which is challenging due to cross-reactions with other flaviviruses and/or absence or low titer of IgM and IgG antibodies at early phase of infection- virus isolation, which is labor intensive, time consuming and requires appropriate containment. Therefore, real-time RT-PCR (rRT-PCR) is an appealing option as a rapid, sensitive and specific method for detection of ZIKV in the early stage of infection. So far, only one rRT-PCR assay has been described in the context of the outbreak in Micronesia in 2007. In this study, we described a one step rRT-PCR for ZIKV which can detect a wider genetic diversity of ZIKV isolates from Asia and Africa. RESULTS The NS5 protein coding regions of African ZIKV isolates were sequenced and aligned with representative flaviviruses sequences from GenBank to design primers and probe from conserved regions. The analytical sensitivity of the assay was evaluated to be 32 genome-equivalents and 0.05 plaque forming unit (pfu). The assay was shown to detect 37 ZIKV isolates covering a wide geographic in Africa and Asia over 36 years but none of the 31 other flaviviruses tested showing high analytical specificity. The rRT-PCR could be performed in less than 3 hours. This method was used successfully to detect ZIKV strains from field-caught mosquitoes. CONCLUSION We have developed a rapid, sensitive and specific rRT-PCR for detection of ZIKV. This assay is a useful tool for detection of ZIKV infection in regions where a number of other clinically indistinguishable arboviruses like dengue or chikungunya co-circulate. Further studies are needed to validate this assay in clinical positive samples collected during acute ZIKV infection

Deployment of the Institut Pasteur de Dakar team to Guinea in the Ebola virus Disease outbreak in West-Africa 2014-2016

First paragraph: The unit of Arbovirus and Haemorrhagic Fever Viruses at the Institut Pasteur de Dakar (IPD), a WHO-approved collaborating Centre was the first laboratory deployed to Conakry in the Ebola virus disease (EVD) outbreak in West-Africa. On 20 March 2014, the IPD laboratory received a letter from the WHO and the Guinean Ministry of Health, informing about a suspected haemorrhagic fever outbreak and difficulties to send collected samples to IPD. They therefore requested the deployment of experts to Guinea for technical support in order to diagnose the haemorrhagic fever of unknown origin. The outbreak was identified by the Institut Pasteur (France) on 21 March 2014 [1,2] in samples shipped to France by a Médecins sans Frontières investigation team

Full-genome characterization and genetic evolution of West African isolates of Bagaza virus

Bagaza virus is a mosquito-borne flavivirus, first isolated in 1966 in Central African Republic. It has currently been identified in mosquito pools collected in the field in West and Central Africa. Emergence in wild birds in Europe and serological evidence in encephalitis patients in India raise questions on its genetic evolution and the diversity of isolates circulating in Africa. To better understand genetic diversity and evolution of Bagaza virus, we describe the full-genome characterization of 11 West African isolates, sampled from 1988 to 2014. Parameters such as genetic distances, N-glycosylation patterns, recombination events, selective pressures, and its codon adaptation to human genes are assessed. Our study is noteworthy for the observation of N-glycosylation and recombination in Bagaza virus and provides insight into its Indian origin from the 13th century. Interestingly, evidence of Bagaza virus codon adaptation to human house-keeping genes is also observed to be higher than those of other flaviviruses well known in human infections. Genetic variations on genome of West African Bagaza virus could play an important role in generating diversity and may promote Bagaza virus adaptation to other vertebrates and become an important threat in human health

Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus

Rabies virus (RABV) remains one of the most important global zoonotic pathogens. RABV causes rabies, an acute encephalomyelitis associated with a high rate of mortality in humans and animals and affecting different parts of the world, particularly in Asia and Africa. Confirmation of rabies diagnosis relies on laboratory diagnosis, in which molecular techniques such as detection of viral RNA by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used.  In this study, two real-time quantitative RT-PCR assays were developed for large-spectrum detection of RABV, with a focus on African isolates. The primer and probe sets were targeted highly conserved regions of the nucleoprotein (N) and polymerase (L) genes.  The results indicated the absence of non-specific amplification and cross-reaction with a range of other viruses belonging to the same taxonomic family, i.e Rhabdoviridae, as well as negative brain tissues from various host species. Analytical sensitivity ranged between 100 to 10 standard RNA copies detected per reaction for N-gene and L-gene assays, respectively. Effective detection and high sensitivity of these assays on African isolates showed that they can be successfully applied in general research and used in diagnostic process and epizootic surveillance in Africa using a double-check strategy

Larval ecology of mosquitoes in sylvatic arbovirus foci in southeastern Senegal

BACKGROUND: Although adult mosquito vectors of sylvatic arbovirus [yellow fever (YFV), dengue-2 (DENV-2) and chikungunya (CHIKV)] have been studied for the past 40 years in southeastern Senegal, data are still lacking on the ecology of larval mosquitoes in this area. In this study, we investigated the larval habitats of mosquitoes and characterized their seasonal and spatial dynamics in arbovirus foci. METHODS: We searched for wet microhabitats, classified in 9 categories, in five land cover classes (agriculture, forest, savannah, barren and village) from June, 2010 to January, 2011. Mosquito immatures were sampled monthly in up to 30 microhabitats of each category per land cover and bred until adult stage for determination. RESULTS: No wet microhabitats were found in the agricultural sites; in the remaining land covers immature stages of 35 mosquito species in 7 genera were sampled from 9 microhabitats (tree holes, fresh fruit husks, decaying fruit husks, puddles, bamboo holes, discarded containers, tires, rock holes and storage containers). The most abundant species was Aedes aegypti formosus, representing 30.2% of the collections, followed by 12 species, representing each more than 1% of the total, among them the arbovirus vectors Ae. vittatus (7.9%), Ae. luteocephalus (5.7%), Ae. taylori (5.0%), and Ae. furcifer (1.3%). Aedes aegypti, Cx. nebulosus, Cx. perfuscus, Cx. tritaeniorhynchus, Er. chrysogster and Ae. vittatus were the only common species collected from all land covers. Aedes furcifer and Ae. taylori were collected in fresh fruit husks and tree holes. Species richness and dominance varied significantly in land covers and microhabitats. Positive associations were found mainly between Ae. furcifer, Ae. taylori and Ae. luteocephalus. A high proportion of potential enzootic vectors that are not anthropophilic were found in the larval mosquito fauna. CONCLUSIONS: In southeastern Senegal, Ae. furcifer and Ae. taylori larvae showed a more limited distribution among both land cover and microhabitat types than the other common species. Uniquely among vector species, Ae. aegypti formosus larvae occurred at the highest frequency in villages. Finally, a high proportion of the potential non-anthropophilic vectors were represented in the larval mosquito fauna, suggesting the existence of unidentified sylvatic arbovirus cycles in southeastern Senegal

Detection of Chikunguny a virus by RT-LAMP

Background A single-tube one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of chikungunya virus (CHIKV) targeting the conserved 6K-E1 target region was developed. The assay was validated with sera collected from a CHIKV outbreak in Senegal in 2015. Methodology/Principal findings A novel design approach by combining Principal Component Analysis and phylogenetic analysis of 110 available CHIKV sequences and the LAMP oligonucleotide design software LAVA was used. The assay was evaluated with an External Quality Assessment panel from the European Network for Diagnostics of "Imported" Viral Diseases and was shown to be sensitive and specific and did not cross-detect other arboviruses. The limit of detection as determined by probit analysis, was 163 molecules, and 100% reproducibility in the assays was obtained for 103 molecules (7/8 repetitions were positive for 102 molecules). The assay was validated using 35 RNA samples extracted from sera, and results were compared with those obtained by quantitative RT-PCR carried out at the Institut Pasteur Dakar, demonstrating that the RT-LAMP is 100% sensitive and 80% specific, with a positive predictive value of 97% and negative predictive value of 100%. Conclusions/Significance The RT-LAMP appeared to show superior performance with material stored for months compared to qRT-PCR and can be therefore recommended for use in infrastructures with poor settings

Phylogeography of Rift Valley Fever Virus in Africa Reveals Multiple Introductions in Senegal and Mauritania

Rift Valley Fever (RVF) virus (Family Bunyaviridae) is an arthropod-borne RNA virus that infects primarily domestic ruminants and occasionally humans. RVF epizootics are characterized by numerous abortions and mortality among young animals. In humans, the illness is usually characterized by a mild self-limited febrile illness, which could progress to more serious complications. RVF virus is widespread and endemic in many regions of Africa. In Western Africa, several outbreaks have been reported since 1987 when the first major one occurred at the frontier of Senegal and Mauritania. Aiming to evaluate the spreading and molecular epidemiology in these countries, RVFV isolates from 1944 to 2008 obtained from 18 localities in Senegal and Mauritania and 15 other countries were investigated. Our results suggest that a more intense viral activity possibly took place during the last century compared to the recent past and that at least 5 introductions of RVFV took place in Senegal and Mauritania from distant African regions. Moreover, Barkedji in Senegal was possibly a hub associated with the three distinct entries of RVFV in West Africa

Rapid molecular assays for the detection of yellow Fever virus in low-resource settings

BACKGROUND Yellow fever (YF) is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The causative agent, the yellow fever virus (YFV), is found in tropical and subtropical areas of South America and Africa. Although a vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Therefore, rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control. METHODOLOGY The aim of this study was to develop a YFV nucleic acid detection method applicable in outbreak investigations and surveillance studies in low-resource and field settings. The method should be simple, robust, rapid and reliable. Therefore, we adopted an isothermal approach and developed a recombinase polymerase amplification (RPA) assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats (real-time with or without microfluidic semi-automated system and lateral-flow assay) to evaluate their application for different purposes. Analytical specificity and sensitivity were evaluated with a wide panel of viruses and serial dilutions of YFV RNA. Mosquito pools and spiked human plasma samples were also tested for assay validation. Finally, real-time RPA in portable format was tested under field conditions in Senegal. CONCLUSION/SIGNIFICANCE The assay was able to detect 20 different YFV strains and demonstrated no cross-reactions with closely related viruses. The RPA assay proved to be a robust, portable method with a low detection limit (<21 genome equivalent copies per reaction) and rapid processing time (<20 min). Results from real-time RPA field testing were comparable to results obtained in the laboratory, thus confirming our method is suitable for YFV detection in low-resource settings