Search

Here, we analyze the complete coding sequences of all recognized tick-borne flavivirus species, including Gadgets Gully, Royal Farm and Karshi virus, seabird-associated flaviviruses, Kadam virus and previously uncharacterized isolates of Kyasanur Forest disease virus and Omsk hemorrhagic fever virus. Significant taxonomic improvements are proposed, e.g. the identification of three major groups (mammalian, seabird and Kadam tick-borne flavivirus groups), the creation of a new species (Karshi virus) and the assignment of Tick-borne encephalitis and Louping ill viruses to a unique species (Tick-borne encephalitis virus) including four viral types (i.e. Western Tick-borne encephalitis virus, Eastern Tick-borne encephalitis virus, Turkish sheep Tick-borne encephalitis virus and Louping ill Tick-borne encephalitis virus). The analyses also suggest a complex relationship between viruses infecting birds and those infecting mammals. Ticks that feed on both categories of vertebrates may constitute the evolutionary bridge between the three distinct identified lineages.

By using degenerate primers deduced from conserved patterns in the flavivirus polymerase gene, a novel RNA virus was discovered in Rhipicephalus ticks sampled from members of the family Bovidae in Senegal. It was named Ngoye virus (NGOV) after the location from which it was isolated. Viral particles could be observed by electron microscopy, but isolation in vertebrate or invertebrate cell lines or by intracerebral infection of newborn mice remained unsuccessful. This is atypical of recognized arboviruses. The characterization of 4176 nt of the non-structural genes revealed that NGOV is a novel flavivirus species. It forms a distinct phylogenetic lineage related distantly to previously identified members of the genus Flavivirus. Analysis of genetic data suggested that the processing of the NGOV polyprotein and the organization of its replication complex are similar to those of flaviviruses. Together with other recent data, these findings suggest that a large number of viruses related distantly to 'classical' arthropod-borne flaviviruses remain to be discovered.

The intention of this study was to investigate the genomics, phylogeny and evolution of the Old World arenaviruses based on sequence data representing the four viral genes. To achieve this aim, we sequenced the complete S and L RNA segments of Ippy virus (IPPYV), Mobala virus (MOBV) and Mopeia virus (MOPV). Full-length sequences of the NP, GPC, Z and L genes were used to reconstruct phylogenetic relationships and to compare resulting tree topologies. Each of the five Old World arenavirus species (namely Lassa virus [LASV], IPPYV, MOBV, MOPV and Lymphocytic choriomeningitis virus [LCMV]) are monophyletic; seven selected strains of LASV showed a similar topology regardless of the gene under analysis; IPPYV rooted the three other African arenaviruses; the four African arenaviruses are rooted by the ubiquitous LCMV; and the tree topologies of the three African arenaviruses other than LASV are identical regardless of the gene used for analysis. No evidence for significant evolutionary events such as intra- or intersegmental recombination was obtained.

A study of malaria morbidity was carried out from November 1994 to October 1995, in a Ferlo village (Barkedji) characterized by a long persistence of the temporary ponds. The objective was to evaluate the repercussions of the strong and long anopheles transmission in humans. A clinical follow-up of a group of residents was conducted at home every 10 days by an investigator trained for taking axillary temperature and making thick smears, when suspecting malaria. Were included in the group, 123 voluntary subjects among whom 50% were children under 10 years old. Any feverish subject (T degree >37 degrees 5) or subject presenting other malaria symptoms (headaches, hot body shivers, sweats, aches...) was regarded as having a malaria attack as well as a parasitemia >2500 P/mm3 in children aged of 0 to 14 years old and 1000 P/mm3 in the oldest. During the study subjects with at least one feverish access, plasmodium infection and malaria attack were 58%, 33% and 22%, respectively. On 172 hyperthermias observed, 49% were accompanied by a circulating parasitemia and 30% corresponded to malaria attack. The feverish subjects (74% vs. 42%), the subjects with parasitemia (51% vs. 16%) and the cases of malaria (34% vs. 10%) were more frequently encountered in children under10 than in the oldest. The cases of malaria attacks were more frequent from November to January (70%). The strong intensity of malaria transmission in Barkedji and the persistence of its temporary ponds until January were sufficient to influence the level of malaria morbidity and consequently the development of an anti-malaria immunity by the indigenous population.

Flavivirus-related sequences have been discovered in the dsDNA genome of Aedes albopictus and Aedes aegypti mosquitoes, demonstrating for the first time an integration into a eukaryotic genome of a multigenic sequence from an RNA virus that replicates without a recognized DNA intermediate. In the Aedes albopictus C6/36 cell line, an open reading frame (ORF) of 1557 aa with protease/helicase and polyprotein processing domains characteristic of flaviviruses was identified. It is closely related to NS1-NS4A genes of the Cell Fusing Agent and Kamiti River virus and the corresponding mRNAs were detected. Integrated sequences homologous to the envelope, NS4B and polymerase genes of flaviviruses were identified. Overall, approximately two-thirds of a flavivirus-like genome were characterized. In the Aedes aegypti A20 cell line, a 492 aa ORF related to the polymerase of the Cell Fusing Agent and Kamiti River virus was identified. These flavivirus-related integrated DNA sequences were detected in laboratory-bred and wild Aedes albopictus and Aedes aegypti mosquitoes, demonstrating that their discovery is not an artefact resulting from the manipulation of mosquito cell lines, since they exist under natural conditions. This finding has major implications regarding evolution, as it represents an entirely different mechanism by which genetic diversity may be generated in eukaryotic cells distinct from accepted processes.

Arenaviruses are rodent-borne negative-stranded bisegmented RNA viruses. Five arenaviruses are etiologic agents of hemorrhagic fever in humans and are potential agents of bioterrorism. They are classified as Biosafety level 4 agents and listed in the category A of the Pathogen Agents edited by the Center for Disease Control and Prevention. To date, evolution and phylogeny of arenaviruses have been based on the analysis of sequences derived from structural genes (small RNA segment) exclusively, due to the lack of sequences available for the large RNA segment. In this study, partial sequences of the polymerase gene were determined for 18 species of arenaviruses and used to investigate phylogenetic relationships. Comparative analysis of topologies obtained from polymerase and structural gene analyses permitted us to determine the evolutionary origin of the major parent of the North American recombinant arenaviruses, and to investigate the role of genetic exchange (reassortment and recombination) in the evolutionary mechanisms driving the evolution of the genus Arenavirus.

Little is known about the genetic relationships between European and other Old-World strains of West Nile virus (WNV) and persistence of WNV North of Mediterranean. We characterized the complete genomes of three WNV strains from France (horse-2000), Tunisia (human-1997) and Kenya (mosquito-1998), and the envelope, NS3 and NS5 genes of the Koutango virus. Phylogenetic analyses including all available full-length sequences showed that: (1) Koutango virus is a distant variant of WNV; (2) the three characterized strains belong to lineage 1, clade 1a; (3) the Tunisian strain roots the lineage of viruses introduced in North America. We established that currently available partial envelope sequences do not generate reliable phylogenies. Accordingly, establishing a large WNV sequence database is pivotal for the understanding of spatial and temporal epidemiology of this virus. For rapid completion of that purpose, colinearized E-NS3-NS5 gene sequences were shown to constitute a valuable surrogate for complete sequences.

Variation at nine microsatellite loci was investigated to understand how Anopheles arabiensis populations survive the dry season in the sahelian region of Senegal. Low estimates of genetic differentiation (F(ST) = 0.012, R(ST) = 0.009) between two populations, 250 km apart, suggested extensive gene flow across this distance. Despite extreme seasonal fluctuation in abundance with dry season minima in which mosquitoes virtually disappeared, allele frequencies remained stable over time in the village of Barkedji from August 1994 to December 1997 (including four rainy seasons and three dry seasons). The effective population size (Ne) was estimated to be 601 with 95% CI (281, 1592), providing strong evidence against annual bottlenecks. Differences in measures of genetic diversity and linkage disequilibrium between the dry and the rainy seasons were not detected. These results suggest that despite extreme minima in local density, An. arabiensis maintains large permanent deme spread out over large area.

From 1993 to 1996, an entomological survey was conducted in the village of Ndiop, Senegal, as part of a research programme on malaria epidemiology and the mechanisms of protective immunity. Mosquitoes were captured on human bait and by indoor spraying. Species from the Anopheles gambiae complex were identified using the polymerase chain reaction, and Plasmodium falciparum infections were detected by enzyme-linked immunosorbent assay for circumsporozoite protein. The vector species identified were A. gambiae (33.9%), A. arabiensis (63.2%), A. melas (0.3%) and A. funestus (2.5%). Similar proportions of A. gambiae (74.2%) and A. arabiensis (73.8%) contained human blood; 27.0% of A. gambiae and 28.3% of A. arabiensis had fed on cattle. The sporozoite rates were similar for A. gambiae (3.2%) and A. arabiensis (3.7%). The annual entomological inoculation rates varied greatly depending on the year. There were 63, 17, 37 and 7 infected bites per person per year in 1993, 1994, 1995 and 1996 respectively. Transmission was highly seasonal, from July to October. A. arabiensis was responsible for 66% of malaria transmission, A. gambiae for 31%, and A. funestus for 3%.

The ecology, population dynamics, and malaria vector efficiency of Anopheles gambiae and An. arabiensis were studied for 2 yr in a Sahelian village of Senegal. Anophelines were captured at human bait and resting indoors by pyrethrum spray. Mosquitoes belonging to the An. gambiae complex were identified by polymerase chain reaction. Of 26,973 females, An. arabiensis represented 79% of the mosquitoes captured and remained in the study area longer than An. gambiae after the rains terminated. There were no differences in nocturnal biting cycles or endophagous rates between An. gambiae and An. arabiensis. Based on an enzyme-linked immunosorbent assay test of bloodmeals, the anthropophilic rate of these 2 vectors were both approximately 60%, when comparisons were made during the same period. Overall, 18% of the resting females had patent mixed bloodmeals, mainly human-bovine. The parity rates of An. gambiae and An. arabiensis varied temporally. Despite similar behavior, the Plasmodium falciparum circumsporozoite protein (CSP) rates were different between An. gambiae (4.1%) and An. arabiensis (1.3%). P. malariae and P. ovale only represented 4% of the total Plasmodium identified in mosquitoes. Transmission was seasonal, occurring mainly during 4 mo. The CSP entomological inoculation rates were 128 bites per human per year for the 1st yr and 100 for the 2nd yr. Because of the combination of a high human biting rate and a low CSP rate, An. arabiensis accounted for 63% of transmission. Possible origin of differences in CSP rate between An. gambiae and An. arabiensis is discussed in relation to the parity rate, blood feeding frequency, and the hypothesis of genetic factors.

We conducted a three-year entomologic study in Dielmo, a village of 250 inhabitants in a holoendemic area for malaria in Senegal. Anophelines were captured on human bait and by pyrethrum spray collections. The mosquitoes belonging to the Anopheles gambiae complex were identified using the polymerase chain reaction. Malaria vectors captured were An. funestus, An. arabiensis, and An. gambiae. Anopheles funestus was the most abundant mosquito captured the first year, An. arabiensis in the following years. The annual entomologic inoculation rates calculated by enzyme-linked immunosorbent assay were 238, 89, and 150 for the first, second, and third years, respectively. Each year there was a peak of transmission at the end of the rainy season, but transmission occurred year round. The heterogeneity of transmission was found at four different levels: 1) the relative vector proportion according to the place and method of capture, 2) the human biting rate and relative proportion of vectors by month and year, 3) the infection rate of each vector by year, and 4) the number of infected bites for all vectors, and for each species, for the year. Our data show that even in areas of intense and perennial transmission, there exist large longitudinal variations and strong heterogeneity in entomologic parameters of malaria transmission. It is important to take these into account for the study of the variations in clinical and biological parameters of human malaria, and to evaluate this relationship, a very thorough investigation of transmission is necessary.

Some informations about malaria transmission, which has until nox difficult to get, can be obtained thanks to the use of molecular biology tools, PCR mainly. In Senegal, we use that technique to solve two kinds of problems: -Identification of species of the Anopheles gambiae complex: PCR technique is useful compared to other diagnostic methods (chromosome pattern, DNA probes, etc.) because it enables quickly and simply identification of captured anopheles from the DNA contained in their legs. The rest of the mosquito is tested by circumsporozoite protein antigen ELISA and blood meal ELISA. The data obtained are used to determine distribution, cycles, trophic preferences and comparative vectorial capacities of Anopheles gambiae, Anopheles arabiensis and Anopheles melas. -Identification in a mosquito blood meal of the individual bitten: we propose to evaluate factors (weight, age, sex, location of bedroom, etc.) which could explain why individuals are more, or less, bitten by a Plasmodium vector. Genetic typing is used on inhabitants'leukocytes DNA and on the leukocyte DNA extracted from the blood meal of resting anopheles. Through the high degree of polymorphism of three (AAAG)n microsatellites markers, we hope, using PCR, to attribute each blood meal to one individual. Statistical analysis will be used to identify attractivity factors and to determine more precisely the inoculation rates for each group rather than the classical rate calculated with male adults volunteers.