Wolbachia transinfections in Culex quinquefasciatus generate cytoplasmic incompatibility.

Culex quinquefasciatus is an important mosquito vector of a number of viral and protozoan pathogens of humans and animals, and naturally carries the endosymbiont Wolbachia pipientis, strain wPip. Wolbachia are used in two distinct vector control strategies: firstly, population suppression caused by mating incompatibilities between mass-released transinfected males and wild females; and secondly, the spread of pathogen transmission-blocking strains through populations. Using embryonic microinjection, two novel Wolbachia transinfections were generated in C. quinquefasciatus using strains native to the mosquito Aedes albopictus: a wAlbB single infection, and a wPip plus wAlbA superinfection. The wAlbB infection showed full bidirectional cytoplasmic incompatibility (CI) with wild-type C. quinquefasciatus in reciprocal crosses. The wPipwAlbA superinfection showed complete unidirectional CI, and therefore population invasion potential. Whereas the wAlbB strain showed comparatively low overall densities, similar to the native wPip, the wPipwAlbA superinfection reached over 400-fold higher densities in the salivary glands compared to the native wPip, suggesting it may be a candidate for pathogen transmission blocking.

Wolbachia strain wAlbA blocks Zika virus transmission in Aedes aegypti.

Transinfections of the maternally transmitted endosymbiont Wolbachia pipientis can reduce RNA virus replication and prevent transmission by Aedes aegypti, and also have the capacity to invade wild-type populations, potentially reaching and maintaining high infection frequencies. Levels of virus transmission blocking are positively correlated with Wolbachia intracellular density. Despite reaching high densities in Ae. aegypti, transinfections of wAlbA, a strain native to Aedes albopictus, showed no blocking of Semliki Forest Virus in previous intrathoracic injection challenges. To further characterize wAlbA blocking in Ae. aegypti, adult females were intrathoracically challenged with Zika (ZIKV) and dengue viruses, and then fed a ZIKV-containing bloodmeal. No blocking was observed with either virus when challenged by intrathoracic injection. However, when ZIKV was delivered orally, wAlbA-infected females showed a significant reduction in viral replication and dissemination compared with uninfected controls, as well as a complete absence of virus in saliva. Although other Wolbachia strains have been shown to cause more robust viral blocking in Ae. aegypti, these findings demonstrate that, in principle, wAlbA could be used to reduce virus transmission in this species. Moreover, the results highlight the potential for underestimation of the strength of virus-blocking when based on intrathoracic injection compared with more natural oral challenges.

Vectorial status of the Asian tiger mosquito Aedes albopictus of La Réunion Island for Zika virus.

La Réunion Island has been the scene of unusually large epidemics of dengue and chikungunya viruses with Aedes albopictus as the sole vector. After experimental oral infection, Ae. albopictus from La Réunion Island can disseminate both dengue and chikungunya viruses but not the Asian genotype of Zika virus, suggesting a strong midgut barrier to dissemination. Autochthonous transmission of the Asian genotype of Zika virus is improbable on La Réunion Island.

Vector competence of Culex antennatus and Anopheles coustani mosquitoes for Rift Valley fever virus in Madagascar.

Culex antennatus (Diptera: Culicidae), Anopheles coustani (Diptera: Culicidae) and Anopheles squamosus/cydippis were found to be infected with Rift Valley fever virus (RVFV) during an epidemic that occurred in 2008 and 2009 in Madagascar. To understand the roles played by Cx. antennatus and An. coustani in virus maintenance and transmission, RVFV vector competence was assessed in each species. Mosquito body parts and saliva of mosquitoes that fed on RVFV-infected blood were tested for RVFV using real-time quantitative polymerase chain reaction (RT-qPCR) assays. Overall, viral RNA was detected in body parts and saliva at 5 days post-infection (d.p.i.) in both species. At 5 d.p.i., infection rates were 12.5% (3/24) and 15.8% (6/38), disseminated infection rates were 100% (3/3) and 100% (6/6), transmission rates were 33.3% (1/3) and 83.3% (5/6), and transmission efficiencies were 4.2% (1/24) and 13.2% (5/38) in Cx. antennatus and An. coustani, respectively. Although RVFV detected in saliva did not propagate on to Vero cells, these results support potential roles for these two mosquito species in the transmission of RVFV.

New evidence for the potential role of Culex pipiens mosquitoes in the transmission cycle of West Nile virus in Tunisia.

Physiological and molecular characteristics of natural populations of Culex pipiens Linnaeus, 1758 (Diptera: Culicidae) were investigated to elucidate how this species is potentially involved in the transmission of West Nile virus in Tunisia. A total of 215 Cx. pipiens females from 11 breeding habitats were analysed in the laboratory to estimate autogeny and stenogamy rates. They were tested individually for the locus CQ11 to distinguish between the two Cx. pipiens forms, pipiens and molestus. All tested Cx. pipiens populations were stenogamous. Females from underground breeding sites were all autogeneous, whereas females from above-ground habitats were mostly anautogeneous. Of all the females tested, 59.7% were identified as pipiens, 22.4% as molestus, and 17.9% as hybrid pipiens/molestus. Furthermore, both Cx. pipiens forms and their hybrids were found to co-occur in sympatry in all sites. The results of this study represent the first evidence that both Cx. pipiens forms and their hybrids are present in Tunisia. Because hybrids able to act as bridge vectors are present in all studied habitats, Tunisia can be considered to have a high degree of receptivity for the establishment of West Nile virus zoonotic cycles.

Zoonotic aspects of vector-borne infections.

Vector-borne diseases are principally zoonotic diseases transmitted to humans by animals. Pathogens such as bacteria, parasites and viruses are primarily maintained within an enzootic cycle between populations of non-human primates or other mammals and largely non-anthropophilic vectors. This 'wild' cycle sometimes spills over in the form of occasional infections of humans and domestic animals. Lifestyle changes, incursions by humans into natural habitats and changes in agropastoral practices create opportunities that make the borders between wildlife and humans more permeable. Some vector-borne diseases have dispensed with the need for amplification in wild or domestic animals and they can now be directly transmitted to humans. This applies to some viruses (dengue and chikungunya) that have caused major epidemics. Bacteria of the genus Bartonella have reduced their transmission cycle to the minimum, with humans acting as reservoir, amplifier and disseminator. The design of control strategies for vector-borne diseases should be guided by research into emergence mechanisms in order to understand how a wild cycle can produce a pathogen that goes on to cause devastating urban epidemics.

[Assessment of the risk of introduction to Tunisia of the Rift Valley fever virus by the mosquito Culex pipiens]. / Estimation du risque d'introduction du virus de la fièvre de la vallée du Rift en Tunisie par le moustique Culex pipiens.

The mosquito Culex pipiens has been involved as vector of the West Nile virus in Tunisia. Its bio-ecological characteristics in combination with some environmental factors have favoured the emergence of this virus in a West-Nile free zone. This leads to question about the potential risk of introducing another arbovirus, the Rift Valley fever (RVF) virus, in Tunisia from neighbouring countries where RVF circulates. In this study, we have evaluated the vector competence of different populations of Cx. pipiens towards two strains of RVF virus, the virulent ZH548 and the avirulent Clone 13 by experimental infections and the genetic differentiation of these populations of Cx. pipiens using four microsatellite loci. We found disseminated infection rates ranging from 0% to 14.7% and a high genetic differentiation among populations without any geographical pattern (no isolation by distance). Thus, although Cx. pipiens is able to sustain an amplification of RVF virus, viral dissemination through mosquito dispersal would be unlikely. However, as RVF is an emerging disease transmitted by several other potential mosquito species (e.g. Ochlerotatus caspius), attention should be maintained to survey livestock and mosquitoes in Tunisia.

Wolbachia modulates Chikungunya replication in Aedes albopictus.

The Aedes albopictus mosquito has been involved as the principal vector of recent major outbreaks due to the chikungunya virus (CHIKV). The species is naturally infected by two strains of Wolbachia (wAlbA and wAlbB). Wolbachia infections are thought to have spread by manipulating the reproduction of their hosts; cytoplasmic incompatibility is the mechanism used by Wolbachia to invade natural populations of many insects including Ae. albopictus. Here, we report a study on the effects of removing Wolbachia from Ae. albopictus on CHIKV replication and examine the consequences of CHIKV infection on some life-history traits (survival and reproduction) of Wolbachia-free Ae. albopictus. We found that Wolbachia-free mosquitoes maintained a highly heterogeneous CHIKV replication compared to Wolbachia-infected individuals. In Wolbachia-infected Ae. albopictus, the regular increase of CHIKV followed by a steady viral load from day 4 post-infection onwards was concomitant with a decline in Wolbachia density. This profile was also detected when examining the two key organs for viral transmission, the midgut and the salivary glands. Moreover, Wolbachia-free Ae. albopictus was not altered in life-history traits such as survival, oviposition and hatching characteristics whether infected or not with CHIKV. We found that Wolbachia is not essential for viral replication, its presence could lead to optimize replication from day 4 post-infection onwards, coinciding with a decrease in Wolbachia density. Wolbachia may regulate viral replication in Ae. albopictus, with consequences on survival and reproduction.

Are Aedes albopictus or other mosquito species from northern Italy competent to sustain new arboviral outbreaks?

The Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae), native to Southeast Asia, has extended its geographical distribution to invade new temperate and tropical regions. This species was introduced in 1990 to Italy and has since become the main pest in urban settings. It was incriminated as a principal vector in the first European outbreak of chikungunya virus (CHIKV) in the province of Ravenna (Italy) in 2007. This outbreak was associated with CHIKV E1-226V, efficiently transmitted by Ae. albopictus. The occurrence of this outbreak in a temperate country led us to estimate the potential of Ae. albopictus to transmit CHIKV and dengue virus (DENV), and to determine the susceptibility to CHIKV of other mosquito species collected in northern Italy. Experimental infections showed that Ae. albopictus exhibited high disseminated infection rates for CHIKV (75.0% in Alessandria; 90.3% in San Lazzaro) and low disseminated infection rates for DENV-2 (14.3% in San Lazzaro; 38.5% in Alessandria). Moreover, Ae. albopictus was able to attain a high level of viral replication, with CHIKV detectable in the salivary glands at day 2 after infection. In addition, the other three mosquito species, Anopheles maculipennis Meigen, Aedes vexans vexans (Meigen) and Culex pipiens L., showed variable susceptibilities to infection with CHIKV, of 0%, 7.7% and 0-33%, respectively. This information on vector competence is crucial in assessing the risk for an outbreak of CHIKV or DENV in Italy.

GloPID-R report on chikungunya, o'nyong-nyong and Mayaro virus, part 5: Entomological aspects.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) chikungunya (CHIKV), o'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group has been established to investigate natural history, epidemiology and clinical aspects of infection by these viruses. Here, we present a report dedicated to entomological aspects of CHIKV, ONNV and MAYV. Recent global expansion of chikungunya virus has been possible because CHIKV established a transmission cycle in urban settings using anthropophilic vectors such as Aedes albopictus and Aedes aegypti. MAYV and ONNV have a more limited geographic distribution, being confined to Africa (ONNV) and central-southern America (MAYV). ONNV is probably maintained through an enzootic cycle that has not been characterized yet, with Anopheles species as main vectors and humans as amplification hosts during epidemics. MAYV is transmitted by Haemagogus species in an enzootic cycle using non-human primates as the main amplification and maintenance hosts, and humans becoming sporadically infected when venturing in or nearby forest habitats. Here, we focused on the transmission cycle and natural vectors that sustain circulation of these viruses in their respective locations. The knowledge of the natural ecology of transmission and the capacity of different vectors to transmit these viruses is crucial to understand CHIKV emergence, and to assess the risk that MAYV and ONNV will expand on wide scale using anthropophilic mosquito species not normally considered primary vectors. Finally, the experts identified knowledge gaps and provided adapted recommendations, in order to address future entomological investigations in the right direction.

GloPID-R report on chikungunya, o'nyong-nyong and Mayaro virus, part 2: Epidemiological distribution of o'nyong-nyong virus.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) chikungunya (CHIKV), o'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group has been established to identify gaps of knowledge about the natural history, epidemiology and medical management of infection by these viruses, and to provide adapted recommendations for future investigations. Here, we present a report dedicated to ONNV epidemiological distribution. Two large-scale ONNV outbreaks have been identified in Africa in the last 60 years, interspersed with sporadic serosurveys and case reports of returning travelers. The assessment of the real scale of ONNV circulation in Africa remains a difficult task and surveillance studies are necessary to fill this gap. The identification of ONNV etiology is made complicated by the absence of multiplex tools in co-circulation areas and that of reference standards, as well as the high cross-reactivity with related pathogens observed in serological tests, in particular with CHIKV. This is a specific obstacle for seroprevalence studies, that necessitate an improvement of serological tools to provide robust results. The scarcity of existent genetic data currently limits molecular epidemiology studies. ONNV epidemiology would also benefit from reinforced entomological and environmental surveillance. Finally, the natural history of the disease deserves to be further investigated, with a specific attention paid to long-term complications. Considering our incomplete knowledge on ONNV distribution, GloPID-R CHIKV, ONNV and MAYV experts recommend that a major effort should be done to fill existing gaps.

Chikungunya-Wolbachia interplay in Aedes albopictus.

A severe Chikungunya (CHIK) outbreak recently hit several countries of the Indian Ocean. On La Réunion Island, Aedes albopictus was incriminated as the major vector. This mosquito species is naturally co-infected with two distinct strains of the endosymbiont Wolbachia, namely wAlbA and wAlbB, which are increasingly attracting interest as potential tools for vector control. A PCR quantitative assay was developed to investigate Wolbachia/mosquito host interactions. We show that Wolbachia densities are slightly decreased in CHIK virus (CHIKV)-infected females. We measured the impact of CHIKV replication on a lysogenic virus: WO bacteriophage. Our data indicate that WO is sheltered by wAlbB, likely at a single copy per bacteria, and that CHIKV replication is not a physiological stress triggering WO entrance into the lytic cycle.

[Aedes albopictus, vector of chikungunya and dengue viruses in Reunion Island: biology and control]. / Aedes albopictus, vecteur des virus du chikungunya et de la dengue a la Réunion: biologie et contrôle.

Chikungunya virus (CHIKV) and dengue virus (DENV) are mosquito-borne viruses transmitted by the Aedes genus. Dengue is considered as the most important arbovirus disease throughout the World. Chikungunya, known from epidemics in continental Africa and Asia, has up to now been poorly studied. It has been recently responsible for the severe 2004-2007 epidemic reported in the Indian Ocean (IO), which has caused several serious health and economic problems. This unprecedented epidemic of the IO has shown severe health troubles with morbidity and death associated, which had never been observed before. The two major vectors of those arboviruses in the IO area are Aedes aegypti and Aedes albopictus. The latest is considered as the main vector in most of the islands of the area, especially in Reunion Island. Ae. albopictus showed strong ecological plasticity. Small disposable containers were the principal urban breeding sites, and preferred natural developmental sites were bamboo stumps and rock holes in peri-urban and gully areas. The virus has been isolated from field collected Ae. albopictus females, and in two out of 500 pools of larvae, demonstrating vertical transmission. Experimental works showed that both Ae. albopictus and Ae. aegypti from west IO islands are efficient vectors of dengue and chikungunya viruses. Since 2006 and all along the epidemic of CHIKV, measures for the control of larvae (temephos then Bacillus thuringiensis) and adults (fenitrothion, then deltamethrine) of Ae. albopictus where applied along with individual and collective actions (by the use of repellents, and removal of breeding sites around houses) in Reunion Island. In order to prevent such epidemics, a preventive plan for arboviruses upsurge is ongoing processed. This plan would allow a quicker response to the threat and adapt it according to the virus and its specific vector.

Short report: microsatellite sequences as markers for population genetic studies of the mosquito Aedes aegypti, the vector of dengue viruses.

We report the isolation of microsatellites from an enriched library of genomic repeated sequences, using a biotin-labeled oligonucleotide bound to streptavidin-coated magnetic particles. Four microsatellites were obtained from a partial library of 120 recombinant clones. This more efficient and rapid method to obtain these specific repeated sequences is preferred to the conventional isolation procedure based on the construction of a genomic library. Microsatellite markers would be promising molecular tools for the study of genetic variability of mosquito populations. Analyses of genetic structure and gene flow would provide information on the distance, direction and rate of dispersal of genes in Aedes aegypti populations. Knowledge on gene dispersal patterns is required to develop vector control strategies.

Aedes aegypti in Tahiti and Moorea (French Polynesia): isoenzyme differentiation in the mosquito population according to human population density.

Genetic differences at five polymorphic isoenzyme loci were analyzed by starch gel electrophoresis for 28 Aedes aegypti samples. Considerable (i.e., high Fst values) and significant (i.e., P values >10(-4)) geographic differences were found. Differences in Ae. aegypti genetic structure were related to human population densities and to particularities in mosquito ecotopes in both Tahiti and Moorea islands. In highly urbanized areas (i.e., the Papeete agglomeration), mosquitoes were highly structured. Recurrent extinction events consecutive to insecticidal treatments during dengue outbreaks tend to differentiate mosquito populations. In less populated zones (i.e., the east coast of Moorea and Tahiti), differences in ecotope characteristics could explain the lack of differentiation among mosquitoes from rural environments such as the east coast of Tahiti where natural breeding sites predominate. When the lowest populated zones such as Tahiti Iti and the west coast of Moorea are compared, mosquito are less differentiated in Moorea. These results will be discussed in relation to the recent findings of variation in mosquito infection rates for dengue-2 virus.

Variation in oral susceptibility to dengue type 2 virus of populations of Aedes aegypti from the islands of Tahiti and Moorea, French Polynesia.

Twenty three samples of Aedes aegypti populations from the islands of Tahiti and Moorea (French Polynesia) were tested for their oral susceptibility to dengue type 2 virus. The high infection rates obtained suggest that the artificial feeding protocol used was more efficient than those previously described. Statistical analysis of the results allowed us to define two distinct geographic areas on Tahiti with respect to the susceptibility of Ae. aegypti: the east coast, with homogeneous infection rates, and the west coast, with heterogeneous infection rates. No geographic differences could be demonstrated on Moorea. The possible mechanisms of this phenomenon are discussed in connection with recent findings on the variability of susceptibility of Ae. aegypti to insecticides.

Population genetic structure and competence as a vector for dengue type 2 virus of Aedes aegypti and Aedes albopictus from Madagascar.

Starch gel electrophoresis was used to assess the polymorphism of 7 isoenzymes in single mosquitoes (field-collected F0 or F1 generation) for Aedes albopictus (8 strains) from northern Madagascar. Mosquitoes of the F2 generation (3 strains of Aedes aegypti and 10 strains of Ae. albopictus) were tested for oral susceptibility to dengue type 2 virus. Aedes aegypti was less susceptible to viral infection than Ae. albopictus. The genetic differentiation was less high between Ae. albopictus populations collected in agglomerations connected by highly frequented roads, indicating that human ground transportation favors mosquito dispersal. These results have implications for the ecology, pattern of migration, and relative importance in epidemic transmission of dengue viruses between the 2 Aedes species.

Aedes aegypti in Brazil: genetically differentiated populations with high susceptibility to dengue and yellow fever viruses.

Aedes aegypti was eliminated from Brazil in 1955, but re-infested the country in the 1970s. Dengue outbreaks have occurred since 1981 and became endemic in several cities in Brazil after 1986. Urban yellow fever has not occurred since 1942, and only jungle yellow fever cases have been reported. A population genetic analysis using isoenzyme variation combined with an evaluation of susceptibility to both yellow fever and dengue 2 viruses was conducted among 23 A. aegypti samples from 13 Brazilian states. We demonstrated that experimental infection rates of A. aegypti for both dengue and yellow fever viruses (YFV) are high and heterogeneous, and samples collected in the endemic and transition areas of sylvatic yellow fever were highly susceptible to yellow fever virus. Boa Vista, a border city between Brazil and Venezuela, and Rio de Janeiro in the Southeast region are considered as the most important entry points for dengue dissemination. Considering the high densities of A. aegypti, and its high susceptibility to dengue and yellow fever viruses, the risk of dengue epidemics and yellow fever urbanization in Brazil is more real than ever.

Aedes aegypti in Ho Chi Minh City (Viet Nam): susceptibility to dengue 2 virus and genetic differentiation.

Aedes aegypti is the principal vector of dengue viruses, responsible for a viral infection that has become a major public health concern in Asia. In Viet Nam, dengue haemorrhagic fever was first detected in the 1960s and is now a leading cause of death in childhood. We studied the variability in competence of Ae. aegypti as a vector for dengue 2 virus and genetic differentiation in this mosquito species. Twenty mosquito samples collected in 1998 in Ho Chi Minh City were subjected to oral infection and isoenzyme polymorphism analysis by starch gel electrophoresis. Ae. aegypti populations from the centre of Ho Chi Minh City were genetically differentiated and their infection rates differed from those of populations from the commuter belt. These results have implications for insecticidal control during dengue outbreaks.

Human transportation influences Aedes aegypti gene flow in Southeast Asia.

Estimates of population genetic organization and gene flow of Aedes aegypti, the main vector of dengue viruses, have provided insights into dengue epidemiology. To assess the role of the vector in the changing pattern of dengue in Southeast Asia, extensive studies on the genetic differentiation of Aedes aegypti have been carried out. Among them, vector differentiation has been estimated for Cambodia, Thailand and South Vietnam using microsatellite markers. Analysis showed that there was less genetic differentiation between mosquito populations from Ho Chi Minh City and Phnom Penh than from either of them and Thailand, suggesting that passive migrations through human transportation help to explain this pattern of differentiation.