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.

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.

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.

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.

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.

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.

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.

[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.

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.

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.

[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.

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.

Factors influencing the population structure of Aedes aegypti from the main cities in Cambodia.

A population genetic analysis was conducted on 47 Aedes aegypti collections from Cambodia. Genetic differentiation at seven polymorphic isoenzyme loci was analysed by starch gel electrophoresis. Low (F(ST)=0.024) but significant (P<10(-6)) differentiation was found when all samples were considered. Whatever the grouping of samples tested, differentiation remained significant but low. The role of human activities (ie insecticide treatments or water storage practices) and environmental factors (ie rainfall) in shaping mosquito differentiation are discussed.

Genetic heterogeneity of the dengue vector Aedes aegypti in Martinique.

In Martinique, Aedes aegypti, the vector of dengue viruses has been the target of insecticide control for more than 35 years. Despite significant control efforts, dengue has become a major disease of public health concern. We conducted a population genetic analysis based on isoenzyme variations combined with an estimation of infection rate to a dengue virus among 26 Ae. aegypti samples. Aedes aegypti samples could be differentiated for their susceptibility to dengue infection (infection rates ranging from 42.8% to 98.6%) and showed important genetic variation (significant F(ST) values).