A chikungunya virus outbreak of unprecedented magnitude is currently ongoing in Indian Ocean territories. In Reunion Island, this alphavirus has already infected about one-third of the human population. The main clinical symptom of the disease is a painful and invalidating poly-arthralgia. Besides the arthralgic form, 123 patients with a confirmed chikungunya infection have developed severe clinical signs, i.e., neurological signs or fulminant hepatitis.

https://hal-pasteur.archives-ouvertes.fr/pasteur-01659363/document

Genome Microevolution of Chikungunya Viruses Causing the Indian Ocean Outbreak

Chikungunya (CHIK) virus is a member of the genus Alphavirus in the family Togaviridae. Serologically, it is most closely related to o’nyong-nyong (ONN) virus and is a member of the Semliki Forest antigenic complex. CHIK virus is believed to be enzootic throughout much of Africa and historical evidence indicates that it spread to other parts of the world from this origin. Strains from Africa and Asia are reported to differ biologically, indicating that distinct lineages may exist. To examine the relatedness of CHIK and ONN viruses using genetic data, we conducted phylogenetic studies on isolates obtained throughout Africa and Southeast Asia. Analyses revealed that ONN virus is indeed distinct from CHIK viruses, and these viruses probably diverged thousands of years ago. Two distinct CHIK virus lineages were delineated, one containing all isolates from western Africa and the second comprising all southern and East African strains, as well as isolates from Asia. Phylogenetic trees corroborated historical evidence that CHIK virus originated in Africa and subsequently was introduced into Asia. Within the eastern Africa and southern Africa/Asia lineage, Asian strains grouped together in a genotype distinct from the African groups. These different geographical genotypes exhibit differences in their transmission cycles: in Asia, the virus appears to be maintained in an urban cycle with Aedes aegypti mosquito vectors, while CHIK virus transmission in Africa involves a sylvatic cycle, primarily with Ae. furcifer and Ae. africanus mosquitoes.

Re-emergence of Chikungunya and O'nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships.

Anopheles gambiae complex mosquitoes are present throughout tropical Africa and its offshore islands. Recent work has shown that at least 6 cryptic or “sibling” species are involved. They comprise 2 salt-water species, A. melas and A. merus, 3 freshwater species—provisionally known as species A, B, and C, and a mineralwater species known as species D. Artificial inter-species crosses yield sterile hybrid males. Rarity of hybridization in nature proves the reproductive isolation and valid genetic barriers between these 6 species.

Morphological identification of most individuals of both saltwater and the mineralwater species is possible for larvae and adult females, using meristic features and other variable dimensions and ratios. Differential identification of the 3 freshwater species relies almost completely on cytotaxonomic methods.

Species A and B occur together in most areas, extending southwards to sub-tropical latitudes and eastward to Mauritius. Proportions of mixed A-B populations may depend directly or indirectly on relative humidity, with A favoured when nocturnal humidities approach saturation. Species B is often absent from areas of highest humidity, but thrives in relatively arid savannas and steppes. Species C and D have relict distributions. Both saltwater species are coastal: melas in West Africa and merus in East Africa and larger islands except Zanzibar; merus also spreads inland.

Apart from species C, which is always zoophilic, all members of the complex are proven or probable vectors of human malaria and filariasis, but with some wide contrasts in their levels of vectorial efficiency. Transmission of some arboviruses (Tataguine, O'nyong-nyong) is associated with species B, and perhaps with A also. Species B may transmit setariasis of cattle; melas and merus may also carry enzootic filariae.

Much of the confusing ecophenotypic plasticity of A. gambiae sensu lato is attributable to the differential biological characteristics of these 6 species with their dissimilar geographical distributions, behavioural contrasts and asynchronous population dynamics. Shifts in the species balance occur regularly between A and B and between freshwater and saltwater populations. Species C does not interchange so much with B under natural conditions, but may survive at high densities after control of A or B.

Additional versatility is caused by genetic polymorphism in some of the species, notably B. This species is the most widespread, and individual females tend to be either endophilic or exophilic, anthropophagic or zoophagic, early biters or late biters, and doubtless other alternatives, according to the arrangement of their floating chromosome inversions.

Control measures have to be considered separately for each of the sibling species. House spraying with residual insecticides against endophilic species A is possibly sufficient to break disease transmission (assuming favourable response of ancillary vectors) and even to eradicate A completely. Efficacy of this method against other species in the complex is reduced by their exophily, which can be enhanced by behaviouristic avoidance due to the extremely low threshold of irritability exhibited by gambiae s.l. adults in general. Genetic polymorphism of species B may lead to true behaviouristic resistance. Larvicidal control of species A and B is beneficial, but made operationally difficult by their tendency to utilize temporary breeding-sites. Effectiveness of DDT and cyclodiene insecticides is further limited by the development of physiological insecticide resistance by species A and B. Adequate control of both saltwater species, and probably the mineralwater species also, can be attained by antilarval measures.

Prospects of reduction of malaria and filariasis where melas, merus and species A and D are principal vectors may be much better than many people imagine. Widespread prevalence and inherent resilience of species B represents an insurmountable public health obstacle at present. Continued research may provide new control methods for integrated use against these mosquitoes. Concepts such as seeding breeding sites with pathogenic microsporidians or fungi, releasing sterile hybrid males or chemosterilized males, and other even more elaborate genetic control techniques, may be of special relevance to control of gambiae complex mosquitoes and diseases they transmit.

Anopheles gambiae complex and disease transmission in Africa

Over the last four decades, a number of arthropod-borne infections have been recognized for the first time. Some have become of considerable public health importance, such as dengue hemorrhagic fever (DHF), and others are spreading geographically and their incidence is increasing. There has been an important recrudescence of several long-known vector-borne diseases. Malaria, leishmaniasis, dengue, and plague have resurged in numerous foci, in some cases where they were thought to be under effective control. In most instances, the appearance of new diseases and syndromes and the resurgence of old can be associated with ecological changes that have favored increased vector densities. Dam construction, irrigation and other development projects, urbanization, and deforestation have all resulted in changes in vector population densities that appear to have enabled the emergence of new diseases and the resurgence of old diseases. Greatly increased human travel has spread infectious agents, introducing them into areas in which they had been hitherto absent. It is essential to understand the factors that caused increased vector densities and hence the transmission of disease to prevent the emergence and resurgence of more diseases, as well as to serve as a basis for effective control.

Emerging and resurging vector-borne diseases

RNA interference (RNAi) is triggered in eukaryotic organisms by double-stranded RNA (dsRNA), and it destroys any mRNA that has sequence identity with the dsRNA trigger. The RNAi pathway in Anopheles gambiae can be silenced by transfecting cells with dsRNA derived from exon sequence of the A. gambiae Argonaute2 (AgAgo2) gene. We hypothesized that RNAi may also act as an antagonist to alphavirus replication in A. gambiae because RNA viruses form dsRNA during replication. Silencing AgAgo2 expression would make A. gambiae mosquitoes more permissive to virus infection. To determine whether RNAi conditions the vector competence of A. gambiae for O'nyong-nyong virus (ONNV), we engineered a genetically modified ONNV that expresses enhanced GFP (eGFP) as a marker. After intrathoracic injection, ONNV-eGFP slowly spread to other A. gambiae tissues over a 9-day incubation period. Mosquitoes were then coinjected with virus and either control beta-galactosidase dsRNA (dsbetagal; note that "ds" is used as a prefix to indicate the dsRNA derived from a given gene throughout) or ONNV dsnsP3. Treatment with dsnsP3 inhibited virus spread significantly, as determined by eGFP expression patterns. ONNV-eGFP titers from mosquitoes coinjected with dsnsP3 were significantly lower at 3 and 6 days after injection than in mosquitoes coinjected with dsbetagal. Mosquitoes were then coinjected with ONNV-eGFP and dsAgAgo2. Mosquitoes coinjected with virus and AgAgo2 dsRNA displayed widespread eGFP expression and virus titers 16-fold higher than dsbetagal controls after 3 or 6 days after injection. These observations provide direct evidence that RNAi is an antagonist of ONNV replication in A. gambiae, and they suggest that the innate immune response conditions vector competence.

https://www.pnas.org/content/pnas/101/49/17240.full.pdf

RNA interference acts as a natural antiviral response to O'nyong-nyong virus (Alphavirus; Togaviridae) infection of Anopheles gambiae

Mosquitoes and bed-bugs were collected in 1959–1960 from places in Uganda and Kenya affected by the o'nyong-nyong epidemic. Tests indicated that 39 of 144 pools containing 5,784 Anopheles funestus , and 15 of 206 pools of 6,933 A. gambiae contained o'nyong-nyong (ONN) virus. No isolation was made from 60 pools containing 1,837 culicine mosquitoes of at least 15 species, or from 13 pools of more than 1,561 bed-bugs. Infected A. funestus and A. gambiae caught in the field maintained ONN virus for at least 20 and 13 days respectively, but limited transmission trials with monkeys and infant mice, through these mosquitoes, were unsuccessful. Transmission was, however, obtained with laboratory-infected mosquitoes of both species. The findings strongly support the conclusions of previous studies in the field that A. funestus was the vector of ONN virus, and that A. gambiae was also involved.

O'nyong-nyong fever: An epidemic virus disease in East Africa VIII. Virus isolations from anopheles mosquitoes

1. 1) Between July and December 1959 four districts in Uganda and Kenya were visited while o'nyong-nyong fever was epidemic. This paper records the results of vector studies made during these visits. 2. 2) Each area visited is briefly described, and an account is given of the biting arthropods encountered in different situations there. 3. 3) The only situation in which the human inhabitants of all districts were bitten severely by arthropods was in huts at night, where only three species of insect were recorded in numbers. These comprised the bed-bug, Cimex hemipterus , and the mosquitoes Anopheles funestus and A. gambiae . 4. 4) Certain known features of the epidemic are considered in relation to the distribution and habits of these insects, and it is concluded that the main vector was A. funestus . 5. 5) This conclusion is briefly discussed, and it is noted that this constitutes the first record of an anopheline mosquito being recognized as the principal vector in an epidemic of virus disease in man.

Philip S. Corbet

Papers

O'nyong-nyong fever: An epidemic virus disease in East Africa VIII. Virus isolations from anopheles mosquitoes

O'Nyong-Nyong fever: an epidemic virus disease in East Africa. IV. Vector studies at epidemic sites.

Recognition of individual nulliparous and parous mosquitoes