Human and plant volatiles; lures for mosquito, vectors of dengue virus and malaria.

Increased outbreaks of mosquito borne diseases like the deadly parasitic disease, malaria and arboviruses like Zika, yellow fever and dengue viruses around the world have led to increased interest in traps that could effectively be used against mosquitoes. For example, a Google search at the time of this writing, asking, 'which is the best way of trapping mosquitoes?' produced 35.5 million search results. Regardless of the interest in the subject, scientists have yet to find a definitive answer to these questions. One area that has been exploited as a potential source of efficient traps for mosquitoes is host odour baits. Since mosquitoes are attracted to their hosts through odours produced by the hosts, it's highly likely that synthetic chemical blends based on host odours could provide a solution. Most mosquito species have 2 hosts: vertebrate animals and vascular plants. Amongst the vertebrates, most diseases spread by mosquitoes are to humans. Considerable research has therefore been conducted on human odours that elicit attraction in mosquitoes, with emphasis on compounds from sweat and skin. Interest on plant volatiles is currently gathering pace because unlike human odours that only attract host seeking female mosquitoes, plant odours can attract both male and female mosquitoes of all gonotrophic stages. This review article concentrates on some of the chemical compounds in human and plant host odours that have shown a potential as attractants to mosquitoes especially Aedes aegypti and Anopheles gambiae s.l.

Seasonal variations of dengue vector mosquitoes in rural settings of Thiruvarur district in Tamil Nadu, India.

BACKGROUND & OBJECTIVES: Mosquitoes are vectors of several important vector-borne diseases (VBDs) like malaria, dengue, chikungunya, Japanese encephalitis (JE) and lymphatic filariasis (LF). Globally, these VBDs are of major public health concern including India. The information on vector mosquitoes from Thiruvarur district in Tamil Nadu state remains largely either unknown or undocumented. The present study was, therefore, undertaken to find out the seasonal variation in mosquitoes with special reference to dengue vectors in rural areas of Thiruvarur district, Tamil Nadu, India. METHODS: Surveillance of immature vector mosquitoes was undertaken from March 2018 to February 2019. The emerged adults were identified to find out the composition of mosquito species prevalent in the district. The seasonal variations of the mosquitoes especially dengue vectors were analysed for summer (March-July) spring (August-November) and winter (December-February) seasons in all the blocks of Thiruvarur district. RESULTS: A total of 4879 mosquitoes emerged from the immature collection and the species identification revealed the prevalence of both vector and non-vector species. Five important mosquito vectors collected were -Aedes albopictus, Ae. aegypti, Culex tritaeniorhynchus, Cx. gelidus, and Cx. quinquefasciatus. Other mosquito species collected were Lutzia fuscana, Anopheles barbirostris, An. subpictus, and Armigeres (Armigeres) subalbatus. During the spring season, the dengue vectors showed high indices of breateau index (BI), ranging from 16 to 120; besides, container index (CI) ranging from14.29 to 68.57 and pupal index (PI) from 53.33 to 295 among the study blocks. The major breeding sites were discarded plastic containers, discarded tyres, open sintex tanks (water storage tanks), cement tanks, discarded fibre box, pleated plastic sheets, tree holes, bamboo cut stumps, coconut spathe, and coconut shells. INTERPRETATION & CONCLUSION: The immature vector surveillance revealed seasonal variations in the entomological indices of Aedes breeding potential. The high indices observed indicate high Aedes breeding density and, therefore, a higher risk for dengue/chikungunya outbreaks in rural areas of Thiruvarur district. The present finding warrants intensive surveillance and follow up vector control measures to avert outbreaks and prevent vector-borne diseases. Health education and the community participation in awareness camps prior to monsoon and societal commitment will help in strengthening source reduction, anti-larval operations and anti-adult measures to tackle vector-borne diseases especially dengue.

De novo profiling of RNA viruses in Anopheles malaria vector mosquitoes from forest ecological zones in Senegal and Cambodia.

BACKGROUND: Mosquitoes are colonized by a large but mostly uncharacterized natural virome of RNA viruses, and the composition and distribution of the natural RNA virome may influence the biology and immunity of Anopheles malaria vector populations. RESULTS: Anopheles mosquitoes were sampled in malaria endemic forest village sites in Senegal and Cambodia, including Anopheles funestus, Anopheles gambiae group sp., and Anopheles coustani in Senegal, and Anopheles hyrcanus group sp., Anopheles maculatus group sp., and Anopheles dirus in Cambodia. The most frequent mosquito species sampled at both study sites are human malaria vectors. Small and long RNA sequences were depleted of mosquito host sequences, de novo assembled and clustered to yield non-redundant contigs longer than 500 nucleotides. Analysis of the assemblies by sequence similarity to known virus families yielded 115 novel virus sequences, and evidence supports a functional status for at least 86 of the novel viral contigs. Important monophyletic virus clades in the Bunyavirales and Mononegavirales orders were found in these Anopheles from Africa and Asia. The remaining non-host RNA assemblies that were unclassified by sequence similarity to known viruses were clustered by small RNA profiles, and 39 high-quality independent contigs strongly matched a pattern of classic RNAi processing of viral replication intermediates, suggesting they are entirely undescribed viruses. One thousand five hundred sixty-six additional high-quality unclassified contigs matched a pattern consistent with Piwi-interacting RNAs (piRNAs), suggesting that strand-biased piRNAs are generated from the natural virome in Anopheles. To functionally query piRNA effect, we analyzed piRNA expression in Anopheles coluzzii after infection with O'nyong nyong virus (family Togaviridae), and identified two piRNAs that appear to display specifically altered abundance upon arbovirus infection. CONCLUSIONS: Anopheles vectors of human malaria in Africa and Asia are ubiquitously colonized by RNA viruses, some of which are monophyletic but clearly diverged from other arthropod viruses. The interplay between small RNA pathways, immunity, and the virome may represent part of the homeostatic mechanism maintaining virome members in a commensal or nonpathogenic state, and could potentially influence vector competence.

First report of Culex flavivirus infection from Culex coronator (Diptera: Culicidae), Colombia.

BACKGROUND: Flaviviruses are important pathogens for humans and animals (Dengue viruses, Yellow fever virus, Zika virus and West Nile virus). Culex flavivirus (CxFV) is an insect-specific virus of the genus Flavivirus, detected in a wide variety of mosquito species. OBJECTIVE: To detect Flavivirus in mosquitoes of a tropical region of the Colombian Caribbean. METHODS: In 2014, an entomological surveillance of arboviruses was conducted in the department of Cordoba area of the Caribbean, Colombia. A total of 8270 mosquitoes were captured as follow: Mansonia (n = 3271/39.5%), Culex (n = 2668/32.26%), Anopheles (n = 840/10.15%), Aedeomyia (n = 411/4.9%), Psorophora (n = 397/4.8%), Coquilletidia (n = 369/4.46%), Uranotaenia (n = 261/3.15%) and Aedes (n = 53/0.6%). All mosquito species were collected in dry tropical forest of the Caribbean area. Universal primers for NS5 gene (958 pb), RT-PCR for flavivirus and sequencing were used for molecular identification of viruses detected. RESULTS: Two pools belonging to Culex coronator were positive for flavivirus RNA sequence by RT-PCR. The sequences of the PCR amplicons, matched that of the Culex flaviviruses, CxFv COL PM_149 (GenBank: KR014201) and CxFv COL PM_212 (GenBank: KT307717). Phylogenetic analysis of the NS5 protein sequences of the Culex flaviviruses sequences with those of reference sequences available in GenBank indicated viruses of Genotype II, closely related to the Brazilian strain, BR_SJRP_01_ (GenBank: KT726939), from Culex sp. The alignment of Culex flavivirus sequences CxFv COL_ PM 212 and CxFv COL_ PM 149 with sequences of strains detected in different geographical regions grouped the strains in a Latin American clade reported in Brazil, Argentina and Mexico. CONCLUSIONS: The present work illustrated that CxFV was circulating among vectors of human pathogenic arboviruses in Colombia, but the impact of CxFV on other flaviviruses which are endemic in the study area still remains to be explored.

Highly focused transcriptional response of Anopheles coluzzii to O'nyong nyong arbovirus during the primary midgut infection.

BACKGROUND: Anopheles mosquitoes are efficient vectors of human malaria, but it is unknown why they do not transmit viruses as well as Aedes and Culex mosquitoes. The only arbovirus known to be consistently transmitted by Anopheles mosquitoes is O'nyong nyong virus (ONNV, genus Alphavirus, family Togaviridae). The interaction of Anopheles mosquitoes with RNA viruses has been relatively unexamined. RESULTS: We transcriptionally profiled the African malaria vector, Anopheles coluzzii, infected with ONNV. Mosquitoes were fed on an infectious bloodmeal and were analyzed by Illumina RNAseq at 3 days post-bloodmeal during the primary virus infection of the midgut epithelium, before systemic dissemination. Virus infection triggers transcriptional regulation of just 30 host candidate genes. Most of the regulated candidate genes are novel, without known function. Of the known genes, a significant cluster includes candidates with predicted involvement in carbohydrate metabolism. Two candidate genes encoding leucine-rich repeat immune (LRIM) factors point to possible involvement of immune protein complexes in the mosquito antiviral response. The primary ONNV infection by bloodmeal shares little transcriptional response in common with ONNV infection by intrathoracic injection, nor with midgut infection by the malaria parasites, Plasmodium falciparum or P. berghei. Profiling of A. coluzzii microRNA (miRNA) identified 118 known miRNAs and 182 potential novel miRNA candidates, with just one miRNA regulated by ONNV infection. This miRNA was not regulated by other previously reported treatments, and may be virus specific. Coexpression analysis of miRNA abundance and messenger RNA expression revealed discrete clusters of genes regulated by Imd and JAK/STAT, immune signaling pathways that are protective against ONNV in the primary infection. CONCLUSIONS: ONNV infection of the A. coluzzii midgut triggers a remarkably limited gene regulation program of mostly novel candidate genes, which likely includes host genes deployed for antiviral defense, as well as genes manipulated by the virus to facilitate infection. Functional dissection of the ONNV-response candidate genes is expected to generate novel insight into the mechanisms of virus-vector interaction.

New genotypes of Liao ning virus (LNV) in Australia exhibit an insect-specific phenotype.

Liao ning virus (LNV) was first isolated in 1996 from mosquitoes in China, and has been shown to replicate in selected mammalian cell lines and to cause lethal haemorrhagic disease in experimentally infected mice. The first detection of LNV in Australia was by deep sequencing of mosquito homogenates. We subsequently isolated LNV from mosquitoes of four genera (Culex, Anopheles, Mansonia and Aedes) in New South Wales, Northern Territory, Queensland and Western Australia; the earliest of these Australian isolates were obtained from mosquitoes collected in 1988, predating the first Chinese isolates. Genetic analysis revealed that the Australian LNV isolates formed two new genotypes: one including isolates from eastern and northern Australia, and the second comprising isolates from the south-western corner of the continent. In contrast to findings reported for the Chinese LNV isolates, the Australian LNV isolates did not replicate in vertebrate cells in vitro or in vivo, or produce signs of disease in wild-type or immunodeficient mice. A panel of human and animal sera collected from regions where the virus was found in high prevalence also showed no evidence of LNV-specific antibodies. Furthermore, high rates of virus detection in progeny reared from infected adult female mosquitoes, coupled with visualization of the virus within the ovarian follicles by immunohistochemistry, suggest that LNV is transmitted transovarially. Thus, despite relatively minor genomic differences between Chinese and Australian LNV strains, the latter display a characteristic insect-specific phenotype.

Identification and genetic analysis of Kadipiro virus isolated in Shandong province, China.

BACKGROUND: Kadipiro virus (KDV) belongs to the Reoviridae family, which consists of segmented, non-enveloped, double-stranded RNA viruses. It has previously been isolated from Culex, Anopheles, Armigeres and Aedes mosquitoes in Indonesia and China. Here, we describe the isolation and characterization of SDKL1625 from Anopheles sinensis mosquitoes in Shandong province, China. METHODS: In this study, we isolated Kadipiro virus in Aedes albopictus C6/36 cell culture and the complete genome sequencing was made by next generation sequencing. RESULTS: We isolated and characterized a Kadipiro virus from Anopheles sinensis mosquitoes in 2016 in Shandong province, China. Nucleotide and amino acid homology analysis of SDKL1625 showed higher levels of sequence identity with QTM27331 (Odonata, China, 2016) than with JKT-7075 (Culex fuscocephalus, Indonesia, 1981). The SDKL1625 has 86-97% amino acid identity with the JKT-7075, 88-99% amino acid identity with the QTM27331. Among the 12 fragments, VP1, VP2, VP4, VP6, VP7, VP9 and VP12 showed high amino acid identity (> 90%) and VP5 showed the lowest identity (86% and 88%). CONCLUSIONS: This is the first identification of KDV from mosquito in China. Virus morphology and genome organization were also determined, which will further enrich our understanding of the molecular biological characteristics of KDV and seadornaviruses.

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.

Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota.

Arboviruses are transmitted by mosquitoes and other arthropods to humans and animals. The risk associated with these viruses is increasing worldwide, including new emergence in Europe and the Americas. Anopheline mosquitoes are vectors of human malaria but are believed to transmit one known arbovirus, o'nyong-nyong virus, whereas Aedes mosquitoes transmit many. Anopheles interactions with viruses have been little studied, and the initial antiviral response in the midgut has not been examined. Here, we determine the antiviral immune pathways of the Anopheles gambiae midgut, the initial site of viral infection after an infective blood meal. We compare them with the responses of the post-midgut systemic compartment, which is the site of the subsequent disseminated viral infection. Normal viral infection of the midgut requires bacterial flora and is inhibited by the activities of immune deficiency (Imd), JAK/STAT, and Leu-rich repeat immune factors. We show that the exogenous siRNA pathway, thought of as the canonical mosquito antiviral pathway, plays no detectable role in antiviral defense in the midgut but only protects later in the systemic compartment. These results alter the prevailing antiviral paradigm by describing distinct protective mechanisms in different body compartments and infection stages. Importantly, the presence of the midgut bacterial flora is required for full viral infectivity to Anopheles, in contrast to malaria infection, where the presence of the midgut bacterial flora is required for protection against infection. Thus, the enteric flora controls a reciprocal protection tradeoff in the vector for resistance to different human pathogens.

A review on test methods for insecticidal fabrics and the need for standardisation.

Insecticidal fabrics are effective personal protective measures against disease vectors and unlike bed nets, these fabrics can provide protection from day-biting mosquitoes and in outdoor environments. The rapid geographical expansion of day-biting mosquitoes and their role in disease transmission necessitate technological interventions, which can be effectively used during the daytime. There is a renewed interest in insecticidal fabrics mainly due to the recent outbreaks and geographical spread of dengue and chikungunya and with the emerging threat of Zika virus infection. Insecticidal fabrics are useful for protection from night-biting mosquitoes and also in situations were sleeping under a bed net is not possible. They are also effective against other biting arthropods like ticks, mites, tsetse flies, sand flies and body lice. Although long-lasting insecticidal fabrics factory-treated with permethrin are now commercially available for military and civilian use, there are no international guidelines for testing their efficacy. The different methods employed so far for testing bioefficacy, washing and quantification of permethrin are compiled in this review. The future prospects and challenges ahead for long-lasting insecticidal fabrics are discussed in the context of the increased threat from day-biting mosquitoes and the diseases transmitted by them. The review focuses on the need for standardisation of the test methods for ensuring adequate bioefficacy and safety to the user. The differences between long-lasting insecticidal nets and long-lasting insecticidal fabrics are elaborated, and the need for a separate registration and licencing procedure for long-lasting insecticidal fabrics is highlighted. A test procedure for insecticidal fabrics is described, which could be used until internationally accepted guidelines are available.

Pathogen-insect interaction candidate molecules for transmission-blocking control strategies of vector borne diseases.

OBJECTIVE: To analyze the current knowledge of pathogen-insect interactions amenable for the design of molecular-based control strategies of vector-borne diseases. MATERIALS AND METHODS: We examined malaria, dengue, and Chagas disease pathogens and insect molecules that participate in interactions during their vectors infection. RESULTS: Pathogen molecules that participate in the insect intestine invasion and induced vector immune molecules are presented, and their inclusion in transmission blocking vaccines (TBV) and in genetically modify insect (GMI) vectors or symbiotic bacteria are discussed. CONCLUSIONS: Disruption of processes by blocking vector-pathogen interactions provides several candidates for molecular control strategies, but TBV and GMI efficacies are still limited and other secondary effects of GMI (improving transmission of other pathogens, affectation of other organisms) should be discarded.

Prevalence of disease vectors in Lakshadweep Islands during post-monsoon season.

BACKGROUND & OBJECTIVES: : Increase of vector-borne diseases (VBDs) in India has posed a question on the situation in Lakshadweep Islands, where VBDs are reported from time-to-time. The present investigation was aimed to assess the faunastic situation of the prevailing vectors along with their breeding sites in different islands of the Lakshadweep. METHODS: : Extensive surveys were carried out from November 2017 to January 2018 (post-monsoon season) randomly in the nine inhabited islands of Lakshadweep for conducting faunastic studies on mosquitoes and to know the basic binomics like breeding and resting preference of mosquitoes. The study islands included, Kavaratti, Agatti, Chetlat, Bitra, Amini, Kadmath, Andrott, Kalpeni and Kiltan. Both immature and adult collections were carried out by standard/appropriate sampling techniques. The obtained data were calculated and analysed in terms of different entomological indices. RESULTS: : A total of 3356 mosquitoes were collected during the study period which comprised of 16 species from nine genera. Out of the 16 species, six belonged to mosquito vectors. The collection included malaria vector, Anopheles stephensi; Japanese encephalitis vector, Culex tritaeniorhynchus; Bancroftian filariasis vector, Cx. quinquefasciatus; Brugian filariasis vector, Mansonia uniformis; and dengue and chikungunya vectors, Stegomya albopicta and St. aegypti. Stegomya albopicta was the most predominant species observed constituting 54% of the catch, followed by Cx. quinquefasciatus, An. stephensi, Cx. tritaeniorhynchus, and St. aegypti constituting 10.5, 6, 3 and 1.2%, respectively. Apart from vector species many non-vectors such as Heizmannia chandi, An. subpictus, An. varuna, Cx. sitiens, Cx. minutissimus, Cx. rubithoracis, Fredwardsius vittatus, Lutzia fuscana, Malaya genurostris and Armigeres subalbatus were also present in the study area. In Kavaratti Island, the capital of Lakshadweep, a non-vector species of sandfly, Sergentomyia (Parrotomyia) babu was observed during the indoor resting collection. The major breeding sites which supported various mosquito species included, discarded plastic containers, tree holes, open sintex tanks (water storage tanks), unused wells, discarded tyres, discarded iron pots, unused and damaged boats, cement tanks, pleated plastic sheets, coral holes, pits and irrigation canals, discarded washing machines, and Colocasia plant leaf axils. Breteau index ranged between 65.3 and 110, CI ranged between 63.64 and 72.41; and HI ranged between 38.46 and 70 among the various islands. INTERPRETATION & CONCLUSION: : Entomological indices such as house index (HI), breteau index (BI) and pupal index (PI) were high in all the nine islands and exceeded the threshold levels specified by WHO, indicating high risk for dengue virus transmission in case of outbreaks. Occurrence of vector as well as non-vector species indicates that the global change in climate is causing notable changes in terms of breeding of vector and non-vector species in the islands. With the reported cases of VBDs and the presence of vectors species in Lakshadweep Islands, a stringent control measure needs to be implemented at the Lakshadweep Islands.

Discovery of new orbiviruses and totivirus from Anopheles mosquitoes in Eastern Australia.

Three new viruses classifiable within the Totivirus and Orbivirus genera were detected from Anopheles mosquito species collected in Eastern Australia. The viruses could not be isolated in C6/36 mosquito cell cultures but were shown to replicate in their mosquito hosts by small RNA analysis. The viruses grouped phylogenetically with other viruses recently detected in insects. These discoveries contribute to a better understanding of commensal viruses in Australian mosquitoes and the evolution of these viruses.

Toxicity of ar-curcumene and epi-ß-bisabolol from Hedychium larsenii (Zingiberaceae) essential oil on malaria, chikungunya and St. Louis encephalitis mosquito vectors.

Mosquitoes act as vectors of key pathogens and parasites. Plant essential oils have been recognized as important sources of biopesticides, which do not induce resistance and have limited toxic effects on human health and non-target organisms. In this research, we evaluated the larvicidal and oviposition deterrence activity of Hedychium larsenii essential oil (EO) and its major compounds ar-curcumene and epi-ß-bisabolol. Both molecules showed high toxicity against early third instars of Anopheles stephensi (LC50=10.45 and 14.68µg/ml), Aedes aegypti (LC50=11.24 and 15.83µg/ml) and Culex quinquefasciatus (LC50=12.24 and 17.27µg/ml). In addition, low doses of ar-curcumene and epi-ß-bisabolol were effective as oviposition deterrents against the three tested mosquito species. Notably, the acute toxicity of H. larsenii oil and its major compounds against the mosquito biocontrol agent Poecilia reticulata was low, with LC50 higher than 1500ppm. Overall, the results from this study revealed that ar-curcumene and epi-ß-bisabolol from the H. larsenii oil can be considered for the development of novel and effective mosquito larvicides.

Mosquito Surveillance for 15 Years Reveals High Genetic Diversity Among West Nile Viruses in Israel.

West Nile Virus (WNV) is endemic in Israel and has been the cause of several outbreaks in recent years. In 2000, a countrywide mosquito survey was established to monitor WNV activity and characterize viral genotypes in Israel. We analyzed data from 7135 pools containing 277 186 mosquitoes collected over the past 15 years and, here, report partial sequences of WNV genomes obtained from 102 of the 336 positive mosquito pools. Phylogenetic analysis demonstrated that cluster 4 and the Mediterranean and Eastern European subtypes of cluster 2 within WNV lineage 1 circulated in Israel, as did WNV lineage 2, highlighting a high genetic diversity of WNV genotypes in our region. As a major crossroads for bird migration between Africa and Eurasia and with a long history of human infection, Israel serves as a resource hub for WNV in Africa and Eurasia and provides valuable information on WNV circulation in these regions.

Merida virus, a putative novel rhabdovirus discovered in Culex and Ochlerotatus spp. mosquitoes in the Yucatan Peninsula of Mexico.

Sequences corresponding to a putative, novel rhabdovirus [designated Merida virus (MERDV)] were initially detected in a pool of Culex quinquefasciatus collected in the Yucatan Peninsula of Mexico. The entire genome was sequenced, revealing 11 798 nt and five major ORFs, which encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The deduced amino acid sequences of the N, G and L proteins have no more than 24, 38 and 43 % identity, respectively, to the corresponding sequences of all other known rhabdoviruses, whereas those of the P and M proteins have no significant identity with any sequences in GenBank and their identity is only suggested based on their genome position. Using specific reverse transcription-PCR assays established from the genome sequence, 27 571 C. quinquefasciatus which had been sorted in 728 pools were screened to assess the prevalence of MERDV in nature and 25 pools were found positive. The minimal infection rate (calculated as the number of positive mosquito pools per 1000 mosquitoes tested) was 0.9, and similar for both females and males. Screening another 140 pools of 5484 mosquitoes belonging to four other genera identified positive pools of Ochlerotatus spp. mosquitoes, indicating that the host range is not restricted to C. quinquefasciatus. Attempts to isolate MERDV in C6/36 and Vero cells were unsuccessful. In summary, we provide evidence that a previously undescribed rhabdovirus occurs in mosquitoes in Mexico.

Chikungunya virus 3' untranslated region: adaptation to mosquitoes and a population bottleneck as major evolutionary forces.

The 3' untranslated genome region (UTR) of arthropod-borne viruses is characterized by enriched direct repeats (DRs) and stem-loop structures. Despite many years of theoretical and experimental study, on-going positive selection on the 3'UTR had never been observed in 'real-time,' and the role of the arbovirus 3'UTR remains poorly understood. We observed a lineage-specific 3'UTR sequence pattern in all available Asian lineage of the mosquito-borne alphavirus, chikungunya virus (CHIKV) (1958-2009), including complicated mutation and duplication patterns of the long DRs. Given that a longer genome is usually associated with less efficient replication, we hypothesized that the fixation of these genetic changes in the Asian lineage 3'UTR was due to their beneficial effects on adaptation to vectors or hosts. Using reverse genetic methods, we examined the functional importance of each direct repeat. Our results suggest that adaptation to mosquitoes, rather than to mammalian hosts, is a major evolutionary force on the CHIKV 3'UTR. Surprisingly, the Asian 3'UTR appeared to be inferior to its predicted ancestral sequence for replication in both mammals and mosquitoes, suggesting that its fixation in Asia was not a result of directional selection. Rather, it may have resulted from a population bottleneck during its introduction from Africa to Asia. We propose that this introduction of a 3'UTR with deletions led to genetic drift and compensatory mutations associated with the loss of structural/functional constraints, followed by two independent beneficial duplications and fixation due to positive selection. Our results provide further evidence that the limited epidemic potential of the Asian CHIKV strains resulted from founder effects that reduced its fitness for efficient transmission by mosquitoes there.

Isolation of Japanese encephalitis virus and a novel insect-specific flavivirus from mosquitoes collected in a cowshed in Japan.

Cattle do not generally appear to develop severe viremia when infected with Japanese encephalitis virus (JEV), and they can be infected without showing clinical signs. However, two cattle in Japan recently died from JEV infection. In this study, we investigated the presence of different species of mosquitoes and flavivirus in a cowshed in the southwest region of Japan. In this cowshed, the two most common species of mosquitoes collected were Culex tritaeniorhynchus (including Culex pseudovishnui) and Anopheles sinensis. We performed virus isolation from the collected mosquitoes and obtained two flaviviruses: JEV and a novel insect-specific flavivirus, tentatively designated Yamadai flavivirus (YDFV). Phylogenetic analysis revealed that all three JEV isolates belonged to JEV genotype I and were closely related to a JEV strain that was isolated from the brains of cattle exhibiting neurological symptoms in Japan. Genetic characterization of YDFV revealed that the full genome RNA (10,863 nucleotides) showed homology with the Culex-associated insect-specific flaviviruses Quang Binh virus (79% identity) and Yunnan Culex flavivirus (78% identity), indicating that YDFV is a novel insect-specific flavivirus.

Distribution and phylogenetic analysis of Culex flavivirus in mosquitoes in China.

Culex flavivirus (CxFV) is an insect-specific virus of the genus Flavivirus. CxFV strains have been isolated from Cx. pipiens, Cx. quinquefasciatus, and other Cx. species in Asia, Africa, North America, Central America and South America. CxFV was isolated for the first time in China in 2006. As this is a novel flavivirus, we explored the distribution and genetic characteristics of Culex flavivirus in China. A total of 46,649 mosquitoes were collected in seven provinces between 2004 and 2012 and were analysed in 871 pools. 29 CxFV RNAs from Cx. pipiens, Cx. tritaeniorhynchus, Anopheles Sinensis, and Culex spp. tested positive for CxFV in real-time RT-PCR. 6 CxFV strains were isolated from Cx. species collected in Shandong, Henan, and Shaanxi provinces, while no virus or viral RNA was detected in samples from Sichuan, Chongqing, Hubei, and Fujian. Phylogenetic analysis of the envelope gene indicated that Chinese strains formed a robust subgroup of genotype 1, together with viruses from the United States and Japan. This study demonstrates that the geographic distribution of CxFV in China is widespread, but geographical boundaries to spread are apparent. Our findings suggest that CxFV can infect various mosquito species in nature.

Genetic divergence of Chikungunya virus plaque variants from the Comoros Island (2005).

Chikungunya virus (CHIKV) from a human sample collected during the 2005 Chikungunya outbreak in the Comoros Island, showed distinct and reproducible large (L2) and small (S7) plaques which were characterized in this study. The parent strain and plaque variants were analysed by in vitro growth kinetics in different cell lines and their genetic similarity assessed by whole genome sequencing, comparative sequence alignment and phylogenetic analysis. In vitro growth kinetic assays showed similar growth patterns of both plaque variants in Vero cells but higher viral titres of S7 compared to L2 in C6/36 cells. Amino acids (AA) alignments of the CHIKV plaque variants and S27 African prototype strain, showed 30 AA changes in the non-structural proteins (nsP) and 22 AA changes in the structural proteins. Between L2 and S7, only two AAs differences were observed. A missense substitution (C642Y) of L2 in the nsP2, involving a conservative AA substitution and a nonsense substitution (R524X) of S7 in the nsP3, which has been shown to enhance O'nyong-nyong virus infectivity and dissemination in Anopheles mosquitoes. The phenotypic difference observed in plaque size could be attributed to one of these AA substitutions. Phylogenetic analysis showed that the parent strain and its variants clustered closely together with each other and with Indian Ocean CHIKV strains indicating circulation of isolates with close evolutionary relatedness in the same outbreak. These observations pave way for important functional studies to understand the significance of the identified genetic changes in virulence and viral transmission in mosquito and mammalian hosts.