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BackgroundUntil recently, arbovirus surveillance is mainly focused on mosquito and tick vectors, resulting in the discovery of several mosquito- and tick-borne arboviruses. However, the role of sandflies in arbovirus transmission and disease has remained largely unexplored. This study sought to isolate and characterize arboviruses from phlebotomine sandflies from selected pastoral ecozones in the North Rift region of Kenya.MethodsSandflies were collected from selected sites in North Rift Kenya between 2015 and 2018. They were sorted and pooled by sex, site, and collection date. The pools were homogenized and inoculated onto Vero cells for virus isolation. The positive pools were analyzed by polymerase chain reaction targeting different arboviruses. The isolates were further characterized by high-throughput sequencing using Illumina Miseq platform.ResultsApproximately 28,226 sandflies translating to 824 pools were sampled from the selected regions. A total of 11 showed reproducible cytopathic effects on Vero cells. We identified five arboviruses: sindbis (n = 4) from Kacheliba and Baringo, Chandipura (n = 4) from Turkana and Baringo, Koutango (n = 1) and Ntepes (n = 1) from Baringo, and Bogoria (n = 1) from Kacheliba. The percent identities of the identified viruses were approximately 80% to 98% compared to known viruses in GenBank, suggesting that some of them could be novel viruses.ConclusionThis study successfully isolated and characterized five arboviruses from sandflies. The findings suggest that sandflies are potential hosts of a wide range of arboviruses and are therefore important vectors to consider in arbovirus surveillance and evaluated for their ability to transmit them. Further studies are needed to determine the public health importance and extent of exposure of these viruses to humans and livestock populations.

The West Nile virus (WNV), primarily transmitted by mosquitoes, is one of the most widespread flaviviruses globally, with past outbreaks occurring in the USA and Europe. Recent studies in parts of Africa, including Kenya, have identified the West Nile virus Koutango lineage (WN-KOUTV) among phlebotomine sandfly populations, however, our understanding of this virus remains limited. This study aimed to characterize WN-KOUTV from phlebotomine sandflies. Sandflies were sampled between 12th -16th March 2021 and 16th -20th March 2023 from six villages each in Baringo and Isiolo Counties, using CDC light traps. Female sandflies were taxonomically identified and pooled based on genus and site of collection. Virus isolation was performed in Vero cells. Viral genomes were determined using next-generation sequencing. Phylogenetic and molecular clock analyses were done to decipher the virus's evolutionary relationships. Comparative analyses of amino acid sequences were performed to determine variations. Protein modeling in Pymol was conducted to elucidate variations in key protein regions. Evolutionary pressure analysis investigated the selection pressures on the virus. In vitro experiments were done to investigate the virus growth kinetics in mammalian Vero E6 and mosquito C6/36 cells. We report the isolation of WN-KOUTV from Salabani in Baringo and Aremet in Isiolo, Kenya. The isolated WN-KOUTVs clustered with previously identified WN-KOUTV strains. Comparative analysis revealed a unique amino acid at NS5 653. The WN-KOUTV lineage as a whole is under purifying selective pressure, with diversifying pressure acting at site NS3 267. The current WN-KOUTV replicated in Vero E6 and C6/36 cells comparable to West Nile virus Lineage 1a, isolated from mosquitoes. Subsequent isolations of WN-KOUTV in phlebotomine sandflies suggest potential vectors, however, vector competence studies would confirm this. Replication in mammalian and insect cell lines suggests there may exist a vector/host relationship. We speculate the close genetic relationship of WN-KOUTV strains from East and West Africa may potentially be enabled by bird migratory routes between the two regions. If proven, this could point to a potential future pandemic pathway for this virus.

Abstract Background Arbovirus surveillance and recurrence of outbreaks in Kenya continues to reveal the re-emergence of viruses of public health importance. This calls for sustained efforts in early detection and characterization of these agents to avert future potential outbreaks. Methods A larval survey was carried out in three different sites in Kwale County, Vanga, Jego and Lunga Lunga. All containers in every accessible household and compound were sampled for immature mosquitoes. In addition, adult mosquitoes were also sampled using CO2-baited CDC light traps and BG-Sentinel traps in the three sites and also in Tsuini. The mosquitoes were knocked down using trimethylamine and stored in a liquid nitrogen shipper for transportation to the laboratory where they were identified to species, pooled and homogenized ready for testing. Results A total of 366 houses and 1730 containers were inspected. The House Index (HI), Container Index (CI) and Breateau Index (BI) for Vanga Island were (3%: 0.66: 3.66) respectively. In Jego, a rural site, the HI, CI and BI were (2.4%: 0.48: 2.4) respectively. In Lunga Lunga, a site in an urban area, the HI, CI and BI were (22.03%: 3.97: 29.7) respectively. The indices suggest that this region is at risk of arbovirus transmission given they were above the WHO threshold (CI > 1, HI > 1% and BI > 5). The most productive containers were the concrete tanks (44.4%), plastic tank (22.2%), claypot (13.3%), plastic drums (8.9%), plastic basins (4%), jerricans (1.2%) and buckets (0.3%). Over 20,200 adult mosquitoes were collected using CDC light traps, and over 9,200 using BG- sentinel traps. These mosquitoes were screened for viruses by inoculating in Vero cells. Eleven Orthobunyavirus isolates were obtained from pools of Ae. pembaensis (4), Ae. tricholabis (1), Cx. quinquefasciatus (3), Culex spp. (1) and Cx. zombaensis (2). Five of the Orthobunyaviruses were sequenced and four of these were determined to be Bunyamwera viruses while one isolate was found to be Nyando virus. One isolate remained unidentified. Conclusions These results indicate circulation of Orthobunyaviruses known to cause diverse grades of febrile illness with rash in humans in this region and highlights the need for continued monitoring and surveillance to avert outbreaks.

Abstract Background Chikungunya virus is an alphavirus, primarily transmitted by Aedes aegypti and Ae. albopictus. In late 2017–2018, an outbreak of chikungunya occurred in Mombasa county, Kenya, and investigations were conducted to establish associated entomological risk factors. Methods Homes were stratified and water-filled containers inspected for immature Ae. aegypti, and larval indices were calculated. Adult mosquitoes were collected in the same homesteads using BG-Sentinel and CDC light traps and screened for chikungunya virus. Experiments were also conducted to determine the ability of Culex quinquefasciatus to transmit chikungunya virus. Results One hundred thirty-one houses and 1637 containers were inspected; 48 and 128 of them, respectively, were positive for immature Ae. aegypti, with the house index (36.60), container index (7.82) and Breteau index (97.71) recorded. Jerry cans (n = 1232; 72.26%) and clay pots (n = 2; 0.12%) were the most and least inspected containers, respectively, while drums, the second most commonly sampled (n = 249; 15.21%), were highly positive (65.63%) and productive (60%). Tires and jerry cans demonstrated the highest and lowest breeding preference ratios, 11.36 and 0.2, respectively. Over 6900 adult mosquitoes were collected and identified into 15 species comprising Cx. quinquefasciatus (n = 4492; 65.04%), Aedes vittatus (n = 1137; 16.46%) and Ae. aegypti (n = 911; 13.19%) and 2 species groups. Simpson’s dominance and Shannon-Wiener diversity indices of 0.4388 and 1.1942 were recorded, respectively. Chikungunya virus was isolated from pools of Ae. aegypti (1) and Cx. quinquefasciatus (4), two of which were males. Minimum infection rates of 3.0 and 0.8 were observed for female Ae. aegypti and Cx. quinquefasciatus, respectively. Between 25 and 31.3% of exposed mosquitoes became infected with CHIKV 7, 14 and 21 days post-exposure. For the experimentally infected Cx. quinquefasciatus mosquitoes, between 13 and 40% had the virus disseminated, with 100% transmission being observed among those with disseminated infection. Conclusions These results demonstrated high risk of chikungunya transmission for residents in the sampled areas of Mombasa. Transmission data confirmed the probable role played by Cx. quinquefasciatus in the outbreak while the role of Ae. vittatus in the transmission of chikungunya virus remains unknown.

BackgroundBunyamwera(BUNV) and Ngari (NGIV) viruses are arboviruses of medical importance globally, the viruses are endemic in Africa, Aedes(Ae) aegypti and Anopheles(An) gambiae mosquitoes are currently competent vectors for BUNV and NGIV respectively. Both viruses have been isolated from humans and mosquitoes in various ecologies of Kenya. Understanding the risk patterns and spread of the viruses necessitate studies of vector competence in local vector population of Ae. simpsoni sl which is abundant in the coastal region. This study sought to assess the ability of Ae. Simpsoni sl mosquitoes abundant at the Coast of Kenya to transmit these viruses in experimental laboratory experiments.MethodsField collected larvae/pupae of Ae. Simpsoni sl mosquitoes from Rabai, Kilifi County, were reared to adults, the first filial generation (F0) females' mosquitoes were orally exposed to infectious blood meal with isolates of the viruses using the hemotek membrane feeder. The exposed mosquitoes were incubated under insectary conditions and sampled on day 7, 14 and 21days post infection to determine susceptibility to the virus infection using plaque assay.ResultsA total of 379 (Bunyamwera virus 255 and Ngari virus 124) Ae. simpsoni sl were orally exposed to infectious blood meal. Overall, the infection rate (IR) for BUNV and NGIV were 2.7 and 0.9% respectively. Dissemination occurred in 5 out 7 mosquitoes with mid-gut infection for Bunyamwera virus and 1 out of 2 mosquitoes with mid-gut infection for Ngari virus. Further, the transmission was observed in 1 out of 5 mosquitoes that had disseminated infection and no transmission was observed for Ngari virus in all days post infection (dpi).ConclusionOur study shows that Ae. simpsoni sl. is a laboratory competent vector for Bunyamwera virus since it was able to transmit the virus through capillary feeding while NGIV infection was restricted to midgut infection and disseminated infection, these finding adds information on the epidemiology of the viruses and vector control plan.

INTRODUCTION:Multiple outbreaks of Rift Valley Fever (RVF) with devastating effects have occurred in East Africa. These outbreaks cause disease in both livestock and humans and affect poor households most severely. Communities living in areas practicing nomadic livestock movement may be at higher risk of infection. This study sought to i) determine the human exposure to Rift Valley fever virus (RVFV) in populations living within nomadic animal movement routes in Kenya; and ii) identify risk factors for RVFV infection in these communities. METHODS:A cross-sectional descriptive study design was used. Samples were collected from the year 2014 to 2015 in a community-based sampling exercise involving healthy individuals aged ≥18 years from Isiolo, Tana River, and Garissa counties. In total, 1210 samples were screened by ELISA for the presence of immunoglobulin IgM and IgG antibodies against RVFV. Positive results were confirmed by plaque reduction neutralization test. RESULTS:Overall, IgM and IgG prevalence for all sites combined was 1.4% (95% CI 0.8-2.3%) and 36.4% (95% CI 33.8-39.2%), respectively. Isiolo County recorded a non-significant higher IgG prevalence of 38.8% than Garissa 35.9% and Tana River 32.2% (Chi square = 2.5, df = 2, p = 0.287). Males were significantly at higher risk of infection by RVFV than females (OR = 1.67, 95% CI 1.17-2.39, p

Between late 2017 and mid-2018, a chikungunya fever outbreak occurred in Mombasa, Kenya that followed an earlier outbreak in mid-2016 in Mandera County on the border with Somalia. Using targeted Next Generation Sequencing, we obtained genomes from clinical samples collected during the 2017/2018 Mombasa outbreak. We compared data from the 2016 Mandera outbreak with the 2017/2018 Mombasa outbreak, and found that both had the Aedes aegypti adapting mutations, E1:K211E and E2:V264A. Further to the above two mutations, 11 of 15 CHIKV genomes from the Mombasa outbreak showed a novel triple mutation signature of E1:V80A, E1:T82I and E1:V84D. These novel mutations are estimated to have arisen in Mombasa by mid-2017 (2017.58, 95% HPD: 2017.23, 2017.84). The MRCA for the Mombasa outbreak genomes is estimated to have been present in early 2017 (2017.22, 95% HPD: 2016.68, 2017.63). Interestingly some of the earliest genomes from the Mombasa outbreak lacked the E1:V80A, E1:T82I and E1:V84D substitutions. Previous laboratory experiments have indicated that a substitution at position E1:80 in the CHIKV genome may lead to increased CHIKV transmissibility by Ae. albopictus. Genbank investigation of all available CHIKV genomes revealed that E1:V80A was not present; therefore, our data constitutes the first report of the E1:V80A mutation occurring in nature. To date, chikungunya outbreaks in the Northern and Western Hemispheres have occurred in Ae. aegypti inhabited tropical regions. Notwithstanding, it has been suggested that an Ae. albopictus adaptable ECSA or IOL strain could easily be introduced in these regions leading to a new wave of outbreaks. Our data on the recent Mombasa CHIKV outbreak has shown that a potential Ae. albopictus adapting mutation may be evolving within the East African region. It is even more worrisome that there exists potential for emergence of a CHIKV strain more adapted to efficient transmission by both Ae. albopictus and Ae.aegypti simultaneously. In view of the present data and history of chikungunya outbreaks, pandemic potential for such a strain is now a likely possibility in the future. Thus, continued surveillance of chikungunya backed by molecular epidemiologic capacity should be sustained to understand the evolving public health threat and inform prevention and control measures including the ongoing vaccine development efforts.

BACKGROUND:Kenya has experienced outbreaks of chikungunya in the past years with the most recent outbreak occurring in Mandera in the northern region in May 2016 and in Mombasa in the coastal region from November 2017 to February 2018. Despite the outbreaks in Kenya, studies on vector competence have only been conducted on Aedes aegypti. However, the role played by other mosquito species in transmission and maintenance of the virus in endemic areas remains unclear. This study sought to determine the possible role of rural Aedes bromeliae and Aedes vittatus in the transmission of chikungunya virus, focusing on Kilifi and West Pokot regions of Kenya. METHODS:Four day old female mosquitoes were orally fed on chikungunya virus-infected blood at a dilution of 1:1 of the viral isolate and blood (10(6.4) plaque-forming units [PFU]/ml) using artificial membrane feeder (Hemotek system) for 45 minutes. The engorged mosquitoes were picked and incubated at 29-30°C ambient temperature and 70-80% humidity in the insectary. At days 5, 7 and 10 post-infection, the mosquitoes were carefully dissected to separate the legs and wings from the body and their proboscis individually inserted in the capillary tube containing minimum essential media (MEM) to collect salivary expectorate. The resultant homogenates and the salivary expectorates were tested by plaque assay to determine virus infection, dissemination and transmission potential of the mosquitoes. RESULTS:A total of 515 female mosquitoes (311 Ae. bromeliae and 204 Ae. vittatus) were exposed to the East/Central/South Africa (ECSA) lineage of chikungunya virus. Aedes vittatus showed high susceptibility to the virus ranging between 75-90% and moderate dissemination and transmission rates ranging from 35-50%. Aedes bromeliae had moderate susceptibility ranging between 26-40% with moderate dissemination and transmission rates ranging from 27-55%. CONCLUSION:This study demonstrates that both Ae. vittatus and Ae. bromeliae populations from West Pokot and Kilifi counties in Kenya are competent vectors of chikungunya virus. Based on these results, the two areas are at risk of virus transmission in the event of an outbreak. This study underscores the need to institute vector competence studies for populations of potential vector species as a means of evaluating risk of transmission of the emerging and re-emerging arboviruses in diverse regions of Kenya.

In April, 2004, chikungunya virus (CHIKV) re-emerged in Kenya and eventually spread to the islands in the Indian Ocean basin, South-East Asia, and the Americas. The virus, which is often associated with high levels of viremia in humans, is mostly transmitted by the urban vector, Aedes aegypti. The expansion of CHIKV presents a public health challenge both locally and internationally. In this study, we investigated the ability of Ae. aegypti mosquitoes from three distinct cities in Kenya; Mombasa (outbreak prone), Kisumu, and Nairobi (no documented outbreak) to transmit CHIKV.Aedes aegypti mosquito populations were exposed to different doses of CHIKV (105.6-7.5 plaque-forming units[PFU]/ml) in an infectious blood meal. Transmission was ascertained by collecting and testing saliva samples from individual mosquitoes at 5, 7, 9, and 14 days post exposure. Infection and dissemination were estimated by testing body and legs, respectively, for individual mosquitoes at selected days post exposure. Tissue culture assays were used to determine the presence of infectious viral particles in the body, leg, and saliva samples. The number of days post exposure had no effect on infection, dissemination, or transmission rates, but these rates increased with an increase in exposure dose in all three populations. Although the rates were highest in Ae. aegypti from Mombasa at titers ≥106.9 PFU/ml, the differences observed were not statistically significant (χ2 ≤ 1.04, DF = 1, P ≥ 0.31). Overall, about 71% of the infected mosquitoes developed a disseminated infection, of which 21% successfully transmitted the virus into a capillary tube, giving an estimated transmission rate of about 10% for mosquitoes that ingested ≥106.9 PFU/ml of CHIKV. All three populations of Ae. aegypti were infectious as early as 5-7 days post exposure. On average, viral dissemination only occurred when body titers were ≥104 PFU/ml in all populations.Populations of Ae. aegypti from Mombasa, Nairobi, and Kisumu were all competent laboratory vectors of CHIKV. Viremia of the infectious blood meal was an important factor in Ae. aegypti susceptibility and transmission of CHIKV. In addition to viremia levels, temperature and feeding behavior of Ae. aegypti may also contribute to the observed disease patterns.

Abstract Background Susceptibility of Ae. aegypti mosquito to dengue virus (DENV) varies geographically and can be influenced by climatic factors such as temperature, which affect the incidence, seasonality and distribution of vector-borne diseases. The first outbreak of dengue fever (DF) in Kenya occured in 1982 in the coastal towns of Malindi and Kilifi. Unlike Nairobi where no active dengue transmission has been reported, DF is currently re-emerging at the Coast causing major outbreaks. This study investigated the vector competence of Ae. aegypti populations from two urban areas, Kilifi (Coast of Kenya) and Nairobi (Central Kenya), for DEN-2 virus and the influence of temperature on the same. Methods Four-day old adult female Ae. aegypti mosquitoes collected as eggs from the two sites were exposed to defibrinated sheep blood mixed with DEN-2 virus (105.08 PFU/ml) using a membrane feeder. Half of the exposed mosquitoes were incubated at high temperature (30°C) and the other half at low temperature (26°C), and every 7 days up to day 21 post-infection 30% of the exposed mosquitoes were randomly picked, individually dissected, separated into abdomen and legs, and tested for midgut and disseminated infection, respectively, including virus quantification by plaque assay using Vero cells. Results Nairobi mosquito populations exhibited significantly higher midgut infection rates (16.8%) compared to the Kilifi population (9%; p = 0.0001). Midgut infection rates among the populations varied with temperature levels with a significantly higher infection rate observed for Nairobi at high (21.3%) compared to low temperature (12.0%; p = 0.0037). Similarly, for the Kilifi population, a significantly higher infection rate was recorded at high (11.6%) relative to low temperature (6.8%; p = 0.0162). It is however, noteworthy that disseminated infection was higher among the Kilifi mosquito population (40.7%) than in Nairobi mosquitoes (10.3%; p

Rift Valley fever (RVF) outbreaks in Kenya have increased in frequency and range to include northeastern Kenya where viruses are increasingly being isolated from known (Aedes mcintoshi) and newly-associated (Ae. ochraceus) vectors. The factors contributing to these changing outbreak patterns are unclear and the population genetic structure of key vectors and/or specific virus-vector associations, in particular, are under-studied. By conducting mitochondrial and nuclear DNA analyses on >220 Kenyan specimens of Ae. mcintoshi and Ae. ochraceus, we uncovered high levels of vector complexity which may partly explain the disease outbreak pattern. Results indicate that Ae. mcintoshi consists of a species complex with one of the member species being unique to the newly-established RVF outbreak-prone northeastern region of Kenya, whereas Ae. ochraceus is a homogeneous population that appears to be undergoing expansion. Characterization of specimens from a RVF-prone site in Senegal, where Ae. ochraceus is a primary vector, revealed direct genetic links between the two Ae. ochraceus populations from both countries. Our data strongly suggest that unlike Ae. mcintoshi, Ae. ochraceus appears to be a relatively recent, single 'introduction' into Kenya. These results, together with increasing isolations from this vector, indicate that Ae. ochraceus will likely be of greater epidemiological importance in future RVF outbreaks in Kenya. Furthermore, the overall vector complexity calls into question the feasibility of mosquito population control approaches reliant on genetic modification.