Genomic surveillance of Rift Valley fever virus: from sequencing to lineage assignment.

Genetic evolution of Rift Valley fever virus (RVFV) in Africa has been shaped mainly by environmental changes such as abnormal rainfall patterns and climate change that has occurred over the last few decades. These gradual environmental changes are believed to have effected gene migration from macro (geographical) to micro (reassortment) levels. Presently, 15 lineages of RVFV have been identified to be circulating within the Sub-Saharan Africa. International trade in livestock and movement of mosquitoes are thought to be responsible for the outbreaks occurring outside endemic or enzootic regions. Virus spillover events contribute to outbreaks as was demonstrated by the largest epidemic of 1977 in Egypt. Genomic surveillance of the virus evolution is crucial in developing intervention strategies. Therefore, we have developed a computational tool for rapidly classifying and assigning lineages of the RVFV isolates. The computational method is presented both as a command line tool and a web application hosted at https://www.genomedetective.com/app/typingtool/rvfv/ . Validation of the tool has been performed on a large dataset using glycoprotein gene (Gn) and whole genome sequences of the Large (L), Medium (M) and Small (S) segments of the RVFV retrieved from the National Center for Biotechnology Information (NCBI) GenBank database. Using the Gn nucleotide sequences, the RVFV typing tool was able to correctly classify all 234 RVFV sequences at species level with 100% specificity, sensitivity and accuracy. All the sequences in lineages A (n = 10), B (n = 1), C (n = 88), D (n = 1), E (n = 3), F (n = 2), G (n = 2), H (n = 105), I (n = 2), J (n = 1), K (n = 4), L (n = 8), M (n = 1), N (n = 5) and O (n = 1) were also correctly classified at phylogenetic level. Lineage assignment using whole RVFV genome sequences (L, M and S-segments) did not achieve 100% specificity, sensitivity and accuracy for all the sequences analyzed. We further tested our tool using genomic data that we generated by sequencing 5 samples collected following a recent RVF outbreak in Kenya. All the 5 samples were assigned lineage C by both the partial (Gn) and whole genome sequence classifiers. The tool is useful in tracing the origin of outbreaks and supporting surveillance efforts.Availability: https://github.com/ajodeh-juma/rvfvtyping.

Evidence of circulation of Orthobunyaviruses in diverse mosquito species in Kwale County, Kenya.

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.

Endemic chikungunya fever in Kenyan children: a prospective cohort study.

BACKGROUND: Chikungunya fever (CHIKF) was first described in Tanzania in 1952. Several epidemics including East Africa have occurred, but there are no descriptions of longitudinal surveillance of endemic disease. Here, we estimate the incidence of CHIKF in coastal Kenya and describe the associated viral phylogeny. METHODS: We monitored acute febrile illnesses among 3500 children visiting two primary healthcare facilities in coastal Kenya over a 5-year period (2014-2018). Episodes were linked to a demographic surveillance system and blood samples obtained. Cross-sectional sampling in a community survey of a different group of 435 asymptomatic children in the same study location was done in 2016. Reverse-transcriptase PCR was used for chikungunya virus (CHIKV) screening, and viral genomes sequenced for phylogenetic analyses. RESULTS: We found CHIKF to be endemic in this setting, associated with 12.7% (95% CI 11.60, 13.80) of all febrile presentations to primary healthcare. The prevalence of CHIKV infections among asymptomatic children in the community survey was 0.7% (95% CI 0.22, 2.12). CHIKF incidence among children < 1 year of age was 1190 cases/100,000-person years and 63 cases/100,000-person years among children aged ≥10 years. Recurrent CHIKF episodes, associated with fever and viraemia, were observed among 19 of 170 children with multiple febrile episodes during the study period. All sequenced viral genomes mapped to the ECSA genotype albeit distinct from CHIKV strains associated with the 2004 East African epidemic. CONCLUSIONS: CHIKF may be a substantial public health burden in primary healthcare on the East African coast outside epidemic years, and recurrent infections are common.

Evolution of mosquito preference for humans linked to an odorant receptor.

Female mosquitoes are major vectors of human disease and the most dangerous are those that preferentially bite humans. A 'domestic' form of the mosquito Aedes aegypti has evolved to specialize in biting humans and is the main worldwide vector of dengue, yellow fever, and chikungunya viruses. The domestic form coexists with an ancestral, 'forest' form that prefers to bite non-human animals and is found along the coast of Kenya. We collected the two forms, established laboratory colonies, and document striking divergence in preference for human versus non-human animal odour. We further show that the evolution of preference for human odour in domestic mosquitoes is tightly linked to increases in the expression and ligand-sensitivity of the odorant receptor AaegOr4, which we found recognizes a compound present at high levels in human odour. Our results provide a rare example of a gene contributing to behavioural evolution and provide insight into how disease-vectoring mosquitoes came to specialize on humans.

Sand Fly-Associated Phlebovirus with Evidence of Neutralizing Antibodies in Humans, Kenya.

We describe a novel virus, designated Ntepes virus (NPV), isolated from sand flies in Kenya. NPV has the characteristic phlebovirus trisegmented genome architecture and is related to, but distinct from, Gabek Forest phlebovirus. Diverse cell cultures derived from wildlife, livestock, and humans were susceptible to NPV, with pronounced permissiveness in swine and rodent cells. NPV infection of newborn mice caused rapid and fatal illness. Permissiveness for NPV replication in sand fly cells, but not mosquito cells, suggests a vector-specific adaptation. Specific neutralizing antibodies were found in 13.9% (26/187) of human serum samples taken at the site of isolation of NPV as well as a disparate site in northeastern Kenya, suggesting a wide distribution. We identify a novel human-infecting arbovirus and highlight the importance of rural areas in tropical Africa for arbovirus surveillance as well as extending arbovirus surveillance to include hematophagous arthropods other than mosquitoes.

Aedes vector-host olfactory interactions in sylvatic and domestic dengue transmission environments.

Interactions between Aedes (Stegomyia) species and non-human primate (NHP) and human hosts govern the transmission of the pathogens, dengue, zika, yellow fever and chikungunya viruses. Little is known about Aedes mosquito olfactory interactions with these hosts in the domestic and sylvatic cycles where these viruses circulate. Here, we explore how the different host-derived skin odours influence Aedes mosquito responses in these two environments. In field assays, we show that the cyclic ketone cyclohexanone is a signature cue for Aedes mosquitoes to detect the NHP baboon, sykes and vervet, whereas for humans, it is the unsaturated aliphatic keto-analogue 6-methyl-5-hepten-2-one (sulcatone). We find that in the sylvatic environment, CO2-baited traps combined with either cyclohexanone or sulcatone increased trap catches of Aedes mosquitoes compared to traps either baited with CO2 alone or CO2 combined with NHP- or human-derived crude skin odours. In the domestic environment, each of these odourants and crude human skin odours increased Aedes aegypti catches in CO2-baited traps. These results expand our knowledge on the role of host odours in the ecologies of Aedes mosquitoes, and the likelihood of associated spread of pathogens between primates and humans. Both cyclohexanone and sulcatone have potential practical applications as lures for monitoring Aedes disease vectors.

Comparison of enzyme-linked immunosorbent assay systems using rift valley fever virus nucleocapsid protein and inactivated virus as antigens.

BACKGROUND: Rift Valley Fever (RVF) is a mosquito-borne viral zoonosis. To detect RVF virus (RVFV) infection, indirect immunoglobulin G (IgG) and immunoglobulin M (IgM) enzyme linked immunosorbent assays (ELISAs) which utilize recombinant RVFV nucleocapsid (RVFV-N) protein as assay antigen, have reportedly been used, however, there is still a need to develop more sensitive and specific methods of detection. METHODS: RVFV-N protein was expressed in Escherichia coli (E. coli) and purified by histidine-tag based affinity chromatography. This recombinant RVFV-N (rRVFV-N) protein was then used as antigen to develop an IgG sandwich ELISA and IgM capture ELISAs for human sera. Ninety six serum samples collected from healthy volunteers during the RVF surveillance programme in Kenya in 2013, and 93 serum samples collected from RVF-suspected patients during the 2006-2007 RVF outbreak in Kenya were used respectively, to evaluate the newly established rRVFV-N protein-based IgG sandwich ELISA and IgM capture ELISA systems in comparison with the inactivated virus-based ELISA systems. RESULTS: rRVFV-N protein-based-IgG sandwich ELISA and IgM capture ELISA for human sera were established. Both the new ELISA systems were in 100% concordance with the inactivated virus-based ELISA systems, with a sensitivity and specificity of 100%. CONCLUSIONS: Recombinant RVFV-N is a safe and affordable antigen for RVF diagnosis. Our rRVFV-N-based ELISA systems are safe and reliable tools for diagnosis of RVFV infection in humans and especially useful in large-scale epidemiological investigation and for application in developing countries.

Evolutionary analyses of Sindbis virus strains isolated from mosquitoes in Kenya.

Sindbis virus (SINV) is a mosquito borne virus maintained in nature in a mosquito-bird cycle, with human outbreaks known to occur in Northern Europe and parts of Africa. We analyzed five SINV strains isolated in Kenya from five different mosquito species and geographic locations between 2007 and 2013. Phylogenetic relationships and evolutionary inferences were performed using maximum likelihood and Bayesian phylogenetic inference approaches. Selection analyses were carried out based on the virus envelope glycoproteins (E1, E2) and non-structural protein (nsP4) genes. Phylogenetic analysis revealed that all the Kenyan SINV isolates belonged to genotype 1 with selection analyses suggesting that SINV E1, E2 and nsP4 protein encoding genes were predominantly evolving under negative selection.

Vector competence of Aedes aegypti in transmitting Chikungunya virus: effects and implications of extrinsic incubation temperature on dissemination and infection rates.

BACKGROUND: Aedes aegypti is a competent arthropod vector of chikungunya virus (CHIKV). The rate at which the virus disseminate in the vector is limited by temperature of their environment which can be an important determinant of geographical and seasonal limits to transmission by the arthropods in the tropics. This study investigated the vector competence of Ae. aegypti for CHIKV at ambient temperature of 32 and 26 °C (Coastal and Western Kenya respectively) reared at Extrinsic Incubation Temperature (EIT) of 32 and 26 °C that resembles those in the two regions. METHODS: Ae. aegypti eggs were collected from coastal and Western Kenya, hatched in the insectary and reared to F1 generation. Four-day old mosquitoes were exposed to CHIKV through a membrane feeding. They were then incubated in temperatures mimicking the mean annual temperatures for Trans-Nzoia (26 °C) and Lamu (32 °C). After every 7, 10 and 13 days post infection (DPI); one third of exposed mosquitoes were sampled and assayed for virus infection and dissemination. RESULTS: The midgut infection rates (MIR) of Ae. aegypti sampled from Coastal Region was significantly (p < 0.05) higher than those sampled from Western Kenya, with no statistical differences observed for the coastal Ae. aegypti at EIT 26 and at 32 °C. The MIR of Ae. aegypti from the Western Region was significantly (p < 0.05) affected by the EIT, with mosquito reared at EIT 32 °C exhibiting higher MIR than those reared at EIT 26 °C. There was a significant (p < 0.05) interactive effects of the region, EIT and DPI on MIR. The disseminated infection rates for the CHIKV in Ae. aegypti in the legs (DIR-L) was higher in mosquitoes sampled from Coast regardless of the EIT while those from Western Kenya, dissemination rates were significantly higher at higher EIT of 32 °C. CONCLUSIONS: Vector competence was higher in mosquito populations reared under high temperatures which weakens the midgut infection barrier. Hence, suggesting Lamu population is more susceptible to CHIKV therefore having a weaker mid gut infection barrier than the Trans Nzoia population. These underscores importance of examining the course of infection at various ambient temperatures and EIT between regions mosquito populations.

Detection of dengue virus serotypes 1, 2 and 3 in selected regions of Kenya: 2011-2014.

BACKGROUND: Dengue fever, a mosquito-borne disease, is associated with illness of varying severity in countries in the tropics and sub tropics. Dengue cases continue to be detected more frequently and its geographic range continues to expand. We report the largest documented laboratory confirmed circulation of dengue virus in parts of Kenya since 1982. METHODS: From September 2011 to December 2014, 868 samples from febrile patients were received from hospitals in Nairobi, northern and coastal Kenya. The immunoglobulin M enzyme linked immunosorbent assay (IgM ELISA) was used to test for the presence of IgM antibodies against dengue, yellow fever, West Nile and Zika. Reverse transcription polymerase chain reaction (RT-PCR) utilizing flavivirus family, yellow fever, West Nile, consensus and sero type dengue primers were used to detect acute arbovirus infections and determine the infecting serotypes. Representative samples of PCR positive samples for each of the three dengue serotypes detected were sequenced to confirm circulation of the various dengue serotypes. RESULTS: Forty percent (345/868) of the samples tested positive for dengue by either IgM ELISA (14.6 %) or by RT-PCR (25.1 %). Three dengue serotypes 1-3 (DENV1-3) were detected by serotype specific RT-PCR and sequencing with their numbers varying from year to year and by region. The overall predominant serotype detected from 2011-2014 was DENV1 accounting for 44 % (96/218) of all the serotypes detected, followed by DENV2 accounting for 38.5 % (84/218) and then DENV3 which accounted for 17.4 % (38/218). Yellow fever, West Nile and Zika was not detected in any of the samples tested. CONCLUSION: From 2011-2014 serotypes 1, 2 and 3 were detected in the Northern and Coastal parts of Kenya. This confirmed the occurrence of cases and active circulation of dengue in parts of Kenya. These results have documented three circulating serotypes and highlight the need for the establishment of active dengue surveillance to continuously detect cases, circulating serotypes, and determine dengue fever disease burden in the country and region.

Detection of West Nile virus in wild birds in Tana River and Garissa Counties, Kenya.

BACKGROUND: West Nile fever virus is a zoonotic arboviral infection maintained in a sylvatic cycle involving mosquito vectors and birds. It is one the arboviruses whose geographical range is expanding because of climate and land use changes that enhance the densities of mosquitoes and promote mosquito-bird-human interactions. We carried out a survey to determine the reservoirs of WNV among wild birds in Tana River and Garissa counties, Kenya. METHODS: Blood samples were obtained from 361 randomly trapped wild birds. Using real-time polymerase chain reaction (PCR), all samples were screened for WNV using gene specific primer sets amplifying a portion of the E region of the genome encoding the envelope protein. RESULTS: Sixty five (65) out of 361 birds screened tested positive for WNV on real-time PCR assay. Sequencing of the selected positive samples reveals that the isolated WNV were most closely related to strains isolated from China (2011). A regression analysis indicated that sampling location influenced the occurrence of WNV while species, age, weight and sex of the birds did not have any effect. CONCLUSIONS: This study provides baseline information on the existing circulation of WNV in this region among wild bird reservoirs that could spill over to the human population and points to the need for implementation of surveillance programs to map the distribution of the virus among reservoirs. Awareness creation about West Nile fever in this region is important to improve its detection and management.

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.

Natural vertical transmission of ndumu virus in Culex pipiens (Diptera: Culicidae) mosquitoes collected as larvae.

Ndumu virus (NDUV) is a member of the family Togaviridae and genus Alphavirus. In Kenya, the virus has been isolated from a range of mosquito species but has not been associated with human or animal morbidity. Little is know about the transmission dynamics or vertebrate reservoirs of this virus. NDUV was isolated from two pools of female Culex pipiens mosquitoes, IJR37 (n = 18) and IJR73 (n = 3), which were collected as larvae on 15 April 2013 from two dambos near the village of Marey, Ijara District, Garissa County, Kenya, and reared to adults and identified to species. These results represent the first field evidence of vertical transmission of NDUV among mosquitoes.

Vector Competence of Selected Mosquito Species in Kenya for Ngari and Bunyamwera Viruses.

Bunyamwera and Ngari viruses have been isolated from a range of mosquito species in Kenya but their actual role in the maintenance and transmission of these viruses in nature remains unclear. Identification of the mosquito species efficient in transmitting these viruses is critical for estimating the risk of human exposure and understanding the transmission and maintenance mechanism. We determined the vector competence of, Aedes aegypti (L.), Culex quinquefasciatus Say, and Anopheles gambiae Giles for transmission of Bunyamwera and Ngari viruses. Ae. aegypti was moderately susceptible to Bunyamwera virus infection at days 7 and 14. Over 60% of Ae. aegypti with a midgut infection developed a disseminated infection at both time points. Approximately 20% more mosquitoes developed a disseminated infection at day 14 compared with day 7. However, while Ae. aegypti was incompetent for Ngari virus, An. gambiae was moderately susceptible to both viruses with dissemination rates more than double by day 14. Cx. quinquefasciatus was refractory to both Bunyamwera and Ngari viruses. Our results underscore the need to continually monitor emergent arboviral genotypes circulating within particular regions as well as vectors mediating these transmissions to preempt and prevent their adverse effects. The genetic mechanism for species specificity and vector competence owing to reassortment needs further investigation.

Ticks and tick-borne viruses from livestock hosts in arid and semiarid regions of the eastern and northeastern parts of Kenya.

Biodiversity and relative abundance of ticks and associated arboviruses in Garissa (northeastern) and Isiolo (eastern) provinces of Kenya were evaluated. Ticks were collected from livestock, identified to species, pooled, and processed for virus isolation. In Garissa, Rhipicephalus pulchellus Gerstacker (57.8%) and Hyalomma truncatum Koch (27.8%) were the most abundant species sampled, whereas R. pulchellus (80.4%) and Amblyomma gemma Donitz (9.6%) were the most abundant in Isiolo. Forty-four virus isolates, comprising Dugbe virus (DUGV; n = 22) and Kupe virus (n = 10; Bunyaviridae: Nirovirus), Dhori virus (DHOV; n = 10; Orthomyxoviridae: Thogotovirus),and Ngari virus (NRIV; n = 2; Bunyaviridae: Orthobunyavirus), were recovered mostly from R. pulchellus sampled in Isiolo. DUGV was mostly recovered from R. pulchellus from sheep and cattle, and DHOV from R. pulchellus from sheep. All Kupe virus isolates were from Isiolo ticks, including R. pulchellus from all the livestock, A. gemma and Amblyomma variegatum F. from cattle, and H. truncatum from goat. NRIV was obtained from R. pulchellus and A. gemma sampled from cattle in Isiolo and Garissa, respectively, while all DHOV and most DUGV (n = 12) were from R. pulchellus sampled from cattle in Garissa. DUGV was also recovered from H. truncatum and Amblyomma hebraeum Koch from cattle and from Rhipicephalus annulatus Say from camel. This surveillance study has demonstrated the circulation of select tick-borne viruses in parts of eastern and northeastern provinces of Kenya, some of which are of public health importance. The isolation of NRIV from ticks is particularly significant because it is usually known to be a mosquito-borne virus affecting humans.

Detection and prevalence of a novel Bandavirus related to Guertu virus in Amblyomma gemma ticks and human populations in Isiolo County, Kenya.

INTRODUCTION: Emerging tick-borne viruses of medical and veterinary importance are increasingly being reported globally. This resurgence emphasizes the need for sustained surveillance to provide insights into tick-borne viral diversity and associated potential public health risks. We report on a virus tentatively designated Kinna virus (KIV) in the family Phenuiviridae and genus Bandavirus. The virus was isolated from a pool of Amblyomma gemma ticks from Kinna in Isiolo County, Kenya. High throughput sequencing of the virus isolate revealed close relatedness to the Guertu virus. The virus genome is consistent with the described genomes of other members of the genus Bandavirus, with nucleotides lengths of 6403, 3332 and 1752 in the Large (L), Medium (M) and Small (S) segments respectively. Phylogenetic analysis showed that the virus clustered with Guertu virus although it formed a distinct and well supported branch. The RdRp amino acid sequence had a 93.3% identity to that of Guertu virus, an indication that the virus is possibly novel. Neutralizing antibodies were detected in 125 (38.6%, 95% CI 33.3-44.1%) of the human sera from the communities in this region. In vivo experiments showed that the virus was lethal to mice with death occurring 6-9 days post-infection. The virus infected mammalian cells (Vero cells) but had reduced infectivity in the mosquito cell line (C636) tested. CONCLUSION: Isolation of this novel virus with the potential to cause disease in human and animal populations necessitates the need to evaluate its public health significance and contribution to disease burden in the affected regions. This also points to the need for continuous monitoring of vector and human populations in high-risk ecosystems to update pathogen diversity.

Monoclonal Antibodies for Rift Valley Fever Virus Nucleocapsid: Application in IgG/IgM ELISA for Sero-Diagnosis.

INTRODUCTION: Rift Valley fever virus (RVFV) belonging to the Phenuiviridae family is responsible for a zoonotic disease called Rift Valley fever (RVF). Currently, RVFV has spread from Africa to Asia, and due to its ability to cause high mortality rates, it has significantly impacted human health and economic development in many societies. Highly specific and sensitive systems for sero-diagnosis of RVFV infection are needed for clinical use. METHOD: BALB/c mice were immunized with recombinant RVFV nucleocapsid (rRVFV-N) protein and the spleen cells fused with SP2/0 myeloma cells to create hybridoma cell lines. The secreted monoclonal antibodies (MAbs) were purified and characterized. Enzyme-linked immunosorbent assay (ELISA) systems for the detection of IgG and IgM using the new MAbs were established and evaluated. Serum samples from 96 volunteers and 93 patients of suspected RVF from Kenya were tested compared with the ELISA systems based on inactivated viruses and the rabbit polyclonal antibody. RESULT: Three monoclonal antibodies against rRVFV-N protein were established. The performance of the MAb-based sandwich IgG ELISA and the IgM capture ELISA perfectly matched the ELISA systems using the inactivated virus or the polyclonal antibody. CONCLUSIONS: Recombinant RVFV-N protein-specific MAbs were developed and they offer useful tools for RVFV studies. The MAb-based ELISA systems for detecting IgG and IgM offer safe and useful options for diagnosing RVFV infections in humans.

Extensive variation and strain-specificity in dengue virus susceptibility among African Aedes aegypti populations.

African populations of the mosquito Aedes aegypti are usually considered less susceptible to infection by human-pathogenic flaviviruses than globally invasive populations found outside Africa. Although this contrast has been well documented for Zika virus (ZIKV), it is unclear to what extent it is true for dengue virus (DENV), the most prevalent flavivirus of humans. Addressing this question is complicated by substantial genetic diversity among DENV strains, most notably in the form of four genetic types (DENV1 to DENV4), that can lead to genetically specific interactions with mosquito populations. Here, we carried out a survey of DENV susceptibility using a panel of seven field-derived Ae. aegypti colonies from across the African range of the species and a colony from Guadeloupe, French West Indies as non-African reference. We found considerable variation in the ability of African Ae. aegypti populations to acquire and replicate a panel of six DENV strains spanning the four DENV types. Although African Ae. aegypti populations were generally less susceptible than the reference non-African population from Guadeloupe, in several instances some African populations were equally or more susceptible than the Guadeloupe population. Moreover, the relative level of susceptibility between African mosquito populations depended on the DENV strain, indicating genetically specific interactions. We conclude that unlike ZIKV susceptibility, there is no clear-cut dichotomy in DENV susceptibility between African and non-African Ae. aegypti. DENV susceptibility of African Ae. aegypti populations is highly heterogeneous and largely governed by the specific pairing of mosquito population and DENV strain.

Mosquito-borne arbovirus surveillance at selected sites in diverse ecological zones of Kenya; 2007 - 2012.

BACKGROUND: Increased frequency of arbovirus outbreaks in East Africa necessitated the determination of distribution of risk by entomologic arbovirus surveillance. A systematic vector surveillance programme spanning 5 years and covering 11 sites representing seven of the eight provinces in Kenya and located in diverse ecological zones was carried out. METHODS: Mosquitoes were sampled bi-annually during the wet seasons and screened for arboviruses. Mosquitoes were identified to species, pooled by species, collection date and site and screened for arboviruses by isolation in cell culture and/or RT-PCR screening and sequencing. RESULTS: Over 450,000 mosquitoes in 15,890 pools were screened with 83 viruses being detected/isolated that include members of the alphavirus, flavivirus and orthobunyavirus genera many of which are known to be of significant public health importance in the East African region. These include West Nile, Ndumu, Sindbis, Bunyamwera, Pongola and Usutu viruses detected from diverse sites. Ngari virus, which was associated with hemorrhagic fever in northern Kenya in 1997/98 was isolated from a pool of Anopheles funestus sampled from Tana-delta and from Aedes mcintoshi from Garissa. Insect only flaviviruses previously undescribed in Kenya were also isolated in the coastal site of Rabai. A flavivirus most closely related to the Chaoyang virus, a new virus recently identified in China and two isolates closely related to Quang Binh virus previously unreported in Kenya were also detected. CONCLUSION: Active transmission of arboviruses of public health significance continues in various parts of the country with possible undetermined human impact. Arbovirus activity was highest in the pastoralist dominated semi-arid to arid zones sites of the country where 49% of the viruses were isolated suggesting a role of animals as amplifiers and indicating the need for improved arbovirus disease diagnosis among pastoral communities.

Ecology and behavior of Anopheles arabiensis in relation to agricultural practices in central Kenya.

Ecological changes associated with anthropogenic ecosystem disturbances can influence human risk of exposure to malaria and other vector-borne infectious diseases. This study in Mwea, Kenya, investigated the pattern of insecticide use in irrigated and nonirrigated agroecosystems and association with the density, survival, and blood-feeding behavior of the malaria vector Anopheles arabiensis. The parity rates of adult An. arabiensis from randomly selected houses were determined by examining their ovaries for tracheal distension, and polymerase chain reaction was used to identify the host blood meals. In addition, structured questionnaires were used to generate data on insecticide use. Anopheles arabiensis densities were highest in irrigated rice agroecosystems, intermediate in irrigated French beans agroecosystems, and lowest in the nonirrigated agroecosystem. Anopheles arabiensis adult survivorship was significantly lower in irrigated rice agroecosystems than in irrigated French beans agroecosystems. The human blood index (HBI) was significantly higher in the nonirrigated agroecosystem compared to irrigated agroecosystems. Moreover, there was marked variation in HBI among villages in irrigated agroecosystems with significantly lower HBI in Kangichiri and Mathangauta compared to Kiuria, Karima, and Kangai. The proportion of mosquitoes with mixed blood meals varied among villages ranging from 0.25 in Kangichiri to 0.83 in Kiuria. Sumithion, dimethoate, and alpha cypermethrin were the most commonly used insecticides. The 1st was used mostly in irrigated rice agroecosystems, and the last 2 were used mostly in irrigated French beans agroecosystems. These findings indicate that agricultural practices may influence the ecology and behavior of malaria vectors and ultimately the risk of malaria transmission.