Transmission Dynamics of Crimean-Congo Haemorrhagic Fever Virus (CCHFV): Evidence of Circulation in Humans, Livestock, and Rodents in Diverse Ecologies in Kenya.

Crimean-Congo haemorrhagic fever virus (CCHFV) is the causative agent of CCHF, a fatal viral haemorrhagic fever disease in humans. The maintenance of CCHFV in the ecosystem remains poorly understood. Certain tick species are considered as vectors and reservoirs of the virus. Diverse animals are suspected as amplifiers, with only scarce knowledge regarding rodents in virus epidemiology. In this study, serum samples from febrile patients, asymptomatic livestock (cattle, donkeys, sheep, and goats), and peridomestic rodents from Baringo (Marigat) and Kajiado (Nguruman) counties within the Kenyan Rift Valley were screened for acute CCHFV infection by RT-PCR and for CCHFV exposure by ELISA. RT-PCR was performed on all livestock samples in pools (5-7/pool by species and site) and in humans and rodents individually. CCHFV seropositivity was significantly higher in livestock (11.9%, 113/951) compared to rodents (6.5%, 6/93) and humans (5.9%, 29/493) (p = 0.001). Among the livestock, seropositivity was the highest in donkeys (31.4%, 16/51), followed by cattle (14.1%, 44/310), sheep (9.8%, 29/295) and goats (8.1%, 24/295). The presence of IgM antibodies against CCHFV was found in febrile patients suggesting acute or recent infection. CCHFV RNA was detected in four pooled sera samples from sheep (1.4%, 4/280) and four rodent tissues (0.83%, 4/480) showing up to 99% pairwise nucleotide identities among each other. Phylogenetic analyses of partial S segment sequences generated from these samples revealed a close relationship of 96-98% nucleotide identity to strains in the CCHFV Africa 3 lineage. The findings of this study suggest active unnoticed circulation of CCHFV in the study area and the involvement of livestock, rodents, and humans in the circulation of CCHFV in Kenya. The detection of CCHF viral RNA and antibodies against CCHFV in rodents suggests that they may participate in the viral transmission cycle.

Analyses of Mosquito Species Composition, Blood-Feeding Habits and Infection with Insect-Specific Flaviviruses in Two Arid, Pastoralist-Dominated Counties in Kenya.

Insect-specific flaviviruses (ISFs), although not known to be pathogenic to humans and animals, can modulate the transmission of arboviruses by mosquitoes. In this study, we screened 6665 host-seeking, gravid and blood-fed mosquitoes for infection with flaviviruses and assessed the vertebrate hosts of the blood-fed mosquitoes sampled in Baringo and Kajiado counties; both dryland ecosystem counties in the Kenyan Rift Valley. Sequence fragments of two ISFs were detected. Cuacua virus (CuCuV) was found in three blood-fed Mansonia (Ma.) africana. The genome was sequenced by next-generation sequencing (NGS), confirming 95.8% nucleotide sequence identity to CuCuV detected in Mansonia sp. in Mozambique. Sequence fragments of a potential novel ISF showing nucleotide identity of 72% to Aedes flavivirus virus were detected in individual blood-fed Aedes aegypti, Anopheles gambiae s.l., Ma. africana and Culex (Cx.) univittatus, all having fed on human blood. Blood-meal analysis revealed that the collected mosquitoes fed on diverse hosts, primarily humans and livestock, with a minor representation of wild mammals, amphibians and birds. The potential impact of the detected ISFs on arbovirus transmission requires further research.

Divergent Hantavirus in Somali Shrews (Crocidura somalica) in the Semi-Arid North Rift, Kenya.

Hantaviruses are zoonotic rodent-borne viruses that are known to infect humans and cause various symptoms of disease, including hemorrhagic fever with renal and cardiopulmonary syndromes. They have a segmented single-stranded, enveloped, negative-sense RNA genome and are widely distributed. This study aimed to investigate the circulation of rodent-borne hantaviruses in peridomestic rodents and shrews in two semi-arid ecologies within the Kenyan Rift Valley. The small mammals were trapped using baited folding Sherman traps set within and around houses, then they were sedated and euthanatized through cervical dislocation before collecting blood and tissue samples (liver, kidney, spleen, and lungs). Tissue samples were screened with pan-hantavirus PCR primers, targeting the large genome segment (L) encoding the RNA-dependent RNA polymerase (RdRp). Eleven of the small mammals captured were shrews (11/489, 2.5%) and 478 (97.5%) were rodents. A cytochrome b gene-based genetic assay for shrew identification confirmed the eleven shrews sampled to be Crocidura somalica. Hantavirus RNA was detected in three (3/11, 27%) shrews from Baringo County. The sequences showed 93-97% nucleotide and 96-99% amino acid identities among each other, as well as 74-76% nucleotide and 79-83% amino acid identities to other shrew-borne hantaviruses, such as Tanganya virus (TNGV). The detected viruses formed a monophyletic clade with shrew-borne hantaviruses from other parts of Africa. To our knowledge, this constitutes the first report published on the circulation of hantaviruses in shrews in Kenya.

Characterization of a Novel Orbivirus from Cattle Reveals Active Circulation of a Previously Unknown and Pathogenic Orbivirus in Ruminants in Kenya.

Arboviruses are among emerging pathogens of public and veterinary health significance. However, in most of sub-Saharan Africa, their role in the aetiologies of diseases in farm animals is poorly described due to paucity of active surveillance and appropriate diagnosis. Here, we report the discovery of a previously unknown orbivirus in cattle collected in the Kenyan Rift Valley in 2020 and 2021. We isolated the virus in cell culture from the serum of a clinically sick cow aged 2 to 3 years, presenting signs of lethargy. High-throughput sequencing revealed an orbivirus genome architecture with 10 double-stranded RNA segments and a total size of 18,731 bp. The VP1 (Pol) and VP3 (T2) nucleotide sequences of the detected virus, tentatively named Kaptombes virus (KPTV), shared maximum similarities of 77.5% and 80.7% to the mosquito-borne Sathuvachari virus (SVIV) found in some Asian countries, respectively. Screening of 2,039 sera from cattle, goats, and sheep by specific RT-PCR identified KPTV in three additional samples originating from different herds collected in 2020 and 2021. Neutralizing antibodies against KPTV were found in 6% of sera from ruminants (12/200) collected in the region. In vivo experiments with new-born and adult mice induced body tremors, hind limb paralysis, weakness, lethargy, and mortality. Taken together, the data suggest the detection of a potentially disease-causing orbivirus in cattle in Kenya. Its impact on livestock, as well as its potential economic damage, needs to be addressed in future studies using targeted surveillance and diagnostics. IMPORTANCE The genus Orbivirus contains several viruses that cause large outbreaks in wild and domestic animals. However, there is little knowledge on the contribution of orbiviruses to diseases in livestock in Africa. Here, we report the identification of a novel presumably disease-causing orbivirus in cattle, Kenya. The virus, designated Kaptombes virus (KPTV), was initially isolated from a clinically sick cow aged 2 to 3 years, presenting signs of lethargy. The virus was subsequently detected in three additional cows sampled in neighboring locations in the subsequent year. Neutralizing antibodies against KPTV were found in 10% of cattle sera. Infection of new-born and adult mice with KPTV caused severe symptoms and lead to death. Together, these findings indicate the presence of a previously unknown orbivirus in ruminants in Kenya. These data are of relevance as cattle represents an important livestock species in farming industry and often is the main source of livelihoods in rural areas of Africa.

Phlebovirus diversity in ticks from livestock in arid ecologies in Kenya.

Phleboviruses are emerging pathogens of public health importance. However, their association with ticks is poorly described, particularly in Africa. Here, adult ticks infesting cattle, goats and sheep were collected in two dryland pastoralist ecosystems of Kenya (Baringo and Kajiado counties) and were screened for infection with phleboviruses. Ticks mainly belonged to the species Rhipicephalus appendiculatus, Hyalomma impeltatum, and Hyalomma rufipes. A fragment of the RNA-dependent RNA polymerase (RdRp) gene was identified in thirty of 671 tick pools, of which twenty-nine were from livestock sampled in Baringo county. Phylogenetic analyses revealed that twenty-five sequences were falling in three clades within the group of tick-associated phleboviruses. The sequences of the three clades showed nucleotide distances 8%, 19% and 22%, respectively, to previously known viruses suggesting that these sequence fragments may belong to three distinct viruses. Viruses of the group of tick-associated phleboviruses have been found in several countries and continents but so far have not been associated with disease in humans or animals. In addition, five sequences were found to group with the sandfly-associated phleboviruses Bogoria virus, Perkerra virus and Ntepes virus recently detected in the same region. Further studies are needed to investigate the transmission and maintenance cycles of these viruses, as well as to assess their potential to infect vertebrates.

Viral diversity and blood-feeding patterns of Afrotropical Culicoides biting midges (Diptera: Ceratopogonidae).

Introduction: Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of arboviral pathogens that primarily affect livestock represented by Schmallenberg virus (SBV), epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV). In Kenya, studies examining the bionomic features of Culicoides including species diversity, blood-feeding habits, and association with viruses are limited. Methods: Adult Culicoides were surveyed using CDC light traps in two semi-arid ecologies, Baringo and Kajiado counties, in Kenya. Blood-fed specimens were analysed through polymerase chain reaction (PCR) and sequencing of cytochrome oxidase subunit 1 (cox1) barcoding region. Culicoides pools were screened for virus infection by generic RT-PCR and next-generation sequencing (NGS). Results: Analysis of blood-fed specimens confirmed that midges had fed on cattle, goats, sheep, zebra, and birds. Cox1 barcoding of the sampled specimens revealed the presence of known vectors of BTV and epizootic hemorrhagic disease virus (EHDV) including species in the Imicola group (Culicoides imicola) and Schultzei group (C. enderleni, C. kingi, and C. chultzei). Culicoides leucostictus and a cryptic species distantly related to the Imicola group were also identified. Screening of generated pools (11,006 individuals assigned to 333 pools) by generic RT-PCR revealed presence of seven phylogenetically distinct viruses grouping in the genera Goukovirus, Pacuvirus and Orthobunyavirus. The viruses showed an overall minimum infection rate (MIR) of 7.0% (66/333, 95% confidence interval (CI) 5.5-8.9). In addition, full coding sequences of two new iflaviruses, tentatively named Oloisinyai_1 and Oloisinyai_2, were generated by next-generation sequencing (NGS) from individual homogenate of Culicoides pool. Conclusion: The results indicate a high genetic diversity of viruses in Kenyan biting midges. Further insights into host-vector-virus interactions as well as investigations on the potential clinical significance of the detected viruses are warranted.

Circulation of Ngari Virus in Livestock, Kenya.

Ngari virus (NRIV) is a mosquito-borne reassortant orthobunyavirus that causes severe febrile illness and hemorrhagic fever in humans and small ruminants. Due to limited diagnostics and surveillance, NRIV has only been detected sporadically during Rift Valley fever virus outbreaks. Little is known on its interepidemic maintenance and geographic distribution. In this study, sera from cattle, goats, and sheep were collected through a cross-sectional survey after the rainy seasons between 2020 and 2021 in two pastoralist-dominated semiarid ecosystems, Baringo and Kajiado counties in Kenya. NRIV was detected in 11 apparently healthy animals (11/2,039, 0.54%) by RT-PCR and isolated in cell culture from seven individuals. Growth analyses displayed efficient replication in cells from sheep and humans in contrast to weak replication in goat cells. NRIV infection of a wide variety of different vector cells showed only rapid replication in Aedes albopictus cells but not in cells derived from other mosquito species or sandflies. Phylogenetic analyses of complete-coding sequences of L, M, and S segments of four viruses showed that the Kenyan sequences established a monophyletic clade most closely related to a NRIV sequence from a small ruminant from Mauritania. NRIV neutralizing reactivity in cattle, goats, and sheep were 41.6% (95% CI = 30 to 54.3), 52.4% (95% CI = 37.7 to 66.6), and 19% (95% CI = 9.7 to 33.6), respectively. This is the first detection of NRIV in livestock in Kenya. Our results demonstrate active and undetected circulation of NRIV in the three most common livestock species highlighting the need for an active one-health surveillance of host networks, including humans, livestock, and vectors. IMPORTANCE Surveillance of vectors and hosts for infection with zoonotic arthropod-borne viruses is important for early detection and intervention measures to prevent outbreaks. Here, we report the undetected circulation of Ngari virus (NRIV) in apparently healthy cattle, sheep, and goats in Kenya. NRIV is associated with outbreaks of hemorrhagic fever in humans and small ruminants. We demonstrate the isolation of infectious virus from several animals as well as presence of neutralizing antibodies in 38% of the tested animals. Our data indicate active virus circulation and endemicity likely having important implications for human and animal health.

Jingmen Tick Virus in Ticks from Kenya.

Jingmen tick virus (JMTV) is an arbovirus with a multisegmented genome related to those of unsegmented flaviviruses. The virus first described in Rhipicephalus microplus ticks collected in Jingmen city (Hubei Province, China) in 2010 is associated with febrile illness in humans. Since then, the geographic range has expanded to include Trinidad and Tobago, Brazil, and Uganda. However, the ecology of JMTV remains poorly described in Africa. We screened adult ticks (n = 4550, 718 pools) for JMTV infection by reverse transcription polymerase chain reaction (RT-PCR). Ticks were collected from cattle (n = 859, 18.88%), goats (n = 2070, 45.49%), sheep (n = 1574, 34.59%), and free-ranging tortoises (Leopard tortoise, Stigmochelys pardalis) (n = 47, 1.03%) in two Kenyan pastoralist-dominated areas (Baringo and Kajiado counties) with a history of undiagnosed febrile human illness. Surprisingly, ticks collected from goats (0.3%, 95% confidence interval (CI) 0.1-0.5), sheep (1.8%, 95% CI 1.2-2.5), and tortoise (74.5%, 95% CI 60.9-85.4, were found infected with JMTV, but ticks collected from cattle were all negative. JMTV ribonucleic acid (RNA) was also detected in blood from tortoises (66.7%, 95% CI 16.1-97.7). Intragenetic distance of JMTV sequences originating from tortoise-associated ticks was greater than that of sheep-associated ticks. Phylogenetic analyses of seven complete-coding genome sequences generated from tortoise-associated ticks formed a monophyletic clade within JMTV strains from other countries. In summary, our findings confirm the circulation of JMTV in ticks in Kenya. Further epidemiological surveys are needed to assess the potential public health impact of JMTV in Kenya.

Outdoor malaria vector species profile in dryland ecosystems of Kenya.

Outdoor biting by anopheline mosquitoes is one of the contributors to residual malaria transmission, but the profile of vectors driving this phenomenon is not well understood. Here, we studied the bionomics and genetically characterized populations of An. gambiae and An. funestus complexes trapped outdoors in three selected dryland areas including Kerio Valley, Nguruman and Rabai in Kenya. We observed a higher abundance of Anopheles funestus group members (n = 639, 90.6%) compared to those of the An. gambiae complex (n = 66, 9.4%) with An. longipalpis C as the dominant vector species with a Plasmodium falciparum sporozoite rate (Pfsp) of 5.2% (19/362). The known malaria vectors including An. funestus s.s. (8.7%, 2/23), An. gambiae (14.3%, 2/14), An. rivulorum (14.1%, 9/64), An. arabiensis (1.9%, 1/52) occurred in low densities and displayed high Pfsp rates, which varied with the site. Additionally, six cryptic species found associated with the An. funestus group harbored Pf sporozoites (cumulative Pfsp rate = 7.2%, 13/181). We detected low frequency of resistant 119F-GSTe2 alleles in An. funestus s.s. (15.6%) and An. longipalpis C (3.1%) in Kerio Valley only. Evidence of outdoor activity, emergence of novel and divergent vectors and detection of mutations conferring metabolic resistance to pyrethroid/DDT could contribute to residual malaria transmission posing a threat to effective malaria control.

Survival rate, blood feeding habits and sibling species composition of Aedes simpsoni complex: Implications for arbovirus transmission risk in East Africa.

Aedes simpsoni complex has a wide distribution in Africa and comprises at least three described sub-species including the yellow fever virus (YFV) vector Ae. bromeliae. To date, the distribution and relative contributions of the sub-species and/or subpopulations including bionomic characteristics in relation to YF transmission dynamics remain poorly studied. In this study conducted in two areas with divergent ecosystems: peri-urban (coastal Rabai) and rural (Rift Valley Kerio Valley) in Kenya, survival rate was estimated by parity in Ae. simpsoni s.l. mosquitoes sampled using CO2-baited BG Sentinel traps. We then applied PCR targeting the nuclear internal transcribed spacer 2 (ITS2), region followed by sequencing and phylogenetic analytics to identify the sibling species in the Ae. simpsoni complex among parous and blood fed cohorts. Our results show that Ae. bromeliae was the most dominant sub-species in both areas, exhibiting high survival rates, human blood-feeding, and potentially, high vectorial capacity for pathogen transmission. We document for the first time the presence of Ae. lilii in Kenya and potentially yet-to-be described species in the complex displaying human feeding tendencies. We also infer a wide host feeding range on rodents, reptile, and domestic livestock besides humans especially for Ae. bromeliae. This feeding trend could likely expose humans to various zoonotic pathogens. Taken together, we highlight the utility of genotype-based analyses to generate precision surveillance data of vector populations for enhanced disease risk prediction and to guide cost-effective interventions (e.g. YF vaccinations).

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.

A previously unreported potential malaria vector in a dry ecology of Kenya.

BACKGROUND: In Kenya, malaria remains a major public health menace equally affecting the semi-arid to arid ecologies. However, entomologic knowledge of malaria vectors in such areas remains poor. METHODS: Morphologically-identified wild-caught Anopheles funestus (s.l.) specimens trapped outdoors from the semi-arid to arid area of Kacheliba, West Pokot County, Kenya, were analysed by PCR and sequencing for species identification, malaria parasite infection and host blood-meal sources. RESULTS: Three hundred and thirty specimens were analysed to identify sibling species of the An. funestus group, none of which amplified using the available primers; two were infected with Plasmodium falciparum and Plasmodium ovale, separately, while 84% (n = 25) of the blood-fed specimens had fed on humans. Mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences of 55 specimens (Plasmodium-positive, blood-fed and Plasmodium-negative) did not match reference sequences, possibly suggesting a previously unreported species, resolving as two clades. CONCLUSIONS: Our findings indicate the existence of yet-to-be identified and described anopheline species with a potential as malaria vectors in Kenya.

Population genetics of Anopheles funestus, the African malaria vector, Kenya.

BACKGROUND: Anopheles funestus is among the major malaria vectors in Kenya and sub-Saharan Africa and has been recently implicated in persistent malaria transmission. However, its ecology and genetic diversity remain poorly understood in Kenya. METHODS: Using 16 microsatellite loci, we examined the genetic structure of An. funestus sampled from 11 locations (n = 426 individuals) across a wide geographical range in Kenya spanning coastal, western and Rift Valley areas. RESULTS: Kenyan An. funestus resolved as three genetically distinct clusters. The largest cluster (FUN1) broadly included samples from western and Rift Valley areas of Kenya with two clusters identified from coastal Kenya (FUN2 and FUN3), not previously reported. Geographical distance had no effect on population differentiation of An. funestus. We found a significant variation in the mean Plasmodium infectivity between the clusters (χ2 = 12.1, df = 2, P = 0.002) and proportional to the malaria prevalence in the different risk zones of Kenya. Notably, there was variation in estimated effective population sizes between the clusters, suggesting possible differential impact of anti-vector interventions in represented areas. CONCLUSIONS: Heterogeneity among Kenyan populations of An. funestus will impact malaria vector control with practical implications for the development of gene-drive technologies. The difference in Plasmodium infectivity and effective population size between the clusters could suggest potential variation in phenotypic characteristics relating to competence or insecticide resistance. This is worth examining in future studies.

Insights into malaria transmission among Anopheles funestus mosquitoes, Kenya.

BACKGROUND: Most malaria vectors belong to species complexes. Sibling species often exhibit divergent behaviors dictating the measures that can be deployed effectively in their control. Despite the importance of the Anopheles funestus complex in malaria transmission in sub-Saharan Africa, sibling species have rarely been identified in the past and their vectoring potential remains understudied. METHODS: We analyzed 1149 wild-caught An. funestus (senso lato) specimens from 21 sites in Kenya, covering the major malaria endemic areas including western, central and coastal areas. Indoor and outdoor collection tools were used targeting host-seeking and resting mosquitoes. The identity of sibling species, infection with malaria Plasmodium parasites, and the host blood meal sources of engorged specimens were analyzed using PCR-based and sequencing methods. RESULTS: The most abundant sibling species collected in all study sites were Anopheles funestus (59.8%) and Anopheles rivulorum (32.4%) among the 1062 successfully amplified specimens of the An. funestus complex. Proportionally, An. funestus dominated in indoor collections whilst An. rivulorum dominated in outdoor collections. Other species identified were Anopheles leesoni (4.6%), Anopheles parensis (2.4%), Anopheles vaneedeni (0.1%) and for the first time in Kenya, Anopheles longipalpis C (0.7%). Anopheles funestus had an overall Plasmodium infection rate of 9.7% (62/636), predominantly Plasmodium falciparum (59), with two infected with Plasmodium ovale and one with Plasmodium malariae. There was no difference in the infection rate between indoor and outdoor collections. Out of 344 An. rivulorum, only one carried P. falciparum. We also detected P. falciparum infection in two non-blood-fed An. longipalpis C (2/7) which is the first record for this species in Kenya. The mean human blood indices for An. funestus and An. rivulorum were 68% (93/136) and 64% (45/70), respectively, with feeding tendencies on a broad host range including humans and domestic animals such as cow, goat, sheep, chicken and pig. CONCLUSIONS: Our findings underscore the importance of active surveillance through application of molecular approaches to unravel novel parasite-vector associations possibly contributed by cryptic species with important implications for effective malaria control and elimination.