Olivia Wesula Lwande

Isolation of Tick and Mosquito-Borne Arboviruses from Ticks Sampled from Livestock and Wild Animal Hosts in Ijara District, Kenya

Tick-borne viruses infect humans through the bite of infected ticks during opportunistic feeding or through crushing of ticks by hand and, in some instances, through contact with infected viremic animals. The Ijara District, an arid to semiarid region in northern Kenya, is home to a pastoralist community for whom livestock keeping is a way of life. Part of the Ijara District lies within the boundaries of a Kenya Wildlife Service-protected conservation area. Arbovirus activity among mosquitoes, animals, and humans is reported in the region, mainly because prevailing conditions necessitate that people continuously move their animals in search of pasture, bringing them in contact with ongoing arbovirus transmission cycles. To identify the tick-borne viruses circulating among these communities, we analyzed ticks sampled from diverse animal hosts. A total of 10,488 ticks were sampled from both wildlife and livestock hosts and processed in 1520 pools of up to eight ticks per pool. The sampled ticks were classified to species, processed for virus screening by cell culture using Vero cells and RT-PCR (in the case of Hyalomma species), followed by amplicon sequencing. The tick species sampled included Rhipicephalus pulchellus (76.12%), Hyalomma truncatum (8.68%), Amblyomma gemma (5.00%), Amblyomma lepidum (4.34%), and others (5.86%). We isolated and identified Bunyamwera (44), Dugbe (5), Ndumu (2), Semliki forest (25), Thogoto (3), and West Nile (3) virus strains. This observation constitutes a previously unreported detection of mosquito-borne Semliki forest and Bunyamwera viruses in ticks, and association of West Nile virus with A. gemma and Rh. pulchellus ticks. These findings provide additional evidence on the potential role of ticks and associated animals in the circulation of diverse arboviruses in northeastern Kenya, including viruses previously known to be essentially mosquito borne.

Seroprevalence of Crimean Congo Hemorrhagic Fever Virus in Ijara District, Kenya

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral disease mainly affecting pastoralists who come in contact with animals infested with Hyalomma ticks, which are the key vectors of CCHF virus (CCHFV). CCHFV has been detected among these ticks in parts of North Eastern Kenya. This study aimed to identify acute cases of CCHF, and to determine the extent of previous exposure to CCHFV in an outpatient population attending Sangailu and Ijara health centers, Ijara District, North Eastern Kenya, presenting with acute febrile illnesses. A total of 517 human serum samples were collected from these patients. The samples were screened for the presence of IgM and IgG antibodies to CCHF using CCCHF-IgG and IgM ELISA test kits. A multivariable logistic regression model was used to investigate the risk factors associated with evidence of exposure to CCHFV. A single patient tested positive for anti-CCHF IgM, while 96 were positive for anti-CCHF IgG. The seroprevalence of CCHFV was 23% in Sangailu and 14% in Ijara. Most exposed persons were aged 40-49 years. The likelihood of exposure was highest among farmers (29%). Age, location, and contact with donkeys were significantly associated with exposure to CCHFV. Acute CCHFV infections could be occurring without being detected in this population. This study confirms human exposure to CCHF virus in Ijara District, Kenya, and identifies several significant risk factors associated with exposure to CCHFV.

Spatio-temporal variation in prevalence of Rift Valley fever: a post-epidemic serum survey in cattle and wildlife in Kenya

Background Rift Valley fever (RVF) is a fatal arthropod-borne zoonotic disease of livestock and humans. Since the identification of RVF in Kenya in the 1930s, repeated epizootics and epidemics coinciding with El Niño events have occurred in several locations in Africa and Saudi Arabia, causing mass deaths of livestock and humans. RVF is of great interest worldwide because of its negative effect on international livestock trade and its potential to spread globally. The latter is due to the increasing incidence of extreme climatic phenomena caused by global warming, as well as to the increase in global trade and international travel. How RVF is maintained and sustained between epidemics and epizootics is not clearly understood, but it has been speculated that wildlife reservoirs and trans-ovarian transmission in the vector may be important. Several studies have examined the role of wildlife and livestock in isolation or in a limited geographical location within the one country over a short time (usually less than a year). In this study, we examined the seroprevalence of anti-RVF antibodies in cattle and several wildlife species from several locations in Kenya over an inter-epidemic period spanning up to 7 years. Methods A serological survey of immunoglobulin G (IgG) antibodies to RVF using competitive ELISA was undertaken on 297 serum samples from different wildlife species at various locations in Kenya. The samples were collected between 2008 and 2015. Serum was also collected in 2014 from 177 cattle from Ol Pejeta Conservancy; 113 of the cattle were in close contact with wildlife and the other 64 were kept separate from buffalo and large game by an electric fence. Results The seroprevalence of RVF virus (RVFV) antibody was 11.6% in wildlife species during the study period. Cattle that could come in contact with wildlife and large game were all negative for RVFV. The seroprevalence was relatively high in elephants, rhinoceros, and buffalo, but there were no antibodies in zebras, baboons, vervet monkeys, or wildebeest. Conclusions Diverse species in conservation areas are exposed to RVFV. RVFV exposure in buffalo may indicate distribution of the virus over wide geographical areas beyond known RVFV foci in Kenya. This finding calls for thorough studies on the epizootology of RVFV in specific wildlife species and locations.

Distribution and abundance of key vectors of Rift Valley fever and other arboviruses in two ecologically distinct counties in Kenya

Background Rift Valley fever (RVF) is a mosquito-borne viral zoonosis of ruminants and humans that causes outbreaks in Africa and the Arabian Peninsula with significant public health and economic consequences. Humans become infected through mosquito bites and contact with infected livestock. The virus is maintained between outbreaks through vertically infected eggs of the primary vectors of Aedes species which emerge following rains with extensive flooding. Infected female mosquitoes initiate transmission among nearby animals, which amplifies virus, thereby infecting more mosquitoes and moving the virus beyond the initial point of emergence. With each successive outbreak, RVF has been found to expand its geographic distribution to new areas, possibly driven by available vectors. The aim of the present study was to determine if RVF virus (RVFV) transmission risk in two different ecological zones in Kenya could be assessed by looking at the species composition, abundance and distribution of key primary and secondary vector species and the level of virus activity. Methodology Mosquitoes were trapped during short and long rainy seasons in 2014 and 2015 using CO2 baited CDC light traps in two counties which differ in RVF epidemic risk levels(high risk Tana-River and low risk Isiolo),cryo-preserved in liquid nitrogen, transported to the laboratory, and identified to species. Mosquito pools were analyzed for virus infection using cell culture screening and molecular analysis. Findings Over 69,000 mosquitoes were sampled and identified as 40 different species belonging to 6 genera (Aedes, Anopheles, Mansonia, Culex, Aedeomyia, Coquillettidia). The presence and abundance of Aedes mcintoshi and Aedes ochraceus, the primary mosquito vectors associated with RVFV transmission in outbreaks, varied significantly between Tana-River and Isiolo. Ae. mcintoshi was abundant in Tana-River and Isiolo but notably, Aedes ochraceus found in relatively high numbers in Tana-River (n = 1,290), was totally absent in all Isiolo sites. Fourteen virus isolates including Sindbis, Bunyamwera, and West Nile fever viruses were isolated mostly from Ae. mcintoshi sampled in Tana-River. RVFV was not detected in any of the mosquitoes. Conclusion This study presents the geographic distribution and abundance of arbovirus vectors in two Kenyan counties, which may assist with risk assessment for mosquito borne diseases.

Detection of Sindbis and Inkoo Virus RNA in Genetically Typed Mosquito Larvae Sampled in Northern Sweden

Abstract Introduction: Mosquito-borne viruses have a widespread distribution across the globe and are known to pose serious threats to human and animal health. The maintenance and dissemination of these viruses in nature are driven through horizontal and vertical transmission. In the temperate climate of northern Sweden, there is a dearth of knowledge on whether mosquito-borne viruses that occur are transmitted transovarially. To gain a better understanding of mosquito-borne virus circulation and maintenance, mosquito larvae were sampled in northern Sweden during the first and second year after a large outbreak of Ockelbo disease in 2013 caused by Sindbis virus (SINV). Materials and Methods: A total of 3123 larvae were sampled during the summers of 2014 and 2015 at multiple sites in northern Sweden. The larvae were homogenized and screened for viruses using RT-PCR and sequencing. Species identification of selected larvae was performed by genetic barcoding targeting the cytochrome C oxidase subunit I gene. Results and Discussion: SINV RNA was detected in mosquito larvae of three different species, Ochlerotatus (Oc.) communis, Oc. punctor, and Oc. diantaeus. Inkoo virus (INKV) RNA was detected in Oc. communis larvae. This finding suggested that these mosquitoes could support transovarial transmission of SINV and INKV. Detection of virus in mosquito larva may serve as an early warning for emerging arboviral diseases and add information to epidemiological investigations before, during, and after outbreaks. Furthermore, our results demonstrated the relevance of genetic barcoding as an attractive and effective method for mosquito larva typing. However, further mosquito transmission studies are needed to ascertain the possible role of different mosquito species and developmental stages in the transmission cycle of arboviruses.

Seroprevalence and Risk Factors of Inkoo Virus in Northern Sweden.

The mosquito-borne Inkoo virus (INKV) is a member of the California serogroup in the family Bunyaviridae, genus Orthobunyavirus. These viruses are associated with fever and encephalitis, although INKV infections are not usually reported and the incidence is largely unknown. The aim of the study was to determine the prevalence of anti-INKV antibodies and associated risk factors in humans living in northern Sweden. Seroprevalence was investigated using the World Health Organization Monitoring of Trends and Determinants in Cardiovascular Disease study, where a randomly selected population aged between 25 and 74 years (N = 1,607) was invited to participate. The presence of anti-INKV IgG antibodies was determined by immunofluorescence assay. Seropositivity for anti-INKV was significantly higher in men (46.9%) than in women (34.8%; P < 0.001). In women, but not in men, the prevalence increased somewhat with age (P = 0.06). The peak in seropositivity was 45–54 years for men and 55–64 years for women. Living in rural areas was associated with a higher seroprevalence. In conclusion, the prevalence of anti-INKV antibodies was high in northern Sweden and was associated with male sex, older age, and rural living. The age distribution indicates exposure to INKV at a relatively early age. These findings will be important for future epidemiological and clinical investigations of this relatively unknown mosquito-borne virus.

Experimental Infection and Transmission Competence of Sindbis Virus in Culex torrentium and Culex pipiens Mosquitoes from Northern Sweden

Abstract Introduction: Sindbis virus (SINV) is a mosquito-borne Alphavirus known to infect birds and cause intermittent outbreaks among humans in Fenno-Scandia. In Sweden, the endemic area has mainly been in central Sweden. Recently, SINV infections have emerged to northern Sweden, but the vectorial efficiency for SINV of mosquito species in this northern region has not yet been ascertained. Objective: Mosquito larvae were sampled from the Umeå region in northern Sweden and propagated in a laboratory to adult stage to investigate the infection, dissemination, and transmission efficiency of SINV in mosquitoes. Materials and Methods: The mosquito species were identified by DNA barcoding of the cytochrome oxidase I gene. Culex torrentium was the most abundant (82.2%) followed by Culex pipiens (14.4%), Aedes annulipes (1.1%), Anopheles claviger (1.1%), Culiseta bergrothi (1.1%), or other unidentified species (1.1%). Mosquitoes were fed with SINV-infected blood and monitored for 29 days to determine the viral extrinsic incubation period. Infection and dissemination were determined by RT-qPCR screening of dissected body parts of individual mosquitoes. Viral transmission was determined from saliva collected from individual mosquitoes at 7, 14, and 29 days. SINV was detected by cell culture using BHK-21 cells, RT-qPCR, and sequencing. Results: Cx. torrentium was the only mosquito species in our study that was able to transmit SINV. The overall transmission efficiency of SINV in Cx. torrentium was 6.8%. The rates of SINV infection, dissemination, and transmission in Cx. torrentium were 11%, 75%, and 83%, respectively. Conclusions: Cx. torrentium may be the key vector involved in SINV transmission in northern Sweden.

Datasets for mapping pastoralist movement patterns and risk zones of Rift Valley fever occurrence

Rift Valley fever (RVF) is a zoonotic disease affecting humans and animals. It is caused by RVF virus transmitted primarily by Aedes mosquitoes. The data presented in this article propose environmental layers suitable for mapping RVF vector habitat zones and livestock migratory routes. Using species distribution modelling, we used RVF vector occurrence data sampled along livestock migratory routes to identify suitable vector habitats within the study region which is located in the central and the north-eastern part of Kenya. Eleven herds monitored with GPS collars were used to estimate cattle utilization distribution patterns. We used kernel density estimator to produce utilization contours where the 0.5 percentile represents core grazing areas and the 0.99 percentile represents the entire home range. The home ranges were overlaid on the vector suitability map to identify risks zones for possible RVF exposure. Assimilating high spatial and temporal livestock movement and vector distribution datasets generates new knowledge in understanding RVF epidemiology and generates spatially explicit risk maps. The results can be used to guide vector control and vaccination strategies for better disease control.

Mosquito-borne Inkoo virus in northern Sweden - isolation and whole genome sequencing

BackgroundInkoo virus (INKV) is a less known mosquito-borne virus belonging to Bunyaviridae, genus Orthobunyavirus, California serogroup. Studies indicate that INKV infection is mainly asymptomatic, but can cause mild encephalitis in humans. In northern Europe, the sero-prevalence against INKV is high, 41% in Sweden and 51% in Finland. Previously, INKV RNA has been detected in adult Aedes (Ae.) communis, Ae. hexodontus and Ae. punctor mosquitoes and Ae. communis larvae, but there are still gaps of knowledge regarding mosquito vectors and genetic diversity. Therefore, we aimed to determine the occurrence of INKV in its mosquito vector and characterize the isolates.MethodsAbout 125,000 mosquitoes were collected during a mosquito-borne virus surveillance in northern Sweden during the summer period of 2015. Of these, 10,000 mosquitoes were processed for virus isolation and detection using cell culture and RT-PCR. Virus isolates were further characterized by whole genome sequencing. Genetic typing of mosquito species was conducted by cytochrome oxidase subunit I (COI) gene amplification and sequencing (genetic barcoding).ResultsSeveral Ae. communis mosquitoes were found positive for INKV RNA and two isolates were obtained. The first complete sequences of the small (S), medium (M), and large (L) segments of INKV in Sweden were obtained. Phylogenetic analysis showed that the INKV genome was most closely related to other INKV isolates from Sweden and Finland. Of the three INKV genome segments, the INKV M segment had the highest frequency of non-synonymous mutations. The overall G/C-content of INKV genes was low for the N/NSs genes (43.8–45.5%), polyprotein (Gn/Gc/NSm) gene (35.6%) and the RNA polymerase gene (33.8%) This may be due to the fact that INKV in most instances utilized A or T in the third codon position.ConclusionsINKV is frequently circulating in northern Sweden and Ae. communis is the key vector. The high mutation rate of the INKV M segment may have consequences on virulence

COMPLETE GENOMIC SEQUENCE OF VIRULENT PIGEON PARAMYXOVIRUS IN LAUGHING DOVES (STREPTOPELIA SENEGALENSIS) IN KENYA

Abstract Following mass deaths of Laughing Doves (Streptopelia senegalensis) in different localities throughout Kenya, internal organs obtained during necropsy of two moribund birds were sampled and analyzed by next generation sequencing. We isolated the virulent strain of pigeon paramyxovirus type-1 (PPMV-1), PPMV1/Laughing Dove/Kenya/Isiolo/B2/2012, which had a characteristic fusion gene motif 110GGRRQKRF117. We obtained a partial full genome of 15,114 nucleotides. The phylogenetic relationship based on the fusion gene and genomic sequence grouped our isolate as class II genotype VI, a group of viruses commonly isolated from wild birds but potentially lethal to Chickens (Gallus gallus domesticus). The fusion gene isolate clustered with PPMV-I strains from pigeons (Columbidae) in Nigeria. The complete genome showed a basal and highly divergent lineage to American, European, and Asian strains, indicating a divergent evolutionary pathway. The isolated strain is highly virulent and apparently species-specific to Laughing Doves in Kenya. Risk of transmission of such a strain to poultry is potentially high whereas the cyclic epizootic in doves is a threat to conservation of wild Columbidae in Kenya.