Origin and evolution of dengue virus type 2 causing outbreaks in Kenya: Evidence of circulation of two cosmopolitan genotype lineages.

Dengue fever (DF) is an arboviral disease caused by dengue virus serotypes 1-4 (DENV 1-4). Globally, DF incidence and disease burden have increased in the recent past. Initially implicated in a 1982 outbreak, DENV-2 recently reemerged in Kenya causing outbreaks between 2011 and 2014 and more recently 2017-8. The origin and the evolutionary patterns that may explain the epidemiological expansion and increasing impact of DENV-2 in Kenya remain poorly understood. Using whole-genome sequencing, samples collected during the 2011-4 and 2017-8 dengue outbreaks were analyzed. Additional DENV-2 genomes were downloaded and pooled together with the fourteen genomes generated in this study. Bioinformatic methods were used to analyze phylogenetic relationships and evolutionary patterns of DENV-2 causing outbreaks in Kenya. The findings from this study have shown the first evidence of circulation of two different Cosmopolitan genotype lineages of DENV-2; Cosmopolitan-I (C-I) and Cosmopolitan-II (C-II), in Kenya. Our results put the origin location of C-I lineage in India in 2011, and C-II lineage in Burkina Faso between 1979 and 2013. C-I lineage was the most isolated during recent outbreaks, thus showing the contribution of this newly emerged strain to the increased DENV epidemics in the region. Our findings, backed by evidence of recent local epidemics that have been associated with C-I in Kenya and C-II in Burkina Faso, add to the growing evidence of expanding circulation and the impact of multiple strains of DENV in the region as well as globally. Thus, continued surveillance efforts on DENV activity and its evolutionary trends in the region, would contribute toward effective control and the current vaccine development efforts.

Sylvatic Mosquito Diversity in Kenya-Considering Enzootic Ecology of Arboviruses in an Era of Deforestation.

As new and re-emerging vector-borne diseases are occurring across the world, East Africa represents an interesting location, being the origin of several arboviruses with a history of urbanization and global spread. Rapid expansion of urban populations and alteration of natural habitats creates the opportunity for arboviruses to host-switch from wild, sylvatic hosts or vectors into urban transmission affecting human populations. Although mosquito surveillance regularly takes place in urban areas of Kenya, for example identifying vectors of dengue virus or malaria viruses, little work has been carried out to determine the distribution and abundance of sylvatic vectors. Here, we describe the mosquito vector species and diversity collected at twelve forest habitats of rural Kenya. We conducted arbovirus screening of over 14,082 mosquitoes (47 species, 11 genera) as 1520 pools, and detected seven viruses (six bunyaviruses, and one flavivirus-bunyavirus co-infection) isolated from pools of Aedes dentatus, Anopheles funestus, Culex annulioris, and Cx. vansomereni. Awareness of sylvatic vector species and their location is a critical part of understanding the ecological foci and enzootic cycling of pathogens that may be of concern to public, animal or wildlife health. As natural ecosystems come under anthropogenic pressures, such knowledge can inform us of the One Health potential for spillover or spillback leading to outbreaks, and assist in vector control strategies.

Enzootic Circulation of Chikungunya Virus in East Africa: Serological Evidence in Non-human Kenyan Primates.

Chikungunya virus (CHIKV) is a globally emerging pathogen causing debilitating arthralgia and fever in humans. First identified in Tanzania (1953), this mosquito-borne alphavirus received little further attention until a 2004 re-emergence in Kenya from an unknown source. This outbreak subsequently spread to the Indian Ocean, with adaptation for transmission by a new urban vector. Under the hypothesis that sylvatic progenitor cycles of CHIKV exist in Kenya (as reported in West Africa, between non-human primates (NHPs) and arboreal Aedes spp. mosquitoes), we pursued evidence of enzootic transmission and human spillover events. We initially screened 252 archived NHP sera from Kenya using plaque reduction neutralization tests. Given an overall CHIKV seroprevalence of 13.1% (marginally higher in western Kenya), we sought more recent NHP samples during 2014 from sites in Kakamega County, sampling wild blue monkeys, olive baboons, and red-tailed monkeys (N = 33). We also sampled 34 yellow baboons near Kwale, coastal Kenya. Overall, CHIKV seropositivity in 2014 was 13.4% (9/67). Antibodies reactive against closely related o'nyong-nyong virus (ONNV) occurred; however, neutralization titers were too low to conclude ONNV exposure. Seroprevalence for the flavivirus dengue was also detected (28%), mostly near Kwale, suggesting possible spillback from humans to baboons. CHIKV antibodies in some juvenile and subadult NHPs suggested recent circulation. We conclude that CHIKV is circulating in western Kenya, despite the 2004 human outbreaks only being reported coastally. Further work to understand the enzootic ecology of CHIKV in east Africa is needed to identify sites of human spillover contact where urban transmission may be initiated.

Yellow fever virus susceptibility of two mosquito vectors from Kenya, East Africa.

Yellow fever is an unpredictable disease of increasing epidemic threat in East Africa. Aedes (Stegomyia) aegypti has never been implicated as a vector in this region and recent outbreaks have involved a newly emerging virus genotype (East African). To better understand the increasing epidemic risk of yellow fever in East Africa, this study is the first to investigate the vector competence for an emerging East African virus genotype in Kenyan A. aegypti sensu latu (s.l) and A. (Stegomyia) simpsoni s.l. mosquito species. Using first filial generation mosquitoes and a low passage yellow fever virus, this study demonstrated that although A. aegypti s.l. is a competent vector, A. simpsoni s.l. is likely a more efficient vector.

Serologic evidence of arboviral infections among humans in Kenya.

Outbreaks of arthropod-borne viral infections occur periodically across Kenya. However, limited surveillance takes place during interepidemic periods. Using serum samples obtained from asymptomatic persons across Kenya in 2000-2004, we assessed (by indirect immunofluorescent assay) prevalence of IgG against yellow fever virus (YFV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV), dengue virus serotypes 1-4 (DENV1-4), and chikungunya virus (CHIKV). Older persons on the Indian Ocean coast were more likely to be seropositive than children inland: YFV = 42% versus 6%, WNV = 29% versus 6%, TBEV = 16% versus 6%, DENV-1 = 63% versus 9%, DENV-2 = 67% versus 7%, DENV-3 = 55% versus 6%, DENV-4 = 44% versus 8%, and CHIKV = 37% versus 20%. Among inland samples, children in lowlands were more likely to be seropositive for CHIKV (42% versus 0%) than children in highlands. In Kenya, transmission of arboviral infection continues between known epidemics and remains common across the country.

The AFHSC-Division of GEIS Operations Predictive Surveillance Program: a multidisciplinary approach for the early detection and response to disease outbreaks.

The Armed Forces Health Surveillance Center, Division of Global Emerging Infections Surveillance and Response System Operations (AFHSC-GEIS) initiated a coordinated, multidisciplinary program to link data sets and information derived from eco-climatic remote sensing activities, ecologic niche modeling, arthropod vector, animal disease-host/reservoir, and human disease surveillance for febrile illnesses, into a predictive surveillance program that generates advisories and alerts on emerging infectious disease outbreaks. The program's ultimate goal is pro-active public health practice through pre-event preparedness, prevention and control, and response decision-making and prioritization. This multidisciplinary program is rooted in over 10 years experience in predictive surveillance for Rift Valley fever outbreaks in Eastern Africa. The AFHSC-GEIS Rift Valley fever project is based on the identification and use of disease-emergence critical detection points as reliable signals for increased outbreak risk. The AFHSC-GEIS predictive surveillance program has formalized the Rift Valley fever project into a structured template for extending predictive surveillance capability to other Department of Defense (DoD)-priority vector- and water-borne, and zoonotic diseases and geographic areas. These include leishmaniasis, malaria, and Crimea-Congo and other viral hemorrhagic fevers in Central Asia and Africa, dengue fever in Asia and the Americas, Japanese encephalitis (JE) and chikungunya fever in Asia, and rickettsial and other tick-borne infections in the U.S., Africa and Asia.

Ability of selected Kenyan mosquito (Diptera: Culicidae) species to transmit West Nile virus under laboratory conditions.

West Nile virus (WNV) is currently active in Kenya as evidenced by the detection of antibodies in birds bled as part of an avian influenza surveillance program in 2009. Although WNV has been isolated from several mosquito species in Kenya, no studies have ever been conducted to determine which of these species are competent vectors of this virus. Therefore, we allowed Kenyan mosquitoes to feed on 2- or 3-d-old chickens that had been infected with a Lineage one strain of WNV 24-48 h earlier. These mosquitoes were tested approximately 2 wk later to determine infection, dissemination, and transmission rates. All five species [Culex quinquefasciatus Say, Culex univittatus Theobald, Culex vansomereni Edwards, Mansonia africana (Theobald), and Mansonia uniformis (Theobald)] were susceptible to infection, but disseminated infections were detected only in the three Culex, and not the two Mansonia species. Culex mosquitoes with a disseminated infection readily transmitted virus by bite, but even when inoculated with WNV, the two Mansonia failed to transmit virus, indicating a salivary gland barrier. These studies indicate that the three Culex species may play a role in the transmission of WNV in Kenya.

Entomologic investigations of a chikungunya virus epidemic in the Union of the Comoros, 2005.

From January to April 2005, an epidemic of chikungunya virus (CHIKV) illness occurred in the Union of Comoros. Entomological studies were undertaken during the peak of the outbreak, from March 11 to March 31, aimed at identifying the primary vector(s) involved in transmission so that appropriate public health measures could be implemented. Adult mosquitoes were collected by backpack aspiration and human landing collection in homes and neighborhoods of clinically ill patients. Water-holding containers were inspected for presence of mosquito larvae. Adult mosquitoes were analyzed by RT-PCR and cultivation in cells for the presence of CHIK virus and/or nucleic acid. A total of 2,326 mosquitoes were collected and processed in 199 pools. The collection consisted of 62.8% Aedes aegypti, 25.5% Culex species, and 10.7% Aedes simpsoni complex, Eretmapodites spp and Anopheles spp. Seven mosquito pools were found to be positive for CHIKV RNA and 1 isolate was obtained. The single CHIKV mosquito isolate was from a pool of Aedes aegypti and the minimum infection rate (MIR) for this species was 4.0, suggesting that Ae. aegypti was the principal vector responsible for the outbreak. This was supported by high container (31.1%), household (68%), and Breteau (126) indices, with discarded tires (58.8%) and small cooking and water storage vessels (31.1%) registering the highest container indices.

Laboratory diagnosis of Ebola hemorrhagic fever during an outbreak in Yambio, Sudan, 2004.

Between the months of April and June 2004, an Ebola hemorrhagic fever (EHF) outbreak was reported in Yambio county, southern Sudan. Blood samples were collected from a total of 36 patients with suspected EHF and were tested by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G and M antibodies, antigen ELISA, and reverse-transcription polymerase chain reaction (PCR) of a segment of the Ebolavirus (EBOV) polymerase gene. A total of 13 patients were confirmed to be infected with EBOV. In addition, 4 fatal cases were classified as probable cases, because no samples were collected. Another 12 patients were confirmed to have acute measles infection during the same period that EBOV was circulating. Genetic analysis of PCR-positive samples indicated that the virus was similar to but distinct from Sudan EBOV Maleo 1979. In response, case management, social mobilization, and follow-up of contacts were set up as means of surveillance. The outbreak was declared to be over on 7 August 2004.

Spatiotemporal distribution of diurnal yellow fever vectors (Diptera: Culicidae) at two sylvan interfaces in Kenya, East Africa.

Yellow fever virus (YFV) remains a significant public health threat in sub-Saharan Africa in which 90% of the estimated 200,000 cases occur annually. In East Africa, human cases of YFV are characterized by unpredictable focal periodicity, lengthy inter-epidemic periods, and a precarious potential for large epidemics. YFV had remained undetected in this region for nearly 40 years until emerging in Kenya in 1992-93 and more recently in Sudan during 2003 and 2005. From an ecological perspective the emergence and epidemiological outcomes associated with YFV, and related vector-borne diseases, are critically dependent upon the underlying vector ecology at a local scale. The study here was aimed at defining the dynamics of important vector interactions at two important sites in Kenya with previous YFV or related arbovirus activity. The temporal abundance, spatial distribution, and human host seeking behavior of diurnal man-landing mosquito species along sylvan interfaces were investigated. A number of YFV vectors were identified including their abundances for the duration of the main rainy season. Spatially, results indicated that the greatest human-mosquito interactions occurred within the forest and decreased across more domesticated biotopes. A discussion of significant differences, ecological associations, and epidemiological implications is included.

Infection and vertical transmission of Kamiti river virus in laboratory bred Aedes aegypti mosquitoes.

Kamiti river virus (KRV) is an insect-only Flavivirus that was isolated from field-collected Ae. macintoshi mosquitoes in 1999, and is closely related to cell fusing agent virus. Both of these viruses belong to the family Flaviviridae, which also contains other viruses of medical importance, such as yellow fever virus, West Nile virus and dengue. Because Ae. macintoshi is the only known natural host to KRV, the main objective of this study was to establish the possibility that other mosquito hosts of the virus exist, by determining its ability to infect Ae. aegypti mosquitoes under laboratory conditions. The study also sought to determine the rates of infection and, subsequently, vertical transmission as a possible means of its maintenance and propagation in nature, given that it neither grows in vertebrate cells or mice. The mosquitoes were infected by the virus either as larvae or adults. Virus assay was done by re-isolation in tissue culture and indirect immunofluoresce assay methods. KRV infected Ae. aegypti mosquitoes, with the observed rates as high as 74 to 96 %. The virus was also transmitted vertically in these mosquitoes. Vertical transmission rates of 3.90 % were observed for the 2nd and 3rd ovarian cycles combined. These results suggest that Ae. aegypti mosquitoes are likely to be infected with KRV in nature, and that vertical transmission is the natural means by which it is maintained and propagated in this host, and possibly others.

Yellow fever outbreak, southern Sudan, 2003.

In May 2003, an outbreak of fatal hemorrhagic fever, caused by yellow fever virus, occurred in southern Sudan. Phylogenetic analysis showed that the virus belonged to the East African genotype, which supports the contention that yellow fever is endemic in East Africa with the potential to cause large outbreaks in humans.

Yellow fever outbreak, Imatong, southern Sudan.

In May 2003, the World Health Organization received reports about a possible outbreak of a hemorrhagic disease of unknown cause in the Imatong Mountains of southern Sudan. Laboratory investigations were conducted on 28 serum samples collected from patients in the Imatong region. Serum samples from 13 patients were positive for immunoglobulin M antibody to flavivirus, and serum samples from 5 patients were positive by reverse transcription-polymerase chain reaction with both the genus Flavivirus-reactive primers and yellow fever virus-specific primers. Nucleotide sequencing of the amplicons obtained with the genus Flavivirus oligonucleotide primers confirmed yellow fever virus as the etiologic agent. Isolation attempts in newborn mice and Vero cells from the samples yielded virus isolates from five patients. Rapid and accurate laboratory diagnosis enabled an interagency emergency task force to initiate a targeted vaccination campaign to control the outbreak.

The effects of a DNA virus infection on the reproductive potential of female tsetse flies, Glossina morsitans centralis and Glossina morsitans morsitans (Diptera: Glossinidae)

Reproductive anomalies associated with the tsetse DNA virus infection in the female tsetse hosts, Glossina morsitans centralis Machado and Glossina morsitans morsitans Westwood, inoculated with the virus during the 3rd instar larval stage were studied and the data compared to those obtained from the control females injected with sterile physiological saline. Virus infected flies had significantly longer first and second pregnancy cycles (P<0.0001) and produced pupae that were of significantly less weight in miligrams (P<0.0001) compared to controls. Transmission of the virus to progeny was not absolute and only 21 per cent of G.m. centralis and 48 per cent of G.m. morsitans first progeny flies from infected females developed salivary gland hypertrophy as a result of transmission from mother to progeny. The virus infected females produced significantly fewere pupae compared to the controls during the experimental period (P<0.00001).