Whole-genome sequence analysis reveals the circulation of multiple SARS-CoV-2 variants of concern in Nairobi and neighboring counties, Kenya between March and July 2021.

The emergence and rapid spread of SARS-CoV-2 variants of concern (VOC) have been linked to new waves of COVID-19 epidemics occurring in different regions of the world. The VOC have acquired adaptive mutations that have enhanced virus transmissibility, increased virulence, and reduced response to neutralizing antibodies. Kenya has experienced six waves of COVID-19 epidemics. In this study, we analyzed 64 genome sequences of SARS-CoV-2 strains that circulated in Nairobi and neighboring counties, Kenya between March 2021 and July 2021. Viral RNA was extracted from RT-PCR confirmed COVID-19 cases, followed by sequencing using the ARTIC network protocol and Oxford Nanopore Technologies. Analysis of the sequence data was performed using different bioinformatics methods. Our analyses revealed that during the study period, three SARS-CoV-2 variants of concern (VOC) circulated in Nairobi and nearby counties in Kenya. The Alpha (B.1.1.7) lineage predominated (62.7%), followed by Delta (B.1.617.2, 35.8%) and Beta (B.1.351, 1.5%). Notably, the Alpha (B.1.1.7) VOC were most frequent from March 2021 to May 2021, while the Delta (B.1.617.2) dominated beginning June 2021 through July 2021. Sequence comparisons revealed that all the Kenyan viruses were genetically similar to those that circulated in other regions. Although the majority of Kenyan viruses clustered together in their respective phylogenetic lineages/clades, a significant number were interspersed among foreign strains. Between March and July 2021, our study's findings indicate the prevalence of multiple lineages of SAR-CoV-2 VOC in Nairobi and nearby counties in Kenya. The data suggest that the recent increase in SARS-CoV-2 infection, particularly in Nairobi and Kenya as a whole, is attributable to the introduction and community transmission of SARS-CoV-2 VOC among the populace. In conclusion, the findings provide a snapshot of the SARS-CoV-2 variants that circulated in Kenya during the study period.

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.

Probable contribution of Culex quinquefasciatus mosquitoes to the circulation of chikungunya virus during an outbreak in Mombasa County, Kenya, 2017-2018.

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

Emergence of a novel chikungunya virus strain bearing the E1:V80A substitution, out of the Mombasa, Kenya 2017-2018 outbreak.

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

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.

Global Outbreaks and Origins of a Chikungunya Virus Variant Carrying Mutations Which May Increase Fitness for Aedes aegypti: Revelations from the 2016 Mandera, Kenya Outbreak.

In 2016, a chikungunya virus (CHIKV) outbreak was reported in Mandera, Kenya. This was the first major CHIKV outbreak in the country since the global reemergence of this virus in Kenya in 2004. We collected samples and sequenced viral genomes from this outbreak. All Kenyan genomes contained two mutations, E1:K211E and E2:V264A, recently reported to have an association with increased infectivity, dissemination, and transmission in the Aedes aegypti vector. Phylogeographic inference of temporal and spatial virus relationships showed that this variant emerged within the East, Central, and South African lineage between 2005 and 2008, most probably in India. It was also in India where the first large outbreak caused by this virus appeared, in New Delhi, 2010. More importantly, our results also showed that this variant is no longer contained to India. We found it present in several major outbreaks, including the 2016 outbreaks in Pakistan and Kenya, and the 2017 outbreak in Bangladesh. Thus, this variant may have a capability of driving large CHIKV outbreaks in different regions of the world. Our results point to the importance of continued genomic-based surveillance and prompt urgent vector competence studies to assess the level of vector susceptibility and virus transmission, and the impact this might have on this variant's epidemic potential and global spread.

Human and entomologic investigations of chikungunya outbreak in Mandera, Northeastern Kenya, 2016.

Chikungunya is a reemerging vector borne pathogen associated with severe morbidity in affected populations. Lamu, along the Kenyan coast was affected by a major chikungunya outbreak in 2004. Twelve years later, we report on entomologic investigations and laboratory confirmed chikungunya cases in northeastern Kenya. Patient blood samples were received at the Kenya Medical Research Institute (KEMRI) viral hemorrhagic fever laboratory and the immunoglobulin M enzyme linked immunosorbent assay (IgM ELISA) was used to test for the presence of IgM antibodies against chikungunya and dengue. Reverse transcription polymerase chain reaction (RT-PCR) utilizing flavivirus, alphavirus and chikungunya specific primers were used to detect acute infections and representative PCR positive samples sequenced to confirm the circulating strain. Immature mosquitoes were collected from water-holding containers indoors and outdoors in the affected areas in northeastern Kenya. A total of 189 human samples were tested; 126 from Kenya and 63 from Somalia. 52.9% (100/189) tested positive for Chikungunya virus (CHIKV) by either IgM ELISA or RT-PCR. Sequence analysis of selected samples revealed that the virus was closely related to that from China (2010). 29% (55/189) of the samples, almost all from northeastern Kenya or with a history of travel to northern Kenya, tested positive for dengue IgM antibodies. Entomologic risk assessment revealed high house, container and Breteau indices of, 14.5, 41.9 and 17.1% respectively. Underground water storage tanks were the most abundant, 30.1%, of which 77.4% were infested with Aedes aegypti mosquitoes. These findings confirm the presence of active chikungunya infections in the northeastern parts of Kenya. The detection of dengue IgM antibodies concurrently with chikungunya virus circulation emphasizes on the need for improved surveillance systems and diagnostic algorithms with the capacity to capture multiple causes of arbovirus infections as these two viruses share common vectors and eco-systems. In addition sustained entomological surveillance and vector control programs targeting most productive containers are needed to monitor changes in vector densities, for early detection of the viruses and initiate vector control efforts to prevent possible outbreaks.

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.

Dengue Outbreak in Mombasa City, Kenya, 2013-2014: Entomologic Investigations.

Dengue outbreaks were first reported in East Africa in the late 1970s to early 1980s including the 1982 outbreak on the Kenyan coast. In 2011, dengue outbreaks occurred in Mandera in northern Kenya and subsequently in Mombasa city along the Kenyan coast in 2013-2014. Following laboratory confirmation of dengue fever cases, an entomologic investigation was conducted to establish the mosquito species, and densities, causing the outbreak. Affected parts of the city were identified with the help of public health officials. Adult Ae. aegypti mosquitoes were collected using various tools, processed and screened for dengue virus (DENV) by cell culture and RT-PCR. All containers in every accessible house and compound within affected suburbs were inspected for immatures. A total of 2,065 Ae. aegypti adults were collected and 192 houses and 1,676 containers inspected. An overall house index of 22%, container index, 31.0% (indoor = 19; outdoor = 43) and Breteau index, 270.1, were observed, suggesting that the risk of dengue transmission was high. Overall, jerry cans were the most productive containers (18%), followed by drums (17%), buckets (16%), tires (14%) and tanks (10%). However, each site had specific most-productive container-types such as tanks (17%) in Kizingo; Drums in Nyali (30%) and Changamwe (33%), plastic basins (35%) in Nyali-B and plastic buckets (81%) in Ganjoni. We recommend that for effective control of the dengue vector in Mombasa city, all container types would be targeted. Measures would include proper covering of water storage containers and eliminating discarded containers outdoors through a public participatory environmental clean-up exercise. Providing reliable piped water to all households would minimize the need for water storage and reduce aquatic habitats. Isolation of DENV from male Ae. aegypti mosquitoes is a first observation in Kenya and provides further evidence that transovarial transmission may have a role in DENV circulation and/or maintenance in the environment.