Assessment of Bunyavirales activity and circulation in humans, livestock, and peri-domestic rodents in diverse ecologies in Kenya, 2020-2022

Abstract

Viruses in the order Bunyavirales are diverse and include zoonotic arthropod- and rodent-borne viruses that cause diseases ranging from mild febrile illnesses to haemorrhagic and/or encephalitis fevers and even death in animals and humans. These viruses contribute significantly to emerging and re-emerging infection threats worldwide as well as neglected tropical diseases as they mainly affect the impoverished and have a long-lasting effect. In tropical and subtropical countries, mainly in Africa, more than 90% of human cases are either undiagnosed or misdiagnosed and treated as other common endemic diseases like malaria. Furthermore, many studies have focused on common arboviruses transmitted by mosquitoes such as the flaviviruses (e.g., yellow fever, Dengue, Chikungunya, Zika viruses), among others, as well as well-known members of the Bunyavirales associated with human outbreaks (Rift Valley Fever virus (RVFV) and Crimean Congo Hemorrhagic Fever virus (CCHFV)). Less known yet potentially harmful arboviruses in the order Bunyavirales are mostly ignored despite being zoonotic or having zoonotic potential with the risk of spreading globally. Despite the associated public and veterinary health, social, and economic importance, the impact of these diseases is largely undetermined due to paucity of active surveillance, poor disease reporting systems, and lack of appropriate diagnosis in affected regions. The prevalence, burden of disease and distribution of most members of the Bunyavirales remains unknown in African countries where they are endemic. Surveillance is thus essential to determine their importance, provide an early warning of their presence, and guide intervention measures to prevent outbreaks. This thesis instituted “One Health” surveillance in selected pastoralist communities of Kenya (Kajiado and Baringo counties) to assess the circulation and viral activity of Bunyavirales among peridomestic rodents, livestock, and humans presenting with febrile illness. Serum samples were analyzed serologically to determine the presence of Bunyavirales neutralizing antibodies as well as culture and molecular methods (RT-PCR and whole genome sequencing and phylogenetic analysis) to isolate and characterise known and novel viruses, respectively using methodologies described in chapter 2. In Chapter 3, we report the findings of the screening experiments including the seroprevalence of RVFV, CCHFV, Ngari virus (NRIV), Ntepes virus (NTPV) and Bunyamwera virus (BUNV) as well as the molecular detection of CCHFV, NTPV; NRIV, BUNV, hantavirus, Shamonda virus (SHAV) and some uncharacterised phleboviruses in livestock and peridomestic rodents. Further characterisation of the most important viruses detected is reported in subsequent chapters (4-7). One of the major findings reported herein includes the isolation and circulation of Ngari virus (NRIV), known to cause outbreaks of haemorrhagic fevers in humans and small ruminants, in apparently healthy cattle, sheep and goats (Chapter 4). Seroprevalence of neutralizing antibodies to the virus ranged 19-52% with growth kinetics on different cells showing efficient replication in cells from sheep and humans and Aedes albopictus but weakly on goat cell lines. Phylogenetic analyses of complete-coding sequences of L, M and S segments of four NRIV isolates showed that the Kenyan viruses clustered with a monophyletic clade that is most closely related to a NRIV sequence from a small ruminant from Mauritania. This is the first detection of NRIV in livestock in Kenya. In Chapter 5, active detection of a Nairovirus, Crimean-Congo haemorrhagic fever (CCHF) virus RNA is demonstrated in sheep and rodent sera as virus exposure is serologically confirmed in these hosts and, cattle, goats, and humans. This virus is the causative agent of CCHF, a fatal viral haemorrhagic fever disease in humans. Among livestock, seroprevalence was lowest in goat (8.1%) and highest in cattle (14%). Phylogenetic analyses of partial sequences of the S segment generated from rodent and sheep revealed a high level of nucleotide sequence identity (96-98%) and a close relationship to other pathogenic strains in the CCHFV Africa 3 lineage. We also report for the first time in Kenya the detection of shrew-borne hantaviruses in Somali shrews (Crocidura somalica) from Baringo county (Chapter 6). The detected hantaviruses closest relative is an African shrew- borne hantavirus, Tanganya viruses (TGNV) with nucleotide identity of 72-80%. Finally, through virus isolation and Next Generation Sequencing (NGS) of samples collected in this study, a novel orbivirus was isolated from cattle which is described in Chapter 7 as an additional finding of this thesis. This followed initial isolation in cell culture from the serum of a clinically sick cow aged 2-3 years, presenting signs of emaciation and lethargy. High throughput sequencing revealed the typical orbivirus genome architecture with ten double-stranded RNA segments and a total size of 18,731 bp. A further detection of Kaptombes virus (KPTV) by RT-PCR in three cattle samples and 5% seroprevalence of neutralizing antibodies to the virus, demonstrate its active circulation. This shows the strength of sequence-independent virus discovery methods to discover new pathogens in surveillance study. Taken together, the findings have provided data on pathogenic bunyavirus prevalence in the two semi-arid regions useful for understanding the virus transmission networks as well as the detection and characterisation of a novel orbivirus. The data herein can be applied in animal health surveillance systems locally to ensure timely and appropriate detection and control as a means of active and continuous animal surveillance and as an early warning sign for zoonotic diseases emergence.