Genetic Characterization of Human Enteroviruses Isolated in Kenya from Respiratory Samples between 2008 and 2011

Abstract

iii WRAIR Walter Reed Army Institute of Research WRP Walter Reed Project μM micro molar ABSTRACT Enteroviruses (EVs) are common viral pathogens infecting humans. They are globally distributed and are estimated to cause infections to about one billion people annually. The disease spectrum associated with these viruses ranges from mild respiratory illnesses to severe complications such as aseptic meningitis, myocarditis and encephalitis. Previous studies have shown that human enteroviruses (HEVs) are among the major etiological agents of acute respiratory illnesses. For instance, a preliminary study in 1980’s involving hospitalized children with severe acute respiratory infections in Kenya showed that HEVs accounted for ~36.5% of all the viral infections. However, despite health concerns posed by the worldwide spread of these viruses, information about individual serotypes of HEVs circulating in Kenya, and the East African region at large is scanty. The aim of this study was to characterize human enteroviruses isolated in Kenya from respiratory samples between 2008 and 2011 using the VP1region of the HEV genome. Archived HEV isolates were propagated in RD cells and total viral RNA extracted from those confirmed positive by indirect immunofluoresence assay (IDFA). Partial VP1 gene was amplified by RT-PCR, followed by nucleotide sequencing. To assign serotypes and determine molecular aspects and evolutionary relationships of the Kenyan isolates, the HEVs nucleotide sequences were compared to those of prototypes and representative homotypic strains retrieved from GenBank. Homology analyses coupled with phylogenetic analyses revealed that the Kenyan isolates belonged to HEV-A, HEV-B and HEV-D species. Overall, 22 different enteroviral serotypes were detected. The majority (72%) of the serotypes were from HEV-B species, followed by HEV-D (21.3%) and HEV-A (6.5%). None of the identified serotypes belonged to HEV-C species. The most frequently detected serotypes were enterovirus-68 (EV68), Coxsackie-virus types –B2, -B1, -B4 and B3. The majority (85%) of HEV-D EV68 isolate strains belonged to clade A while a minority belonged to clades B and C. Subsequent analyses revealed that a high proportion of amino acid variations present in the VP1 region of the Kenyan isolates were conserved among respective homotypic contemporaneous strains retrieved from GenBank. Besides, the Kenyan isolates clustered closely with sequences of these global strains. Overall, findings from this work indicate that although the Kenyan isolates were genetically disparate from their respective prototypes, they were closely related to global strains. The study further demonstrated existence of high genetic diversity among HEVs that circulated in Kenya during 2008 to 2011. Moreover, viruses belonging to HEV-B and HEV-D species played key role in respiratory enteroviral infections experienced in the country during this period. Furthermore, the absence of disparate mutations in the VP1 region of most Kenyan isolates suggests worldwide transmissibility of these viruses. Our data highlights the need for serotype-based surveillance of HEVs in the country, to provide knowledge on circulating serotypes. Whole genome studies ought to be carried out to establish whether there are recombinations among the viruses in Kenya, and provide more insight into their molecular epidemiology and evolutionary dynamics.