Genetic characterization of influenza A(H3N2) viruses circulating in coastal Kenya, 2009-2017

Background Influenza viruses evolve rapidly and undergo immune driven selection, especially in the hemagglutinin (HA) protein. We report amino acid changes affecting antigenic epitopes and receptor‐binding sites of A(H3N2) viruses circulating in Kilifi, Kenya, from 2009 to 2017. Methods Next‐generation sequencing (NGS) was used to generate A(H3N2) virus genomic data from influenza‐positive specimens collected from hospital admissions and health facility outpatients presenting with acute respiratory illness to health facilities within the Kilifi Health and Demographic Surveillance System. Full‐length HA sequences were utilized to characterize A(H3N2) virus genetic and antigenic changes. Results From 186 (90 inpatient and 96 outpatient) influenza A virus‐positive specimens processed, 101 A(H3N2) virus whole genomes were obtained. Among viruses identified in inpatient specimens from 2009 to 2015, divergence of circulating A(H3N2) viruses from the vaccine strains A/Perth/16/2009, A/Texas/50/2012, and A/Switzerland/9715293/2013 formed 6 genetic clades (A/Victoria/208/2009‐like, 3B, 3C, 3C.2a, 4, and 7). Among viruses identified in outpatient specimens from 2015 to 2017, divergence of circulating A(H3N2) viruses from vaccine strain A/Hong Kong/4801/2014 formed clade 3C.2a, subclades 3C.2a2 and 3C.2a3, and subgroup 3C.2a1b. Several amino acid substitutions were associated with the continued genetic evolution of A(H3N2) strains in circulation. Conclusions Our results suggest continuing evolution of currently circulating A(H3N2) viruses in Kilifi, coastal Kenya and suggest the need for continuous genetic and antigenic viral surveillance of circulating seasonal influenza viruses with broad geographic representation to facilitate prompt and efficient selection of influenza strains for inclusion in future influenza vaccines

Human metapneumovirus prevalence and patterns of subgroup persistence identified through surveillance of pediatric pneumonia hospital admissions in coastal Kenya, 2007–2016

Background Human metapneumovirus (HMPV) is an important respiratory pathogen that causes seasonal epidemics of acute respiratory illness and contributes significantly to childhood pneumonia. Current knowledge and understanding on its patterns of spread, prevalence and persistence in communities in low resource settings is limited. Methods We present findings of a molecular-epidemiological analysis of nasal samples from children < 5 years of age admitted with syndromic pneumonia between 2007 and 2016 to Kilifi County Hospital, coastal Kenya. HMPV infection was detected using real-time RT-PCR and positives sequenced in the fusion (F) and attachment (G) genes followed by phylogenetic analysis. The association between disease severity and HMPV subgroup was assessed using Fisher’s exact test. Results Over 10 years, 274/6756 (4.1%) samples screened were HMPV positive. Annual prevalence fluctuated between years ranging 1.2 to 8.7% and lowest in the recent years (2014–2016). HMPV detections were most frequent between October of one year to April of the following year. Genotyping was successful for 205/274 (74.8%) positives revealing clades A2b (41.0%) and A2c (10.7%), and subgroups B1 (23.4%) and B2 (24.9%). The dominance patterns were: clade A2b between 2007 and 11, subgroup B1 between 2012 and 14, and clade A2c in more recent epidemics. Subgroup B2 viruses were present in all the years. Temporal phylogenetic clustering within the subgroups for both local and global sequence data was seen. Subgroups occurring in each epidemic season were comprised of multiple variants. Pneumonia severity did not vary by subgroup (p = 0.264). In both the F and G gene, the sequenced regions were found to be predominantly under purifying selection. Conclusion Subgroup patterns from this rural African setting temporally map with global strain distribution, suggesting a well-mixed global virus transmission pool of HMPV. Persistence in the local community is characterized by repeated introductions of HMPV variants from the global pool. The factors underlying the declining prevalence of HMPV in this population should be investigated

Epidemiological and evolutionary dynamics of influenza B virus in coastal Kenya as revealed by genomic analysis of strains sampled over a single season

The genomic epidemiology of influenza B virus (IBV) remains understudied in Africa despite significance to design of effective local and global control strategies. We undertook surveillance throughout 2016 in coastal Kenya, recruiting individuals presenting with acute respiratory illness at nine outpatient health facilities (any age) or admitted to the Kilifi County Hospital (&amp;lt;5-year-old). Whole genomes were sequenced for a select 111 positives; 94 (84.7%) of B/Victoria lineage and 17 (15.3%) of B/Yamagata lineage. Inter-Lineage reassortment was detected in 10 viruses; nine with B/Yamagata backbone but B/Victoria NA and NP segments and one with a B/Victoria backbone but B/Yamagata PB2, PB1, PA and MP segments. Five phylogenomic clusters were identified among the sequenced viruses; (i) pure B/Victoria clade 1A (n = 93, 83.8%), (ii) reassortant B/Victoria clade 1A (n = 1, 0.9%), (iii) pure B/Yamagata clade 2 (n = 2, 1.8%), (iv) pure B/Yamagata clade 3 (n = 6, 5.4%) and (v) reassortant B/Yamagata clade 3 (n = 9, 8.1%). Using divergence dates and clustering patterns in the presence of global background sequences, we counted up to 29 independent IBV strain introductions into the study area (∼900 km2) in 2016. Local viruses, including the reassortant B/Yamagata strains, clustered closely with viruses from neighbouring Tanzania and Uganda. Our study demonstrated that genomic analysis provides a clearer picture of locally circulating IBV diversity. The high number of IBV introductions highlights the challenge in controlling local influenza epidemics by targeted approaches e.g. sub-population vaccination or patient quarantine. The finding of divergent IBV strains co-circulating within a single season emphasizes why broad immunity vaccines are the most ideal for influenza control in Kenya

Spread and evolution of respiratory syncytial virus A genotype ON1, coastal Kenya, 2010–2015

In February 2012, the novel respiratory syncytial virus (RSV) group A, genotype ON1, was detected in Kilifi County, coastal Kenya. ON1 is characterized by a 72-nt duplication within the highly variable G gene (encoding the immunogenic attachment surface protein). Cases were diagnosed through surveillance of pneumonia in children at the county hospital. Analysis of epidemiologic, clinical, and sequence data of RSV-A viruses detected over 5 RSV seasons (2010/2011 to 2014/2015) indicated the following: 1) replacement of previously circulating genotype GA2 by ON1, 2) an abrupt expansion in the number of ON1 variants detected in the 2014/2015 epidemic, 3) recent accumulation of amino acid substitutions within the ON1 duplicated sequence, and 4) no clear evidence of altered pathogenicity relative to GA2. The study demonstrates the public health importance of molecular surveillance in defining the spread, clinical effects, and evolution of novel respiratory virus variants

Genomic epidemiology and evolutionary dynamics of respiratory syncytial virus group B in Kilifi, Kenya, 2015-17

Respiratory syncytial virus (RSV) circulates worldwide, occurring seasonally in communities, and is a leading cause of acute respiratory illness in young children. There is paucity of genomic data from purposively sampled populations by which to investigate evolutionary dynamics and transmission patterns of RSV. Here we present an analysis of 295 RSV group B (RSVB) genomes from Kilifi, coastal Kenya, sampled from individuals seeking outpatient care in 9 health facilities across a defined geographical area (∼890 km2), over 2 RSV epidemics between 2015 and 2017. RSVB diversity was characterized by multiple virus introductions into the area and co-circulation of distinct genetic clusters, which transmitted and diversified locally with varying frequency. Increase in relative genetic diversity paralleled seasonal virus incidence. Importantly, we identified a cluster of viruses that emerged in the 2016/17 epidemic, carrying distinct amino-acid signatures including a novel non-synonymous change (K68Q) in antigenic site ∅ in the Fusion protein. RSVB diversity was additionally marked by signature non-synonymous substitutions that were unique to particular genomic clusters, some under diversifying selection. Our findings provide insights into recent evolutionary and epidemiological behaviors of RSV group B, and highlight possible emergence of a novel antigenic variant, which has implications on current prophylactic strategies in development

Comparative analysis of spatial-temporal patterns of human metapneumovirus and respiratory syncytial virus in Africa using genetic data, 2011-2014

Background: Human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are leading causes of viral severe acute respiratory illnesses in childhood. Both the two viruses belong to the Pneumoviridae family and show overlapping clinical, epidemiological and transmission features. However, it is unknown whether these two viruses have similar geographic spread patterns which may inform designing and evaluating their epidemic control measures. Methods: We conducted comparative phylogenetic and phylogeographic analyses to explore the spatial-temporal patterns of HMPV and RSV across Africa using 232 HMPV and 842 RSV attachment (G) glycoprotein gene sequences obtained from 5 countries (The Gambia, Zambia, Mali, South Africa, and Kenya) between August 2011 and January 2014. Results: Phylogeographic analyses found frequently similar patterns of spread of RSV and HMPV. Viral sequences commonly clustered by region, i.e., West Africa (Mali, Gambia), East Africa (Kenya) and Southern Africa (Zambia, South Africa), and similar genotype dominance patterns were observed between neighbouring countries. Both HMPV and RSV country epidemics were characterized by co-circulation of multiple genotypes. Sequences from different African sub-regions (East, West and Southern Africa) fell into separate clusters interspersed with sequences from other countries globally. Conclusion: The spatial clustering patterns of viral sequences and genotype dominance patterns observed in our analysis suggests strong regional links and predominant local transmission. The geographical clustering further suggests independent introduction of HMPV and RSV variants in Africa from the global pool, and local regional diversification

Spatial-temporal distribution and sequence diversity of Group A human respiratory syncytial viruses in Kenya preceding the emergence of ON1 genotype

Background Human respiratory syncytial virus (HRSV) is a major cause of severe viral acute respiratory illness and contributes significantly to severe pneumonia cases in Africa. Little is known about its spatial–temporal distribution as defined by its genetic diversity. Methods A retrospective study conducted utilizing archived nasopharyngeal specimens from patients attending outpatient clinics in hospitals located in five demographically and climatically distinct regions of Kenya; Coast, Western, Highlands, Eastern and Nairobi. The viral total RNA was extracted and tested using multiplex real time RT-PCR (reverse transcriptase polymerase chain reaction). A segment of the G-gene was amplified using one-step RT-PCR and sequenced by Sanger di-deoxy method. Bayesian analysis of phylogeny was utilized and subsequently median joining methods for haplotype network reconstruction. Results Three genotypes of HRSVA were detected; GA5 (14.0%), GA2 (33.1%), and NA1 (52.9%). HRSVA prevalence varied by location from 33% to 13.2% in the Highlands and the Eastern regions respectively. The mean nucleotide diversity (Pi[π]) varied by genotype: highest of 0.018 for GA5 and lowest of 0.005 for NA1. A total of 58 haplotypes were identified (GA5 10; GA2 20; NA1 28). These haplotypes were introduced into the population locally by single haplotypes and additional subsidiary seeds amongst the GA2 and the NA1 haplotypes. Conclusions HRSVA was found across all the regions throughout the study period and comprised three genotypes; GA5, GA2, and NA1 genotypes. The genotypes were disproportionately distributed across the regions with GA5 gradually increasing toward the Western zones and decreasing toward the Eastern zones of the country

Human Coronavirus NL63 Molecular Epidemiology and Evolutionary Patterns in Rural Coastal Kenya.

Background: Human coronavirus NL63 (HCoV-NL63) is a globally endemic pathogen causing mild and severe respiratory tract infections with reinfections occurring repeatedly throughout a lifetime. Methods: Nasal samples were collected in coastal Kenya through community-based and hospital-based surveillance. HCoV-NL63 was detected with multiplex real-time reverse transcription PCR, and positive samples were targeted for nucleotide sequencing of the spike (S) protein. Additionally, paired samples from 25 individuals with evidence of repeat HCoV-NL63 infection were selected for whole-genome virus sequencing. Results: HCoV-NL63 was detected in 1.3% (75/5573) of child pneumonia admissions. Two HCoV-NL63 genotypes circulated in Kilifi between 2008 and 2014. Full genome sequences formed a monophyletic clade closely related to contemporary HCoV-NL63 from other global locations. An unexpected pattern of repeat infections was observed with some individuals showing higher viral titers during their second infection. Similar patterns for 2 other endemic coronaviruses, HCoV-229E and HCoV-OC43, were observed. Repeat infections by HCoV-NL63 were not accompanied by detectable genotype switching. Conclusions: In this coastal Kenya setting, HCoV-NL63 exhibited low prevalence in hospital pediatric pneumonia admissions. Clade persistence with low genetic diversity suggest limited immune selection, and absence of detectable clade switching in reinfections indicates initial exposure was insufficient to elicit a protective immune response