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.

Endemic chikungunya fever in Kenyan children: a prospective cohort study.

BACKGROUND: Chikungunya fever (CHIKF) was first described in Tanzania in 1952. Several epidemics including East Africa have occurred, but there are no descriptions of longitudinal surveillance of endemic disease. Here, we estimate the incidence of CHIKF in coastal Kenya and describe the associated viral phylogeny. METHODS: We monitored acute febrile illnesses among 3500 children visiting two primary healthcare facilities in coastal Kenya over a 5-year period (2014-2018). Episodes were linked to a demographic surveillance system and blood samples obtained. Cross-sectional sampling in a community survey of a different group of 435 asymptomatic children in the same study location was done in 2016. Reverse-transcriptase PCR was used for chikungunya virus (CHIKV) screening, and viral genomes sequenced for phylogenetic analyses. RESULTS: We found CHIKF to be endemic in this setting, associated with 12.7% (95% CI 11.60, 13.80) of all febrile presentations to primary healthcare. The prevalence of CHIKV infections among asymptomatic children in the community survey was 0.7% (95% CI 0.22, 2.12). CHIKF incidence among children < 1 year of age was 1190 cases/100,000-person years and 63 cases/100,000-person years among children aged ≥10 years. Recurrent CHIKF episodes, associated with fever and viraemia, were observed among 19 of 170 children with multiple febrile episodes during the study period. All sequenced viral genomes mapped to the ECSA genotype albeit distinct from CHIKV strains associated with the 2004 East African epidemic. CONCLUSIONS: CHIKF may be a substantial public health burden in primary healthcare on the East African coast outside epidemic years, and recurrent infections are common.

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.

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.

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 ON1, 2) an abrupt expansion in the number of ON1 variants detected in the 2014/2015 epidemic, 3) recently 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.

Molecular Evolutionary Dynamics of Respiratory Syncytial Virus Group A in Recurrent Epidemics in Coastal Kenya.

UNLABELLED: The characteristic recurrent epidemics of human respiratory syncytial virus (RSV) within communities may result from the genetic variability of the virus and associated evolutionary adaptation, reducing the efficiency of preexisting immune responses. We analyzed the molecular evolutionary changes in the attachment (G) glycoprotein of RSV-A viruses collected over 13 epidemic seasons (2000 to 2012) in Kilifi (n = 649), Kenya, and contemporaneous sequences (n = 1,131) collected elsewhere within Kenya and 28 other countries. Genetic diversity in the G gene in Kilifi was dynamic both within and between epidemics, characterized by frequent new variant introductions and limited variant persistence between consecutive epidemics. Four RSV-A genotypes were detected in Kilifi: ON1 (11.9%), GA2 (75.5%), GA5 (12.3%), and GA3 (0.3%), with predominant genotype replacement of GA5 by GA2 and then GA2 by ON1. Within these genotypes, there was considerable variation in potential N-glycosylation sites, with GA2 and ON1 viruses showing up to 15 different patterns involving eight possible sites. Further, we identified 15 positively selected and 34 genotype-distinguishing codon sites, with six of these sites exhibiting both characteristics. The mean substitution rate of the G ectodomain for the Kilifi data set was estimated at 3.58 × 10(-3) (95% highest posterior density interval = 3.04 to 4.16) nucleotide substitutions/site/year. Kilifi viruses were interspersed in the global phylogenetic tree, clustering mostly with Kenyan and European sequences. Our findings highlight ongoing genetic evolution and high diversity of circulating RSV-A strains, locally and globally, with potential antigenic differences. Taken together, these provide a possible explanation on the nature of recurrent local RSV epidemics. IMPORTANCE: The mechanisms underlying recurrent epidemics of RSV are poorly understood. We observe high genetic diversity in circulating strains within and between epidemics in both local and global settings. On longer time scales (∼7 years) there is sequential replacement of genotypes, whereas on shorter time scales (one epidemic to the next or within epidemics) there is a high turnover of variants within genotypes. Further, this genetic diversity is predicted to be associated with variation in antigenic profiles. These observations provide an explanation for recurrent RSV epidemics and have potential implications on the long-term effectiveness of vaccines.

Successive Respiratory Syncytial Virus Epidemics in Local Populations Arise from Multiple Variant Introductions, Providing Insights into Virus Persistence.

UNLABELLED: Respiratory syncytial virus (RSV) is a global respiratory pathogen of humans, with infection occurring characteristically as recurrent seasonal epidemics. Unlike influenza viruses, little attention has been paid to the mechanism underlying worldwide spread and persistence of RSV and how this may be discerned through an improved understanding of the introduction and persistence of RSV in local communities. We analyzed 651 attachment (G) glycoprotein nucleotide sequences of RSV B collected over 11 epidemics (2002 to 2012) in Kilifi, Kenya, and contemporaneous data collected elsewhere in Kenya and 18 other countries worldwide (2002 to 2012). Based on phylogeny, genetic distance and clustering patterns, we set out pragmatic criteria to classify local viruses into distinct genotypes and variants, identifying those newly introduced and those locally persisting. Three genotypes were identified in the Kilifi data set: BA (n = 500), SAB1 (n = 148), and SAB4 (n = 3). Recurrent RSV epidemics in the local population were composed of numerous genetic variants, most of which have been newly introduced rather than persisting in the location from season to season. Global comparison revealed that (i) most Kilifi variants do not cluster closely with strains from outside Kenya, (ii) some Kilifi variants were closely related to those observed outside Kenya (mostly Western Europe), and (iii) many variants were circulating elsewhere but were never detected in Kilifi. These results are consistent with the hypothesis that year-to-year presence of RSV at the local level (i.e., Kilifi) is achieved primarily, but not exclusively, through introductions from a pool of variants that are geographically restricted (i.e., to Kenya or to the region) rather than global. IMPORTANCE: The mechanism by which RSV persists and reinvades local populations is poorly understood. We investigated this by studying the temporal patterns of RSV variants in a rural setting in tropical Africa and comparing these variants with contemporaneous variants circulating in other countries. We found that periodic seasonal RSV transmission at the local level appears to require regular new introductions of variants. However, importantly, the evidence suggests that the source of new variants is mostly geographically restricted, and we hypothesize that year-to-year RSV persistence is at the country level rather than the global level. This has implications for control.

Local evolutionary patterns of human respiratory syncytial virus derived from whole-genome sequencing.

UNLABELLED: Human respiratory syncytial virus (RSV) is associated with severe childhood respiratory infections. A clear description of local RSV molecular epidemiology, evolution, and transmission requires detailed sequence data and can inform new strategies for virus control and vaccine development. We have generated 27 complete or nearly complete genomes of RSV from hospitalized children attending a rural coastal district hospital in Kilifi, Kenya, over a 10-year period using a novel full-genome deep-sequencing process. Phylogenetic analysis of the new genomes demonstrated the existence and cocirculation of multiple genotypes in both RSV A and B groups in Kilifi. Comparison of local versus global strains demonstrated that most RSV A variants observed locally in Kilifi were also seen in other parts of the world, while the Kilifi RSV B genomes encoded a high degree of variation that was not observed in other parts of the world. The nucleotide substitution rates for the individual open reading frames (ORFs) were highest in the regions encoding the attachment (G) glycoprotein and the NS2 protein. The analysis of RSV full genomes, compared to subgenomic regions, provided more precise estimates of the RSV sequence changes and revealed important patterns of RSV genomic variation and global movement. The novel sequencing method and the new RSV genomic sequences reported here expand our knowledge base for large-scale RSV epidemiological and transmission studies. IMPORTANCE: The new RSV genomic sequences and the novel sequencing method reported here provide important data for understanding RSV transmission and vaccine development. Given the complex interplay between RSV A and RSV B infections, the existence of local RSV B evolution is an important factor in vaccine deployment.

Rapid spread and diversification of respiratory syncytial virus genotype ON1, Kenya.

Respiratory syncytial virus genotype ON1, which is characterized by a 72-nt duplication in the attachment protein gene, has been detected in >10 countries since first identified in Ontario, Canada, in 2010. We describe 2 waves of genotype ON1 infections among children admitted to a rural hospital in Kenya during 2012. Phylogenetic analysis of attachment protein gene sequences showed multiple introductions of genotype ON1; variants distinct from the original Canadian viruses predominated in both infection waves. The genotype ON1 dominated over the other group A genotypes during the second wave, and some first wave ON1 variants reappeared in the second wave. An analysis of global genotype ON1 sequences determined that this genotype has become considerably diversified and has acquired signature coding mutations within immunogenic regions, and its most recent common ancestor dates to ≈2008-2009. Surveillance of genotype ON1 contributes to an understanding of the mechanisms of rapid emergence of respiratory viruses.

Examining strain diversity and phylogeography in relation to an unusual epidemic pattern of respiratory syncytial virus (RSV) in a long-term refugee camp in Kenya.

BACKGROUND: A recent longitudinal study in the Dadaab refugee camp near the Kenya-Somalia border identified unusual biannual respiratory syncytial virus (RSV) epidemics. We characterized the genetic variability of the associated RSV strains to determine if viral diversity contributed to this unusual epidemic pattern. METHODS: For 336 RSV positive specimens identified from 2007 through 2011 through facility-based surveillance of respiratory illnesses in the camp, 324 (96.4%) were sub-typed by PCR methods, into 201 (62.0%) group A, 118 (36.4%) group B and 5 (1.5%) group A-B co-infections. Partial sequencing of the G gene (coding for the attachment protein) was completed for 290 (89.5%) specimens. These specimens were phylogenetically analyzed together with 1154 contemporaneous strains from 22 countries. RESULTS: Of the 6 epidemic peaks recorded in the camp over the period, the first and last were predominantly made up of group B strains, while the 4 in between were largely composed of group A strains in a consecutive series of minor followed by major epidemics. The Dadaab group A strains belonged to either genotype GA2 (180, 98.9%) or GA5 (2, < 1%) while all group B strains (108, 100%) belonged to BA genotype. In sequential epidemics, strains within these genotypes appeared to be of two types: those continuing from the preceding epidemics and those newly introduced. Genotype diversity was similar in minor and major epidemics. CONCLUSION: RSV strain diversity in Dadaab was similar to contemporaneous diversity worldwide, suggested both between-epidemic persistence and new introductions, and was unrelated to the unusual epidemic pattern.

TheiaEuk: a species-agnostic bioinformatics workflow for fungal genomic characterization.

Introduction: The clinical incidence of antimicrobial-resistant fungal infections has dramatically increased in recent years. Certain fungal pathogens colonize various body cavities, leading to life-threatening bloodstream infections. However, the identification and characterization of fungal isolates in laboratories remain a significant diagnostic challenge in medicine and public health. Whole-genome sequencing provides an unbiased and uniform identification pipeline for fungal pathogens but most bioinformatic analysis pipelines focus on prokaryotic species. To this end, TheiaEuk_Illumina_PE_PHB (TheiaEuk) was designed to focus on genomic analysis specialized to fungal pathogens. Methods: TheiaEuk was designed using containerized components and written in the workflow description language (WDL) to facilitate deployment on the cloud-based open bioinformatics platform Terra. This species-agnostic workflow enables the analysis of fungal genomes without requiring coding, thereby reducing the entry barrier for laboratory scientists. To demonstrate the usefulness of this pipeline, an ongoing outbreak of C. auris in southern Nevada was investigated. We performed whole-genome sequence analysis of 752 new C. auris isolates from this outbreak. Furthermore, TheiaEuk was utilized to observe the accumulation of mutations in the FKS1 gene over the course of the outbreak, highlighting the utility of TheiaEuk as a monitor of emerging public health threats when combined with whole-genome sequencing surveillance of fungal pathogens. Results: A primary result of this work is a curated fungal database containing 5,667 unique genomes representing 245 species. TheiaEuk also incorporates taxon-specific submodules for specific species, including clade-typing for Candida auris (C. auris). In addition, for several fungal species, it performs dynamic reference genome selection and variant calling, reporting mutations found in genes currently associated with antifungal resistance (FKS1, ERG11, FUR1). Using genome assemblies from the ATCC Mycology collection, the taxonomic identification module used by TheiaEuk correctly assigned genomes to the species level in 126/135 (93.3%) instances and to the genus level in 131/135 (97%) of instances, and provided zero false calls. Application of TheiaEuk to actual specimens obtained in the course of work at a local public health laboratory resulted in 13/15 (86.7%) correct calls at the species level, with 2/15 called at the genus level. It made zero incorrect calls. TheiaEuk accurately assessed clade type of Candida auris in 297/302 (98.3%) of instances. Discussion: TheiaEuk demonstrated effectiveness in identifying fungal species from whole genome sequence. It further showed accuracy in both clade-typing of C. auris and in the identification of mutations known to associate with drug resistance in that organism.

Accelerating bioinformatics implementation in public health.

We have adopted an open bioinformatics ecosystem to address the challenges of bioinformatics implementation in public health laboratories (PHLs). Bioinformatics implementation for public health requires practitioners to undertake standardized bioinformatic analyses and generate reproducible, validated and auditable results. It is essential that data storage and analysis are scalable, portable and secure, and that implementation of bioinformatics fits within the operational constraints of the laboratory. We address these requirements using Terra, a web-based data analysis platform with a graphical user interface connecting users to bioinformatics analyses without the use of code. We have developed bioinformatics workflows for use with Terra that specifically meet the needs of public health practitioners. These Theiagen workflows perform genome assembly, quality control, and characterization, as well as construction of phylogeny for insights into genomic epidemiology. Additonally, these workflows use open-source containerized software and the WDL workflow language to ensure standardization and interoperability with other bioinformatics solutions, whilst being adaptable by the user. They are all open source and publicly available in Dockstore with the version-controlled code available in public GitHub repositories. They have been written to generate outputs in standardized file formats to allow for further downstream analysis and visualization with separate genomic epidemiology software. Testament to this solution meeting the requirements for bioinformatic implementation in public health, Theiagen workflows have collectively been used for over 5 million sample analyses in the last 2 years by over 90 public health laboratories in at least 40 different countries. Continued adoption of technological innovations and development of further workflows will ensure that this ecosystem continues to benefit PHLs.

Rhinovirus dynamics across different social structures.

Rhinoviruses (RV), common human respiratory viruses, exhibit significant antigenic diversity, yet their dynamics across distinct social structures remain poorly understood. Our study delves into RV dynamics within Kenya by analysing VP4/2 sequences across four different social structures: households, a public primary school, outpatient clinics in the Kilifi Health and Demographics Surveillance System (HDSS), and countrywide hospital admissions and outpatients. The study revealed the greatest diversity of RV infections at the countrywide level (114 types), followed by the Kilifi HDSS (78 types), the school (47 types), and households (40 types), cumulatively representing >90% of all known RV types. Notably, RV diversity correlated directly with the size of the population under observation, and several RV type variants occasionally fuelled RV infection waves. Our findings highlight the critical role of social structures in shaping RV dynamics, information that can be leveraged to enhance public health strategies. Future research should incorporate whole-genome analysis to understand fine-scale evolution across various social structures.

Origins and Evolution of Seasonal Human Coronaviruses.

Four seasonal human coronaviruses (sHCoVs) are endemic globally (229E, NL63, OC43, and HKU1), accounting for 5-30% of human respiratory infections. However, the epidemiology and evolution of these CoVs remain understudied due to their association with mild symptomatology. Using a multigene and complete genome analysis approach, we find the evolutionary histories of sHCoVs to be highly complex, owing to frequent recombination of CoVs including within and between sHCoVs, and uncertain, due to the under sampling of non-human viruses. The recombination rate was highest for 229E and OC43 whereas substitutions per recombination event were highest in NL63 and HKU1. Depending on the gene studied, OC43 may have ungulate, canine, or rabbit CoV ancestors. 229E may have origins in a bat, camel, or an unsampled intermediate host. HKU1 had the earliest common ancestor (1809-1899) but fell into two distinct clades (genotypes A and B), possibly representing two independent transmission events from murine-origin CoVs that appear to be a single introduction due to large gaps in the sampling of CoVs in animals. In fact, genotype B was genetically more diverse than all the other sHCoVs. Finally, we found shared amino acid substitutions in multiple proteins along the non-human to sHCoV host-jump branches. The complex evolution of CoVs and their frequent host switches could benefit from continued surveillance of CoVs across non-human hosts.

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.

Evolution of respiratory syncytial virus genotype BA in Kilifi, Kenya, 15 years on.

Respiratory syncytial virus (RSV) is recognised as a leading cause of severe acute respiratory disease and deaths among infants and vulnerable adults. Clinical RSV isolates can be divided into several known genotypes. RSV genotype BA, characterised by a 60-nucleotide duplication in the G glycoprotein gene, emerged in 1999 and quickly disseminated globally replacing other RSV group B genotypes. Continual molecular epidemiology is critical to understand the evolutionary processes maintaining the success of the BA viruses. We analysed 735 G gene sequences from samples collected from paediatric patients in Kilifi, Kenya, between 2003 and 2017. The virus population comprised of several genetically distinct variants (n = 56) co-circulating within and between epidemics. In addition, there was consistent seasonal fluctuations in relative genetic diversity. Amino acid changes increasingly accumulated over the surveillance period including two residues (N178S and Q180R) that mapped to monoclonal antibody 2D10 epitopes, as well as addition of putative N-glycosylation sequons. Further, switching and toggling of amino acids within and between epidemics was observed. On a global phylogeny, the BA viruses from different countries form geographically isolated clusters suggesting substantial localized variants. This study offers insights into longitudinal population dynamics of a globally endemic RSV genotype within a discrete location.

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 nine health facilities across a defined geographical area (∼890 km2), over two 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 nonsynonymous change (K68Q) in antigenic site ∅ in the Fusion protein. RSVB diversity was additionally marked by signature nonsynonymous substitutions that were unique to particular genomic clusters, some under diversifying selection. Our findings provide insights into recent evolutionary and epidemiological behaviors of RSVB, and highlight possible emergence of a novel antigenic variant, which has implications on current prophylactic strategies in development.

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 (<5 years old). Whole genomes were sequenced for a selected 111 positives; 94 (84.7%) of B/Victoria lineage and 17 (15.3%) of B/Yamagata lineage. Inter-lineage reassortment was detected in ten 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 twenty-nine 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, for example, sub-population vaccination or patient quarantine. The finding of divergent IBV strains co-circulating within a single season emphasises why broad immunity vaccines are the most ideal for influenza control in Kenya.

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.

Peripheral blood mononuclear cell transcriptomes reveal an over-representation of down-regulated genes associated with immunity in HIV-exposed uninfected infants.

HIV-exposed uninfected (HEU) infants are disproportionately at a higher risk of morbidity and mortality, as compared to HIV-unexposed uninfected (HUU) infants. Here, we used transcriptional profiling of peripheral blood mononuclear cells to determine immunological signatures of in utero HIV exposure. We identified 262 differentially expressed genes (DEGs) in HEU compared to HUU infants. Weighted gene co-expression network analysis (WGCNA) identified six modules that had significant associations with clinical traits. Functional enrichment analysis on both DEGs and the six significantly associated modules revealed an enrichment of G-protein coupled receptors and the immune system, specifically affecting neutrophil function and antibacterial responses. Additionally, malaria pathogenicity genes (thrombospondin 1-(THBS 1), interleukin 6 (IL6), and arginine decarboxylase 2 (ADC2)) were down-regulated. Of interest, the down-regulated immunity genes were positively correlated to the expression of epigenetic factors of the histone family and high-mobility group protein B2 (HMGB2), suggesting their role in the dysregulation of the HEU transcriptional landscape. Overall, we show that genes primarily associated with neutrophil mediated immunity were repressed in the HEU infants. Our results suggest that this could be a contributing factor to the increased susceptibility to bacterial infections associated with higher morbidity and mortality commonly reported in HEU infants.