Estimating prevalence of Enterovirus D111 in human and non-human primate populations using cross-sectional serology.

Enteroviruses primarily affect young children with a varying severity of disease. Recent outbreaks of severe respiratory and neurological disease due to EV-D68 and EV-A71, as well as atypical hand-foot-and-mouth-disease due to CVA6, have brought to light the potency of enteroviruses to emerge as severe human pathogens. Enterovirus D111 (EV-D111) is an enteric pathogen initially detected in Central Africa in human and wildlife samples and was recently detected in environmental samples. The natural history and epidemiology of EV-D111 are poorly studied. Here, the presence of serum neutralizing antibodies to EV-D111 was estimated in human and wildlife samples from five countries. We report high prevalence of neutralizing antibodies measured against EV-D111 in human populations (range, 55-83 %), a proxy for previous infection, which indicates active virus circulation in absence of detection in clinical cases and a high number of undiagnosed infections. Notably, seroprevalence in samples from the UK varied by age and was higher in children and older adults (1-5 and >60 years old), but lower in ages 11-60. EV-D111 seroprevalence in apes and Old World monkeys was 50 % (33-66 %), which also suggests prior exposure and supports existing knowledge of enterovirus circulation in wild and captive apes and Old World monkeys. Generally, reported cases of infection likely underestimate the prevalence of infection particularly when the knowledge of community transmission is limited. Continued serologic surveillance and detection of EV-D111 in clinical and environmental samples will allow for a more robust assessment of EV-D111 epidemiology.

Changes in transmission of Enterovirus D68 (EV-D68) in England inferred from seroprevalence data.

The factors leading to the global emergence of Enterovirus D68 (EV-D68) in 2014 as a cause of acute flaccid myelitis (AFM) in children are unknown. To investigate potential changes in virus transmissibility or population susceptibility, we measured the seroprevalence of EV-D68-specific neutralising antibodies in serum samples collected in England in 2006, 2011, and 2017. Using catalytic mathematical models, we estimate an approximately 50% increase in the annual probability of infection over the 10-year study period, coinciding with the emergence of clade B around 2009. Despite such increase in transmission, seroprevalence data suggest that the virus was already widely circulating before the AFM outbreaks and the increase of infections by age cannot explain the observed number of AFM cases. Therefore, the acquisition of or an increase in neuropathogenicity would be additionally required to explain the emergence of outbreaks of AFM. Our results provide evidence that changes in enterovirus phenotypes cause major changes in disease epidemiology.

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.

Whole genome sequencing of two human rhinovirus A types (A101 and A15) detected in Kenya, 2016-2018.

Background: Virus genome sequencing is increasingly utilized in epidemiological surveillance. Genomic data allows comprehensive evaluation of underlying viral diversity and epidemiology to inform control. For human rhinovirus (HRV), genomic amplification and sequencing is challenging due to numerous types, high genetic diversity and inadequate reference sequences. Methods: We developed a tiled amplicon type-specific protocol for genome amplification and sequencing on the Illumina MiSeq platform of two HRV types, A15 and A101. We then assessed added value in analyzing whole genomes relative to the VP4/2 region only in the investigation of HRV molecular epidemiology within the community in Kilifi, coastal Kenya. Results: We processed 73 nasopharyngeal swabs collected between 2016-2018, and 48 yielded at least 70% HRV genome coverage. These included all A101 samples (n=10) and 38 (60.3%) A15 samples.  Phylogenetic analysis revealed that the Kilifi A101 sequences interspersed with global A101 genomes available in GenBank collected between 1999-2016. On the other hand, our A15 sequences formed a monophyletic group separate from the global genomes collected in 2008 and 2019. An improved phylogenetic resolution was observed with the genome phylogenies compared to the VP4/2 phylogenies. Conclusions: We present a type-specific full genome sequencing approach for obtaining HRV genomic data and characterizing infections.

Seroprevalence and Virologic Surveillance of Enterovirus 71 and Coxsackievirus A6, United Kingdom, 2006-2017.

Enterovirus A71 (EV-A71) and coxsackievirus A6 (CVA6) cause hand, foot and mouth disease (HFMD) and are occasionally linked to severe neurologic complications and large outbreaks worldwide. We estimated EV-A71 and CVA6 seroprevalence using cross-sectional age-stratified samples collected in 2006, 2011, and 2017. Seroprevalences of EV-A71 and CVA6 increased from 32% and 54% at 6-11 months to >75% by 10 years of age. Antibody titers declined after 20 years, which could indicate infrequent re-exposure in older populations. Age profiles for acquiring infections and mean titers were comparable in the 3 testing years, despite the marked increase in incidence of CVA6-related HFMD from 2010. The uncoupling of changes in disease severity from the infection kinetics of CVA6 as we inferred from the seroprevalence data, rather than incidence of infection over the 11-year study period, provides further evidence for a change in its pathogenicity.

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.

Trends and Intensity of Rhinovirus Invasions in Kilifi, Coastal Kenya, Over a 12-Year Period, 2007-2018.

BACKGROUND: Rhinoviruses (RVs) are ubiquitous pathogens and the principal etiological agents of common cold. Despite the high frequency of RV infections, data describing their long-term epidemiological patterns in a defined population remain limited. METHODS: Here, we analyzed 1070 VP4/VP2 genomic region sequences sampled at Kilifi County Hospital on the Kenya coast. The samples were collected between 2007 and 2018 from hospitalized pediatric patients (<60 months of age) with acute respiratory illness. RESULTS: Of 7231 children enrolled, RV was detected in 1497 (20.7%) and VP4/VP2 sequences were recovered from 1070 samples (71.5%). A total of 144 different RV types were identified (67 Rhinovirus A, 18 Rhinovirus B, and 59 Rhinovirus C) and at any month, several types co-circulated with alternating predominance. Within types, multiple genetically divergent variants were observed. Ongoing RV infections through time appeared to be a combination of (1) persistent types (observed up to 7 consecutive months), (2) reintroduced genetically distinct variants, and (3) new invasions (average of 8 new types annually). CONCLUSIONS: Sustained RV presence in the Kilifi community is mainly due to frequent invasion by new types and variants rather than continuous transmission of locally established types/variants.

Enrichment approach for unbiased sequencing of respiratory syncytial virus directly from clinical samples.

Background: Nasopharyngeal samples contain higher quantities of bacterial and host nucleic acids relative to viruses; presenting challenges during virus metagenomics sequencing, which underpins agnostic sequencing protocols. We aimed to develop a viral enrichment protocol for unbiased whole-genome sequencing of respiratory syncytial virus (RSV) from nasopharyngeal samples using the Oxford Nanopore Technology (ONT) MinION platform. Methods: We assessed two protocols using RSV positive samples. Protocol 1 involved physical pre-treatment of samples by centrifugal processing before RNA extraction, while Protocol 2 entailed direct RNA extraction without prior enrichment. Concentrates from Protocol 1 and RNA extracts from Protocol 2 were each divided into two fractions; one was DNase treated while the other was not. RNA was then extracted from both concentrate fractions per sample and RNA from both protocols converted to cDNA, which was then amplified using the tagged Endoh primers through Sequence-Independent Single-Primer Amplification (SISPA) approach, a library prepared, and sequencing done. Statistical significance during analysis was tested using the Wilcoxon signed-rank test. Results: DNase-treated fractions from both protocols recorded significantly reduced host and bacterial contamination unlike the untreated fractions (in each protocol p<0.01). Additionally, DNase treatment after RNA extraction (Protocol 2) enhanced host and bacterial read reduction compared to when done before (Protocol 1). However, neither protocol yielded whole RSV genomes. Sequenced reads mapped to parts of the nucleoprotein (N gene) and polymerase complex (L gene) from Protocol 1 and 2, respectively. Conclusions: DNase treatment was most effective in reducing host and bacterial contamination, but its effectiveness improved if done after RNA extraction than before. We attribute the incomplete genome segments to amplification biases resulting from the use of short length random sequence (6 bases) in tagged Endoh primers. Increasing the length of the random nucleotides from six hexamers to nine or 12 in future studies may reduce the coverage biases.

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.

Surveillance of respiratory viruses among children attending a primary school in rural coastal Kenya.

Background: Respiratory viruses are primary agents of respiratory tract diseases. Knowledge on the types and frequency of respiratory viruses affecting school-children is important in determining the role of schools in transmission in the community and identifying targets for interventions. Methods: We conducted a one-year (term-time) surveillance of respiratory viruses in a rural primary school in Kilifi County, coastal Kenya between May 2017 and April 2018. A sample of 60 students with symptoms of ARI were targeted for nasopharyngeal swab (NPS) collection weekly.  Swabs were screened for 15 respiratory virus targets using real time PCR diagnostics. Data from respiratory virus surveillance at the local primary healthcare facility was used for comparison. Results: Overall, 469 students aged 2-19 years were followed up for 220 days. A total of 1726 samples were collected from 325 symptomatic students; median age of 7 years (IQR 5-11). At least one virus target was detected in 384 (22%) of the samples with a frequency of 288 (16.7%) for rhinovirus, 47 (2.7%) parainfluenza virus, 35 (2.0%) coronavirus, 15 (0.9%) adenovirus, 11 (0.6%) respiratory syncytial virus (RSV) and 5 (0.3%) influenza virus.  The proportion of virus positive samples was higher among lower grades compared to upper grades (25.9% vs 17.5% respectively; χ 2 = 17.2, P -value <0.001). Individual virus target frequencies did not differ by age, sex, grade, school term or class size. Rhinovirus was predominant in both the school and outpatient setting. Conclusion: Multiple respiratory viruses circulated in this rural school population.  Rhinovirus was dominant in both the school and outpatient setting and RSV was of notably low frequency in the school. The role of school children in transmitting viruses to the household setting is still unclear and further studies linking molecular data to contact patterns between the school children and their households are required.

Molecular Epidemiology of Human Rhinovirus From 1-Year Surveillance Within a School Setting in Rural Coastal Kenya.

BACKGROUND: Human rhinovirus (HRV) is the most common cause of the common cold but may also lead to more severe respiratory illness in vulnerable populations. The epidemiology and genetic diversity of HRV within a school setting have not been previously described. The objective of this study was to characterize HRV molecular epidemiology in a primary school in a rural location of Kenya. METHODS: Between May 2017 and April 2018, over 3 school terms, we collected 1859 nasopharyngeal swabs (NPS) from pupils and teachers with symptoms of acute respiratory infection in a public primary school in Kilifi County, coastal Kenya. The samples were tested for HRV using real-time reverse transcription polymerase chain reaction. HRV-positive samples were sequenced in the VP4/VP2 coding region for species and genotype classification. RESULTS: A total of 307 NPS (16.4%) from 164 individuals were HRV positive, and 253 (82.4%) were successfully sequenced. The proportion of HRV in the lower primary classes was higher (19.8%) than upper primary classes (12.2%; P < .001). HRV-A was the most common species (134/253; 53.0%), followed by HRV-C (73/253; 28.9%) and HRV-B (46/253; 18.2%). Phylogenetic analysis identified 47 HRV genotypes. The most common genotypes were A2 and B70. Numerous (up to 22 in 1 school term) genotypes circulated simultaneously, there was no individual re-infection with the same genotype, and no genotype was detected in all 3 school terms. CONCLUSIONS: HRV was frequently detected among school-going children with mild acute respiratory illness symptoms, particularly in the younger age groups (<5-year-olds). Multiple HRV introductions were observed that were characterized by considerable genotype diversity.

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.

Recombination Analysis of Non-Poliovirus Members of the Enterovirus C Species; Restriction of Recombination Events to Members of the Same 3DPol Cluster.

Enteroviruses (EVs) are highly prevalent viruses worldwide. Recombination is known to occur frequently in EVs belonging to species Enterovirus A, Enterovirus B, and Enterovirus C. Although many recombinant vaccine-derived poliovirus (VDPV) strains have been reported, our knowledge on recombination in non-polio EVs in the species Enterovirus C is limited. Here, we combined a dataset consisting of 11 newly generated full-length Enterovirus C sequences and 180 publicly available sequences to study recombination dynamics in non-polio EVs. To identify recombination patterns, maximum likelihood phylogenetic trees of different genomic regions were constructed, and segregation analyses were performed. Recombination was observed between members of the same 3DPol cluster, but was rarely observed between members of different clusters. We hypothesize that this restriction may have arisen through their different compartmentalization in respiratory and enteric tracts related to differences in cellular tropisms so that the opportunity to recombine may not be available.

Transmission and evolutionary dynamics of human coronavirus OC43 strains in coastal Kenya investigated by partial spike sequence analysis, 2015-16.

Human coronavirus OC43 (HCoV-OC43) is a major contributor to seasonal outbreaks of acute respiratory illness (ARI). The origins of locally circulating HCoV-OC43 strains and characteristics of their genetic diversity are unknown for most settings despite significance to effective HCoV control strategies. Between December 2015 and June 2016, we undertook ARI surveillance in coastal Kenya in nine outpatients and one inpatient health facility (HF). Ninety-two patient samples tested HCoV-OC43 positive and forty (43.5%) were successfully sequenced in spike (S) gene region (2,864 long, ∼70%). Phylogenetic analysis confirmed co-circulation of two distinct HCoV-OC43 clades that closely clustered with genotype G (n = 34, 85%) and genotype H (n = 6, 15%) reference strains. Local viruses within the same clade displayed low genetic diversity yielding identical sequences in multiple HF. Furthermore, the newly sequenced Kenyan viruses showed close phylogenetic relationship to other contemporaneous sampled strains (2015-16) including those originating from distant places (e.g. USA and China). Using a genetic similarity threshold of 99.1 per cent at nucleotide level, the HCoV-OC43 strains sampled globally between 1967 and 2019 fell into nine sequence clusters. Notably, some of these clusters appeared to have become extinct, or occurred only sporadically in a few geographical areas while others persisted globally for multiple years. In conclusion, we found that HCoV-OC43 strains spread rapidly both locally and across the globe with limited genetic evolution in the spike gene. Full-genome sequences that are spatio-temporally representative are required to advance understanding of the transmission pathways of this important human respiratory pathogen.

Correction to: Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia.

Following the publication of this article [1], it was noted that due to a typesetting error the figure legends were paired incorrectly.

Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia.

BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients.

Genome Sequences of Human Coronavirus OC43 and NL63, Associated with Respiratory Infections in Kilifi, Kenya.

Coding-complete genomes of two human coronavirus OC43 strains and one NL63 strain were obtained by metagenomic sequencing of clinical samples collected in 2017 and 2018 in Kilifi, Kenya. Maximum likelihood phylogenies showed that the OC43 strains were genetically dissimilar and that the NL63 strain was closely related to NL63 genotype B viruses.

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.

Increase in Enterovirus D68 Infections in Young Children, United Kingdom, 2006-2016.

We determined the change in seroprevalence of enterovirus D68 (EV-D68) in the United Kingdom in age-stratified cohorts from 2006 to 2016, the period during which EV-D68 emerged as a cause of severe respiratory disease occasionally leading to paralysis. Infections were acquired primarily in infants and young children, and incidence was markedly higher in 2016.