HighlightsSequence variation at probe target site inhibited detection of a new RSV-B variant.RSV-B virus diversity was consistent with real-time RT-PCR sensitivity.Reduced PCR insensitivity could underestimate disease prevalence in clinical settings.Regular check of primer and probe target sites for rapidly evolving viruses is key.

The respiratory syncytial virus (RSV) group A variant with the 72-nucleotide duplication in the G gene, genotype ON1, was first detected in Kilifi in 2012 and has almost completely replaced circulating genotype GA2 strains. This replacement suggests some fitness advantage of ON1 over the GA2 viruses in Kilifi, and might be accompanied by important genomic substitutions in ON1 viruses. Close observation of such a new virus genotype introduction over time provides an opportunity to better understand the transmission and evolutionary dynamics of the pathogen. We have generated and analysed 184 RSV-A whole-genome sequences (WGSs) from Kilifi (Kenya) collected between 2011 and 2016, the first ON1 genomes from Africa and the largest collection globally from a single location. Phylogenetic analysis indicates that RSV-A circulation in this coastal Kenya location is characterized by multiple introductions of viral lineages from diverse origins but with varied success in local transmission. We identified signature amino acid substitutions between ON1 and GA2 viruses’ surface proteins (G and F), polymerase (L), and matrix M2-1 proteins, some of which were positively selected, and thereby provide an enhanced picture of RSV-A diversity. Furthermore, five of the eleven RSV open reading frames (ORFs) (G, F, L, N, and P) formed distinct phylogenetic clusters for the two genotypes. This might suggest that coding regions outside of the most frequently studied G ORF also play a role in the adaptation of RSV to host populations, with the alternative possibility that some of the substitutions are neutral and provide no selective advantage. Our analysis provides insight into the epidemiological processes that define RSV spread, highlights the genetic substitutions that characterize emerging strains, and demonstrates the utility of large-scale WGS in molecular epidemiological studies.

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. Electronic supplementary material The online version of this article (10.1186/s12879-019-4381-9) contains supplementary material, which is available to authorized users.

Short tandem repeat (STR) variation is an often overlooked source of variation between genomes. STRs comprise about 3% of the human genome sequence, are highly polymorphic, and are known to cause not only Mendelian disease, but extensively affect gene expression. Nevertheless, their contribution to common disease is not well-understood, but recent software tools designed to genotype STRs using short read sequencing data are beginning to address this. Here, we compare software that genotypes common STRs and detect rarer STR expansions genome-wide, with the aim of applying them to population-scale genome sequences. By using the Genome-In-A-Bottle (GIAB) consortium and 1000 Genomes Project sequencing data, we can compare performance in terms of sequence length, depth, computing resources needed, genotyping accuracy and number of STRs genotyped. To ensure broad applicability of our findings, we also measure performance against a set of clinical samples with known STR expansions, and a set of STRs commonly used for forensic identification. By using analysis of Mendelian inheritance patterns and comparison with capillary electrophoresis genotypes, we find that both HipSTR and GangSTR perform well in genotyping common STRs, with GangSTR outperforming HipSTR for genotyping call rate and memory usage. Analysis for expanded STRs showed ExpansionHunter denovo (EHdn) and STRetch outperformed GangSTR, but EHdn used considerably less processor time and memory compared to STRetch. Analysis on shared genomic sequence data provided by the GIAB consortium allows future performance comparisons of new software approaches on a common set of data, facilitating comparisons and allowing researchers to choose the best software that fulfils their needs.

BackgroundHuman 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.ResultsWe 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.ConclusionThis is the first genomic characterization of HMPV genomes from African patients.