Metagenomic sequencing at the epicenter of the Nigeria 2018 Lassa fever outbreak

Abstract

The 2018 Nigerian Lassa fever season saw the largest ever recorded upsurge of cases, raising concerns over the emergence of a strain with increased transmission rate. To understand the molecular epidemiology of this upsurge, we performed, for the first time at the epicenter of an unfolding outbreak, metagenomic nanopore sequencing directly from patient samples, an approach dictated by the highly variable genome of the target pathogen. Genomic data and phylogenetic reconstructions were communicated immediately to Nigerian authorities and the World Health Organization to inform the public health response. Real-time analysis of 36 genomes and subsequent confirmation using all 120 samples sequenced in the country of origin revealed extensive diversity and phylogenetic intermingling with strains from previous years, suggesting independent zoonotic transmission events and thus allaying concerns of an emergent strain or extensive human-to-human transmission.

Figure 1.. Phylogenetic reconstruction of the S segment data.

The circular tree includes 96 sequences from 2012 to 2017, 88 sequences from 2018 and sequences available from GenBank. The rectangular tree focuses on the genotype II clade (in blue in the circular tree) that includes most of the 2018 sequences. The six genotypes are indicated with different colors and roman symbols. Bootstrap support >90% is indicated with a small grey circle at the middle of their respective branches. The color strip highlights the human LASV sequences obtained from previous years (light grey), sequences obtained from rodent samples (dark grey) and 2018 sequences as light pink for the first seven sequences generated in Nigeria, magenta for the remaining 28 sequences analysed on-site, and purple for the remaining finalised in Europe. The same color code is used in the genotype II rectangular tree. Bootstrap values >80% are shown for the major genotype II lineages.

Figure 2.. Assessing the potential for direct linkage between pairs of 2018 sequences in the S segment.

The maximum clade credibility tree summarizes a Bayesian evolutionary inference for the genotype II sequences in the S segment. A time scale and a marginal posterior distribution for the time to the most recent common ancestor are shown to the left. The size of the internal node circles reflects posterior probability support values. 2018 sequences clustering as pairs are indicated in purple; the number of substitutions between them is indicated at their respective tips. A posterior estimate of the evolutionary rate and probability distributions for observing a given number of substitutions during a human-tohuman transmission event are shown as insets. The distribution represented by grey bars is based on the mean evolutionary rate estimate and a mean estimate for the generation time whereas the light blue distribution is based on upper estimates and also incorporates an upper estimate for the MinION sequencing error (Supplementary Methods). At the bottom, clusters of sequences for which human-to-human transmission cannot be excluded according to the upper estimates of generation time are indicated. A pair of identical sequences (137–138) that was retrospectively found to be derived from the same patient is marked with a grey box. One pair (096–115) was still disregarded as potential transmission chain due to 21 differences in L segment (Fig. S9). The temporal signal prior to BEAST inference was explored in Fig. S10.