Assessing Genome-Wide Diversity in European Hantaviruses through Sequence Capture from Natural Host Samples

Abstract

Research on the ecology and evolution of viruses is often hampered by the limitation of sequence information to short parts of the genomes or single genomes derived from cultures. In this study, we use hybrid sequence capture enrichment in combination with high-throughput sequencing to provide efficient access to full genomes of European hantaviruses from rodent samples obtained in the field. We applied this methodology to Tula (TULV) and Puumala (PUUV) orthohantaviruses for which analyses from natural host samples are typically restricted to partial sequences of their tri-segmented RNA genome. We assembled a total of ten novel hantavirus genomes de novo with very high coverage (on average >99%) and sequencing depth (average >247×). A comparison with partial Sanger sequences indicated an accuracy of >99.9% for the assemblies. An analysis of two common vole (Microtus arvalis) samples infected with two TULV strains each allowed for the de novo assembly of all four TULV genomes. Combining the novel sequences with all available TULV and PUUV genomes revealed very similar patterns of sequence diversity along the genomes, except for remarkably higher diversity in the non-coding region of the S-segment in PUUV. The genomic distribution of polymorphisms in the coding sequence was similar between the species, but differed between the segments with the highest sequence divergence of 0.274 for the M-segment, 0.265 for the S-segment, and 0.248 for the L-segment (overall 0.258). Phylogenetic analyses showed the clustering of genome sequences consistent with their geographic distribution within each species. Genome-wide data yielded extremely high node support values, despite the impact of strong mutational saturation that is expected for hantavirus sequences obtained over large spatial distances. We conclude that genome sequencing based on capture enrichment protocols provides an efficient means for ecological and evolutionary investigations of hantaviruses at an unprecedented completeness and depth.

Genetic Diversity of Puumala orthohantavirus in Rodents and Human Patients in Austria, 2012–2019

“…There have been several methods published to statistically approach phylogeographic analyses (i.e., establishing genetic-geographic correlations with or without temporal resolution). For PUUV, it has been established that genetic diversity can largely be attributed to the isolation-by-distance pattern of divergence, owing to the relatively small dispersal ranges of the enzootic host [9,25,26,38]. Our data also support the strong correlation between genetic and geographic distance on a small scale for PUUV, using cophenetic distance as the measure of phylogenetic distance.…”

“…This may account for the lower genetic diversity under the isolation-by-distance assumption (c.f. Figure S1 to similar curves in [25,26]). Even so, others have identified potentially three distinctive phylogeographic groups of PUUV-ALAD (by partial L segment [11]), and we show that at least two genotypes exist in Austria-identifiable by a conserved predicted amino acid substitution in the nucleocapsid protein, with clear separation at a cophenetic distance of <0.07 (i.e., below the maximum diversity value for ALAD of~0.1).…”

“…Phylogeographic relationships were tested by estimating the correlation between genetic distance and geographic distance using Mantel's Z, based on a likelihood function comparing pairwise matrices over 10 5 permutations. This method has previously been used to test the geographic-genetic correlation for zoonotic RNA viruses [21][22][23] including PUUV [24][25][26].…”

Geographical Distribution and Genetic Diversity of Bank Vole Hepaciviruses in Europe

“…These isolation-by-distance patterns are similar to the patterns observed in bank vole-associated PUUV [ 36 ] or common vole-associated Tula orthohantavirus [ 41 ]. These associations can be explained by host dispersal occurring only at a local scale and the restriction of the accumulation of mutations in viral strains within local vole populations [ 42 , 43 ]. Over larger geographic distances, virus populations in each BvHV species thus evolve independently.…”

How Bank Vole-PUUV Interactions Influence the Eco-Evolutionary Processes Driving Nephropathia Epidemica Epidemiology—An Experimental and Genomic Approach

“…But amplification processes introduce errors that can be tempered by technical replicates for example [ 59 ]. In the future, integrating technics enabling the enrichment of viral matrices should help developing more efficient and accurate assessment of within-host viral diversity [ 60 , 61 ].…”