Bones hold the key to DNA virus history and epidemiology

Abstract

DNA in human skeletal remains represents an important historical source of host genomic information and potentially of infecting viruses. However, little is known about viral persistence in bone. We searched ca. 70-year-old long bones of putative Finnish casualties from World War II for parvovirus B19 (B19V) DNA, and found a remarkable prevalence of 45%. The viral sequences were exclusively of genotypes 2 (n = 41), which disappeared from circulation in 1970´s, or genotype 3 (n = 2), which has never been reported in Northern Europe. Based on mitochondrial and Y-chromosome profiling, the two individuals carrying B19V genotype 3 were likely from the Soviet Red Army. The most recent common ancestor for all genotypes was estimated at early 1800s. This work demonstrates the forms of B19V that circulated in the first half of the 20(th) century and provides the first evidence of the suitability of bone for exploration of DNA viruses.

Figure 1. Correlation of B19V DNA copy numbers determined by Pan-B19V and VP qPCRs.

Genomic B19V DNA was quantified by means of two in-house quantitative PCRs targeting distinct conserved regions of the viral genome (NS and VP). Represented are in the y-axis the genomic copies/1 μg of total DNA of individual samples as determined by the Pan-B19V qPCR. In the x-axis, genomic copies/1 μg of total DNA of individual samples as determined by VP-qPCR. In squares are represented the copy numbers of the five samples that were positive by the VP-qPCR only.

Figure 2. B19V phylogenetic tree.

Maximum likelihood tree of the combined NS/VP region constructed using optimal substitution models (Kimura 2-parameter + invariant sites). Sample dates (acute infection; plasma) or predicted infection dates (assumed at 9 years of age; tissue) are shown on labels. Bootstrap re-sampling was used to determine robustness of groupings; values of 70% or greater shown.

Figure 3. Genetic diversity over time.

The genetic distance from the putative ancestral root from the maximum likelihood tree (Fig. 2) for each sample is plotted with the corresponding sampling date (circles). Dotted line: line of best fit for these points corresponding to a simple substitution rate estimate of 1 × 10^–4 substitutions/site/year. Solid line (mean) and shaded region (95% HPD): overall substitution rate estimate from BEAST analysis: 2.1–2.2 × 10^–4 (1.1–3.2 × 10^–4) substitutions/site/year.

Figure 4. Time to most recent common ancestors of B19V genotypes.

Estimated time to most recent common ancestor of genotypes 1 (pink), 2 (blue) and 3 (orange), as well as that of the whole tree (grey) using several models with BEAST. Models 1–3, and 7–9 additionally use the HKY nucleotide substitution model with site to site rate variation (HKYG4) and models 4–6 and 10–12 use the SRD06 codon partitioned substitution model. Models 1–6 also use a constant population coalescent model (‘constPop’), whereas models 7–12 use a Skygrid flexible effective population size prior model with 10 bins over 300 years (‘skygrid’). The model quoted in the main text is model 4, with the SRD06 substitution model, strict clock and constant population size.