After the European colonization of the Americas there was a dramatic population collapse of the Indigenous inhabitants caused in part by the introduction of new pathogens. Although there is much speculation on the etiology of the Colonial epidemics, direct evidence for the presence of specific viruses during the Colonial era is lacking. To uncover the diversity of viral pathogens during this period, we designed an enrichment assay targeting ancient DNA (aDNA) from viruses of clinical importance and applied it on DNA extracts from individuals found in a Colonial hospital and a Colonial chapel (16th c. – 18th c.) where records suggest victims of epidemics were buried during important outbreaks in Mexico City. This allowed us to reconstruct three ancient human parvovirus B19 genomes, and one ancient human hepatitis B virus genome from distinct individuals. The viral genomes are similar to African strains, consistent with the inferred morphological and genetic African ancestry of the hosts as well as with the isotopic analysis of the human remains, suggesting an origin on the African continent. This study provides direct molecular evidence of ancient viruses being transported to the Americas during the transatlantic slave trade and their subsequent introduction to New Spain. Altogether, our observations enrich the discussion about the etiology of infectious diseases during the Colonial period in Mexico.

Dental samples (premolars and molars) from archeological remains in Mexico were superficially cleaned with NaClO (70%) and ethanol (70%) and later exposed to UV light for 1.5 minutes. The teeth were sectioned from the crown and fragmented by mechanical pressure. aDNA was extracted from 200 mg of teeth powder by EDTA and proteinase K treatment followed by DNA purification by binding to silica in the presence of high concentrations of guanidinium thiocyanate (https://www.nature.com/articles/nprot.2007.247). NGS libraries were constructed following the protocol in Meyer and Kircher 2010 (https://doi.org/10.1101/pdb.prot5448). Capture-enrichment was performed on the indexed libraries using custom designed biotinylated probes to pull-down viral aDNA (hybridization at 60ºC during 48h).  Enriched libraries were amplified by PCR (18-20 cycles), pooled and deep sequenced on an Illumina NextSeq550 (2x75 mid-output). Resulting sequencing reads were merged (>11bp overlap) and trimmed with AdapterRemoval 1.5.4. Overlapping reads (>30 bp in length, quality filter >30) were kept and mapped to the human genome (hg19) using BWA 0.7.13. Unmapped reads were mapped to HBV or B19V publicly available genomes using BWA. Genome sequences with the most hits (for HBV) or that were covered > 50% (B19V) were selected as reference sequence. Unmapped reads were aligned to the correspondent reference sequence using BWA. The alignment was rescaled for damaged sites using mapDamage 2.0 and a consensus genome was produced using SAMtools 1.9.

FASTQ files contain the reads mapped to HBV (Ref: GQ331046) or B19V (Ref: AB550331) and used to construct ancestral consensus genomes.