The arrival of European colonists to the Americas, beginning in the 15^th century, contributed to the spread of new viruses amongst Indigenous people. This led to massive outbreaks of disease, and millions of deaths that caused an important Native population to collapse. The exact viruses that caused these outbreaks are unknown, but smallpox, measles, and mumps are all suspected.

During these times, traders and colonists forcibly enslaved and displaced millions of people mainly from the West Coast of Africa to the Americas. The cruel, unsanitary, and overcrowded conditions on ships transporting these people across the Atlantic contributed to the spread of infectious diseases onboard. Once on land, infectious diseases spread quickly, partly due to the poor conditions that enslaved and ndigenous people were made to endure. Native people were also immunologically naïve to the newly introduced pathogens, making them susceptible to severe or fatal outcomes. The new field of paleovirology may help scientists identify the viruses that were circulating in the first years of colonization and trace how viruses arrived in the Americas.

Using next-generation DNA sequencing and other cutting-edge techniques, Guzmán-Solís et al. extracted and enriched viral DNA from skeletal remains dating back to the 16^th century. These remains were found in mass graves that were used to bury epidemic victims at a colonial hospital and chapel in what is now Mexico City. The experiments identified two viruses, human parvovirus B19 and a human hepatitis B virus. These viral genomes were recovered from human remains of first-generation African people in Mexico, as well as an individual who was an Indigenous person.

Although the genetic material of these ancient viruses resembled pathogens that originated in Africa, the study did not determine if the victims died from these viruses or another cause. On the other hand, the results indicate that viruses frequently found in modern Africa were circulating in the Americas during the slave trade period of Mexico. Finally, the results provide evidence that colonists who forcibly brought African people to the Americas participated in the introduction of viruses to Mexico. This constant influx of viruses from the old world, led to dramatic declines in the populations of Indigenous people in the Americas.

European colonization in the Americas resulted in a frequent genetic exchange mainly between Native American populations, Europeans, and Africans (Aguirre-Beltrán, 2005; Rotimi et al., 2016; Salas et al., 2004). Along with human migrations, numerous new species were introduced to the Americas including bacterial and viral pathogens, which played a major role in the dramatic population collapse that afflicted the immunologically naïve Indigenous inhabitants (Acuña-Soto et al., 2004; Lindo et al., 2016). Among these pathogens, viral diseases, such as smallpox, measles, and mumps, have been proposed to be responsible for many of the devastating epidemics during the Colonial period (Acuña-Soto et al., 2004). Remarkably, the pathogen(s) responsible for the deadliest epidemics reported in New Spain (the Spanish viceroyalty that corresponds to Mexico, Central America, and the current US southwest states) remains unknown and is thought to have caused millions of deaths during the 16th century (Acuña-Soto et al., 2004). Indigenous populations were drastically affected by these mysterious epidemics, generically referred to as Cocoliztli (‘pest’ in Nahuatl), followed by Africans and to a lesser extent European people (Acuña-Soto et al., 2004; Malvido and Viesca, 1982; Somolinos d’Árdois, 1982). Accounts of the 1576 Cocoliztli epidemic were described in autopsy reports of victims treated at the ‘Hospital Real de San José de los Naturales’ (HSJN) (Malvido and Viesca, 1982; Wesp, 2017), the first hospital in Mexico dedicated specifically to treat the Indigenous population (Malvido and Viesca, 1982; Wesp, 2017; Figure 1a and b).

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The study of ancient viral genomes has revealed important insights into the evolution of specific viral families (Barquera et al., 2020; Duggan et al., 2016; Düx et al., 2020; Kahila Bar-Gal et al., 2012; Krause-Kyora et al., 2018; Mühlemann et al., 2018a; Mühlemann et al., 2018a; Mühlemann et al., 2018b; Neukamm et al., 2020; Pajer et al., 2017; Patterson Ross et al., 2018; Xiao et al., 2013), as well as their interaction with human populations (Spyrou et al., 2019). To explore the presence of viral pathogens in circulation during epidemic periods in New Spain, we leveraged the vast historical and archeological information available for the Colonial HSJN. These include the skeletal remains of over 600 individuals recovered from mass burials associated with the hospital’s architectural remnants (Figure 1b). Many of these remains were retrieved from burial contexts suggestive of an urgent and simultaneous disposal of the bodies, as in the case of an epidemic (Meza, 2013; Wesp, 2017). Prior bioarcheological research has shown that the remains of a number of individuals in the HSJN collection displayed dental modifications and/or morphological indicators typical of African ancestry (Meza, 2013), consistent with historical and archeological research that documents the presence of a large number of both free and enslaved Africans and their descendants in Colonial Mexico (Aguirre-Beltrán, 2005). Indeed, a recent paleogenomics study reported a sub-Saharan African origin of three individuals from this collection (Barquera et al., 2020).

Here we describe the recovery and characterization of viral pathogens that circulated in New Spain during the Colonial period, using ancient DNA (aDNA) techniques (Figure 1—figure supplement 1). For this work, we sampled skeletal human remains recovered from the HSJN where archeological context suggest victims of epidemics were buried (Meza, 2013) and from ‘La Concepcion’ chapel, one of the first catholic conversion centers in New Spain (Moreno-Cabrera et al., 2015; Figure 1a). We report the reconstruction of ancient hepatitis B virus (HBV) and human parvovirus B19 (B19V) genomes recovered from these remains, providing a direct molecular evidence of human viral pathogens of African origin being introduced to New Spain during the transatlantic slave trade.

We sampled the skeletal remains from two archeological sites, a Colonial Hospital and a Colonial chapel in Mexico City (Figure 1a and b). For the HSJN, 21 dental samples (premolar and molar teeth) were selected based on previous morphometric analyses and dental modifications that suggested an African ancestry (Hernández-Lopez and Negrete, 2012; Karam-Tapia, 2012; Meza, 2013; Ruíz-Albarrán, 2012). The African presence in the Indigenous Hospital might reflect an urgent response to an epidemic outbreak since hospitals treated patients regardless of the origin of the affected individuals during serious public health crises (Meza, 2013). Dental samples of five additional individuals were selected (based on their conservation state) from ‘La Concepción’ chapel (COY), which is located 10 km south of the HSJN in Coyoacán, a Pre-Hispanic Indigenous neighborhood that became the first Spanish settlement in Mexico City after the fall of Tenochtitlan (Moreno-Cabrera et al., 2015). Following strict aDNA protocols, we processed these dental samples to isolate aDNA for next-generation sequencing (NGS) (Figure 1—figure supplement 1, Materials and methods). Tooth roots (which are vascularized) can be a good source of pathogen DNA (Key et al., 2017), especially in the case of viruses that are widespread in the bloodstream during systemic infection. Accordingly, a number of previous studies have successfully recovered ancient viral DNA from tooth roots (Barquera et al., 2020; Krause-Kyora et al., 2018; Mühlemann et al., 2020; Mühlemann et al., 2018a; Mühlemann et al., 2018b).

Metagenomic analyses with MALT (Herbig et al., 2018) (Materials and methods) on the NGS data using the Viral NCBI RefSeq database as a reference (Pruitt et al., 2007) revealed 17 samples containing at least one normalized hit to viral DNA (abundances were normalized to the smallest library since each sample had different number of reads) (Materials and methods), particularly similar to Hepadnaviridae, Herpesviridae, Parvoviridae, and Poxviridae viral families (Figure 1c, Figure 1—figure supplement 2, Materials and methods). These viral hits revealed the potential to recover ancient viral genomes from these samples. We selected 12 samples for further screening (Figure 1c, Figure 1—figure supplement 3) based on the DNA concentration of the NGS library and the quality of the hits to a clinically important virus (HBV, B19V, papillomavirus, smallpox).

To isolate and enrich the viral DNA fraction in the sequencing libraries, biotinylated single-stranded RNA probes designed to capture sequences from diverse human viral pathogens were synthesized (Supplementary file 1A). The selection of the viruses included in the capture design considered the following criteria: (1) DNA viruses previously retrieved from archeological human remains (i.e., hepatitis B virus, human parvovirus B19, variola virus), (2) representative viruses from families capable of integrating into the human genome (i.e., Herpesviridae, Papillomaviridae, Polyomaviridae, Circoviridae), or (3) RNA viruses with a DNA intermediate (i.e., Retroviridae). Given the size constraints of the probe kit, only a couple of genes were selected from some viral families (Materials and methods, Supplementary file 1A). Additionally, a virus-negative aDNA library, which showed no hits to any viral family included in the capture assay (except for a frequent Poxviridae-like region identified as an Alu repeat; Tithi et al., 2018) was captured and sequenced as a negative control (HSJN177) to estimate the efficiency of our capture assay. Only one post-capture library had an ~100-fold increase of Hepadnaviridae-like hits (HBV), while three more libraries had an ~50–200-fold increase of Parvoviridae-like hits (B19V) (Figure 1c, Supplementary file 1B), compared to their corresponding pre-capture libraries (Materials and methods). In contrast, the captured negative control (HSJN177) presented a negligible enrichment of these viral hits (Figure 1c, Supplementary file 1B).

Independently, a metagenomic analysis using Kraken2 (Wood et al., 2019) and Pavian (Breitwieser and Salzberg, 2020) was performed on the non-human (unmapped) reads as part of a different study (Bravo-Lopez et al., 2020). Our samples presented bacterial constituents of human oral and soil microbiota at different proportions between the samples (Figure 1—figure supplements 4–7). Although no lethal bacterial pathogen was retrieved, some ancient dental pathogens (Tannerella forsythia) were reconstructed and described in more detail by Bravo-Lopez et al., 2020 (Figure 1—figure supplements 4–7).

We verified the authenticity of the reads mapped to HBV (BWA) or B19V (BWA/blastn) in the enriched libraries (Materials and methods) by querying the reads against the non-redundant (nr) NCBI database using megaBLAST (Altschul et al., 1990). This step was performed to avoid including in the genome assembly reads that were mapped by BWA or blastn as HBV or B19V, but with a similar identity to a different taxon in the nr database (and absent in DS1; Materials and methods). Therefore, we only retained reads for which the top hit was to either B19V or HBV (Supplementary file 1C). To confirm the ancient origin of these viral reads, we evaluated the misincorporation damage patterns using the program mapDamage 2.0 (Jónsson et al., 2013), which revealed an accumulation of C to T mutations towards their 5′ terminal site with an almost symmetrical G to A pattern on the 3′ end (Figure 2a, Figure 2—figure supplement 1), as expected for aDNA (Briggs et al., 2007). Three ancient B19V genomes were reconstructed (Figure 2b, Supplementary file 1C) with sequence coverages between 92.37% and 99.1%, and average depths of 2.98–15.36× along their single-stranded DNA (ssDNA) coding region, which excludes the double-stranded DNA (dsDNA) hairpin regions at each end of the genome (Luo and Qiu, 2015). These dsDNA inverse terminal repeats (ITRs) displayed considerably higher depth values (<218×) compared to the coding region consistent with the better postmortem preservation of dsDNA compared to ssDNA (Lindahl, 1993; Figure 2b). In addition, we reconstructed one ancient HBV genome (Figure 2c, Supplementary file 1C) at 30.8× average depth and with a sequence coverage of 89.9%, including its ssDNA region at a reduced depth (<10×).

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The reconstructed ancient HBV genome shows a 6 nucleotide (nt) insertion in the core gene, which is characteristic of the genotype A (Kramvis, 2014). Further phylogenetic analyses (Materials and methods) revealed that the Colonial HBV genome clustered with modern sequences corresponding to sub-genotype A4 (previously named A6) (Pourkarim et al., 2014; Figure 3a, Figure 3—figure supplement 1). The genotype A (HBV/GtA) has a broad diversity in Africa reflecting its long history in this continent (Kostaki et al., 2018; Kramvis, 2014), while the sub-genotype A4 has been recovered uniquely from African individuals in Belgium (Pourkarim et al., 2010) and has never been found in the Americas. Regarding the three Colonial B19V genomes from individuals HSJN240, COYC4, and HSJNC81 (C81A), these were phylogenetically closer to modern B19V sequences belonging to genotype 3 (Figure 3b, Figure 3—figure supplements 2–3). This B19V genotype is divided into two sub-genotypes: 3a, which is mostly found in Africa, and 3b, which is proposed to have spread outside Africa recently (Hübschen et al., 2009). The viral sequences from the individuals HSJN240 and COYC4 are similar to sub-genotype 3b genomes sampled from immigrants (Morocco, Egypt, and Turkey) in Germany (Schneider et al., 2008; Figure 3b, Figure 3—figure supplements 2–3) while the sequence of the individual HSJNC81 is more similar to a divergent sub-genotype 3a strain (Figure 3b, Figure 3—figure supplements 2–3) retrieved from a child with severe anemia born in France (Nguyen et al., 1999). These observations support the African origin of the reconstructed Colonial viral genomes.

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In order to use our reconstructed viral genomes as molecular fossils to recalibrate each virus phylogeny and perform evolutionary inferences, we first needed to estimate if the phylogenetic relationships among B19V or HBV genomes had a temporal structure (i.e., sufficient genetic change between sampling times to reconstruct a statistical relationship between genetic divergence and time) (Rambaut et al., 2016). In the context of viruses, temporal structure is canonically tested with a root-to-tip distance and date randomization analyses (see Firth et al., 2010; Rieux and Balloux, 2016). Similarly to previous studies (Krause-Kyora et al., 2018; Patterson Ross et al., 2018), we found little or no temporal structure for this HBV phylogeny containing all genotypes (R² = 0.1351; correlation coefficient = 0.3676) (Figure 3—figure supplement 5a-c). The complex evolution of HBV may not be prone to an appropriate genetic dating since multiple inter-genotype recombination and cross-species transmission (Human-Ape) events (Krause-Kyora et al., 2018) occurred throughout its evolution. Since the entire genotype A has been identified as a recombinant genotype before (Mühlemann et al., 2018a), we analyzed it independently and identified a stronger temporal signal within this genotype (R² = 0.722; correlation coefficient = 0.8498) (Figure 3—figure supplement 5d-f). In the case of B19V, we identified a temporal structure when including all three genotypes (R² = 0.3837; correlation coefficient = 0.6194) (Figure 3—figure supplement 6a-c), in agreement with previous studies (Mühlemann et al., 2018b). Furthermore, we corroborated this temporal structure was not an artifact by a set of tip-dated randomized analyses (Rieux and Balloux, 2016), where none of the 95% highest posterior density (HPD) intervals of the clock rate overlapped with the correctly dated dataset (Figure 3—figure supplement 7).

Given its strong temporal structure, we then performed a dated coalescent phylogenetic analysis for B19V (Supplementary file 1D). We inferred a median substitution rate for B19V of 1.03 × 10^–5 (95% HPD: 8.66 × 10^–6–1.21 × 10^–5) s/s/y under a strict clock and a constant population prior, and a substitution rate of 2.62 × 10^–5 (95% HPD: 1.50 × 10^–5–3.98 × 10^–5) s/s/y under a relaxed log normal clock and a constant population prior. The divergence times from the most recent common ancestor of genotypes 1, 2, and 3 under a strict clock were 7.19 (95% HPD: 6.98–7.46), 2.11 (95% HPD: 1.83–2.51), and 3.64 (95% HPD: 3.04–4.33) ka, respectively. The inferred substitution rates and divergence times from the most recent common ancestor for genotypes 1 and 2 were similar to previous estimations (Mühlemann et al., 2018b) that included much older sequences, while the divergence of genotype 3 was subtly older since no other ancient genotype 3 had been reported previously.

Next, we used the shotgun data generated to determine the mitochondrial haplogroup of the hosts, as well as their autosomal genetic ancestry using the 1000 Genomes Project (1000 Genomes Project Consortium, 2015) as a reference panel (Figure 4a, Supplementary file 2A). The nuclear genetic ancestry analysis showed that all three HSJN individuals, from which the reconstructed viral genomes were isolated, fall within African genetic variation in a principal component analysis (PCA) plot (Figure 4a), while their mitochondrial aDNA belong to the L haplogroup, which has high frequency in African populations (Supplementary file 2A, Figure 2—figure supplement 2). Additionally, we performed ⁸⁷Sr/⁸⁶Sr isotopic analysis on two of the HSJN individuals using tooth enamel as well as phalanx (HSJN240) or parietal bone (HSJNC81) to provide insights on the places of birth (adult enamel) and where the last years of life were spent (phalanx/parietal). The ⁸⁷Sr/⁸⁶Sr ratios measured on the enamel of the individual HSJNC81 (0.71098) and HSJN240 (0.71109) are similar to average ⁸⁷Sr/⁸⁶Sr ratios found in soils and rocks from West Africa (average of 0.71044, Figure 4—figure supplement 1, Supplementary file 2), as well as to ⁸⁷Sr/⁸⁶Sr ratios described in first-generation Africans in the Americas (Barquera et al., 2020; Bastos et al., 2016; Fricke et al., 2020; Price et al., 2012; Schroeder et al., 2009). In contrast, the ⁸⁷Sr/⁸⁶Sr ratios on the parietal and phalange bones from the HSJNC81 (0.70672) and HSJN240 (0.70755) show lower values similar to those observed in the Trans Mexican Volcanic Belt where the Mexico City Valley is located (0.70420–0.70550, Figure 4—figure supplement 1, Supplementary file 2). Moreover, radiocarbon dating of HSJN240 (1442–1608 CE, years calibrated for 1σ) and HSJN194 (1472–1625 CE, years calibrated for 1σ) (Supplementary file 2A, Figure 4—figure supplement 2) indicates that these individuals arrived during the first decades of the Colonial period, when the number of enslaved individuals arriving from Africa was particularly high (Aguirre-Beltrán, 2005).

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Strikingly, Colonial individual COYC4, who was also infected with an African B19V strain, clusters with present-day Mexicans and Peruvians from the 1000 Genomes Project (Figure 4a). An ADMIXTURE (Alexander and Lange, 2011) analysis with these data confirmed a Native American genetic component (Figure 4b), as expected for an indigenous individual. The B19V ancient genome from the individual COYC4 is the first genotype 3 genome obtained from a non-African individual and suggests that following the introduction from Africa, the virus (B19V) spread and infected people of different ancestries during the Colonial period.

In this study, we reconstructed one HBV and three B19V ancient genomes from four different individuals using NGS, metagenomics, and in-solution targeted enrichment methods (Figure 2b,c, Figure 1—figure supplement 1). Several lines of evidence support the ancient nature of these viral sequences, in contrast to environmental contamination or a capture artifact. First, our negative control was not enriched for B19V or HBV hits in our capture sequencing (Figure 1c). For those samples that showed an enrichment in viral sequences after capture, the reads covered the reference genomes almost in their entirety and displayed deamination patterns at the terminal ends of the reads, as expected for aDNA (Figure 2a). Moreover, it is important to notice that B19V and HBV are blood-borne human pathogens that are not present in soil or the environment, and that DNA from these viruses had never been extracted before in the aDNA facilities used for this study.

The recovery of aDNA from B19V, which has a ssDNA genome (with dsDNA terminal repeats), in previous studies (Mühlemann et al., 2018b) as well as in our samples is noteworthy considering the NGS libraries were constructed using dsDNA as a template. Therefore, we would not expect to recover the ssDNA from B19V with this library building method. However, it is known that dsDNA intermediates form during the B19V replication cycle (Ganaie and Qiu, 2018), and that throughout the viral infection the replicating genomes are present in both the ssDNA and dsDNA forms. The sequences we retrieved must therefore correspond to the cell-free dsDNA replication intermediates. This is coherent with the peculiar coverage pattern on the B19V genome, where the dsDNA hairpins at its terminal sites and are highly covered, reflecting a better stability of these regions over time (Figure 2b). Similarly, the partially circular dsDNA genome from HBV was poorly covered at the ssDNA region (Figure 2c), which also goes through a dsDNA phase during replication, a similar coverage is reported in three previous ancient HBV genomes (Krause-Kyora et al., 2018). Although HBV and B19V are also capable of integrating into the human host genome (Yuen et al., 2018; Janovitz et al., 2017), the uneven read coverage observed for all reconstructed viruses (higher coverage in dsDNA regions) suggests that these sequences do not correspond to integration events. If the B19V or HBV reads we recovered derived from integrated sequences in the human genome, we would expect an even coverage along the reference viral genome, which is not the case. Further analyses would be needed to determine if the aDNA retrieved in this and other studies comes from systemic circulating virions or from systemic cell-free DNA intermediates (Cheng et al., 2019) produced after viral replication in the bone marrow or liver for B19V and HBV, respectively (Broliden et al., 2006; Yuen et al., 2018).

The ancient B19V genomes were assigned to genotype 3. This genotype is most prevalent in West Africa (Ghana: 100%, n = 11; Burkina Faso: 100%, n = 5) (Candotti et al., 2004; Hübschen et al., 2009; Rinckel et al., 2009) and a potential African origin has been suggested (Candotti et al., 2004). It has also been sporadically found outside of Africa (Jain et al., 2015)^,(Candotti et al., 2004; Rinckel et al., 2009) in Brazil (50%, n = 12) (Freitas et al., 2008; Sanabani et al., 2006), India (15.4%, n = 13) (Jain et al., 2015), France (11.4%, n = 79) (Nguyen et al., 1999; Servant et al., 2002), and the USA (0.85%, n = 117) (Rinckel et al., 2009) as well as in immigrants from Morocco, Egypt, and Turkey in Germany (6.7%, n = 59) (Schneider et al., 2008). Two other genotypes, 1 and 2, exist for this virus. Genotype 1 is the most common and is found worldwide, while the almost extinct genotype 2 is mainly found in elderly people from Northern Europe (Pyöriä et al., 2017). Ancient genomes from genotypes 1 and 2 have been recovered from Eurasian samples, including a genotype 2 B19V genome from a 10th-century Viking burial in Greenland (Mühlemann et al., 2018b). ⁸⁷Sr/⁸⁶Sr isotopes on individuals from this burial revealed that they were immigrants from Iceland (Mühlemann et al., 2018b), suggesting an introduction of the genotype 2 to North America during Viking explorations of Greenland.

While serological evidence indicates that B19V currently circulates in Mexico, only the presence of genotype 1 has been formally described using molecular analyses (Valencia Pacheco et al., 2017). Taken together, our results are consistent with an introduction of the genotype 3 to New Spain as a consequence of the transatlantic slave trade imposed by European colonization. This hypothesis is supported by the ⁸⁷Sr/⁸⁶Sr isotopic analysis, which suggests that the individuals from the HSJN with B19V (HSJN240, HSJNC81) were born in West Africa and spent their last years of life in New Spain (Figure 4—figure supplement 1). Furthermore, the radiocarbon analysis for individuals HSJN240 and HSJN194 (Figure 4—figure supplement 2) support this notion as they correspond to the Early Colonial period, during which the number of enslaved Africans arriving was higher compared to later periods (Aguirre-Beltrán, 2005). Remarkably, a B19V genome belonging to the genotype 3 was recovered from an individual (COYC4) with 100% Indigenous ancestry (Figure 4b). COY4 was excavated in an independent archeological site 10 km south of the HSJN (Figure 1a), supporting the notion that viral transmissions between African individuals and Native Americans occurred during the Colonial period in Mexico City.

The HBV genotype A is highly diverse in Africa, reflecting its long evolutionary history, and likely originated somewhere between Africa, the Middle East, and Central Asia (Kostaki et al., 2018). The introduction of the genotype A from Africa to the Americas has been proposed based on phylogenetic analysis of modern strains from Brazil (Freitas et al., 2008; Kostaki et al., 2018) and Mexico (Roman et al., 2010), and more precisely of the sub-genotype A1 using sequences from Martinique (Brichler et al., 2013), Venezuela (Quintero et al., 2002), Haiti (Andernach et al., 2009), and Colombia (Alvarado-Mora et al., 2012). Recently, a similar introduction pattern was proposed for the quasi genotype A3 based on an ancient HBV genome recovered from an ancient African individual sampled in Mexico (Barquera et al., 2020). The origin of the sub-genotype A4 is controversial since the apparent African origin is based on modern sequences recovered from African immigrants living in Europe (Pourkarim et al., 2010). The Colonial ancient HBV genome reconstructed in our work represents the first ancient A4 linked to the transatlantic slave trade (Figure 3a, Figure 3—figure supplement 1), and the only report of this sub-genotype in the Americas, further supporting its African origin. The introduction of pathogens from Africa to the Americas has been proposed for other human-infecting viruses such as smallpox (Mandujano-Sánchez et al., 1982; Somolinos d’Árdois, 1982), based on historical records; or yellow fever virus (Bryant et al., 2007), HTLMV-1 (Gadelha et al., 2014), hepatitis C virus (genotype 2) (Markov et al., 2009), and human herpes simplex virus (Forni et al., 2020) based on phylogenetic analysis of modern strains from Afro-descendant or admixed human populations.

Although we cannot assert where exactly the African-born individuals in this study contracted B19V or HBV (Africa, America, or the Middle Passage) nor if the cause of their deaths can be attributed to such infections, the identification of ancient B19V and HBV in contexts associated with Colonial epidemics in Mexico City is still relevant in light of their paleopathological marks and the clinical information available for the closest sequences in the phylogenetic analyses. Notably, individual HSJNC81 displayed cribra orbitalia in the eye sockets and porotic hyperostosis on the cranial vault (Figure 4—figure supplement 3). The reconstructed ancient B19V genome from this individual is closest to the V9 strain, which was isolated from an infant with severe anemia and G6PD deficiency (Nguyen et al., 1999; GenBank: AJ249437; Figure 3b). The HSJN skeletal collection has a notably higher rate of cribra orbitalia and porotic hyperostosis compared to other Colonial archeological sites, marks that were proposed to be caused by an unknown infectious disease (Castillo-Chavez, 2000). These skeletal indicators are caused by irregular hematopoiesis in the bone marrow and are typically associated with genetic anemias such as thalassemia and sickle cell anemia (Angel, 1966), as well as to nutritional stress or parasitic infections (Walker et al., 2009). It is acknowledged that B19V infection can cause severe or even fatal anemia due to the low level of hemoglobin in individuals with other blood disorders, such as thalassemia, sickle cell anemia, malaria, or iron deficiency (Broliden et al., 2006; Heegaard and Brown, 2002). Therefore, since B19V infects precursors of the erythroid lineage (Broliden et al., 2006), it is possible that the morphological changes found in HSJNC81 might be the result of a severe anemia caused or enhanced by a B19V infection. With our data we cannot discard the simultaneous presence of a genetic disease since the loci for thalassemia, sickle cell anemia, and G6PD deficiency were not covered with our human-mapped NGS data. Nevertheless, the identification of ancient B19V in a Colonial context is noteworthy considering several recent reports reveal that measles-like cases were actually attributable to B19V (De Los Ángeles Ribas et al., 2019; Rezaei et al., 2016) or rubella (Anderson et al., 1985; Davidkin et al., 1998; De Los Ángeles Ribas et al., 2019; Rezaei et al., 2016), which produce a similar kind of rash and fever. Therefore, it is possible that B19V might have been responsible for some of the numerous cases attributed to measles that were described in early 16th-century Mexico (Acuña-Soto et al., 2004; Mandujano-Sánchez et al., 1982; Wesp, 2017), in particular historical records that document the treatment of an outbreak of measles at the HSJN in 1531 CE (Meza, 2013). Our study, however, does not reject the notorious role that measles played during the Colonial outbreaks (as it is strongly supported by historical records), but provides evidence of the presence of B19V during the Colonial period in Mexico City to facilitate discussions about the paradigmatic etiology of the supposed measles epidemics reported in historical records (Malvido and Viesca, 1982; Mandujano-Sánchez et al., 1982; Somolinos d’Árdois, 1982). This hypothesis requires additional comprehensive studies aimed at characterizing the presence of measles and rubella viruses from ancient remains, a task that currently poses difficult technical challenges given that RNA is known to degrade rapidly. In fact, most ancient viral RNA genomes have been recovered only from formalin-fixed tissue (Düx et al., 2020; Xiao et al., 2013).

Furthermore, historical records of the autopsies of the victims of the 1576 CE Cocoliztli epidemic treated at the HSJN describe the presence of enlarged hard liver and jaundice (Acuña-Soto et al., 2002; Acuña-Soto et al., 2004; Malvido and Viesca, 1982; Marr and Kiracofe, 2000; Somolinos d’Árdois, 1982) as well as a black spleen and lungs and heart with yellow liquid and black blood (Acuña-Soto et al., 2000; Malvido and Viesca, 1982; Somolinos d’Árdois, 1982). This is noteworthy given that both HBV and B19V viruses proliferate in the liver and are associated with hepatitis and jaundice (Broliden et al., 2006; Yuen et al., 2018). The radiocarbon dating of individuals HSJN194 (HBV) and HSJN240 (B19V) suggests that these individuals died between 1472–1625 CE and 1442–1608 CE (years calibrated for 1σ), respectively (Figure 4—figure supplement 2), which overlaps with the period of time when the hepatitis symptoms were reported in the autopsies after the 1576 Cocoliztli epidemic at the HSJN (Acuña-Soto et al., 2004; Marr and Kiracofe, 2000; Somolinos d’Árdois, 1982). However, additional analyses are needed before being able to establish a link between these viruses and the wide array of symptoms described for Cocoliztli. Currently, technological limitations prevent the direct identification of ancient RNA viruses in bone or dental remains. However, future studies, with larger sample sizes from different contexts associated with the outbreak, should explore a wider range of pathogens previously suggested as potential causative agents, like arthropod-borne pathogens (malaria, yellow fever virus, and dengue virus) (Marr and Kiracofe, 2000) or hemorrhagic fever RNA viruses (Acuña-Soto et al., 2004).

Furthermore, it is important to acknowledge that both viruses have also been previously identified in aDNA datasets not necessarily associated with disease or epidemic contexts (Kahila Bar-Gal et al., 2012; Krause-Kyora et al., 2018; Mühlemann et al., 2018a; Patterson Ross et al., 2018). Additionally, our data is not sufficient to elucidate the age when the individuals acquired the viruses or if it is related to their cause of death.

In the case of HSJN194, we cannot establish if he acquired HBV vertically or horizontally, nor if this individual presented an acute or chronic infection. Finally, although our data does not allow us to associate these viruses to a specific epidemic outbreak, the identification of HBV and B19V in Post-Contact remains opens up new opportunities for investigating their presence in similar contexts and expand our knowledge on their evolution and potential link to disease in Colonial Mexico. This type of research is particularly relevant when considering previous hypotheses favoring the synergistic action of different types of pathogens in these devastating Colonial epidemics (Somolinos d’Árdois, 1982).

It is important to emphasize that our findings should be interpreted with careful consideration of the historical and social context of the transatlantic slave trade. This cruel episode in history involved the forced displacement of millions of individuals to the Americas (ca. 250,000 to New Spain; Aguirre-Beltrán, 2005) under inhumane, unsanitary, and overcrowded conditions that, with no doubt, favored the spread of infectious diseases (Mandujano-Sánchez et al., 1982). Therefore, the introduction of these and other pathogens from Africa to the Americas should be attributed to the brutal and harsh conditions of the Middle passage that enslaved Africans were subjected to by traders and colonizers, and not to the African peoples themselves. Moreover, the adverse life conditions for enslaved Africans and Native Americans, especially during the first decades after colonization, surely favored the spread of diseases and emergence of epidemics (Mandujano-Sánchez et al., 1982). Integrative and multidisciplinary approaches are thus needed to understand this phenomenon in full.

In summary, our study provides direct aDNA evidence of HBV and B19V introduced to the Americas from Africa during the transatlantic slave trade. The isolation and characterization of these ancient HBV and B19V genomes represent an important contribution to the ancient viral genomes reported in the Americas (Barquera et al., 2020; Duggan et al., 2020; Schrick et al., 2017). Our results expand our knowledge on the viral agents that were in circulation during Colonial epidemics like Cocoliztli, some of which resulted in the catastrophic collapse of the immunologically naïve Indigenous population. Although we cannot assign a direct causality link between HBV and B19V and Cocoliztli, our findings confirm that these potentially harmful viruses were indeed circulating in individuals found in archeological contexts associated with this epidemic outbreak. Further analyses from different sites and samples will help understand the possible role of these and other pathogens in Colonial epidemics, as well as the full spectrum of pathogens that were introduced to the Americas during European colonization.

Reconstructed genomes from this study are available in Genbank under accession number MT108214, MT108215, MT108216, MT108217. NGS reads used to reconstruct ancient viral genomes reported in this study are available in Dryad (https://doi.org/10.5061/dryad.5x69p8d2s).

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Author details

1. Axel A Guzmán-Solís

   Laboratorio Internacional de Investigación sobre el Genoma Humano, Universidad Nacional Autónoma de México, Querétaro, Mexico

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   None

   "This ORCID iD identifies the author of this article:" 0000-0001-6878-206X

2. Viridiana Villa-Islas

   Laboratorio Internacional de Investigación sobre el Genoma Humano, Universidad Nacional Autónoma de México, Querétaro, Mexico

   Contribution

   Data curation, Formal analysis, Methodology, Visualization, Writing – review and editing

   Competing interests

   None

3. Miriam J Bravo-López

   Laboratorio Internacional de Investigación sobre el Genoma Humano, Universidad Nacional Autónoma de México, Querétaro, Mexico

   Contribution

   Data curation, Methodology, Visualization, Writing – review and editing

   Competing interests

   none

4. Marcela Sandoval-Velasco

   Section for Evolutionary Genomics, The Globe Institute, Faculty of Health, University of Copenhagen, Copenhagen, Denmark

   Contribution

   Conceptualization, Funding acquisition, Resources, Writing – review and editing

   Competing interests

   None

5. Julie K Wesp

   Department of Sociology and Anthropology, North Carolina State University, Raleigh, United States

   Contribution

   Conceptualization, Funding acquisition, Investigation, Resources, Supervision, Writing – review and editing

   Competing interests

   None

6. Jorge A Gómez-Valdés

   Escuela Nacional de Antropología e Historia, Mexico City, Mexico

   Contribution

   Resources, Supervision

   Competing interests

   None

   "This ORCID iD identifies the author of this article:" 0000-0001-6996-2732

7. María de la Luz Moreno-Cabrera

   Instituto Nacional de Antropología e Historia, Mexico City, Mexico

   Contribution

   Resources

   Competing interests

   None

8. Alejandro Meraz

   Instituto Nacional de Antropología e Historia, Mexico City, Mexico

   Contribution

   Resources

   Competing interests

   None

9. Gabriela Solís-Pichardo

   Laboratorio Universitario de Geoquímica Isotópica (LUGIS), Instituto de Geología, Universidad Nacional Autónoma de México, Mexico City, Mexico

   Contribution

   Formal analysis, Methodology, Writing – review and editing

   Competing interests

   None

10. Peter Schaaf

    LUGIS, Instituto de Geofísica, Universidad Nacional Autónoma de México, Mexico City, Mexico

    Contribution

    Formal analysis, Methodology, Writing – review and editing

    Competing interests

    None

11. Benjamin R TenOever

    Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, United States

    Contribution

    Resources, Writing – review and editing

    Competing interests

    None

    "This ORCID iD identifies the author of this article:" 0000-0003-0324-3078

12. Daniel Blanco-Melo

    1. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, United States
    2. Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, United States

    Contribution

    Conceptualization, Data curation, Funding acquisition, Supervision, Visualization, Writing – original draft, Writing – review and editing

    For correspondence

    dblancom@fredhutch.org

    Competing interests

    None

    "This ORCID iD identifies the author of this article:" 0000-0002-0256-5019

13. María C Ávila Arcos

    Laboratorio Internacional de Investigación sobre el Genoma Humano, Universidad Nacional Autónoma de México, Querétaro, Mexico

    Contribution

    Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing

    For correspondence

    mavila@liigh.unam.mx

    Competing interests

    None

    "This ORCID iD identifies the author of this article:" 0000-0003-1691-1696

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