Elise N. Paietta

Communities living in biodiversity hotspots are at increased risk of pathogen exposure and spread due to insufficient access to nutrition, clean water, and sanitation. Viruses, in particular, have high epidemic and spillover potential, and widespread health impacts, yet, viral diversity and dynamics remain understudied in many high-risk regions. In Madagascar, a hotspot with unparalleled biodiversity, socioeconomic constraints, and a heavy burden of infectious diseases, ongoing healthcare initiatives provide opportunities to survey circulating viral diversity. Here, we used a metagenomic workflow to characterize the viromes of oral/nasal swabs from febrile individuals visiting a mobile healthcare clinic in southeastern Madagascar, of which 49% were malaria-positive ( Plasmodium falciparum ). We characterized sequences of human-infecting viruses from twelve families (Adenoviridae, Anelloviridae, Orthoherpesviridae, Papillomaviridae, Polyomaviridae , Astroviridae, Coronaviridae, Matonaviridae, Orthomyxoviridae, Paramyxoviridae, Picornaviridae, and Sedoreoviridae ). These viral sequences generally showed high similarity to known lineages circulating globally, but multiple viruses considered rare or emerging were identified (e.g. enterovirus C105, enterovirus C117). While we did not detect a significant association between malaria status and pathogenic virus presence, younger individuals had higher odds of having a pathogenic virus. Nonpathogenic viruses in Anelloviridae were prevalent and diverse with richness ranging from 0 to 70. Individuals with P. falciparum or a pathogenic virus tended to share more anelloviruses. We also detected viruses that are protist-associated, plant-infecting, or with unknown hosts. Overall, our work documents extensive viral diversity in Madagascar, highlighting how metagenomic workflows can be used to build viral genomic resources for pathogen surveillance in regions with high infectious disease burden.

Given that some DNA viruses have been found to exhibit virus–host co-evolution and establish lifelong infection, mammals with unique evolutionary histories in island ecosystems likely host exceptionally diverse viruses. Madagascar is inhabited by endemic non-human primate and rodent lineages interacting with expansive populations of introduced non-native rodents across the island. Using a viral metagenomic workflow on 189 oral swabs of lemurs and rodents in southeastern Madagascar, we characterized genomic sequences of DNA viruses in the families Adenoviridae , Circoviridae , Orthoherpesviridae , Papillomaviridae , Parvoviridae and Polyomaviridae and assessed their phylogenetic relationships to known viruses. Endemic lemurs and tufted-tailed rats displayed particularly novel DNA viral diversity mirroring the geographic isolation and subsequently rich evolutionary history of their hosts. Notably, we provide the first coding-complete sequences in lemurs of herpesviruses, polyomaviruses, adeno-associated viruses and circoviruses. In contrast, the DNA viral communities of black rats in Madagascar were similar to those found in globally distributed black and brown rat populations, given their broad geographic spread and relatively recent introduction to the island. Given the scarcity of viral research in natural populations of lemurs and rodents in Madagascar despite the island’s exceptional biodiversity and escalating anthropogenic pressures, this study provides a genomic and phylogenetic foundation for DNA viruses infecting Malagasy lemurs and rodents.

Anelloviruses are circular, negative-sense single-stranded DNA viruses that have remarkable diversity and ubiquity across mammals. However, few studies have attempted to determine anellovirus diversity and dynamics across a biodiverse landscape. Madagascar offers a unique opportunity to study anellovirus diversity, with speciose, endemic mammalian lineages that have evolved in geographic isolation for millions of years. These endemic animals frequently interact with more recently introduced populations of non-native small mammals. From oral swab samples taken from natural populations of lemurs, rodents and shrews in the Manombo Special Reserve and surrounding area in southeastern Madagascar, we determined the complete genomes of anelloviruses from black rats ( n =647 genomes), Webb’s tufted-tailed rats ( n =2), mouse lemurs ( n =4), a woolly lemur ( n =1) and a house shrew ( n =3). We observed distinct anellovirus lineages in the endemic lemurs and tufted-tailed rats, which we infer to be the result of their long-term geographic isolation in Madagascar. Lemur-infecting anelloviruses, in particular, do not cluster with other primate-infecting anelloviruses. In contrast, anellovirus diversity in widespread, non-native rodents (i.e. black rats) was similar to that of closely related, globally dispersed rodent species, concordant with their later introduction to Madagascar. With our large anellovirus dataset from black rats, we examined anellovirus alpha- and beta-diversity and viral co-occurrence networks. Black rat anellovirus populations showed high intra-individual variation, but the overall pool of circulating anelloviruses was consistent across age classes and sexes. Adult relative to juvenile/subadult black rats harboured richer anellovirus populations and were more connected within the co-occurrence network. Proximity between individuals and greater intra-individual viral diversity were also linked to more virus-sharing between black rats.

Madagascar, one of the largest islands and located off Africa’s eastern coast, is recognized as one of the world’s top biodiversity hotspots, harboring incredible plant and animal endemism alongsid...

Few studies have addressed viral diversity in lemurs despite their unique evolutionary history on the island of Madagascar and high risk of extinction. Further, while a large number of studies on animal viromes focus on fecal samples, understanding viral diversity across multiple sample types and seasons can reveal complex viral community structures within and across species. Groups of captive lemurs at the Duke Lemur Center (Durham, NC, USA), a conservation and research center, provide an opportunity to build foundational knowledge on lemur-associated viromes. We sampled individuals from seven lemur species, i.e., collared lemur (Eulemur collaris), crowned lemur (Eulemur coronatus), blue-eyed black lemur (Eulemur flavifrons), ring-tailed lemur (Lemur catta), Coquerel’s sifaka (Propithecus coquereli), black-and-white ruffed lemur (Varecia variegata variegata), and red ruffed lemur (Varecia rubra), across two lemur families (Lemuridae, Indriidae). Fecal, blood, and saliva samples were collected from Coquerel’s sifaka and black-and-white ruffed lemur individuals across two sampling seasons to diversify virome biogeography and temporal sampling. Using viral metagenomic workflows, the complete genomes of anelloviruses (n = 4), cressdnaviruses (n = 47), caudoviruses (n = 15), inoviruses (n = 34), and microviruses (n = 537) were determined from lemur blood, feces, and saliva. Many virus genomes, especially bacteriophages, identified in this study were present across multiple lemur species. Overall, the work presented here uses a viral metagenomics approach to investigate viral communities inhabiting the blood, oral cavity, and feces of healthy captive lemurs.

The Papillomaviridae are a family of vertebrate-infecting viruses of oncogenic potential generally thought to be host species- and tissue-specific. Despite their phylogenetic relatedness to humans, there is a scarcity of data on papillomaviruses (PVs) in speciose non-human primate lineages, particularly the lemuriform primates. Varecia variegata (black-and-white ruffed lemurs) and Varecia rubra (red ruffed lemurs), two closely related species comprising the Varecia genus, are critically endangered with large global captive populations. Varecia variegata papillomavirus (VavPV) types −1 and −2, the first PVs in lemurs with a fully identified genome, were previously characterized from captive V. variegata saliva. To build upon this discovery, saliva samples were collected from captive V. rubra with the following aims: (1) to identify PVs shared between V. variegata and V. rubra and (2) to characterize novel PVs in V. rubra to better understand PV diversity in the lemuriform primates. Three complete PV genomes were determined from V. rubra samples. Two of these PV genomes share 98% L1 nucleotide identity with VavPV2, denoting interspecies infection of V. rubra by VavPV2. This work represents the first reported case of interspecies PV infection amongst the strepsirrhine primates. The third PV genome shares Papillomaviridae family, likely representing a novel genus. Future work diversifying sample collection (i.e., lemur host species from multiple genera, sample type, geographic location, and wild populations) is likely to uncover a world of diverse lemur PVs.

The diversity of viruses identified from the various niches of the human oral cavity—from saliva to dental plaques to the surface of the tongue—has accelerated in the age of metagenomics. This rapid expansion demonstrates that our understanding of oral viral diversity is incomplete, with only a few studies utilizing passive drool collection in conjunction with metagenomic sequencing methods. For this pilot study, we obtained 14 samples from healthy staff members working at the Duke Lemur Center (Durham, NC, USA) to determine the viral diversity that can be identified in passive drool samples from humans. The complete genomes of 3 anelloviruses, 9 cressdnaviruses, 4 Caudoviricetes large bacteriophages, 29 microviruses, and 19 inoviruses were identified in this study using high-throughput sequencing and viral metagenomic workflows. The results presented here expand our understanding of the vertebrate-infecting and microbe-infecting viral diversity of the human oral virome in North Carolina (USA).