A strategy to estimate unknown viral diversity in mammals

Abstract

The majority of emerging zoonoses originate in wildlife, and many are caused by viruses. However, there are no rigorous estimates of total viral diversity (here termed "virodiversity") for any wildlife species, despite the utility of this to future surveillance and control of emerging zoonoses. In this case study, we repeatedly sampled a mammalian wildlife host known to harbor emerging zoonotic pathogens (the Indian Flying Fox, Pteropus giganteus) and used PCR with degenerate viral family-level primers to discover and analyze the occurrence patterns of 55 viruses from nine viral families. We then adapted statistical techniques used to estimate biodiversity in vertebrates and plants and estimated the total viral richness of these nine families in P. giganteus to be 58 viruses. Our analyses demonstrate proof-of-concept of a strategy for estimating viral richness and provide the first statistically supported estimate of the number of undiscovered viruses in a mammalian host. We used a simple extrapolation to estimate that there are a minimum of 320,000 mammalian viruses awaiting discovery within these nine families, assuming all species harbor a similar number of viruses, with minimal turnover between host species. We estimate the cost of discovering these viruses to be ~$6.3 billion (or ~$1.4 billion for 85% of the total diversity), which if annualized over a 10-year study time frame would represent a small fraction of the cost of many pandemic zoonoses.

Importance: Recent years have seen a dramatic increase in viral discovery efforts. However, most lack rigorous systematic design, which limits our ability to understand viral diversity and its ecological drivers and reduces their value to public health intervention. Here, we present a new framework for the discovery of novel viruses in wildlife and use it to make the first-ever estimate of the number of viruses that exist in a mammalian host. As pathogens continue to emerge from wildlife, this estimate allows us to put preliminary bounds around the potential size of the total zoonotic pool and facilitates a better understanding of where best to allocate resources for the subsequent discovery of global viral diversity.

FIG 1

Phylogenetic tree (ML) of PMV large gene (RdRp). Alignment length, 534 bp of nucleotide sequence. PgPMV-1 to -10 were discovered in this study. PgPMV-11 is Nipah virus. The number of samples that tested positive for each respective virus in urine (U) and throat (T) is indicated in parentheses. *, published bat PMV sequences. Novel viruses detected in this study are identified with the prefix Pg (Pteropus giganteus) and were assigned accession numbers KC692403 to KC692412

FIG 2

Phylogenetic tree (ML) of AdV polymerase. Alignment length, 301 bp of nucleotide sequence. PgAdV-1 to -14 were discovered in this study. The number of samples that tested positive for each respective virus in urine (U), throat (T), feces (F), and roost urine (RU) is indicated in parentheses. *, published bat AdV sequences. Viruses detected in this study are identified with the prefix Pg (Pteropus giganteus) and were assigned accession numbers KC692417 to KC692430

FIG 3

Phylogenetic tree (ML) of AstV RdRp. Alignment length, 320 bp of nucleotide sequence. PgAstV-1 to -8 were discovered in this study. The number of samples that tested positive for each respective virus in urine (U), throat (T), and roost urine (RU) is indicated in parentheses. *, published bat AstV sequences. Viruses detected in this study are identified with the prefix Pg (Pteropus giganteus) and were assigned accession numbers KC692431 to KC692437

FIG 4

Phylogenetic tree (ML) of CoV RdRp. Alignment length, 310 bp of nucleotide sequence. PgCoV-1 to -4 were discovered in this study. The number of samples that tested positive for each respective virus in urine (U), throat (T), and roost urine (RU) is indicated in parentheses. *, published bat CoV sequences. Bat coronaviruses cluster based on the host family (indicated). ~, HKU2 seems anomalously positioned as it was detected in Rhinolophus sinicus, which is unrelated to bats from the families Vespertilionidae or Molossidae. The reason for this is unknown. Viruses detected in this study are identified with the prefix Pg (Pteropus giganteus), and were assigned accession numbers KC692413 to KC692416. IBV, infectious bronchitis virus; MHV, mouse hepatitis virus; PHEV, porcine hemagglutinating encephalomyelitis virus; HCoV, human CoV; BtCoV, bat CoV; FIPV, feline infectious peritonitis virus; TGEV, transmissible gastroenteritis coronavirus; PEDV, porcine epidemic diarrhea virus.

FIG 5

Phylogenetic tree (ML) of PyV VP1 (major capsid protein). Alignment length, 320 bp of nucleotide sequence. PgPyV-1 to -3 were discovered in this study. The number of samples that tested positive for each respective virus in urine (U) and throat (T) is indicated in parentheses. *, published bat PyV sequences. Viruses detected in this study are identified with the prefix Pg (Pteropus giganteus) and were assigned accession numbers KC692400 to KC692402.

FIG 6

Phylogenetic tree (ML) of HV polymerase. Alignment length, 211 bp of nucleotide sequence. PgHV-1 to -13 were discovered in this study. The number of samples that tested positive for each respective virus in urine (U), throat (T), feces (F), and roost urine (RU) is indicated in parentheses. *, published bat HV sequences. Viruses detected in this study are identified with the prefix Pg (Pteropus giganteus) and were assigned accession numbers KC692438 to KC692450.

FIG 7

Phylogenetic tree (ML) of BoV NS1. Alignment length, 287 bp of nucleotide sequence. PgBoV-1 and -2 were detected in this study. The number of samples that tested positive for each respective virus in urine (U), throat (T), feces (F), and roost urine (RU) is indicated in parentheses. Viruses detected in this study are identified with the prefix Pg (Pteropus giganteus) and were assigned accession numbers KC692451 to KC692452.

FIG 8

Viral discovery curve. A subset of 1,092 samples were tested for all nine viral families, and 44 viruses (out of a total of 55) are represented. The 11 viruses not considered were PgHV-2, -5, -6, and -9; PgAdV-1 and -10, PgAstV-4, -5, -6, and -8, and PgBoV-1. Black line, the rarefaction curve; red line, collector curve showing accumulation of novel viruses over samples tested; blue line, Chao2 estimator at every sample point, with arrow indicating 95% confidence intervals; gray lines, ICE and Jackknife estimators at every sample point; dashed vertical lines, required sampling effort to discover an arbitrary proportion of the total diversity (including 100%); horizontal line, total estimated diversity, 58 viruses, and effort required to discover 100% of the estimated diversity, 7,079 samples.

FIG 9

Coinfection matrix of the 55 viruses identified or discovered in P. giganteus. Heat map of coinfections is presented in lower left section. Data used to generate the heat map (numbers of pairwise incidences) are presented in upper right section.