A Strategy To Estimate Unknown Viral Diversity in Mammals

Anthony, Simon J.; Epstein, Jonathan H.; Murray, Kris A.; Navarrete-Macias, Isamara; Zambrana-Torrelio, Carlos M.; Solovyov, Alexander; Ojeda-Flores, Rafael; Arrigo, Nicole C.; Islam, Ariful; Ali Khan, Shahneaz; Hosseini, Parviez; Bogich, Tiffany L.; Olival, Kevin J.; Sanchez-Leon, Maria D.; Karesh, William B.; Goldstein, Tracey; Luby, Stephen P.; Morse, Stephen S.; Mazet, Jonna A. K.; Daszak, Peter; Lipkin, W. Ian

Viral discovery curves and estimates of viral richness. Asymptotic viral richness was estimated from observed detections using three statistical models, Chao2, ICE, and Jackknife (10) . To ensure internal consistency, only those samples screened for the full complement of nine viral families/genera were included (n ϭ 1,092), which accounted for 44/55 viruses identified in this study. The relative frequency of these viruses is presented in Fig. S1 in the supplemental material. Of the 1,092 samples included, 766 were negative for all viruses. There were 595 viral detections from 326 positive samples, with 167 samples containing Ͼ1 virus. When all 44 viruses were considered, the accumulative discovery curve began to show signs of saturation (Fig. 8) . The Chao2 estimator demonstrated asymptotic behavior as early as 500 samples (Ͻ50% of tested samples) and was highly stable (low variance) by 1,000 samples (Fig. 8) . The total viral richness within our sample population was estimated to be 58 viruses (limited to viral families tested for), and the required sampling effort to discover all 58 was estimated to be 7,079 samples. Asymptotic estimates of viral richness were also calculated individually for PMVs, AstVs, HVs, and AdVs (see Fig. S2 in the supplemental material). For HVs, AstVs, and PMVs, estimates were stable or stabilizing and showed that most of the predicted viral diversity had been identified. For AdVs, none of the estimators stabilized, probably due to a high singleton-to-doubleton ratio (i.e., the rate of discovery of new viruses was still high). Individual assessments were not performed for CoVs, PyVs, or BoVs because a lack of single or double detections prevented meaningful estimates (10) .