Mitochondrial DNA Sequencing and BLAST Analysis Reveal Visual Misidentification of Nonhuman Primates Suspected to have Died of Yellow Fever in Brazil in 2017–2020

Abstract

Epidemics have significant impacts on primate populations. In 2016, yellow fever spread rapidly to the most densely populated states of Brazil, resulting in the death of hundreds of humans and thousands of nonhuman primates. The Yellow Fever Surveillance Program (Programa de Vigilância da Febre Amarela) implemented by the Brazilian government was designed to prevent and control yellow fever outbreaks. In 2020, the regional reference laboratory at the Oswaldo Cruz Institute in Rio de Janeiro state in Brazil, responsible for yellow fever diagnosis, gave us access to 24 DNA samples from capuchin monkeys, which had tested negative for yellow fever virus. The samples were all from sites in Brazil where Sapajus nigritus is the only species of capuchin monkey present. We attempted to sequence two mitochondrial molecular markers commonly used for Cebidae, but five samples did not amplify. In this study, we performed additional DNA amplification and the Basic Local Alignment Search Tool (BLAST) to confirm the species identity of the 19 samples, which amplified and identify the taxon of the five samples that had failed to amplify. Among the 19 samples correctly identified as Sapajus, one was not Sapajus nigritus but another Sapajus species. Of the five samples that previously failed to amplify, three were similar to Callicebus nigrifrons, one was similar to Alouatta guariba clamitans, and one failed to amplify successfully for any marker. These findings show an error rate of 5/24 (21%) in the original taxonomic identification. Misidentification occurred at the genus level as well as the species level. Our findings highlight the importance of precise taxonomic classification in obtaining reliable data on the consequences of the yellow fever epidemic for primates.