Baseline molecular surveillance of monkeypox virus in wildlife and exotic animals during the pre- and early-2022 outbreak in Thailand

Background and Aim: The global outbreak of monkeypox (mpox) in 2022 raised major concerns regarding the potential establishment of new animal reservoirs outside endemic regions. Although human-to-human transmission was the primary driver of the outbreak, reverse zoonotic transmission (spillback) from infected humans to animals could lead to long-term viral persistence in wildlife populations. Thailand, a non-endemic country affected during the 2022 outbreak, has extensive human–animal interfaces involving free-ranging wildlife, exotic pets, and urban scavenging mammals, which may facilitate cross-species transmission. The present study aimed to perform molecular surveillance for mpox virus (MPXV) DNA in selected high-risk animal populations in Thailand using archival samples collected during the pre-outbreak (2019) and early-outbreak (2022) periods to establish baseline evidence of viral presence or absence. Materials and Methods: A total of 1,248 animals, including 593 free-ranging cynomolgus macaques (Macaca fascicularis), 436 exotic pets, and 219 garbage-scavenging small mammals, were included in this cross-sectional surveillance study. Swab samples (oral, nasal, buccal, or rectal) collected during 2019 and 2022 were retrieved from archival storage. Viral DNA was extracted using a commercial genomic DNA kit, and detection of MPXV was performed by real-time polymerase chain reaction targeting the B7R gene, a conserved region specific to MPXV. Samples were pooled in groups of 4–5 to increase screening efficiency. Descriptive statistics were used to determine prevalence, and the rule-of-three method and binomial probability model were applied to estimate confidence limits and surveillance power in the absence of positive results. Results: All 1,248 samples tested negative for MPXV DNA. The studied population represented 29 animal species across multiple ecological settings, including urban, peri-urban, and rural environments in several provinces of Thailand. Based on the sample size, the upper 95% confidence limit for prevalence was estimated to be <0.24%. Power analysis indicated a 99.81% probability of detecting at least one positive case if the true prevalence had been ≥0.5%, confirming strong surveillance sensitivity. Conclusion: This study provides the first ecological baseline evidence of the absence of MPXV in wildlife, exotic pets, and garbage-scavenging mammals in Thailand before and during the early phase of the 2022 global outbreak. The findings support the importance of One Health-based surveillance integrating wildlife, domestic animals, and environmental interfaces to prevent spillback events and the establishment of new reservoirs in non-endemic regions. Continued longitudinal monitoring using both molecular and serological approaches is essential for early detection of emerging zoonotic threats. Keywords: animal surveillance, exotic pets, molecular detection, monkeypox virus, One Health, real-time PCR, wildlife reservoirs, zoonotic spillback.