Development of Quantitative Real-Time PCR and Loop-Mediated Isothermal Amplification Assays for the Surveillance and Diagnosis of Herpes B Virus Infection

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Abstract
Herpes B virus (BV) is a zoonotic virus which can be transmitted from macaques to humans, which is often associated with high mortality rates. Because macaques often exhibit asymptomatic infections, individuals who come into contact with these animals face unexpected risks of BV infections. A serological test is widely performed to investigate BV infections. However, the assay’s sensitivity and specificity appeared to be inadequate, and it does not necessarily indicate ongoing viral shedding. Here, we developed LAMP and qPCR assays aiming to detect BVs with a high sensitivity and specificity in various macaque species and validated them using oral swab samples collected from 97 wild cynomolgus macaques living in Thailand. Our LAMP and qPCR assays detected more than 50 and 10 copies of the target sequences per reaction, respectively. The LAMP assay could detect BV within 25 min, indicating its advantages for the rapid detection of BV. Collectively, our findings indicated that both assays developed in this study exhibit advantages and usefulness for BV surveillance and the diagnosis of BV infections in macaques. Furthermore, for the first time, we determined the partial genome sequences of BVs detected in cynomolgus macaques in Thailand. Phylogenetic analysis revealed the species-specific evolution of BV within macaques.
Funding Information
  • Japan Agency for Medical Research and Development (JP23fm0208101, JPJSCCB20220009, RTA6280010)
  • Japan Society for the Promotion of Science (JP23fm0208101, JPJSCCB20220009, RTA6280010)
  • Research Fund Senior Scholar (JP23fm0208101, JPJSCCB20220009, RTA6280010)

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