Worawat DangsagulKriengsak RuchusatsawatApiwat TawatsinDon ChangsomPirom NoisumdaengSukontip PutchakarnChayawat PhatihattakornPrasert AuewarakulPilaipan PuthavathanaSiriraj HospitalThailand Ministry of Public HealthMahidol UniversityThammasat University2022-08-042022-08-042021-07-01PLoS ONE. Vol.16, No.7 July (2021)193262032-s2.0-85111766347https://repository.li.mahidol.ac.th/handle/20.500.14594/79332Zika virus (ZIKV) was isolated from the archival urine, serum, and autopsy specimens by intrathoracic inoculation of Toxorhynchitis splendens and followed by three blind sub-passaging in C6/36 mosquito cells. The virus isolates were identified using an immunofluorescence assay and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). This study analyzed 11 ZIKV isolates. One isolate (0.6%) was obtained from 171 urine samples, eight (8.7%) from 92 serum samples and two from tissues of an abortive fetus. After propagation in C6/36 cells, ZIKV was titrated by plaque and focus forming unit (FFU) assays in Vero cell monolayers, and viral genomes were determined via real-time and digital RT-PCR. Plaque and FFU assay quantitations were comparable, with the amount of infectious viruses averaging 106−107 PFU or FFU/ml. Real-time RT-PCR semi-quantified the viral genome numbers, with Ct values varying from 12 to 14. Digital RT-PCR, which precisely determines the numbers of the viral genomes, consistently averaged 10-100 times higher than the number of infectious units. There was good correlation between the results of these titration methods. Therefore, the selection of a method should be based on the objectives of each research studies.Mahidol UniversityMultidisciplinaryArticleSCOPUS10.1371/journal.pone.0255314