Abstract
Zika 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.
Highlights
Zika virus (ZIKV) was first isolated from a captive rhesus monkey on a canopy in Uganda during surveillance for Yellow fever in 1947 [1]
Virus isolation was the gold standard method for viral disease diagnosis for many years until it was replaced by molecular techniques which identify viruses at the level of type, subtype and clade
Viral isolates are essential for studying their biology, e.g., morphology, size and structure of the viral particles, pathogenesis, drug sensitivity, and for vaccine development and evaluation
Summary
Zika virus (ZIKV) was first isolated from a captive rhesus monkey on a canopy in Uganda during surveillance for Yellow fever in 1947 [1]. Zika fever was found in three human cases in Nigeria [2]. The first documented outbreak of Zika fever was in 2007 on the Western Pacific island of Yap in the Federated States of Micronesia [3]. This was followed by an epidemic in French Polynesia in the South Pacific in 2013 and 2014, which had an unusual number of cases complicated with Guillain-Barre syndrome [4]. In March 2015, ZIKV emerged in Brazil and included the highest incidence of congenital microcephaly ever reported [5].
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