Zika virus isolation, propagation, and quantification using multiple methods

Autor:	Prasert Auewarakul, Don Changsom, Chayawat Phatihattakorn, Worawat Dangsagul, Pirom Noisumdaeng, Pilaipan Puthavathana, Kriengsak Ruchusatsawat, Sukontip Putchakarn, Apiwat Tawatsin
Jazyk:	angličtina
Rok vydání:	2021
Předmět:	RNA viruses 0301 basic medicine Physiology Cell Lines Artificial Gene Amplification and Extension Urine Disease Vectors Pathology and Laboratory Medicine Polymerase Chain Reaction Mosquitoes Genome Zika virus Medical Conditions Chlorocebus aethiops Medicine and Health Sciences Viral Genomics Multidisciplinary medicine.diagnostic_test biology Zika Virus Infection Eukaryota Genomics Isolation (microbiology) Body Fluids Insects Infectious Diseases Medical Microbiology Viral Pathogens Viruses Viral Genome RNA Viral Medicine Biological Cultures Autopsy Pathogens Anatomy Research Article Arthropoda Science 030106 microbiology Surgical and Invasive Medical Procedures Genome Viral Microbial Genomics Real-Time Polymerase Chain Reaction Research and Analysis Methods Immunofluorescence Microbiology Virus 03 medical and health sciences Virology Genetics medicine Animals Humans Molecular Biology Techniques Vero Cells Microbial Pathogens Molecular Biology Biology and life sciences Flaviviruses Inoculation Organisms Zika Virus Reverse Transcriptase-Polymerase Chain Reaction biology.organism_classification Invertebrates Insect Vectors Species Interactions Culicidae 030104 developmental biology Vero cell Zoology Entomology
Zdroj:	PLoS ONE, Vol 16, Iss 7, p e0255314 (2021) PLoS ONE
ISSN:	1932-6203
Popis:	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.
Databáze:	OpenAIRE
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