A sensitive virus yield assay for evaluation of Antivirals against Zika Virus.
Despite the rapid spread of Zika virus (ZIKV) infection and associated neurological complications in the America's, prophylactic or therapeutic countermeasures are not currently available. This is mostly due to the fact that until recently there was no presumed need for medical intervention since there was no association between ZIKV infection and significant human morbidity. Consequently, there are currently no tools due mostly to the lack of sensitive cell based assays amenable for identification of ZIKV inhibitors. To address this unmet need we have developed a cell based virus yield assay suitable for testing antivirals against Zika virus. Using bioinformatics, several isolates of ZIKV from the Americas, Africa, and Asia were analyzed for sequence similarity. The alignment data were then used to design primers targeting a ZIKV genomic region that was highly conserved among all the ZIKV isolates. Subsequently, primers were used in a sensitive, quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay to detect ZIKV RNA. The qRT-PCR assay was found to be highly sensitive (lower limit of detection between-10-100 copies) and reproducible. Evaluation of the primers and probes used for ZIKV against another flavivirus (Dengue virus) demonstrated specificity of detection. To evaluate potential of qRT-PCR assay as an antiviral screening tool against ZIKV, Vero cells pretreated with Type I Interferons (IFN α) were infected with virus, followed by measurement of ZIKV RNA found in the cell culture supernatants using qRT-PCR assay. Dose-dependent antiviral activity of Type I Interferons and mycophenolic acid (MPA) against Zika virus in this cell culture system was confirmed using qRT-PCR. Due to reproducible assay performance, qPCR associated higher sensitivity and short duration of the assay time, this novel cell based assay will be very useful for confirming the activity of antivirals against ZIKV.
Department of Infectious Diseases Research, Drug Development, Southern Research, Frederick, MD, United states.
Scott
S
Goebel
Department of Infectious Diseases Research, Drug Development, Southern Research, Frederick, MD, United states.
Beth
B
Snyder
Department of Infectious Diseases, Drug Discovery Division, Southern Research, Birmingham, AL, United states.
Timothy
T
Sellati
Department of Infectious Diseases, Drug Discovery Division, Southern Research, Birmingham, AL, United states.
Mohammad
M
Saeed
Department of Infectious Diseases Research, Drug Development, Southern Research, Frederick, MD, United states.
Roger
R
Ptak
Department of Infectious Diseases Research, Drug Development, Southern Research, Frederick, MD, United states.
Michael
M
Murray
Department of Infectious Diseases, Drug Discovery Division, Southern Research, Birmingham, AL, United states.
Robert
R
Bostwick
Department of Infectious Diseases Research, Drug Development, Southern Research, Birmingham, AL, United states.
Jonathan
J
Rayner
Department of Infectious Diseases Research, Drug Development, Southern Research, Frederick, MD, United states.
Fusataka
F
Koide
Department of Infectious Diseases Research, Drug Development, Southern Research, Frederick, MD, United states. Electronic address: rkalkeri@southernresearch.org.
Raj
R
Kalkeri
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D017931:DNA Primers; D003716:Dengue Virus / Q000235:genetics; D055808:Drug Discovery / Q000379:methods; D016679:Genome, Viral; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D057230:Limit of Detection; D008826:Microbial Sensitivity Tests / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014709:Vero Cells; D000071244:Zika Virus / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification
D000998:Antiviral Agents; D017931:DNA Primers; D016898:Interferon-alpha; D012367:RNA, Viral
CP assay; Quantitative RT-PCR; Virus yield assay; Zika virus