Quantifying ZIKV RNA in Maternal-Fetal Tissues

Viral RNA load was assessed in tissues from the dam, fetus, and placenta using a ZIKV-specific RT-qPCR assay. Fetal and maternal organs were immersed in RNA-later immediately upon harvest and were then weighed and homogenized in RLT solution (Qiagen) using a bead-beater apparatus (Precellys). RNA was extracted from tissues using the RNeasy kit (Qiagen) and from sera using the ZR Viral RNA extraction kit (Zymo Research). From tissues, 400 ng of total RNA was used to generate cDNA using the iScript select cDNA synthesis kit (Bio-Rad) according to manufacturer’s protocols for gene-specific primers. Viral RNA was quantified using the Taqman Universal Master Mix (Applied Biosystems) and an Applied Biosystems 7300 RT-PCR machine with primers that correspond to residues conserved in both the FSS13025 and Brazil Fortaleza genome (GenBank numbers KU955593.10, KX811222.1).^{49 (link)} To adhere to stringent guidelines, Ct (cycle threshold) values >38 were deemed as not reliably detected and were not reported. Copy number sensitivity, as determined using a standard curve from diluted known quantities of ZIKV genome, was 25 copies/qPCR reaction.

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Adams Waldorf K.M., Nelson B.R., Stencel-Baerenwald J.E., Studholme C., Kapur R.P., Armistead B., Walker C.L., Merillat S., Vornhagen J., Tisoncik-Go J., Baldessari A., Coleman M., Dighe M.K., Shaw D.W., Roby J.A., Santana-Ufret V., Boldenow E., Li J., Gao X., Davis M.A., Swanstrom J.A., Jensen K., Widman D.G., Baric R.S., Medwid J.T., Hanley K.A., Ogle J., Gough G.M., Lee W., English C., Durning W.M., Thiel J., Gatenby C., Dewey E.C., Fairgrieve M.R., Hodge R.D., Grant R.F., Kuller L., Dobyns W.B., Hevner R.F., Gale M J.r, & Rajagopal L. (2018). Congenital Zika Virus Infection as a Silent Pathology With Loss of Neurogenic Output in the Fetal Brain. Nature medicine, 24(3), 368-374.

Publication 2018

RLT solution RNeasy kit ZR Viral RNA extraction kit iScript select cDNA synthesis kit Taqman Universal Master Mix 7300 RT-PCR machine

Assay Cdna Fetal Gene Genome Maternal Placenta Primers Rt pcr Sensitivity Sera Synthesis Tissues Viral rna Zikv

Corresponding Organization :

Other organizations : University of Washington, Infectious Disease Research Institute, University of North Carolina at Chapel Hill, New Mexico State University, Allen Institute, Allen Institute for Brain Science

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Variable analysis

independent variables

Viral RNA load

dependent variables

Viral RNA load in tissues from the dam, fetus, and placenta

control variables

Cycle threshold (Ct) values >38 were deemed as not reliably detected and were not reported
Copy number sensitivity was 25 copies/qPCR reaction

controls

Positive control: Diluted known quantities of ZIKV genome used to generate a standard curve
Negative control: Not explicitly mentioned

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