Vaccine shedding and genetic stability
VIRUS SHEDDING
Vaccine-virus shedding was analyzed by RT-qPCR of nasal swabs collected on days 1 (before vaccination) and days 8, 15, and 29 after vaccination. A regular sized flocked nylon swab was used to collect nasal secretion from one nostril, placed in 1-ml universal transport medium (VTM, Copan), and stored at −80°C until sample testing. RNA was extracted from 140 μl (group 2) or 560 μl (group 1) of nasal swab by the QIAmp Viral RNA Mini Kit (QIAGEN) and eluted in 40 μl of buffer. The primers targeted the trailer region of PIV5 genome to quantify PIV5-RSV vaccine virus genomic RNA. The PCR reaction mix included Luna Universal probe One-Step RT-qPCR (New England BioLabs), PIV5 trailer region primers/probe (sequence information is available upon request), and 8 μl of RNA in a QuantStudio 3 instrument (Thermo Fisher Scientific). The assay had a lower LOD of 4.6 logVaccine virus shedding samples with RNA genome levels greater than 5.0 log