Experiments followed the infectivity
of HuNoV, as well as the signal decay of the intact viral capsid and
RNA genome in creek water (15-1, 15-2, 20-1, and 20-2;Table 1 and Figure S1 ). For all experiments, creek water was inoculated with HuNoV
GII.4 Sydney strain such that the starting concentration was between
2.4 × 105 ± 4.3 × 104 and 7.1
× 105 ± 3.5 × 104 gene copies
per mL (gc/mL) of HuNoV (error is the standard deviation). Water samples
were aseptically divided into 1 mL aliquots and stored in the dark
at 15 °C (15-1 and 15-2) or 20 °C (20-1 and 20-2). One 1
mL aliquot was sacrificed at each time point (Table 1 ); time points were generally 7 days apart
and the duration of the experiments was 5 and 28 days. Experiments
20-1 and 20-2 were each completed with replicate 1 mL aliquots at
each time point (Figure S1 ). At each time
point, each sacrificed aliquot was divided into two: one subaliquot
was used to evaluate HuNoV infectivity in HIE cells and the other
to evaluate HuNoV decay using five different RT-qPCR assays. Additional
details can be found in theSI .
The
concentrations of one short segment of gRNA commonly used to quantify
HuNoV in the environment (89 nt, hereafter referred to as “ORF”),
and four different long segments (∼500 nt) of gRNA were measured
at each time point using RT-qPCR. The combined damage incurred by
the long genome segments was extrapolated to estimate that of the
whole genome.31 (link) For the short-genome segment
dsDNA assay, the R2 and efficiency of
the master standard curve were 0.99 and 84.3%, respectively (Table S7 ). Across all long genome segment dsDNA
assays used on experimental samples, the R2 and efficiency of the master standard curves ranged from 0.93 to
1 and 76.6 to 100.1%, respectively (Table S7 ). In two experiments (15-2 and 20-2), RT-qPCR was completed with
samples pretreated with RNase I (Thermo Fisher Scientific; enzyme-treated
RT-qPCR; ET-RT-qPCR) to eliminate gRNA not protected within an intact
viral capsid.42 (link)−44 (link) The details of these methods are provided below.
Positive HuNoV stool samples were limited and so different HuNoV
stool filtrates were used for experiments 15-1 and 15-2 (accession
#MK764019) and experiments 20-1 and 20-2 (accession #OL913976). Although
the genomic sequences of these HuNoV strains have 97.7% pairwise nucleotide
identity and were genotyped as GII.4 Sydney, separate RT-qPCR assays
targeting long segments of each genome had to be designed for use
with each (see details below).
of HuNoV, as well as the signal decay of the intact viral capsid and
RNA genome in creek water (15-1, 15-2, 20-1, and 20-2;
GII.4 Sydney strain such that the starting concentration was between
2.4 × 105 ± 4.3 × 104 and 7.1
× 105 ± 3.5 × 104 gene copies
per mL (gc/mL) of HuNoV (error is the standard deviation). Water samples
were aseptically divided into 1 mL aliquots and stored in the dark
at 15 °C (15-1 and 15-2) or 20 °C (20-1 and 20-2). One 1
mL aliquot was sacrificed at each time point (
and the duration of the experiments was 5 and 28 days. Experiments
20-1 and 20-2 were each completed with replicate 1 mL aliquots at
each time point (
point, each sacrificed aliquot was divided into two: one subaliquot
was used to evaluate HuNoV infectivity in HIE cells and the other
to evaluate HuNoV decay using five different RT-qPCR assays. Additional
details can be found in the
The
concentrations of one short segment of gRNA commonly used to quantify
HuNoV in the environment (89 nt, hereafter referred to as “ORF”),
and four different long segments (∼500 nt) of gRNA were measured
at each time point using RT-qPCR. The combined damage incurred by
the long genome segments was extrapolated to estimate that of the
whole genome.31 (link) For the short-genome segment
dsDNA assay, the R2 and efficiency of
the master standard curve were 0.99 and 84.3%, respectively (
assays used on experimental samples, the R2 and efficiency of the master standard curves ranged from 0.93 to
1 and 76.6 to 100.1%, respectively (
samples pretreated with RNase I (Thermo Fisher Scientific; enzyme-treated
RT-qPCR; ET-RT-qPCR) to eliminate gRNA not protected within an intact
viral capsid.42 (link)−44 (link) The details of these methods are provided below.
Positive HuNoV stool samples were limited and so different HuNoV
stool filtrates were used for experiments 15-1 and 15-2 (accession
#MK764019) and experiments 20-1 and 20-2 (accession #OL913976). Although
the genomic sequences of these HuNoV strains have 97.7% pairwise nucleotide
identity and were genotyped as GII.4 Sydney, separate RT-qPCR assays
targeting long segments of each genome had to be designed for use
with each (see details below).
