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\begin{document}

\title{Variation in Severity of Symptoms Associated with Two Snow Mountain
Virus Inocula}



\author[1]{Pengbo Liu}%
\author[1]{Hongyan Qu}%
\author[2]{Nadine Rouphael}%
\author[3]{Mark Mulligan}%
\author[1]{Yuke Wang}%
\author[1]{Christine Moe}%
\affil[1]{Emory University School of Public Health}%
\affil[2]{Emory University Department of Medicine}%
\affil[3]{New York University Grossman School of Medicine}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: Norovirus (NoV) is a leading cause of epidemic non-bacterial
acute gastroenteritis in young children and adults globally. Snow
Mountain Virus (SMV), the prototype of genogroup II and genotype II NoV,
has been used in three human challenge studies to examine the
infectivity, pathogenicity, and immune response to NoV. Methods: This is
a secondary data analysis. Clinical and laboratory data from two
previously completed SMV human challenge trials using two different
inocula (Inoculum 1 and Inoculum 2) were analyzed to compare
infectivity, illness (including modified Vesikari severity scores of
gastroenteritis in those subjects with clinical symptoms), viral
shedding, and serum IgG conversion. SMV Inoculum 2 is a
second-generation inoculum prepared from a stool sample collected from a
study subject who was infected with SMV Inoculum 1. Results: Of 15
subjects orally challenged with SMV Inoculum 1 between 2000 and 2002,
nine were infected, and seven presented with acute gastroenteritis. Of
33 subjects orally challenged with SMV Inoculum 2 between 2016 and 2018,
25 were infected, and nine presented with acute gastroenteritis. There
were no statistically significant differences in overall infection and
illness rates between subjects challenged with Inoculum 1 vs. Inoculum
2. However, subjects infected with Inoculum 1 experienced more severe
clinical symptoms of acute gastroenteritis and had higher severity
scores (6.00 vs. 2.94, P = 0.003) compared with those infected with
Inoculum 2. We also observed that infection with Inoculum 2 resulted in
longer viral shedding compared with Inoculum 1. This analysis also
indicated that secretor-positive subjects had more severe
gastroenteritis than secretor-negative subjects. Among ill subjects, no
association was observed between challenge dose and severity of acute
gastroenteritis. Conclusions: Understanding the differences between
these two SMV inocula is critical for NoV vaccine evaluation because
illness and viral shedding are two important outcomes in NoV challenge
studies to determine vaccine efficacy. Using a less pathogenic inoculum
for a vaccine trial will require more participants to meet the target
reduction in illness when evaluating the efficacy of candidate vaccines.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Norovirus (NoV) is a leading cause of epidemic non-bacterial acute
gastroenteritis in children under five years and adults worldwide {[}1,
2{]}. In the United States, NoV infection is associated with an
estimated 71,000 hospitalizations and 21 million total illnesses per
year {[}3{]}. NoV infection is characterized by the acute onset of
vomiting, diarrhea, nausea, abdominal cramps, and/or fever, which
generally last for 48-72 hours. NoV transmission occurs through
contaminated water, food, hands, and environmental surfaces, and person
to person by the fecal-oral route {[}4{]}.

The NoV genome is a linear, positive-sense, single-stranded RNA, and
these viruses are classified into at least ten genogroups (GI-GX) and 49
genotypes based on the major structural protein (VP1) amino acid
sequence diversity {[}5{]}. Only genogroups I (GI), GII, and GIV have
been associated with human gastroenteritis. NoV GII is further divided
into 22 genotypes {[}6{]}. Snow Mountain Virus (SMV) is the prototype
strain of genogroup II genotype II NoV.

Human challenge studies with Norwalk virus (NV, the prototype of NoV
genogroup I and genotype I) demonstrated a strong association between NV
infection and secretor status as determined by the \emph{FUT2} gene
{[}7, 8{]}. \emph{FUT} 2 encodes an \selectlanguage{greek}α\selectlanguage{english}(1,2) fucosyltransferase that is
responsible for the synthesis of H antigen, and individuals with H
antigen expression are considered secretor positive. Previous human
challenge studies indicated that secretor-negative individuals do not
become infected with NV regardless of the dose {[}7{]}, but the
relationship between secretor status and genogroup II NoV infection is
less clear. We observed SMV infection and illness in both
secretor-positive and secretor-negative subjects challenged with SMV
{[}9{]}. Secretor-negative subjects are not completely protected from
GII.4 infections {[}10{]}.

The human challenge model has been used to study the pathogenesis and
immunology of NoV infection, and the efficacy of NoV vaccine candidates.
We have conducted two SMV human challenge studies, one between 2000 to
2002 with a first generation SMV inoculum {[}11{]} and the second
between 2016 to 2018 with a second generation SMV inoculum {[}9{]}.

The objectives of this analysis were to compare infection (defined by
serum IgG conversion and/or detection of SMV RNA in stool by RT-PCR or
RT-qPCR) and illness (defined by diarrhea, vomiting, and other clinical
symptoms) among subjects challenged with the first SMV inoculum {[}11{]}
prepared by Dolin et al. around 1980 {[}12{]} and the second SMV
inoculum prepared in 2009 by Dr. Baric {[}9{]}. This analysis also
examined the severity of illness and the duration of the viral shedding
among human volunteers challenged with the two SMV inocula. The results
from this study will contribute to our understanding of SMV infectivity
and pathogenesis.

\textbf{MATERIALS AND METHODS}

\emph{SMV human challenge study 1} {[}11{]}: 15 healthy human subjects
were challenged with one dose of SMV inoculum, ranging from 31.7 to 3.17
\selectlanguage{ngerman}× 10\textsuperscript{5} genome copies quantitated by RT-qPCR {[}13{]}.
Subjects remained in the hospital clinical research unit for five days
after challenge. Stool, serum, vomitus, saliva samples and clinical
information were collected daily. Subjects were also followed up on days
7, 15, 30, and 45 post-challenge for stool and serum sample collection
{[}11{]}.

\emph{SMV human challenge study 2} {[}9{]}: The study consisted of four
sequential cohorts and a placebo group. In cohorts 1 through 3 (all
subjects were secretor positive), individuals per cohort were challenged
with 1.2 x 10\textsuperscript{4} genome equivalent copies (GEC) (cohort
1), 1.2 x 10\textsuperscript{6} GEC (cohort 2) and 1.2 x
10\textsuperscript{7} GEC (cohort 3) of SMV. Cohort 4 only included
secretor-negative subjects, and they were challenged with a dose of 1.2
x 10\textsuperscript{7} GEC. Subjects remained in the hospital clinical
research unit for five days after challenge. Stool, serum, vomitus,
saliva samples and clinical information were collected. Subjects were
also followed up on days 7, 15, 30, and 45 post-challenge for stool and
serum sample collection. For this secondary data analysis, a total of 33
subjects who received the SMV inoculum and completed the study {[}9{]}
were included in this study.

All the laboratory assays, including detection of anti-SMV IgG in serum,
detection of secretor status, and quantification of SMV RNA in stool
have been described previously {[}9, 11{]}. SMV infection was defined as
RNA detection in any post-challenge stool sample by RT-PCR or specific
RT-qPCR with CT values \emph{\textless{}} 40 in duplicate reactions
and/or anti-SMV serum IgG conversion by ELISA in any post-challenge
serum sample vs. pre-challenge serum sample. Illness was defined as
those infected with SMV who presented with diarrhea \selectlanguage{english}[?]3 loose or liquid
stools or \selectlanguage{english}[?]300 g of loose or liquid stools in any continuous 24-h
period, or one or more vomiting episodes during the inpatient period and
with one other clinical sign or symptom such as fever, abdominal cramps,
nausea, headache, chills, fatigue, or myalgia.

\textbf{STATISTICAL ANALYSIS}

The databases from the two SMV human challenge studies were merged into
a single database for analyses. Data were analyzed using SAS 9.4 (SAS,
Cary, NC) for Windows. Categorical data were analyzed using a chi-square
test or Fisher's exact test. Continuous variables were analyzed using
the t test or Mann-Whitney U test. A P-value of \textless{}0.05 was
considered significant. Severity scores of illness were calculated using
an 18-point numerical scoring system, which was modified from Ruuska and
Vesikari's score system {[}14{]}. The scoring system used information on
the duration of diarrhea and vomiting, the maximum number of episodes of
diarrhea and vomiting within a 24- hour period, and whether the subject
experienced certain clinical signs or symptoms, including headache,
fever, chill, fatigue, nausea, abdominal cramp, and myalgia (``yes'',
``more than one'' or ``no''). Fever was further graded at four levels
(\selectlanguage{english}[?]37.0degC, 37.1degC-38.4degC, 38.5degC-38.9degC, \selectlanguage{english}[?]39degC) by the clinical staff.
Table 1 shows the severity score criteria of the modified Vesikari score
system {[}14{]}.

\textbf{RESULTS}

Analysis of the demographic characteristics of the subjects in the two
SMV trials indicated no significant differences in sex, age, and
secretor status between subjects challenged with SMV Inoculum 1 and
Inoculum 2 (Table 2).

We compared infection and illness rates between the subjects who
received SMV Inoculum 1 (n=15) to those who received SMV Inoculum 2
(n=33). Overall, SMV infection occurred in 9 of 15 (60\%) subjects
challenged with Inoculum 1 and in 25 of 33 (75.7\%) subjects challenged
with Inoculum 2. Illness occurred in 7 of 15 (46.7\%) subjects
challenged with Inoculum 1 and in 9 of 33 (27.3\%) subjects challenged
with Inoculum 2. There were no statistically significant differences
between the infection (60.0\% vs. 75.7\%) and illness (46.7\% vs.
27.3\%) rates in the two trials (Table 3).

In addition to examining the infection and illness rates following SMV
challenge, we explored possible differences in severity of illness in
the 16 infected subjects who met our definition of illness (Table 4).
Subjects infected with SMV Inoculum 1 had a mean severity score of 6.00
(95\% CI: 4.97, 7.03{]}, whereas the mean severity score for subjects
infected with SMV inoculum 2 was significantly lower 2.94 (95\% CI:
1.74, 4.14, P = 0.003) (Table 4, Figure 1). Furthermore, our analysis
indicates that Inoculum 1 was associated with more severe acute
gastroenteritis even at doses more than 100-fold lower than the doses
used for the Inoculum 2 challenge. In addition, we found
secretor-positive subjects had significantly higher mean modified
Vesikari severity scores compared to secretor-negative subjects (P
\textless{} 0.001) at the dose of 1.2x10\textsuperscript{7} genome
copies (Figure 1). When we combined all data from both inocula and
different challenge doses, we did not find a significant association
between log\textsubscript{10} inoculum dose and modified Vesikari scores
for either inoculum (Inoculum 1 and 2, P = 0.951 and P = 0.905,
respectively).

The RT-PCR results (Table 5) indicated that the NV positive rate was not
significantly different in subjects infected with Inoculum 1 vs.
inoculum 2 in stool samples collected during the first three days post
challenge, but subjects infected with Inoculum 1 had significantly more
(P \textless{} 0.001) PCR-positive stools during days 4-6 post challenge
(46.6\%) compared to none of the subjects infected with Inoculum 2
having PCR-positive stool samples during that period of time. When
shedding duration was compared between these two groups, SMV RNA was
detected in three stool samples (9.1\%) from days 15-45 post-challenge
from subjects challenged with Inoculum 2, but no subject challenged with
Inoculum 1 shed virus at 15 and 45 days post-challenge.

Finally, we compared anti-SMV serum IgG conversion (\textgreater{}4-fold
vs. pre-challenge) in subjects challenged with Inoculum 1 and Inoculum 2
(Table 5). During days 1 - 3 post challenge, none of subjects in both
groups showed anti-SMV serum IgG conversion. During days 4 - 6
post-challenge, eight subjects (57.1\%) infected with Inoculum 1 showed
anti-SMV serum IgG conversion compared to none of the subjects infected
with Inoculum 2 (P \textless{} 0.001) during that period. Most serum IgG
conversion occurred between 15 and 30 days post challenge, but there was
no significant difference between the two trials in the overall
proportion of subjects with seroconversion.

\textbf{DISCUSSION}

This analysis compared the infectivity and virulence of two SMV inocula
used in two different human challenge trials by examining infection
rates, illness rates, severity of illness, viral shedding, and IgG
seroconversion among subjects challenged with a single dose of one of
these inocula. Inoculum 1 was prepared at NIH sometime between 1977 and
1979 from the stool filtrate of a subject infected in the original Snow
Mountain Virus outbreak in Colorado in December 1976. This safety-tested
inoculum was first used in a human challenge study with 12 subjects
conducted at NIH by Dolin et al. {[}12{]} and then again in a human
challenge study with 15 subjects conducted between 2000-2002 at the
University of North Carolina-Chapel Hill {[}11{]}. Inoculum 2 was
prepared from the stool filtrate of an infected subject in the 2000-2002
human challenge trial and was then used in a human challenge study with
44 subjects at Emory University from 2016-2018 {[}9{]}. The goal of this
analysis is to examine whether the infectivity, pathogenicity, and
virulence of the SMV changed due to passage in the human host by
assessing differences in the outcome measures of the two more recent
challenge trials.

Both inocula were associated with similar overall infection rates as
measured by the proportion of challenged subjects who developed
infection and illness after challenge in each trial. However, the median
infectious dose (ID\textsubscript{50}) of Inoculum 2 was about 100-fold
higher than that estimated for Inoculum 1 {[}13{]}. Comparing the
severity scores of infected subjects with clinical symptoms indicated
that subjects infected with SMV Inoculum 2 had less severe acute
gastroenteritis and more delayed viral shedding even though these
subjects were challenged with a much higher dose than the subjects who
were infected with Inoculum 1. These results are consistent with what we
recently reported {[}15{]} that subjects challenged with a
first-generation NV inoculum (8FIIa, prepared in 1971) had significantly
higher severity scores of acute gastroenteritis but shorter duration of
viral shedding compared with those challenged with a second-generation
NV inoculum (8FIIb, prepared in 1997 from the stool filtrate of a
subject infected with the first NV inoculum).

Viral shedding and clinical illness are two important outcomes evaluated
in NoV challenge trials. Based on outbreak investigations and human
challenge studies, most infected subjects experience clinical symptoms
along with viral shedding in their feces during the first several days
of infection {[}16{]}. Symptoms usually resolve after 48-72 hours,
however viral shedding can continue for up to three weeks {[}16{]} and
even longer in immunocompromised subjects {[}17, 18{]}. In this study,
all subjects infected with SMV Inoculum 1 shed virus for up to six days,
but 3\% of subjects infected with Inoculum 2 had viral shedding between
days 15 to 45 days post challenge. We hypothesize that the less severe
clinical illness, but possibly longer SMV shedding, observed in subjects
infected with Inoculum 2 may be associated with viral mutations that
reduced the virulence of the inoculum, rather than host factors or
laboratory assessment methods. However, it is possible that the RT-qPCR
assay used to measure viral shedding in the more recent challenge trial
with Inoculum 2 is more sensitive and better able to detect prolonged
shedding compared to the conventional RT-PCR assay used in the trial
with Inoculum 1 {[}19{]}.

As the prototype of NoV GII.2, SMV is associated with a small percentage
of NoV outbreaks, and little is known about the mutation of this virus.
Swanstrom et al., {[}20{]} reported that the sequence of the P2 domain
of SMV strains collected between 1976 and 2010 has evolved over time but
less extensively than has been reported for GII.4 NoV strains. Mutations
in the surface protein could significantly alter the antigenicity of
representative strains, which could also change viral function,
pathogenesis, transmission, and infectivity. Studies of other RNA
viruses indicate that viral mutations can be deleterious or favorable to
the pathogens in terms of infectivity and virulence. Both host and viral
factors may explain the change in infectivity and virulence that we
observed between the two SMV inocula. Some host factors, including
acquired and innate immunity prior to challenge and secretor status, can
affect the likelihood of NoV infection and illness. Other host factors,
including age, sex, and race/ethnicity may or may not impact the risk of
NoV infection and illness. Rouphael et al {[}9{]} reported that
prechallenge anti-SMV serum IgG concentration, carbohydrate-binding
blockade antibody, and salivary IgA were not associated with infection
with Inoculum 2. Given the evidence from viral evolution of other RNA
viruses such as influenza and SARS-CoV-2 {[}21-23{]}, we hypothesize
that the less severe symptoms associated with the second generation SMV
inoculum in this study may be due to intra-host SMV mutations during the
course of infection in the subject who was the original source of
Inoculum 2 and possibly further inter-host mutations in the subjects who
were infected with Inoculum 2 after challenge.

This analysis is the first to compare the clinical outcomes associated
with two different SMV inocula used in two human challenge trials
{[}9,11{]}. We examined detailed clinical and laboratory data collected
from well-controlled human challenge studies with nearly identical
protocols that included follow-up sample collection and analyses to
carefully characterize infection, illness, and immune response. The
limitations of this analysis include small sample size in both studies
that may not provide enough power to detect a significant difference in
some outcomes, retrospective analysis of two studies that span over 20
years and had different study populations, and slightly different
laboratory methods for measuring viral shedding and serum antibodies.
Finally, we were not able to assess how differences between inocula
preparation in the late 1970's (Inoculum 1) vs. 1997 (Inoculum 2) and
storage may have affected the infectivity, pathogenicity, and virulence
of these two inocula. The time between inoculum preparation and the
challenge trials that provided the data we analyzed was approximately 20
years for both inocula: Inoculum 1 (1979-2000) and Inoculum 2
(1997-2016).

Understanding the difference in the severity of illness associated with
these two SMV inocula is critical for those who plan to use second
generation NoV inoculum to evaluate new NoV vaccine candidates in
vaccination-challenge trials. As we observed for the NV inocula, the
second generation SMV Inoculum 2 was associated with a higher
ID\textsubscript{50} and less severe clinical illness when compared with
the first generation SMV inoculum. If second generation NoV inoculum
does not elicit expected symptomatic illness rates in challenged
subjects, it will become more complex and costly to conduct
vaccination-challenge trials because they may require larger numbers of
study subjects and challenges with higher doses of inoculum in order to
assess the efficacy of a candidate vaccine to reduce illness.

\textbf{Funding:} This study was supported by the National Center for
Advancing Translational Sciences of the National Institutes of Health
(UL1TR002378); the National Institute of Allergy and Infectious Disease
(R01 AI148260); and the Division of Microbiology and Infectious Diseases
to the Emory Vaccine and Treatment Evaluation Units (VTEU):
HHSN272200800005C, HHSN272201300018I, HHSN27200003, and HHSN27200018

Table 1: Modified Vesikari score (18-point scale) {[}14{]} for
SMV-infected subjects

with clinical symptoms\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Clinical symptoms & Clinical symptoms & Clinical symptoms &
Points\tabularnewline
\midrule
\endhead
Duration of diarrhea days & & &\tabularnewline
0 & 0 & 0 & 0\tabularnewline
1 & 1 & 1 & 1\tabularnewline
2-3 & 2 & 2 & 2\tabularnewline
[?]4 & 3 & 3 & 3\tabularnewline
Maximum number of diarrhea stools/24h & Maximum number of diarrhea
stools/24h & Maximum number of diarrhea stools/24h &\tabularnewline
0 & 0 & 0 & 0\tabularnewline
1-3 & 1 & 1 & 1\tabularnewline
4-5 & 2 & 2 & 2\tabularnewline
[?]6 & 3 & 3 & 3\tabularnewline
Duration of vomiting days & Duration of vomiting days & Duration of
vomiting days &\tabularnewline
0 & 0 & 0 & 0\tabularnewline
1 & 1 & 1 & 1\tabularnewline
2 & 2 & 2 & 2\tabularnewline
[?]3 & 3 & 3 & 3\tabularnewline
Maximum number of vomiting episodes /24h & Maximum number of vomiting
episodes /24h & Maximum number of vomiting episodes /24h
&\tabularnewline
0 & 0 & 0 & 0\tabularnewline
1-3 & 1 & 1 & 1\tabularnewline
4-5 & 2 & 2 & 2\tabularnewline
[?]6 & 3 & 3 & 3\tabularnewline
Chills & Chills & Chills &\tabularnewline
No & 0 & 0 & 0\tabularnewline
Yes & 2 & 2 & 2\tabularnewline
Headache, Nausea, Abdominal cramp, and Myalgia & Headache, Nausea,
Abdominal cramp, and Myalgia & Headache, Nausea, Abdominal cramp, and
Myalgia &\tabularnewline
Yes[?]1 & Yes[?]1 & 1 & 1\tabularnewline
No\textless{}1 & No\textless{}1 & 0 & 0\tabularnewline
Fever & Fever & Fever &\tabularnewline
[?]37.0C\selectlanguage{ngerman}° & 0 & 0 & 0\tabularnewline
37.1-38.4C\selectlanguage{ngerman}° & 1 & 1 & 1\tabularnewline
38.5-38.9C\selectlanguage{ngerman}° & 2 & 2 & 2\tabularnewline
[?]39C\selectlanguage{ngerman}° & 3 & 3 & 3\tabularnewline
\bottomrule
\end{longtable}

Table 2: Characteristics of subjects challenged with SMV Inoculum 1 and
Inoculum 2\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& No. (\%) of subjects challenged with & No. (\%) of subjects challenged
with &\tabularnewline
\midrule
\endhead
Characteristics & Inoculum 1 (N=15) & Inoculum 2 (N=33) &
P\tabularnewline
Sex & & & 0.48\textsuperscript{a}\tabularnewline
Male & 7(46.7) & 20(60.6) &\tabularnewline
Female & 8(53.3) & 13(39.4) &\tabularnewline
Race/Ethnicity & & & 0.019\textsuperscript{b}\tabularnewline
African American & 4(26.7) & 22(66.6) &\tabularnewline
White & 11(73.3) & 9(27.3) &\tabularnewline
Other & 0(0) & 2(6.1) &\tabularnewline
Secretor Status & & & 0.81\textsuperscript{b}\tabularnewline
Positive & 11(73.3) & 25(75.7) &\tabularnewline
Negative & 4(26.7) & 8(24.3) &\tabularnewline
Age (years) & 30.7(9.3)\textsuperscript{c} &
32.9(9.3)\textsuperscript{c} & 0.35\textsuperscript{d}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a}Chi-square P-value

\textsuperscript{b}Fisher's exact test P\emph{-} value

\textsuperscript{c}Mean (Standard deviation)

\textsuperscript{d}Two-sample \emph{t-} test P-value

Table 3: Comparison of infection and illness in subjects challenged with
SMV Inoculum 1

and Inoculum 2\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& Inoculum 1 (N=15) & Inoculum 2 (N=33) & &\tabularnewline
\midrule
\endhead
Characteristics & No. (\%) & No. (\%) & Total &
P\textsuperscript{a}\tabularnewline
Infection\textsuperscript{b} & 9 (60.0) & 25 (75.7) & 34(70.8) &
0.54\tabularnewline
Illness\textsuperscript{c} & 7 (46.7) & 9 (27.3) & 16(33.3) &
0.41\tabularnewline
\bottomrule
\end{longtable}

SMV: Snow Mountain Virus

\textsuperscript{a}Chi-square test P-value

\textsuperscript{b}Infection was defined as SMV RNA detection in any
post-challenge stool sample by specific RT-PCR or RT-qPCR and/or
anti-SMV serum IgG conversion by ELISA in any post-challenge serum
sample

\textsuperscript{c}Illness was defined as with diarrhea [?]3 loose or
liquid stools or [?]300 g of loose or liquid

stools in any continuous 24-h period or one or more vomiting episodes
during the inpatient period and

with one additional clinical sign or symptom such as fever, abdominal
cramps, nausea, headache, chills, fatigue, or myalgia

Table 4: Comparison of mean illness severity score between subjects
infected with

Inoculum 1 and Inoculum 2\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& & & Modified Vesikari Score 95\% CI & Modified Vesikari Score 95\% CI
& Modified Vesikari Score 95\% CI &\tabularnewline
\midrule
\endhead
Inoculum & N & No. Illness & Mean & SD & 95\% CI &
P\textsuperscript{a}\tabularnewline
1 & 15 & 7 & 6.00 & 1.85 & (4.97, 7.03) & 0.003\tabularnewline
2 & 33 & 9 & 2.94 & 3.43 & (1.74, 4.14) &\tabularnewline
\bottomrule
\end{longtable}

CI: Confidence interval

\textsuperscript{a}Mann-Whitney P value indicating the probability of a
statistically significant difference

in mean severity scores between subjects with illness challenged with
Inoculum 1 and Inoculum 2

Table 5. PCR detection of SMV RNA in post-challenge stool samples and
anti-SMV IgG seroconversion in post-challenge serum samples from human
subjects challenged with SMV Inoculum 1 and Inoculum 2\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
& Inoculum 1 (N=15) & Inoculum 1 (N=15) & Inoculum 2 (N=33) & Inoculum 2
(N=33) &\tabularnewline
\midrule
\endhead
& No & No. Positive\textsuperscript{a} (\%) & No & No.
Positive\textsuperscript{b} (\%) & P\tabularnewline
RT-PCR results & & & & &\tabularnewline
Day 1-3 & 15 & 9(60.0) & 33 & 23(69.7) &
0.60\textsuperscript{c}\tabularnewline
Day 4-6 & 15 & 7(46.6) & 32 & 0(0) &
\textless{}0.001\textsuperscript{d}\tabularnewline
Day 15 & 15 & 0(0) & 32 & 1(3.1) & -\tabularnewline
Day 30 & 13 & 0(0) & 31 & 1(3.2) & -\tabularnewline
Day 45 & 2 & 0(0) & 28 & 1(3.6) & -\tabularnewline
Serum IgG conversion\textsuperscript{e} & & & & &\tabularnewline
Day 1-3 & 15 & 0(0) & 33 & 0(0) & -\tabularnewline
Day 4-6 & 14 & 8(57.1) & 32 & 0(0) &
\textless{}0.001\textsuperscript{c}\tabularnewline
Day 15 & 15 & 8(53.3) & 32 & 13(40.6) &
0.41\textsuperscript{c}\tabularnewline
Day 30 & 14 & 7(50.0) & 31 & 15(48.4) &
0.92\textsuperscript{c}\tabularnewline
Day 45 & 1 & 1(100) & 29 & 13(48.3) &
0.96\textsuperscript{d}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a}RT-PCR positive was defined as visible SMV-specific
amplified PCR product on agarose gel in the study with Inoculum 1

\textsuperscript{b}RT-qPCR positive was defined as CT
values\emph{\textless{}} 40 in duplicate reactions in the study with
Inoculum 2

\textsuperscript{c}Chi-square test

\textsuperscript{d}Fisher's exact test

``-`` Statistical analysis was not performed due to small sample size

\textsuperscript{e}Serum conversion was defined as anti-SMV serum IgG
conversion (\textgreater{}4 fold) by ELISA in any post-challenge serum
sample vs. pre-challenge serum sample\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1. Association between the modified Vesikari severity score of
gastroenteritis and log\textsubscript{10} challenge dose of SMV inoculum
(genome copies). Simple linear regressions were performed for subjects
in two challenge studies and fitted line (in blue) and confidence
interval (in gray) are presented.

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