Peer review of: Human norovirus targets enteroendocrine epithelial cells in the small intestine (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
This study investigates the cell tropism of a human norovirus during symptomatic 
infection of a highly immunocompromised pediatric patient. The findings are largely 
consistent with a previous study of multiple biopsy patients (Karandikar et al. 2016 
J Gen Virol 97(9):2291-2300), showing viral antigen detected in both intestinal 
epithelial cells and subepithelial cells including macrophages and dendritic cells. 
One novel observation in this study is the detection of viral negative sense RNA in 
enteroendocrine cells. 
 
Major concerns: 
1) The conclusions drawn by the authors are not entirely supported by the data. For 
example, the authors state repeatedly that negative sense viral RNA was detected 
nearly exclusively in intestinal epithelial cells. This is not supported by their data 
(see below). Furthermore, the authors fail to perform quantitative analysis of their 
data which would provide a more complete picture of the prevalence of specific cell 
types targeted by a human norovirus. 
2) The authors draw broad conclusions about norovirus cell tropism from studies of 
a single severely immunocompromised patient. Acknowledging the inherent 
difficulties in obtaining human tissue, it is understandable that a large sample set 
was not available. However, this does not diminish the need to be cautious and 
narrow about interpreting results from n = 1. The authors should emphasize this 
point in the manuscript because it could significantly alter the types and functional 
state of the cell types present in this tissue biopsy compared to the normal cellular 
milieu which could in turn impact the tropism of a virus infecting in this 
environment. 
 
Specific comments: 
1) The manuscript presents a biased view of human norovirus cell tropism. While 
the authors cite four studies that reported detection of viral antigen in immune cells 
in their introduction, they seem to dismiss these as relevant targets but no 
explanation for this dismissal is given. Similarly, in their discussion they state that 
“Although viral antigen was abundant in the lamina propria during acute infection, 
we could not demonstrate compelling evidence for active replication in immune 
cells.” Yet they detected abundant VP1 protein and positive sense viral RNA, and 
less abundant but still convincing levels of negative sense viral RNA (Fig. 3 and 
Extended data Fig. 4) and viral nonstructural protein (Extended data Fig. 3), in 
subepithelial cells. It is unclear why these observations are not compelling yet 
detection of the same viral replication intermediates are compelling evidence for 
epithelial cell infection. 

2) The authors fail to quantify the number of infected cells in any of their 
experiments. One assumes they selected representative images for presentation, 
but quantification across multiple fields of view and across serial sections stained 
independently will substantially increase confidence in the authors’ conclusions. 
3) Based on the images presented in Fig. 2D-F and Extended data Fig. 3, I am not 
convinced that viral negative sense RNA and nonstructural proteins were localized 
predominantly in epithelial cells. Fig. 2 images show few cells positive for negative 
sense RNA and it is unclear that a majority are epithelial cells. In panel 2D, there is 
no clear villous so it is difficult to interpret tissue architecture. In panel 2E, there 
are three positive cells, one of which is underneath the epithelium suggesting it is 
an immune or stromal cell. Similarly, in Extended data Fig. 3, there appear to be 
cells positive for nonstructural proteins in the lamina propria but without 
quantification it is hard to fully glean their prevalence compared to epithelial cell-
positive cells. 
4) I have the same concern with Fig. 3 and Extended data Fig. 4 – there appear to 
be similar numbers of epithelial and subepithelial cells positive for negative strand 
in multiple representative images although the authors state that signal is 
predominantly in epithelial cells. 
5) It is interesting that the authors found evidence for infection of enteroendocrine 
cells which has not previously been reported. It would be informative to describe 
the frequency of this infection event. The authors state that there were virus-
negative CgA-positive cells and virus-positive CgA-negative cells. What were the 
frequencies of each of these? In other words, what proportion of enteroendocrine 
cells are infected by the virus and what proportion of virus-positive cells are 
enteroendocrine cells in this patient? Considering the title of the manuscript and the 
significance presented in the abstract pertain to this finding, these data should be 
analyzed in a more rigorous manner. The same applies to M cells and tuft cells – 
how many M/tuft cells were visualized and what percentage of those were positive 
for negative sense RNA? 
6) In Extended Data Fig. 6, the authors test for co-localization of VP1, a 
macrophage marker, and cytokeratin. They conclude this pattern is “consistent with 
active scavenger activity during the acute infection.” However, they also show that 
this is not a uniform observation since VP1 is detected in the lamina propria in the 
absence of abundant cytokeratin (Extended Data Fig. 7). Moreover, lamina propria 
macrophages continuously function to phagocytose apoptotic debris from terminally 
differentiated epithelial cells even in the absence of viral infection. Detecting 
cytokeratin in an intestinal macrophage does not prove absence of viral infection 
when viral antigens are detected inside the cell, nor does it prove that the viral 
antigens were acquired by phagocytosis instead of infection. Yet the title of this 
subsection is “Dendritic cells and macrophages process large amounts of norovirus 
antigen during acute infection.” This is misleading and not indicated by the data. 
7) The B and T cell data included in the manuscript are not sufficient to draw the 

conclusion that these cell types do not support active viral replication. First, they 
did not show results from co-staining of a B cell marker and viral antigen or viral 
genomes. Second, while the data presented in Fig. 6 D-E suggest that VP1 protein 
is frequently adjacent to, but not co-localized with, T cells markers, these low-
magnification images are insufficient to conclusively rule this out. Moreover, why 
did the authors not test viral genome localization in B and T cells like they did for 
other cell types? The authors should also discuss the observation here of inapparent 
T cell infection versus the findings in Karandikar et al. where viral antigen was 
frequently detected in T cells. Finally, can the authors discuss the implications of 
this patient being on lymphocyte inhibitors (tacrolimus and mycophenolate) at the 
time of illness and endoscopy in relation to their observations? 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The manuscript titled “Human Norovirus Targets Enteroendocrine Epithelial Cells in 
the Small Intestine” describes the cellular tropism in the gastrointestinal tract of a 
human norovirus in an acutely infected pediatric patient. Using unique tissue 
samples obtained from sectional biopsies, Green et al demonstrate norovirus capsid 
antigen and positive sense RNA in both the epithelium as well as the laminia propria 
of the small intestine but not in the colon. They extend this finding by determining 
that active viral replication (detected by the negative sense RNA) was localized 
predominately to the epithelium or near the epithelium with little to no staining in 
the lamina propria. This finding was confirmed by detection of non-structural 
proteins in the same locations. Using cell type specific identifiers, the associate 
expression of enteroendocrine cell markers and negative sense RNA was found. 
They see no association of capsid proteins or viral RNA with either CD3 T cells or 
CD20 B cells. They provide interesting and novel assessments, using confocal 
microscopy, of the intracellular distribution patterns of structural, non-structural, 
positive and negative sense RNAs in the infected cells. This is a very carefully 
performed and well written study in which all reagents were validated in several 
ways including the use of intestinal biopsies from norovirus-negative patients. The 
quality of the data and images is exceptional. The detection of the viral genome is 
new and was an important tool in this study and suggests that detection of viral 
antigen in cells in the lamina propria does not automatically indicate that viral 
replication is occurring in the cell. The conclusion of this study have significant 
implications for defining the pathogenesis of norovirus. The pathogenesis of human 
norovirus has remained mostly elusive since the identification of the virus mainly 
due to the lack of relevant clinical specimens in which the questions surrounding 
pathogenesis could be addressed. There is only one paper at this time that has 
looked at norovirus in intestinal biopsies from humans and stained for cell types. 

Karandikar et al (2016) examined biopsies from immunocompromised patients in 
which norovirus is a chronic infection and was able to detect norovirus capsid 
proteins in enterocytes, macrophages, T cells, and dendritic cells while non-
structural proteins indicated replication only in enterocytes (Karankikar et al 2016). 
B cells are also thought to be a target for the virus based on infection of in vitro 
transformed human cell lines (Jones et al 2014). Recent work using untransformed 
human intestinal organoid cultures has implied that the differentiated enterocyte is 
the target of human norovirus (Ettayebi et al 2016). In the murine norovirus model, 
in which the disease differs substantially from the human situation, M cells, 
macrophages, dendritic cells, B cells, T cells, and tuft cells have been shown to be 
infected instead of enterocytes or enteroendocrine cells (Grau et al 2017, Gonzalez-
Hernandez et al 2015, Wilen et al 2018). Thus, the true tropism of human norovirus 
as it commonly occurs in acute infection of the human host is still a matter of much 
debate. Given this ongoing debate, publication of well conducted studies that look 
at different proposed cell types that support norovirus replication will provide clear 
answers that are critical to move studies on norovirus biology forward. This report 
by Green et al confirms and significantly extends the previously published work of 
Karandikar et al in chronically infected patients by clearly demonstrating both the 
enteroendocrine cell and the enterocyte exhibit evidence of infection and viral 
replication during acute infection associated with norovirus disease. This work 
provides a significant piece of information that will change the course of the debate 
on the target of norovirus replication. The association of norovirus with the 
enteroendocrine cell is novel and the relevance of this finding remains to be 
determined. Overall, this manuscript provides valuable insight into interactions 
between norovirus and the cells of the human intestine and is an exciting step in 
developing targets to either treat or prevent the infection. 
 
 
Minor Comments: 
 
• It would be great to have included co-staining for negative sense RNA or non 
structural proteins along with the dendritic cell, macrophage, B and T cell markers 
to really demonstrate limited/no viral replication was occurring in these cell 
populations 
 
• This reviewer realizes the difficulty of obtaining the clinical sample during acute 
infection but additional patient samples would provide more confidence in the 
results. 
 
• Was there any attempt to determine exactly what type of enteroendocrine cells 
was infected? Clearly not all are infected including enteroendocrine cells close to 
each other so is there something special about the infected enteroendocrine cell? 

 
 
 
Reviewer #3: 
Remarks to the Author: 
Green and colleagues performed a careful analysis of intestinal biopsies obtained 
from a patient who was status/post small intestinal transplantation and who 
subsequently became infected with a GII.4 Sydney/2012-like virus. They performed 
stains using capsid-specific, noroviral non-structural protein-specific, and positive- 
and negative-sense viral RNA specific probes, as well as probes targeting various 
cell-specific proteins. In some instances confocal microscopy was used to capture 
images. The authors found evidence of replication (expression of non-structural 
proteins, presence of negative strand RNA) occurred primarily in enterocytes but 
not in immune cells. 
 
A major question in the field has been whether murine norovirus is a representative 
model of human norovirus infection, including which types of cells are infected in 
vivo. The work by Green and colleagues builds on previous data to clearly 
document the presence of viral proteins and positive and negative strands of RNA in 
epithelial cells, including enterocytes and (for the first time) enteroendocrine cells. 
Capsid protein and positive sense viral RNA, potentially representing virus, are also 
found in the lamina propria, but negative sense RNA and non-structural proteins 
were either not observed or less clearly present (extended data figure 3E) in the 
lamina propria. These data provide strong support to the conclusion that human 
norovirus is distinct in its in vivo tropisms from that of murine norovirus. 
 
A few points should be addressed to strengthen the manuscript: 
Many enterocytes have only the VP1 capsid or positive sense genome present. If 
these findings don’t represent active infection, how do these protein or +RNA get 
into the cell in sufficient amounts to stain? Is the negative sense RNA detection as 
sensitive as detection of positive sense RNA and or viral VP1? If less sensitive, what 
implications does this have for failure to detect evidence of replication in immune 
cells? Consider adding this as a limitation of the study. 
 
It would be worthwhile to show a video file of CD20-positive cells and VP1-stained 
tissue, as is done for CD4-positive cells in video 3. Figures 6A and 6B suggest the 
possibility that some B cells within the GALT also are VP1-antigen positive. This 
should be clarified, especially if the findings are similar to those observed with the 
CD4 staining. 
 
Figure 1 – the bar in each panel represents what length? The size the bar 
represents should be noted in all figures, as is done in Figure 2. 

 
Figure 2 – the DAPI staining looks to be more purple than blue. 
 
Figure 2c – the orientation of the tissue is difficult to ascertain. Is the lumen at the 
upper left and do the intensely staining (red) cells represent enterocytes (appears 
to be from the text)? Please note the site of the lumen for clarity. 
 
Figure 3B – is the -RNA at the bottom of the panel on the other side of the villus? It 
is not clear whether it is associated with the epithelium or the lamina propria. 
Please clarify. 
 
Line 269 – it is not clear from the figures shown that the negative sense RNA is 
perinuclear. 
 
Supplementary Figure 2 legend – “magnified insets of B. and C. mark a CgA-
positive cell proximal to epithelial cells containing” Do the authors mean ‘proximate 
t0 epithelial cells’ (i.e., near such cells)? 
 
I could not get supplementary video files 1 and 3 to play.