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Review 1: "Interferons and Tuft Cell Numbers are Bottlenecks for Persistent Murine Norovirus Infection"

The reviewer also raised questions about potential confounding effects from mouse-to-mouse re-infection that weren't fully addressed in the study.

Published onJul 01, 2024
Review 1: "Interferons and Tuft Cell Numbers are Bottlenecks for Persistent Murine Norovirus Infection"
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key-enterThis Pub is a Review of
Interferons and tuft cell numbers are bottlenecks for persistent murine norovirus infection
Interferons and tuft cell numbers are bottlenecks for persistent murine norovirus infection
Description

Abstract Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractable in vivo model for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3’ position of the NS4 gene and used this pool as our viral inoculum for in vivo infections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts.

RR:C19 Evidence Scale rating by reviewer:

  • Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review: This paper is an interesting and well-executed study of viral population dynamics for an important enteric virus using an in vivo mouse model. It is presented in a cohesive manner and findings are largely congruent with the data. A major strength of the study is development of barcoded MNV, and the application of this novel tool to define the most important bottlenecks in this infection. The authors rigorously characterize this new virus in cell culture prior to use in vivo, increasing confidence in the results.

Mice infected with the barcoded MNV pool are used to compare barcode identities across tissue sites. The authors find that establishment of infection in the first five days is a bottleneck controlled by type III interferon and the number of susceptible epithelial cells in the intestine. Spread of infection to the spleen is also a bottleneck that is separately controlled by type I and II interferons. Persistent infection in the intestine is an additional bottleneck that is associated with further restriction in barcode diversity over time. These data identify important restriction points and the data are convincing.

Unexpectedly, the authors find that there is not always overlap between viral barcodes at different sites within a single host (e.g. between small and large intestinal tissues or between intestine and spleen). This suggests that there may be multiple independently-sustained regions of infection. A minor limitation of this system is the relatively few barcodes used and their somewhat unequal proportion (Fig. 1D) which decreases the power of the analyses. The conclusions of the paper are appropriately circumspect and future studies with larger numbers of barcodes may be needed to quantitate the full extent of this phenotype.

One additional discussion point we would like clarified is how mouse-to-mouse re-infection via fecal-oral route from shed virus might be affecting the population dynamics. If bottlenecks restrict viral diversity, a significant confounding variable could be re-infection from co-housed mice. This might be reflected in shed virus and intestinally localized virus coming from different “rounds” of infection. In this way, different barcodes could be seen to “cycle” through the intestinal track. A simple test of this using available data might be to see if the specific barcodes present in a tissue change over time, or are static (eg, in fig. 3a are the specific barcodes seen in 5dpi and 21dpi shared or unique for each mouse). A related point is that the authors should describe how the mice are housed. It is unclear if they are co-housed, and whether corporophagy is restricted; these factors could influence the results. In the future, it would be interesting to see how differential housing and preventing of coprophagy in infected mice affects the results.

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