Vesicle-Cloaked Noroviruses

Submitted by Lyndsi Leprowse

The topic of this discussion are Caliciviruses, and this discussion goes into specific detail about that of a certain member of the Caliciviridae family, called Norovirus. The family of Caliciviridae consists of seven species that are divided among five genera, which are: lagoviruses, neboviruses, noroviruses, sapoviruses, and vesiviruses1. Of the five genera in this family, only two infect humans, one of which is the Norovirus. According to the book, in developing countries 200,000 deaths per year are caused by noroviruses, and nearly 1,000 in the US, all in children that are under five years old1. The Caliciviridae family is made up of non-enveloped viruses that are icosahedral, and are 27-40 nanometers in diameter1.

The genome of the Norovirus is polyadenylate, and it is a positive-sense single-stranded RNA that is roughly 7.3-7.5 kb in size1. The 3’ end is polyadenylated, and the 5’ end of the genome has the virus protein VPg attached1. VPg then acts as a cap substitute and recruits factors that initiate translation of mRNAs1. As for how this virus can be transmitted, the most common causes of transmission are the faecal-oral route, inhalation of aerosols from vomit, and point source outbreaks from contaminated food and water1. Noroviruses are also more likely to occur during the winter, however, different Caliciviruses can be seen at all times of the year1.

The paper, Vesicle-Cloaked Virus Clusters Are Optimal Units for Inter-organismal Viral Transmission, discusses how both rotaviruses and noroviruses might enter a cell. While this paper discusses both Rotavirus and Norovirus, I will mainly only be going into detail about the Norovirus. The purpose of this paper was to determine how exactly Rotavirus and Norovirus are shed from the body. It is well known that Caliciviruses are shed in the body through the faecal matter, however, this paper is trying to determine exactly how the Rotavirus and Norovirus are shed and then passed onto the next host after being shed.

While this study shows how these two viruses are released, it is important to note that the two viruses are also very different in size. For this study, it was determined that both rotaviruses and noroviruses are released by extracellular vesicles. However, since the size of the norovirus is anywhere from 27-40 nanometers in diameter, and rotaviruses are around 75 nanometers in diameter, it has been determined that the rotaviruses are released in extracellular vesicles, while noroviruses are released by exosomes. However, exosomes are actually a type of extracellular vesicle.

To begin their experiment, stool samples from infected patients were taken and incubated using TIM-4-coupled beads2. After incubating, the sample was run on an SDS-PAGE/western analysis using anti-human norovirus VP1 antibody. After analyzing picture A in figure 3 of the article, it can be seen that the stool sample had the presence of the human norovirus capsid protein VP1 that was associated with the phosphatidylserine (PS) vesicles2. Once the infection was found within the stool sample, the sample was then placed under a negative-stain electron micrograph and these images show that the norovirus was contained within a small exosome2.

Norovirus vesicles.gif

Figure 1: This figure is an example of how a TIM4 antibody attaches to the VP13.

From the data found from the first two images in figure 3 of the paper, it was concluded that the human norovirus was shed non-lytically into the human stool by escaping inside of exosomes that came from the infected host2. To determine whether or not this information was accurate, more studies needed to be done. To continue to research this, the cells of a mouse phage called RAW264.7 were infected in culture with non-lytic murine norovirus (MNV-1). The purpose of this experiment was to test the permeability of the membrane of the mouse phage. The results of this test showed that approximately one hundred percent of the MNV-1 cells left the RAW264.7 cells without breaking the membrane2.

After the results of the third experiment confirmed the first two experiments, the next experiment was done using extracellular MNV-1 that was enriched in small exosome size PS vesicles, and were then centrifuged at 104 x g, and 105 x g. Both the supernatant and the pellet from both of these centrifugation techniques were then incubated with either Annexin V-coupled magnetic beads (ANX) or control (CTL) magnetic beads, and then run on a SDS-PAGE or western blot analysis using anti-murine norovirus antibody VP1, which was also the antibody that was used during the first SDS-PAGE/western analysis2. From the results that are shown in figure 3, it can be determined that the norovirus is not actually present in the supernatant, but it is present in the pellet, and it also seems to show that the presence is more noticeable at 105 x g. This indicates that it is possible that whatever was found at 104 x g was actually debris. Just as with the first two figures, they took the results from the SDS-PAGE/ western of the 105 Annexin V bead and observed them through a negative-stain electron micrograph, and it revealed small exosome-size vesicles containing MNV-1 particles2.

MNV-1 were then treated with acute levels of GW4869, and the MNV-1 levels were then reduced, which was similar to multivesicular body (MVB) derived exosomes2. The replication from this experiment was not affected, and the lipid analysis of the vesicles from MALDI-TOF/MS showed that there was a presence of bis(monoacylglycerol)phosphate (BMP), which is a lipid enriched in MVBs and MVB-derived exosomes2. They then took the sample to test whether or not the exosomes containing the norovirus were infectious. To do this, they inoculated RAW264.7 cells and human enteroid cell cultures with either human norovirus or MNV-1-containing exosomes2. An increase in human norovirus genome copies was measured after the vesicle inoculum was washed off of the enteroids, and this showed that the exosomes were infectious, as well as the MNV-1-containing exosomes when inoculated into new RAW264.7 cells2.

From this information, another test was done to determine the infectivity of MNV-1 without the receptor. It was found, however, that without the MNV-1 receptor, CD300lf, the cells could not be infected. CD300lf is a member of a family of PS receptors2. This result was tested to rule out a simple vesicle membrane-host plasma membrane fusion as a way of delivering the virus to the host2.  The cells were then inoculated with CellBrite Fix 488-labeled fluorescent MNV-1 exosomes, and then z-sectioning showed that there was approximately a 50% decrease in the cells that were treated with the anti-CD300lf antibodies compared to those that were treated with the immunoglobulin G (IgG) antibodies2. The study then suggests that CD300lf could serve a dual purpose: the first being to allow exosome internalization into an endocytic compartment through the interaction with the vesicle PS lipids, and also binding to the MNV capsids and mediating genome transfer into the cytosol2.

The results of this study showed that rotaviruses and noroviruses that were not surrounded by a vesicle were less likely to cause a problem, whereas if the viruses were in larger populations inside vesicles, have a higher chance of infection rate and overcoming replication barriers. This result shows that if viruses are clustering together in higher populations inside of vesicles and then moving from host to host, then infection is more likely to occur. Also, if the viruses are clustering inside of vesicles then they are more likely to use more than just vesicles, and more than these two viruses are likely to spread this way as well. The results of this showed that in order to disrupt the clustering among viruses, antiviral therapeutics need to be used2.

References

  1. Oxford, J., Kellam, P., and Collier, L. (2016). Human Virology. New York: Oxford University Press
  2. Santiana, M., Ghosh, S., Ho, B.A., et al. (2018). Vesicle-Cloaked Virus Clusters Are Optimal Units for Inter-organismal Viral Transmission. Cell Host & Microbe 24, 208-220.
  3. Graff, J. Virology. September 25, 2018.

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