Cell: New study pinpoints the entrance and exit of the new coronavirus inside the human nose

Someone just coughed
at you.
On the
plane.
At
a dinner party.
When
queuing at the supermarket.
If there is a "morning after" nasal spray that eliminates the ability of respiratory viruses to colonize your nose and throat, that's
fine.

In a new study, Dr.
Peter Jackson, professor of pathology and professor of microbiology and immunology at Stanford University School of Medicine, and Raul Andino, Ph.
D.
, professor of microbiology and immunology at the University of California, San Francisco, and colleagues brought this possibility closer to reality
by identifying the routes that SARS-CoV-2 --- the coronavirus that causes COVID-19 --- into and out of cells in our nasal passages 。 The findings were published in the January 5, 2023 issue of the journal Cell in the paper "SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming.
"

Jackson said, "Our upper respiratory tract is not only a launching pad for lung infections, but also for infection to others
.
”

It's like a river that crosses it

The nose and airways are lined with epithelial tissue consisting mainly of three cell types--- basal cells, goblet cells and multiciliated cells, which make up about
---80% of all cells in the nasal epithelium.
Polycilia cells form a protective barrier that prevents viruses from entering the airways
.

Jackson and his colleagues focused on two structures found on multiciliated epithelial cells: cilia and microvilli
.
While both structures are well known, they have not previously been found to be related to
how viruses enter or leave cells within the airways.

Cilia are noodle-like appendages
that grow from various cells to the outer surface.
A single nasal epithelial cell may carry up to 400 of these whip-like lines on its nasal-facing surface, all of which are beating
in harmony and continuously.
They have a thin layer of protein called mucin on top of them, and a layer of mucus
on top of them.

Jackson said mucin molecules can connect with each other to form a network that resembles an elastic, three-dimensional chain fence, preventing larger viruses such as SARS-CoV-2 from entering upper respiratory tract cells
.
The mucus coat captures viral particles, bacteria, environmental debris, and cell-breaking up waste and keeps the cells located underneath moist
.

The cilia of the epithelial cells of the upper respiratory tract pass through this layer of mucus, and their synchronized beating creates a kind of wave, like a slow-flowing river, pushing the mucus and the particles it traps forward to where
it can be coughed up or swallowed and digested.

Another common feature of almost all animal cells is microvilli, small finger-like spines
that extend from the surface of the cell.
Microvilli can grab and transport subcellular granules and vesicles
.

You say goodbye, I say hello

To get a closer look at what happens during neoviral infections, Jackson and his colleagues used a sophisticated tissue culture method to generate what they call airway epithelial organoids that mimic normal airways
.
Although lacking vascular and immune cells, these organoids fully reproduce the structure of the nasal epithelium, including an intact mucus-mucin layer and well-developed polycilia cells
.

These authors inoculated these cultures in the same Petri dish as the SARS-CoV-2 culture
.
Through light and electron microscopy and immunochemical staining, they monitor viral entry, replication, and exit
of epithelial cells.

Only polycilia cells are infected
.
Electron microscopy showed that the virus initially attached only to
cilia.
After 6 h of incubation of epithelial organoids with SARS-CoV-2, many viral particles spread from apex down on both sides
of the cilia.
Even 24 h after inoculation, the virus replicates
in only a few cells.
It took 48 hours for large-scale replication
to occur.
It also takes a full day or two for SARS-CoV-2 in real life to begin full replication
.

By lowering levels of a protein that is essential for cilia formation in nasal epithelial cells, cilia are depleted, severely slowing SARS-CoV-2 infection
.
Jackson said, "It is clear that human polycilia nasal epithelial cells are the main entry point
for SARS-CoV-2 in nasal epithelial tissue.
”

Image from Cell, 2023, doi:10.
1016/j.
cell.
2022.
11.
030
.

The authors speculate that the delay in infection is due to the airway mucus-mucin barrier
that the virus must cross.
To test this, they treated airway epithelial organoids
with a mucin-selective enzyme that disrupts the mucin network.
Jackson said it accelerated the virus's entry rate within 24 hours from "almost undetectable to" to "easily detectable," and he concluded that eliminating mucin from this reticular structure could prevent it from blocking SARS-CoV-2's ability to
infect airway epithelial organoids.

In patients with a very rare disease--- primary ciliary dyskinesia---, their ability to beat is affected or no longer synchronized, and mucus flow loses its directionality
.

In the airway epithelial organoids produced from these patients, the virus attaches to cilia similar to what is seen in normal cells
.
After 24 h of seeding, cell infection rates were also similar
to those of normally infected cells.
Microvilli on the cell surface look normal
.
But after 48 hours, SARS-CoV-2 infected far fewer cells overall--- it could only infect cells in its immediate vicinity--- suggesting that once SARS-CoV-2 began replicating inside infected cells, the virus relied on adequate mucus flow to help it spread
throughout the upper respiratory tract.

A previous study has shown that ACE2 receptors, as classic SARS-CoV-2-bound cell surface molecules, are concentrated on the cilia of nasal epithelial cells (Nature Communications, 2020, doi:10.
1038/s41467-020-19145-6).

The new study shows that SARS-CoV-2 binds
to the cilia of epithelial cells through this receptor.

From there, Jackson says, the virus may slide across this mucus-mucin barrier in one of two ways: either by jumping from one side of the cilia in a hopscotch fashion, from one ACE2 molecule to another until it reaches the body of the cell, where it fuses with the cell membrane and crawls in; Either it enters the cilia in the form of a wedge and takes an internal freight elevator to reach the body
of the cell.

"Once the virus slips through this barrier, it can replicate
freely in the cells underneath," he said.
”

The authors also found that SARS-CoV-2, once inside nasal epithelial cells, induces the activity of intracellular enzymes, causing microvilli to expand and branch, like crazy cactus plants, until their tips protrude out of the mucus barrier
.
Their number increases
.
24 hours after inoculation, many altered microvilli--- often less than half the length of cilia--- have turned into huge, branched, dendritic structures about or larger in size than cilia, which are decorated with attached viral particles that can enter the mucus-mucin barrier, where they can drift along the mucus, infecting other, more distant cells
.

The authors identified enzymes in nasal epithelial cells that are turned on in large quantities by SARS-CoV-2 infection, leading to this shift
in microvilli.
Inhibiting these enzymes stops this alteration and greatly reduces the spread of the virus to other cells
.

One spray to combine them all?

Jackson and his colleagues observed similar results
when cultured with airway epithelial organoids --- the currently circulating respiratory syncytial virus and the less common parainfluenza virus — and BA.
1 (a subvariant of the Omicron variant of SARS-CoV-2) with airway epithelial organoids.

Omicron was more contagious and, as expected, it infected multiciliated epithelial cells
in airway tissue faster than older strains used for other SARS-CoV-2 experiments.
But inhibiting the entry or exit of the virus in airway cells has still proven effective, even against this highly contagious SARS-CoV-2 variant
.

Jackson said these viral entry mechanisms may be a common feature
of many respiratory viruses.

These findings identify new targets for nasal drugs, and by impeding cilia movement or microvilli giganticization, may even prevent unknown respiratory viruses from establishing themselves in the nose or throat of humans
.

Jackson said the substances used in these experiments may be optimized for nasal sprays shortly after exposure to respiratory viruses, or as prophylactic drugs
.

"Delaying the entry, exit or spread of the virus with a topical, short-term drug will help our immune system catch up and arrive in time to stop a full-blown infection and, hopefully, limit future pandemics
," he said.
(Biovalley Bioon.
com)

Resources:

1.
Chien-Ting Wu et al.
 SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming.
Cell, 2023, doi:10.
1016/j.
cell.
2022.
11.
030.

2.
Scientists pinpoint COVID-19 virus's entry and exit ports inside our noses

https://med.
stanford.
edu/news/all-news/2023/01/covid-virus-infection-nasal.
html