SARS-CoV-2 Entry: The Potential EV Hitchhiker

You can find the original article here, and the Ya-Wen Chen Lab (Icahn Medical School at Mount Sinai) here.

Introduction

The COVID-19 pandemic, caused by SARS-CoV-2, is an event whose modern-day impact precludes introduction. In December 2019, an outbreak of SARS-CoV-2 emerged from a cluster of patients in Wuhan, situated in China’s Hubei province (1). By November 2025, upwards of 7.1 million deaths worldwide had been attributed to the pandemic, especially among adults above the age of 65 (2). SARS-CoV-2 infection is usually characterized, within the first 0-7 days post-infection, by its constitutional symptoms (the generic, body-wide symptoms associated with “normal” disease progression). In the vast majority of infections, this is followed by a gradual return to health following bodily elimination of the virus. For certain groups of individuals (especially older adults, immunocompromised individuals, and those with underlying pulmonary or respiratory illness), however, internal dysregulation or severe COVID-19 disease may manifest instead. This is usually due to the emergence of a “cytokine storm” (essentially an overproduction of molecules that cause inflammation), and SARS-CoV-2 infection of cell types outside of its normal hosts. There is also “Long COVID”, a poorly understood condition wherein patients experience internal dysregulation months to years after virus elimination (2). Overall, SARS-CoV-2 creates a very diverse set of illnesses and conditions, and the reasons for this remain poorly understood. Given these discrepancies, there has been a major focus in the scientific enterprise over the last several years to fully understand and characterize SARS-CoV-2 infection mechanisms, with the hope of producing more effective and specific therapies for affected individuals, especially given the large number of coronaviruses circulating in mammal populations. This paper, published from the Ya-Wen Chen Lab at the Icahn Medical School at Mount Sinai, explores a poorly understood but intriguing aspect of SARS-CoV-2 infection: how it can infect cells that do not possess its attachment receptors (molecules on cells that the virus uses to gain entry).

An Upheaval in SARS-CoV-2 Entry

Viruses build their outer proteins into super-specific shapes, and in the world of proteins, shape determines what you can do and what you can interact with. By doing so, viruses, under evolutionary pressure, design their outer proteins to specifically and strongly interact with host cells' outer proteins. This allows them to gain entry to these cells and determines which cells they can interact with. In the case of SARS-CoV-2, established models affirmed that the virus interacts with Angiotensin Converting Enzyme 2, or ACE2, on pulmonary epithelial cells (cells that comprise the surfaces of much of the lungs. This places the virus in an endosome (an internal cellular compartment) that then showers the virus with acids that allow it to release its genetic material. An alternate entry mechanism, however, involves TMPRSS2, a protein that changes SARS-CoV-2’s outer protein shapes, which in turn allows the virus’s outer membrane to fuse with the membrane of the host cell. 

The latter explanation generally assumes that these proteins are on the surface of cells, since the virus needs to access them first in order to gain entry. However, imaging of the cells reveals that TMPRSS2 is almost exclusively found on the inside of cells, near but not breaching the surface:

Fig. 1A, C: These results show that TMPRSS2 exists inside the cells. To the left, Figure 1A shows images of cells with dyes that stain TMPRSS2 and EPCAM, a surface protein. As can be seen, TMPRSS2 exists in an inner band within the EPCAM signals, indicating that it exists inside the cells, rather than on the surface. On the right, we can see the results of flow cytometry, where the further along an axis a signal is, the greater the amount of protein is in that sample. In the surface samples, we can see high levels of EPCAM-APC but low levels of TMPRSS2. In the inner samples, we observe high levels of both signals, indicating that TMPRSS2 is exclusively intracellular.

Fig. 2A and Fig. S5A: These results show that TMPRSS2 exists in extracellular vesicles or EVs (which are small compartments that carry stuff out of cells). To the left, there are more images showing TMPRSS2 in green and common “markers” of EVs in red. As can be seen, there is significant overlap between these signals. To the right, we can see that a great proportion of these EV markers are in the same places as TMPRSS2 (and vice versa), indicating that TMPRSS2 may exist inside of EVs.

Fig 3A-B: These results show that TMPRSS2 is expressed on the surface of pulmonary epithelial cells in diseased lungs. In A, we see that healthy lung cells have low levels of TMPRSS2 compared to EPCAM, indicating little to no TMPRSS2 is on their surfaces. In B, we see that amongst patients with idiopathic pulmonary fibrosis (IPF), fibrosis, or interstitial lung disease (ILD), there are small but nonzero levels of TMPRSS2 on cell surfaces

From the above results, the authors begin to challenge the latter SARS-CoV-2 entry mechanism. Only in diseased lungs is TMPRSS2 on the surface, since in healthy lung cells, TMPRSS2 remains in extracellular vesicles. EVs are notable in that they tend to be absorbed by other cells, especially phagocytic cells (cells that engulf and consume given materials). Based on these findings, then, the authors began to look for cells that contain TMPRSS2 but aren’t capable of producing the molecule, as they may be absorbing it through these EVs.

A Striking Discovery: SARS-CoV-2 can enter Cell Types that do not Produce TMPRSS2

In their search, the authors find that alveolar macrophages, or AMs, and endothelial cells (ECs) expressed TMPRSS2 on their surfaces despite not creating the genetic material needed to produce TMPRSS2. Alveolar macrophages are phagocytic cells that patrol the alveoli for incoming disease threats. Endothelial cells are cells that comprise the lining of blood vessels, and do not normally express TMPRSS2 at all. However, the authors find that some of these cells nevertheless have TMPRSS2 on their surfaces:

Fig 5A-B: These results show that, in some diseased lungs, endothelial cells and alveolar macrophages possess TMPRSS2 on their surfaces, despite these cell types being incapable of doing so. We can see in A and in B that TMPRSS2 levels are high in both cell populations. 

Fig 6C, E: These results show that, within purified alveolar macrophages and endothelial cells, adding the aforementioned EVs caused the sudden appearance of TMPRSS2 on their surfaces. In C and E, we can see that adding EVs caused a massive increase in the TMPRSS2 signal. This perhaps explains why, in certain cases, cells have TMPRSS2 on their surfaces even if they are incapable of producing it, and a potential way in which they then acquire TMPRSS2. 

Fig 7B-C: These results show that adding EVs to stem cells increases their susceptibility to SARS-CoV-2 infection. We can see in both panels that viral titers (concentrations) are massively increased when EVs are added. Stem cells are cells that haven’t developed into any given cell type, so they shouldn’t normally produce TMPRSS2 since they haven’t developed in pulmonary epithelial cells. However, by adding EVs, SARS-CoV-2 was able to infect them nonetheless, suggesting that the EVs were installing TMPRSS2 onto their surfaces, and thus allowing the virus to gain entry.

The aggregate of these experiments reveals a striking new mechanism through which SARS-CoV-2 gains access to cells. Extracellular vesicles can carry TMPRSS2 and install it onto the surface of phagocytic cells, expanding the range of cells SARS-CoV-2 can infect. This newfound host diversity may begin to explain some of the greater discrepancies regarding COVID-19 disease, especially within severe COVID-19. As aforementioned, this installation of TMPRSS2 onto cell surfaces occurs more frequently in diseased lungs, providing potential directions regarding COVID-19’s abnormal pathologies (the manifestation of disease) in such cases. One major question that remains is whether this release of EVs is a deliberate mechanism of SARS-CoV-2, meant to prolong or intensify infection, or if this release is a hijacked or deliberate mechanism of the body. The authors posit that these EVs may serve as “decoys”, meant to absorb viruses so that they cannot enter actual cells. It’s also possible, as the authors also consider, that SARS-CoV-2 deliberately “hitchhikes” within the EVs and waits for them to be absorbed by cells, which would allow the viruses to enter undetected. More experiments in these regards will be needed to meaningfully explore these avenues, but this paper nonetheless explores a fascinating aspect of SARS-CoV-2 infection. In exploring such questions, it may be possible to create better treatments against coronaviruses, especially for vulnerable individuals, and to better understand SARS-CoV-2 infection. Coronaviruses are diverse and circulate in animal populations, so there is an omnipresent threat that a new virus may emerge in humans, as has occurred a few times this century (such as SARS-CoV in 2006, MERS-CoV in 2011, and of course, SARS-CoV-2 in 2019). By studying SARS-CoV-2 in its entirety, we can better safeguard ourselves against such new diseases, improving outcomes for untold numbers of people as we continue in our ever-advancing day.

References

N.A., (2024, July 4).  CDC Museum COVID-19 Timeline. Center for Disease Control,

    https://www.cdc.gov/museum/timeline/covid19.html#print.

Zhu, Y., Sharma, L., & Chang, D. (2023). Pathophysiology and clinical management of 

coronavirus disease (COVID-19): a mini-review.Frontiers in immunology, 14, https://doi.org/10.3389/fimmu.2023.1116131. 

Rea-Moreno, M., Tian, L., Tavakol, T.N. et al. (2026). Unveiling alternate pathways for 

SARS-CoV-2 infection via extracellular vesicle-mediated transfer of ACE2 and TMPRSS2. Nature Communications, https://doi.org/10.1038/s41467-026-71680-w. 

Inapparent maternal ZIKV infection impacts fetal brain development and postnatal behavior

You can find the original article here, and the Daniels Lab can be found here. 

Zika virus became a household name in 2015, when an epidemic began in Brazil and spread throughout the Americas (1). Zika is spread through mosquito bites, and in adults it causes mild illness. The primary concern with Zika is not its impact on adults, but its effects on developing fetuses. When a pregnant woman is infected with Zika virus, the virus can cross the placenta and infect the fetus in a process known as vertical transmission. This can cause severe birth defects known collectively as congenital Zika syndrome, or CZS. CZS is as dramatic as it is rare: an estimate from the CDC in 2017 estimated that one in twenty children exposed to Zika during their mother’s pregnancy would develop birth defects (2). What happens to the other nineteen children? This paper, out of Brian Daniels’ lab at Rutgers University, looks into how milder Zika virus infections reshape the developing brain and can potentially have effects that reach far into adulthood.

           

A Mouse Model for Mild Zika

            As with many other diseases, we study Zika virus through observing the course of infection in mice. Zika virus presents a unique challenge, however. Adult mice are not naturally susceptible to Zika virus infection due to differences between their immune system and ours. Traditionally, the solution to this problem was to use immunodeficient mice. While this allowed Zika virus to take hold, the resulting disease progression looks very different to what we see in most humans. The authors of this paper take a different approach. They use a specialized lineage of mice genetically altered to have a crucial immune response regulator, Stat2, replaced with the human analogue. These mice are susceptible to Zika virus because of their humanized immune systems without having to be immunocompromised, allowing for more nuanced studies like this one to go forward.

            The authors show their refinement of this mouse model in Figure 1. Their goal here is “inapparent maternal Zika infection”: infection with Zika virus that is successful but does not produce severe symptoms in the mother or the pups. This is achieved through exposing the mothers to Zika virus during the middle of their pregnancy, rather than in earlier stages. The resulting procedure has the researchers infecting the mothers at E12.5 (roughly equivalent to the late first trimester in humans) and collecting samples for analysis at E18.5 (late second to early third trimester).

            The most important elements of Figure 1 are the experiments showing that the pups of infected mothers are not showing severe symptoms of Zika infection. This figure shows that few fetuses are resorbed regardless of Zika virus exposure; resorption occurs in mice when a fetus is not viable, similar to miscarriage in humans. Additionally, the researchers show that there is no difference between the brain weights of fetuses with and without Zika virus exposure. This shows a lack of microcephaly, a classic sign of fetal Zika virus exposure that involves a newborn’s head being much smaller than normal. Taken together, these confirm that the infant mice are “clinically normal” and allow the researchers to look at the less-obvious effects of Zika virus exposure. Having confirmed that their mouse model works the way they want it to, the researchers move forward.

Figure 1I-J: This data shows that the fetuses of mice infected with Zika virus following this paper’s protocol are viable at the same rate as uninfected mice. The authors show data from two timepoints, E15.5 and E18.5. Black bars represent fetuses resorbed by the mother, a process that occurs in mice when a fetus is not viable similar to miscarriage in humans. There is no significant difference between resorption in Zika virus-infected mothers and mothers not infected.

Figure 1K: Microcephaly (small heads) can occur as a result of Zika virus in a fetus, so the authors checked brain weights to confirm that their mice were not experiencing this symptom. The bars show the average brain weights of mice exposed to Zika virus as fetuses and those not exposed, and show that there is no significant difference between the two. This demonstrates that microcephaly is not a problem in the Zika virus-exposed fetuses.

 

The transcriptome: a snapshot of activity

            Much of this paper revolves around analysis of the cellular transcriptome. While the genome is a collection of all the genes in a cell, the transcriptome provides a record of all the genes that are being expressed; that is, what is turned on and affecting the cell. This paper is looking at the transcriptome of mouse pup brains and how it is different if the mice have been exposed to Zika virus through their mothers. They find that there is a shift in activity briefly around E18.5, the timepoint used here as an analogue to late-second and early-third trimester pregnancies. Figure 2 shows this divergence: the transcriptomes of Zika virus-exposed mice and healthy mice overlap at E15.5 (1^st trimester) and at P21 (early infancy), but diverge at E18.5.

Figure 2B: These three charts visualize the transcriptomes of Zika virus-exposed and unexposed fetal mice at three timepoints (E15.5, E18.5, and P21). The exposed and unexposed samples overlap except at E18.5, suggesting that this is when the viral infection is affecting which genes are active in the cell.

            The next step after identifying if and when the transcriptomes are different is finding out how they differ. This paper does this using gene ontology enrichment analysis, which analyzes a transcriptome sample and identifies the functions of the genes found to be active. The results from this analysis show that there is a difference in the regulation of genes associated with synapse development between Zika virus-exposed and healthy mice. Synapses are connections between neurons that allow for communication between individual cells, and proper synapse formation is essential for healthy brain function. Abnormal expression of genes related to synapse function in fetal mice exposed to Zika virus suggests that these mice may have issues with their brain function, particularly since these differences are seen right as the brain is beginning to come together in earnest. It’s also important to remember that these differences are in mice who appear “clinically normal” – this is the first sign in the paper that Zika-exposed children might have underlying health issues that aren’t immediately obvious.

 

Figure 2D: This table shows the results of an initial gene ontology analysis, which looks at the functions of the genes that have different activation levels between healthy and Zika virus-exposed mice. The lines highlighted in blue show genes associated with synapse function and development, suggesting that the brain cells exposed to Zika virus will form connections with other cells differently.

 

            The next four figures build on this finding by narrowing the focus to two specific types of cells that have the greatest change in activity and studying them in depth.

 

Beyond the transcriptome: problems in later life

            Although finding these changes to cellular activity is interesting, it may be just a temporary shift as the cells deal with a brief visit from a virus. Do the cells return to normal after their encounter with Zika virus, or are there effects that last beyond the brief window of observed transcriptomic change? Figure 7 provides compelling evidence for the latter.

            The authors study the brains of P30 (early infancy) mice and observe differences in the number of excitatory synapses in the hippocampus when the mouse was exposed to Zika virus. Excitatory synapses increase the likelihood that a neuron they are connected to fires, or transmits an electrical signal. They’re necessary for normal brain function, but too many can cause a condition called excitotoxicity. Excitotoxicity happens when a neuron is damaged by too many excitatory signals, and it is a factor in neurodegenerative diseases and stroke. More excitatory synapses in the brains of Zika-exposed mice suggests that these mice are more prone to these kinds of brain damage.

Figure 7B: Here, the authors show a significant increase in the amount of excitatory synapses in the hippocampi of mice exposed to Zika virus as fetuses. This is done through measuring levels of Homer1, a fluorescent tag that marks excitatory synapses.

            The authors show how an increase in excitatory neurons might result in brain damage by testing how susceptible to stroke their Zika-exposed mice are. This is done by inducing strokes in the mice and recording how severe the stroke is. Compared with nonexposed mice, Zika-exposed mice have much more severe strokes, including a death rate twice as high and no “mild” strokes.

Figure 7H and 7I: The authors show a scale for grading seizure severity in 7H and apply it to the seizures observed in their mice in 7I. Mice exposed to Zika virus as fetuses have a greater risk of death from induced seizure, and they did not have any observed mild seizures.

            Finally, the authors test how the behavior of Zika-exposed pups differs from their unexposed counterparts as adults. These differences seem to be divided between sexes: female Zika-exposed mice show differences in movement distance, movement speed, and behaviors exhibited that aren’t present in the male mice. Although mapping mouse behavior onto human diseases is difficult, the fact that there are differences after the mice are fully grown is a concerning finding that shows how long Zika virus exposure can have an effect.

            The authors began this study asking if there are health outcomes of Zika virus exposure during development that aren’t immediately obvious at birth, and they come away from it with strong evidence that there are. Although mice are far from humans (no matter how tailored the model may be), these results are concerning and suggest that some children may have slipped through the cracks of the medical system without awareness of their underlying risk for neurological conditions. The authors suggest keeping a closer watch on children exposed to Zika virus before they are born and following up with them as they grow. Do these children have more strokes than is typical as they grow? Are there higher rates of learning disabilities or memory issues among them? While there is no guarantee that there will be, it might be better to be overcautious to ensure that these children grow up to be as healthy as possible.  

Figure 8B and 8C: 8B shows differences in distance traveled, while 8C shows differences in velocity (speed) between female mice exposed to Zika virus as fetuses (red line) and unexposed mice. The authors do not see a difference in exposed and unexposed males.

Figure 8E: This graph shows behaviors that have different likelihoods in female mice exposed to Zika virus as fetuses and unexposed females. Gray dots show behaviors that are equally likely in either mouse, red dots above the trendline show behaviors more likely in unexposed mice, and blue dots below the trendline show behaviors more likely in Zika virus-exposed mice.

 

Citations

Chou, T. et al. (2026). Inapparent maternal ZIKV infection impacts fetal brain development and         postnatal behavior. PLOS Pathogens 22(1): e1013850.             https://doi.org/10.1371/journal.ppat.1013850

1.     Hennessey, M., Fischer, M., & Staples, J. E. (2016, January 29). Zika virus spreads to new areas – Region of the Americas, May 2015-January 2016. CDC Morbidity and Mortality Weekly Report. http://dx.doi.org/10.15585/mmwr.mm6503e1.

2.     Shapiro-Mendoza, C. K. et al. (2017, June 16). Pregnancy outcomes after maternal Zika virus infection during pregnancy – U.S. Territories, January 1, 2016 – April 25, 2017. CDC Morbidity and Mortality Weekly Report. http://dx.doi.org/10.15585/mmwr.mm6623e1