Virus World

The continued evolution of SARS-CoV-2 requires persistent monitoring of its subvariants. Omicron subvariants are responsible for the vast majority of SARS-CoV-2 infections worldwide, with XBB and BA.2.86 sublineages representing more than 90% of circulating strains as of January 2024. In this study, we characterized the functional properties of Spike glycoproteins from BA.2.75, CH.1.1, DV.7.1, BA.4/5, BQ.1.1, XBB, XBB.1, XBB.1.16, XBB.1.5, FD.1.1, EG.5.1, HK.3 BA.2.86 and JN.1. We tested their capacity to evade plasma-mediated recognition and neutralization, ACE2 binding, their susceptibility to cold inactivation, Spike processing, as well as the impact of temperature on Spike-ACE2 interaction.

We found that compared to the early wild-type (D614G) strain, most Omicron subvariants Spike glycoproteins evolved to escape recognition and neutralization by plasma from individuals who received a fifth dose of bivalent (BA.1 or BA.4/5) mRNA vaccine and improve ACE2 binding, particularly at low temperatures. Moreover, BA.2.86 had the best affinity for ACE2 at all temperatures tested. We found that Omicron subvariants Spike processing is associated with their susceptibility to cold inactivation. Intriguingly, we found that Spike-ACE2 binding at low temperature was significantly associated with growth rates of Omicron subvariants in humans. Overall, we report that Spikes from newly emerged Omicron subvariants are relatively more stable and resistant to plasma-mediated neutralization, present improved affinity for ACE2 which is associated, particularly at low temperatures, with their growth rates.

Preprint in bioRxiv (Jan. 23, 2024):

https://doi.org/10.1101/2024.01.20.576353 

ACE2 is the indispensable entry receptor for SARS-CoV and SARS-CoV-2. Because of the COVID-19 pandemic, it has become one of the most therapeutically targeted human molecules in biomedicine. ACE2 serves two fundamental physiological roles: as an enzyme, it alters peptide cascade balance; as a chaperone, it controls intestinal amino acid uptake. ACE2’s tissue distribution, affected by co-morbidities and sex, explains the broad tropism of coronaviruses and the clinical manifestations of SARS and COVID-19. ACE2-based therapeutics provide a universal strategy to prevent and treat SARS-CoV-2 infections, applicable to all SARS-CoV-2 variants and other emerging zoonotic coronaviruses exploiting ACE2 as their cellular receptor.

Published in Cell (March 2, 2023):

https://doi.org/10.1016/j.cell.2023.01.039

An enzyme that helps COVID-19 (coronavirus) infect the body also plays a role in inflammation and patient outcomes in inflammatory bowel disease (IBD), according to a new study led by Cedars-Sinai. The findings raise the possibility that anti-inflammatory drug therapies for IBD may aid recovery from coronavirus. The multisite study, led by Cedars-Sinai and published today in the journal Gastroenterology, focused on angiotensin-converting enzyme 2 (ACE2), which normally plays a crucial health role by activating a hormone that helps regulate blood pressure. But in COVID-19 infections, the SARS-CoV-2 virus binds to ACE2 and uses it to invade and infect cells, "hijacking" them to spread the virus.  To learn more about how ACE2 affects the body, investigators examined its role in Crohn's disease and ulcerative colitis—two types of IBD that can cause inflammation and scarring (fibrosis) in the digestive tract along with diarrhea, cramping and loss of appetite. "We chose these disorders because COVID-19, while known for attacking the lungs, frequently causes gastrointestinal symptoms," said Dermot P. McGovern, MD, Ph.D., the Joshua L. and Lisa Z. Greer Chair in Inflammatory Bowel Disease Genetics and senior author of the new study. "It was important for us to understand how COVID-19 might affect IBD patients who are treated with anti-inflammatory medications. Also, there is increasing evidence that the GI tract may serve as an alternate route for uptake of SARS-COV-2 in general."

By examining records of nearly 1,000 patients at Cedars-Sinai, Washington University in St. Louis, Missouri, and multiple other centers across North America, the team found that levels of ACE2 in the small bowel were lower in Crohn's patients and higher in the colons of ulcerative colitis patients than they were in patients without IBD. The differing ACE2 levels were associated with poorer outcomes and more severe disease in the IBD patients.  "We saw that the effect of ACE2 depended on both its specific location in the gastrointestinal tract and the specific disease involved," said McGovern, professor of Medicine and Biomedical Sciences. "So, this enzyme was a double-edged sword." In both types of IBD, treatment with infliximab, an anti-inflammatory drug, normalized the levels of ACE2 and was associated with improved disease outcomes in patients. This finding suggests these drugs, commonly used in autoimmune diseases, also might improve outcomes in COVID-19, the investigators said.

"Overall, our study supports the potential paradoxical function of ACE2 in inflammation and COVID-19," McGovern explained. "Individuals with higher ACE2 expression may be at increased risk of infection with SARS-CoV-2. But judging from our discoveries of how ACE2 works in IBD, this enzyme likely has anti-inflammatory and anti-fibrotic functions that also could help certain COVID-19 patients recover from the virus." Further research is needed to delineate the processes involving ACE2 and what they might mean for treating COVID-19 patients, he said. In support of that effort, the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health recently awarded a two-year grant of $677,036 to McGovern to examine overlaps in the mechanisms that drive inflammation in IBD and COVID-19.

Original study published in Gastroenterology (Nov. 5, 2020):

https://doi.org/10.1053/j.gastro.2020.10.041

Humans are not the only species facing a potential threat from SARS-CoV-2, the novel coronavirus that causes COVID-19, according to a new study from the University of California, Davis. An international team of scientists used genomic analysis to compare the main cellular receptor for the virus in humans -- angiotensin converting enzyme-2, or ACE2 -- in 410 different species of vertebrates, including birds, fish, amphibians, reptiles and mammals.  ACE2 is normally found on many different types of cells and tissues, including epithelial cells in the nose, mouth and lungs. In humans, 25 amino acids of the ACE2 protein are important for the virus to bind and gain entry into cells. 

The researchers used these 25 amino acid sequences of the ACE2 protein, and modeling of its predicted protein structure together with the SARS-CoV-2 spike protein, to evaluate how many of these amino acids are found in the ACE2 protein of the different species. "Animals with all 25 amino acid residues matching the human protein are predicted to be at the highest risk for contracting SARS-CoV-2 via ACE2," said Joana Damas, first author for the paper and a postdoctoral research associate at UC Davis. "The risk is predicted to decrease the more the species' ACE2 binding residues differ from humans." About 40 percent of the species potentially susceptible to SARS-CoV-2 are classified as "threatened" by the International Union for Conservation of Nature and may be especially vulnerable to human-to-animal transmission. The study was published Aug. 21 in the Proceedings of the National Academy of Sciences. 

"The data provide an important starting point for identifying vulnerable and threatened animal populations at risk of SARS-CoV-2 infection," said Harris Lewin, lead author for the study and a distinguished professor of evolution and ecology at UC Davis. "We hope it inspires practices that protect both animal and human health during the pandemic."

Endangered species predicted to be at risk

Several critically endangered primate species, such as the Western lowland gorilla, Sumatran orangutan and Northern white-cheeked gibbon, are predicted to be at very high risk of infection by SARS-CoV-2 via their ACE2 receptor. Other animals flagged as high risk include marine mammals such as gray whales and bottlenose dolphins, as well as Chinese hamsters. Domestic animals such as cats, cattle and sheep were found to have a medium risk, and dogs, horses and pigs were found to have low risk for ACE2 binding. How this relates to infection and disease risk needs to be determined by future studies, but for those species that have known infectivity data, the correlation is high. In documented cases of SARS-COV-2 infection in mink, cats, dogs, hamsters, lions and tigers, the virus may be using ACE2 receptors or they may use receptors other than ACE2 to gain access to host cells. Lower propensity for binding could translate to lower propensity for infection, or lower ability for the infection to spread in an animal or between animals once established. Because of the potential for animals to contract the novel coronavirus from humans, and vice versa, institutions including the National Zoo and the San Diego Zoo, which both contributed genomic material to the study, have strengthened programs to protect both animals and humans. "Zoonotic diseases and how to prevent human to animal transmission is not a new challenge to zoos and animal care professionals," said co-author Klaus-Peter Koepfli, senior research scientist at Smithsonian-Mason School of Conservation and former conservation biologist with the Smithsonian Conservation Biology Institute's Center for Species Survival and Center for Conservation Genomics. "This new information allows us to focus our efforts and plan accordingly to keep animals and humans safe." 

The authors urge caution against overinterpreting the predicted animal risks based on the computational results, noting the actual risks can only be confirmed with additional experimental data. The list of animals can be found here. Research has shown that the immediate ancestor of SARS-CoV-2 likely originated in a species of bat. Bats were found to be at very low risk of contracting the novel coronavirus via their ACE2 receptor, which is consistent with actual experimental data.  Whether bats directly transmitted the novel coronavirus directly to humans, or whether it went through an intermediate host, is not yet known, but the study supports the idea that one or more intermediate hosts was involved. The data allow researchers to zero in on which species might have served as an intermediate host in the wild, assisting efforts to control a future outbreak of SARS-CoV-2 infection in human and animal populations.

The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor, and is a major determinant of host range and a dominant target of neutralizing antibodies.

Here we experimentally measure how all amino-acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD’s surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open analysis pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations observed during viral surveillance.

Preprint available at bioRxiv (June 17, 2020):

https://doi.org/10.1101/2020.06.17.157982

Researchers in British Columbia have identified a set of gene variants that are likely to influence disease progression from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection to COVID-19 disease. They have identified a rare set of variants in specific amino acid residues on the protein angiotensin-converting enzyme 2 (ACE2) that affect the viral spike protein’s ability to bind to and enter human cells. A spike protein is a key structural protein on the surface of SARS-CoV and SARS-CoV2 (the causative agent in COVID-19) virions that enables interaction with human cell receptors and fusion with the host cell membrane....

For their study, the team used the gnomAD database – a resource cataloging genetic coding variants for 141,456 adults. They assessed entries on ACE2 in the database and downloaded files predicting missense variants in the protein. The database cataloged 242 coding missense variants, 15 of which were predicted to be located at or close to the ACE2 binding site for SARS-CoV-2 spike protein. On aggregating the frequency of these variants, the team estimated that they occur in approximately 3.9 per 1,000 males and 8.5 per 1,000 females. However, the distribution of these rare alleles is not even across subpopulations, and the only common one was rs41303171 (thought to encode p.Asn720Asp), which occurred in 1.7% of males and 3.2% of females in the database. 

Although the occurrence of this allele was frequent enough to be identified in genome-wide association studies, it does not lie in the ACE2 domain thought to be bound by SARS-CoV-2 and was therefore not considered a worthwhile candidate for this interaction. The second most common variant was rs4646116 (thought to encode p.Lys26Arg), which was predicted to lie next to the ACE2-Spike protein interface.

Preprint of the original study available at bioRxiV (April 14, 2020):

https://www.biorxiv.org/content/10.1101/2020.04.05.026633v1

The most distinctive comorbidities of 32 non-survivors from a group of 52 intensive care unit patients with novel coronavirus disease 2019 (COVID-19) in the study by Xiaobo Yang and colleagues were cerebrovascular diseases (22%) and diabetes (22%). Another study included 1099 patients with confirmed COVID-19, of whom 173 had severe disease with comorbidities of hypertension (23·7%), diabetes mellitus (16·2%), coronary heart diseases (5·8%), and cerebrovascular disease (2·3%). In a third study, of 140 patients who were admitted to hospital with COVID-19, 30% had hypertension and 12% had diabetes. Notably, the most frequent comorbidities reported in these three studies of patients with COVID-19 are often treated with angiotensin-converting enzyme (ACE) inhibitors; however, treatment was not assessed in either study.

Human pathogenic coronaviruses (severe acute respiratory syndrome coronavirus [SARS-CoV] and SARS-CoV-2) bind to their target cells through angiotensin-converting enzyme 2 (ACE2), which is expressed by epithelial cells of the lung, intestine, kidney, and blood vessels. The expression of ACE2 is substantially increased in patients with type 1 or type 2 diabetes, who are treated with ACE inhibitors and angiotensin II type-I receptor blockers (ARBs). Hypertension is also treated with ACE inhibitors and ARBs, which results in an upregulation of ACE2.  ACE2 can also be increased by thiazolidinediones and ibuprofen. These data suggest that ACE2 expression is increased in diabetes and treatment with ACE inhibitors and ARBs increases ACE2 expression. Consequently, the increased expression of ACE2 would facilitate infection with COVID-19. We therefore hypothesise that diabetes and hypertension treatment with ACE2-stimulating drugs increases the risk of developing severe and fatal COVID-19.

We suggest that patients with cardiac diseases, hypertension, or diabetes, who are treated with ACE2-increasing drugs, are at higher risk for severe COVID-19 infection and, therefore, should be monitored for ACE2-modulating medications, such as ACE inhibitors or ARBs. Based on a PubMed search on Feb 28, 2020, we did not find any evidence to suggest that antihypertensive calcium channel blockers increased ACE2 expression or activity, therefore these could be a suitable alternative treatment in these patients.

Published at the Lancet Respiratory Medicine (Feb. 24, 2020):

https://doi.org/10.1016/S2213-2600(20)30079-5

In Europe, the pandemic triggered in 2020 by the SARS-CoV-2 coronavirus is now largely under control. But why this virus is able to spread so efficiently remains unclear. A team of researchers led by Dr. Simone Backes, Dr. Gerti Beliu and Prof. Dr. Markus Sauer of the Julius Maximilians University of Würzburg (JMU) has now shown in a publication in "Angewandte Chemie" that some previous assumptions need to be reconsidered. For example, the virus does not bind with several surface proteins simultaneously to several receptors of the cell to be infected. This assumption has previously been an attempt to explain how viruses increase their infectivity. Binding to a single receptor also does not lead to the subsequent docking of further receptors to the virus. The Würzburg research group has now provided evidence that a single virus binds to a single receptor, opening the door for a highly efficient infection.

What could only be speculated about

SARS-CoV-2 carries an average of 20 - 40 spike proteins on its surface. With these, it binds to ACE2 receptors in the membrane of its target cells, for example in the nose and throat of humans. When these receptors are blocked with antibodies, the cell can no longer be infected. Making the ACE2 receptors and their interaction with the viral spike proteins visible microscopically has not been possible so far. Therefore, much was left to speculation - such as whether the viruses bind to multiple receptors with multiple spikes to facilitate entry into the cell. It was also considered that the receptors are present in the membrane in pairs or groups of three rather, so that they can bind more efficiently to the trimeric spike proteins. Or that they are only combined into such groups after binding to a spike protein. Both depend strongly on the density of the ACE2 receptors in the membrane.

Super-resolution microscopy made it clear

The Würzburg researchers wanted to elucidate this mystery: They labeled antibodies with dyes to make the receptors visible and countable. To do this, they used various cell lines that are used as model systems for SARS-CoV infection, and the single-molecule sensitive super-resolution microscopy method dSTORM, developed in Markus Sauer's research group. It turned out that Vero cells, for example, which are often used as a model for SARS-CoV-2 infection, only have one to two ACE2 receptors per square micrometre of cell membrane. This is very few: "In other membrane receptors, this number is often between 30 and 80," Sauer added. "The average distance between neighbouring ACE2 receptors is about 500 nanometres. It is thus much larger than a virus particle, which measures only 100 nanometres," says Backes. The idea that a virus particle with multiple spike proteins can bind to multiple receptors simultaneously is therefore very unlikely, she adds.

ACE2 receptors are always single

The following open question: Are the receptors also present as pairs or groups of three in the membrane? "No. They only occur there singly. And it stays that way even when a viral spike protein has bound to them," says Beliu, group leader at the Rudolf Virchow Center. For an infection, it is sufficient if a single spike binds to a single receptor. With these results, the JMU team was able to disprove many of the original hypotheses about the interaction of viral particles with multiple ACE2 receptors. It also showed that host cells with higher ACE2 expression are more easily to infect, as expected. However, the lipid composition of the membrane and other factors also influence infection efficiency.

What is next?

The JMU team wants to gather as much detailed knowledge as possible about the cell entry mechanism of coronaviruses in order to better understand the infection process. This could ultimately contribute to better prevention and the development of better drugs against COVID-19. Next, the Würzburg researchers want to analyse the entry mechanism with high-resolution light sheet microscopy.

Published March 2023:

https://doi.org/10.1002/anie.202300821 

Salk researchers and collaborators show how the protein damages cells, confirming COVID-19 as a primarily vascular disease. Scientists have known for a while that SARS-CoV-2’s distinctive “spike” proteins help the virus infect its host by latching on to healthy cells. Now, a major new study shows  that they also play a key role in the disease itself.  The paper, published on April 30, 2021, in Circulation Research, also shows conclusively that COVID-19 is a vascular disease, demonstrating exactly how the SARS-CoV-2 virus damages and attacks the vascular system on a cellular level. The findings help explain COVID-19’s wide variety of seemingly unconnected complications, and could open the door for new research into more effective therapies. “A lot of people think of it as a respiratory disease, but it’s really a vascular disease,” says Assistant Research Professor Uri Manor, who is co-senior author of the study. “That could explain why some people have strokes, and why some people have issues in other parts of the body. The commonality between them is that they all have vascular underpinnings.”

Salk researchers collaborated with scientists at the University of California San Diego on the paper, including co-first author Jiao Zhang and co-senior author John Shyy, among others. While the findings themselves aren’t entirely a surprise, the paper provides clear confirmation and a detailed explanation of the mechanism through which the protein damages vascular cells for the first time. There’s been a growing consensus that SARS-CoV-2 affects the vascular system, but exactly how it did so was not understood. Similarly, scientists studying other coronaviruses have long suspected that the spike protein contributed to damaging vascular endothelial cells, but this is the first time the process has been documented. In the new study, the researchers created a “pseudovirus” that was surrounded by SARS-CoV-2 classic crown of spike proteins, but did not contain any actual virus. Exposure to this pseudovirus resulted in damage to the lungs and arteries of an animal model—proving that the spike protein alone was enough to cause disease. Tissue samples showed inflammation in endothelial cells lining the pulmonary artery walls.

The team then replicated this process in the lab, exposing healthy endothelial cells (which line arteries) to the spike protein. They showed that the spike protein damaged the cells by binding ACE2. This binding disrupted ACE2’s molecular signaling to mitochondria (organelles that generate energy for cells), causing the mitochondria to become damaged and fragmented.

Previous studies have shown a similar effect when cells were exposed to the SARS-CoV-2 virus, but this is the first study to show that the damage occurs when cells are exposed to the spike protein on its own. “If you remove the replicating capabilities of the virus, it still has a major damaging effect on the vascular cells, simply by virtue of its ability to bind to this ACE2 receptor, the S protein receptor, now famous thanks to COVID,” Manor explains. “Further studies with mutant spike proteins will also provide new insight towards the infectivity and severity of mutant SARS CoV-2 viruses.” The researchers next hope to take a closer look at the mechanism by which the disrupted ACE2 protein damages mitochondria and causes them to change shape.

Original findings published in Circulation Research (March 31, 2021):

https://doi.org/10.1161/CIRCRESAHA.121.318902

Viruses enter cells and initiate infection by binding to their cognate cell surface receptors. The expression and distribution of viral entry receptors therefore regulates their tropism, determining the tissues that are infected and thus disease pathogenesis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus known to co-opt the peptidase angiotensin-converting enzyme 2 (ACE2) for cell entry.

The interaction between SARS-CoV-2 and ACE2 is critical to determining both tissue tropism and progression from early SARS-CoV-2 infection to severe coronavirus disease 2019 (COVID-19). Understanding the cellular basis of SARS-CoV-2 infection could reveal treatments that prevent the development of severe disease, and thus reduce mortality....

Published in Science (July 31, 2020):

https://doi.org/10.1126/science.abc6156

Background: Effect of angiotensin converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARB) on outcomes in patients with coronavirus disease 2019 (COVID-19) is uncertain. Available evidence is limited to a few retrospective observational studies with small number of patients.

Methods: We did a meta-analysis to assess the effect of ACEi/ARB in patients with COVID-19 on severity of disease, risk for hospitalisation, and death compared to those not on ACEi/ARB. We searched the Cochrane library, PubMed, Embase, ClinicalTrial.gov and medRxiv for studies published until 21.04.2020. Inclusion criteria included all studies with patients with confirmed COVID-19 either taking, or not taking, ACEi/ARB. Depending on degree of heterogeneity, fixed or random effect model was selected to calculate effect size (Odds ratio).

Findings: Five studies were eligible for meta-analysis. These included 308 patients on ACEi/ARB, and 1172 not on ACEi/ARB. Compared to patients with COVID-19 not on ACEi/ARB, there was a statistically significant 44% reduction in odds of developing severe disease (OR: 0.56; 95% CI: 0.34-1.89, I2=68.15), and 62% reduction in odds of death (OR: 0.38; 95% CI: 0.19-0.74, I2=0.000) in those on ACEi/ARB. There was a non-significant 19% (OR 0.81; 95% CI: 0.42-1.55, I2: 0.000) reduction in odds of hospitalisation among those on ACEi/ARB.

Interpretation: It is safe to use ACEi/ARB in patients with COVID-19 requiring these medications for associated comorbidities. Although limited by confounding factors typical of a meta-analysis of retrospective observational studies, our data suggests that use of these medications may reduce risk of developing severe disease and death.

Preprint Available at medRxiv (April 28, 2020):

https://www.medrxiv.org/content/10.1101/2020.04.23.20076661v1

COVID-19, caused by the new pandemic coronavirus, is strangely—and tragically—selective. Only some infected people get sick, and although most of the critically ill are elderly or have complicating problems such as heart disease, some killed by the disease are previously healthy and even relatively young. Researchers are now gearing up to scour the patients’ genomes for DNA variations that explain this mystery. The findings could be used to identify those most at risk of serious illness and those who might be protected, and they might also guide the search for new treatments. 

The projects range from ongoing studies with DNA for many thousands of participants, some now getting infected with the coronavirus, to new efforts that are collecting DNA from COVID-19 patients in hard-hit places such as Italy. The goal is to compare the DNA of people who have serious cases of COVID-19 (which stands for coronavirus disease 2019)—but no underlying disease like diabetes, heart or lung disease—with those with mild or no disease. “We see huge differences in clinical outcomes and across countries. How much of that is explained by genetic susceptibility is a very open question,” says geneticist Andrea Ganna of the University of Helsinki’s Institute for Molecular Medicine Finland (FIMM).

It’s hard to predict what will pop out from these gene hunts, some researchers say. But there are obvious suspects, such as the gene coding for the cell surface protein angiotensin-converting enzyme 2 (ACE2), which the coronavirus uses to enter airway cells. Variations in the ACE2 gene that alter the receptor could make it easier or harder for the virus to get into cells, says immunologist Philip Murphy of the National Institute of Allergy and Infectious Diseases, whose lab identified a relatively common mutation in another human cell surface protein, CCR5, that makes some people highly resistant to HIV.