Paul Has Measles is a free children's book about viruses and vaccines available in 16 languages (link). Paul Stays Home is a free children's book about COVID-19 (link).

The spike glycoprotein of SARS-CoV-2 contains a cleavage site for host cell proteases called furins. Deciphering the role of this cleavage site during infection is important for understanding the origin of the pandemic virus and its disease pattern in humans.

Back in February it was not known if the furin site in the SARS-CoV-2 was cleaved by cell proteases, and whether its presence is required for infectivity. Both questions have now been answered.

Spike cleavage sites

The figure shows amino acids at cleavage sites in the spike glycoproteins of various CoVs. A furin site is present in the spike glycoprotein of HCoV-OC43, HCoV-HKU1, MERS-CoV and SARS-CoV 2. It is called a multibasic site because it contains multiple basic (arginine) amino acids. The spike glycoproteins of HCoV-NL63, HCoV-229E, and SARS-CoV do not contain this multibasic cleavage site. Neither do SARS-related CoVs found in bats, including RaTG13, the virus with the closest overall genome sequence identity with SARS-CoV-2.

To study cleavage and function of the furin site, various CoV spike glycoproteins were engineered into vesicular stomatitis virus particles. This manipulation allowed the study of infected cells without the need for a BSL3 facility. The spike glycoprotein of SARS-CoV-2 was efficiently cleaved, while that of SARS-CoV or RaTG13 was not. Furthermore, when the SARS-CoV-2 furin site was exchanged with the corresponding sequence from SARS-CoV or RaTG13, no cleavage was observed. That cleavage was mediated by furins was verified by using specific protease inhibitors.

Cleavage of CoV spike glycoproteins is required for fusion of the viral and cell membranes upon entry. VSV harboring the spike of SARS-CoV-2 caused fusion of a human lung cell line; substitution of the furin cleavage site with the corresponding sequence from SARS-CoV or RaTG13 prevented cell fusion. However, VSV harboring the spike of SARS-CoV did cause fusion of these lung cells, due to cleavage by a different protease. These observations demonstrate that the furin cleavage site in the spike glycoprotein is essential for entry of SARS-CoV-2 into lung cells. In contrast, a monobasic cleavage site is sufficient for entry of SARS-CoV.

The activation of the spike glycoproteins of SARS-CoV-2 and MERS-CoV are therefore similar. They both must first be cleaved by furins followed by cleavage by a different cell protease, TMPRSS2.

An interesting question is the origin of the furin cleavage site it SARS-CoV-2. Its closest relative, the bat isolate RaTG13, does not have this site. Nor do any of the other bat SARS-like CoVs or the pangolin CoVs that have been isolated. However recently a newly isolated bat SARS-like CoV, RmYN02, was shown to contain a poly basic amino acid insertion in the spike glycoprotein. This observation supports the hypothesis that the furin cleavage site in SARS-CoV-2 arose by recombination among bat viruses in nature.

Paul Stays Home

Paul Stays HomeFrom the authors of Paul Has Measles comes Paul Stays Home, an illustrated book about COVID-19 and SARS-CoV-2 for children.

Paul is sad because he can’t go out. He can’t see his friends or visit his grandparents. Like everyone else, he has to wait until the coronavirus pandemic is over. What is the coronavirus? How is it spread? How can we take care of ourselves and our families?

Paul Stays Home is written by Susana López, Selene Zárate, and Marth Yocupicio, with illustrations by Eva Lobatón.

A pdf of Paul Stays Home can be downloaded free of charge in the following languages:

English (click here
Espagnol (click here)
Francais (click here)

Vincent, Kathy and Rich explain the Jenner Institute’s SARS-CoV-2 vaccine, the NIH decision to stop the Remdesivir study, and answer listener questions.

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From the Nipah Virus International Conference in Singapore, Vincent speaks with Richard Hatchett, CEO of CEPI, about its mission to stimulate and accelerate the development of vaccines against emerging infectious diseases.

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By David Tuller, DrPH

Today, May 12th, is International Awareness Day for Chronic Immunological and Neurological Diseases (CIND)—often shortened to International ME (or ME/CFS) and Fibromyalgia Awareness Day. Besides ME, other diseases included in the CIND group, per the May 12th International Awareness Day site, are chronic fatigue syndrome, Gulf War Syndrome and multiple chemical sensitivity. The date was chosen because it is the birthday of Florence Nightingale, who apparently suffered from an ME-like condition.

[continue reading…]

Daniel Griffin provides a weekly clinical update on COVID-19, then Michael Schmidt discusses how dentistry can be safely practiced during a pandemic, followed by answers to listener questions.

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By David Tuller, DrPH

I met Valerie Eliot Smith a year or so before I published my 15,000-word investigation of the PACE trial. As an experienced lawyer familiar with how libel and related torts are handled in the UK, she provided invaluable advice on legal issues. (She and her husband also suggested the name “Trial By Error,” for which I remain indebted.)

Valerie routinely posts her sharp commentary about legal issues and ME at her blog, Law and Health. Earlier this week, she posted a blog about the impact of the pandemic on the current guideline review that the National Institute for Health and Care Excellence (NICE) is conducting, as well as what that might mean for patients and the possibility of litigation. (The discussion is based mainly on UK law.)

Below is the first half of Valerie’s post.


COVID-19, NICE and ME: towards litigation?
MAY 5, 2020

[continue reading…]

coronavirusThe moment a preprint emerges describing a new patient isolate of SARS-CoV-2, with a change in the genome sequence, the world seems to explode with concern about a new viral ‘strain’. I want to explain why such angst is misguided and in the process explain exactly what is a virus strain and a virus isolate.

In science, word usage matters. And sadly, even virologists often do not use their terms properly. I’ve written about it before.

When SARS-CoV-2 is isolated from a COVID-19 patient, that virus is called an isolate. The origin of the term is clear: the virus has been isolated from a patient.

These virus isolates are all the same strain of SARS-CoV-2. They are not different strains, even if they have changes in their genome sequences. A virus strain is an isolate with a different biological property, such as binding to a different receptor, or having a distinctly different stability at higher temperatures, to give just two of many possible examples.

There is only one strain of SARS-CoV-2. The first virus isolate, taken from a Wuhan patient in December 2019, is the same strain as the most recent isolate taken anywhere else in the world in May 2020. So far no one has shown that any of these virus isolates differ in any fundamental property.

I can hear some of you shouting, but isn’t a nucleotide change enough to make a strain? The answer is a resounding NO. Every virus expelled by an infected individual differs from the next by many base changes. It would be foolish and of little utility to call each patient isolate a strain. That term is reserved only for special changes that confer a new property to the virus.

No doubt you have heard reports of different SARS-CoV-2 strains, but I assure you they are likely wrong. Some time ago it was claimed in China that there were ‘L’ and ’S’ strains with distinct pathogenicity in humans. Wrong. You will also hear that there are eight circulating strains of the virus. Wrong. These are all isolates. None have been shown to have a distinct biological property, no matter what the preprints claim.

Most of these claims are in preprints and, if the scientific review process does its job, most of them will simply be reports of new genome sequences with no associated biological changes.

The most recent offender is a preprint claiming that SARS-CoV-2 with an amino acid change in the spike glycoprotein (D614G) increases the transmissibility of the virus. The claim that this amino acid change increases viral transmission is unsubstantiated and likely incorrect. There is no doubt that viruses with the D614G change are emerging in different geographical regions of the world. Until proven otherwise, their emergence is likely due to the founder effect. Let’s say a virus with D614G emerges during replication in a person’s respiratory tract. If viruses with that change infect the next person, and the next, and so on, then the D614G change will predominate. The change is simply a single nucleotide polymorphism of little consequence. It is the noise produced by error-prone RNA synthesis by the virus. Viruses with D614S are simply virus isolates. They are not strains of SARS-CoV-2.

Because of the founder effect, showing that a particular mutation increases viral transmission in humans is very difficult. Many such claims have been made for other viruses in the past, but none have been proven. One that comes to mind is a single amino acid that emerged in the Ebolavirus glycoprotein early in the 2015 West Africa outbreak and was subsequently found in all isolates. No proof emerged that it was not simply a founder effect.

I would also caution that making claims that SARS-CoV-2 is becoming more transmissible ignores the fact that the virus is already exceedingly transmissible among humans. For an amino acid change such as D614S to be positively selected, as opposed to being maintained as a consequence of the founder effect, requires selective pressure. For such an already highly transmissible virus, the nature of such selection pressure is difficult to discern.

The perfect storm

Cytokine Stormby Gertrud U. Rey

A small subset of people are more vulnerable to severe complications and death resulting from SARS-CoV-2 infection. What causes this vulnerability?

An increasing body of evidence suggests that patients suffering from severe COVID-19 often have one or more pre-existing conditions (co-morbidities). In an effort to describe the co-morbidities of COVID-19 patients requiring hospitalization, a recent case series tracked the course of disease in 5,700 patients in 12 different New York City hospitals over a period of five weeks. The study revealed that the median age of the hospitalized patients was 63, and that 57% of these patients had hypertension, 34% had diabetes, and 42% were obese. Interestingly, the vast majority of the patients (88%) had two or more of these co-morbidities.

While these underlying diseases are clearly risk factors for severe COVID-19, the exact mechanisms responsible for the morbidity and mortality in the affected patients are unclear. Nonetheless, the overwhelming amount of literature continuously emerging on this topic provides some insight.

Sentinel cells of the innate immune system can sense the presence of viruses never seen before and trigger a cascade of events that mobilizes immune cells such as macrophages, neutrophils, and dendritic cells to the site of infection. Once there, these immune cells produce pro-inflammatory signaling proteins known as cytokines, which then cue other responses and prime adaptive T and B cells for future functions. A primary wave of cytokines includes type I interferons, which stimulate a signaling cascade that ultimately limits viral replication. As the infection progresses and the virus is cleared, the recruited immune cells and resulting cytokines typically recede and the patient recovers. However, in some cases the immune reaction continues, leading to an excessive inflammatory response often referred to as a “cytokine storm.”

In an effort to define some of the key players in SARS-CoV-2-associated cytokine storms, a group in France analyzed the cytokine responses in fifty COVID-19 patients experiencing varying disease severity. The authors found that patients with severe disease had persistently high levels of virus in the blood – a condition known as viremia, and a significantly impaired type I interferon response. In contrast, patients with mild to moderate disease exhibited low viremia and a high type I interferon response. In parallel, patients with severe disease had increased levels of the pro-inflammatory cytokines tumor necrosis factor-⍺ and interleukin-6 (IL-6), both of which are major promoters of fever and other tissue changes that occur in response to cytokine production. The authors suggest that the acute respiratory distress syndrome seen in patients with severe COVID-19 may be triggered by a combined effect of these pro-inflammatory cytokines and impaired type I interferon responses.

The authors also note that critically ill patients produced lower levels of T cells, an effect that aligns with observations that SARS-CoV-2 can infect and kill T cells. Because T cells are crucial for directly killing infected host cells and regulating and/or suppressing the immune response, this finding would further explain why patients with severe disease have more inflammation.

Increased trafficking of pro-inflammatory cytokines can also lead to dysfunctions in blood clotting. Normal blood clotting typically involves the aggregation of clotting factors to limit excessive bleeding and trap microorganisms. SARS-CoV-2 infection can cause thrombosis, the formation of small clots throughout the lungs, which can restrict blood flow and thus oxygenation of the blood in the lungs. Sometimes, pieces of the clots can break off, lodge in vessels, and cause life-threatening obstruction of blood flow to the brain or other vital organs, thereby compromising their function. The digestion of clots releases small protein fragments called D-dimers, which are being used as biomarkers of severe disease and are associated with an increased risk of death in COVID-19 patients. As clots form and break down, the supply of clotting factors is depleted, leading to leaky blood vessels and movement of fluid into the lungs and other tissues.

As we gain a better understanding of SARS-CoV-2 pathogenesis, it may be necessary to track disease progression in high-risk patients by monitoring the presence of biomarkers like tumor necrosis factor-⍺, IL-6, and D-dimers. It is possible that co-morbidities associated with severe COVID-19 also prevent production of type I interferon and exacerbate inflammation. For example, obesity can lead to reduced type I interferon responses. The severity of disease in patients with comorbidities could be mitigated with administration of interferon and/or anti-inflammatory therapies that target IL-6 or tumor necrosis factor-⍺. Clinical trials are currently underway to evaluate the benefits of IL-6 targeted therapy in the context of COVID-19. Also, if D-dimers are detected in a timely manner, patients can be treated with heparin or other blood thinners to prevent thrombosis, which might improve COVID-19 outcomes. Further studies are needed to identify additional drugs that can be used to target key inflammatory markers in SARS-CoV-2 infection.

All things considered, only about 95% of severe COVID-19 cases are accounted for by old age or the co-morbidities described above. There is a small subset of individuals under the age of 50 who become very ill and die from SARS-CoV-2 infection even in the absence of obvious co-morbidities. Experts believe that certain genetic factors may predispose younger, previously healthy individuals to severe disease. For a review of this topic, I recommend this interview with Jean-Laurent Casanova of Rockefeller University.

[Cindy Leifer provides an excellent explanation of blood clotting in the context of SARS-CoV-2 infection on episode 30 of Immune.]

The TWiV team summarizes serology-based tests for SARS-CoV-2, lack of effect of ACE inhibitors or ARBs on COVID-19 severity, and answers listener questions.

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