virology blog

SARS-CoV-2 related viruses from bats in Laos

23 September 2021 by Vincent Racaniello Leave a Comment

Various SARS-CoV-2 like viruses have been isolated from bats in China, Thailand, and Japan, but none have a spike protein that can bind ACE2 and allow entry into human cells. Sampling of bats in Laos has now revealed the presence of such viruses.

The genome of a virus called RaTG13, from Rhinolophus affinis bats in China, is the closest to SARS-CoV-2 (although infectious RaTG13 has never been isolated). However the spike receptor binding domain (RBD) encoded in this genome has low sequence similarity with that of SARS-CoV-2 and its affinity for ACE2 is very limited. RaTG13 is clearly not the proximal ancestor of SARS-CoV-2.

Sampling of 645 bats from limestone caves in Northern Laos (see map) yielded three Sarbecovirus genomes, called BANAL-52, -103, and -236, with high sequence similarity to SARS-CoV-2 and RaTG13. These three viral genomes were obtained from three different Rhinolophus species. Of the 17 amino acids that interact with the receptor binding domain of ACE2, 16 are conserved between SARS-CoV-2 and BANAL-52 or -103, and 15/17 conserved with BANAL-236. In contrast, only 11/17 RBD amino acids are conserved among SARS-CoV-2 and RaTG13. In other words, the RBD encoded in these BANAL genomes are closer to SARS-CoV-2 than that of any other known bat virus.

Binding affinity assays done with purified proteins revealed that the BANAL-52/103 and -236 spikes bind ACE2 with affinities in the low nanomolar range, comparable to reported values for the SARS-CoV-2 spike.

Lentiviral particles with the spike protein from BANAL-236 were able to bind and enter human cells producing ACE2. Entry of this virus was blocked by human sera containing antibodies to SARS-CoV-2 but not by control sera.

Infectious BANAL-236 virus was recovered from a bat fecal swab after inoculation of Vero cells, providing a rare virus isolate for a bat SARS-CoV-2 like virus. The virus will be useful for studying the biology of infection.

Recombination analysis demonstrated that the SARS-CoV-2 genome is a mosaic of at least five different genomes, including BANAL-52, -103, and -236, and the previously published RmYN02, RpYN06, and RaTG13, the latter all discovered in China. The implication of these observations is clear: SARS-CoV-2 likely arose from recombination of viruses circulating in different species of Rhinolophus bats in the limestone caves of South China and Southeast Asia. Because the RBD of the BANAL isolates can mediate binding to and entry into cells that produce ACE2, no host to host passage need be hypothesized to explain increased RBD affinity in an intermediate host before spillover into humans.

The spike proteins encoded in the BANAL isolates do not have the furin cleavage site found in the protein from SARS-CoV-2. Such sites might be present in viruses within these bat communities, but were missed due to insufficient sampling. Alternatively, it is possible that selection for the furin cleavage site occurred after spillover into humans or another intermediate host.

These findings further emphasize and demonstrate that SARS-CoV-2 came from Nature, not from a lab.

Trial By Error: Advocates Propose Fixes to Dysfunctional US Coding System for ME, CFS and ME/CFS

20 September 2021 by David Tuller 9 Comments

By David Tuller, DrPH

I have been focused lately on the continuing saga of the hijacked-and-still-unpublished ME/CFS clinical guidelines (HSUME/CFSCG) from Britain’s National Institute for Health and Care Excellence (NICE). The fate of the HSUME/CFSCG will apparently be addressed at an October so-called “roundtable” meeting of NICE and representatives from relevant interest groups. (I’m not sure what makes it a “roundtable” meeting or what the word “roundtable” is supposed to convey about the tenor of these HSUME/CFSCG proceedings.)

Anyway, while I’ve been focused on the HSUME/CFSCG, other stuff has been happening—like deliberations over proposed changes in how the US medical system should code the disease or diseases included under the constructs of ME, CFS, and ME/CFS. The US uses its own adapted version of the International Classification of Diseases (ICD), with is now in its tenth revision and continues to get updated.

A zero time point is essential

16 September 2021 by Vincent Racaniello 3 Comments

The recent release of grant materials from EcoHealth Alliance pertaining to their research with the Wuhan Institute of Virology has been used to demonstrate that gain of function research was funded by the NIH. This conclusion is correct, although not all the experiments done would fall into this category.

One of the figures from the released grant materials shows the results of two experiments using recombinant SARS-like coronaviruses that were constructed in the laboratory. As I described previously, after the SARS-CoV pandemic of 2003, wildlife sampling efforts in China revealed many SARS-like coronaviruses in bats. To assess the potential of these viruses for infecting humans, their spike protein encoding genes were substituted into the SARS-like CoV WIV1.

The reproduction of these recombinant viruses were assessed in two experimental systems. ACE2 transgenic mice were inoculated with the recombinant viruses to assess their pathogenic potential, by measuring weight loss. The results (figure below, left panel) show that only one recombinant virus, carrying the spike gene from SHC014, caused more rapid weight loss than did WIV-1. Hence, SCH014 has a gain of function. However the recombinant virus 4231 caused the same rate of weight loss as WIV-1 and therefore does not have a gain of function. Recombinant virus WIV-16 caused no weight loss – its curve resembles that of uninfected mice – and therefore this virus has a loss of function.

The second experiment is not, in my opinion, proof that any of these recombinant viruses have gained a new function. The experiment was to infect ACE2 transgenic mice with WIV1 and the three recombinant viruses, and then remove lung tissue at different times after infection and determine viral RNA loads. The first problem with this experiment is that RT-PCR was used to measure viral RNA loads, which does not directly measure viral infectivity. Therefore it cannot be concluded that any of the viruses reproduced to higher levels than WIV1. More problematic is that there is no time zero – the measurement of lung tissues begins at day 2. Consequently, one cannot reliably compare viral RNA loads of any of the recombinant viruses with WIV1. When doing kinetic experiments, a time zero is ALWAYS needed, so you can see how much virus was actually instilled in the mice. It gives you a baseline from which to compare subsequent time points. Even though the authors state the amount of virus added to the mice, they do not know how much was actually added. Operator error, binding to tissues, and a host of other factors can interfere early in the infection. For example, it is possible that the viral RNA loads on day 0 were the same as in subsequent time points, which showed little change over the course of the experiment. One cannot conclude from this experiment anything about the viral RNA loads without a time zero. No viruses gained any function in this experiment.

Unfortunately, many expert virologists do not have a problem with the absence of time zero. When I spoke to a reporter about these results, and told her my interpretation, she told me other ‘experts’ did not agree with me. You will find many examples of published time courses without a time zero – they are worthless.

Why the difference between virology experts? I was trained to always have a time zero; the others were not. They continue to get away with it so they believe it is correct. It is not proper research practice and no amount of arguing will make it right.

Trial By Error: An Updated Letter to the NICE Chief Executive About the Unpublished ME/CFS Guideline

15 September 2021 by David Tuller 20 Comments

By David Tuller, DrPH

I sent the following letter today to Professor Gillian Leng, chief executive of the National Institute for Health and Care Excellence (NICE). It was a follow-up to the letter I sent on September 1st about the agency’s decision to delay publication of it new ME/CFS guidelines. The letter has now been signed by more than 150 experts and more than 100 UK and international charities, support groups and other relevant organizations.

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Trial By Error: More Science-as-Promotion from the GET Campaigners

14 September 2021 by David Tuller 13 Comments

By David Tuller, DrPH

Professor Trudie Chalder, Professor Peter White and like-minded members of the CBT/GET ideological brigades have appeared desperate in the last year to promote their favored interventions, publishing one shoddy paper after another. This stream of sewage has seemed intended to influence the new ME/CFS clinical guidelines that Britain’s National Institute for Health and Care Excellence (NICE) has been developing since 2017. NICE called off the scheduled August 18^th publication of the new guidelines, which formally reject the longstanding treatment paradigm for ME/CFS, because of fierce objections from the cabal of CBT/GET true believers.

Gain of function research explained

9 September 2021 by Vincent Racaniello 12 Comments

The term ‘gain of function’ is perhaps one of the most misunderstood in the scientific lexicon. I would like to explain what the phrase means from the perspective of a scientist who has done gain of function research for the past 40 years.

Gain of function (GoF) research gives an organism a new property or enhances an existing one. The organism can be a virus, bacterium, fungus, rodent, bird, fish or anything that can be experimentally manipulated (technically whales and elephants could be included in the definition but it would be very difficult to to GoF research on them).

Many have the impression that GoF research involves making an organism more deadly – for example, increasing the capacity of a virus to cause disease. That impression is incorrect. Certainly GoF research might lead to a more dangerous organism, but most of the time that is not the goal.

There are two broad approaches to GoF research. I’ll illustrate them with viruses but the principles could apply to any organism. In one approach, a virus is passaged in a host until a virus with different properties is obtained. An example is the adaptation of a strain of poliovirus – the type 2 Lansing strain – to replicate in mice and paralyze them. The Lansing strain does not infect mice, but an investigator passed the virus 99 times from mouse to mouse and ended up with a new strain that could now paralyze the mice. The new version of the virus had a new property – it could now infect mice. This experiment was GoF research.

Another way to do GoF research is to use recombinant DNA technology to engineer changes in the genome of the organism. In experiments done in my laboratory, we took a small piece of the genome of the mouse-adapted Lansing strain of poliovirus – coding for just eight amino acids – and spliced it into the genome of another poliovirus that is unable to infect mice. The recombinant virus from this experiment had a new property – the ability to infect mice. This experiment would also be classified as GoF.

An illustration of how GoF can be done on mice is our creation of transgenic mice susceptible to poliovirus. Mice cannot be infected with the virus because they do not produce the cell receptor for poliovirus. We introduced the human poliovirus receptor gene into the mouse germline, leading to mice that produce poliovirus receptor. After infection, these poliovirus receptor transgenic mice can be infected with poliovirus and develop paralysis. The mice have a new property – susceptibility to poliovirus infection. The mice are a product of a GoF experiment.

GoF research may have a myriad of useful outcomes. Do you want to make a different tasting beer? Modify an enzyme in the yeast used for fermentation. But in the past 30 years GoF research has received a bad name. The catalyst was a series of experiments on highly pathogenic avian H5N1 influenza viruses. These viruses rarely infect humans and do not transmit well among people. In experiments to understand what limited transmission, the virus was genetically modified and passaged among ferrets. The result was a virus that could transmit among ferrets by respiratory droplets. These GoF experiments were met with criticism, entirely unwarranted as the passaged viruses had lost their virulence for ferrets! Nevertheless since then a dark cloud has unjustifiably hung over all GoF research.

GoF research has been in the press again recently as a consequence of the COVID-19 pandemic. After the SARS-CoV pandemic of 2003, wildlife sampling efforts in China revealed many SARS-like coronaviruses in bats. To assess the potential of these viruses for infecting humans, their spike protein encoding genes were substituted into the SARS-like CoV WIV1. These recombinant viruses reproduced in human airway cells – no different from WIV1 – but at least one caused more severe disease in mice. Consequently these are GoF experiments. Some have suggested that such GoF work gave rise to SARS-CoV-2 in a lab, but this notion is impossible, as none of these viruses are close enough to be a precursor of the current pandemic virus.

The production of recombinant coronaviruses to assess pandemic potential was carried out in several laboratories, all funded by the NIH. Recently Dr. Anthony Fauci told Congress that the NIH did not fund GoF coronavirus research. The press has suggested that he lied, but the truth is that his definition of GoF research is that it only involves passaged of organisms in animals. This interpretation is not correct but being wrong does not mean you are lying.

I want readers to understand that the goals of GoF research are laudable, and only a small subset has the potential to harm humans. Consequently these experiments are highly regulated and carried out under high levels of biological containment. GoF is not a dirty word.

Trial By Error: Jennie Spotila’s Annual Fact-Check of NIH Spending on ME/CFS Research

7 September 2021 by David Tuller 5 Comments

By David Tuller, DrPH

There is a lot going on in the ME and ME/CFS world that I don’t get around to. That’s why I’m always grateful that Jennie Spotila always deconstructs the numbers on the annual spending claims from the National Institutes of Health. Last month, on her blog Occupy M.E., Spotila submitted the NIH’s 2020 numbers to her usual sharp analysis. I am reposting it in full here with her permission.

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Innately Immune

2 September 2021 by Gertrud U. Rey 5 Comments

by Gertrud U. Rey

It is still not entirely clear why children are less susceptible to severe COVID-19. Early hypotheses included the possibility that children may have a stronger innate immune response, which is the response that occurs upon an initial encounter with a pathogen. Results from a recent study support this hypothesis.

To clarify why children have an enhanced ability to control a SARS-CoV-2 infection, the authors of the study collected nasal samples from SARS-CoV-2-negative and SARS-CoV-2-positive children and adults ranging in age from 4 weeks to 77 years. The presence of viral RNA in samples from SARS-CoV-2-positive participants was confirmed by PCR. The samples were also analyzed for the presence of different cell types using single cell RNA sequencing, a method that reveals the RNA expression profiles of individual cells. The authors detected 33 different cell types in the upper respiratory tract of all tested individuals, including 21 immune and 12 epithelial cell subtypes. The differences in the cell compositions of children and adults were quite dramatic – while nasal samples from healthy adults rarely contained immune cells, samples from children contained high levels of almost every immune cell subset, with neutrophils representing a substantial portion of the cell population. Neutrophils are an essential part of the innate immune system because they accumulate quickly at a site of infection, where they ingest pathogens and recruit and activate other immune cells.

Despite this difference in cell composition in the nasal mucosa of children and adults, the expression level of ACE2 (the SARS-CoV-2 binding target), was similar in both age groups. This result is contrary to previous suggestions that children may express less ACE2, but it is consistent with reports indicating that the frequency of SARS-CoV-2 infection in children is similar to that of adults.

Sentinel cells of the innate immune system recognize invading pathogens by sensing structurally conserved molecular motifs in infectious microbes. This sensing occurs through various pattern recognition receptors on or in the immune cells present in most tissues, like the well-characterized RIG-I-like receptors. Together with two other proteins, MDA5 and LGP2, RIG-I-like receptors detect the presence of viral RNA inside our cells and trigger a cascade of events that mobilize 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. Sensing of viral RNA by RIG-I and MDA5 initiates the production of a cytokine called interferon, a signaling protein that triggers downstream protective defenses.

When the authors compared the baseline expression of RIG-I-, MDA5-, and LGP2-encoding genes in the upper respiratory tract epithelial cells of healthy children and adults, they found that healthy children expressed significantly higher levels of these genes compared to healthy adults and SARS-CoV-2-positive adults who were in the early phase of infection. Samples from healthy children also contained a subpopulation of cytotoxic T cells that was absent in adults, and these T cells produced high levels of interferon gamma, a cytokine that inhibits viral replication and induces macrophages to engulf and digest pathogens.

When children became infected with SARS-CoV-2, they produced significantly higher levels of interferon gamma compared to SARS-CoV-2-positive adults, both in the early and later phases of infection. This observation is particularly interesting when considering that impaired interferon responses are a hallmark of severe COVID-19 and that SARS-CoV-2 is highly susceptible to interferon treatment. Furthermore, samples from SARS-CoV-2-infected children contained a subpopulation of SARS-CoV-2-specific memory T cells that was nearly absent in adults, suggesting that children might have an increased ability to respond to future SARS-CoV-2 reinfections.

The increased numbers of innate immune cells and increased expression of pattern recognition receptor genes in the upper airways of children may facilitate a more efficient innate response to SARS-CoV-2 infection, leading to reduced viral replication and faster clearance of virus. This type of innate immune response seems to be delayed in older adults, and in an effort to “catch up,” may result in excessive inflammation, thereby ultimately causing more severe damage. Although there are likely more factors at play, this study brings us one step closer to understanding why COVID-19 is generally less severe in children.

Trial By Error: While NICE Waffles, US Specialists Publish New Clinical Guidelines for ME/CFS

1 September 2021 by David Tuller 20 Comments

By David Tuller, DrPH

Two weeks ago, NICE abruptly announced that it was putting the brakes on publication of its new ME/CFS guidelines—a move precipitated by fierce opposition from key members of the GET/CBT ideological brigades in the British medical establishment. Then last week, Mayo Clinic Proceedings, a well-known journal, published a different set of ME/CFS guidelines that—like the unpublished NICE document—specifically advises against the traditional GET/CBT approach.

Trial By Error: A Letter Urging NICE to Publish ME/CFS Guideline Without Delay

1 September 2021 by David Tuller 54 Comments

By David Tuller, DrPH

This morning, I sent the following letter to Professor Gillian Leng, the chief executive of the National Institute for Health and Care Excellence.

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Professor Gillian Leng CBE
Chief Executive
National Institute for Health and Care Excellence

Dear Professor Leng,

Please find below a letter urging NICE to publish the new ME/CFS guideline, signed by scientists, clinicians, academics and other experts from the UK, the US, and other countries.

I would be remiss not to mention another guideline for the illness, which was published last week by Mayo Clinic Proceedings. This document –“Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Essentials of Diagnosis and Management”–was written by the US ME/CFS Clinician Coalition. In a statement that highlights the larger context of the current debate, the authors note that “the United States and other governments as well as major health care organizations have recently withdrawn graded exercise and cognitive-behavioral therapy as the treatment of choice for patients with ME/CFS.”

Best–David Tuller