Sigh, yes, the 'COVID virus' is real

Ian M Mackay, PhD
Virology Down Under
Tue, 06 Oct 2020 16:24 UTC

There has been talk out thar in the wildlands of Twitter from people who don't believe the evidence that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is real.

Has the 'the COVID virus' ever been isolated in cell culture, visualised by electron microscopy, reacted with antibodies, genetically sequenced and otherwise characterised in many samples collected from people with coronavirus disease 19 (COVID-19) all over the world during the past nine months?

Sigh, yes, and yes the 'COVID virus' is real. Here are a few of the scientific endeavours that show this virus has been isolated from clinically diagnosed, ill and laboratory-confirmed human COVID-19 cases.

© CDC.gov

The spikes that adorn the outer surface of the virus, which impart the look of a corona surrounding the virion, when viewed electron microscopically. These spikes determine which animal it can infect.

Studies that successfully isolated the virus from the sick human January 2020¹

Peng Zhou and colleagues from the Wuhan Institute of Virology grew a novel coronavirus (later to be called SARS-CoV-2) in cell culture (isolated) from bronchoalveolar lavage fluid collected from an ill and RT-PCR-positive female patient (ICU-06 in Spike tree=WIV04/2019 virus). The patient developed no IgM antibodies for Legionella pneumophilia, Mycoplasma pneumoniae, Chlamydia pneumoniae, respiratory syncytial virus, adenovirus, Rickettsia, influenza A virus, influenza B virus and parainfluenza virus.¹

© Wuhan Institute of Virology

Extended Table 2. The samples from ICU-06 had some of the highest viral loads (estimated from the low threshold cycle values [CT]) among the patients tested.

The virus was called nCoV-2019 BetaCoV/Wuhan/WIV04/2019 and grew in Vero E6 (grivet, or African green monkey kidney cell line which lack genes to encode type I interferon, so they mount a defective antiviral response) and Huh-7 (human adult liver cancer-derived cell line) cells. Virus-induced cellular changes (cytopathic effect or CPE) were observed.

Vero E6 cells are shown at 24 h after infection with (a) mock virus) or (b) SARS-CoV-2. Mock-virus-infected (c) or SARS-CoV-2-infected (d) samples were stained with rabbit serum raised against recombinant SARSr-CoV Rp3N protein (red; able to cross react) and DAPI (blue; a counterstain). The experiment was conducted twice independently but with similar results.

Whole-genome sequencing (WGS) was used to identify the unique genetic sequence of the cultured virus and a specific real-time reverse transcription-polymerase chain reaction (RT-rPCR) designed to allow screening of more sensitive and rapid screening of more samples. The RT-rPCR was tested against human endemic CoVs (229E, OC43, HKU1) as well as MERS-CoV, SARS-CoV and others, and was found to be highly specific for SARS-CoV-2.

Complete Spike (S) gene tree (WIV04/ICU06 is the SARS-CoV-2 variant that was isolated in culture)

The authors also saw viral particles. Electron microscopy (EM) was used to visualise virus in cultured Vero E6 cells after they had been inoculated with the patent sample.

Viral particles shown in ultrathin cut sections of prepared virus-positive Vero E6 cell cultures, using electron microscopy at 200 kV. The inset shows the viral particles in an intra-cytosolic vacuole, likley on their way to thw surface and out of the infected cell.[1]

They also showed that the antibodies from five infected people could neutralise (=prevent) infection of Vero E6 after incubated dilutions of patient sera with a cultured virus preparation and added to cultures of uninfected cells. An antibody made in horses against the original SARS-CoV could also neutralise the virus, however serum from two healthy people in Wuhan could not.

February 2020²

Jeong-Min Kim and colleagues from South Korea inoculated RT-rPCR-positive patient nasopharyngeal and oropharyngeal samples onto Vero cells. After most inoculated (but not mock-inoculated) cells were showing CPE, they were harvested and tested using the two RT-rPCRs (Corman et al. RdRp and E; these detect viruses in the genus Sarbecovirus). RT-rPCR testing found an increase in viral RNA, which the team estimated at 10-70-fold.

The study also examined three-day post-inoculation cells using EM. They found virus-like particles in vesicles within the infected cells.

To confirm that the virus was indeed SARS-CoV-2, the authors conducted WGS, naming the SARS-CoV-2 variant they'd isolated BetaCoV/Korea/KCDC03/2020:

February 2020⁴

This is a method for culturing SARS-CoV-2 from patients' samples. It doesn't delve into virus characterisation though. Nonetheless, the image below shows CPE on a monolayer (=single cell layer) of African green monkey kidney Vero C1008, clone E6 cells (ATCC®-CRL-1586).

The CPE was confirmed as being due to SARS-CoV-2 by RT-PCR and sequencing.

An example of a SARS-CoV-2-infected monolayer culture of Vero E6 cells demonstrating focal CPE. Source: Dr. Alyssa Pyke, Public Health Virology Laboratory, Forensic and Scientific Services, Queensland. 08FEB2020.[4]

An example of an uninfected monolayer culture of Vero E6 cells with no CPE despite growing under the same conditions. Source: Dr. Alyssa Pyke, Public Health Virology Laboratory, Forensic and Scientific Services, Queensland. 08FEB2020.[4]

March 2020³

Leon Caly and a crew from Australia isolated the virus from an ill traveller from Wuhan. The virus was detected using an in-house (designed by this team) RT-rPCR. The nasopharyngeal swab was used to inoculate the Vero/SLAM cell line.

Growth was seen both by the CPE induced and the use of that specific RT-rPCR to show an increase in the presence of SARS-CoV-2 RNA (decrease in C_T)

Cell-free liquid from infected cultures (called supernatant) and cells from the cultures were each examined using EM, finding particles with characteristic coronavirus morphology (=shape) and the same particles inside vesicles within the cells.

To further characterise the amplified agent, WGS identified the genome and names this virus BetaCoV/Australia/VIC/01/2020.

August 2020¹³

Steffen Klein and colleagues used the SARS-CoV-2 variant called BavPat1/2020 to grow in VeroE6, HEK293T (human embryonic kidney cell line expressing the SV40 T antigen), Calu3 (from adult lung adenocarcinoma) and A549 (from human adult lung carcinoma) cells - some made to express the SARS-CoV-2 receptor molecule (ACE2). They knew that what they were seeing below was SARS-CoV-2 because they infected these cells with a known and well-characterised SARS-CoV-2 virus.¹³

Single virus particles and the structures on them could be seen using cryo-electron tomography. Virus particles can be seen caught in the act of budding out off infected cells and from double-membrane vesicles (little bubbles enveloped in lipid) inside cells. This gives us wonderful insight into the SARS-CoV-2 moving around as it leaves the cell for new lands to plunder.

The authors could even measure the distance between spike protein trimers (a spike 'protein' is actually 3 proteins in a trimer, sticking out of the envelope of each virion) on SARS-CoV-2 virion!

October 2020⁶

Beata Turoňová and team took some of the SARS-CoV-2 BavPat1/2020 isolate, grew it in Vero E6 cell culture, passaging (infecting, harvesting, infecting a fresh flask of cells and so on) five times. They took this virus and centrifuged all the uninfected cells and cell debris and made stocks into a pellet, and collected and froze the liquid above the inactivate (the supernatant).

Some of this stock was later thawed and used to infect a fresh new flask of Vero E6 cells. After six hours the cultures were fixed (treated with a chemical to kill the cells and inactivate the virus while retaining the cell and virus structures - like freezing them in time). This preparation was clarified twice by centrifugation (now a pretty pure preparation of virus + salty solution). This was then layered gently onto a cushion of sucrose (20% weight per volume) in a special tube. The tubes were centrifuged at very high speed (30,000 rpm) in an ultracentrifuge and the pellet - consisting of virus (any less dense material like small particles of cell debris couldn't travel through the more dense sucrose cushion) - was suspended in a tenth of a millilitre of saline.

They now had a very pure preparation. There are no Vero E6 cells or bacteria or broken up pieces left, just the tiny virus particles.¹⁶

This was then prepared for cryo-electron tomography studies which allowed the investigators to view and produces images of single virions and even individual projecting spike protein trimers.

Some amazing detail can be seen below. A preparation of a virus can't get much more "purified" than this.

The detail is so exquisite that the investigators were able to measure the size of parts of the spike protein molecules on a single virion.

NOTE ON ANTIBODY CROSS-REACTIONS

You can see here and elsewhere that there are cross-reactivities among commercial antibodies which allow them to be used to detect SARS-CoV-2. This isn't a problem for general diagnostics though, because SARS-CoV (or any bat-related SARS virus) isn't circulating in humans anywhere anymore. Also, we know that the SARS-CoV-2 detections we make using RT-PCR aren't from a different virus because of all the genetic sequencing that's been done to show it is SARS-CoV-2 that's present in sick COVID-19 patients.

Evidence of SARS-CoV-2 isolation and visualisation in other ways

Below is a brief list of some major feel-good SARS-CoV-2 discovery announcements that came to us through the mainstream media or University websites.

1. Melbourne scientists first to grow and share novel coronavirus
2. China coronavirus: Hong Kong researchers have already developed vaccine but need time to test it, expert reveals
3. China CDC developing novel coronavirus vaccine
4. Coronavirus: Scientists isolate virus responsible for deadly Covid-19 outbreak
5. I study viruses: How our team isolated the new coronavirus to fight the global pandemic

© Hong Kong National Microbiology Data Centre

Individual SARS-CoV-2 virus particles.

Free-to-use images of the SARS-CoV-2 virus

These images are made public by the US Centers for Disease Control ND prevention via their Public Health Image Library (PHIL). A fantastic resource for virus images. The exact details of how the viruses were prepared aren't present, nor the ways in which SARS-CoV-2 was confirmed. That may be a problem for you. However, I'm sure they were prepared and confirmed using the exact same methods as those listed above.

Transmission electron microscopic image of an isolate from the first U.S. case of COVID-19, formerly known as 2019-nCoV (SARS-CoV-2). The spherical viral particles, colourized blue, contain cross-sections through the viral genome, seen as black dots.
Source: https://phil.cdc.gov/Details.aspx?pid=23354

Electron microscopic image of a negatively stained particle of SARS-CoV-2, causative agent of COVID-19. Note the prominent spikes from which the coronavirus gets its name for “corona”, or “crown-like”.
http://phil.cdc.gov/Details.aspx?pid=23640

Thin section electron microscopic image of SARS-CoV-2, the causative agent of COVID-19. Spherical virus particles contain black dots, which are cross-sections through the viral nucleocapsid. In the cytoplasm of the infected cell, clusters of particles are found within the membrane-bound cisternae of the rough endoplasmic reticulum/Golgi area.
https://phil.cdc.gov/Details.aspx?pid=23640

Electron microscopic image of a negatively stained particle of SARS-CoV-2, causative agent of COVID-19. Note the prominent spikes from which the coronavirus gets its name for “corona”, or “crown-like”.
Source: https://phil.cdc.gov/Details.aspx?pid=23641

Transmission electron microscopic image of an isolate from the first U.S. case of COVID-19, formerly known as 2019-nCoV. The spherical extracellular viral particles contain cross-sections through the viral genome, seen as black dots.
Source: https://phil.cdc.gov/Details.aspx?pid=23336

Postulating into failure

Koch's postulates - derived from his work on bacteria - were formally proposed at a lecture by Koch in 1890.

For context, that's before we had ever visualised a human or plant virus, before organ or cell culture of viruses, before sequencing of viral genes or genomes and before we had labelled antibodies we could use as probing tools to show viral proteins in tissues. It was known that this toxic stuff (virus, from Latin, translates to 'slimy liquid, poison') was smaller than bacteria because it passed through filters that stopped them, and yet could still cause disease. Discovery of human viruses came later still (Yellow fever virus, 1901-1921).

Koch's postulates were never intended to be rigidly applied, even then. In fact, trying to strictly adhere to them probably delayed the discovery of viruses.¹¹

So let's move away from the 1890s... this revision by Fredericks and Relman (from Fenner and White's Medical Virology - Fifth Edition) is more much more relevant to recent decades:

How do we know the SARS-CoV-2 in these earlier studies wasn't the flu or something else that made the patients sick?

Fair enough question. Let's look at one variant of SARS-CoV-2 because it's mentioned a couple of times in the studies above. BetaCoV/Germany/BavPat1/2020 p.1 has been made available for researchers via reliable and professional providers of such biological materials (linked above). It was collected January 28th from a hospitalized male patient from Munich who was described as the index case of the Bavaria cluster in Germany.¹⁴ He was initially suffering from cough, fever and diarrhoea. The SARS-CoV-2 variant from this patient is listed on the sequence database, GISAID, as hCoV-19/Germany/BY-ChVir-929/2020 (with its unique identified EPI_ISL_406862). It was sequenced from the original sample, not undergoing any cell culturing first. Obviously, after this, the virus was cultured and made available as a resource for other scientists to use - which it clearly has been and put to very good use.

SARS-CoV-2 was confirmed in all of these cases by careful testing at two different laboratories. All patients in this Munich-based cluster¹⁴ were also tested for all other important respiratory viruses using sensitive PCR-based tests. These included:

• HCoV-HKU1
• HCoV-OC43
• HCoV-NL63
• HCoV-229E
• influenza virus A
• influenza virus B
• rhinoviruses
• enteroviruses
• respiratory syncytial virus
• human parainfluenza viruses 1, 2, 3 and 4
• human metapneumovirus
• adenoviruses
• human bocavirus

No concurrent infections were identified. The initial case in the cluster, who provided the sputum that was sequenced, also seroconverted at day 10 after symptom onset showing the body's immune response to the viral invader. He was still shedding RT-PCR detectable viral RNA in the sputum and stool, but not nasal swabs, 21 days after onset.¹⁴

All of this sums up as: the studies done on this virus are studies of this virus. These were not accidentally studies of some other (completely unknown beforehand, by the way) virus. And the sequence was not "tainted" by culturing; the SARS-CoV-2 entire genetic makeup was determined from material taken directly from the patient who was sick due to this virus and not another virus, at the time. They were also sick with an acute respiratory illness traditionally typical of a viral cause. It's a tight case.

Do we know if SARS-CoV-2 causes disease though?

Well, yeah. I mean, if we overlook the many cases of COVID-19 patients harbouring SARS-CoV-2 and no other virus and if we willfully ignore for a moment the strong links with COVID-19 signs, symptoms and progress and virus presence and replication kinetics, there are also animal studies showing that introducing this pathogen causes disease.

One good example,⁷ because it reproduces some of the disease we see in humans, is the primate model. In one study of different monkeys, Macaca mulatta (and to a lesser extent, Macaca fascicularis) were found to reproduce aspects of human COVID-19. After being given a dose of virus, a rise in temperature was noted as was the loss of body weight and chest X-rays found lung abnormalities. Nasal swabs taken over time found a peak in viral RNA and RNA was found at lower levels but in the blood of most animals at two days post-infection. Among two animals examined at necropsy, viral RNA could be detected in the lung, trachea, bronchi, spleen, stomach, rectum, bladder, uterus as well as in hilar and mesenteric lymph nodes. Only two tissues still tested positive for viral RNA at late.

Another primate study⁸ administered SARS-CoV-2 via the nose and throat to Macaca fascicularis (did not use a virus-free control group). Only one monkey developed symptoms (cold-like) and all developed antibodies to a region of the spike protein and to the nucleocapsid protein, by day 14. Infectious virus could be recovered from throat (up to day 2) and nasal (up to day 4) swabs using Vero E6 cells, as could viral RNA. RNA was also detected in one rectal swab. SARS-CoV-2 proteins were identified in lung cells (type I and a few type II pneumocytes) at autopsy of three out of four animals.

So why do some think SARS-CoV-2 isn't real or hasn't been properly "purified"?

I don't really know - and it seems many of those who belong to this group aren't sure either. But I strongly suspect a lot of this is due to the wording of Koch's postulates. Where they said "repeatedly grown in pure culture", some with no experience in the area have taken it upon themselves to endow the word "pure" with some sort of celestial perfection that was never meant to be. Much like the postulates themselves were never meant to be adhered to with such ardent fervour. This is yet another conspiracy theory to which people have decided to attach themselves.

Some of the concern could come from papers like this one¹² which notes that researchers need to be careful when growing a virus in culture because the process can result in genetic changes or mutation. Very true. Cell culture cells often originate from an animal that is different to the one in which the virus originated so there can be pressure on the virus to produce mutant variants which are better adapted to the new cellular environment. But today we can sequence the new virus's entire genome - the code for all it is and can be - before (or in parallel or after if we dip into the original samples several times) we try and culture the virus. That way we have our "baseline" genetic code against which we can compare the cell culture code. Indeed scientists like to do this to see how the virus is changing and explain why new behaviours in culture may occur.

The earliest virus discoveries were made simply by noting that the thing causing disease could not be filtered out of a liquid using a very fine porcelain (Chamberland) filter, the way bacteria could be. Basic stuff but the foundation of virology.⁹

The disease could be passed along by injecting fluids or ground-up tissues from an infected plant or animal into a new plant or animal and watching disease develop. All very crude yet accepted at the time as evidence for a "virus" being present even if that couldn't be characterised at the time. As we can see in the Table below, acceptance has changed with technology alongside the flexible careful thinking of experts in the field. To be clear, that expertise is not in play among those who refuse to believe SARS-CoV-2 is a real virus.

Those "filterable agents" were in preparations that were no more "purified" than a virus preparation obtained from a cell culture supernatant today, and yet they were enough to create the field of virology.

How changing technology has changed our ability to find viruses. As the technology becomes better, so the world of virology has opened up to us as the failings of each preceding method are overcome. From Woolhouse et al.[10]

Since then we've been able to add many new tools to the characterisation process - visualising individual virions and their component parts, determining the unique sequence of their entire genetic code and measuring the development of a constellation of immune responses to the virus.

There is - as you can see for yourself here I hope - an embarrassment of rich evidence for the existence of SARS-CoV-2 and its links to COVID-19.

Some final thoughts

I know there will be comments below (I'll make sure to publish some of the less offensive ones ) to the extent that 'I don't care what y'all say, I ain't seen no evidence, make it empirical, abide by the Koch'...or something.

And that's all well and good.

There is evidence and some of the most significant parts of it are listed above.

It convinces me. If it doesn't convince you, there is nothing more I can do except ask what it would take to convince you and in the meantime assure you that you are wrong on this one. Yes, the 'COVID virus' is real.

References

1. Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin and then nature.com
2. Identification of Coronavirus Isolated from a Patient in Korea with COVID-19
3. Isolation and rapid sharing of the 2019 novel coronavirus (SARS‐CoV‐2) from the first patient diagnosed with COVID‐19 in Australia
4. Culture of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; f.2019-nCoV)
5. Causation and Disease: The Henle-Koch Postulates Revisited
6. In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges & sciencemag.org
7. Comparison of nonhuman primates identified the suitable model for COVID-19
8. Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model
9. CONTRIBUTIONS TO THE PATHOLOGY OF EXPERIMENTAL VIRUS ENCEPHALITIS. I. AN EXOTIC STRAIN OF ENCEPHALITOGENIC VIRUS
10. Human viruses: discovery and emergence
11. Virology: From Contagium Fluidum to Virome, Enquist LW and Racaniello VR, Chapter 1, Fields Virology, 6th Edition, Volume I. Editors Knipe and Howley.
12. Amending Koch's postulates for viral disease: When "growth in pure culture" leads to a loss of virulence
13. SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography
14. Virological assessment of hospitalized patients with COVID-2019