The Initial Isolation and Purification of the Rona - Part Two

The Prelude

Before diving into the yawn-inducing analysis of the science behind the alleged discovery of the rona, let’s set the scene a little. It helps to bear in mind the chain of events that led to the speedy identification of the rona, so here’s a quick summary of what happened in the years preceding 2020:

2001 – Baric et al publish patent ‘Methods for producing recombinant coronavirus’. After this point, Baric and colleagues made a great many coronaviruses using similar reverse genetics systems.
2002-2003 – alleged SARS outbreak occurs; a new type of respiratory disease is said to cause around 800 deaths. A newly discovered virus is given the name ‘SARS-CoV’. Three virus isolates are sequenced and patented by America, China and Hong Kong; this is followed by a staggering amount of research into coronaviruses, especially spike proteins.
2013 – another coronavirus is blamed for causing a new respiratory illness. This time it’s MERS-CoV, and again, around 800 people die, and this leads to a fresh surge of vax development.
2014 – the Scripps Institute ‘solve the spike’ of coronavirus, with a pre-fusion design, using SARS, MERS and HKU-1; a moratorium is imposed in the US, limiting the use of such viruses in research.
2015 – several companies begin to produce genetic MERS-CoV vaccines, and some of them are tested on humans.
2017 – the new spike design[i] is patented, and the moratorium is lifted.
2018 – NIAID advertises the spike for commercial use, noting it can be used with a mRNA platform.
2019 – Event 201 and the Wuhan Military Games take place in October.

Official Timeline of the ‘Discovery’ of SARS-CoV-2

27 December, 2019 – the first patient with an unknown form of pneumonia is admitted to hospital in Wuhan.
30 December – a doctor called Li Wenliang messages other doctors to warn them about the new illness.
3 January, 2020 – China announces the discovery of a new type of coronavirus that’s highly similar to SARS-CoV. The sequences are published on GISAID.
5 January – the first death is reported and Li Wenliang and his parents get pneumonia.
10 January – Zhang et al from the Shanghai lab submit the genome for Wuhan-Hu-1 to GenBank (accession number MN908947).
12 January – the Shanghai lab is closed for rectification.
13 January – Moderna finalizes the sequence for mRNA-1273; Drosten et al release the RT-PCR test protocol for the rona; Moderna and the NIH sign an amendment to their “research collaboration agreement” re MERS and Nipah virus.
14 January – the Wuhan-Hu-1 genome on GenBank is “replaced by 2”
16 January – Drosten’s RT-PCR test protocol is published by the WHO.
17 January – the final version of Wuhan-Hu-1 is made available on GenBank. This version is 3 and it’s the one that most of the vax now contain. The database entry notes the record has been curated by staff at the US National Center for Biotechnology Information; the reference sequence is said to be identical to MN908947. “Annotation was added using homology to SARSr-CoV NC_004718.3” (i.e. they used Tor2, the Canadian SARS reference genome from 2003 to help them finish off the genome).
20 January – the ‘landmark’ paper by the WIV is submitted to Nature – the authors claim to have discovered a bat coronavirus which matches the genome of the rona really well. The paper isn’t published until the 3^rd of February, at the same time as the paper by the Shanghai team.
21 January – Corman, Drosten, et al, submit a paper describing how they designed a PCR test for the rona.
22 January – the US CDC take samples from a man suspected of being infected with the rona and create an isolate; their paper does not mention purification, and nor do subsequent papers that also describe creating isolates.
24 January – three papers about the rona are published by Chinese researchers: Zhu et al, ‘A novel coronavirus from patients with pneumonia in China, 2019’; Huang et al, ‘Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China’; Chan et al, ‘A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: A study of a family cluster.’
27 January – the sequence for RaTG13 is uploaded by China (the CAS Key Laboratory of Special Pathogens); it’s said to be from a bat in Yunnan, collected in 2013.
3 February – both Shanghai and WIV[ii] publish their findings in Nature.
12 February – “Baric’s lab in North Carolina is the only one in the US known to be trying to re-create the virus completely from ordered DNA parts.”
“By March 4, the U.S. Food and Drug Administration had greenlit the ModeRNA vaccine for human trials. At about the same time, Pfizer and BioNTech spoke with Graham about using the 2P mutation in their vaccine. Because their work was patented and widely published, other drugmakers—including Novavax and Johnson & Johnson—also based their candidates on the design.”
16 March – Moderna inject their first dose.
26 March – researchers discover a pangolin coronavirus with a RBD in the spike that fits with the rona, which is taken to mean the rona jumped from a bat to a pangolin and then somehow to humans. It’s yet another remarkable discovery – just like the WIV finding RaTG13 to ‘explain’ the bat origin, the researchers had decided to re-examine the viruses already stored in the lab, and found the magic answer.

Here’s what they did to isolate and purify specimens

The first isolate and genetic sequencing data came from China CDC, followed by deep metatranscriptomic sequencing performed by researchers at the Shanghai lab, who put together the first full genome of the rona, and then there were a series of experiments and another isolation performed by the team at the WIV. The following summaries take the researchers at their word, and are merely ‘translations’ of their own accounts of the tests they performed, i.e. they’re not meant to be any kind of endorsement or validation!

CHINA CDC – an isolate, some images of particles, and genetic sequencing

The team from China CDC was led by George F. Gao, who attended Event 201, so that’s something to bear in mind! The team published a paper describing their findings on the 20^th of February, 2020 (‘A Novel Coronavirus from Patients with Pneumonia in China, 2019’, Zhu et al). The paper says they leapt into action on the 31^st of December, 2019, when “several local health facilities reported clusters of patients with pneumonia of unknown cause”. They had four lower respiratory tract samples from people connected to the Huanan Seafood Market.

“Virus isolation from the clinical specimens was performed with human airway epithelial cells and Vero E6 and Huh-7 cell lines. The isolated virus was named 2019-nCoV. To determine whether virus particles could be visualized in 2019-nCoV–infected human airway epithelial cells, mock-infected and 2019-nCoV–infected human airway epithelial cultures were examined with light microscopy daily and with transmission electron microscopy 6 days after inoculation. Cytopathic effects were observed 96 hours after inoculation on surface layers of human airway epithelial cells; a lack of cilium beating was seen with light microcopy in the center of the focus (Figure 2). No specific cytopathic effects were observed in the Vero E6 and Huh-7 cell lines until 6 days after inoculation.”

No pathogens could be found in any of the samples, but they all tested positive using a pan-coronavirus RT-PCR test that can detect specific sequences found in all coronaviruses (the consensus RdRp region).
To isolate the virus, samples were “centrifuged to remove cellular debris” then used to infect human airway epithelial cells.
Cells that showed a cytopathic effect were collected, inactivated, and then ultracentrifuged in order to sediment virus particles.
This created an “enriched supernatant” which was used for analysis with a transmission electron microscope.
“RNA extracted from bronchoalveolar-lavage fluid from the patients was used as a template to clone and sequence a genome using a combination of Illumina sequencing and nanopore sequencing.”
More than 20,000 viral reads were obtained, and they were about 85% the same as a bat SARS-like CoV called ZC45.
Results from mock-infected cells were also obtained to serve as a control.
They said they’d identified three distinct strains of the virus and called it 2019-nCoV; these strains all aligned with ZC45 and the viral particles had a typical crown-like appearance.

How the Shanghai team purified the sample

“Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. …. this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients.”

Researchers based at the Shanghai Public Health Clinical Center in China published their findings in a paper called, ‘A new coronavirus associated with human respiratory disease in China‘ (Wu et al). Since most viruses consist of RNA, the Shanghai team ‘purified’ the sample they’d received by extracting all the RNA, and then they analysed it by performing deep meta-transcriptomic sequencing. This is a relatively new genetic technique that uses RNA sequencing (RNA-Seq) to work out how many RNA transcripts are in any given organism or biological sample. It involves removing everything except the mRNA which is only about 3% of the total content of an RNA sample and it allows researchers to perform de novo transcriptome assembly in organisms whose genomes have not been sequenced before. De novo transcriptome assembly is a next-generation sequencing technology which has been used on chickpeas and crocodile brain, and all sorts of other organisms that do not have a reference genome (i.e. previously documented sequences).

According to Wikipedia,

“De novo transcriptome assembly is often the preferred method [for] studying non-model organisms, since it is cheaper and easier than building a genome, and reference-based methods are not possible without an existing genome. The transcriptomes of these organisms can thus reveal novel proteins and their isoforms that are implicated in such unique biological phenomena.”

How the WIV isolated and purified the rona

The Wuhan Institute of Virology (WIV) performed the most comprehensive research and their paper is considered the landmark paper on the discovery of the rona. The WIV team was led by Zeng Li Shi, the woman who spent years doing gain-of-function research with Ralph Baric, and their paper (‘A pneumonia outbreak associated with a new coronavirus of probable bat origin’, Zhou et al, 2020) describes the procedures they followed:

“We next successfully isolated the virus (called 2019-nCoV BetaCoV/Wuhan/WIV04/2019) from both Vero E6 and Huh7 cells using the BALF sample of patient ICU-06.”

Washings from the lungs of patient ICU-06 were obtained as a specimen, then added to two types of cell culture. Vero E6 cells are from a monkey kidney, and Huh7 cells were obtained from the cancerous liver of a Japanese man.

“The PCR-positive BALF sample from ICU-06 patient was spun at 8,000g for 15 min, filtered and diluted 1:2 with DMEM supplemented with 16 μg ml−1 trypsin before it was added to the cells.”

Before adding the ICU-06 specimen to the cells, they confirmed it belonged to the genus known as ‘coronavirus’ (it had certain features, such as specific molecular sequences, which identify it as belonging to the family of coronaviruses).
The sample was purified by spinning and filtering it.
DMEM was added to help keep it alive, and trypsin was added to break down (lyse) the cells.

“After incubation at 37 °C for 1 h, the inoculum was removed and replaced with fresh culture medium containing antibiotics and 16 μg ml−1 trypsin. The cells were incubated at 37 °C and observed daily for cytopathogenic effects.”

This nutrient solution was refreshed after incubating the sample for a while. Antibiotics were also added to prevent growth of bacteria. (The scientists were looking for viruses because all patients had been treated with antibiotics in the hospital and their health did not improve.)
Then they popped it back in the incubator and kept checking to see what was happening to the cells.

“Clear cytopathogenic effects were observed in cells after incubation for three days.”

After three days, the (infected) cells started to die off.

“The identity of the strain WIV04 was verified in Vero E6 cells by immunofluorescence microscopy using the cross-reactive viral N antibody….”

Having isolated a new strain of the rona from a patient using monkey cells, the isolate is given the name WIV04.
The scientists then checked their findings by using immunofluorescence microscopy. This technique involved using a fluorescent dye to label any antibodies in the specimen that matched the N gene of the recently sequenced virus, and analysing it with a special microscope.
Some of the patient samples were found to contain antibodies that could neutralize 2019-nCoV; this test has been used for decades to ‘confirm’ presence of a virus.

They then performed deep metagenomic sequencing of WIV04 and found most of the reads mapped to 2019-nCoV (the genome sequenced by Shanghai). When they viewed the cells using an electron microscope, the “infected cells displayed a typical coronavirus morphology”, just like the ones reported by the China CDC.

Other points to note:

All patients were tested for the following respiratory pathogens: Legionella pneumophilia, Mycoplasma pneumoniae, Chlamydia pneumoniae, respiratory syncytial virus, adenovirus, Rickettsia, influenza A virus, influenza B virus and parainfluenza virus.
“mock-transfected cells were used as controls” and they did not exhibit a cytopathic effect; see pictures ‘a’ and ‘b’ here (“Vero E6 cells are shown at 24 h after infection with mock virus”)

Criticism of the sequencing performed by WIV

In ‘SARS-Cov-2 Natural or Artificial, That is the Question‘, Alejandro Sousa criticized the methods reported by the WIV – “using only 1,378 reads from random amplification to conduct de novo assembly and getting near complete genome coverage … for such a large RNA virus genome (near 30K in total length) is beyond miracle. Meanwhile, for regions with high chance of mutations such as spike protein open reading frame, very deep coverage of raw reads is often needed to ensure the accuracy of sequencing data…. The accuracy of the full genome sequences obtained in this study should be seriously challenged.”

Notes:

[i]And somehow or other, that same year, Curevac had the same ‘two prolines in the spike’ solution in their patent for a coronavirus vax based on mRNA. The design also appears to have been trialled with Moderna during their collaboration with the NIAID.

[ii] The WIV team say they’ve discovered a virus called RaTG13 which explains the genome of the rona, i.e. that it came from bats. The problem with this virus was that the RBM was rather different to the rona… so scientists theorized there ‘must have been’ an intermediate host – another species into which the bat virus had jumped, and mutated a bit, before it then jumped into humans.

READ PART 1

Image: Pixabay