“We did not obtain an electron micrograph showing the degree of purification.”
Replying Author: Wan Beom Park
This paper is often cited by many as proof that “SARS-COV-2” exists and has been “isolated” from a sick patient. The problem is, they only read the title of the study. Had they read the study itself, it would be obvious that what Park et al. call “isolation” of a “virus” is the exact opposite of separating the “virus” from everything else. As usual, they used the same exact cell culture soup recipe full of foreign animal DNA, antibiotics/antifungals, “nutrients,” chemicals, etc. that was used in the previous “Coronavirus” papers. Adding various ingredients together with the unpurified sample taken from a sick patient and incubating it for days until nonspecific cell damage called Cytopathic Effects (CPE) is observed is not isolation. It is nothing more than unpurified witches brew full of millions of similar particles, as can be seen from the quote above by lead researcher Wan Beom Park admitting they did not purify anything when obtaining their “virus.” Highlights from his study below:
Virus Isolation from the First Patient with SARS-CoV-2 in Korea
“Here, we report the isolation of SARS-CoV-2 using Vero cells from a patient entering Korea from Wuhan, China.
The patient with the first laboratory-confirmed SARS-CoV-2 infection in Korea is published previously.6 Briefly, a 35-year-old woman developed fever, chill, and myalgia on January 18, 2020, and arrived at the Incheon airport from Wuhan on the next day. After laboratory-confirmed diagnosis of SARS-CoV-2 infection, she developed nasal congestion, cough, and sputum. Oxygen supplementation was started on day 4 of her illness, and her oxygen requirement increased to 6 L/min on day 7 of illness. Fever persisted for ten days and her maximum body temperature during her illness was 38.9°C on day 7 of illness.
The patient’s oropharyngeal samples were obtained by using UTM™ kit containing 1 mL of viral transport media (Copan Diagnostics Inc., Murrieta, CA, USA) on day 7 of her illness. We inoculated monolayers of Vero cells (ATCC ® CCL-81™) with the samples and cultured the cells at 37°C in a 5% carbon dioxide atmosphere. Until 5 days after inoculation, cytopathic effects were not distinct, which is compatible with the previous findings that no specific cytopathic effects were observed in the Vero E6 cells until 6 days after inoculation in the report about first isolation of SARS-CoV-2.3 Five days after inoculation, we did blind passage of culture supernatant into T-25 culture flask (ThermoFisher Scientific Inc., Waltham, MA, USA) with monolayers of Vero cells, and cytopathic effects consisting of rounding and detachment of cells were observed in the whole area of the T-25 flask 3 days after the first blind passage (Fig. 1A and B).
In order to observe virus particles, Vero cell monolayer showing the cytopathic effects was fixed as previously described.7 It was cut on ultramicrotome (RMC MT-XL; RMC Boeckeler, Tucson, AZ, USA) at 65 nm. Ultrathin sections were stained with saturated 4% uranyl acetate and 1% lead citrate before examination with a transmission electron microscope (JEM-1400; JEOL USA Inc., Peabody, MA, USA) at 80 kV. Spherical particles with crown-like spikes ranging 66 to 81 nm in diameter were observed within the cytoplasmic vesicles and in the extracellular space adjacent to cell membrane (Fig. 1C and D).
For whole genome sequencing of the virus isolate (BetaCoV/Korea/SNU01/2020), culture supernatant of Vero cells infected was used for RNA extraction. RNA was extracted by using QIAamp viral RNA mini kit (QIAGEN, Valencia, CA, USA), according to the manufacturer’s instructions. RNA libraries were prepared using TruSeq Stranded Total RNA Kit (catalog No. 20020596; Illumina, San Diego, CA, USA) according to the manufacturer protocol. Sequencing was performed on an Illumina Nextseq 500 platform, produced on average a total of 150 million reads, 150 bp per sample, as per the manufacturer’s instructions in Macrogen Inc. (Seoul, Korea).8,9,10 FASTQ was used to trim the adapter and remove low quality bases and reads. Qualified reads were mapped to NC_045512, a SARS-CoV-2 genome reference using Burrows-Wheeler Aligner (v0.7.12-r1039), and a bam file was produced.11 In this bam file, the variation was confirmed by comparing with genome using SAMtools (v1.3.1).12 For genome-base phylogeny analysis, 37 strains including BetaCoV/Korea/KCDC03/2020 were used in combination with BetaCoV/Korea/SNU01/2020. The sequences used for analysis were downloaded from NCBI (http://www.ncbi.nlm.nih.gov/) and GISAID (http://www.gisaid.org). The 37 strain genomes were multiple-sequence aligned using MAFFT (v7.450), a sequence alignment tool, and were used to generate phylogenetic tree.13 Phylogenetic analysis of the aligned sequence was performed with 1,000 bootstrap replicates using MEGAX and a general time-reversible model used as the nucleotide substitution model.14
Next-generation sequencing of BetaCoV/Korea/SNU01/2020 (GenBank accession no. MT039890) revealed 9 mutations compared to the NC_045512 reference genome isolated from Wuhan (Table 1). Most of the mutations in our isolate consisted of 70% alternative genes and 30% reference genes (NC_045512). Five variants were found in ORF1ab, one variant in S gene, two variants in ORF3a, and one variant in E gene. Of the nine mutations, six also showed changes in amino acids. When comparing our isolate with the one isolated from the Korea Centers for Disease Control and Prevention (BetaCoV/Korea/KCDC03/2020), 12 variants including the above 9 mutations were found. These mutations may occur by cell culture-adaptation in that our culture isolate was obtained after first blind passage, or by micro-evolution of SARS-CoV-2 before acquisition in Wuhan. Because those genome sequences are quite homologous each other, it is difficult to validate these two hypothesis.”
“In summary, we isolated SARS-CoV-2 using Vero cells from the first laboratory-confirmed SARS-CoV-2-infected patient in Korea. Phylogenetic analyses of the whole genome sequences showed that it clustered with other SARS-CoV-2 reported from Wuhan, China.”
- Park et al. “isolated” their “virus” using Vero cell (African Green Monkey kidneys) culture
- The case is considered the first labratory-confirmed case in Korea
- They took an oropharyngeal (throat) sample and immediately placed it in Viral Transport Media (ingredients not listed)
- This sample was inoculated directly onto the Vero cells
- They did not observe any CPE after 5 days so they did a first blind passage into a new flask of Vero cells and 3 days later observed CPE
- The process of blind passaging cells can damage/alter the sample as detailed here:
- In order to observe “virus,” the Vero cells showing CPE (to which they admitted was not purified) were fixed and stained for EM imaging in order to find the spherical crown-like particles they wanted to see
- For whole-genome sequencing, RNA was extracted from the same unpurified Vero cell sample
- “Qualified” reads were then mapped to an already prepared “SARS-COV-2” reference genome
- There were 9 “mutations” found in the Korea “virus” that differed from the Wuhan reference “virus”
- When comparing to a genome from the Korean CDC, they found that their genome had 12 “mutations” including the 9 from the Wuhan reference
- They admit that the culturing process may have created these “mutations” through cell-culture adaptation as they obtained their genome after the first blind passage
The problem for papers like this, beyond the fact that the researchers have completely inverted the meaning of the word ISOLATION, is that they do not perform the neccessary controls to find out if the CPE they observe in the cell culture soup is created by a “virus” or from the cell-culturing process itself. The researchers here at least admit they are unable to determine if the “mutations” in their genome were created by the culturing process. The process of blind passaging is known to cause cell-culture adaptations and alter the end results of the experiment. If they took it a step further, they would also realize that they can never prove that what they created through their chemistry experiments in a lab was ever in the original sample to begin with. This is why it is absolutely necessary to purify/isolate the particles believed to be “virus” DIRECTLY from the UNALTERED patient sample and not from experimental soup in a lab.