On March 7, 2020, the CDC released their study on the “isolation” and characterization of “SARS-COV-2” from the first US patient identified by PCR testing on January 22nd, 2020. As with every single paper before it, the CDC relies on unpurified cell cultures to claim “isolation” of a new “Coronavirus.” They utilize the same unproven molecular tricks to create a genetic blueprint in a computer database for something they have never seen or observed in reality. There are some interesting wrinkles I will point out with this particular study that put it squarely into unreliable territory right along with all the others as well. Highlights below:
Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient
“The public health community requires viral lysates to serve as diagnostic references, and the research community needs virus isolates to test anti-viral compounds, develop new vaccines, and perform basic research. In this manuscript, we describe isolation of virus from the first US COVID-19 patient and described its genomic sequence and replication characteristics. We have made the virus isolate available to the public health community by depositing into two virus reagent repositories.”
RESULTS and DISCUSSION
“A patient was identified with confirmed COVID-19 in Washington State on January 22, 2020 with cycle threshold (Cts) of 18–20 (nasopharyngeal(NP)) and 21–22 (oropharyngeal (OP)) (1). The positive clinical specimens were aliquoted and refrozen inoculation into cell culture on January 22, 2020. We first observed cytopathic effect (CPE) 2 days post inoculation and harvested viral lysate on day 3 post inoculation (Figure 1B and and1C).1C). Fifty μl of P1 viral lysates were used for nucleic acid extraction to confirm the presence of SARS-CoV-2 using the CDC molecular diagnostic assay (1). The Cts of three different nucleic acid extractions ranged from 16.0–17.1 for N1, 15.9–17.1 for N2 and 16.2–17.3 for N3, confirming isolation of SARS-CoV-2. A Ct of less than 40 is considered positive. The extracts were also tested for the presence of 33 additional different respiratory pathogens with the fast track 33 assay. No other pathogens were detected. Identity was additionally supported by thin section electron microscopy (Figure 1D). We observed a morphology and morphogenesis characteristic of coronaviruses.
Isolates from the first passage of an OP and an NP specimen were used for whole genome sequencing. The genomes from the NP specimen (Genbank accession MT020880) and OP specimen (Genbank accession MT020881) matched each other 100%. The isolates also matched the corresponding clinical specimen 100% (Genbank accession MN985325).
After the second passage, OP and NP specimens were not cultured separately. Virus isolate was passaged two more times in Vero CCL-81 cells, and titrated by TCID50. The titers of the third and fourth passages were 8.65 × 106 and 7.65 × 106 TCID50 per mL, respectively.
We subsequently generated a fourth passage stock of SARS-CoV-2 on VeroE6 cells, another fetal rhesus monkey kidney cell line. Viral RNA from SARS-CoV-2 passage four stock was sequenced and confirmed to have no nucleotide mutations compared with the original reference sequence (Genbank accession MN985325). Both SARS-CoV and MERS-CoV had been found to grow well on VeroE6 and Vero CCL81 respectively (12, 13). To establish a plaque assay and determine the preferred Vero cell type for quantification, we titered our passage four stock on VeroE6 and VeroCCL81. Following infection with a dilution series, we found that SARS-CoV-2 replicated in both Vero cell types; however, the viral titers were slightly higher in VeroE6 cells than Vero CCL81 (Figure 2A). In addition, plaques were more distinct and visible on Vero E6 (Figure 2B). As early as 2 days post inoculations, VeroE6 cells produced distinct plaques visible with neutral red staining. In contrast, Vero CCL81 produced less clear plaques and was most easily quantitated with neutral red 3 days post inoculation. On the individual plaque monolayers, SARS-CoV-2 infection of Vero E6 cells produced cytopathic effect with areas of cell clearance (Figure 2C). In contrast, Vero CCL81 had areas of dead cells that had fused to form plaques, but the cells did not clear. Together, the results suggest that VeroE6 may be the best choice for amplification and quantification, but both Vero cell types support amplification and replication of SARS-CoV-2.
As research is initiated to study and respond to SARS-CoV-2, information about cell lines and types susceptible to infection is needed. Therefore, we examined the capacity of SARS-CoV-2 to infect and replicate in several common primate and human cell lines, including human adenocarcinoma cells (A549), human liver cells (HUH7.0), and human embryonic kidney cells (HEK-293T), in addition to Vero E6 and Vero CCL81. We also examined an available big brown bat kidney cell line (EFK3B) for SARS-CoV-2 replication capacity. Each cell line was inoculated with at high MOI and examined 24 hours post infection (Figure 3A). No cytopathic effect was observed in any of the cell lines except in Vero cells which grew to >107 PFU at 24 hours post infection. In contrast, both HUH7.0 and 293T cells showed only modest viral replication and A549 cells were incompatible with SARS-CoV-2 infection. These results are consistent with previous susceptibility findings for SARS-CoV and suggest other common culture systems including MDCK, HeLa, HEP-2, MRC-5 cells, and embryonated eggs are unlikely to support SARS-CoV-2 replication (14–16). In addition, SARS-CoV-2 failed to replicate in the bat EFK3B cells which are susceptible to MERS-CoV. Together, the results indicate that SARS-CoV-2 maintain a similar profile to SARS-CoV in terms of susceptible cell lines.
Having established robust infection with SARS-CoV-2 in several cell types, we next evaluated the cross reactivity of SARS-CoV antibodies against the SARS-CoV-2. Cell lysates from infected cell lines were probed for protein analysis; we found that polyclonal sera against the SARS-CoV spike and nucleocapsid proteins recognize SARS-CoV-2 (Figure 3B & C). The N protein, highly conserved across the group 2B family, retains >90% amino acid identity between SARS-CoV and SARS-CoV-2. Consistent with the replication results (Figure 3A), SARS-CoV-2 showed robust N protein in both Vero cell types, less protein in HUH7.0 and 293T, and minimal signal in A549 and EFK3B cells (Figure 3B). Similarly, the SARS-CoV spike antibody also recognized SARS-CoV-2 spike protein, indicating cross reactivity (Figure 3C). Consistent with SARS CoV, several cleaved and uncleaved forms of the SARS-CoV-2 spike protein. Notably, the cleavage pattern to the the SARS spike positive control from Calu3 cells, a respiratory cell line, varies slightly and could signal differences between proteolytic cleavage of the spike proteins between the two viruses due to predicted insertion of a furin cleavage site in SARS-CoV-2 (10). However, differences in cell type and conditions complicate this interpretation and indicate the need to further study in equivalent systems. Overall, the protein expression data from SARS-CoV N and S antibodies recapitulate replication findings and indicate that SARS-CoV reagents can be utilized to characterize SARS-CoV-2 infection.”
METHODS
Specimen collection
“Virus isolation from patient samples was deemed to be non-human subjects research by CDC National Center for Immunizations and Respiratory Diseases (research determination 0900f3eb81ab4b6e) Clinical specimens from the first identified US case of COVID-19 acquired during travel to china, were collected as described (1). Nasopharyngeal (NP) and oropharyngeal (OP) swabs in 2 to 3 mL viral transport media were collected on day 3 post-symptom onset for molecular diagnosis and frozen. Confirmed PCR- positive specimens were aliquoted and refrozen until virus isolation was initiated.
Cell culture, limiting dilution, and isolation
Vero CCL-81 cells were used for isolation and initial passage. Vero E6, Vero CCL-81, HUH 7.0, 293T, A549, and EFKB3 cells were cultured in Dulbecco’s minimal essential medium (DMEM) supplemented with heat inactivated fetal bovine serum(5 or 10%) and antibiotic/antimyotic (GIBCO). Both NP an OP swabs were used for virus isolation. For the isolation, limiting dilution, and passage 1 of the virus, 50 μl serum free DMEM was pipetted into columns 2–12 of a 96-well tissue culture plate. One-hundred μl clinical specimens were pipetted into column 1, and then serially diluted 2-fold across the plate. Vero cells were trypsinized and resuspended in DMEM + 10% FBS + 2X Penicillin-Streptomycin + 2X antibiotic − antimycotic + 2 X amphotericin B at 2.5 × 105 cells / ml. One hundred μl of cell suspension were added directly to the clinical specimen dilutions and mixed gently by pipetting. The inoculated cultures were grown in a humidified 37°C incubator with 5% CO2 and observed for cytopathic effect (CPE) daily. Standard plaque assays were used for SARS-CoV-2 based on both SARS-CoV and MERS-CoV protocols (19, 20).
When CPE were observed, the cell monolayers were scrapped with the back of a pipette tip. Fifty μl of the viral lysate were used for total nucleic acid extraction for confirmatory testing and sequencing. Fifty μl of virus lysate was used to inoculate a well of a 90% confluent 24-well plate.
Inclusivity / Exclusivity testing
From the wells in which CPE were observed, confirmatory testing was performed using using CDC’s rRT-PCR assay and full genome sequencing (1) The CDC molecular diagnostic assay targets three portions of the N gene, and all three must be positive to be considered positive (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-detection-instructions.html) and (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html). To confirm that no other respiratory viruses were present, Fast Track respiratory pathogen 33 testing was performed (FTD diagnostics).
Whole genome sequencing.
Thirty-seven pairs of nested PCR assays spanning the genome were designed based on the reference sequence, Genbank Accession No. NC045512. Nucleic acid was extracted from isolates and amplified by the 37 individual nested PCR assays. Positive PCR amplicons were used individually for subsequent Sanger sequencing and also pooled for library preparation using a ligation sequencing kit (Oxford Nanopore Technologies), subsequently for Oxford Nanopore MinION sequencing. Consensus Nanopore sequences were generated using minimap 2.17 and samtools 1.9. Consensus sequences by Sanger sequences were generated from both directions using Sequencher 5.4.6, and were further confirmed by consensus sequences generated from nanopore sequencing.”
In Summary:
- On January 22nd, 2020, a patient was confirmed positive by the CDC PCR test
- Nasopharyngeal (NP) and oropharyngeal (OP) samples were inoculated onto cell cultures and frozen “viral” lysates from cell culture were used for nucleic acid extraction using the CDC PCR test to confirm “SARS-COV-2”
Quick Detour:
The CDC’s PCR test, heavily relied upon for the results from their study, was initially recalled due to producing too many false-positives which was later blamed on contamination problems and a lack of proper quality control:
CDC Report: Officials Knew Coronavirus Test Was Flawed But Released It Anyway
“But NPR has learned the results of that final quality control test suggested something troubling — it said the kit could fail 33% of the time.
Under normal circumstances, that kind of result would stop a test in its tracks, half a dozen public and private lab officials told NPR. But an internal CDC review obtained by NPR confirms that lab officials decided to release the kit anyway. The revelation comes from a CDC internal review, known as a “root-cause analysis,” which the agency conducted to understand why an early coronavirus test didn’t work properly and wound up costing scientists precious weeks in the early days of a pandemic.”
“In addition to learning of the early warning, reviewers determined the Respiratory Viruses Diagnostic Laboratory, run by a highly regarded scientist named Stephen Lindstrom, was beset with problems, including “process failures, a lack of appropriate recognized laboratory quality standards, and organizational problems related to the support and management of a laboratory supporting an outbreak response,” the review said.”
“Compounding the problem, officials said, was the fact that the CDC had not established specific benchmarks for the test. There was not, for example, an agency directive that said the test needed to be correct some specific percentage of the time before it could be released.”
The CDC created and knowingly sent out faulty tests which were generating positive results even for negative control samples including WATER:
CDC coronavirus test kits were likely contaminated, federal review confirms
“The false positives arose during testing of “negative control” samples that contained highly purified water and no genetic material. That aspect of testing was essential to confirm that results would be reliable and not skewed by contamination.”
“Before they were shipped out, the test kits “began showing issues with negative controls showing positive results,” the review said. However, the kits were not vetted in advance with standard quality control and quality assurance, or “QC/QA,’’ procedures, according to the review.”
Of course, this was in February 2020 so it was very early in the “pandemic” and mistakes are sure to occur. The CDC quickly identified and corrected the mistake with their revised test…or did they? Not so, according to this WHO review published 4 months later:
Performance Assessment of SARS-CoV-2 PCR Assays Developed by WHO Referral Laboratories
“The E Charité and N2 US CDC assays were positive for all specimens, including negative samples and negative controls (water). These false-positive results were explored (details below), but the sensitivity of these assays was not further assessed.”
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355678/
It seems needless to say that any results stemming from the CDC PCR test which continually pegs negative controls and water as positive for “SARS-COV-2” should be immediately questioned and considered invalid.
End Detour.
- They observed “Coronavirus-like” morphology/morphogenesis in their unpurified EM images
- Unpurified “isolates” from the first passage cell culture of both NP and OP swabs were used for whole-genome sequencing
- After second passage, both NP and OP samples were cultured together in Vero cells
- They generated “viral” stocks from fourth passaged VeroE6 (fetal rhesus monkey kidney) cells
- VeroE6 cells were determined to produce the “virus” the easiest
- They decided to test whether “SARS-COV-2” would replicate in other primate and HUMAN cell lines
- No cytopathic effect (CPE) occurred in any of the other cell lines used, only Vero cells
- They determined that the “virus” could not infect/replicate in HUMAN nor many other common cell lines including MDCK, HeLa, HEP-2, MRC-5 cells, and embryonated eggs nor in Bat EFK3B cells
- Next, they evaluated the cross reactivity of “SARS-COV-1” antibodies against the “SARS-COV-2” and found that “SARS1” polyclonal sera recognized “SARS2”
- They also found that “SARS1” spike antibody recognized “SARS2” spike protein, indicating cross reactivity (thus proving that supposedly specific antibodies are in fact nonspecific in the process)
- It is admitted that differences in cell type and conditions can complicate interpretations
- Their antibody experiments indicated, fortunately enough for their purposes, that “SARS1” reagents can be utilized to characterize “SARS2” infection
- The NP and OP swabs were immediately placed in “viral” transport media
- VeroE6 cells were added to DMEM media along with fetal bovine serum and several antibiotics/antimycotics
- VeroE6 cells were trypsinized and suspended in added DMEM, FBS, 2x penicillin-streptomycin, 2x amphotericin B
- The VeroE6 concoction was added to the NP/OP samples and mixed together
- This unpurified creation was used for total nucleic acid extraction, confirmatory testing, and sequencing
- For Whole-Genome Sequencing, ONLY 37 base pairs were used yet the “SARS-COV-2” genome consists of 30,000 base pairs:
“Thirty thousand base pairs make up the (relatively tiny) SARS-CoV-2 genome. A singular genome holds limited information.”
https://www.google.com/amp/s/www.genengnews.com/news/mining-the-sars-cov-2-genome-for-answers/amp/
Dr. Tom Cowan did a brilliant breakdown of why this is an issue:
“… we find that rather than having isolated the virus and sequencing the genome from end to end, they found 37 base pairs from unpurified samples using PCR probes. This means they actually looked at 37 out of the approximately 30,000 of the base pairs that are claimed to be the genome of the intact virus. They then took these 37 segments and put them into a computer program, which filled in the rest of the base pairs.
“To me, this computer-generation step constitutes scientific fraud. Here is an equivalency: A group of researchers claim to have found a unicorn because they found a piece of a hoof, a hair from a tail, and a snippet of a horn.
“They then add that information into a computer and program it to re-create the unicorn, and they then claim this computer re-creation is the real unicorn. Of course, they had never actually seen a unicorn so could not possibly have examined its genetic makeup to compare their samples with the actual unicorn’s hair, hooves and horn.”
In this CDC study, we have unpurified cell cultures, faulty PCR tests/data, evidence “SARS-COV-2” can not infect human cells but only Vero cells, and a genome made up almost entirely by consensus computer-algorithms.
To say that the CDC committed scientific FRAUD would be an understatement.
ViroLIEgy 
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