Viral Transport Media

Viral Transport Media is used to “preserve” a “virus” after the sample has been taken from the sick person. It is intended to keep the “virus” in a stable condition while it is transported to laboratories in order to be tested, cultured, sequenced, etc. Looking at two of the original “SARS-COV-2” papers for what was used as a VTM sheds quite a bit of light on this hoax:

From the Zhou paper “A pneumonia outbreak associated with a new coronavirus of probable bat origin:”

“For swabs, 1.5 ml DMEM CONTAINING 2% FBS was added to each tube.”

“THE VIRAL TRANSPORT MEDIUM was composed of Hank’s balanced salt solution (pH 7.4) containing BSA (1%), AMPHOTERICIN (15 μg ml−1), PENICILLIN G (100 units ml−1) AND STREPTOMYCIN (50 μg ml−1).”

https://www.nature.com/articles/s41586-020-2012-7

From the Kim paper “Identification of Coronavirus Isolated from a Patient in Korea with COVID-19:

“Oropharyngeal samples were diluted with VIRAL TRANSFER MEDIUM CONTAINING nasopharyngeal swabs and ANTIBIOTICS (NYSTADIN, PENICILLIN-STREPTOMYCIN 1:1 dilution)”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7045880/

Knowing what we already know about the disastrous effects of DMEM, Fetal Bovine Serum, and Antibiotics on cell cultures should be enough to question the results of these studies. Keep in mind that adding VTM is done to the sample BEFORE the Cell Culture. It is already contaminated by toxic chemicals and then it is added to African Green Monkey Kidney cells which is further bombarded by cell altering chemicals/contaminants.

However, VTM not only effects the cells during the culturing process, it also effects the degradation of RNA for PCR testing and sequencing purposes even in instances where culturing does not occur. From a study in June 2020:

The impact of viral transport media on PCR assay results for the detection of nucleic acid from SARS-CoV-2 and other viruses

“The stability of SARS-Cov-2 RNA was assessed in several commercially produced transport media and an in-house solution. Coronavirus RNA was rapidly destroyed in the commercial transport media though the deleterious effects on intact virus were limited. Similar results were obtained for a Type A influenza virus. There was reduced detection of both virus and nucleic acid when a herpesvirus sample and purified DNA were tested. Collectively these data showed that the commercial viral transport media contained nucleases or similar substances and may seriously compromise diagnostic and epidemiological investigations.

“Recommendations to include foetal bovine serum as a source of protein to enhance the stabilising properties of viral transport media are contraindicated. Almost all commercial batches of foetal bovine serum contain pestiviruses and at times other bovine viruses. In addition to the potential for there to be nucleases in the transport medium, the presence of these viruses and other extraneous nucleic acid in samples may compromise the interpretation of sequence data.

“The results of these studies clearly indicate that the commercially prepared VTM solutions have had an adverse impact on the ability to detect both SARS-CoV-2 and influenza RNA. The results for the RNA preparations diluted in the commercial VTMs would suggest that there are components of these VTMs that have prevented the detection of the RNA in these samples. As the detection of the XIPC was not affected, we propose that these results provide unequivocal evidence of the presence of a nuclease(s) or similar substance in these VTMs.”

“It is also important to recognise that these observations reflect the outcome of contact between viral nucleic acid and VTM for less than 1 hour in each instance. The levels of nucleic acid that were destroyed after almost immediate addition to the VTMs were not insignificant. With a concentration of RNA that gave a Ct value of approximately 21 in both PBS and VTM-1, this represents a reduction of approximately ten thousand-fold and cannot be ignored. While it might be argued that the adverse impact on whole virus appeared to be slight, free nucleic acid and perhaps whole virus was destroyed at levels that could be of diagnostic relevance (20). While the impact on RNA viral genomes is likely to be markedly greater than on DNA sequences, the outcome cannot be predicted as secondary structure may also have an influence (20) and the speed and severity of the impact may vary depending on the nucleic acid target, as shown by the differences between the results for the two RNA viruses and the XIPC. Further, it cannot be assumed that the target nucleic acid will always be protected by nucleoprotein. Degradation will occur during the course of an infection and also under conditions where sample collection, transport and storage are sub-optimal.”

“As whole genome nucleic acid sequencing is now often undertaken on many original, uncultured patient samples, the presence of these viruses and other extraneous nucleic acid in samples may reduce the sensitivity of sequencing protocols and complicate the interpretation of sequence data. The nucleases present may also have an impact on the quantity of RNA available for sequencing. Furthermore, the inclusion of fbs presents a biosecurity risk for the movement of animal pathogens between countries and renders such VTM solutions unsuitable for many animal disease diagnostic or research applications.”

https://www.biorxiv.org/content/10.1101/2020.06.09.142323v1.full

It is clear that VTM is a toxic mixture currently being used in the transport of patient samples. Are there other solutions that are less toxic to the samples and cells that can be used as VTM?

PHOSPHATE BUFFERED SALINE:

Other media has been used to transport specimens during this “pandemic” such as Phosphate Buffered Saline, which is already used for other aspects of Cell Culture such as washing and dilution:

“PBS (phosphate buffered saline) is a balanced salt solution used for a variety of cell culture applications, such as washing cells before dissociation, transporting cells or tissue, diluting cells for counting, and preparing reagents.”

https://www.thermofisher.com/order/catalog/product/10010023#/10010023

PBS is considered a relatively safe and non-toxic salt solution for most cells. However, even this relatively “non-toxic” solution can alter the cells during the culturing process so it’s use as a transport media should be questioned as well:

“THE EFFECT OF TIME AND DILUENTS ON CELL CULTURE IS NOT WELL UNDERSTOOD.”

“Furthermore, LENGTH OF TIME samples were incubated in phosphate buffered saline also CONTRIBUTED TO THE OBSERVED DROP IN CELL VIABILITY.”

“THIS REPORT PRESENTS THE ADVERSE EFFECTS OF, and alternatives for, CELL CULTURE SAMPLES DILUTED IN PHOSPHATE BUFFERED SALINE (PBS). LOWER VIABILITY AND GREATER VARIABILITY WAS OBSERVED WITH PBS DILUTED SAMPLES. Furthermore, the viability of PBS diluted samples CONTINUOUSLY DECREASED OVER TIME AND AT A FASTER RATE THAN THE OTHER CONDITIONS. This phenomenon was observed with multiple cell lines and different culture systems.”

“Therefore, care needs to be taken when preparing viability samples with diluents TO ENSURE THE RESULTS ARE ACCURATE AND REPRESENTATIVE OF THE CULTURE.”

“Cell culture sample dilution is sometimes necessary to preserve the working volume and/or to extend assay measurement range. Therefore, IT IS IMPERATIVE TO IDENTIFY ALTERNATIVE DILUENTS THAT DO NOT INADVERTENTLY DECREASE VIABILITY.”

“A systematic investigation has revealed that CELL CULTURE SAMPLES DILUTED IN PBS COULD INADVERTENTLY LOWER THE VIABILITY WHEN MEASURED USING AUTOMATED CELL COUNTERS. The effect of PBS on viability can be consistently reproduced, and is independent of process scale, cell line, operator and automated cell counter used. In addition, THE NEGATIVE IMPACT OF PBS ON THE VIABILITY IS PROPORTIONAL TO THE SAMPLE INCUBATION TIME, AND INVERSELY PROPORTIONAL TO THE TCC.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338812/

From a Cell Counting Manual:

“However resuspension in PBS BUFFER RESULTS IN A DRASTIC LOSS OF VIABILITY AND SIGNIFICANT REDUCTION IN AVERAGE CELL DIAMETER. There is also some minor reduction in cell concentration which may be expected from losses during resuspension. Spinning and resuspending a second time in PBS HAS A FURTHER NEGATIVE IMPACT ON CELL VIABILITY AND CELL SIZE.”

“It is recommended that prior to running a cell culture on a complete plate that some initial evaluation is performed to determine the robustness of the cells involved AND THEIR ABILITY TO TOLERATE THE SAMPLE PREPARATION METHODS AND PROLONGED EXPOSURE OUTSIDE THE INCUBATOR ENVIRONMENT. Even engineered
cells such as CHO cells or immortal HELA cells, which are generally selected to be quite durable, CAN BE EFFECT BY STRESS OR PARTICULAR SAMPLE PREPARATION CONDITIONS.”

https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.beckman.com/gated-media%3FmediaId%3D%257B06D69A51-A996-456C-9E06-6E8A23E2D79C%257D&ved=2ahUKEwiOwIqvqvvuAhXUHc0KHe25A5sQFjADegQIBBAG&usg=AOvVaw0fqNRJAJ-l6vIFhB3g1eXk

It appears even the relatively “harmless” PBS has an adverse effect on cell cultures including lower viability, greater variability, and a decrease in cell size. It is admitted that the length of time and effect of PBS on cell cultures is not well known, which seems to be a common theme regarding the interactions of all of the various chemicals, antibiotics, nutrients, serums, etc. on the samples and cell cultures.

Trust the conclusions of these “studies” at your own risk.

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