Creating the Cytopathic Effect

“Cytopathic effect (CPE), structural changes in a host cell resulting from viral infection. CPE occurs when the infecting virus causes lysis (dissolution) of the host cell or when the cell dies without lysis because of its inability to reproduce.”

https://www.britannica.com/science/cytopathic-effect

I’ve already gone through some of the various factors (bacteria, amoeba, parasites, chemical additives) which can be a cause of the CPE said to be specific to “viral” invasion. I’ve also outlined many of the numerous problems regarding the use of cell cultures as proof of “virus” isolation in previous posts. However, this syllabus on cell culture techniques that I came across was full of additional insight on causes of CPE as well as some interesting admissions regarding cell cultures so I felt the need to share it. Below are some highlights along with a summary at the end. I recommend reading the whole syllabus sometime as it is a long but very worthwhile read.

A few things to think about while reading:

• Note the various forms of contamination in cell culture
• Look at the many admittances to CPE being caused by factors other than a “virus”
• Notice that kidney cells are considered sanctuaries of “viruses”
• Look for the different ways they try to bring about CPE with cell-altering tricks (longer culturing times, blind cultures, or changing cell lines)
• Think about the numerous assumptions which are made during the “isolation” process

Hopefully, this information may shed some new light for some of you as well:

THE ART OF ANIMAL CELL CULTURE FOR VIRUS ISOLATION

Cell Culture Techniques

“To paraphrase recent statements by a colleague, “All cells cultured in vitro are angry; they are outside of their normal environment and maintained under artificial conditions, surrounded by physiologically incorrect concentrations of all things important to their well-being. No wonder it is so difficult to have well-behaved cell cultures”! Without adequate training and preparation, cell culture as an art and science becomes sloppy, and data generated by such practices are questionable. We have often not been able to repeat the results of others, and they have not been able to repeat ours, due to a difference in the cells used in our experiments. In some cases, the cells are not what they should be, in other cases, the cells are contaminated with adventitious agents that confound the results, and sometimes, the cells have changed, either through differentiation or genetic instability.”

COMPLICATIONS ARISING FROM THE USE OF PRIMARY CELLS FOR VIRUS ISOLATION

“Primary cells (Table 1), which are non-immortalized cells taken directly from a living organism, are often used in clinical laboratories for the isolation of various viruses. For example, primary monkey kidney cells, which in the USA are obtained from rhesus or other macaques or from various African “green” monkey species, are used for the isolation of echo and other picorna viruses, and human parainfluenza and other paramyxoviruses. Primary cells are especially useful for diagnostic virology because some viruses are easier to isolate (or can only be isolated) in them. However, primary cells often harbor latent viruses that become reactivated once the cells are separated from kidneys and propagated in vitro, or, contain viruses that produce a persistent but subclinical infection of the host. The latter viruses may not cause significant (if any) pathology in vivo, where the cells exist in an environment with a functional immune system. But outside of the host and away from the immune system, the cells may be fully permissive and the same virus cause highly cytopathic effects (CPE). Unfortunately, some primary cells may also harbor viruses that can replicate in the host cells without causing easily recognized CPE, and also in the indicator cells used for their isolation (or detection) in vitro. Unwanted viruses in primary cells cause various complications relevant to the isolation of a target virus, including:

They might quickly overtake a cell culture, reducing the chances of isolating the target virus.

They may cause CPE identical to those of the target virus, thus causing a false positive preliminary assessment.

They are obvious sources of contamination that complicate the isolation of the target virus in “PURE” form.

They may pose a biosafety risk to laboratory workers.

Noteworthy, primary cells can harbor contaminating agents other than viruses. For example, mycoplasma species are present in most animals, and are prevalent on the surfaces of the respiratory tract. Moreover, mycoplasma species exist as intracellular and extracellular varieties. For reasons not yet entirely clear, kidneys are “sanctuaries” for viruses. For this reason, we often hunt for new viruses in kidney cells sourced from exotic species (J. Lednicky, unpublished).”

“Primary cells also have a finite lifespan, and should be used with minimal passages in vitro. Otherwise, senescence of the cells can be mistaken for CPE caused by viruses.”

“A common mistake is to assume that primary cells obtained from the suppliers are certified to be virus free. In reality, this is not the case. For example, the donors of primary human cells sold in the USA are examined (by serology) for antibodies to Hepatitis B and C viruses, and to HIV, following United States Food and Drug Administration (USFDA) guidelines, and if that information is not available, the cells are checked by PCR or other methods for the same viruses. [The USFDA is an agency of the United States Department of Health and Human Services responsible for protecting and promoting public health through the regulation and supervision of biopharmaceuticals, blood transfusions, cosmetics, dietary supplements, electromagnetic radiation emitting devices (ERED), food safety, medical devices, over-the-counterpharmaceutical drugs (medications), tobacco products, prescription, vaccines and veterinary products]. However, additional tests for other adventitious agents have not been mandated by the USFDA, and it may be impractical to check for the presence of many other agents with regard to cost and representative sampling reasons. Thus, commercially supplied human primary cells are sold with an advisory statement indicating the cells should be considered as potentially infected, and that biosafety practices be used when working with the cells.”

“Cell deterioration in primary cells due to improper cell growth media formulation can also be confused for CPE caused by viruses.”

“We have noted cell deterioration due to l-glutamine deficiency, and to improper dosage of antifungal agents in the growth media, among a few batches of commercially bought primary cells. Similarly, commercial media formulations for primary human cells often include additives such as epinephrine, human recombinant epidermal growth factor, hydrocortisone, insulin, transferrin, and others; a mistake in the amounts of some of these biomolecules added to the cell growth media can adversely affect cell viability.”

“Thus, primary cells are useful for the isolation of some viruses, but should be used with caution because: (a) they can contain adventitious agents, and (b) cell deterioration due to one of many different reasons can be mistaken for virus-induced CPE.”

ADVENTITIOUS VIRUSES IN CELL-LINES

“It is not uncommon to receive virus-contaminated cell lines from suppliers, and this is especially true for cells obtained through inter-laboratory transfer. One problem is that the cells may have become infected with bovine viruses (from serum) that replicate relatively slowly (i.e., the time it takes for them to complete a replication cycle and form progeny virions is higher than that of the cell population doubling time). These contaminating viruses are referred to as “adventitious” viruses (i.e., they are viruses that should not be present).

Many times, the adventitious viruses go unnoticed, and the deterioration of the cells is attributable to some type of “folklore” prevalent among cell culture practitioners.”

“Apart from sera, contaminating viruses can also be traced to laboratory workers, to animal-sourced enzymes used for cell culture (such as porcine trypsin), and to other biological used for cell culture. A recent compilation of bovine and porcine viruses that may contaminate bovine serum and porcine trypsin is available in ref. 59. As new viruses are discovered, awareness of their possible presence in biologicals like sera and trypsin draws more interest and attention.”

“We recently traced a filtered amino acid supplement as the source of a contaminating reovirus, and learned from some industry colleagues they had made the same finding. However, as typical of these cases, the findings are not published and thus the information not widely disseminated.”

“In some cases, unusual bacteria, and even some single-celled eukaryotic microorganisms cause cell contamination problems that are attributed to viruses. This is because many people engaged in cell culture have little experience with the detection and identification of these types of organisms.”

“It is also distressing when one performs electron microscopy and discovers that more than one virus is present in the specimen being viewed (or worse, only the wrong virus is visualized). Contaminated cell lines are a main reason gene expression studies can vary significantly between laboratories.”

PARADIGMS FOR VIRUS ISOLATION

“Cultures should not be considered negative for virus isolation if CPE are not detected. A second measure should be considered and well-thought criteria should be developed before rejecting a “negative” culture. For example, would CPE form if the cultures were held for a longer period of time?“

“Electron microscopy should be performed using material from spent media to detect liberated virions, and also, on a sample of the infected cells (often, the number of liberated virions is too low to be easily visualized through electron microscopy, and virus infection is determined only by examining the infected cells themselves).”

“Some viruses require “adaptation” prior to adequate replication in cultured cells and the formation of CPE. In the past, the process referred to as “blind culture” was performed when virus was suspected but CPE inapparent. A popular version of this method is to periodically remove samples from a culture of presumably infected cells, and to inoculate that into a new batch of cells. This process is repeated four times. An adjunct to former process is to split the infected cells (if confluent) into a larger flask or into several flasks and allow the cells to replicate. This may make CPE apparent if actively replicating cells are optimal for the detection of the CPE caused by a particular virus.”

“During the primary isolation of virus from clinical or environmental specimens, many laboratories routinely filter specimens though a 0.45µm filter prior to inoculation of cell cultures. This filtration step is performed to remove bacteria, fungi, and other potential microbial contaminants, and non-living particulates. A problem with this filtration step is that many viruses are pleomorphic and some have long, filamentous forms that may exceed 0.45 µm. This includes some influenza viruses, and morbilliviruses. Also, in clinical specimens, many viruses are attached to cellular and other debris, and are trapped by the filter. We recommend the inoculation of two batches of cells; one with a filtered aliquot, the other unfiltered, of the virus specimen. Unfortunately, it is not uncommon these days for bacteria in clinical specimens (such as normal flora that are contaminants of naopharyngeal swabs) to be resistant to penicillin and streptomycin; we prefer to use an antibiotic mixture that includes neomycin in addition to penicillin and streptomycin.”

CLOSING REMARKS

“Due to the complexities of cell culture, and the nature of the biomaterials used, it is not possible to consistently attain the same end results at all times. Moreover, viruses constantly mutate, and so the “rules of the game” can change. Therefore, the practice of cell culture for virus isolation is part art, part science, and part luck.”

https://www.alexandriarepository.org/syllabus/cell-culture-techniques/

Alternate link:

https://www.google.com/url?sa=t&source=web&rct=j&url=https://fdocuments.in/amp/document/the-art-of-animal-cell-culture-for-virus-isolation-the-art-of-animal-cell.html&ved=2ahUKEwjv6uKypoX2AhUDCjQIHaBoDTQQFnoECAkQAQ&usg=AOvVaw1qmwdzFVN-1250U7dP3Ntj

In Summary:

• “All cells cultured in vitro are angry; they are outside of their normal environment and maintained under artificial conditions, surrounded by physiologically incorrect concentrations of all things important to their well-being”
• The author states that his lab has been unable to reproduce the cell culture experiments of others and vice versa
• Primary cells, which are non-immortalized cells taken directly from a living organism, are often used in clinical laboratories for the isolation of various “viruses”
• These cells are harbors of “viruses”
• The latent “viruses” may not cause CPE and go unnoticed
• They might quickly overtake a cell culture, reducing the chances of isolating the target “virus”
• They may create identical CPE to the target “virus”
• They may make isolating the target “virus” in a “PURE” (his quotations, not mine) form difficult (i.e. impossible)
• These cells also harbor contaminates other than “viruses”
• Kidney cells (ex. Vero cells) are sanctuaries of “viruses” and are used to hunt for new “viruses”
• Senescence of the cells (deterioration caused by aging) can be mistaken for CPE
• A common mistake is assuming primary cells bought from suppliers are “virus-free”
• Additional tests for other adventitious agents in primary cells have not been mandated by the USFDA
• Commercially supplied human primary cells are sold with an advisory statement indicating the cells should be considered as potentially infected (in other words, they admit these come contaminated)
• Cell deterioration in primary cells due to improper cell growth media formulation can also be confused for CPE caused by “viruses”
• Mistakes in cell growth media formulations can impact cell viability
• Primary cells should be used with caution due to:
  1. Contamination from adventitious agents
  2. Cell deterioration can be mistaken for CPE
• It is not uncommon to receive “virus-contaminated” cell lines from suppliers
• He claims cells may have become infected with bovine “viruses” from serum
• These contaminating “viruses” are referred to as “adventitious viruses” (i.e., “viruses” that should not be present)
• Adventitious “viruses” go unnoticed, and the deterioration of the cells is attributable to some type of “folklore”
• In other words, they see CPE that does not line up with the target “virus” so they assume there must be some other “virus” present which caused it
• Contaminating “viruses” can also be traced to laboratory workers, to animal-sourced enzymes used for cell culture (such as porcine trypsin), and to other biological used for cell culture
• As new “viruses” are discovered, awareness of their possible presence in biologicals like sera and trypsin draws more interest and attention
• In other words, they have no clue whether or not there are undiscovered “viruses” in their sera, culture, media, etc. and will use these invisible contaminants as an excuse for unintended CPE
• Unusual bacteria, and even some single-celled eukaryotic microorganisms cause cell contamination problems that are attributed to “viruses”
• The author admits that it is distressing when one performs electron microscopy and discovers that more than one “virus” is present in the specimen being viewed (or worse, only the wrong “virus” is visualized) which obviously implies that the samples were neither purified nor isolated
• Contaminated cell lines are a main reason gene expression studies can vary significantly between laboratories
• Cultures should not be considered negative for “virus isolation” if CPE are not detected (thus destroying the importance of CPE as an identifier of “viruses”)
• He asks the question “Could CPE form if the cultures were held for a longer period of time?” thus demonstrating that the culture process is the cause of the CPE, not a “virus”
• Often the number of liberated “virions” is too low to be easily visualized through electron microscopy, and “virus” infection is determined only by examining the infected cells themselves
• In other words, they often can not find any “virus” visually even if there is CPE indicative of one
• Some “viruses” require “adaptation” prior to adequate replication in cultured cells and the formation of CPE once again confirming that the cell culture process itself causes this effect, not “viruses”
• In the past, the process referred to as “blind culture” was performed when “virus” was suspected but CPE inapparent
• In other words, they sub-culture until the cell starts to die and they get the effect they want in order to claim “virus”
• A problem with the filtration step is that many “viruses” are pleomorphic and some have long, filamentous forms that may exceed 0.45 µm
• It is not uncommon these days for bacteria in clinical specimens (such as normal flora that are contaminants of naopharyngeal swabs) to be resistant to penicillin and streptomycin
• Due to the complexities of cell culture, and the nature of the biomaterials used, it is not possible to consistently attain the same end results at all times

It is clear to see based on the numerous statements in this syllabus that the culture conditions have a profound impact on the results of the cell culture experiments. This stretches far beyond the various forms of contamination that are sure to affect the cells. The deterioration of the cells due to aging and/or due to the nature of the biochemicals used causes the exact same cytopathic effect (CPE) said to be caused by “viruses.” Yet though they know this, it is often the invisible “viruses” getting the blame even when they can not be found or seen. The researchers assume other “viruses” are present and causing the effect.

Even worse is when no CPE is observed. Instead of stating no “viruses” are present in the sample, the author states that this should not necessarily be the conclusion. He ponders whether further culturing will bring about the CPE if it is done over a longer period of time. This leads to a process known as blind passaging which is where the cells are divided and put into new petri dishes with fresh cell altering media/chemicals. This process is repeated multiple times until they finally achieve the CPE result that they are looking for. It is well known that cells react negatively to environmental/physical stressors and the process of blind passaging is highly stressful on them. This leads to cell deterioration. Virologists are literally creating the effect they want to see through their culturing process. Once they get their CPE, virologists get to claim a “virus” is present even if they are unable to obtain an EM image of the suspected “virus.”

This is scientific fraud being perpetrated on a massive scale. They know it and they admit it in their work. They hope no one notices.

It’s far past time we all take notice and start to hold them accountable.