Do Cytopathic Effects Prove “Viral” Replication?
I want to preface this article by stating that the cell culture experiment has been repeatedly shown to be a pseudoscientific set-up. I have already written about the logical fallacies built into its design and demonstrated how it fails to adhere to the scientific method
Crucially, the experiment does not test a hypothesis derived from an observed natural phenomenon. There is no valid independent variable—no purified, isolated “viral” particles—prior to the experiment.
The dependent variable, the cytopathic effect (CPE), is a non-specific, laboratory-induced artifact that can be explained by multiple causes without invoking a fictional one.
Despite this, the claim that CPE equals the presence of a “replicating virus” became the cornerstone of virology. My aim here is to show that this core assumption was never proven, was baseless from the start, and has been falsified by virology’s own experiments and methods.
The advent of tissue culture techniques in the early twentieth century marked a watershed moment for the fledgling field of virology, enabling researchers to generate effects in the lab that they could then attribute to their invisible entities.
Before tissue and cell culture techniques existed, “viruses” were merely a shifting concept defined purely in negative terms. As Thomas Rivers noted, they were identified by what they were not:
- invisible to ordinary microscopy,
- not recoverable through filtration,
- unable to propagate without living cells.
With the rise of tissue culture in the early 20th century, virology finally gained something visible—but only in the form of cell damage. It is crucial to note, however, that tissue and cell cultures are not natural biological systems.
They are artificial laboratory constructs made from dying or weakened animal and human tissues kept alive through nutrient broths, fetal bovine serum, antibiotics, antifungals, and various chemical additives.
Nothing about these mixtures resembles the internal environment of a living organism. Any effects observed within such systems reflect the breakdown, toxicity, and stress of the culture conditions themselves—not a naturally occurring process.
In other words, tissue culture did not emerge from observing a phenomenon in nature and seeking its cause; instead, the phenomenon was created first, and the cause was assumed afterward.
The most striking of these lab-created phenomena were morphological changes such as rounding, detachment, syncytia formation, and eventual cell death—changes that virologist John Franklin Enders later termed cytopathic effects (CPE) in the late 1940s.
These changes were quickly interpreted as proof of “viral replication.”
According to virologist Thomas Weller, who worked with Enders during the polio investigations, the so-called “viruses” induced degenerative changes in the cells in which they multiplied, changes detectable morphologically or biochemically.
From the outset, these visible alterations were interpreted as direct evidence of “viral” replication, establishing a central assumption: a “virus” infects a cell, it replicates, and that replication produces CPE.
This untested assumption quickly became foundational for “virus isolation,” identification, and quantification moving forward, with the Encyclopedia of Virology defining CPE as when “viruses kill or morphologically modify their host cells when they multiply.”
Similarly, Fields Virology notes that “one of the classic ways of detecting virus replication in cells was the observation of changes in cell structure, or CPEs, resulting from virus infection.”
By the late 1980s, this assumption was still firmly entrenched. In the book Veterinary Virology, Frank Fenner described CPE as “visible evidence of cell damage” that occurs when “viruses” kill the cells that they replicate in:
“Many viruses kill the cells in which they replicate, so that infected cell monolayers gradually develop visible evidence of cell damage, as newly formed virions spread to involve more and more cells in the culture. These changes are known as cytopathic effects (CPE), and the responsible virus is said to be cytopathogenic.”
Similarly, Altmeyer’s Encyclopedia—a reference for medical professionals—defines CPE as the “effect of viral replication on the host cell itself.”
Taken together, these definitions show how deeply entrenched the CPE = “viral replication” assumption became. The observation of CPE was no longer treated as a phenomenon requiring explanation, but rather as an indirect marker of both the presence and multiplication of the invisible entity.
Yet the critical question remained unasked: does this lab-created phenomenon actually demonstrate that “viruses” were present and replicating, or was it simply assumed to? If other evidence surfaced that refuted the CPE-equals-replication dogma, would the framework of virology even allow it to be acknowledged?
Let’s take a look.
Classical Demonstration: Enders and the “Proof” of “Viral” Replication
The work of Enders, Weller, and Robbins in the late 1940s exemplifies the historical reliance on CPE as proof of “viral replication.” According to Grafe’s A History of Experimental Virology (1994, Ch. 4), the aim of Enders’ group was to prove “viral multiplication” of the “poliovirus.”
They observed that samples alleged to harbor “poliovirus” that were inoculated into human embryonal tissue produced morphological changes which were inhibited by specific “immune” serum:
“A very significant improvement that proved crucial for quantitative virology was introduced by Enders and colleagues in 1949. F.Robbins had been able to show in tissue-culture experiments in 1948 that the Lansing strain of poliovirus, which was adapted to the mouse, would multiply in a culture of human, embryonal, intestinal tissue. In an analogous procedure, virus were also successfully cultured in human embryonal nerve, skin, connective and muscle tissue.
Proof of virus multiplication was obtained in subsequent infection experiments with mice and monkeys. Far more important, however, were the changes in cell morphology they could trace, and the discovery of the unstainability of infected cell nuclei. In a 1950 study, these two scientists, together with Weller, presented the “cytopathogenic effects” which were later referred to as the cytopathic effect (CPE) of cytopathogenic viruses.”
“Proof that the poliovirus, which until that time had been strictly classified as a neurotropic virus, could multiply in vitro in nonneural tissue triggered analogous tissue-culture trials with other types of virus. It should be mentioned that the aim of the Enders group was to demonstrate the multiplication of the poliovirus in the intestinal tract.”
Crucially, however, these experiments never involved purified and isolated “viral” particles. Instead, tissue homogenates alleged to contain “poliovirus” were added to cultures of human embryonal tissues, and the subsequent cellular degeneration was interpreted as “viral multiplication.” This leap—from correlation to causation—was never empirically validated.
These observations were nonetheless interpreted as confirmation that the presumed “virus” caused the observed CPE and were further used to quantify “virus” content, identify “viral” strains, and test “antiviral” substances.
The trio were awarded with the Nobel Prize in 1954 based upon their polio findings, with the prize motivation being “for their discovery of the ability of poliomyelitis viruses to grow in cultures of various types of tissue.”
The Nobel committee’s wording makes it clear that their interpretation of what the researchers observed (CPE in cultured cells) was framed as evidence of “viral growth/replication.”
By enshrining this interpretation as a “discovery,” the award effectively erased the distinction between assumption and proof, thereby reinforcing the assumption that CPE was synonymous with “viral” growth.
From that moment on, virology proceeded under a framework where visible cellular injury in culture was treated as definitive evidence of “viral replication.”
However, is this truly what Enders et al. obseeved?
Enders’ Initial Caution and Contradictions
In their 1949 paper Cultivation of the Lansing Strain of Poliomyelitis Virus in Cultures of Various Human Embryonic Tissues, Enders noted morphological differences between inoculated and uninoculated cultures after prolonged cultivation: the nuclei of cells in inoculated fragments showed a marked loss of staining properties, while cells in control cultures appeared normal. Yet, rather than leaping to a definitive conclusion, Enders wrote:
“Since the amount of material which has been studied is as yet relatively small, one cannot conclude that the changes observed in the inoculated cultures were caused by the virus.”
This admission is striking. Enders openly acknowledged that correlation did not establish causation, and that the observed cellular abnormalities might have alternative explanations. At this early stage, the link between CPE and “viral replication” was not treated as fact, but as a working hypothesis awaiting further confirmation.
But only a year later, in the 1950 paper Cytopathogenic Effect of Poliomyelitis Viruses In Vitro on Human Embryonic Tissues, Enders and his colleagues made a sweeping reversal, concluding that the cytopathic effects they observed “left no doubt” the poliomyelitis “virus” was multiplying in cells and that CPE could serve as a reliable marker for identifying “viruses.”
This leap—from tentative observation to claimed certainty—became the foundation of modern virology:
‘These phenomena are of interest from two general points of view. First. they leave no doubt that poliomyelitis virus in vitro can multiply in cells other than those of the nervous system and cause profound injury of such cells.
Secondly, they provide criteria by which the presence of the virus can be recognized in vitro and hence may afford a basis of technics for isolating virus from patients or animals, for the quantitative assay of virus, for serologic typing and possibly for the screening of chemotherapeutic and antibiotic substances.
Further study will be required of the reliability and practicability of the application of these phenomena to such ends.”
However, only a few years later, and in the same year he received the Nobel Prize for “proving” that “viruses replicate” in tissue cultures and published his influential measles study that also showed CPE in the “uninfected” cultures, Enders published a revealing paper Cytopathology of Virus Infections: Particular Reference to Tissue Culture Studies.
In it, Enders acknowledged that the central evidence—cytopathic effects (CPE)—was not specific to “viruses.” He admitted that:
- CPE can be caused by “many noxious agents,” not just “viruses.”
- Inclusion bodies are not conclusive evidence, since chemicals and unknown factors can produce them as well.
- Culture conditions and donor variables drive the outcomes, meaning age, species, and prior handling of tissue could dictate whether CPE appeared.
- Conclusions about “viral” causation were only “tentative.”
Enders wrote:
“The phenomena mentioned above under Group 1 changes may be evoked by many noxious agents. Accordingly, they cannot alone be considered as necessarily the result of viral activity. To prove this certain control procedures (serial cultivation, prevention of changes by homologous antibody, etc.) must be applied. Familiarity, however, with the effects of a specific virus in a given cell system often enables the observer to conclude tentatively that this virus is responsible.”
“Of morphological indices of viral injury, the formation of inclusion bodies (Group 2 above) is the most characteristic, although again this process cannot be accepted as conclusive evidence of viral activity since certain chemical as well as other unknown factors may condition its development.
Inclusion bodies were the first cytopathic changes to be sought for in vitro and employed as criteria of infection. As indices of viral multiplication, however, they are less useful than the changes of Group 1, because these structures can be unmistakably demonstrated only in stained preparations.”
“Cytopathogenicity in vitro is influenced by factors some of which are known while many remain to be defined. At the outset a few of those now recognized will be mentioned as an introduction to the review of recorded observations on the behavior of individual agents. Of primary importance is the species from which the cells are derived.
Analogous to the host range of a virus is its cytopathogenic range in cultivated cells. But correlation between susceptibility of the organism and its cells in vivo does not always exist. For although this correlation frequently obtains, the tissues of a susceptible species occasionally fail to support viral multiplication while the converse of this situation also occurs.
The age of the donor of tissue may influence cytopathogenicity. Just as young animals are frequently more susceptible to infection so their tissues may be more vulnerable to injury by the virus, yet again this correlation is not invariable. Most of the pertinent data indicate that acquired immunity to viral infections is not reflected by an increased cellular resistance, a fact advantageous from the technical point of view since it eliminates concern over the immunologic status of the donor animal.
The intensity and degree of cytopathic injury may vary according to the strain of virus or the conditions under which it has been propagated prior to its study in tissue culture.
The investigator should be prepared to encounter such variations in the study of a number of representatives of a viral species. Moderate or weak cytopathogenicity may sometimes be enhanced by serial passage in vitro.”
In other words, at the exact moment virology was being elevated as having proven “viral replication,” its leading figure openly conceded that the evidence was ambiguous, confounded, and reliant on assumption.
Yet two years later in 1956, Enders framed things differently. In his Gordon Wilson Lecture, Observations on Certain Viruses Causing Exanthematous Diseases in Man, he explained that the “shortcomings” of tissue culture were overcome not by isolating and independently demonstrating a causal agent, but by observing degenerative changes in cells after exposure to suspect material:
“In this respect the shortcomings of the tissue culture have in many instances been overcome by demonstration of the fact that the majority of viruses so far examined produce, as they multiply, degenerative changes in the cells. These changes are often apparent within a few hours or a few days after the viral inoculum is introduced and frequently are sufficiently characteristic to permit a tentative identification of the virus under examination. Such effects are now referred to as “cytopathic changes” or as “cytopathogenic effects of the virus”.
Although cytopathic changes were noted earlier by various investigators, especially by Ivanovics and Hyde in 1932 and Huang in 1942, their full significance was not generally appreciated until 1950, when it was demonstrated that the viruses of poliomyelitis were highly cytopathogenic in cultures of a variety of several human tissues.
Since that time results obtained with a large number of viruses and a variety of cells from various species show that exhibition of cytopathogenicity in vitro affords criteria of viral multiplication as reliable as the production of signs of infection in animals. Moreover, such criteria may be often more conveniently and accurately observed.”
Although he had previously acknowledged that CPE was nonspecific and could be due to multiple factors, Enders now elevated visual degeneration in artificial cell systems to the status of proof.
He hailed the observed cell death as “criteria of viral multiplication as reliable as the production of signs of infection in animals.”
These laboratory artifacts were treated as equivalent to disease itself, without first demonstrating an independently verified causal agent.
This is taken from a long document. See the rest here substack.com
Header image: Boston University