Cytopathic Effects in Uninoculated Cultures
CPE is not dependent on the addition of a host sample
Virology’s Lack of Control is an excellent article about the presence of cytopathic effects in uninoculated cultures.
Mike Stone outlines how the cell culture method used in virology was refuted by John Enders and other observers.
The failed results of Enders (1954) was confirmed by various other researchers over the years, as described by a 1961 study.
“During the last 6 years there have been many reports of isolation of simian “foamy” virus from “uninoculated” kidney cell cultures of rhesus and cynomolgus monkeys (Enders and Peebles 1954; Rustigian et al, 1955; Henle and Deinhardt, 1955; Hotta and Evans, 1956; Brown, 1957; Falke, 1958; Ruckle, 1958a; Endo et al, 1959) and from African monkeys and baboons (Hsiung et al, 1958; Lepine and Paccaud, 1957). Similar virus was found by Weller (1956) in “spontaneously” degenerating (uninoculated) cultures of monkey testis.”
The same was reported in a 1959 publication.
“Of the two tissue culture systems first used successfully by Enders and Peebles (1954), rhesus or cynomolgus monkey kidney is the easier for most laboratories to obtain. Unfortunately, early in the course of this work it was found that agents which induced cytopathic effects superficially resembling that of measles virus occurred in uninoculated cultures. The original observations have since been confirmed and extended by Rustigian et al. (1955), Ruckle (1958), and Brown (1957).”
The authors concluded that the cell culture method can not demonstrate the existence of a measles virus because the same effects are seen in uninoculated cultures.
“In addition to the foamy viruses, other agents that are identical to measles virus in terms of their serological relationships, cytopathological effects and range of tissue culture susceptibility have been found in uninoculated cultures (Ruckle, 1956; Brown, 1957). In view of these complications, cultures of monkey kidney cannot be considered a suitable tool for the isolation or propagation of measles virus. Even if, by careful serological and cytological tests, one identifies an agent grown in monkey kidney as measles virus, there can be no certainty that it did not derive from the cultures themselves.”
Some people might defend John Enders by claiming he could differentiate the cytopathic effects in the cultures because “internuclear changes typical of the measles agents” was not observed in the uninoculated culture.
However, researchers have demonstrated that intranuclear inclusions are non-specific, as described by a 1937 publication entitled Intranuclear Inclusion Bodies in the Tissue Reactions Produced by Injections of Certain Foreign Substances.
“From their general appearance and also from their morphological characteristics it would seem that the inclusions produced by the substances mentioned are not distinguishable from those that occur in virus-containing tissues ... In view of this, it is evident that no disease can be classified as a virus disease on evidence of finding in pathological examination of tissues intranuclear inclusion bodies unless it can be proved that they are associated with a virus.”
Intranuclear changes attributed to “measles” do occur in uninoculated cultures as well. At the Federal Proceedings in 1956, Jonas Salk reported of the findings of Gisela Ruckle, a virologists who discovered intranuclear inclusions identical to measles in uninoculated cultures. The uninoculated cultures were also immunologically indistinguishable from human measles virus.
“During these studies two different types of transmissible agents have been found in uninoculated monkey kidney tissue cultures. One resembles the so-called ‘foamy virus’ and the other produces intranuclear inclusions that are indistinguishable from that produced by the agent obtained from human measles. Each of these agents has been encountered on six different occasions and under circumstances where the possibility of contamination with human measles virus could be excluded … Cross neutralization tests reveal that the ‘foamy virus’ is different from the monkey intranuclear inclusion agent and that the latter is immunologically indistinguishable from human measles virus.”
In her 1958 publication entitled Studies with the monkey-intra-nuclear-inclusion-agent (MINIA) and foamy-agent derived from spontaneously degenerating monkey kidney cultures Gisela Ruckle studied this uninoculated “agent” further.
“Early in the study of measles virus in monkey kidney tissue, a second agent was encountered in uninoculated monkey kidney cultures, which was, in its cytopathic capacity, indistinguishable from measles virus and provisionally referred to as monkey-intra-nuclearinclusion-agent (MINIA) … This paper describes studies to investigate the tissue culture behavior of MINIA and foamy-agent, their immunologic relationship to each other and to measles virus.”
She observed cytopathic effects identical with those attributed to measles in control cultures, as well as in cultures inoculated with material other than from measles patients. She stated that the changes seen in the uninoculated cultures had previously only been associated with measles virus.
“The examination of the stained preparations of one batch prepared on June 23, 1955, revealed the presence of cytopathic changes identical with those induced by measles virus in the control, as well as in the cultures inoculated with clinical material other than from measles patients … A greater number of uninoculated cultures derived from cell batches prepared on June 29, 30 and July 8, were examined and also showed changes, in a number of cultures, which had been associated until this time only with measles virus.”
She concluded that the alleged “agent” called MINIA was indistinguishable from measles virus.
“MINIA produces identical cytopathic changes in monkey kidney cultures as does measles virus … The immunological properties of 8 MINIA strains investigated were indistinguishable and identical with those of human measles virus.”
In the second part of her study entitled Studies with the Monkey-Intra-Nuelear-Inclusion-Agent (MINIA) and Foamy-Agent she describes how the exact same effects attributed to measles virus are seen in uninoculated cultures.
“The recovery of MINIA and foamy-agent from spontaneously degenerating cultures of monkey kidney tissue has been previously reported. MINIA has been shown to be immunologically indistinguishable from human measles virus and to produce the same unique cytopathic changes in human kidney, monkey kidney and human amniotic membrane cell cultures … MINIA and foamy-agent are agents which have been recovered from spontaneously degenerating monkey kidney cultures. Tissue-culture behavior and immunological properties of the both agents were distinct and MINIA was identified as being indistinguishable from measles virus.”
The same conclusions were confirmed in her 1962 publication.
“The preceding studies show that the 2 agents, measles virus and MINIA, behave identically with respect to their biological, chemical, antigenic, and epidemiological properties and can be considered as homogeneous agents.”
In a 1968 study, measles intranuclear changes appeared in uninoculated control cultures. Without any evidence, the authors claim that the uninoculated culture must have been “contaminated” with a measles virus. Convenient rescue device highlighting the pseudoscience of virology.
“To our surprise, measles virus intranuclear and intracytoplasmic eosinophilic inclusions occurred in both inoculated and uninoculated control HEK cultures. Thus, the adenovirus stock derived from the commercially made HEK cultures was inadvertently contaminated with a measles virus.”
In 1957, Lenora Brown observed cellular degenerations (CPE) occur spontaneously in cell cultures prepared from the kidneys of normal, healthy monkeys.
“In recent years, with the increased use of tissue cultures prepared from monkey kidney cells, a new group of viruses has come into recognition. The presence of these agents is made known by the cytopathic effect which they produce in uninoculated or control cultures. The combined observations of workers in several laboratories indicate that these agents, as yet unclassified, are unaccountably present in the kidney tissues (or blood elements) of the apparently normal, healthy monkeys from which the cultures are derived.”
[…]
“SUMMARY AND CONCLUSIONS
A cellular degeneration of peculiar type, featuring the formation of “blisters” or “foamy” patches and of multinucleated giant cells, has been seen to occur spontaneously in tissue cultures prepared from the kidneys of apparently normal, healthy rhesus (Macaca mulatto) or cynomologus (Macaca irus mindanensis (Mearns)) monkeys”
Henle and Deinhardt (1955) observed cytopathic effects indistinguishable to mumps “virus” in uninoculated cultures.
“The use of monkey kidney cells for virus isolations presents certain problems. In the above studies, 11 batches of kidney cells were employed. In cultures of one of these, large “giant cells” appeared in control tubes after 11 days of incubation similar to those produced by the “foamy agent”(3,4). Prior to the 11th day, tubes inoculated with saliva from a mumps patient had shown lesions which were attributed to the presence of mumps virus. Since the cytopathogenic effects induced by these 2 agents thus far proved to be indistinguishable…”
In 1956, Susumu Hotta obtained cytopathic effects in uninoculated control cultures.
“A cytopathogenic agent recovered from an apparently normal kidney tissue culture
A cytopathogenic agent was originally encountered in a control uninoculated culture tube of rhesus kidney tissue 7 days after the beginning of incubation.”
Monkeys were not the only species whose kidneys were prone to spontaneous disintegration. In a 1970 study, it was reported that uninoculated goat kidneys were frequently breaking down.
“We found that goat kidney cells were also highly susceptible to measles virus, but uninoculated cultures also developed cytopathic effects frequently.”
Chimpanzee kidneys suffered the same fate in a 1963 study called Chimpanzee Kidney Tissue for Growth and Isolation of Viruses.
“During prolonged incubation (2 weeks or more) of uninoculated chimpanzee kidney tissue cultures, the cells frequently exhibited changes similar to changes caused by the growth of viruses.”
The authors suspected “adventitious viruses” and not the experimental procedure itself.
“The presence of adventitious viruses in some uninoculated chimpanzee kidney tissue cultures is suspected.”
This idea is unfounded, considering that even innocuous substances have been shown to induce cytopathic effects.
In a study published in 1962 it was found that yeast extract increases cytopathic effects and causes degenerative changes in both uninoculated and inoculated cultures.
“Experiments with yeast extract. Wittler, Cary, and Lindberg (1956) reported that yeast extract speeds up the growth of PPLO in tissue cultures and increases the cytopathic effect. When yeast extract (Difco) at a concentration of 0.5% was present in our cultures at the time of inoculation, both the control and the inoculated cultures showed degenerative changes within 1 week, probably because of toxicity of the extract for the cells.”
Fluids from people with “non-infectious” diseases have also shown to induce cytopathic effects.
“We have found that CSFs from patients with certain psychiatric syndromes, including schizophrenia, produce a cytopathic effect (CPE) when inoculated into stationary tissue cultures .. This CPE resembles that produced by certain viruses but no cytopathic agent has yet been established in tissue culture”
In 1969, Anthony Nesburn observed CPE in uninoculated rabbit cell cultures.
“An agent which possesses the physical, chemical, cytopathic, histological, and electron microscopic attributes of a herpes group virus was isolated from an uninoculated batch of primary rabbit kidney cell cultures.”
A study published in 1984 attempted to search for a viral etiology of inflammatory bowel disease. Cell cultures were used, but the authors concluded that the cytopathic effects observed were probably caused by non-replicating cytotoxic factor.
“Intestinal tissue filtrates induce cytopathic effects in inoculated cell cultures, but the effect we observed is non-specific … Our results suggest that the observed cytopathic effect was caused by a non-replicating cytotoxic factor.”
In 1989, virologists attempted to culture a rabies “virus.” However, their uninoculated cultures exhibited cytopathic effects. The uninoculated tested negative for “rabies virus.” However, mice inoculated into the brain with the fluids of the uninoculated culture died.
“Cytopathic effects were detected in ERA virus-inoculated as well as uninoculated FBC’s. Immunoflorescent antibody testing of uninoculated FBC’s provided no evidence for the presence of rabies virus. However, mice inoculated intracranially with supenatant fluid from uninoculated FBC’s died.”
A 1970 publication demonstrated a clear indication that the experimental procedure itself create the cytopathic effects.
Kidney cells from healthy puppies were cultivated without the addition of an alleged “infected” sample. Yet, cytopathic effects were observed.
“Puppies serving as donors of kidney tissues for the cell cultures were derived from a closed colony of apparently healthy beagle dogs. The primary canine kidney cells were placed on maintenance medium consisting of Eagle’s minimal essential medium plus 2% fetal calf serum on the day after receipt of the monolayer cultures. Foci of CPE consisting of rounded cells and plaques (Fig. IA) appeared at 14 days after initiation of the cultures. The CPE progressed to involve 50 to 75% of the cell sheet by the end of the third week of cultivation.”
When virologists uninoculated control cultures exhibit cytopathic effects, they often blame “viral” contamination and not the experimental procedure itself. This can be seen in a 1970 study were an “adventitious virus” was blamed when the uninoculated cultures exhibited CPE.
“While attempting to isolate bovine rhinoviruses in BEK cell cultures, occasional controls were noted to degenerate spontaneously. The BEK cultures were incubated in roller drums at 33 C for isolation of bovine rhinoviruses. The maintenance medium contained Eagle basal medium with Earle salts, 2% normal rabbit serum, and the usual concentrations of antibiotics. On primary isolation, occasional uninoculated control cultures showed focal cytopathic effect (CPE) after 12 to 14 days of incubation.”
The idea of “endogenous viral contamination” goes back to 1955 when Rustigian et al. observed cytopathic effects in uninoculated cultures. Since then, virologists have been claiming to discover many viruses when their uninoculated cultures exhibit cytopathic effects.
“In recent years, there have been increasing numbers of reports concerning the recognition of latent virus infections in tissues of primates as well as nonprimates … As a result of the extensive use of primate cell cultures, a great number of simian viruses have been recovered from a variety of monkeys, baboons, and marmosets.
[…]
“The isolation of virus-like agents from monkey kidney tissue cultures was first reported by Rustigian et al. in 1955 (89). Subsequently, as a result of the extensive use of monkey kidney cell cultures, especially in the preparation of virus vaccines, a great number of simian viruses have been recovered as endogenous tissue contaminants by Hull and associates (56- 58).”
[…]
“On the basis of certain biological properties, especially cytopathic effect (CPE), simian viruses were originally divided into four groups by Hull et al. (57). Other biological properties, including plaque morphology, host-cell susceptibility, and hemagglutinin production, have also been used for grouping these viruses.”
[…]
“Recognition and characterization of simian viruses in cell cultures are of practical importance, since monkey tissues often harbor a variety of viruses.”
This convenient rescue device of endogenous viral presence have frequently been used by virologists in order justify their pseudoscientific methodologies. In his book Contamination in Tissue Culture, Jørgen Fogh outlines the widespread usage of this rescue device.
“Very many animal species have hidden virus infections, that is, viruses that appear to exist in close association with particular tissues or organs of the species involved, without causing overt symptoms. It follows that primary cultures prepared from almost any tissue may be found infected with viruses in vitro simply because the organ from which they were prepared was infected in vivo (Hsiung, 1968). In this way, primary cultures from chickens (Burke et al, 1965), chimpanzees (Rogers et al., 1967), dogs (Smith et al., 1970), ducks (Luginbuhl, 1968), guinea pigs (Hsiung and Kaplow, 1969), hamsters (Stenback et al., 1966), horses (Hsiung et al., 1969), man (Hsiung, 1968), rabbits (Nesburn, 1969), rats (Melendez et al., 1967), monkeys (Hsiung, 1968), and swine (Huygelen and Peetermans, 1967) have all been found to yield viruses spontaneously.”
When virologists don’t see the results they want, they often play around and add reagents until they do. In the case of the polio virus in Vero cells, scientists weren’t able to see the CPE they wanted until they added trypsin. Of course, they blame it on “virus.”
“When monkey kidneys are infected in vivo with polio virus, no cytopathic effect can be observed and no virus can be recovered following subsequent perfusion of the kidney or homogenization of its tissue. However, if parts of an infected kidney found negative for virus by such tests are reduced to cell suspensions by trypsin treatment and used to set up conventional monolayer cultures, cell destruction eventually takes place, accompanied by the appearance of fully infective virus. When the kidney cells are removed from their in vivo situation to an alien environment, they somehow acquire susceptibility to polio virus.”
A study from 1973 published in the American Society for Microbiology stated that it had been known for a long time that uninoculated cultures frequently exhibit cytopathic effects.
“It has been recognized for a long time that uninoculated cell cultures may show spontaneously the cytopathological effects (CPE) of a latent virus infection … The spontaneous occurrences of simian foamyvirus CPE in kidney cell cultures of a number of monkey species is frequently observed.”
The presence of a viral agent in cell cultures was detected through the observation of cytopathic effects, or in the case of certain viruses, the detection of hemadsorption (the adherence of red blood cells to the surface of something). However, hemadsorption also occures in uninoculated cultures, as described by a 1961 publication.
“The presence of a viral agent in inoculated cell cultures may be detected either by observing the cytopathic effect, i.e., cellular degeneration, in the inoculated cultures or, in the case of certain myxoviruses which do not give rise to distinct cellular degeneration, by the addition of red blood cells to the cell sheet and observance of hemadsorption. It is necessary to distinguish between cellular destruction due to specific viral effect and that due to toxicity of the inoculum … In employing hemadsorption as an indicator of the presence of viral agents, it must be kept in mind that certain simian viruses occur as natural contaminants of monkey kidney cell cultures, and that these may cause hemadsorption.”
The identification of the specific ‘viral agent’ was through the use of neutralization tests. The cell culture breakdown product was mixed with different protein brews and then added to cell cultures, and depending on what results happened to be seen in the cell cultures, a virus was determined.
“Identification of viral agents recovered in tissue culture systems is usually accomplished by the neutralization test. For this procedure, a standard amount of the unknown virus, usually 100 TCD50, is mixed with known immune serums, and after a suitable incubation period the serum-virus mixtures are inoculated into cell cultures in order to determine which, if any, of the immune serums is able to prevent cytopathogenesis or hemadsorption. A modification of the neutralization test which has proved particularly useful for the identification of viral agents is the metabolic inhibition test in which multiplication or nonmultiplication of the virus is detected colorimetrically. If the virus is specifically neutralized by an immune serum, cellular metabolism is unimpaired, and the medium is converted from alkaline to acid, as indicated by a shift in color of the phenol red indicator from red to yellow. For this procedure, the cells, virus and serum are all added to the test on the same day, eliminating the need for preliminary preparation of monolayer cell cultures in tubes.”
However, this methodology has been falsified due to control studies. In a 1968 publication entitled Latent virus infections in primate tissues with special reference to simian viruses, G. D. Hsiung et al. tested the surrogate markers, i.e. CPE and neutralization tests, on uninoculated normal cell cultures.
To their surprise, positive results from their surrogate markers were frequently observed in normal uninoculated cells.
“Much to our surprise, an unusually high percentage of cultures that were considered “normal” showed virus infection.”
The same effects attributed to viruses are seen in cell cultures even though no assumed infected material is added.
If virologists observe CPE in uninoculated cultures, they are likely going to throw the cultures in the trash and repeat the experiment until desired results are obtained, as stated by Laboratory Techniques in Virology in 1984:
“First, examine the control wells. If CPE occurs in control wells, test is invalidated and must be repeated”
Typical distraction from the real cause of disease.
That's why they find the sequences and antibodies in healthy people.
But the sick people get the label they pre defined in order to cover up for the real causes.
Example: polio blamed for paralysis caused by ddt and arsenic based pesticides .
Really well put together Vil.