The Ridiculous Ruse of the Rampaging Rhinovirus

I love the doctors—they are dears;

But must they spend such years and years

Investigating such a lot

Of illnesses which no one’s got,

When everybody, young and old,

Is frantic with the common cold?

And I will eat my only hat

If they know anything of that!”

Herbert AP. The common cold. In: Look back and laugh. Methuen; London: 1960. pp. 115–117.

In the 1950’s, virologists had no clue as to what could potentially be causing all of the common cold symptoms people were regularly experiencing. In fact, it was estimated that less than 30% of the respiratory diseases could be linked to anything of bacterial and of “viral” origin. However, the 1950’s was also an explosive decade in the discovery of “viruses:”

“Up to nine new human virus species have been detected each year since the 1950s, and this is projected to continue in coming decades. The factors driving the discovery of human viruses remain to be elucidated…”

https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009079

While it is claimed that the factors driving this “virus” discovery spree are unknown, it can be linked to the cell culture technique introduced by John Franklin Enders in 1954. Because of this technique, virologists were able to mix together the human sample with numerous other substances in a petri dish and claim the isolation of a new “virus” nearly at will. Big name “viruses” such as polio, varicella zoster, measles, respiratory syncytial “virus,” adenovirus, the paramyxoviruses, and more were said to be “isolated” in this manner.

It shouldn’t come as any surprise then to discover that through the use of Enders cell culture technique, the elusive main cause of the common cold was also said to be discovered in the form of “rhinoviruses” in 1956. The discovery was credited to a man named Winston Price, who fittingly named his “virus” the JH “virus” after Johns Hopkins, the institute where the “isolation” was said to have occurred. Unfortunately for rhinoceroses everywhere, as the “virus” is said to infect the nasal passages, it was later renamed the “rhinovirus” after the prefix rhino which means nose.

My initial intent was to provide only a breakdown of Price’s original 1956 “rhinovirus” paper to see if the isolation claim held up. However, upon researching the current knowledge regarding the “rhinovirus,” it became clear that there was quite the fictional narrative woven around his paper in the proceeding decades to cover up the numerous holes left in its wake. Thus I have broken this post down into two sections. I will provide a look at the claims made in Price’s original paper and then show how the “rhinovirus” morphed throughout the following decades in order to try and patch up the holes left by Price’s lack of evidence.

What you will find in Price’s paper is the usual cell culture tricks as mentioned above. He took nasopharyngeal swabs from patients and immediately placed them in monkey kidney maintenance media thus nullifying any claims of purification (free of contaminants/foreign materials) and isolation (separated from everything else within the sample). The samples were frozen in an alcohol-dry ice mixture, thawed, and then added to Hela cells taken from a woman with cervical cancer. However, even with this toxic mixture, degeneration of the cells could not be shown in over 240 hours along with 3 blind passages.

With this failure, Price decided to use monkey kidney cells instead to find his “virus.”  He added 3 percent inactivated horse serum, 5 percent beef embryo extract, and 92 percent 199 solution which contained 100 units of penicillin and streptomycin per one ml. of media. This of course destroyed the monkey kidney cells (as the antibiotics alone would do) and Price was able to claim that his “virus” grew within this toxic unpurified culture. Oddly enough, he admitted that the cytopathogenic effect (CPE) could usually be observed more readily in the antibiotic heavy 199 medium without the above additions of horse and beef extracts. However, he also noted that “viruses” could be “isolated” (i.e. show CPE) in richer medium (what that entailed was not specified) and not just with plain medium 199. However, he left the significance of this evidence as unclear. Price even stated that the JH “virus” had a very long incubation period before producing a cytopathogenic effect in monkey kidney cells. It required a period of 25 days, along with a blind passage, in order for the CPE to show up on the tenth to sixteenth day of the second passage. It should be clear that as all virologists do, Price played with his recipe until he got the desired dish.

Interestingly, the rest of the evidence for the JH “virus” relied solely upon serological (i.e. indirect, non-specific anyibody) results. It should not have to be pointed out that one can not use fictional antibodies in order to prove the existence of a fictional “virus,” but as is often the case in these papers, that is exactly what was done. However, even when using this method, Price admitted that no complement-fixing (CF) antigen could be prepared from monkey cells infected with the JH agent. There are many other reasons to question these antibody results which we will cover in a paper published well after this initial discovery. No other evidence, such as electron microscope images of the particles assumed to be his “virus,” was presented in his paper.

For some reason, Price ignored the results of his own animal experiments which showed that his JH “virus” was not pathogenic. He tried numerous ways to infect mice and other animals without success. He inoculated intranasally, intracerebrally, or intraperitoneally twenty 1-day-old mice or ten 10-day-old hamsters and no signs of disease were noted over the 21-day observation period. Similar negative results were obtained when ten 15-17-gm. mice or six young adult guinea pigs were inoculated intraperitoneally. Finally, the intranasal inoculation of ferrets gave negative results with the animals showing normal fever curves and no signs of illness.

Price concluded that the importance of the JH agent in causing illness in the civilian population as well as other problems dealing with his “virus” were still under investigation. In other words, he had no definitive claim to having isolated a pathogenic “virus” at all. Price was seemingly unconvinced that he had isolated the common cold “virus” as he stated that in view of the similarity between the mild respiratory infection associated with the JH “virus” and the common cold, he hoped that the study of the JH “virus” may lead to some clues as to how the common cold “virus” may be isolated:

THE ISOLATION OF A NEW VIRUS ASSOCIATED WITH
RESPIRATORY CLINICAL DISEASE IN HUMANS

“The isolation of the RI-APC group of respiratory viruses through the use of tissue culture methods has greatly stimulated interest in this field.

In this paper we wish to report the isolation of a new respiratory virus (JH), which from all available data is responsible for a clinical disease in the human population.

RESULTS

Isolation of Virus.– Naso-pharyngeal washings were collected in monkey kidney maintenance media. They were shell frozen in ampules in an alcohol-dry ice mixture and stored at -500 C. When isolation attempts were made the ampule was quickly thawed and 0.1 ml. of the washing was inoculated into tubes of Hela cells as described by Hilleman and Werner.’ Our methods follow theirs exactly except the maintenance media of Eagle’ was substituted. The virus which will be described in this paper did not cause degeneration of the Hela cells within 240 hours, even after 3 blind passages. The twenty isolations on which most of this work has been done elicited titers of approximately 106 to 107 tissue culture infecting doses per 1.0 ml. of the infected monkey kidney cells. The agent multiplies in monkey kidney cells grown in 3 per cent inactivated horse serum, 5 per cent beef embryo extract, and 92 per cent 199 solution. This media contains 100 units of penicillin and streptomycin per one ml. of media. Monkey kidney cells are destroyed by the virus.

The cytopathogenic effect of this agent can usually be observed more readily in 199 medium without the above additions. However, in a few primary isolations this virus was isolated in the richer medium and not in plain 199 medium. The significance of these observations is not clear at the present time.

Epidemiological and Clinical Data.- Although detailed epidemiological and clinical data will be reported elsewhere, brief mention will be made here of certain of the data.

The virus has been isolated from human respiratory cases in all seasons of the year from both adults and children. It has been isolated from two large respiratory outbreaks, as well as from individuals clinically ill with respiratory infections who came to the Johns Hopkins Hospital or to the Baltimore city hospitals for treatment. The main clinical features with which this agent is associated are malaise and coryza and mild sore throat. A low-grade fever may or may not be present. There are no physical findings to suggest lower respiratory involvement. The duration of clinical symptoms averages about three days. Bacteriological analysis of the throat and nasopharynx swabs of individuals ill with the above syndrome show normal flora. Antibody surveys on various age groups carried out so far have shown that this virus is relatively widespread, about 20 per cent of the individuals over eight years of age showing the presence of neutralizing antibodies. Complement-fixing antibodies also can be shown to be present for this agent in the sera of individuals.

Serological Characteriatics of Etiological Agent.- Table 1 shows a typical neutralization test using acute-convalescent sera of individuals from whom the virus has been isolated who were clinically ill with the symptoms described above. It can be readily seen that there is a large increase in the neutralizing antibody level.

These tests were carried out as described above using 10 to 50 TCDN. All isolations of the agent seem to be immunologically similar as determined by neutralization tests.

Table 2 shows that there was no significant increase in titer against this virus in acute-convalescent sera of the following infections: influenza, RI-APC, Q fever, Rocky Mountain spotted fever, scarlet fever, streptococcal sore throat, rheumatic fever, and poliomyelitis. All these sera showed very high increases in titer in the convalescent sera against the homologous etiological agents. Furthermore, acute-convalescent sera from various other diseases shown in Table 2 showed no increase in neutralizing titer in the convalescent sera to the JH agent. All these data indicate that the increase in neutralizing titer is specific.

No complement-fixing (CF) antigen could be prepared from monkey cells infected with the JH agent which reacted with standard RI-APC sera using methods which could readily detect RI-APC CF antigen in Hela cells infected with a RI-APC virus.’ The JH preparation used for these CF experiments titered out to approximately 106 tissue-culture-infecting doses. Standard sera to RI-APC types 1-7 which neutralized 1,000 tissue-culture-destroying doses of these viruses did not neutralize 10 tissue-culture-destroying doses of the JH agents, all tests being carried out in monkey cells under the same conditions.

The acute-convalescent sera of the individuals shown in Table 1 did not show any hemagglutination inhibition rise to influenza A (PR8), influenza A’ (FW-1-50), influenza B (B-1-11), influenza C (strain 1233), or a strain of swine influenza. None of the sera showed rises to the RI-APC CF antigen supplied by Microbiological Associates. None of the convalescent sera showed evidence of cold or streptococcus MG agglutinins, or a rise in streptolysin 0 antibodies.

Other Characteristics of the JH Agent.- The agent can be passed in series in monkey cells without loss of titer at least 10 times, after which no further attempts were made to pass the agent. When the JH suspension, which titered about 10-‘in monkey cells, was inoculated intranasally, intracerebrally, or intraperitoneally into twenty 1-day-old mice or ten 10-day-old hamsters, no overt signs of disease were noted over the 21-day observation period. Similar negative results were obtained when ten 15-17-gm. mice or six young adult guinea pigs were inoculated intraperitoneally. The baby mice showed no lesions in their thoracic or abdominal viscera when some were sacrificed 7 or 12 days following inoculation. Intranasal inoculation of ferrets gave negative results, the animals showing normal fever curves and showing no overt signs of illness. Two blind passages of various organs and tissues under all the above conditions failed to show any evidence that the JH agent had multiplied.

Inoculation of the JH suspensions which contained about 102 and 105 TCD60 into 12-day-old chick embryos by the amniotic and allantoic routes failed to reveal evidence of multiplication even after three blind passages, the fluids being harvested 72 hours after inoculation and titered in monkey cells. The amniotic and allantoic fluids did not agglutinate chicken, human “O”, or guinea pig red cells at room temperature or at 40 C.

The JH virus does not appear related to the ECHO group of viruses. The JH virus shows a very different cytopathogenic effect than the ECHO viruses in monkey kidney cells. It is not neutralized by antisera prepared to ECHO viruses 1, 2, 3, 6, 5, 7, 8, and 9. The JH virus also has very peculiar nutritional requirenments which are different from those reported for any ECHO virus.

The JH virus is not neutralized by antisera prepared to Coxsackie A types 1, 2, 5, 6, 8, 9, 10 or B types 1, 2, 3, 4 or 5. The cytopathogenic effect of the JH agent in monkey kidney cells is also different from that associated with Coxsackie viruses.

Attempts To Grow JH Agent on Artificial Medium.- Infected monkey tissue culture preparations which titered out to approximately 10-5 in monkey cells were inoculated into Brewers’ thioglycolate medium and sheep, human, and rabbit blood agar plates. These cultures were inoculated both aerobically and anaerobically, and none showed growth after 12 days at 300 C. The JH agent could be filtered through standard bacteriological sterile filters which completely removed all
Staphylococcus aureus present in prepared suspensions.

DISCUSSION

The data presented in this paper indicate that the agent described is a new virus responsible for clinical illness in the civilian population. This statement is based on the following facts: (1) the isolation of the agent from individuals ill with a particular respiratory clinical syndrome; (2) the rise in antibodies to the agent in such people and the failure to show a rise in antibody to the agent in many other human respiratory as well as other viral, bacterial, rickettsial, and chronic diseases; (3) the failure to isolate the agent from 1,100 individuals not showing clinical signs of respiratory illness in the same seasons and from the same age group and in the same state as the ill individuals from whom the virus was isolated: (4) the relationship between antibody level and clinical disease in certain respiratory outbreaks. Thus, in one large respiratory outbreak where the JH virus was isolated from many sick individuals, there was a definite correlation between the individuals’ blood sera neutralizing antibody titers to the JH virus one month previous to the outbreak and their developing the clinical signs of respiratory infection during the epidemic. The higher the titer to the JH virus previous to the respiratory outbreak, the less chance a person had of developing overt illness. No such relationship was found for influenza A’ or RI-APC antibodies. Furthermore, 16 individuals who developed respiratory infections during this epidemic did not show the presence of the JH virus in their nasal pharyngeal washings taken one month previous to their respiratory infections. However, from 11 of these individuals the JH virus was isolated from their nasal pharyngeal washings when they were ill during the respiratory outbreak, 10 of the 11 individuals showing significant rises in neutralizing antibodies to the JH virus. Such longitudinal studies as these where the same individuals are studied for a long period of time with test samples being taken at frequent intervals, greatly help in deciding whether a virus is the cause of a disease.

The viruses responsible for the major cause of respiratory disease in the civilian population have not as yet been identified. In our extensive field-laboratory study over the last three years, less than 30 per cent of the respiratory illnesses could be identified as being caused by a known microparasite, of either bacterial or viral origin.’

The importance of the JH agent in causing illness in the civilian population as well as other problems dealing with this virus are under investigation and will be described elsewhere. The virus seems fairly widespread, as our surveys carried out so far indicate that about 20 per cent of the population over eight years of age have antibodies to this agent.

Clinically, the virus described in this paper is associated with a very mild upper respiratory infection. The major difference between this illness and the common cold is that the JH virus is usually associated with low grade fever, as well as slight malaise, coryza, mild sore throat, and in some cases a cough. However, in view of the similarity between the mild respiratory infection associated with the JH virus and the common cold we hope that study of the JH virus may lead to some clues as to how the common cold virus may be isolated. In this connection it should be pointed out that the JH virus has a very long incubation period before producing a cytopathogenic effect in monkey kidney cells, particularly on primary isolation. Thus, a period of 25 days, as well as a blind passage at this time, has been found to be required in many of the isolations, the cytopathogenic effect showing up on the tenth to sixteenth day of the second passage.”

doi: 10.1073/pnas.42.12.892

It should be very clear that Winston Price never isolated any “virus.” Not a single purification step was carried out either directly on the sample or after cell culture. The steps to aquire any sign of CPE required a ridiculously long 25 day incubation period, followed by a culture-altering blind passage, which in turn was followed by another 10-16 days of incubation. No EM images were obtained of any assumed “rhinovirus” particles whatsoever. The various animal experiments performed all failed to prove pathogeniticity of the cultured soup as none of the animals became sick. The only evidence Price could attempt to claim as proof were the result of indirect non-specific antibody tests. Perhaps this complete and utter lack of direct evidence of any “virus” is precisely why Price thought of his JH “virus” paper as providing clues on how to isolate a common cold “virus,” rather than actually presenting evidence for one.

In any case, Price’s “virus” soon became the face of the common cold. It is said to be the cause 80% of the cold symptoms experiemced during any particular season. Throughout the proceeding decades, more evidence was gathered in order to solidufy Price’s “virus” as the main culprit for the common cold and attempt to fill in the gaps of the missing evidence from his original paper. Was this a successful endeavor? You can be the judge.

In 2013, a review of the evidence for “rhinoviruses” came out detailing the long and complex history of Price’s “virus” after its creation. The paper pointed out that it has been over sixty years since his 1956 paper and a “cure” for the common cold is still non-existent. There are no “antivirals” nor any vaccines that can be used and treatment is typically supportive and aimed at symptom relief only. Why has the search for a “cure” resulted in the complete lack of efficacy of the treatments as well as toxicities produced by them? 

After Price’s initial “rhinovirus” discovery in 1956, it was established that there was not only one “rhinovirus,” but three distinct species. The first two, HRV-A and B, were the main ones for most of its 60+ year history when over 100 different serotypes were “discovered” in the 60’s and 70’s.  However, the twosome became a threesome with the arrival of molecular virology in the early 2000’s and the discovery of HRV-C. Unlike HRV-A and B, HRV-C was said to be unable to grow in cell culture, thus it evaded identification until more advanced molecular technologies came along. In other words, HRV-C exists only as a genome. This “discovery” led to a further 50+ serotypes added to the original 100+ serotypes for a grand total of around 160 different serotypes associated with the “rhinovirus.” Each serotype is said to have different surface proteins thus making them antigenically distinct. This means that there is no common antigen among these different “rhinoviruses” which makes antigen and antibody results useless in terms of diagnosis. It also provides the excuse for why a vaccine can not be created as well. Keep in mind that Price’s original paper relied on specific antibody results in order to claim the existence of his “isolated virus.” If the antigen and antibody tests for diagnosis are useless due to the inability to find a common antigen, how could Price’s original antibody evidence be trusted? Obviously his results could not be validated which led to the excuse of the 160+ different serotypes currently said to exist to cover up these failures. To add even more fuel to the fire, the “rhinovirus” antibodies cross-react with “enteroviruses” (EV’s) which solidifies the fact that these results are entirely unspecific.

As for Price’s cell culture evidence, it is said now that the primary monkey kidney cells used by Price will only support the growth of some strains. Other cell lines such as human fetal embryonic lung fibroblast cell lines, certain HeLa cell clones, and human embryonic kidney cell lines are now the most commonly used for HRV culture. In fact, it is recommended to combine different cell lines to produce results. This combination of methods to generate CPE may be due to the fact that while CPE is usually visible in most cell lines, there are CPE-negative strains of the “rhinovirus.” In other words, virologists can not produce the desired effect every time they perform the cell culture. They also claim that presumptive identification is usually made on the basis of CPE in appropriate cell lines, yet the CPE appearance is the same or very similar to that of “enteroviruses.” This means that the CPE that virologists can sometimes see is not specific for “rhinoviruses” and can also be found in the culture of “enteroviruses.” To top it all off, HRVs are said to be fastidious (i.e. difficult and demanding) in vitro, and due to the specific conditions required for optimal culture, “isolation” rates are generally low.

It can be seen that the two main criteria used by Price to claim his “isolated virus,” mainly the antibody and cell culture results, were shown to be entirely unreliable and non-specific over the proceeding decades. This means that his paper fails spectacularly as a claim for the discovery of the “rhinovirus.” There is also other recent evidence that invalidate the claims in his paper as well. First, with the advent of molecular virology and PCR detection, asymptomatic “rhinovirus” cases have been increasingly documented in up to 32% of all cases. Thus, the “viral” RNA said to belong to the “rhinovirus” is found in entirely healthy people. This means that the “rhinovirus” can be found in both sick and healthy individuals thus failing Koch’s first Postulate for proving a microbe as a cause of disease. Also interesting to note is that early “rhinovirus” PCR identification in the 1980’s had to be confirmed with dideoxy sequencing due to cross-reactivity with human DNA.

Second, the role of the “virus” itself and the host immune response in HRV pathogenicity and symptomatic illness is still under debate. HRV has not been shown to cause direct cytopathology in the upper airways, which is rather odd for a “virus” said to cause primarily upper respiratory disease. Common symptoms of “rhinovirus” include rhinorrhea, nasal congestion, sore throat, cough, headache, fevers, and malaise, all of which it shares with “coronaviruses.” When compared to patients with “coronavirus-associated” colds, there is no difference in respiratory symptom severity or duration. Thus, there are no specific symptoms differentiating any of the “viruses” said to cause the common cold.

There are other interesting tidbits about the “rhinovirus” contained within the highlights from this 2013 paper that should, at the very least, cast doubt in the mind of any casual observer about the existence of any “rhinovirus:”

Human Rhinoviruses

INTRODUCTION

“Human rhinoviruses (HRVs) were first discovered in the 1950s in an effort to identify the etiology of the common cold. Nearly 60 years later, the search for a “cure” for the common cold virus is still ongoing. Worldwide and nearly year-round, HRV is the most common cause of upper respiratory tract infection (URI), leading to considerable economic burdens in terms of medical visits and school and work absenteeism (1–4). However, while once thought to cause relatively benign upper respiratory tract illness, HRVs are now linked to exacerbations of chronic pulmonary disease, asthma development, and, more recently, severe bronchiolitis in infants and children as well as fatal pneumonia in elderly and immunocompromised adults. Our enhanced understanding of the spectrum of illness of HRVs draws largely from advances in molecular methods that have facilitated the detection and characterization of HRV groups and strains. Indeed, a growing number of clinical laboratories are adopting multiplex PCR-based assays for the detection of respiratory viruses that include HRVs (5).

There are currently no approved antiviral agents for the prevention or treatment of HRV infection. Clinical trials of antiviral therapies have been limited by drug toxicities, drug interactions, and a lack of efficacy when applied to the natural setting. Efforts at vaccine development are hindered by the existence of more than 100 HRV serotypes with high-level sequence variability in the antigenic sites. The treatment of HRV infection remains primarily supportive, including over-the-counter products aimed at symptom relief.”

Serotypes/Genotypes

“Until recently, HRVs were classified into two species, HRV-A and -B, based on phylogenetic sequence criteria. Clinical specimens in the 1960s and 1970s yielded approximately 100 different HRV strains (known as the reference or prototype set), which were subsequently serotyped (6). Partial sequencing of viral capsid-encoding regions, noncoding regions, and a limited number of complete genomes led to a division of the original 99 strains into two species: HRV-A (containing 74 serotypes) and HRV-B (containing 25 serotypes). To understand further the molecular and evolutionary biology of the virus and aid in epidemiological investigations, the sequencing of the full genomes of these 99 serotypes was recently completed (8).

The development of highly sensitive molecular techniques for the identification of HRV in clinical specimens led to the identification and designation of a novel species, HRV-C, by the International Committee on Taxonomy of Viruses in 2009 (9). HRV-C strains do not grow in standard cell culture, likely postponing their discovery; therefore, a genetically based classification system was developed. HRV-C strains have a genomic organization similar to that of HRV-A and HRV-B; however, there are several distinct characteristics supporting their classification as a new species. To date, at least 50 different types of HRV-C have been identified by using a threshold of a 13% nucleotide difference in VP1 or at least a 10% nucleotide difference in the VP4/VP2 region if the VP1 sequence is unavailable (10, 11).”

“While other respiratory viruses, such as influenza virus and respiratory syncytial virus (RSV), cause a destruction of airway epithelial cells, HRV is seldom associated with cytopathology of the upper respiratory tract. Using light and scanning electron microscopy of nasal biopsy specimens from subjects with natural colds, Winther et al. found that epithelial cells were sloughed; however, the epithelial cell lining and borders remained structurally intact (26). A similar preservation of cell morphology and composition was observed for the nasal epithelium during studies of experimental HRV infection, where the amount of viral shedding did not correlate with the severity of symptoms (19, 27).”

Animal Models

“The development of small-animal models is useful to understand further the pathogenesis of HRV infection in both the upper and lower airways; however, there are no known murine rhinoviruses.”

“In conclusion, in vitro and clinical data indicate that HRVs are a significant upper and lower respiratory tract pathogen. However, the relative roles of the virus itself and the host immune response in HRV pathogenicity and symptomatic illness are still under debate. In the upper airways, HRV has not been shown to cause direct cytopathology; clinical symptoms are likely the result of local and systemic immune responses.”

Asymptomatic infections. 

“With the increasing use of molecular methods of viral detection, asymptomatic HRV infection has been noted to be relatively common, particularly in children. The frequent detection of HRV in asymptomatic individuals may also reflect one of several states: prolonged virus shedding after a symptomatic respiratory illness has resolved; mild, unrecognized symptoms; or the incubation period prior to the onset of symptoms. In children less than 4 years old, rates of asymptomatic infection range from 12 to 32% (87–91) and tend to be higher in the youngest age groups (91).”

“Common symptoms include rhinorrhea, nasal congestion, sore throat, cough, headache, subjective fevers, and malaise. Compared to patients with coronavirus-associated colds, there is no difference in respiratory symptom severity or duration (96).”

Antigen Detection and Serology

“There is no common antigen among HRVs, and an increasingly large number of serotypes have been described; therefore, antigen detection assays are not used for routine detection. Antibodies are measured in both serum and nasal secretions by neutralization, plaque reduction, complement fixation, and enzyme-linked immunosorbent assays (ELISAs) in research settings (104, 186). As with antigen assays, however, the lack of a common antigen across all strains of HRV makes the detection of antibody responses impractical for diagnostic purposes.”

“HRVs were originally isolated from primary monkey kidney cells, although these cells will support the growth of only some strains (181). Human fetal embryonic lung fibroblast cell lines, certain HeLa cell clones, and human embryonic kidney cell lines are most commonly used for HRV culture in clinical laboratories. In a recent study, HRV-infected susceptible HeLa cells were shown to be a good model compared to HBECs for the study of viral RNA synthesis, translation, protein processing, intracellular protein localization, and disruption of host cell functions (188). In a study of HRV recovery from nasal wash specimens, WI-38 and an HRV-susceptible HeLa clone (with high levels of ICAM-1 expression) were found to be the most sensitive cell types for HRV culture (183). As noted by other investigators (189), those authors suggested a combination of different cell lines as the best approach for the optimal recovery of HRV.”

“CPE is usually visible in most cell lines, although CPE-negative strains have been reported (195). Morphological changes are easiest to observe in fibroblast lines and include foci of small and large rounded, refractile cells with pyknotic nuclei and cellular debris (181).

Conventional cultures should be incubated for up to 14 days, and presumptive identification is usually made on the basis of CPE in appropriate cell lines, although the appearance is the same or very similar to that of enteroviruses (EVs).”

“Again, the absence of a common antigen makes the development of reliable and specific immunofluorescence reagents difficult. Centrifugation-enhanced cultures can be stained prior to the development of CPE with some EV detection reagents that cross-react with HRV; however, laboratories are left without a specific diagnosis.”

Molecular Methods

Conventional and real-time RT-PCR. 

“HRVs are somewhat fastidious in vitro, and due to the specific conditions required for optimal culture, isolation rates have been generally low except in large reference virology laboratories. Furthermore, since the viruses were believed to cause only “common colds,” most laboratories did not consider the detection and identification of HRV infections worth their time and effort. In the late 1980s, methods for PCR-based assays capable of detecting HRV in primary respiratory samples were reported (203–206). The use of these early molecular tests resulted in an increased HRV detection rate. Although identification had to be confirmed with dideoxy sequencing due to cross-reactivity with human DNA, these tests shortened the time to diagnosis from up to 2 weeks to a few days. However, HRV testing continued to be performed primarily in specialized laboratories under limited clinical circumstances or, for specific studies, for many years.”

“Multiple RT-PCR and real-time RT-PCR techniques have been developed for the detection of HRV since the late 1980s. In general, most of these assays target the 5′UTR, a region highly conserved among all HRVs and EVs, causing cross-reactivity between assays for the two viruses and making their differentiation difficult. RT-PCR tests that can differentiate HRV from EV by amplicon size (207, 208), restriction fragment length polymorphism (RFLP) (206), hybridization with HRV-specific probes (205), RT-PCR followed by sequencing (209), and real-time RT-PCR (210) have also been reported. Since the International Committee on Taxonomy of Viruses reclassified all HRVs into the EV genus, however, the reporting of diagnostic test results as HRV or EV without further testing has become acceptable.

Quantitative real-time RT-PCR assays. 

While some reports have indicated a correlation between higher viral loads and symptomatic disease (172, 211), which makes quantitative testing attractive for verifying the clinical relevance of positive results, there are currently no commercially available quantitative molecular HRV tests. Additionally, due to the intertypic variability of HRV, there is no standard for the quantification of all HRV types, although some studies have used a chimeric HRV internal standard to limit assay variability between types (212). Other challenges that arise when attempting to provide an accurate and relevant quantification include factors such as the sample type and collection procedure, test methodology, age of the patient, and immune status. All these factors affect viral load and the potential standardization of assays and collectively impact the clinical interpretation of quantitative HRV data such that generally applicable guidelines are not feasible with any reasonable level of confidence.”

“As data accumulated on the involvement of HRV in more serious clinical disease, the importance of the inclusion of assays for the virus in these new panels became clear. In turn, as these new assays have been introduced into clinical practice, more data have emerged on the high incidence of HRV infection, resulting in the further awareness of the widespread and sometimes serious disease manifestations. Additionally, some HRVs, such as the group C viruses, are uncultivable by routine culture techniques. Many studies have now demonstrated that HRV infection can lead to an influenza-like illness, lower respiratory tract infections, chronic infections, and secondary bacterial infections, especially in immunocompromised patients (132), children with asthma (218–221), and adults with COPD (152).”

Social Distancing and Respiratory Masks

“Behavioral strategies such as social distancing and respiratory mask application have been evaluated primarily in the context of pandemic influenza A virus and influenza-like illness prevention. Social distancing includes school closures and the avoidance of public gatherings. An assessment of the effectiveness of social distancing in real-world settings is challenging due to the lack of randomization, inadequate reporting, and changing interventions over time (266, 267). Broderick et al. evaluated febrile respiratory illness (FRI) transmission rates in a military training setting where some units were open to potentially infectious convalescents and some units were closed to the entry of potentially infected individuals (268). They found that there was no significant difference in FRI rates between open and closed units; however, any effect of social distancing may have been mitigated by the finding that there was also considerable environmental pathogen contamination in the housing units. Such studies highlight the complexity of systematic evaluations of population-based interventions in natural settings, rather than disprove their efficacy. Indeed, computer simulations have demonstrated that social distancing can be an effective public health prevention measure, reducing epidemic attack rates by as much as 90%, depending on the infectivity of the pathogen (269, 270). The use of masks, particularly among health care workers, is an established effective intervention to reduce respiratory virus transmission (266). It remains uncertain whether N95 respirators confer additional protection over surgical masks, and the degree of difference may vary by pathogen.”

Vaccination

“To date, there have been no HRV vaccines evaluated in clinical trials. Challenges to vaccine development include the presence of more than 100 different HRV serotypes, the lack of epidemiological data to identify the most commonly circulating HRV strains, the incomplete understanding of antigenic differences between the recently discovered HRV-C species and known serotypes, and limited animal models of HRV infection to understand viral pathogenesis (282).”

https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC3553670/

In Summary:

• The “isolation” of the RI-APC group of respiratory “viruses” through the use of tissue culture methods greatly stimulated interest in this field
• Naso-pharyngeal washings were collected in monkey kidney maintenance media
• They were shell frozen in ampules in an alcohol-dry ice mixture and stored at -500 C
• When “isolation” attempts were made the ampule was quickly thawed and 0.1 ml. of the washing was inoculated into tubes of Hela cells as described by Hilleman and Werner
• The methods followed Hilleman/Werner exactly except the maintenance media of Eagle’ was substituted

Quick Detour:

The method that Price followed from Hilleman/Werner is detailed below. As can be seen, the sample is added to the usual soup of foreign animal material mixed with cancer cells and antibiotics.

Materials and methods. Tissue culture. Tube and bottle cultures of HeLa cells (human epidermoid carcinoma) were prepared by the general method of Scherer, Syverton, and Gey except that tryptic digestion was not employed. The bottle (32 oz prescription size)
and tube (16 x 150 mm) cultures were fed with nutrient fluid consisting of pooled human serum, 25 parts; chick embryo extract, 7 parts (2 parts 50% chick embryo extract and 5 parts 50% chick embryo extract ultrafiltrate); Hanks balanced salt solution, 68 parts, plus sufficient penicillin and streptomycin to give a final concentration of 50 units of each per ml. In order to remove the human serum contained in the nutrient fluid, the contents of the bottles and tubes were washed 3 times, immediately before use, with 20 or 1.0 ml, respectively, of 5% chicken serum in Hanks-Simms solution containing antibiotics. Finally, 30 ml (bottles) or 0.6 ml (tubes) of maintenance solution (10 parts pooled chicken serum; 5 parts 50% chick embryo extract ultrafiltrate; 85 parts Hanks balanced salt solution and antibiotics) were added to the washed cultures. At time of use, the bottle cultures contained about 6-10 million cells and were 3-5 days old; the tube cultures contained about 60-80 thousand cells and were 1-3 days old. The bottles and tubes were kept tightly stoppered prior to and following inoculation with the material under study and were incubated at 36°C in a stationary position.

Isolation of strains of respiratory illness (Rl) agents. Throat washings (collected in veal infusion broth and stored frozen at -70°C) from a patient with PAP (case 67) in the epidemic were centrifuged briefly to remove gross particles and then inoculated into roller tube cultures of adult human tracheal epithelium prepared by the general method of Enders. A cytopathogenic agent (RI-67) was recovered in first passage in such tissue and this organism was readily propagated in HeLa cells following 3 additional passages in tracheal cultures. All further isolations were attempted by direct inoculation into the HeLa culture medium.”

doi: 10.3181/00379727-85-20825.

Nothing pure nor isolated about that concoction.

End Detour.

• The “virus” in this paper did not cause degeneration of the Hela cells within 240 hours, even after 3 blind passages
• The agent multiplied in monkey kidney cells grown in 3 percent inactivated horse serum, 5 percent beef embryo extract, and 92 percent 199 solution
• This media contained 100 units of penicillin and streptomycin per one ml. of media
• The cytopathogenic effect of this agent can usually be observed more readily in 199 medium without the above additions
• However, in a few primary isolations this “virus” was “isolated” in the richer medium and not in plain 199 medium
• The significance of these observations was not clear
• The main clinical features with which this agent was associated are malaise and coryza and mild sore throat and a low-grade fever may or may not be present
• There are no physical findings to suggest lower respiratory involvement
• Antibody surveys on various age groups carried out were said to have shown that this “virus” was relatively widespread, about 20 percent of the individuals over eight years of age showing the presence of neutralizing antibodies
• Complement-fixing antibodies also could be shown to be present for this agent in the sera of individuals
• Remember, it was later revealed that there are no specific antibodies for HRV as there are supposedly over 100 different serotypes
• No complement-fixing (CF) antigen could be prepared from monkey cells infected with the JH agent which reacted with standard RI-APC sera using methods which could readily detect RI-APC CF antigen in Hela cells infected with a RI-APC “virus”
• The agent was said to be able to be passed in series in monkey cells without loss of titer at least 10 times, after which no further attempts were made to pass the agent
• When the JH suspension, which titered about 10-‘in monkey cells, was inoculated intranasally, intracerebrally, or intrapertoneally into twenty 1-day-old mice or ten 10-day-old hamsters, no overt signs of disease were noted over the 21-day observation period
• Similar negative results were obtained when ten 15-17-gm. mice or six young adult guinea pigs were inoculated intraperitoneally
• Intranasal inoculation of ferrets gave negative results, the animals showing normal fever curves and showing no overt signs of illness
• Two blind passages of various organs and tissues under all the above conditions failed to show any evidence that the JH agent had multiplied
• Inoculation of the JH suspensions which contained about 10^2 and 10^5 TCD60 into 12-day-old chick embryos by the amniotic and allantoic routes failed to reveal evidence of multiplication even after three blind passages
• The JH “virus” also had very peculiar nutritional requirenments which are different from those reported for any ECHO “virus” (i.e. it’s all about the recipe used to create the desired effect)
• Infected monkey tissue culture preparations which titered out to approximately 10-5 in monkey cells were inoculated into Brewers’ thioglycolate medium and sheep, human, and rabbit blood agar plates were inoculated both aerobically and anaerobically, and none showed growth after 12 days at 300 C
• The researchers claimed the discovery of a new “virus” based on 4 points:
  1. The “isolation” of the agent from individuals ill with a particular respiratory clinical syndrome (however nothing was in fact isolated)
  2. The rise in antibodies to the agent in such people and the failure to show a rise in antibody to the agent in many other human respiratory as well as other “viral,” bacterial, rickettsial, and chronic diseases (the antibodies were not specific)
  3. The failure to “isolate” the agent from 1,100 individuals not showing clinical signs of respiratory illness in the same seasons and from the same age group and in the same state as the ill individuals from whom the “virus” was isolated (they failed to “isolate” any “virus” from those with symptoms so…)
  4. The relationship between antibody level and clinical disease in certain respiratory outbreaks (again, non-specific antibodies)
• In other words, not a single bit of DIRECT evidence of any “virus” and the INDIRECT evidence is non-specific
• The “viruses” responsible for the major cause of respiratory disease in the civilian population had not been identified
• The researchers stated that in their extensive field-laboratory study over the last three years, less than 30 percent of the respiratory illnesses could be identified as being caused by a known microparasite, of either bacterial or “viral” origin
• The importance of the JH agent in causing illness in the civilian population as well as other problems dealing with this “virus” was still under investigation and was to be described elsewhere
• In view of the similarity between the mild respiratory infection associated with the JH “virus” and the common cold, they hoped that study of the JH “virus” may lead to some clues as to how the common cold “virus” may be isolated
• In this connection it should be pointed out that the JH “virus” has a very long incubation period before producing a cytopathogenic effect in monkey kidney cells, particularly on primary isolation
• Thus, a period of 25 days, as well as a blind passage at this time, had been found to be required in many of the isolations, the cytopathogenic effect showing up on the tenth to sixteenth day of the second passage

• Human “rhinoviruses” (HRVs) were first discovered in the 1950s in an effort to identify the etiology of the common cold
• 70 years later, the search for a “cure” for the common cold “virus” is still ongoing
• While once thought to cause relatively benign upper respiratory tract illness, HRVs are now linked to exacerbations of chronic pulmonary disease, asthma development, and, more recently, severe bronchiolitis in infants and children as well as fatal pneumonia in elderly and immunocompromised adults
• Our enhanced understanding of the spectrum of illness of HRVs draws largely from advances in molecular methods that have facilitated the detection and characterization of HRV groups and strains
• In other words, while HRV was only thought to cause the common cold, this new link to more severe disease was “discovered” through the use of PCR
• There are currently no approved antiviral agents for the prevention or treatment of HRV infection
• Clinical trials of antiviral therapies have been limited by:
  1. Drug toxicities
  2. Drug interactions
  3. Lack of efficacy when applied to the natural setting
• Efforts at vaccine development are hindered by the existence of more than 100 HRV serotypes with high-level sequence variability in the antigenic sites (i.e. the antibody responses are not specific)
• Clinical specimens in the 1960s and 1970s yielded approximately 100 different HRV strains (known as the reference or prototype set), which were subsequently serotyped
• Partial sequencing of “viral” capsid-encoding regions, noncoding regions, and a limited number of complete genomes led to a division of the original 99 strains into two species:
  1. HRV-A (containing 74 serotypes)
  2. HRV-B (containing 25 serotypes)
• The development of highly sensitive molecular techniques for the identification of HRV in clinical specimens led to the identification and designation of a novel species, HRV-C, by the International Committee on Taxonomy of Viruses in 2009
• HRV-C strains do not grow in standard cell culture, likely postponing their discovery; therefore, a genetically based classification system was developed
• To date, at least 50 different types of HRV-C have been identified
• While other respiratory “viruses,” such as influenza “virus” and respiratory syncytial “virus” (RSV), cause a destruction of airway epithelial cells, HRV is seldom associated with cytopathology of the upper respiratory tract
• A similar preservation of cell morphology and composition was observed for the nasal epithelium during studies of experimental HRV infection, where the amount of “viral” shedding did not correlate with the severity of symptoms
• The development of small-animal models is useful to understand further the pathogenesis of HRV infection in both the upper and lower airways; however, there are no known murine “rhinoviruses”
• The relative roles of the “virus” itself and the host immune response in HRV pathogenicity and symptomatic illness are still under debate
• In the upper airways, HRV has not been shown to cause direct cytopathology; clinical symptoms are likely the result of local and systemic immune responses
• With the increasing use of molecular methods of “viral” detection, asymptomatic HRV infection has been noted to be relatively common, particularly in children
• In children less than 4 years old, rates of asymptomatic infection range from 12 to 32% (87–91) and tend to be higher in the youngest age groups
• Common symptoms include:
  • Rhinorrhea
  • Nasal congestion
  • Sore throat
  • Cough
  • Headache
  • Subjective fevers
  • Malaise
• Compared to patients with “coronavirus-associated colds,” there is no difference in respiratory symptom severity or duration
• There is no common antigen among HRVs, and an increasingly large number of serotypes have been described; therefore, antigen detection assays are not used for routine detection
• The lack of a common antigen across all strains of HRV makes the detection of antibody responses impractical for diagnostic purposes
• HRVs were originally isolated from primary monkey kidney cells, although these cells will support the growth of only some strains (i.e. they can not create the desired CPE every time)
• The most commonly used cell lines for HRV culture in clinical laboratories include:
  1. Human fetal embryonic lung fibroblast cell lines (cells taken from an aborted female fetus)
  2. Certain HeLa cell clones (cloned cervical cancer cells)
  3. Human embryonic kidney cell lines (cells taken from an aborted female fetus)
• In a study of HRV recovery from nasal wash specimens, WI-38 and an HRV-susceptible HeLa clone (with high levels of ICAM-1 expression) were found to be the most sensitive cell types for HRV culture
• As noted by other investigators, those authors suggested a combination of different cell lines as the best approach for the optimal recovery of HRV
• In other words, instead of using monkey kidneys, the best approach is to mix aborted fetal cells with cervical cancer cells to create HRV
• CPE is usually visible in most cell lines, although CPE-negative strains have been reported
• Conventional cultures should be incubated for up to 14 days, and presumptive identification is usually made on the basis of CPE in appropriate cell lines, although the appearance is the same or very similar to that of “enteroviruses” (EVs)
• Centrifugation-enhanced cultures can be stained prior to the development of CPE with some EV detection reagents that cross-react with HRV; however, laboratories are left without a specific diagnosis
• This means that the CPE seen in cell cultures is not specific to HRV, does not always occur with HRV, is identical to that seen with EV, and EV detection in culture cross-reacts with HRV
• HRVs are somewhat fastidious in vitro, and due to the specific conditions required for optimal culture, isolation rates have been generally low except in large reference virology laboratories
• PCR was used to identify HRV in the late 1980’s, although identification had to be confirmed with dideoxy sequencing due to cross-reactivity with human DNA
• Multiple RT-PCR and real-time RT-PCR techniques have been developed for the detection of HRV since the late 1980s with most of these assays targeting the 5′UTR, a region highly conserved among all HRVs and EVs, causing cross-reactivity between assays for the two “viruses” and making their differentiation difficult
• Since the International Committee on Taxonomy of Viruses reclassified all HRVs into the EV genus, however, the reporting of diagnostic test results as HRV or EV without further testing has become acceptable
• In other words, if during your experiments you find that you keep encountering the same results with two different “viruses,” instead of admitting you may be on the wrong path, it’s OK to reclassify the genus of one “virus” so that the contradictory results are now acceptable to diagnose both “viruses”
• There are currently no commercially available quantitative molecular HRV tests
• Additionally, due to the intertypic variability of HRV, there is no standard for the quantification of all HRV types
• Other challenges that arise when attempting to provide an accurate and relevant quantification include factors such as:
  1. The sample type and collection procedure
  2. Test methodology
  3. Age of the patient
  4. Immune status
• All these factors affect “viral” load and the potential standardization of assays and collectively impact the clinical interpretation of quantitative HRV data such that generally applicable guidelines are not feasible with any reasonable level of confidence
• As data (based on molecular techniques) accumulated on the involvement of HRV in more serious clinical disease, the importance of the inclusion of assays for the “virus” in these new panels became clear
• In turn, as these new assays have been introduced into clinical practice, more data have emerged on the high incidence of HRV infection, resulting in the further awareness of the widespread and sometimes serious disease manifestations
• Additionally, some HRVs, such as the group C “viruses,” are uncultivable by routine culture techniques
• As they have started including non-specific HRV assays into PCR testing and finding cases of HRV in more serious disease, the “virus” long said to only cause mild upper respiratory disease is now being implicated in more severe lower respiratory disease, all based on PCR results
• An assessment of the effectiveness of social distancing in real-world settings is challenging due to the lack of randomization, inadequate reporting, and changing interventions over time
• Broderick et al. evaluated febrile respiratory illness (FRI) transmission rates in a military training setting where some units were open to potentially infectious convalescents and some units were closed to the entry of potentially infected individuals and they found that there was no significant difference in FRI rates between open and closed units
• Such studies highlight the complexity of systematic evaluations of population-based interventions in natural settings, rather than disprove their efficacy (excuses excuses…)
• Computer simulations have demonstrated that social distancing can be an effective public health prevention measure, reducing epidemic attack rates by as much as 90%, depending on the infectivity of the pathogen
• In other words, REAL world studies showed social distancing to be ineffective however ARTIFICIAL computer simulations proved they work…so I guess it’s a draw according to virologists…?
• To date, there have been no HRV vaccines evaluated in clinical trials
• Challenges to vaccine development include:
  1. The presence of more than 100 different HRV serotypes
  2. The lack of epidemiological data to identify the most commonly circulating HRV strains
  3. The incomplete understanding of antigenic differences between the recently discovered HRV-C species and known serotypes
  4. Limited animal models of HRV infection to understand “viral” pathogenesis

There is a saying that when a doctor doesn’t know the cause of disease, he will claim it is a “virus.” If one were to read through the original studies of any so-called “virus,” it is obvious that this saying pertains to virologists as well. Regarding the “rhinovirus,” we have zero evidence of any “virus” whatsoever. There were no purified/isolated particles taken directly from sick humans anywhere in Winston Price’s original 1956 paper. Instead, we are presented with the usual cell culture trick of human fluids combined with foreign animal material and antibiotics. The cultures required long incubation periods with blind passaging in order to produce non-specific CPE which was later said to be identical to that seen with “enteroviruses.” As not every culture produces CPE, we are now told that there are non-CPE producing strains in order to cover up for this contradictory evidence.

The antibody results provided by Price as the hallmark evidence for his “virus” was later shown to be non-specific as these results cross-react with “enteroviruses.” To cover up this equally contradictory evidence, it was “discovered” that there are 160+ different serotypes of “rhinoviruses” which do not share common antigens as they each have their own surface proteins. This conveniently means that antigen and antibody tests do not work for diagnosing “rhinovirus.” However, it should also show that the antibody results are meaningless and are not valid as indirect proof for the existence of any “rhinovirus.” Even the PCR tests were said to cross-react with human DNA which should immediately throw doubt on the validity of any “rhinovirus” genome.

In any way you break it down, the “rhinovirus” is the perfect culprit for doctors to blame when they don’t know the cause of disease. They simply have to look for symptoms of the common cold for diagnosis as antibody and antigen tests do not work. “Viral” cultures do not need to produce CPE to be considered successful as there are CPE-negative strains. There are no vaccines nor any treatments available so the only option is symptom suppression. No proof of “virus” is required in order to diagnose the “rhinovirus” as noted here:

“Clinical signs and symptoms of the common cold, by definition, are similar regardless of the infectious etiology. Accordingly, if findings from a thorough history and physical examination are consistent with a viral etiology and no complications are noted, an aggressive workup is rarely necessary. Differentiation of one virus from another or one rhinovirus (RV) serotype from another on the basis of clinical presentation is difficult.”

https://emedicine.medscape.com/article/227820-workup#c1

When you don’t have to find a “virus” in order to diagnose a “virus,” perhaps that is the very reason why the “rhinovirus” is the most common cause of the common cold.