The Purification Problem

It is increasingly clear that purification of a “virus” is impossible. There are too many contaminants, variables, unknowns, and nanoparticles of similar shape/size to be able to say with certainty that the particles assumed to be a “virus” in a cell culture are the same ones imaged by TEM or for which the genome sequence is said to be based upon. In order to shed further light on this problem, let’s look at two different moments in time to see what the state of purification methods were as well as what, if any, purification was considered possible. The first source is from a 1975 book which inadvertently makes the case that complete purification/isolation of particles assumed to be “viruses” is impossible:

Purification of Viruses. In: Chemistry of Viruses. Springer Study Edition.

“Each virus poses an individual purification problem that is related to the properties of the virus, the nature of the host, and the culture conditions. Consequently, it is not possible to outline a purification procedure that will work with equal effectiveness for all viruses.”

“In these terms, purity means the degree of freedom from nonviral components, or, conversely, the extent to which viral particles show gross physicochemical homogeneity. No single test is sufficient to establish this type of purity, but a consistent answer from each of several tests establishes the degree of homogeneity of the preparation in question and hence the reliance to be placed on analytical data and other results obtained with such a preparation.”

“The lower limit of contaminant detectable by either sedimentation analysis or electrophoresis is variable, and is dependent upon the nature of the material and the circumstances of the test. As usually applied in testing virus preparations, these methods cannot be expected to detect less than a few percent of contaminant (Sharp 1953). For many purposes, it is satisfactory to measure purity to this degree, but as the tools for chemical and biological analyses become sharper and sharper, it will be increasingly necessary to remember the limitations of sedimentation and electrophoresis measurements.”

“The electron microscope can be used to examine directly the physical homogeneity of a virus preparation. Under favorable conditions it is possible to detect an impurity present in a concentration of as little as 1 percent of the virus (Williams 1954). It is obvious, of course, that impurities will escape detection if they have the same size and shape as the virus particles, or if they are below the size resolved by the microscope. Also, particles present in small number but large in mass are easily overlooked, owing to sampling difficulties (Lauffer 1951).”

“In summary, no single criterion of purity is sufficient to establish the homogeneity of a preparation of virus. This must be done by applying critically as many tests as possible (see Knight 1974).”

https://www.google.com/url?sa=t&source=web&rct=j&url=https://link.springer.com/content/pdf/10.1007/978-3-642-85899-4_2.pdf&ved=2ahUKEwi04tPs_7DvAhWOZs0KHZp1BC0QFjANegQIFxAC&usg=AOvVaw0Fx51ebi9lAsov0RyK1wyl

This second source is from August 2020 and it deals primarily with attempts to separate EV’s from “viruses:”

Purification Methods and the Presence of RNA in Virus Particles and Extracellular Vesicles

“The fields of extracellular vesicles (EV) and virus infections are marred in a debate on whether a particular mRNA or non-coding RNA (i.e., miRNA) is packaged into a virus particle or copurifying EV and similarly, whether a particular mRNA or non-coding RNA is contained in meaningful numbers within an EV. Key in settling this debate, is whether the purification methods are adequate to separate virus particles, EV and contaminant soluble RNA and RNA: protein complexes. Differential centrifugation/ultracentrifugation and precipitating agents like polyethylene glycol are widely utilized for both EV and virus purifications. EV are known to co-sediment with virions and other particulates, such as defective interfering particles and protein aggregates.”

“For RNA viruses as well, RNAs other than full-length genomic RNA have been reported in mature virion preparations. Is it possible that EV may have contaminated the virion preparations in these studies? Has there been enough experimental evidence to rule out EV contamination?“

“Proteins, DNA, mRNA, miRNA, and other non-coding RNAs were found enclosed in these small, membrane-enclosed exosomes and microvesicles [1] (Figure 1B). This makes EV conceptionally and biochemically similar to viruses [1,18] (Figure 1C). The formation and egress of microvesicles and exosomes share similarities to virus biogenesis, such as HIV [24] and enveloped hepatitis A viruses (HAV) [25] (Figure 1), respectively. In fact, viruses have been thought of as emerging from exosomes or vice versa [26]. Any EV can play an analogous role to a virus particle in the functional transfer materials from one cell to the next, regardless of the class.”

How do RNAs Co-Purify with Viruses and EV by Different Purification Methods?

“Viruses and EV are purified by similar techniques (Table 1) [18,27]. Historically, differential centrifugation and ultracentrifugation have been the most widely used methods for concentrating viruses and EV [28]. RNAs co-purify with viruses and EV in the form of (a) non-encased extracellular RNAs and (b) co-contamination of RNA encased in EV and virions. For cleaner purification, RNase treatment are always recommended to remove non-EV encapsulated extracellular RNAs. Co-purified RNAs within a heterogeneous mixture can obscure definitive and functional research on EV as well as viruses. Separating virions from EV, however, is much more challenging. Thus, we will discuss how such contamination—if any—can be removed by different purification methods.”

“When the differences are small, i.e., between viruses, EV and protein aggregates [30], a gradient medium (i.e., sucrose, iodixanol, sorbitol, cesium chloride, etc.) is needed to increase the separation efficiency [29]. Often the viruses’ densities and buoyancies so closely overlap with exosomes’, that even separation via density gradients is impractical [18,31–33] and leads to co-isolation of their encased RNAs.”

“Polyethylene glycol (PEG) Precipitation – PEG has long been used to “precipitate” and purify viruses [34,35]. It is the main reagent in several commercial kits for exosome purification [18,35]. The method consists of a precipitation step followed by low-speed centrifugation. PEG precipitation offers little separation efficacies, cannot separate viruses from EV [36], and often co-precipitates other macromolecule contaminants like RNA, DNA, and protein aggregates [30,35,37,38]. Exosomes isolated by commercial kits are likely to be contaminated by viruses, proteins, non-EV associated nucleic acids, and other extracellular debris [36]. This includes any molecules stuck to the outside of the EV rather than being carried inside. Many of these contaminations may carry RNAs.”

“Chromatography- Size-exclusion chromatography (SEC), ion-exchange chromatography (IEC), and affinity chromatography (AC) are commonly used chromatography methods for virus purification [41–43].”

“Both viruses and EV can be purified by SEC and IEC [18,40] but virus-EV cross-contamination is difficult to avoid [30]. Thus, SEC and IEC will most likely co-isolate the encased RNAs contaminants.”

“Separating EV from virus particles, particularly exosomes and microvesicles, has proven to be a considerable hurdle in the field of host–pathogen interactions. Chugh et al. [36] and Bess et al. [77] showed that virions and EV co-sedimented in various isolation techniques due to their similar size, density, and sedimental velocity [31]. Other studies [25,27,31,32,36,48,77] also showed that neither differential centrifugation nor commercial exosome precipitation reagents separate virions from EV.”

“Since it is near impossible to separate EV from virions by biochemical methods, the absence of EV is typically demonstrated by the absence of EV protein markers. For instance, Cliffe et al. [8] checked the purity of their virion preparations by transmission electron microscopy, but no image was included in the manuscript. Lin et al. [9] performed a Western blot and did not detect the exosome markers CD63 or CD81 in the purified virions, concluding that miRNAs were present in virions. However, whether the Western blot had the required level of sensitivity is unknown. In contrast to Lin et al., Chugh et al. [36] showed that for the same virus, the majority of miRNA are carried by EV rather than virions. Herpesvirus can switch between latent and lytic phases [78]. The RNA profiles are very different [79–81]. It is not clear whether the phase of the virus played a role in the discrepancy between Lin et al. [9] and Chugh et al. [36]. The majority of the studies in Table 2 [4–7,10–13,15] did not investigate possible EV contaminations. The concept of EV transferring functional nucleic acids has only gained traction recently [82], so it is not surprising that studies before 2010 did not consider this possibility.

The problem becomes more difficult when considering that as virus-infected cells not only release virions with virus-derived RNAs, they also release EV filled with virus-encoded RNAs at the same time, as well as various species of defective interfering particles. Hence, we would expect EV to contain viral RNA under most circumstances. EV emanating from cells infected with HIV, hepatitis C virus (HCV), and various human herpesvirus viruses (HHV) can have virus-encoded RNAs present within them [83–85]. In the case of KSHV, viral miRNAs are present predominantly within exosomes, rather than mature virions [36]. Additionally, picornavirus like the EMCV and HAV can traffic the entire virion into EV [25].”

“Still, better and more carefully validated purification methods are necessary to prepare cleaner virion and EV preparations before many of the proposed biological functions that have been associated with EV can be accepted. Affinity reagents, in particular, reveal surprising heterogeneity amongst EV. Describing and limiting the increasing complexity of EV may seem burdensome, but it is essential for establishing biological relevance.”

https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.mdpi.com/1999-4915/12/9/917/pdf&ved=2ahUKEwi8x6SNvcnxAhWGW80KHfVPB3EQFjAMegQIERAC&usg=AOvVaw3kccThKbbHOPZasZ_5KBWb

In Summary:

• Each “virus” poses an individual purification problem related to the properties of the “virus,” the nature of the host, and the culture conditions
• It is not possible to outline a purification procedure that will work with equal effectiveness for all “viruses”
• Purity means the degree of freedom from “nonviral” components
• No single test is sufficient to establish this type of purity
• The lower limit of contaminant detectable by either sedimentation analysis or electrophoresis is variable and these methods cannot be expected to detect less than a few percent of contaminant
• Impurities will escape detection if they have the same size and shape as the “virus” particles, or if they are below the size resolved by the microscope
• Particles present in small number but large in mass are easily overlooked
• No single criterion of purity is sufficient to establish the homogeneity of a preparation of “virus”
• It needs to be established whether purification methods are adequate to separate “virus” particles, EV and contaminant soluble RNA and RNA: protein complexes
• EV are known to co-sediment with “virions” and other particulates, such as defective interfering particles and protein aggregates
• Two important questions are asked:
  1. Is it possible that EV may have contaminated the “virion” preparations in these studies?
  2. Has there been enough experimental evidence to rule out EV contamination?
• EV’s are conceptionally and biochemically similar to “viruses”
• The formation and egress of microvesicles and exosomes share similarities to “virus” biogenesis
• “Viruses” have been thought of as emerging from exosomes or vice versa and any EV can play an analogous role to a “virus” particle
• RNAs co-purify with “viruses” and EV in the form of (a) non-encased extracellular RNAs and (b) co-contamination of RNA encased in EV and “virions”
• Separating “virions” from EV is much more challenging
• Often the “viruses” densities and buoyancies so closely overlap with exosomes’, that even separation via density gradients is impractical and leads to co-isolation of their encased RNAs
• PEG precipitation offers little separation efficacies, cannot separate “viruses” from EV, and often co-precipitates other macromolecule contaminants like RNA, DNA, and protein aggregates
• Exosomes isolated by commercial kits are likely to be contaminated by “viruses,” proteins, non-EV associated nucleic acids, and other extracellular debris
• This includes any molecules stuck to the outside of the EV rather than being carried inside and many of these contaminations may carry RNAs
• “Virus”-EV cross-contamination is difficult to avoid
• SEC and IEC will most likely co-isolate the encased RNAs contaminants
• Separating EV from “virus” particles, particularly exosomes and microvesicles, has proven to be a considerable hurdle in the field of host–pathogen interactions
• Chugh et al. and Bess et al. showed that “virions” and EV co-sedimented in various isolation techniques due to their similar size, density, and sedimental velocity
• Other studies also showed that neither differential centrifugation nor commercial exosome precipitation reagents separate “virions” from EV
• It is near impossible to separate EV from “virions” by biochemical methods
• The majority of the studies did not investigate possible EV contaminations
• The concept of EV transferring functional nucleic acids has only gained traction recently, so it is not surprising that studies before 2010 did not consider this possibility
• It is expected for EV to contain “viral” RNA under most circumstances
• Better and more carefully validated purification methods are necessary to prepare cleaner “virion” and EV preparations before many of the proposed biological functions that have been associated with EV can be accepted

We have a source from 1975 showing that complete purification/isolation of “viral” particles is impossible and we have a source 46 years later saying the same thing. It seems that even with modern day advancements in technology, there just is no possible way to completely purify/isolate particles believed to be “viruses.” This should make it obvious that every single virology paper is scientific fraud as without purification/isolation of the particles believed to be “viruses,” there never was a valid independent variable to manipulate in order to show that any particles were actual causes of disease. This means we can throw out any “virus” study ever as claims were made for which they could never back up with an isolated physical entity. We can throw out any EM images of “viruses” as there is no possible way to be certain the particles imaged are the ones claimed to cause disease. We can throw out any “viral” genome evidence as there is no realistic way it can be stated that the RNA came from a single source.

While we are at it, we can also throw out virology as a science as well.