Van Der Hoek’s “Coronavirus” NL63 Paper (2004)

Coronavirus data flow numbers art concept. Pandemic COVID virus IT safe blue background. Statistics epidemia pneumonia vector illustration.

“I have the hypothesis that some viruses may be beneficial. I have no proof. It is only a hypothesis, simply because some viruses fail to be associated with disease, so these viruses may be beneficial, but this has not been investigated yet. Work on this topic is ongoing in my group.”

-Lia Van Der Hoek

The above quote by Lia Van Der Hoek is a very candid and revealing insight into the mind of a virologist. When discussing her views on “viruses,” Van Der Hoek made a claim about her hypothesis for which she had no evidence which she had based on the lack of evidence of disease caused by the “discovery” of some “viruses.” Making claims and hypotheses about the presence and nature of “viruses” without any evidence is par for course for Van Der Hoek as she did exactly that back in 2004 with her paper on the “discovery” of the new “coronavirus” NL63.

With the dawn of molecular virology in the early 2000’s, “virus” hunters such as Van Der Hoek uncovered many new “viruses” based solely on genomic sequencing without the use of tissue, organ, or cell cultures. There was no new disease prompting the search for these “viruses,” just the thrill of the molecular hunt. Today, researchers take unpurified samples from animals, the soil, the ocean, etc., use computer algorithms to create a genomic sequence, put the sequence in a database, call it a day and move on to find the next “virus-by-random-letters” wherever they may find it.

Lia Van Der Hoek

In the case of “coronavirus” NL63 in 2003-4, Van Der Hoek relied on a sample from a 7-month old infant with mild illness to capture her targeted “virus.” She recounted this “discovery” process in a recent interview from May 2021:

Q&A: How to track down a new virus – and link it to disease

How did you go about finding the NL63 virus?

“My identification of a new coronavirus started with a child seen in a hospital in the Netherlands who was sick with bronchitis and conjunctivitis. The child was not severely ill and was sent home and monitored. But (the hospital) collected a sample (from the child’s nose). 

There was a suspicion that it was an infection with RSV (respiratory syncytial virus), yet when the lab tried to detect RSV this turned out negative. Then all the other respiratory virus tests were done and these were also negative. The laboratory from the Municipal Health Service of Amsterdam asked me to see which virus was infecting the cells. I found a coronavirus and named it NL63 in April 2003.

How do you go about identifying a virus?

Back then there was no next generation sequencing like today. I had to develop my own techniques to find new viruses, when I started hunting for them in 2001. At the time, we had no clue about the proteins or genetic code of unknown viruses.

First, we know viruses are small, so you select out things that are 100 or 200 nanometres. Then I multiplied DNA or RNA present in a sample. After that, I used restriction enzymes from bacteria that chop up specific small genetic sequences. Comparing the code of these small fragments with the viruses we already know then shows whether the virus may be a new one.

How did you work out it was a coronavirus?

Once I (did the sequencing), I discovered that the code resembled other coronaviruses, but was different enough to be new.

In that same year, there was an outbreak in China, Hong Kong, with an overflow to Canada. It took two to three months to find the virus that caused that epidemic, which was SARS. When I first got the sequence of NL63, I saw that it was similar to the (SARS) virus and I knew immediately that this was important.

There were only three known human coronaviruses before NL63, the other common cold viruses OC43 and 229E, and SARS coronavirus.

How does your approach differ from that of other virus hunters?

Today, virus hunters can easily sequence hundreds of unknown viruses by taking samples from the oceans or soils or animals. Most virus discoverers usually publish on a new virus, and how it looks, and then continue on to the next virus. Searching for unknown viruses is not difficult, but finding new relevant viruses is. That is why I only search for new viruses when I am in contact with medical doctors that see a disease in patients and that disease seems to be infectious.”

https://ec.europa.eu/research-and-innovation/en/horizon-magazine/qa-how-track-down-new-virus-and-link-it-disease

It’s all in silico these days.

As can be seen by Van Der Hoek’s comments, genomics has quickly become the new go-to indirect method relied upon to claim unseen theoretical “viruses” are present in unpurified samples. “Virus” hunters can easily pull up random A,C,T,G’s whenever they want and compare them to the “viruses” in the genomic database in order to claim relation to other “viruses” based solely on the sequence generated. Everything can be done in silico, i.e. by means of computer modeling and simulation.

However, in Van Der Hoek’s case, since genomics and molecular virology was in its infancy and relatively new in the early 2000’s, she had to create her own method in order to “discover” her new “virus.” This method was known as VIDISCA. A very nice breakdown of this process was presented in the following article:

Confirmed: PCR Tests CANNOT Detect SARS-CoV-2, Cause Of COVID19

“The authors state that “the identification of unknown pathogens using molecular biology tools is difficult because the target sequence is not known so that PCR-specific initiators cannot be designed“. What they used is a tool they developed themselves called VIDISCA which, they claim, does not require prior knowledge of the sequence!

Is that possible?

Let’s see how it works: first the culture is prepared and it is assumed that a virus is present due to the evidence of “cytopathic effect”. The novelty introduced by this method is that “restriction enzymes” are added, enzymes that cut the nucleic acid molecules at certain locations and always by the same length.

In this way, if after the action of these enzymes they observe many fragments of DNA or RNA that are the same or very similar, they deduce that it comes from a virus, since the host genome would present random cuts, while the virus genome presents a large number of copies that are the same due to the replication of the virus.

And is such a deduction correct? Of course not!

This assumption (which adds to the previous assumption that there is a virus) does not take into account that there are “virus-like particles”, “retrovirus-like particles”, “endogenous retroviruses”, “exosomes”, “extracellular” particles and even mitochondrial DNA.

In denial, there are a multitude of particles that possess the same reproductive characteristics in large quantities as “viruses” and therefore can falsify results by producing large numbers of identical copies when cut by enzymes as recognised in an article on the VIDISCA technique entitled Enhanced bioinformatic proSling of VIDISCA libraries for virus detection and Discovery. It was published in volume 263 of Virus Research on April 2, 2019, and its authors-Cormac M. Kinsella et al.-recognise that “no redundancy is expected in the VIDISCA insert from the host background nucleic acid except in the case of ‘virus-like’ characteristics, i.e., high copy numbers as in mitochondrial DNA.”

Confirmed: PCR Tests CANNOT Detect SARS-CoV-2, Cause of COVID19

Creating New “Viruses” with VIDISCA

As can be seen, Van Der Hoek relied on many assumptions such as “viruses” being present in the sample as well as how the similar lengths of the DNA/RNA fragments resulting from cutting the nucleic acid by enzymes equaled a “virus” rather than coming from any of the similar particles known to be present in the unpurified sample as well. There are numerous particles with “virus-like” characteristics which can ultimately falsify results. Thus, everything relating to NL63 depends on how accurate her VIDISCA method is and whether or not it can actually sequence unknown “viruses.” Relying on these unproven assumptions means VIDISCA is not off to a good start.

Further evidence against this method was presented by Van Der Hoek in 2011, seven years after the “discovery” of NL63, when she described the limitations of VIDISCA:

A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples

“Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor.”

“Respiratory tract infection is the most common cause of hospitalization of children below the age of 5 years [1][2]. In 5–40% of these hospitalizations no infectious agent can be identified but it is suspected that a viral infection is involved [3][5]. In these cases a yet unknown virus might be the cause of respiratory illness.”

“In the VIDISCA assay viral genomes (which are (reverse-) transcribed into double stranded DNA) are digested with restriction enzymes. The enzymes digest short (4 nucleotides) recognition sequences that are present in virtually all viruses. After ligation of adaptors, the digested fragments are PCR amplified with adaptor-specific primers. The assay is user-friendly however the sensitivity of the assay is low. At least 1 E6 genome copies/ml of a virus in a background that is low in competitor RNA/DNA are needed. These conditions are generally only met when virus culture supernatant is used. In clinical respiratory samples like nasopharyngeal swabs in universal transport medium (UTM) various amounts of competitor RNA/DNA from disrupted cells/bacteria can be present. Ribosomal RNA, which is ∼80% of the total cellular RNA, is one of the biggest problems due to its high copy number and its stability within ribosomes. In particular RNA viruses are difficult to discover since in these cases a reverse transcription is needed, which will enable rRNA to act as competiting nucleic acid sequences.”

“Respiratory samples contain non-viral nucleic acids that interfere in virus discovery techniques like VIDISCA. It is relatively easy to decrease the influence of background bacterial or human DNA and mRNA by centrifugation and DNase/RNase treatment, but ribosomal RNA (rRNA) is difficult to eliminate because the ribosomal proteins protect the rRNA inside the ribosomes.”

“The original VIDSICA method described in 2004 is based on amplification after digestion with 2 restriction enzymes (Hinp1-I and MseI[6]. Investigation of human rRNAs revealed that 28S rRNA contains a very high number of Hinp1-I recognition sites (85, see table 3), but relatively low frequency of MseI restriction sites. The high frequency of HinP1-I digestion in 28S rRNA and the generation of a massive amount of small digested fragments likely interferes in the VIDISCA-ligation.”

“In figure 1 it is shown that the sensitivity of VIDISCA reaches 1 E6 viral genome copies/ml. Although this is an improvement, this detection limit might be too low to detect viruses directly in clinical samples. The concentration of respiratory viruses in nasopharyngeal swabs is in the main below 1 E6 copies/ml, and we can assume that a yet unknown virus will be present in similar concentrations. Thus additional improvement of the VIDISCA-sensitivity is needed.”

“Sequence independent amplification methods, such as VIDISCA and random-PCR, can identify viral sequences without prior knowledge of a viral genome. Unfortunately, the detection of unknown viral pathogens in respiratory clinical material is difficult with these sequence independent virus discovery methods because of low viral load and high background nucleic acids in these samples. During the last years sequence independent virus discovery techniques were mostly used with virus culture supernatant, as they contain high concentrations of viral genomes [6][12], or to discover previously unknown DNA viruses [13][15]. So far no study has been able to identify novel human respiratory RNA viruses with sequence independent amplification techniques. Thus sequence independent amplification techniques like VIDISCA have to be optimized to allow discovery without requiring a culture amplification step.”

“The authors also address a large amount of unknown sequences present in their data set. We also observed the presence of unknown sequences within our data set. It could be that these sequences are derived from yet unknown viruses, or it could be that the sequences are part of a genomic sequence from a known organism, e.g. a bacterium of which not the complete genomic sequence is present in the Genbank databases. Thus care should be taken to assign sequences as potentially viral, since so many organisms have not been fully sequenced.”

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016118

“Purification.” 🤣

According to Van Der Hoek, the discovery of unknown pathogens in NP swabs is difficult due to the high concentration of ribosomal RNA. VIDISCA has a low sensitivity thus it needs a lot of “virus” present in the sample to be able to detect it. This can only be done through cell culturing which is a process that does not produce purified/isolated “viruses” but instead a toxic mixture of human/animal DNA, fetal bovine serum, antibiotics, “nutrients,” and various other additives. The “viral” transport media with NP swabs adds RNA/DNA which acts as competing nucleic acids. She admits that respiratory samples contain many “non-viral” nucleic acids which interferes with VIDISCA and that this interference is difficult to eliminate.

The 2004 VIDISCA method Van Der Hoek used to “discover” NL63 relied upon amplification after digestion with 2 restriction enzymes (Hinp1-I and MseI). However, it was found that human rRNAs contain a very high number of Hinp1-I recognition sites and relatively low frequency of MseI restriction sites which led Van Der Hoek to conclude that the high frequency of HinP1-I digestion and the generation of a massive amount of small digested fragments likely interferes in the VIDISCA-ligation. She ultimately concluded that no studies have shown that techniques like VIDISCA can detect unknown RNA “viruses” without the use of cell culturing and that due to various unknown sequences in genomic data sets, care should be taken to label any sequences as “potentially viral.”

It is apparent that the VIDISCA method was a new and unproven process which relied upon numerous assumptions to generate a sequence which Van Der Hoek claimed as a new “coronavirus.” Is the generation of random A,C,T,G’s from unpurified culture supernatant enough to prove a new “virus?” Van Der Hoek seemed to believe so. Below are highlights from her 2004 NL63 “discovery” paper:

Identification of a new human coronavirus

“Three human coronaviruses are known to exist: human coronavirus 229E (HCoV-229E), HCoV-OC43 and severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV). Here we report the identification of a fourth human coronavirus, HCoV-NL63, using a new method of virus discovery. The virus was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis. The complete genome sequence indicates that this virus is not a recombinant, but rather a new group 1 coronavirus. The in vitro host cell range of HCoV-NL63 is notable because it replicates on tertiary monkey kidney cells and the monkey kidney LLC-MK2 cell line. The viral genome contains distinctive features, including a unique N-terminal fragment within the spike protein. Screening of clinical specimens from individuals suffering from respiratory illness identified seven additional HCoV-NL63-infected individuals, indicating that the virus was widely spread within the human population.

To date, there is still a variety of human diseases with unknown etiology. A viral origin has been suggested for many of these diseases, emphasizing the importance of a continuous search for new viruses1,2,3. Major difficulties are encountered, however, when searching for new viruses. First, some viruses do not replicate in vitro, at least not in the cells that are commonly used in viral diagnostics. Second, for those viruses that do replicate in vitro and cause a cytopathic effect (CPE), the subsequent virus identification methods may fail. Antibodies raised against known viruses may not recognize the cultured virus, and virus-specific PCR methods may not amplify the new viral genome. To solve both problems, we developed a new method for virus discovery based on the cDNA-amplified restriction fragment–length polymorphism technique (cDNA-AFLP4). Here we report the identification of a new coronavirus using this method of Virus-Discovery-cDNA-AFLP (VIDISCA).”

“The new coronavirus that we present here was isolated from a child suffering from bronchiolitis and conjunctivitis. This was not an isolated case, as we identified the virus in clinical specimens from seven additional individuals, both infants and adults, during the last winter season. We also resolved the complete sequence of the viral genome, which revealed several unique features.

Results

Virus isolation from a child with acute respiratory disease

In January 2003, a 7-month-old child was admitted to the hospital with coryza, conjunctivitis and fever. Chest radiography revealed typical features of bronchiolitis. A nasopharyngeal aspirate specimen was collected 5 d after the onset of disease (sample NL63). Diagnostic tests for respiratory syncytial virus, adenovirus, influenza viruses A and B, parainfluenza virus types 1, 2 and 3, rhinovirus, enterovirus, HCoV-229E and HCoV-OC43 yielded negative results. The clinical sample was subsequently inoculated onto human fetal lung fibroblasts, tertiary monkey kidney cells (Cynomolgus monkey) and HeLa cells. CPE was detected exclusively on tertiary monkey kidney cells, and was first noted 8 d after inoculation. The CPE was diffuse, with a refractive appearance in the affected cells followed by cell detachment. More pronounced CPE was observed upon passage onto the monkey kidney cell line LLC-MK2, with overall cell rounding and moderate cell enlargement (Supplementary Fig. 1 online). Additional subcultures on human fetal lung fibroblasts, rhabdomyosarcoma cells and Vero cells remained negative for CPE. Immunofluorescence assays to detect respiratory syncytial virus, adenovirus, influenza viruses A and B, and parainfluenza virus types 1, 2 and 3 remained negative. Acid lability and chloroform sensitivity tests indicated that the virus was most likely enveloped, and did not belong to the picornavirus group23.

Virus discovery by the VIDISCA method

Identification of unknown pathogens using molecular biology tools is difficult because the target sequence is not known, so genome-specific PCR primers cannot be designed. To overcome this problem, we developed the VIDISCA method based on the cDNA-AFLP technique4. The advantage of VIDISCA is that prior knowledge of the sequence is not required, as the presence of restriction enzyme sites is sufficient to guarantee PCR amplification. The input sample can be either blood plasma or serum, or culture supernatant. Whereas cDNA-AFLP starts with isolated mRNA, VIDISCA begins with a treatment to selectively enrich for viral nucleic acid, including a centrifugation step to remove residual cells and mitochondria (Fig. 1a). A DNase treatment is also used to remove interfering chromosomal and mitochondrial DNA from degraded cells (viral nucleic acid is protected within the viral particle). Finally, by choosing frequently cutting restriction enzymes, the method can be fine-tuned such that most viruses will be amplified. We were able to amplify viral nucleic acids in EDTA-treated plasma from a person with hepatitis B viral infection, and from a person with an acute parvovirus B19 infection (Fig. 1b). The technique can also detect HIV-1 in cell culture, demonstrating its capacity to identify both RNA and DNA viruses (Fig. 1b).

The supernatant of the CPE-positive LLC-MK2 culture NL63 was analyzed by VIDISCA. The supernatant of uninfected cells was used as a negative control. After the second PCR amplification step, unique and prominent DNA fragments were present in the test sample but not in the control (1 of 16 selective PCR reactions is shown in Fig. 1c). These fragments were cloned and sequenced. Thirteen of 16 fragments showed sequence similarity to members of the coronavirus family, but significant sequence divergence with known coronaviruses was apparent in all fragments, indicating that we had identified a new coronavirus. The sequences of the 13 VIDISCA fragments are provided in Supplementary Figure 2 online.

Detection of HCoV-NL63 in patient specimens

To show that HCoV-NL63 originated from the nasopharyngeal aspirate of the child, we designed a diagnostic RT-PCR that specifically detects HCoV-NL63. This test confirmed the presence of HCoV-NL63 in the clinical sample. The sequence of the RT-PCR product of the 1b gene was identical to that of the virus identified upon in vitro passage in LLC-MK2 cells (data not shown).

Having confirmed that the cultured coronavirus originated from the child, the question remained as to whether this was an isolated clinical case, or whether HCoV-NL63 is circulating in humans. To address this question, we used two diagnostic RT-PCR assays to examine respiratory specimens of hospitalized individuals and those visiting the outpatient clinic between December 2002 and August 2003 (Fig. 2). We identified seven additional individuals carrying HCoV-NL63 (Table 1). Sequence analysis of the PCR products indicated the presence of a few characteristic point mutations in several samples, suggesting that several viruses with different molecular markers may be cocirculating (Fig. 3 and Supplementary Fig. 3 online). At least five of the HCoV-NL63-positive individuals suffered from respiratory tract illness; the clinical data of two individuals was not available. Including the index case, five of the patients were children less than 1 year old, and three patients were adults. Two adults were likely to be immunosuppressed, as one of them was a bone marrow transplant recipient and the other an HIV-positive patient suffering from AIDS, with very low CD4+ cell counts (Table 1). No clinical data was available for the third adult. One patient was coinfected with respiratory syncytial virus (no. 72), and the HIV-infected patient (no. 466) carried Pneumocystis carinii. No other respiratory agent was found in the other patients, suggesting that the respiratory symptoms were caused by HCoV-NL63. All positive samples were collected during the last winter season, with a detection frequency of 7% in January 2003. None of the 306 samples collected in the spring and summer of 2003 contained HCoV-NL63 (P < 0.01 by two-tailed t test).”

“To determine whether the HCoV-NL63 genome organization shares these characteristics, we constructed a cDNA library with purified virus stock as input material. A total of 475 genome fragments were analyzed, with an average coverage of seven sequences per nucleotide. Specific PCR reactions were designed to fill in gaps and to sequence regions with low-quality sequence data. We combined this with 5′ and 3′ rapid amplification of cDNA ends to resolve the complete HCoV-NL63 genome sequence.

The RNA genome of HCoV-NL63 consists of 27,553 nucleotides and a poly-A tail. With a GC content of 34%, HCoV-NL63 has the lowest GC content among the Coronaviridae, which range from 37–42% (ref. 24). ZCurve software was used to identify the ORFs25, and the genome configuration was portrayed using the similarity with known coronaviruses as a guide (Fig. 4a and Supplementary Table 1 online).”

“We next aligned the sequence of HCoV-NL63 with the complete genomes of other coronaviruses. The percentage nucleotide identity was determined for each gene and is listed in Table 2. All genes except the M gene shared the highest identity with HCoV-229E. To confirm that HCoV-NL63 is a new member of the group 1 coronaviruses, we conducted phylogenetic analysis using the nucleotide sequence of the 1a, 1b, S, M and N genes (Fig. 4b). For each gene analyzed, HCoV-NL63 clustered with the group 1 coronaviruses. The 1a, 1b and S genes of HCoV-NL63 are most closely related to those of HCoV-229E. However, further inspection revealed a subcluster of HCoV-NL63, HCoV-229E and PEDV. Phylogenetic analysis could not be performed for the ORF3 and E genes because the regions were too variable or too small for analysis, respectively. Bootscan analysis by the Simplot software version 2.5 (ref. 28) found no signs of recombination (data not shown).”

Discussion

In this study we present a detailed description of a new human coronavirus. Thus far, only three human coronaviruses have been characterized if we include SARS-CoV; further characterization of HCoV-NL63 as the fourth member will provide important insight into the variation among human coronaviruses. HCoV-NL63 is a member of the group 1 coronaviruses and is most closely related to HCoV-229E, but the differences between them are prominent. First, they share on average only 65% sequence identity. Second, a single gene, ORF3, in HCoV-NL63 takes the place of the 4A and 4B genes of HCoV-229E. Third, the 5′ region of the S gene of HCoV-NL63 contains a large in-frame insertion of 537 nucleotides. The N-terminal region of the S protein has been implicated in binding to aminopeptidase N (group I coronaviruses) and sialic acid31,32,33, so the 179–amino acid insert in HCoV-NL63 might be involved in receptor binding and may explain the tropism of this virus in cell culture. However, the aminopeptidase N receptor-binding domain of the HCoV-229E S protein has been mapped to amino acids (407–547 ref. 33), so it seems unlikely that the insertion will be directly involved in binding to aminopeptidase N. Fourth, whereas HCoV-229E is fastidious in cell culture with a narrow host range, HCoV-NL63 replicates efficiently in monkey kidney cells.”

“The common cold–causing virus HCoV-229E can cause more serious respiratory disease in infants and immunocompromised patients36,37. Our data indicate that HCoV-NL63 causes acute respiratory disease in children below the age of 1 year, and in immunocompromised adults. To date, no known viral pathogen can be identified in a substantial portion of respiratory disease cases in humans (20–30%; ref. 38). Several assays have been used to diagnose coronavirus infections. Traditionally, an antibody test is implemented to measure a rise in titers of antibodies to the human coronaviruses HCoV-229E or HCoV-OC43 (ref. 12). Antibodies to HCoV-NL63 might cross-react with HCoV-229E, given that these viruses are members of the same serotype. If this were the case, HCoV-NL63 infections might have been misdiagnosed as HCoV-229E. Molecular biology tools such as RT-PCR assays39,40 were designed to selectively detect the human coronaviruses HCoV-229E and HCoV-OC43, but these assays will not detect HCoV-NL63. Even the RT-PCR assay that was designed to amplify all known coronaviruses40 is not able to amplify HCoV-NL63 because of several mismatches with the primer sequences. The availability of the complete HCoV-NL63 genome sequence means that these diagnostic assays can be substantially improved.

Our results indicate that HCoV-NL63 is present in a significant number of respiratory tract illnesses of unknown etiology. HCoV-NL63 was detected in patients suffering from respiratory disease, with a frequency of up to 7% in January 2003. The virus was not detected in more recent samples collected in the spring and summer of 2003, which correlates with the fact that human coronaviruses tend to be transmitted predominantly in the winter season12. Future experiments with more sensitive diagnostic tools should yield a more accurate picture of the prevalence of this virus and its association with respiratory disease.

Methods

VIDISCA method.

The virus was cultured on LLC-MK2 cells. Details of virus culture and patient descriptions are available in Supplementary Methods online. To remove residual cells and mitochondria, 110 μl of virus culture supernatant was spun for 10 min at maximum speed (13,500 r.p.m.) in an Eppendorf microcentrifuge. To remove chromosomal DNA and mitochondrial DNA from the lysed cells, 100 μl of supernatant was transferred to a fresh tube and treated with DNase I for 45 min at 37 °C (Ambion). Nucleic acids were extracted as described41. A reverse transcription reaction was performed with random hexamer primers (Amersham Bioscience) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT; Invitrogen). Second-strand DNA synthesis was carried out with Sequenase II (Amersham Bioscience), without further addition of a primer. A phenol-chloroform extraction was followed by ethanol precipitation.

cDNA-AFLP was performed essentially as described4, with some modifications. The double-stranded DNA was digested with the HinP1I and MseI restriction enzymes (New England Biolabs). MseI and HinP1I anchors (see below) were subsequently added, along with 5U ligase enzyme (Invitrogen) in the supplied ligase buffer, for 2 h at 37 °C.”

https://www.nature.com/articles/nm1024#MOESM5

Oh look! A modern day “virus!”

In Summary:

  • NL63 came from a nasal sample from a 7-month old infant that was sent home from the doctor’s office for suffering a mild case of bronchitis and conjunctivitis
  • There was a suspicion that it was an infection with RSV (respiratory syncytial “virus”), but lab tests turned out negative
  • Then all the other respiratory “virus” tests were done and those were also negative
  • Since there was no next generation sequencing back then, Van Der Hoek developed her own techniques to find new “viruses” when she started hunting for them in 2001
  • She stated that at the time,  they had no clue about the proteins or genetic code of unknown “viruses”
  • Van Der Hoek’s Guide to Discovering “Coronaviruses:”
    1. According to Van Der Hoek, since she knew “viruses” are small, she selected out things that were 100 or 200 nanometres
    2. She then multiplied DNA or RNA present in the sample
    3. After that, she used restriction enzymes from bacteria that chop up specific small genetic sequences
    4. She compared the code of these small fragments with known “viruses” to show whether the “virus” was a new one
    5. Once she (did the sequencing), Van Der Hoek discovered that the code resembled other “coronaviruses,” but was different enough to be new
    6. When she first got the sequence of NL63, Van Der Hoek saw that it was similar to the (SARS) “virus” and she knew immediately that it was important
  • Today, “virus” hunters can easily sequence hundreds of unknown “viruses” by taking samples from the oceans or soils or animals
  • Most “virus” discoverers usually publish on a new “virus,” how it looks, and then continue on to the next “virus”
  • According to Van Der Hoek, searching for unknown “viruses” is not difficult, but finding new relevant “viruses” is
  • Because PCR requires a known genome, Van Der Hoek created VIDISCA which was said to be able to sequence a genome without a reference
  • The VIDISCA method simplified:
    1. The culture is prepared and it is assumed that a “virus” is present due to the evidence of “cytopathic effect”
    2. The novelty introduced by this method is that “restriction enzymes” are added, enzymes that cut the nucleic acid molecules at certain locations and always by the same length
    3. After the action of these enzymes, if they observe many fragments of DNA or RNA that are the same or very similar, they deduce that it comes from a “virus” since the host genome would present random cuts, while the “virus” genome presents a large number of copies that are the same due to the replication of the “virus”
  • These assumptions do not take into account that there are “virus-like particles”, “retrovirus-like particles”, “endogenous retroviruses”, “exosomes”, “extracellular” particles and even mitochondrial DNA
  • There are a multitude of particles that possess the same reproductive characteristics in large quantities as “viruses” and therefore can falsify results by producing large numbers of identical copies when cut by enzymes
  • The authors of a 2019 study claimed “no redundancy is expected in the VIDISCA insert from the host background nucleic acid except in the case of ‘virus-like’ characteristics, i.e., high copy numbers as in mitochondrial DNA”
  • VIDISCA is a “virus” discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR
  • Despite claiming to have discovered NL63 from the NP swab of an infant, Van Der Hoek admits in 2011 that direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor
  • It is claimed that in 5–40% of these hospitalizations no infectious agent can be identified but it is suspected that a “viral” infection is involved
  • In the VIDISCA assay, “viral” genomes (which are (reverse-) transcribed into double stranded DNA) are digested with restriction enzymes
  • The enzymes digest short (4 nucleotides) recognition sequences that are (assumed) present in virtually all “viruses”
  • After ligation of adaptors, the digested fragments are PCR amplified with adaptor-specific primers
  • The assay is user-friendly however the sensitivity (ability of a test to correctly identify those with the disease; true positive rate) of the assay is low
  • At least 1 E6 genome copies/ml of a “virus” in a background that is low in competitor RNA/DNA are needed yet these conditions are generally only met when “virus” culture supernatant is used
  • In clinical respiratory samples like nasopharyngeal swabs in universal transport medium (UTM) various amounts of competitor RNA/DNA from disrupted cells/bacteria can be present
  • In particular, RNA “viruses” (of which NL63 is) are difficult to discover since in these cases a reverse transcription is needed, which will enable rRNA to act as competiting nucleic acid sequences
  • Respiratory samples contain “non-viral” nucleic acids that interfere in “virus” discovery techniques like VIDISCA
  • Ribosomal RNA (rRNA) is difficult to eliminate because the ribosomal proteins protect the rRNA inside the ribosomes
  • The original VIDSICA method described in 2004 (as used to “discover NL63) is based on amplification after digestion with 2 restriction enzymes (Hinp1-I and MseI)
  • The high frequency of HinP1-I digestion in 28S rRNA and the generation of a massive amount of small digested fragments likely interferes in the VIDISCA-ligation
  • It was shown in 2011 that the sensitivity of VIDISCA reaches 1 E6 “viral” genome copies/ml
  • Van Der Hoek states that although this is an improvement, this detection limit might be too low to detect “viruses” directly in clinical samples
  • The concentration of respiratory “viruses” in nasopharyngeal swabs is in the main below 1 E6 copies/ml, and so they assume that a yet unknown “virus” will be present in similar concentrations
  • She admits additional improvement of the VIDISCA-sensitivity is needed
  • The detection of unknown “viral” pathogens in respiratory clinical material is difficult with these sequence independent “virus” discovery methods because of low “viral” load and high background nucleic acids in these samples
  • So far no study has been able to identify novel human respiratory RNA “viruses” with sequence independent amplification techniques
  • Thus sequence independent amplification techniques like VIDISCA have to be optimized to allow discovery without requiring a culture amplification step (in other words, VIDISCA requires culturing of “virus” which means any genome is taken from unpurified non-isolated culture supernatant)
  • Van Der Hoek admits that they also observed the presence of unknown sequences within her data set in this study
  • She speculated that it could be that these sequences are derived from yet unknown “viruses,” or it could be that the sequences are part of a genomic sequence from a known organism, e.g. a bacterium of which not the complete genomic sequence is present in the Genbank databases
  • Thus care should be taken to assign sequences as potentially “viral,” since so many organisms have not been fully sequenced
  • Van Der Hoek reported the identification of a fourth human “coronavirus,” HCoV-NL63, using a new method of “virus” discovery she created called VIDISCA
  • The “virus” was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis
  • The complete genome sequence indicated to her that this “virus” was not a recombinant, but rather a new group 1 “coronavirus”
  • The in vitro host cell range of HCoV-NL63 is notable because it replicates on tertiary monkey kidney cells and the monkey kidney LLC-MK2 cell line
  • To date, there is still a variety of human diseases with unknown etiology
  • A “viral” origin has been suggested for many of these diseases, which emphasized to her the importance of a continuous search for new “viruses”
  • Major difficulties are encountered, however, when searching for new “viruses” such as:
    1. Some “viruses” do not replicate in vitro, at least not in the cells that are commonly used in “viral” diagnostics
    2. For those “viruses” that do replicate in vitro and cause a cytopathic effect (CPE), the subsequent “virus” identification methods may fail
    3. Antibodies raised against known “viruses” may not recognize the cultured “virus”
    4. “Virus-specific” PCR methods may not amplify the new “viral” genome
  • Van Der Hoek claimed this was not an isolated case as she identified the “virus” in clinical specimens from seven additional individuals, both infants and adults, through genomic sequencing
  • The clinical sample (NL63) was inoculated onto human fetal lung fibroblasts, tertiary monkey kidney cells (Cynomolgus monkey) and HeLa cells
  • CPE was detected exclusively on tertiary monkey kidney cells, and was first noted 8 d after inoculation
  • More pronounced CPE was observed upon passage onto the monkey kidney cell line LLC-MK2, with overall cell rounding and moderate cell enlargement
  • Additional subcultures on human fetal lung fibroblasts, rhabdomyosarcoma cells and Vero cells remained negative for CPE
  • Identification of unknown pathogens using molecular biology tools is difficult because the target sequence is not known, so genome-specific PCR primers cannot be designed
  • To overcome this problem, she developed the VIDISCA method based on the cDNA-AFLP technique
  • She claims that the advantage of VIDISCA is that prior knowledge of the sequence is not required, as the presence of restriction enzyme sites is sufficient to guarantee PCR amplification (an assumption)
  • The input sample can be either blood plasma or serum, or culture supernatant
  • The following steps are incorporated:
    1. VIDISCA begins with a treatment to selectively enrich for “viral” nucleic acid, including a centrifugation step to remove residual cells and mitochondria
    2. A DNase treatment is also used to remove interfering chromosomal and mitochondrial DNA from degraded cells (it is assumed “viral” nucleic acid is protected within the “viral” particle)
    3. Finally, by choosing frequently cutting restriction enzymes, the method can be fine-tuned such that most “viruses” will be amplified
  • The supernatant of the CPE-positive LLC-MK2 culture NL63 was analyzed by VIDISCA
  • After the second PCR amplification step, unique and prominent DNA fragments were present in the test sample but not in the control
  • These fragments were cloned and sequenced
  • Thirteen of 16 fragments showed sequence similarity to members of the “coronavirus” family, but significant sequence divergence with known “coronaviruses” was apparent in all fragments, indicating to Van Der Hoek thar she had identified a new “coronavirus”
  • To show that HCoV-NL63 originated from the nasopharyngeal aspirate of the child, she designed a diagnostic RT-PCR that specifically detects HCoV-NL63
  • In other words, Van Der Hoek created a sequence from cell culture supernatant, designed a PCR test for her created sequence, and then used her own test to verify that the created sequence was present in the sample
  • The sequence of the RT-PCR product of the 1b gene was identical to that of the “virus” identified upon in vitro passage in LLC-MK2 cells but sadly the data was not shown
  • Having “confirmed” that the cultured “coronavirus” originated from the child (where else would it have come from?), the question remained as to whether this was an isolated clinical case, or whether HCoV-NL63 is circulating in humans
  • To address this question, she used two diagnostic RT-PCR assays to examine respiratory specimens of hospitalized individuals and those visiting the outpatient clinic between December 2002 and August 2003
  • She identified seven additional individuals carrying HCoV-NL63
  • Sequence analysis of the PCR products indicated the presence of a few characteristic point mutations in several samples, suggesting that several “viruses” with different molecular markers may have been cocirculating
  • One patient was coinfected with respiratory syncytial “virus” and the HIV-infected patient carried Pneumocystis carinii
  • No other respiratory agent was found in the other patients, suggesting that the respiratory symptoms were caused by HCoV-NL63
  • During genome sequencing, specific PCR reactions were designed to fill in gaps and to sequence regions with low-quality sequence data which were combined with 5′ and 3′ rapid amplification of cDNA ends to resolve the complete HCoV-NL63 genome sequence
  • ZCurve software was used to identify the ORFs, and the genome configuration was portrayed using the similarity with known “coronaviruses” as a guide
  • She aligned the sequence of HCoV-NL63 with the complete genomes of other “coronaviruses”
  • All genes except the M gene shared the highest identity with HCoV-229E (this reference sequence was obtained from the infectious HCoV-229E cDNA clone (Inf-1) that is based on the 1973-deposited laboratory-adapted prototype strain of HCoV-229E VR-740)
  • The 1a, 1b and S genes of HCoV-NL63 were most closely related to those of HCoV-229E
  • Phylogenetic analysis could not be performed for the ORF3 and E genes because the regions were too variable or too small for analysis, respectively
  • Bootscan analysis by the Simplot software version 2.5 found no signs of recombination (data not shown again)
  • HCoV-NL63 is considered a member of the group 1 “coronaviruses” and is most closely related to HCoV-229E, but the differences between them are prominent:
    1. They share on average only 65% sequence identity
    2. A single gene, ORF3, in HCoV-NL63 takes the place of the 4A and 4B genes of HCoV-229E
    3. The 5′ region of the S gene of HCoV-NL63 contains a large in-frame insertion of 537 nucleotides
    4. Whereas HCoV-229E is fastidious in cell culture with a narrow host range, HCoV-NL63 replicates efficiently in monkey kidney cells
  • Her data indicated that HCoV-NL63 causes acute respiratory disease in children below the age of 1 year, and in immunocompromised adults
  • Antibodies to HCoV-NL63 might cross-react with HCoV-229E, given that these “viruses” are members of the same serotype
  • If this were the case, HCoV-NL63 infections might have been misdiagnosed as HCoV-229E
  • Molecular biology tools such as RT-PCR assays were designed to selectively detect the human “coronaviruses” HCoV-229E and HCoV-OC43, but these assays will not detect HCoV-NL63
  • Even the RT-PCR assay that was designed to amplify all known “coronaviruses” is not able to amplify HCoV-NL63 because of several mismatches with the primer sequences
  • The results indicated that HCoV-NL63 was present in a significant number of respiratory tract illnesses of unknown etiology (based on finding only 8 cases…)
  • Future experiments with more sensitive diagnostic tools should yield a more accurate picture of the prevalence of this “virus” and its association with respiratory disease
  • In other words, this study is a less accurate picture with less accurate diagnostic tools which did not prove the presence, prevalence, nor association of a new “coronavirus” with respiratory disease
  • The double-stranded DNA was digested with the HinP1I and MseI restriction enzymes
  • Remember, in 2011 Van Der Hoek admitted thar the high frequency of HinP1-I digestion in 28S rRNA and the generation of a massive amount of small digested fragments likely interferes in the VIDISCA-ligation
It’s fitting that all pictures of the in silico NL63 are all CGI.

The use of unproven technologies like VIDISCA as in the case of NL63 highlights the fatal flaws currently dominating virology: the over reliance on molecular tests and data as proof of “virus” discovery. In this paper, Van Der Hoek used her own unproven technique, the VIDISCA method, which she claimed can sequence the genome of an unknown “virus.” The problem is that this sequence did not come from purified/isolated particles. It came directly from unpurified monkey kidney cell culture supernatant that was mixed with the nasopharyngeal aspirate from a 7-month old baby (among other things). Van Der Hoek claimed the discovery of a new “Coronavirus” based solely on the sequence as there were no electron microscope images of any new “Coronavirus,” just letters in a database. She did not present any indirect antibody results to tie this “coronavirus” to the previously discovered ones. She assumed, based on sequencing data, that NL63 was serologically similar to 229E, the genome of which came from a 2001 synthetic cloned copy of a 1973 lab-adapted strain. There were no attempts to prove pathogenicity with any purified/isolated particles assumed to be “virus.” Instead, Van Der Hoek created her own PCR test to look in clinical samples from patients the year prior in order to fabricate the existence of seven previous “cases” of her “virus.”

In essence, all Van Der Hoek did was create her own “discovery” method which was later revealed to be heavily flawed in order to claim the indirect discovery of a new “virus” with nothing physical backing it up, just a bunch of A’s, C’s, T’s, and G’s in a computer database. This paper is the perfect representation of the SCAM called Molecular Virology. Van Der Hoek’s 2021 quote “I have no proof, it is only a hypothesis” should be written in giant bold letters above every single virology paper past and present.

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