Woo “Coronavirus” HKU1 Paper (2005)

In 2005, Patrick Woo claimed the discovery of another new “coronavirus” which he named HKU1. This was the third new human “coronavirus” identified since 2003 after 30 years of apparent hibernation with the discovery of the first two strains in the 1960’s. On top of the human “coronaviruses” popping up at the turn of the century, many animal “coronaviruses” were also making their presence known. The dawn of molecular virology was seemingly bringing about many new “viral discoveries” which were filling up the genomic databases in record speeds. This is how Woo described the situation in a 2009 article:

More and More Coronaviruses: Human Coronavirus HKU1

“Before 2003, there were only 10 coronaviruses with complete genomes available, including only two human coronaviruses, human coronavirus 229E (HCoV-229E) and human coronavirus OC43 (HCoV-OC43). These two human coronaviruses were discovered in the 1960s, with HCoV-229E being a group 1 coronavirus and HCoV-OC43 a group 2 coronavirus [17, 34]. After the SARS epidemic, up to December 2008, 16 novel coronaviruses were discovered and their complete genomes sequenced. Among these 16 previously unrecognized coronaviruses were two more human coronaviruses, human coronavirus NL63 (HCoV-NL63) and human coronavirus HKU1 (HCoV-HKU1) [37,39], ten other mammalian coronaviruses and four avian coronaviruses [7,16,23,24,27,28,33,40,41,44,46]. HCoV-NL63 is a group 1 coronavirus whereas HCoV-HKU1 is a group 2 coronavirus. In just a few years after their discoveries, numerous reports throughout the world had described the presence of HCoV-NL63 and HCoV-HKU1 in patients with respiratory infections in their corresponding countries.” 

“The discovery of SARS coronavirus marked the beginning of the race of coronavirus hunting in humans and animals. In just a few years, the number of “coronavirus” papers found by Medline search has doubled and the number of coronaviruses with complete genomes available has tripled. With this increase in the number of coronaviruses and genomes, we are starting to appreciate the diversity of coronaviruses. Moreover, comprehensive and user-friendly databases for efficient sequence retrieval and the ever-improving bioinformatics tools have further enabled us to improve our understanding of the phylogeny and genomics of coronaviruses [19]. With the increasing number of coronaviruses, more and more closely related coronaviruses from distantly related animals have been observed. The most notable example related to human coronaviruses is the clustering of HCoV-OC43, bovine coronavirus and porcine hemagglutinating encephalomyelitis virus. With more and more coronaviruses discovered, we will be able to understand the origin of the various human coronaviruses, and more importantly, the secret behind their mechanisms of interspecies transmission.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185465/

While we will return to Woo’s 2009 article for a few revelations later, it is apparent from these excerpts that with the emergence and increased use of genomic sequencing technology, an explosion of new “viruses” came about. These once hidden and invisible entities were, well…still invisible…but now able to be “seen” as random letters on a computer screen and stored in large databases. These computer-algorithm generated A,C,T,G’s could now be compared and contrasted with other random letters so that newly revealed “connections” could be made. A fresh indirect method for claiming the existence of these notoriously difficult to find “viruses” was created with the advent of genomic analysis. Whereas in the past, a tissue, organ, and/or cell culture was required in order to claim indirect evidence of a “virus,” now all that was needed was some unpurified nasopharyngeal aspirates and a PCR kit and virologists could poop out a new “virus” genome at will.

Fortunately, we have Patrick Woo’s insight to guide us in regards to this eruption of genomic sequencing. He is the perfect man to know about how to create a “virus” out of thin air using nothing but genomic analysis. He did exactly that in 2005 with HKU1. Presented below are highlights from his study:

Characterization and Complete Genome Sequence of a Novel Coronavirus, Coronavirus HKU1, from Patients with Pneumonia

Abstract

“Despite extensive laboratory investigations in patients with respiratory tract infections, no microbiological cause can be identified in a significant proportion of patients. In the past 3 years, several novel respiratory viruses, including human metapneumovirus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and human coronavirus NL63, were discovered. Here we report the discovery of another novel coronavirus, coronavirus HKU1 (CoV-HKU1), from a 71-year-old man with pneumonia who had just returned from Shenzhen, China. Quantitative reverse transcription-PCR showed that the amount of CoV-HKU1 RNA was 8.5 to 9.6 × 10⁶ copies per ml in his nasopharyngeal aspirates (NPAs) during the first week of the illness and dropped progressively to undetectable levels in subsequent weeks. He developed increasing serum levels of specific antibodies against the recombinant nucleocapsid protein of CoV-HKU1, with immunoglobulin M (IgM) titers of 1:20, 1:40, and 1:80 and IgG titers of <1:1,000, 1:2,000, and 1:8,000 in the first, second and fourth weeks of the illness, respectively. Isolation of the virus by using various cell lines, mixed neuron-glia culture, and intracerebral inoculation of suckling mice was unsuccessful. The complete genome sequence of CoV-HKU1 is a 29,926-nucleotide, polyadenylated RNA, with G+C content of 32%, the lowest among all known coronaviruses with available genome sequence. Phylogenetic analysis reveals that CoV-HKU1 is a new group 2 coronavirus. Screening of 400 NPAs, negative for SARS-CoV, from patients with respiratory illness during the SARS period identified the presence of CoV-HKU1 RNA in an additional specimen, with a viral load of 1.13 × 10⁶ copies per ml, from a 35-year-old woman with pneumonia. Our data support the existence of a novel group 2 coronavirus associated with pneumonia in humans.”

“In this study, we report the discovery of a novel group 2 coronavirus in the nasopharyngeal aspirates (NPAs) of patients with pneumonia. The complete genome of the coronavirus was sequenced and analyzed. Based on the findings of this study, we propose that this new virus be designated coronavirus HKU1 (CoV-HKU1).

MATERIALS AND METHODS

Index patient, clinical specimens, and microbiological tests.

NPAs were collected from the index patient weekly from the first till the fifth week of illness, stool and urine were collected in the first and second weeks, and sera were collected in the first, second, and fourth weeks. The NPAs were assessed by direct antigen detection for influenza A and B viruses, parainfluenza virus types 1, 2, and 3, respiratory syncytial virus, and adenovirus by immunofluorescence (46) and were cultured for conventional respiratory viruses on MDCK (canine kidney), LLC-Mk2 (rhesus monkey kidney), HEp-2 (human epithelial carcinoma), and MRC-5 (human lung fibroblast) cells. In addition, FRhK-4 (rhesus monkey kidney), A-549 (lung epithelial adenocarcinoma), BSC-1 (African green monkey kidney), CaCO2 (human colorectal adenocarcinoma), Huh-7 (human hepatoma), and Vero E6 (African green monkey kidney) cells were added to the routine panel of cell lines. Reverse transcription (RT)-PCR for influenza A virus, human metapneumovirus, and SARS-CoV was performed directly on the NPAs (25). Serological assays for antibodies against Mycoplasma, Chlamydia, Legionella, and SARS-CoV were performed by using SERODIA-MYCO II (Fujirebio Inc., Tokyo, Japan), Chlamydia pneumoniae MIF immunoglobulin G (IgG) (Focus technologies, Cypress, Calif.), indirect immunofluorescence (MRL; San Diego, Calif.), and our recently developed enzyme-linked immunosorbent assay (ELISA), respectively (45).

RNA extraction.

Viral RNA was extracted from the NPA, urine, and fecal specimens by using the QIAamp Viral RNA Mini kit (QIAgen, Hilden, Germany). The RNA pellet was resuspended in 10 μl of DNase-free, RNase-free double-distilled water and was used as the template for RT-PCR.

RT-PCR of the pol gene of coronaviruses, using conserved primers and DNA sequencing.

A 440-bp fragment of the RNA-dependent RNA polymerase (pol) gene of coronaviruses was amplified by RT-PCR with conserved primers (5′-GGTTGGGACTATCCTAAGTGTGA-3′ and 5′-CCATCATCAGATAGAATCATCATA-3′) designed by multiple alignment of the nucleotide sequences of available pol genes of known coronaviruses. RT was performed by using the SuperScript II kit (Invitrogen, San Diego, Calif.). The PCR mixture (50 μl) contained cDNA, PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 3 mM MgCl₂, 0.01% gelatin), 200 μM (each) deoxynucleoside triphosphates, and 1.0 U of Taq polymerase (Boehringer, Mannheim, Germany). The mixtures were amplified in 40 cycles of 94°C for 1 min, 48°C for 1 min, and 72°C for 1 min and a final extension at 72°C for 10 min in an automated thermal cycler (Perkin-Elmer Cetus, Gouda, The Netherlands). The PCR products were gel purified using the QIAquick gel extraction kit (QIAgen, Hilden, Germany). Both strands of the PCR products were sequenced twice with an ABI Prism 3700 DNA analyzer (Applied Biosystems, Foster City, Calif.), using the two PCR primers. The sequences of the PCR products were compared with known sequences of the pol genes of coronaviruses in the GenBank database.

Complete genome sequencing and genome analysis.

The complete genome of CoV-HKU1 was amplified and sequenced by using the RNA extracted from the NPAs as a template. The RNA was converted to cDNA by a combined random-priming and oligo(dT) priming strategy. As the initial results obtained from sequencing the 440-bp fragment revealed that the polymerase (Pol) of CoV-HKU1 is homologous to those of other group 2 coronaviruses, the cDNA was amplified by degenerate primers designed by multiple alignment of the genomes of murine hepatitis virus (MHV) (GenBank accession no. AF201929), HCoV-OC43 (GenBank accession no. NC_005147), bovine coronavirus (BCoV) (GenBank accession no. NC_003045), rat sialodacryoadenitis coronavirus (SDAV) (GenBank accession no. AF207551), equine coronavirus NC99 (ECoV) (GenBank accession no. AY316300), and porcine hemagglutinating encephalomyelitis virus (PHEV) (GenBank accession no. AY078417) and additional primers designed from the results of the first and subsequent rounds of sequencing. These primer sequences are available on request. The 5′ end of the viral genome was confirmed by rapid amplification of cDNA ends using the 5′/3′ rapid amplification of cDNA ends kit (Roche, Mannheim, Germany). Sequences were assembled and manually edited to produce a final sequence of the viral genome. The nucleotide sequence of the genome and the deduced amino acid sequences of the open reading frames (ORFs) were compared to those of other coronaviruses. Phylogenetic tree construction was performed by using the PileUp method with GrowTree (Genetics Computer Group, Inc.). Prediction of signal peptides and their cleavage sites was performed by using SignalP (21). Protein family analysis was performed by using PFAM and InterProScan (1, 2). Prediction of transmembrane domains was performed by using TMpred and TMHMM (11, 32). PHDhtm was also used when there was disagreement between the results obtained by using TMpred and TMHMM (3). Potential N-glycosylation sites were predicted by using ScanProsite (7).

Quantitative RT-PCR.

For real-time quantitative PCR assays, cDNA was amplified in SYBR Green I fluorescence reactions (Roche) (23). Briefly, 20 μl of reaction mixtures containing 2 μl of cDNA, 3.5 mM MgCl₂, and 0.25 M (each) forward and reverse specific primers (5′-GGTTGGGATTATCCTAAATGTGA-3′ and 5′-CCATCATCACTCAAAATCATCATA-3′) were subjected to thermal cycling at 95°C for 10 min followed by 50 cycles of 95°C for 10 s, 55°C for 4 s, and 72°C for 18 s, using a Light cycler (Roche). A plasmid with the target sequence was used to generate the standard curve. At the end of the assay, PCR products (440-bp fragment of pol) were subjected to a melting curve analysis (65 to 95°C, 0.1°C/s) to confirm the specificity of the assay.”

ELISA with recombinant N protein of CoV-HKU1.

“Sera from 100 healthy blood donors were used to set up a baseline for the N protein ELISA-based IgG and IgM antibody tests. The ELISA-based IgG and IgM antibody tests were modified from our previous publication (45). Briefly, each well of a Nunc (Roskilde, Denmark) immunoplate was coated with purified His₆-tagged recombinant N protein (20 ng for IgG and 80 ng for IgM) for 1 h and then blocked in phosphate-buffered saline with 5% skim milk. The serum samples obtained from the patient during the first, second, and fourth weeks of the illness were serially diluted and were added to the wells of the His₆-tagged recombinant N protein-coated plates in a total volume of 100 μl and incubated at 37°C for 2 h. After five washes with washing buffer, 100 μl of diluted horseradish peroxidase-conjugated goat antihuman IgG (1:4,000) and mouse antihuman IgM (1:1,000) antibodies (Zymed Laboratories Inc., South San Francisco, Calif.) was added to the wells and incubated at 37°C for 1 h. After washing with washing buffer five times, 100 μl of diluted 3,3′,5,5′-tetramethylbenzidine (Zymed Laboratories, Inc.) was added to each well and incubated at room temperature for 15 min. One hundred microliters of 0.3 M H₂SO₄ was added, and the absorbance at 450 nm of each well was measured. Each sample was tested in duplicate, and the mean absorbance for each serum was calculated.”

RESULTS

Index patient and microbiological tests.

“A 71-year-old Chinese man was admitted to hospital in January 2004 because of fever and productive cough with purulent sputum for 2 days. He had a history of pulmonary tuberculosis more than 40 years ago complicated by cicatrization of the right upper lobe and bronchiectasis with chronic Pseudomonas aeruginosa colonization of airways. He was a chronic smoker and also had chronic obstructive airway disease, hyperlipidemia, and asymptomatic abdominal aortic aneurysm. He had just returned from Shenzhen, China, 3 days before admission. A chest radiograph showed patchy infiltrates over the left lower zone. NPA for direct antigen detection of respiratory viruses, RT-PCR of influenza A virus, human metapneumovirus, and SARS-CoV, and viral cultures were negative. After the virus was determined to be a coronavirus, the NPAs were inoculated into RD (human rhabdomyosarcoma), I13.35 (murine macrophage), L929 (murine fibroblast), HRT-18 (colorectal adenocarcinoma), and B95a (marmoset B-lymblastoid) cell lines and mixed neuron-glia culture. No cytopathic effect was observed. Quantitative RT-PCR, using the culture supernatants and cell lysates to monitor the presence of viral replication, also showed negative results. Moreover, intracerebrally inoculated suckling mice remained healthy after 14 days. Sputum was negative for bacterial and mycobacterial pathogens. Paired sera for antibodies against Mycoplasma, Chlamydia, Legionella, and SARS-CoV were negative. His symptoms improved, and he was discharged after 5 days of hospitalization.”

“ORF 2 (nucleotide position 21773 to 22933) encodes the predicted HE glycoprotein with 386 amino acids. HE is present in group 2 coronaviruses and influenza C virus. The HE of CoV-HKU1 has 50 to 57% amino acid identities with the HE of other group 2 coronaviruses (Table 1 and Fig. 2). PFAM and InterProScan analysis of the ORF shows that amino acid residues 1 to 349 of the predicted protein constitute a member of the hemagglutinin esterase family (PFAM accession no. PF03996and INTERPRO accession no. IPR007142). Furthermore, PFAM and InterProScan analysis shows that amino acid residues 122 to 236 of the predicted protein constitute the hemagglutinin domain of the HE fusion glycoprotein family (PFAM accession no. PF02710and INTERPRO accession no. IPR003860).”

ORF 3 (nucleotide position 22942 to 27012) encodes the predicted S glycoprotein (PFAM accession no. PF01601) with 1,356 amino acids. The S protein of CoV-HKU1 has 60 to 61% amino acid identities with the S proteins of other group 2 coronaviruses but less than 35% amino acid identities with the S proteins of non-group 2 coronaviruses (Table 1 and Fig. 2). InterProScan analysis predicts it as a type I membrane glycoprotein. Important features of the S protein of CoV-HKU1 are depicted in Fig. 4. PrositeScan of the S protein of CoV-HKU1 revealed 28 potential N-linked glycosylation (12 NXS and 16 NXT) sites. SignalP analysis revealed a signal peptide probability of 0.909, with a cleavage site between residues 13 and 14. By multiple alignments with the S proteins of other group 2 coronaviruses, a potential cleavage site located after RRKRR, between residues 760 and 761, where S will be cleaved into S1 and S2, was identified. Immediately upstream to RRKRR, there is a series of five serine residues that are not present in any other known coronaviruses (Fig. 4). Most of the S protein (residues 15 to 1300) is exposed on the outside of the virus, with a transmembrane domain at the C terminus (TMHMM analysis of the ORF shows one transmembrane domain at positions 1301 to 1356), followed by a cytoplasmic tail rich in cysteine residues. Two heptad repeats, located at residues 982 to 1083 (HR1) and 1250 to 1297 (HR2), identified by multiple alignments with other coronaviruses, are present. The receptor for S protein binding in MHV and HCoV-OC43 are CEACAM1 and sialic acid, respectively (15, 41, 43). While the three conserved regions (sites I, II, and III) and amino acid residues (Thr⁶², Thr²¹², Tyr²¹⁴, and Tyr²¹⁶) in the N-terminal of the MHV S protein important for receptor-binding activity (33) are present in CoV-HKU1 (Fig. 4), the amino acid residues on the S protein of HCoV-OC43 that are important for receptor binding are not well defined. Further experiments should be performed to delineate the receptor for CoV-HKU1.

ORF 4 (nucleotide position 27051 to 27380) encodes a predicted protein with 109 amino acids. This ORF overlaps with the ORF that encodes the E protein. PFAM analysis of the ORF shows that the predicted protein is a member of the coronavirus nonstructural protein NS2 family (PFAM accession no. PF04753). TMpred and TMHMM analysis does not reveal any transmembrane helix. This predicted protein of CoV-HKU1 has 44 to 51% amino acid identities with the corresponding proteins of other group 2 coronaviruses.

ORF 5 (nucleotide position 27373 to 27621) encodes the predicted E protein with 82 amino acids. The E protein of CoV-HKU1 has 54 to 60% amino acid identities with the E proteins of other group 2 coronaviruses but less than 35% amino acid identities with the E proteins of non-group 2 coronaviruses (Table 1 and Fig. 2). PFAM and InterProScan analysis of the ORF shows that the predicted E protein is a member of the nonstructural protein NS3/small envelope protein E family (PFAM accession no. PF02723). SignalP analysis predicts the presence of a transmembrane anchor (probability 0.995). TMpred analysis of the ORF shows two transmembrane domains at positions 16 to 34 and 39 to 59, and TMHMM analysis of the ORF shows two transmembrane domains at positions 10 to 32 and 39 to 58, consistent with the anticipated association of the E protein with the viral envelope.

ORF 6 (nucleotide position 27633 to 28304) encodes the predicted M protein with 223 amino acids. The M protein of CoV-HKU1 has 76 to 84% amino acid identities with the M proteins of other group 2 coronaviruses but less than 40% amino acid identities with the M proteins of non-group 2 coronaviruses (Table 1 and Fig. 2). PFAM analysis of the ORF shows that the predicted M protein is a member of the coronavirus matrix glycoprotein family (PFAM accession no. PF01635). SignalP analysis predicts the presence of a transmembrane anchor (probability, 0.926). TMpred analysis of the ORF shows three transmembrane domains at positions 21 to 42, 53 to 74, and 77 to 98. TMHMM analysis of the ORF shows three transmembrane domains at positions 20 to 39, 46 to 68, and 78 to 100. The N-terminal 19 to 20 amino acids are located on the outside, and the C-terminal 123- to 125-amino-acid hydrophilic domain is located on the inside of the virus.

ORF 7 (nucleotide position 28320 to 29645) encodes the predicted N protein (PFAM accession no. PF00937) with 441 amino acids. The N protein of CoV-HKU1 has 57 to 68% amino acid identities with the N proteins of other group 2 coronaviruses but less than 40% amino acid identities with the N proteins of non-group 2 coronaviruses (Table 1 and Fig. 2).

ORF 8 (nucleotide position 28342 to 28959) encodes a hypothetical protein (N2) of 205 amino acids within the ORF that encodes the predicted N protein. PFAM analysis of the ORF shows that the predicted protein is a member of the coronavirus nucleocapsid I protein family (PFAM accession no. PF03187). This hypothetical N2 protein of CoV-HKU1 has 32 to 39% amino acid identities with the N2 proteins of other group 2 coronaviruses. This protein has been shown to be nonessential for viral replication in MHV (5).”

Purification of His₆-tagged recombinant N protein and Western blot analysis.

“To produce recombinant N protein of CoV-HKU1, the recombinant N protein was expressed in Escherichia coli and subsequently purified. The purified recombinant N protein was separated on sodium dodecyl sulfate-polyacrylamide gels followed by Western blot analysis with serum samples. Several prominent immunoreactive bands were visible for serum samples collected during the second and fourth weeks of the patient’s illness (Fig. 6, lanes 2 and 3). The sizes of the largest bands were about 53 kDa, consistent with the expected size of 52.8 kDa for the full-length His6-tagged recombinant N protein, whereas the other bands were probably its degradation products. Only very faint bands were observed for serum samples obtained from the patient during the first week of the illness (Fig. 6, lane 1) and two healthy blood donors (Fig. 6, lanes 4 and 5).

ELISA using recombinant N protein of CoV-HKU1.

An ELISA-based antibody test was developed with this recombinant N protein for the detection of specific antibodies against this protein. Box titration was carried out with serial dilutions of recombinant N protein coating antigen (in one axis) and serum (in the other axis) obtained from the fourth week of the patient’s illness.”

Screening of NPAs during the SARS period.

“Among the 400 NPAs that were negative for SARS-CoV by RT-PCR, obtained during the SARS period in 2003, one was positive for RNA of CoV-HKU1. The NPA was obtained from a 35-year-old, previously healthy woman with pneumonia of unknown etiology in March 2003, 10 months earlier than the index case. There was no direct relationship or contact between the two cases. The detection of several unique features upon sequencing confirmed the presence of CoV-HKU1. Sequencing of the 2,784-bp fragment that encodes Pol revealed 87 base (3.1%) and seven (0.8%) amino acid differences between the Pol of this virus and that of the virus from the index patient.”

DISCUSSION

“We report the characterization and complete genome sequence of a novel coronavirus detected in the NPAs of patients with pneumonia. The clinical significance of the virus in the index patient was made evident by the high viral loads in the patient’s NPAs during the first week of his illness, which coincided with his acute symptoms. The viral load decreased during the second week of the illness and was undetectable in the third week. In addition, the fall in viral load was accompanied by the recovery from the illness and development of a specific antibody response to the recombinant N protein of the virus. The fact that the present virus could not be recovered from cell cultures could be related to the lack of a susceptible cell line for CoV-HKU1 or the inherently low recovery rate of some coronaviruses. Many decades after the recognition of HCoV-229E and HCoV-OC43, the other non-SARS human respiratory coronaviruses known to cause pneumonia at low frequencies (27, 35, 40), there are still only a few primary virus isolates available, and organ culture is required for primary isolation of HCoV-OC43. In our experience, SARS-CoV can be recovered only from less than 20% of patients with serologically and RT-PCR-documented SARS-CoV pneumonia. After the discovery of CoV-HKU1 in the index patient, we conducted a preliminary study on 400 NPAs that were collected last year during the SARS period. Among these 400 NPAs, CoV-HKU1 was detected in one specimen, with a viral load comparable to that of the index patient. These results suggested that CoV-HKU1 is not only an incidental finding in an isolated patient but a previously unrecognized coronavirus associated with pneumonia.”

“The prevalence of CoV-HKU1 in humans as a cause of respiratory tract infections remains to be determined. HCoV-OC43, HCoV-229E, and probably HCoV-NL63 are endemic in humans. On the other hand, isolation of SARS-CoV-like coronavirus from civet cats and the absence of a resurgent SARS epidemic in 2004 apart from sporadic laboratory-acquired cases imply that SARS-CoV probably originated from animals. For CoV-HKU1, the detection of its existence in the NPAs of two patients almost 1 year apart suggests that it may have been endemic in humans, or alternatively, it may originally have been an animal coronavirus but may have crossed the species barrier in the past few years. In the serological experiments, Western blot analysis revealed that the serum samples of the two healthy blood donors showed some antigen-antibody reaction with the purified N protein of CoV-HKU1 (Fig. 6). It is not known whether these were due to cross-reaction between the N protein of CoV-HKU1 and that of HCoV-OC43, since these two proteins showed 58% amino acid identity, or due to past infections by CoV-HKU1. Further clinical, seroepidemiological, and phylogenetic studies would be required to determine the relative importance of CoV-HKU1 compared to other respiratory tract viruses in causing upper and lower respiratory tract infections, its seroprevalence, and the origin of the virus.”

https://jvi.asm.org/content/79/2/884

In Summary:

• Despite extensive laboratory investigations in patients with respiratory tract infections, no microbiological cause can be identified in a significant proportion of patients
• Woo et al reported the discovery of another novel “coronavirus,” coronavirus HKU1 (CoV-HKU1), from one 71-year-old man with pneumonia who had just returned from Shenzhen, China
• He developed increasing serum levels of “specific” antibodies against the recombinant nucleocapsid protein of CoV-HKU1

Quick Detour:

Ever wonder what is meant by recombinant? Essentially, these are synthetic lab-created proteins that are based off of computer-generated sequences:

“Recombinant DNA molecules are DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.”

https://en.m.wikipedia.org/wiki/Recombinant_DNA

What are recombinant proteins?

“Recombinant proteins are proteins encoded by recombinant DNA that has been cloned in an expression vector that supports expression of the gene and translation of messenger RNA. Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein. Recombinant protein is a manipulated form of native protein, which is generated in various ways in order to increase production of proteins, modify gene sequences, and manufacture useful commercial products.

How are recombinant proteins made?

Recombinant protein production begins at the genetic level, where coding sequence for the protein of interest is first isolated and cloned into an expression plasmid vector. Most recombinant proteins for therapeutic use are from humans but are expressed in microorganisms such as bacteria, yeast, or animal cells in culture.”

https://www.enzolifesciences.com/science-center/technotes/2020/january/why-do-we-need-recombinant-proteins?/

These proteins are not coming from a purified/isolated “virus,” they are coming from sequences in a computer database that were then synthesized in a lab. In other words, showing an antibody rise to synthetic lab-created recombinant nucleocapsid protein means absolutely nothing.

End Detour.

• Isolation of the “virus” by using various cell lines, mixed neuron-glia culture, and intracerebral inoculation of suckling mice was unsuccessful (this should be the end of the study right there…no “virus” = no proof)
• Screening of 400 NPAs, negative for “SARS-CoV,” from patients with respiratory illness during the “SARS” period identified the presence of CoV-HKU1 RNA in an additional specimen (in other words, 1 out of 400 attempts)
• Woo claims his data supports the existence of a novel group 2 “coronavirus” associated with pneumonia in humans (based off of one man with a history of lung problems and a positive PCR test of one woman with pneumonia)
• Nasopharyngeal aspirates (NPAs) were collected from the index patient weekly from the first till the fifth week of illness, stool and urine were collected in the first and second weeks, and sera were collected in the first, second, and fourth weeks
• The NPAs were assessed by direct antigen detection for influenza A and B “viruses,” parainfluenza “virus” types 1, 2, and 3, respiratory syncytial “virus,” and adenovirus by immunofluorescence
• They were cultured for conventional respiratory “viruses” on MDCK (canine kidney), LLC-Mk2 (rhesus monkey kidney), HEp-2 (human epithelial carcinoma), and MRC-5 (human lung fibroblast) cells
• In addition, FRhK-4 (rhesus monkey kidney), A-549 (lung epithelial adenocarcinoma), BSC-1 (African green monkey kidney), CaCO2 (human colorectal adenocarcinoma), Huh-7 (human hepatoma), and Vero E6 (African green monkey kidney) cells were added to the routine panel of cell lines
• “Viral” RNA was extracted from the NPA, urine, and fecal specimens by using the QIAamp “Viral” RNA Mini kit (no attempts to purify/isolate “vitus” particles first)
• A 440-bp fragment of the RNA-dependent RNA polymerase (pol) gene of “coronaviruses” was amplified by RT-PCR with conserved primers designed by multiple alignment of the nucleotide sequences of available pol genes of known “coronaviruses”
• In other words, the conserved primers were designed off of alignments to sequences from previous unpurified and non-isolated “viruses”
• The mixtures were amplified in 40 cycles of 94°C for 1 min, 48°C for 1 min, and 72°C for 1 min and a final extension at 72°C for 10 min in an automated thermal cycler (remember, according to Fauci, anything over 35 cycles is dead nucleotides)
• The sequences of the PCR products were compared with known sequences of the pol genes of “coronaviruses” in the GenBank database (which means the accuracy of HUK1 relies on the accuracy of previous “coronavirus” genomes which all came from unpurified and non-isolated sources and relied on each other for accuracy…see the problem?)
• The complete genome of CoV-HKU1 was amplified and sequenced by using the RNA extracted from the NPAs as a template (which were never purified/isolated so there would be no way to know what the source(s) of the RNA truly is/are)
• Since the initial results obtained from sequencing the 440-bp fragment revealed that the polymerase (Pol) of CoV-HKU1 is homologous to those of other group 2 “coronaviruses,” the cDNA was amplified by degenerate primers designed by multiple alignment of the genomes of:
  1. Murine hepatitis “virus” (MHV)
  2. HCoV-OC43
  3. Bovine “coronavirus” (BCoV)
  4. Rat sialodacryoadenitis “coronavirus” (SDAV)
  5. Equine “coronavirus” NC99 (ECoV)
  6. Porcine hemagglutinating encephalomyelitis “virus” (PHEV)
• Sequences were assembled and manually edited to produce a final sequence of the “viral” genome
• In other words, the genome of HKU1 is a combination of mouse, human, cow, rat, horse, and pig “viral” genomes
• The nucleotide sequence of the genome and the deduced (to derive as a conclusion from something known or assumed; infer) amino acid sequences of the open reading frames (ORFs) were compared to those of other “coronaviruses”
• Prediction of transmembrane domains was performed by using TMpred and TMHM
• PHDhtm was also used when there was disagreement between the results obtained by using TMpred and TMHMM
• Potential N-glycosylation sites were predicted by using ScanProsite
• For quantitative RT-PCR, 20 μl of reaction mixtures containing 2 μl of cDNA, 3.5 mM MgCl2 and 0.25 M (each) forward and reverse specific primers were subjected to thermal cycling at 95°C for 10 min followed by 50 cycles of 95°C for 10 s, 55°C for 4 s, and 72°C for 18 s, using a Light cycler (50 cycles this time…)
• For ELISA antibody testing, each well of a Nunc (Roskilde, Denmark) immunoplate was:
  1. Coated with purified His6-tagged recombinant N protein (20 ng for IgG and 80 ng for IgM) for 1 h and then blocked in phosphate-buffered saline with 5% skim milk
  2. The serum samples obtained from the patient during the first, second, and fourth weeks of the illness were serially diluted and were added to the wells of the His6-tagged recombinant N protein-coated plates in a total volume of 100 μl and incubated at 37°C for 2 h
  3. After five washes with washing buffer, 100 μl of diluted horseradish peroxidase-conjugated goat antihuman IgG (1:4,000) and mouse antihuman IgM (1:1,000) antibodies was added to the wells and incubated at 37°C for 1 h.
  4. After washing with washing buffer five times, 100 μl of diluted 3,3′,5,5′-tetramethylbenzidine was added to each well and incubated at room temperature for 15 min
  5. One hundred microliters of 0.3 M H2SO4 was added, and the absorbance at 450 nm of each well was measured
• In other words, ELISA tests were a giant orgy of human blood mixed with skim milk, synthetic recombinant proteins, phosphate buffered saline, horseradish peroxidase-conjugated goat antihuman IgG, mouse antihuman IgM, and various other chemicals and these Frankenstein results are supposed to mean something
• The HUK1 sequence came from a 71-year-old Chinese man who was admitted to a hospital in January 2004 because of fever and productive cough with purulent sputum for 2 days
• He had a history of pulmonary tuberculosis more than 40 years ago complicated by cicatrization of the right upper lobe and bronchiectasis with chronic Pseudomonas aeruginosa colonization of airways
• He was a chronic smoker and also had chronic obstructive airway disease, hyperlipidemia, and asymptomatic abdominal aortic aneurysm (obviously it was an unknown “virus” causing disease this time, not the years of horrible health choices and chronic disease)
• After the “virus” was determined to be a “coronavirus,” (Woo fails to mention how this was determined) the NPAs were inoculated into RD (human rhabdomyosarcoma), I13.35 (murine macrophage), L929 (murine fibroblast), HRT-18 (colorectal adenocarcinoma), and B95a (marmoset B-lymblastoid) cell lines and mixed neuron-glia culture yet no cytopathic effect was observed
• Quantitative RT-PCR, using the culture supernatants and cell lysates to monitor the presence of “viral” replication, also showed negative results
• Intracerebrally inoculated suckling mice remained healthy after 14 days

Quick Detour:

I’ve edited out much of the fictional genomic analysis and comparisons so feel free to read through those in the linked study but I did include some excerpts, specifically regarding predicted and hypothetical proteins.

Predicted Proteins

Predicted proteins structures is defined as “the prediction of the three-dimensional structure of a protein from its amino acid sequence—that is, the prediction of its secondary and tertiary structure from primary structure”

https://www.biologicscorp.com/blog/protein-structure-prediction-methods-introduction/#.Ybos5upMF-E

This is all done computationally in a few ways:

“The problem of protein structure prediction has been approached through three main routes: 1) computer simulation based on empirical energy calculations, 2) knowledge based approaches using information derived from structure-sequence relationships from experimentally determined protein 3-D structures; and 3) hierarchical methods. Each approach has its merits and limitations.”

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/protein-structure-prediction

Hypothetical Proteins

As for hypothetical proteins, they are “those that are predicted to be expressed in an organism, but no evidence of their existence is known.”

https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-018-2554-y

Let’s see how many predicted and hypothetical proteins make up HUK1:

1. ORF 2 (nucleotide position 21773 to 22933) encodes the predicted HE glycoprotein with 386 amino acids.
   • PFAM and InterProScan analysis of the ORF shows that amino acid residues 1 to 349 of the predicted protein constitute a member of the hemagglutinin esterase family
   • Furthermore, PFAM and InterProScan analysis shows that amino acid residues 122 to 236 of the predicted protein constitute the hemagglutinin domain of the HE fusion glycoprotein family
2. ORF 3 (nucleotide position 22942 to 27012) encodes the predicted S glycoprotein with 1,356 amino acids
   • The S protein of CoV-HKU1 has 60 to 61% amino acid identities with the S proteins of other group 2 “coronaviruses” but less than 35% amino acid identities with the S proteins of non-group 2 “coronaviruses”
   • InterProScan analysis predicts it as a type I membrane glycoprotein
   • By multiple alignments with the S proteins of other group 2 “coronaviruses,” a potential cleavage site located after RRKRR, between residues 760 and 761, where S will be cleaved into S1 and S2, was identified
   • Two heptad repeats, located at residues 982 to 1083 (HR1) and 1250 to 1297 (HR2), identified by multiple alignments with other “coronaviruses,” were present
   • Woo claimed that further experiments should be performed to delineate the receptor for CoV-HKU1 (which experiments that would be exactly, who knows…?)
3. ORF 4 (nucleotide position 27051 to 27380) encodes a predicted protein with 109 amino acids
   • This ORF overlaps with the ORF that encodes the E protein
   • PFAM analysis of the ORF shows that the predicted protein is a member of the “coronavirus” nonstructural protein NS2 family
   • This predicted protein of CoV-HKU1 has 44 to 51% amino acid identities with the corresponding proteins of other group 2 “coronaviruses”
4. ORF 5 (nucleotide position 27373 to 27621) encodes the predicted E protein with 82 amino acids
   • The E protein of CoV-HKU1 has 54 to 60% amino acid identities with the E proteins of other group 2 “coronaviruses” but less than 35% amino acid identities with the E proteins of non-group 2 “coronaviruses”
   • PFAM and InterProScan analysis of the ORF shows that the predicted E protein is a member of the nonstructural protein NS3/small envelope protein E family
   • SignalP analysis predicts the presence of a transmembrane anchor (probability 0.995)
5. ORF 6 (nucleotide position 27633 to 28304) encodes the predicted M protein with 223 amino acids
   • The M protein of CoV-HKU1 has 76 to 84% amino acid identities with the M proteins of other group 2 “coronaviruses” but less than 40% amino acid identities with the M proteins of non-group 2 “coronaviruses”
   • PFAM analysis of the ORF shows that the predicted M protein is a member of the “coronavirus” matrix glycoprotein family
   • SignalP analysis predicts the presence of a transmembrane anchor (probability, 0.926)
6. ORF 7 (nucleotide position 28320 to 29645) encodes the predicted N protein with 441 amino acids
   • The N protein of CoV-HKU1 has 57 to 68% amino acid identities with the N proteins of other group 2 “coronaviruses” but less than 40% amino acid identities with the N proteins of non-group 2 “coronaviruses”
7. ORF 8 (nucleotide position 28342 to 28959) encodes a hypothetical protein (N2) of 205 amino acids within the ORF that encodes the predicted N protein
   • PFAM analysis of the ORF shows that the predicted protein is a member of the “coronavirus” nucleocapsid I protein family
   • This hypothetical N2 protein of CoV-HKU1 has 32 to 39% amino acid identities with the N2 proteins of other group 2 “coronaviruses.”
   • This (hypothetical) protein has been shown to be nonessential for “viral” replication in MHV

Looks like we have at least 6 predicted proteins with one hypothetical protein contained within a predicted protein. Seriously, you can’t make this stuff up…oh…no wait, that’s exactly what they did.

End Detour.

• To produce recombinant N protein of CoV-HKU1 for western blot analysis, the recombinant N protein was expressed in Escherichia coli and subsequently “purified”
• Very faint bands were observed for serum samples obtained from the patient during the first week of the illness and two healthy blood donors
• An ELISA-based antibody test was developed with this recombinant N protein for the detection of specific antibodies against this protein
• In other words, Woo created a synthetic protein said to belong to a “virus” and then used that synthetic protein to create an antibody test in order to detect it
• Among 400 NPAs that were negative for “SARS-CoV” by RT-PCR, one was positive for RNA of CoV-HKU1
• The NPA was obtained from a 35-year-old, previously healthy woman with pneumonia of unknown etiology in March 2003, 10 months earlier than the index case
• Woo claimed that the detection of several unique features upon sequencing confirmed the presence of CoV-HKU1
• Sequencing of the 2,784-bp fragment that encodes Pol revealed 87 base (3.1%) and seven (0.8%) amino acid differences between the Pol of this “virus” and that of the “virus” from the index patient
• Woo stated that the fall in “viral” load in the index patient was accompanied by the recovery from the illness and development of a specific antibody response to the recombinant N protein of the “virus”
• He felt the fact that the present “virus” could not be recovered from cell cultures could be related to the lack of a susceptible cell line for CoV-HKU1 or the inherently low recovery rate of some “coronaviruses”
• In his experience, “SARS-CoV” can be recovered only from less than 20% of patients with serologically and RT-PCR-documented “SARS-CoV” pneumonia
• Based on the results of only 2 patients, Woo felt that this suggested that CoV-HKU1 was not only an incidental finding in an isolated patient but a previously unrecognized “coronavirus” associated with pneumonia
• The prevalence of CoV-HKU1 in humans as a cause of respiratory tract infections remains to be determined
• For CoV-HKU1, the detection of its existence in the NPAs of two patients almost 1 year apart suggested to Woo that it may have been endemic in humans, or alternatively, it may originally have been an animal “coronavirus” but may have crossed the species barrier in the past few years
• In the serological experiments, Western blot analysis revealed that the serum samples of the two healthy blood donors showed some antigen-antibody reaction with the “purified” (recombinant) N protein of CoV-HKU1
• It was not known whether these were due to cross-reaction between the N protein of CoV-HKU1 and that of HCoV-OC43, since these two proteins showed 58% amino acid identity, or due to past infections by CoV-HKU1
• Further clinical, seroepidemiological, and phylogenetic studies would be required to determine the relative importance of CoV-HKU1 compared to other respiratory tract “viruses” in causing upper and lower respiratory tract infections, its seroprevalence, and the origin of the “virus”

Here is the list of evidence Partick Woo used in order to claim the existence of HKU1 in 2005:

1. One genome sequence from the unpurified nasopharyngeal aspirate of a 71-year-old man

Here is the list of evidence Patrick Woo did not have for the existence of HKU1 in 2005:

1. No cultured “virus” even in numerous attempted cell lines
2. No proof of pathogenicity even after intracranial inoculation in suckling mice
3. No specific antibodies
4. No evidence of transmissibility
5. No evidence for disease prevalence
6. No large sample size

Referring back to Patrick Woo’s 2009 article from the beginning, we can see how his evidence changed in the passing four years:

“After human coronaviruses OC43, 229E and NL63, human coronavirus HKU1 (HCoV-HKU1) is the fourth human coronavirus discovered. HCoV-HKU1 is a group 2a coronavirus that is still not cultivable.”

“Using HCoV-HKU1 S pseudotyped virus, human alveolar epithelial A549 cells were shown to be the most susceptible cell line [4]. Using an A549 cDNA expression library transduced into the non-permissive, baby hamster kidney cell line BHK-21 for detecting proteins that bind HCoV-HKU1 S_1-600 glycoprotein, independent clones with inserts encoding HLA-C were fished out. Further experiments also suggested that HLA-C is involved in the attachment of HCoV-HKU1 to A549 cells and is a potential candidate to facilitate cell entry [4].”

“In our studies on animal coronaviruses, no HCoV-HKU1 was detected in screening more than 10,000 animal specimens in a variety of mammalian and avian species [23, 40, 41, 44]. The dn/ds ratios of all ORFs in the genomes of HCoV-HKU1 are low. Therefore, HCoV-HKU1 is stably evolving in humans, probably the only known reservoir. Similar to other respiratory viruses, HCoV-HKU1 is presumably transmitted through exchange of respiratory secretions.”

“The seroprevalence of HCoV-HKU1 antibody varied widely in different studies that used different antigens and methodologies for antibody detection. In a recent study on seroepidemiology of HCoV-HKU1 using Escherichia coli BL21 expressed recombinant S-based enzyme-linked immunosorbent assay (ELISA) and line immunoassay, it was observed that the seroprevalence of HCoV-HKU1 antibody increased from 0% in patients who were <10 years old to a plateau of 22% in patients who were 31 to 40 years old.”

“Similar to other human coronaviruses, HCoV-HKU1 is associated with both upper and lower respiratory tract infections. Respiratory tract infections associated with HCoV-HKU1 are indistinguishable from those associated with other respiratory viruses.”

“For antibody detection, E. coli BL21- and baculovirus-expressed recombinant N of HCoV-HKU1 has been used for IgG and IgM detection in sera of patients and normal individuals, using Western blot and ELISA.”

“Although HCoV-HKU1 is still not cultivable, a neutralization antibody test was also developed recently using HCoV-HKU1 pseudotyped virus [5].”

In 2009, Patrick Woo’s evidence consisted of a still uncultivable “virus” that relied on artificial lab-created pseudotyped “viruses” (replication-defective “viral” particles formed with a structural and enzymatic core from one “virus” and the envelope glycoprotein of another) to glean information about his nonexistent “virus.” The serological results based on synthetic recombinant proteins varied widely. In over 10,000 animal samples, he could not find one species harboring his “coronavirus” which told him that humans were the primary reservoir. Woo presumed that his “virus” travelled through respiratory secretions like other “viruses” and associated it with upper and lower respiratory disease even though symptomatically, it was indistinguishable from other respiratory “viruses.” In other words, 2009 Woo had as much evidence as 2005 Woo…nada.

Even in 2021, researchers still admit not much is known about HKU1:

“There is no viral sequences relating HCoV-HKU1 to other animal species, save the relationship it has with rodent-associated viruses (Wang et al. 2015; Woo et al. 2005). Just like HCoV-NL63, the mechanism of transmission of HCoV-HKU1 to humans is not documented with no zoonotic reservoir so far identified.”

http://www.scielo.org.za/scielo.php?script=sci_arttext&pid=S0030-24652021000100003

No CPE observed, no “virus” was isolated from numerous cell and mixed neuron-glia cultures, quantitative RT-PCR results for “viral” replication were negative, the intracerebrally inoculated suckling mice remained healthy, no EM images were obtained, there remained an unknown mechanism for human transmission, and the sample size was one 71 year old man with a history of respiratory disease.

Where is the evidence of a new “coronavirus” again?