Creating the “Coronavirus” OC43 Genome

In 1967, it was claimed a new “Coronavirus” labelled OC43 was discovered and “isolated.” This was supposedly done through tissue cultures using tracheas from 5-9 month old aborted fetuses which were collected and stored in Hanks Salt Solution along with 10% fetal bovine serum as well as penicillin and streptomycin. Frgaments were partially covered in Leibovitz Medium, bovine albumin, glutamine, penicillin, and streptomycin. This cultured goo was later “transferred” to suckling mice brain cultures. This is all detailed here:

1967 OC43 Preliminary Paper:

https://viroliegy.com/2021/12/09/mcintosh-preliminary-coronavirus-oc43-paper-1967/

1967 OC43 Discovery Paper:

https://viroliegy.com/2021/12/10/mcintosh-coronavirus-oc43-discovery-paper-1967/

As genomic sequencing was not around when this “isolation” was done in the 1960’s, it wasn’t until 2005 that a genome for OC43 came into existence. To achieve this, the researchers used genomes for the bovine “Coronavirus” and the MHV mice “virus” as reference genomes due to their supposed genetic similarity to OC43. However, knowing that OC43 was never properly purified nor isolated and was mixed with bovine and mice DNA, one has to wonder about these similarities. Could it be that this genetic connection between the animal and human “viruses” have had something to do with the bovine albumin used to cover the OC43 tissues in as well as the fetal bovine serum used as a storage solution? Or maybe these similarities could be due to passaging this mixture of human/bovine goo numerous times into suckling mice brain cultures? These would be reasonable assumptions to make. Yet according to the study which sequenced the OC43 genome, the use of cow and mice blood/tissues had nothing to do with these genetic similarities. These came about due to a theoretical leap from animal “virus” to man based on the entirely hypothetical and assumption-filled Molecular Clock theory. Highlights and summary below:

Complete Genomic Sequence of Human Coronavirus OC43: Molecular Clock Analysis Suggests a Relatively Recent Zoonotic Coronavirus Transmission Event

ABSTRACT

“Coronaviruses are enveloped, positive-stranded RNA viruses with a genome of approximately 30 kb. Based on genetic similarities, coronaviruses are classified into three groups. Two group 2 coronaviruses, human coronavirus OC43 (HCoV-OC43) and bovine coronavirus (BCoV), show remarkable antigenic and genetic similarities. In this study, we report the first complete genome sequence (30,738 nucleotides) of the prototype HCoV-OC43 strain (ATCC VR759). Complete genome and open reading frame (ORF) analyses were performed in comparison to the BCoV genome. In the region between the spike and membrane protein genes, a 290-nucleotide deletion is present, corresponding to the absence of BCoV ORFs ns4.9 and ns4.8. Nucleotide and amino acid similarity percentages were determined for the major HCoV-OC43 ORFs and for those of other group 2 coronaviruses. The highest degree of similarity is demonstrated between HCoV-OC43 and BCoV in all ORFs with the exception of the E gene. Molecular clock analysis of the spike gene sequences of BCoV and HCoV-OC43 suggests a relatively recent zoonotic transmission event and dates their most recent common ancestor to around 1890. An evolutionary rate in the order of 4 × 10⁻⁴ nucleotide changes per site per year was estimated. This is the first animal-human zoonotic pair of coronaviruses that can be analyzed in order to gain insights into the processes of adaptation of a nonhuman coronavirus to a human host, which is important for understanding the interspecies transmission events that led to the origin of the severe acute respiratory syndrome outbreak.”

“Before the 2002-to-2003 severe acute respiratory syndrome (SARS) epidemic, coronaviruses were somewhat neglected in human medicine, but they have always been of considerable importance in animal health. Coronaviruses infect a variety of livestock, poultry, and companion animals, in whom they can cause serious and often fatal respiratory, enteric, cardiovascular, and neurologic diseases (25). Most of our understanding about the molecular pathogenic properties of coronaviruses has been achieved by the veterinary virology community.

HCoV-OC43 (ICTVdb code 19.0.1.0.006) and HCoV-229E (ICTVdb code 19.0.1.0.005) were isolated in 1967 from volunteers at the Common Cold Unit in Salisbury, United Kingdom. HCoV-OC43 was initially propagated on ciliated human embryonic tracheal and nasal organ cultures (42). HCoV-OC43 and HCoV-229E are responsible for 10 to 30% of all common colds, and infections occur mainly during the winter and early spring (38).”

“HCoV-OC43 and BCoV (ICTVdb code 03.019.0.01.002) show remarkable antigenic and genetic similarities (23, 29, 36, 44, 52, 63, 65). They both have hemagglutinating activity by attaching to the N-acetyl-9-O-acetylneuraminic acid moiety on red blood cells (33). BCoV causes severe diarrhea in newborn calves. The complete nucleotide sequences of different BCoV strains are known, but only fragments of the HCoV-OC43 genome had been determined previously. In this paper, we report the complete HCoV-OC43 sequence (30,738 bases) and the comparative characterization and evolutionary relationship of the BCoV-HCoV-OC43 pair. This is the first animal-human zoonotic pair of coronaviruses that can be analyzed in order to gain insights into the processes of adaptation of a nonhuman coronavirus to a human host.

MATERIALS AND METHODS

Preparation of HCoV-OC43 RNA. 

An HCoV-OC43 strain (VR759) was obtained from the American Type Culture Collection (ATCC). The ATCC VR759 strain originated from a volunteer with a common cold-like illness at the Common Cold Unit in Salisbury, United Kingdom (42). HCoV-OC43 was propagated in a human rhabdomyosarcoma (RD) cell line, obtained from the European Collection of Cell Cultures (ECACC). The supernatant was harvested after 7 days of incubation at 33°C, and RNA was isolated by using the QIAamp viral RNA kit (QIAGEN, Westburg, The Netherlands). A real-time quantitative reverse transcription PCR (RT-PCR) (Taqman; Perkin-Elmer Applied Biosystems, Foster City, Calif.) was developed to determine the number of RNA copies present in the supernatant.

Sequencing of the HCoV-OC43 genome. 

To determine the HCoV-OC43 genomic sequence, a set of overlapping RT-PCR products (average size, 1.5 kb) encompassing the entire genome was generated. For both RT-PCR and sequencing, oligonucleotide primers were designed in regions that were conserved between the BCoV and MHV genomes. The forward PCR primer in the 5′-terminal sequence (OC43F1 [5′-GATTGTGAGCGATTTGC-3′]) was based on the HCoV-OC43 5′ untranslated region partial sequence (H. Y. Wu, J. S. Guy, D. Yoo, R. Vlasak, and D. A. Brian, unpublished data; GenBank accession number AF523847). To generate RT-PCR products containing the exact 3′-terminal sequence, we used oligonucleotide OC43R74 (5′-TTTTTTTTTTGTGATTCTTCCA-3′) based on the conserved 3′-end sequence of all known group 2 coronaviruses. By using 150 sequencing primers, sequencing in both directions was performed on an ABI Prism 3100 genetic analyzer (Perkin-Elmer Applied Biosystems) using the BigDye terminator cycle sequencing kit (version 3.1). Chromatogram sequencing files were inspected with Chromas 2.2 (Technelysium, Helensvale, Australia), and contigs were prepared by using SeqMan II (DNASTAR, Madison, Wis.).

DNA and protein sequence analyses. 

ORF analysis was performed by using the NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Potential 3C-like protease cleavage sites were identified by using the NetCorona 1.0 server (30). DNA and protein similarity searches were performed using the NCBI WWW-BLAST (basic local alignment search tool) server on the GenBank DNA database, release 118.0 (2). Pairwise nucleotide and protein sequence alignments were performed by using FASTA algorithms in the ALIGN program on the GENESTREAM network server (http://www2.igh.cnrs.fr) at the Institut de Génétique Humaine in Montpellier, France (47). Maizel-Lenk dot matrix plots were calculated using the pairwise FLAG 1.0 (fast local alignment for gigabases) algorithm at the server of the Biomedical Engineering Center of the Industrial Technology Research Institute in Hsinchu City, Taiwan (http://bioinformatics.itri.org.tw/prflag/prflag.php). Multiple sequence alignments were prepared by using CLUSTALW (58) and CLUSTALX, version 1.82 (59) and were manually edited in GENEDOC (46). Phylogenetic analyses were conducted by using MEGA, version 2.1 (34).

Evolutionary rate analyses and timing of the most recent common ancestor. 

The relationship between isolation date and genetic divergence was investigated using a linear regression, based on a maximum-likelihood tree, as implemented in the Path-O-Gen software, kindly provided by Andrew Rambaut (University of Oxford, Oxford, United Kingdom). Evolutionary rates and divergence times were estimated by using maximum likelihood in the TipDate software package, version 1.2 (49), and Bayesian inference in BEAST, version 1.03 (kindly made available by A. J. Drummond and A. Rambaut, University of Oxford; http://evolve.zoo.ox.ac.uk/beast/). The molecular clock hypothesis was tested by using the likelihood ratio test.”

HCoV-OC43 sequence similarity to other group 2 coronaviruses. 

“The sequence similarity among HCoV-OC43, BCoV, CRCoV, PHEV, ECoV, MHV, and SDAV was investigated by pairwise alignments of the corresponding ORFs and their proteins (Table (Table2).2). HCoV-OC43 showed the highest percentage of similarity to BCoV in all ORFs except for the HCoV-OC43 E gene, which showed 99.6% identity on the nucleotide level and 98.8% identity on the protein level to the PHEV E gene. Maizel-Lenk dot matrix plots illustrate the similarity between HCoV-OC43 and BCoV (Fig. (Fig.33).”

“Based on the nucleotide sequence coding for the spike protein, a maximum-likelihood phylogenetic tree was constructed for HCoV-OC43 and several BCoV strains for which the date of isolation was known (Table (Table3;3; Fig. Fig.5).5). HEC4408, a coronavirus isolated in 1988 from a child with acute diarrhea, was also included in the analysis and has actually been shown to be a BCoV (66). The time to the most recent common ancestor (TMRCA) of HCoV-OC43 and BCoV was dated by three methods (Fig. (Fig.6).6). Linear regression of root-to-tip divergence versus sampling time situates the TMRCA of HCoV-OC43 and BCoV in 1891. The maximum-likelihood estimate for TMRCA is 1873, with a 95% confidence interval of 1815 to 1899. The Bayesian coalescent approach dates TMRCA around 1890 (95% highest posterior density interval, 1859 to 1912). This estimate was highly consistent under different demographic models, including an exponential-growth model, which resulted in a TMRCA around 1893 (95% confidence interval, 1866 to 1918). The evolutionary rate of BCoV was also calculated by these three methods (Table (Table4).4). A maximum-likelihood evolutionary rate of 4.3 × 10⁻⁴ substitutions per site per year was estimated (95% confidence interval, 2.7 × 10⁻⁴ to 6.0 × 10⁻⁴). A likelihood ratio test indicated that the molecular clock hypothesis could not be rejected (P = 0.10).

DISCUSSION

“We report in this paper the first complete genome sequence (30,738 nucleotides) of the prototype HCoV-OC43 strain (VR759). Until now, only partial sequence fragments of the structural protein genes of HCoV-OC43 were available in GenBank, leaving the greater 5′ part of the genome to be determined. The recent discovery of a new human coronavirus, SARS-CoV, necessitates a better understanding of the genomic structure and evolution of other known coronaviruses in order to gain insights in how this new type could have emerged.

The prototype HCoV-OC43 strain (ATCC VR759) is a laboratory strain that, since its isolation in 1967, has been passaged 7 times in human embryonic tracheal organ culture, followed by 15 passages in suckling mouse brain cells and an unknown number of passages in human rectal tumor HRT-18 cells and/or Vero cells. During the passage history, it is likely that a number of mutations have accumulated. It would be interesting to analyze the complete nucleotide sequence of contemporary HCoV-OC43 strains that are free from in vitro expansion mutations.

Nucleotide and amino acid similarity percentages were determined for the major HCoV-OC43 ORFs and those of other group 2 coronaviruses (BCoV, CRCoV, PHEV, ECoV, MHV, and SDAV). For all HCoV-OC43 ORFs, the highest similarity demonstrated was that to the corresponding BCoV ORFs, except for the HCoV-OC43 E gene, which showed 99.6% identity on the nucleotide level and 98.8% identity on the amino acid level with the PHEV E gene. Based on the high similarity between HCoV-OC43 and PHEV in E, and between HCoV-OC43 and BCoV in all the other major ORFs, some hypotheses concerning the origin of HCoV-OC43 can be put forward. Adaptation of BCoV to a human host and a recombination event between BCoV and PHEV leading to a new type of coronavirus with a different species specificity could both have been responsible for the emergence of a new human coronavirus.

Phylogenetic analysis of coronavirus ORF1b replicase protein sequences confirms the presence of three coronavirus group clusters and a separate branch for SARS-CoV, which seems to be most closely related to group 2 coronaviruses (21, 54). HCoV-OC43 and BCoV cluster together, demonstrating the close relationship between the two viruses. There is in fact more divergence between the different MHV strains or between the different IBV strains than between HCoV-OC43 and BCoV. The close relationship between HCoV-OC43 and BCoV on the genetic level has also been shown to correspond to a close antigenic relationship: by using monoclonal antibodies directed against the BCoV S protein, common antigenic determinants for BCoV, HCoV-OC43, and PHEV have been demonstrated (62, 63). A phylogenetic tree was also constructed for the spike gene of HCoV-OC43 and several BCoV isolates for which the date of isolation could be traced. Different molecular clock calculations situate the most recent common ancestor of HCoV-OC43 and the different BCoV isolates around 1890. We suggest that around 1890, BCoV might have jumped the species barrier and became able to infect humans, resulting in the emergence of a new type of human coronavirus (HCoV-OC43), a scenario similar to the origin of the SARS outbreak. Indisputable evidence for the bovine-to-human direction of the interspecies transmission event, instead of a human-to-bovine direction, is not available. However, we consider the occurrence of a 290-nucleotide deletion (corresponding to the absence of BCoV ns4.9 and ns4.8) in HCoV-OC43 relative to the BCoV genome to be a potential supporting argument, as this additional sequence fragment in BCoV is also present in MHV and SDAV. Consequently, we assume that a deletion from BCoV to HCoV-OC43 rather than an insertion in the opposite direction took place during evolution, and thus, we hypothesize that the interspecies transmission event occurred from bovines to humans.

Nevertheless, it is possible that two other group 2 coronaviruses, CRCoV and PHEV, might have played a role in the emergence of HCoV-OC43. CRCoV appears to be very closely related to BCoV and HCoV-OC43 (16), and for the HCoV-OC43 E gene, the highest percentage of similarity was found with the PHEV E gene, suggesting a possible recombination event. To elucidate the evolutionary relationship of HCoV-OC43 and BCoV with CRCoV and PHEV, complete genome sequence data of CRCoV and PHEV would be required. Molecular dating has frequently been used to investigate the origin of viral epidemics (31, 40, 48). The reliability of such an analysis is dependent on the validity of the molecular clock hypothesis, which assumes that the evolutionary rate is roughly constant in the lineages of a phylogenetic tree. Although this assumption is frequently violated for viral sequence data (28), a molecular clock test indicated that this hypothesis could not be rejected for the coronavirus data set investigated here.

Interestingly, around the period in which the BCoV interspecies transmission would probably have taken place, a human epidemic ascribed to influenza was spreading around the world. The 1889-1890 pandemic probably originated in Central Asia (3) and was characterized by malaise, fever, and pronounced central nervous system symptoms (53). A significant increase in case fatality with increasing age was observed. Absolute evidence that an influenza virus was the causative agent of this epidemic was never obtained, due to the lack of tissue samples from that period. However, postepidemic analysis in 1957 of the influenza antibody pattern in sera of people who were 50 to 100 years old indicated that H2N2 influenza antibodies might have originated from the 1889-1890 pandemic (45). However, it is tempting to speculate about an alternative hypothesis, that the 1889-1890 pandemic may have been the result of interspecies transmission of bovine coronaviruses to humans, resulting in the subsequent emergence of HCoV-OC43. The dating of the most recent common ancestor of BCoV and HCoV-OC43 to around 1890 is one argument. Another argument is the fact that central nervous system symptoms were more pronounced during the 1889-1890 epidemic than in other influenza outbreaks. It has been shown that HCoV-OC43 has neurotropism and can be neuroinvasive (4).

Maximum-likelihood phylogenetic analysis of the spike gene of HCoV-OC43 and several BCoV strains for which the date of isolation is known indicates that these strains evolved according to a molecular clock. An evolutionary rate on the order of 4 × 10⁻⁴ nucleotide change per site per year was estimated, and this rate was highly consistent across the different methods used. This rate falls within the range reported for other RNA viruses, including SARS-CoV (14, 50, 51).

This study provides evidence for viral promiscuity, a phenomenon that has already been reported for several animal coronaviruses, including BCoV, for which the potential to infect other species, including humans, has already been described (26, 66). The isolation of the SARS-CoV from masked palm civets and raccoon dogs indicates that this new type of coronavirus was also enzootic in an animal species before suddenly emerging as a virulent virus for humans. The characterization of the BCoV-HCoV-OC43 pair presented in this study provides insights into the process of adaptation of a nonhuman coronavirus to a human host, which is important for understanding the interspecies transmission events that led to the origin of the SARS outbreak.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC544107/

In Summary:

• Based on genetic similarities, “coronaviruses” are classified into three groups
• Two group 2 “coronaviruses,” human “coronavirus” OC43 (HCoV-OC43) and bovine “coronavirus” (BCoV), show remarkable antigenic and genetic similarities
• In this study, the researchers report the first complete genome sequence (30,738 nucleotides) of the prototype HCoV-OC43 strain (ATCC VR759)
• Complete genome and open reading frame (ORF) analyses were performed in comparison to the BCoV genome
• The highest degree of similarity is demonstrated between HCoV-OC43 and BCoV in all ORFs with the exception of the E gene
• Molecular clock analysis of the spike gene sequences of BCoV and HCoV-OC43 suggests a relatively recent zoonotic transmission event and dates their most recent common ancestor to around 1890
• Most of our understanding about the molecular pathogenic properties of “coronaviruses” has been achieved by the veterinary virology community
• HCoV-OC43 was initially propagated on ciliated human embryonic tracheal and nasal organ cultures
• HCoV-OC43 and BCoV show remarkable antigenic and genetic similarities
• The complete nucleotide sequences of different BCoV strains are known, but only fragments of the HCoV-OC43 genome had been determined previously
• This is the first animal-human zoonotic pair of “coronaviruses” that can be analyzed in order to gain insights into the processes of adaptation of a nonhuman “coronavirus” to a human host
• HCoV-OC43 was propagated in a human rhabdomyosarcoma (RD) cell line, obtained from the European Collection of Cell Cultures (ECACC)
• Quick side note: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence and the RD cell line used for the OC43 genome was derived directly from biopsy specimens of a 7-year-old female with a pelvic RMS previously treated with cyclophosphamide and radiation and found to have refractory disease
• It is grown in Eagle’s Medium with 10% fetal bovine serum https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3713458/
• The supernatant was harvested after 7 days of incubation at 33°C, and RNA was isolated by using the QIAamp “viral” RNA kit
• A real-time quantitative reverse transcription PCR (RT-PCR) was developed to determine the number of RNA copies present in the supernatant
• For both RT-PCR and sequencing, oligonucleotide primers were designed in regions that were conserved between the BCoV (cow) and MHV (mice) genomes
• The forward PCR primer in the 5′-terminal sequence was based on the HCoV-OC43 5′ untranslated region partial sequence (H. Y. Wu, J. S. Guy, D. Yoo, R. Vlasak, and D. A. Brian, unpublished data)
• To generate RT-PCR products containing the exact 3′-terminal sequence, they used oligonucleotide OC43R74 based on the conserved 3′-end sequence of all known group 2 “coronaviruses”
• In other words, the researchers assumed a “virus” was in the sample, cultured it in human cancer cells, extracted RNA from the unpurified cell culture supernatant, and developed primers based on cow and mice “coronaviruses” as well as previous OC43 partial sequences from other researchers and conserved regions from all group 2 “coronaviruses” thus creating one mess of a genome from multiple parts
• Potential 3C-like protease cleavage sites were identified by using the NetCorona 1.0 server
• DNA and protein similarity searches were performed using the NCBI WWW-BLAST (basic local alignment search tool) server on the GenBank DNA database
• Pairwise nucleotide and protein sequence alignments were performed by using FASTA algorithms in the ALIGN program on the GENESTREAM network server at the Institut de Génétique Humaine in Montpellier, France
• Maizel-Lenk dot matrix plots were calculated using the pairwise FLAG 1.0 (fast local alignment for gigabases) algorithm at the server of the Biomedical Engineering Center of the Industrial Technology Research Institute in Hsinchu City, Taiwan
• Multiple sequence alignments were prepared by using CLUSTALW and CLUSTALX, version 1.82 and were manually edited in GENEDOC
• The relationship between isolation date and genetic divergence was investigated using a linear regression, based on a maximum-likelihood tree, as implemented in the Path-O-Gen software
• Evolutionary rates and divergence times were estimated by using maximum likelihood in the TipDate software package, version 1.2
• The molecular clock hypothesis was tested by using the likelihood ratio test
• Based on the nucleotide sequence coding for the spike protein, a maximum-likelihood phylogenetic tree was constructed for HCoV-OC43 and several BCoV strains for which the date of “isolation” was known
• The time to the most recent common ancestor (TMRCA) of HCoV-OC43 and BCoV was dated by three methods
• Linear regression of root-to-tip divergence versus sampling time situates the TMRCA of HCoV-OC43 and BCoV in 1891
• The maximum-likelihood estimate for TMRCA is 1873, with a 95% confidence interval of 1815 to 1899
• The Bayesian coalescent approach dates TMRCA around 1890 (95% highest posterior density interval, 1859 to 1912)
• This estimate was highly consistent under different demographic models, including an exponential-growth model, which resulted in a TMRCA around 1893 (95% confidence interval, 1866 to 1918).
• The evolutionary rate of BCoV was also calculated by these three methods
• In other words, they used computer algorithms, models, and estimates to come up with likely dates that these unpurified seqeuncing creations hypothetically crossed species
• Until this 2005 study, only partial sequence fragments of the structural protein genes of HCoV-OC43 were available in GenBank, leaving the greater 5′ part of the genome to be determined
• They state that the discovery of “SARS-CoV” necessitates a better understanding of the genomic structure and evolution of other known “coronaviruses” in order to gain insights in how this new type could have emerged
• The prototype HCoV-OC43 strain (ATCC VR759) is a laboratory strain that, since its “isolation” in 1967, has been passaged 7 times in human embryonic tracheal organ culture, followed by 15 passages in suckling mouse brain cells and an unknown number of passages in human rectal tumor HRT-18 cells and/or Vero cells
• During the passage history, it is likely that a number of mutations have accumulated (i.e. what they worked with in 2005 would in no way compare or be similar to the OC43 goo “isolated” in 1967)
• The researchers state that it would be interesting to analyze the complete nucleotide sequence of contemporary HCoV-OC43 strains that are free from in vitro expansion mutations
• For all HCoV-OC43 ORFs, the highest similarity demonstrated was that to the corresponding BCoV ORFs, except for the HCoV-OC43 E gene, which showed 99.6% identity on the nucleotide level and 98.8% identity on the amino acid level with the PHEV E gene
• Based on the high similarity between HCoV-OC43 and PHEV in E, and between HCoV-OC43 and BCoV in all the other major ORFs, some hypotheses concerning the origin of HCoV-OC43 can be put forward
• Adaptation of BCoV to a human host and a recombination event between BCoV and PHEV leading to a new type of “coronavirus” with a different species specificity could both have been responsible for the emergence of a new human “coronavirus“
• HCoV-OC43 and BCoV cluster together, demonstrating the close relationship between the two (human and cow) “viruses”
• There is in fact more divergence between the different MHV (mice) strains or between the different IBV (chicken) strains than between HCoV-OC43 and BCoV (maybe because the mice and chicken strains weren’t cultured together…?)
• The close relationship between HCoV-OC43 and BCoV on the genetic level has also been shown to correspond to a close antigenic relationship: by using monoclonal antibodies directed against the BCoV S protein, common antigenic determinants for BCoV, HCoV-OC43, and PHEV have been demonstrated (in other words, the antibodies cross-react and are therefore not specific)
• The researchers suggest that around 1890, BCoV might have jumped the species barrier and became able to infect humans, resulting in the emergence of a new type of human “coronavirus” (HCoV-OC43)
• Indisputable evidence for the bovine-to-human direction of the interspecies transmission event, instead of a human-to-bovine direction, is not available
• Consequently, they assume that a deletion from BCoV to HCoV-OC43 rather than an insertion in the opposite direction took place during evolution, and thus, they hypothesize that the interspecies transmission event occurred from bovines to humans
• Nevertheless, the researchers claim it is possible that two other group 2 “coronaviruses,” CRCoV (dog) and PHEV (horse), might have played a role in the emergence of HCoV-OC43
• To elucidate the evolutionary relationship of HCoV-OC43 and BCoV with CRCoV and PHEV, complete genome sequence data of CRCoV and PHEV would be required
• The researchers state that the reliability of such an analysis is dependent on the validity of the molecular clock hypothesis, which assumes that the evolutionary rate is roughly constant in the lineages of a phylogenetic tree
• Although this assumption is frequently violated for “viral” sequence data, a molecular clock test indicated that this hypothesis could not be rejected for the “coronavirus” data set investigated here
• The researchers attempt to make the connection to a “flu” that occurred around the period in which the BCoV interspecies transmission would probably have taken place
• At that time, a human epidemic ascribed to influenza was spreading around the world
• The 1889-1890 pandemic probably originated in Central Asia and was characterized by malaise, fever, and pronounced central nervous system symptoms
• A significant increase in case fatality with increasing age was observed
• Absolute evidence that an influenza “virus” was the causative agent of this epidemic was never obtained, due to the lack of tissue samples from that period (thus it left the door open, in their easily swayed minds, to the possible cow-to-human “coronavirus” jump)
• Antibody testing of 50 to 100 year olds in 1957 “revealed” H2N2 reactions, yet it was tempting to speculate about an alternative hypothesis, that the 1889-1890 pandemic may have been the result of interspecies transmission of bovine “coronaviruses” to humans, resulting in the subsequent emergence of HCoV-OC43
• The researchers conclude that their study provided evidence for “viral” promiscuity, a phenomenon that has already been reported (i.e. hypothesized) for several animal “coronaviruses,” including BCoV, for which the potential to infect other species, including humans, has already been described

To anyone looking at this logically, it is clear to see no “virus” was ever purified/isolated before the creation of the OC43 genome in 2005. This is what is admitted in regards to in the “isolation” of this “virus” in the past leading up to its sequencing:

“The prototype HCoV-OC43 strain (ATCC VR759) is a laboratory strain that, since its isolation in 1967, has been passaged 7 times in human embryonic tracheal organ culture, followed by 15 passages in suckling mouse brain cells and an unknown number of passages in human rectal tumor HRT-18 cells and/or Vero cells.”

The “virus” itself is nothing more than human embryo tissue and cancer cell cultured goo combined with bovine and mice DNA (and even african green monkeys judging by the use of Vero cells). The genome is a mixture of all of these sources stitched together off of cow/mice “coronavirus” genome templates. The researchers used conserved regions from all Group 2 “coronaviruses” to generate RT-PCR products and then somehow seemed surprised that the genome created for OC43 had a similarity to all of them with the highest similarity to BCoV which was used as a template for both RT-PCR and sequencing. The researchers then claim a relation between all of these “viruses” and a zoonotic leap based on an unproven Molecular Clock hypothesis when the only “leaps” that occurred where in the cell culture dish, the numerous unfounded assumptions made from the computer-algorithm guided results, and the attempt to cram all of this unrelated indirect evidence into an unproven hypothetical narrative. Sadly, insane leaps in logic along with ridiculous assumptions based on unproven hypotheses and inhumane experimentation is par for the course with virology.