Zaki MERS “Coronavirus” Paper (2012)

Remember the horrendous MERS “Coronavirus” outbreak of 2012? Don’t feel bad if it doesn’t ring a bell as most people outside of Saudi Arabia remain unaware of it. This epidemic-that-never-was resulted in only 882 deaths associated with it globally. Apparently, the MERS “virus” didn’t like to travel as 804 of those deaths were in Saudi Arabia. The “virus” also seems rather shy as there has been no MERS outbreak ever since the “virus” made its presence known. Fortunately, we can thank the work of one man for bringing this “virus” to the attention of some of the public in order to stop it in its tracks: Dr. Ali Mohamed Zaki. He was kind enough to do an interview in 2014 breaking down his discovery process for the MERS “virus:”

The story of the first MERS patient

How did the MERS-CoV story unfold?

“On the 13th of June, 2012, a patient, commonly known now as patient zero, was admitted to my hospital. He suffered from severe respiratory symptoms that quickly progressed to acute pneumonia and renal failure and died 11 days later. 

While the patient was alive, I was called on by a pulmonary specialist and urologist who were treating him. I collected blood and sputum samples (the sputum sample of patient zero happened to be number 38 in my regular serial order of samples, so I am going to refer to it as sample #38). I had an indirect immunofluorescent assay that uses the sputum sample as an unknown antigen, which can then be tested against a panel of known antibodies induced by a group of commonly encountered respiratory viral pathogens. All of them yielded negative results with sample #38. 

By law, I had to submit this sample together with all test results to the Saudi Ministry of Health. Polymerase chain reaction (PCR) test results came back as negative for influenza H1N1. 

I was almost certain that this sample had some sort of virus. 

Repeated inoculation of sample #38 into two types of cells, Vero and LLC-MK2, consistently yielded cytopathic effects (CPE) that are typical of viral-induced cellular damage. 

Under the microscope, inoculated cells exhibited syncytium formations, which are large multinucleated cells that result from fusion of multiple viral infected cells. The patient was dead by that time and nobody was interested to carry on investigating the mysterious causative agent that killed patient zero. My scientific curiosity was not satisfied and I decided to pursue investigations on my own. 

How did that happen? When did you pick up the trail again? 

It was around mid-July when I had strong suspicions that this could have been a case of paramyxovirus infection. The acute clinical picture of patient zero and his subsequent quick death, coupled with negative immunofluorescent and H1N1 PCR results and the syncytium formation, led me to suspect the deadly Nipah or Hendra viruses, both are paramyxoviruses carried by bats. I decided to test sample #38 by PCR against pan-paramyxovirus primers targeting conserved regions across all genomes of viruses that belong to the paramyxovirus family. 

I went through published literature and identified two sets of pan-paramyxovirus primers, one of them happened to be designed by the Dutch scientists at the Erasmus Medical Center (EMC) who later on confirmed the novel coronavirus in sample #38. I ordered these primers from a German company. To my disappointment, all my pan-paramyxovirus PCRs yielded negative results. 

Thinking that my reaction conditions might have been suboptimal, I contacted the scientists at EMC asking for technical help. I offered to send them some RNA extracted from sample #38 to be tested in their lab using the assay that they had developed. I was told that the technician is on a vacation and they can’t test my sample until August. I was not willing to waste any more time. 

I went ahead and tested sample #38 for the presence of hantavirus, a fatal zoonotic disease carried by rodents. Again, sample #38 was hantavirus-negative. 

Coronavirus was another respiratory agent, for which sample #38 had not been tested. I ordered pan-coronavirus primers targeting conserved region within the viral polymerase gene from the same German company, and bingo, I got the expected 440 base-pair band. 

The first thing that jumped to my mind was SARS; the coronavirus that caused a massive global outbreak in 2003. I ordered primers specific for SARS, and to my surprise, the PCR was negative. I repeated these PCRs several times to confirm. I could consistently reproduce the pan-coronavirus positive result, but it was not SARS. This was the first evidence that I could be dealing with a novel human coronavirus that had not been described before. 

How did you move forward with these results?

I hesitated to announce my findings because I knew that nobody would believe such a significant result from a small lab in a private Saudi hospital; I needed a second opinion from an independent reputable lab. I was also concerned that I might lose my intellectual rights if I had kept circulating my sample around. By that time, the Dutch scientists at EMC were willing to test my RNA sample based on my preliminary findings that this could be a novel human coronavirus. 

They did confirm my initial findings and asked me to send them a small portion of patient zero’s sample because they want to do some more testing and they were running out of RNA. I didn’t have any mechanism to ship a live virus sample while maintaining the cold chain during transit. So, I filtered the sputum sample and mixed the filtrate with Vero cells, packaged the tightly capped tube in appropriate biohazard containers and shipped it with a private carrier at room temperature as a diagnostic sample. It worked. They received it in the Netherlands and managed to recover the live virus, the first genetic analysis of this novel virus published in New England Journal of Medicine.”

So, you rushed back to Cairo, leaving everything, including your own  discovery. Did the hospital ask you to return back to pursue your work?

“No, they asked me to send them a resignation letter; they closed down my lab and autoclaved all sample #38 material. If not for the sample I had sent to the Dutch lab, it would have been lost forever.

How did you manage to get the NEJM paper out with your Dutch collaborators after you returned to Cairo?

My ProMED message served as an irrefutable record that protected my intellectual rights for this discovery. I had kept all the clinical data of patient zero. EMC scientists amplified the virus sample that I sent them and managed to produce a full genome sequence for this novel coronavirus. They also did the phylogenetic analysis that was published in the paper. I wrote the bulk of this manuscript and offered the two Egyptian clinicians who had examined patient zero to be co-authors on this paper and they refused. They felt that having their names on this paper might bring them trouble and they might lose their jobs too. The paper was quickly accepted and published in early November.”

https://www.natureasia.com/en/nmiddleeast/article/10.1038/nmiddleeast.2014.134

According to Zaki, he had a patient that was suffering from pneumonia. Blood and sputum samples were all negative for the primary suspects. After having been mechanically ventilated and treated with numerous renal toxic drugs for 11 days, the patient succumbed to respiratory and renal failure. Zaki’s colleagues were not interested in carrying on an investigation into the “mysterious causative agent” that killed patient zero, possibly because unlike Zaki, they understood the real cause. However, Zaki believed that there was an unknown “virus” somewhere in those samples so he set out to investigate on his own. He eventually called upon the help of the Dutch EMC after making sure his intellectual rights were secure. Of course, his independent actions made Zaki a fugitive from the Saudi government who were none too happy with his heroic “virus-humting” deeds. It’s a good thing he kept going, as if he hadn’t, Zaki claimed the “virus” would have been lost forever and thus, the nearly 2,600 cases since its 2012 discovery would have remained hidden to the world. Presented below are highlights from his rebellious paper:

Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia

Case Report

“A 60-year-old Saudi man was admitted to a private hospital in Jeddah, Saudi Arabia, on June 13, 2012, with a 7-day history of fever, cough, expectoration, and shortness of breath. He had no history of cardiopulmonary or renal disease, was receiving no long-term medications, and did not smoke. The physical examination revealed a body-mass index (the weight in kilograms divided by the square of the height in meters) of 35.1, a blood pressure of 140/80 mm Hg, a pulse of 117 beats per minute, a temperature of 38.3°C, and a respiratory rate of 20 breaths per minute.”

“On day 1, treatment was started with oseltamivir, levofloxacin, piperacillin–tazobactam, and micafungin. On day 4, treatment with meropenem was started, since Klebsiella pneumoniae that was sensitive to meropenem was detected on bronchoscopy and tracheal lavage performed on day 2. Staphylococcus aureus, which was sensitive to a wide range of antimicrobials, was collected from a sputum sample collected on admission. Acinetobacter was detected in a tracheal aspirate sample collected on the day of death. No other pathogens were detected in respiratory specimens, and no bacterial growth was detected from blood samples.

On the day after admission, the patient was transferred to an intensive care unit, where he underwent intubation for mechanical ventilation. Laboratory findings obtained on admission showed normal white-cell counts, except for a relatively high percentage of neutrophils (92.5%) and a low percentage of lymphocytes (4.3%) (Table 1). Levels of liver enzymes, blood urea nitrogen, and creatinine were within the normal ranges. Somewhat elevated liver enzymes were detected on day 7 and later, with levels of alanine aminotransferase of 20 IU, 78 IU, and 47 IU per liter on days 1, 7, and 8, and levels of aspartate aminotransferase of 33 IU and 96 IU per liter on days 1 and 8, respectively. The patient tested negative for the human immunodeficiency virus; testing was not performed for pneumocystis pneumonia.

Starting on day 3 after admission, levels of blood urea nitrogen and creatinine progressively increased. Starting on day 8, the white-cell count began to rise, reaching a peak of 23,800 cells per cubic millimeter on day 10, with neutrophilia, persistent lymphopenia, and progressive thrombocytopenia. Arterial oxygen saturation ranged from 78 to 98% (Table 1). On day 11 after admission (June 24, 2012), the patient died of progressive respiratory and renal failure. A postmortem examination was not performed.

Methods

CLINICAL SPECIMENS AND VIRAL CULTURE

Blood samples were collected in vacutainers with and without EDTA. Sputum samples were collected in sterile cups, after which virus transport medium was added; samples were stirred and centrifuged at 2000 rpm for 10 minutes. Supernatant was transferred to a new sterile tube and used to inoculate Vero and LLC-MK2 cells by adsorption for 1 hour at room temperature, after which 2% fetal bovine serum in minimal essential medium Eagle was added. Flasks were incubated in a carbon dioxide incubator at 37°C and observed daily for 15 days for cytopathic changes with change of medium every 3 days.

VIRAL DIAGNOSTICS

Respiratory epithelial cells from sputum were washed three times in phosphate-buffered saline (PBS), resuspended in 1 ml of PBS, and spotted on Teflon-coated slides. Slides were left to air-dry and then fixed for 10 minutes in chilled acetone. Slides were tested by indirect immunofluorescence for influenza A virus, influenza B virus, parainfluenza viruses types 1 to 3, adenovirus, and respiratory syncytial virus with the use of a Bartels Viral Respiratory Screening and Identification Kit, as described by the manufacturer (Trinity Biotech). The same procedure was used to detect viral antigens in inoculated cells after cytopathic effects had been observed. To this end, cells were scraped from tissue-culture flasks, and cells with media were transferred to a sterile centrifuge tube and prepared as described for respiratory epithelial cells from sputum. Supernatant from sputum as well as from experimentally inoculated cell cultures that displayed cytopathic effects (and uninfected cultures as negative controls) was extracted with the use of a High Pure Viral Nucleic Acid Kit, as described by the manufacturer (Roche). Extracted nucleic acids were tested by reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay designed to detect all known paramyxoviruses,^6,7 coronaviruses,^3,8 and enteroviruses⁹ and by real-time PCR for adenoviruses.¹⁰

VIRAL GENOME SEQUENCING

To sequence the PCR fragments of the pan-coronavirus PCR,³ amplicons were purified from the gel and sequenced with the use of a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a 3130XL Genetic Analyzer (Applied Biosystems), according to the manufacturer’s instructions. To further characterize the virus genome, we used a random-amplification deep-sequencing approach. Supernatant was cleared from cellular debris by low-speed centrifugation, and virus was filtered through a 0.45-μm centrifugal filter unit (Millipore) to minimize bacterial background. We used OmniCleave endonuclease (Epicenter) to remove free DNA and RNA, according to the manufacturer’s protocol. Viral RNA was extracted from supernatants in infected cell cultures with the use of a High Pure RNA Isolation Kit (Roche). To remove mammalian ribosomal RNA, we used Ribo-Zero rRNA Removal Kit RZH110424 (Epicenter), according to the manufacturer’s protocol. RNA underwent reverse transcription with the use of circular permuted primers¹¹ that were extended with random hexamer sequences. DNA was amplified by means of PCR with the circular permuted primers.

We sequenced the amplified fragments using the Roche 454 GS FLX sequencing platform. A fragment library was created according to the manufacturer’s protocol without DNA fragmentation (GS FLX Titanium Rapid Library Preparation, Roche). The emulsion-based clonal amplification PCR (Amplification Method Lib-L) and GS junior sequencing run was performed according to the manufacturer’s instructions (Roche). The sequence reads were trimmed at 30 nucleotides from the 3′ and 5′ ends to remove all primer sequences. Sequence reads from the GS FLX sequencing data were assembled into contig maps (a set of overlapping DNA segments) with the use of CLC Genomics software, version 4.6.1 (CLC Bio). Using the 454 sequencing platform, we obtained approximately 90% of the virus genome sequence. Subsequently, specific primers were designed to amplify overlapping fragments of approximately 800 bp by means of PCR. These PCR products were purified from the gel and sequenced with the use of a BigDye Terminator v3.1 Cycle Sequencing Kit and a 3130XL Genetic Analyzer, according to the manufacturer’s instructions.

Results

DETECTION OF A CORONAVIRUS

The day 1 sputum sample tested negative by indirect immunofluorescence assays for influenza A and B viruses, parainfluenza viruses types 1 to 3, respiratory syncytial virus, and adenovirus. However, for a sputum sample obtained on admission, inoculation in LLC-MK2 and Vero cells resulted in cytopathic changes suggestive of virus replication (Figure 2A). Cytopathic changes consisted of syncytium formation in LLC-MK2 cells at low pH and rounding and detachment of cells at neutral or alkaline pH in Vero and LLC-MK2 cells. On passage of the culture supernatant to fresh cells, the same cytopathic effects were observed within 5 days. Virus was not isolated from a blood sample collected on admission or from a tracheal aspirate sample collected 4 days after admission.

Indirect immunofluorescence assays for the detection of influenza A and B viruses, parainfluenza viruses types 1 to 3, respiratory syncytial virus, and adenovirus were performed with the infected cell cultures, but again with negative results. In contrast, when these slides were incubated with serum samples collected from the patient 10 and 11 days after admission, the samples reacted strongly when dilutions of 1:20 were tested on immunofluorescence assay specific for IgG antibodies. No attempts were made to detect virus-specific IgM antibodies. In contrast, 2400 control serum samples collected from persons seeking medical attention at the Dr. Soliman Fakeeh Hospital in Jeddah from 2010 through 2012 remained negative in this assay. These data suggested that antibodies to an unknown virus had developed in the patient, although such antibodies were not detectable in the general population over the previous 2 years.

Real-time PCR assays specific for adenovirus, enterovirus, human metapneumovirus, and human herpesvirus types 1 to 3 yielded negative results with the use of nucleic acids extracted from the inoculated cell-culture supernatants. Furthermore, family-wide PCR assays that can detect all known paramyxoviruses^6,7 also yielded negative results. However, family-wide PCR assays for the detection of coronaviruses^3,8 yielded PCR fragments of the expected sizes.

GENETIC ANALYSIS OF A NOVEL CORONAVIRUS

The PCR fragments of the pan-coronavirus PCR³ were sequenced. This sequence corresponded with a conserved region of open reading frame 1b of the replicase gene of a coronavirus. Reference coronavirus genome sequences were downloaded from GenBank and aligned with the amplified fragment of the newly discovered virus, hereafter called HCoV-EMC (for Erasmus Medical Center). A maximum-likelihood tree was constructed to infer the phylogenetic relationships (Figure 2B).”

“To further characterize the virus, approximately 90% of the virus genome sequence was obtained on sequence analysis with the use of the 454 platform. Subsequently, specific primers were designed to amplify overlapping PCR fragments of approximately 800 bp each for conventional Sanger sequencing. The nearly full-length sequence was obtained (GenBank accession number, JX869059), for which final annotation remained in progress at the time of this report.”

“We compared the open reading frame 1ab gene product of HCoV-EMC with those of the other betacoronaviruses, HKU4 and HKU5, to test whether HCoV-EMC might belong to one of these known species or whether it represents a new species within the genus. The International Committee on Taxonomy of Viruses (ICTV) considers that viruses sharing more than 90% of sequence identity in the conserved replicase domains belong to the same species.¹ This 90% identity threshold serves as the sole species demarcation criterion. Since the identity of amino acid sequences in these conserved domains of open reading frame 1ab between HCoV-EMC and HKU4 and HKU5 was less than 80%, we concluded that HCoV-EMC represented a novel betacoronavirus species, although such classification requires formal ICTV approval.”

Discussion

“The first decade of the 21st century has witnessed an increase in the number of coronaviruses that have been identified, along with a corresponding increase in the number of coronavirus genomes that have been sequenced. Such increases were due to the discovery of the SARS coronavirus, which resulted in a global outbreak of pneumonia in 2003 that affected persons in approximately 30 countries and resulted in about 800 deaths.¹² Before 2003, only two human coronaviruses were known, HCoV-229E and HCoV-OC43, both discovered in the 1960s.^13,14 After the emergence of the SARS-CoV in 2003, two additional human coronaviruses were discovered, HCoV-NL63 and HCoV-HKU1.^15-17 Here we report the isolation and characterization of the sixth coronavirus that apparently may infect humans.

On the basis of genetic data, the ICTV has identified four virus clusters within the Coronavirinae subfamily, of which three represent approved genera; alphacoronavirus, betacoronavirus, and gammacoronavirus. The five known human coronaviruses all belong to the genera alphacoronavirus (HCoV-229E and HCoV-NL63) and betacoronavirus (HCoV-OC43, HCoV-HKU1, and SARS-CoV).^2-4,13-16,18 HCoV-EMC is the first human coronavirus in lineage C of the betacoronavirus genus. Its closest relatives are coronaviruses HKU4 and HKU5, isolated from Tylonycteris pachypus and Pipistrellus abramus bats, respectively.¹⁷

As compared with other coronaviruses, HCoV-EMC was isolated and propagated relatively easily in Vero and LLC-MK2 cells. The only other human coronaviruses that replicate well in these monkey-cell lines are SARS-CoV and HCoV-NL63, which both use human angiotensin-converting enzyme 2 as their receptor. We hypothesize that one or more species of animals, possibly bats, were the reservoir host of this new coronavirus. Saudi Arabia harbors numerous bat species, including pipistrellus bats, which were found to carry BatCoV-HKU5 in Asia.

The patient’s findings on chest radiography together with the clinical symptoms indicated acute respiratory distress syndrome (ARDS) with multiorgan dysfunction syndrome (MODS), similar to what has been described in severe cases of influenza and SARS.^19-21 These pneumonic changes did not respond to antibacterial treatment.²² The patient was treated with oseltamivir for the possibility of infection with the H1N1 swine flu virus. Hematologic changes were evident in this patient in the form of lymphopenia, neutrophilia, and late thrombocytopenia. Abnormal hematologic variables were also quite common among patients with SARS. Lymphopenia was the most common finding in a cohort of 157 patients with SARS. In those patients, postmortem findings showed lymphopenia in various lymphoid organs with no features of bone marrow failure or reactive hemophagocytic syndrome.²³ The patient also had progressive impairment of renal function, similar to what had been described in some patients with SARS and possibly attributed to direct infection of renal tissue by the virus. The renal impairment in this case started on the 9th day of symptoms and progressed over the course of the patient’s illness.

No symptoms were observed in the hospital among doctors and nurses caring for the patient, which suggests that the disease did not spread readily. However, staff members were not tested for antibodies against the virus for confirmation. Now that the genome sequence of HCoV-EMC has become available and rapid diagnostic tests specific for HCoV-EMC have been developed,²⁴ thorough epidemiologic investigations are warranted. Such studies should initially focus on identifying the original source of the virus (including bats and other animal species) and potential transmission events between the infected patient and direct contacts. The development of serologic assays for surveillance studies is important.

Three months after the hospitalization of the patient in Jeddah, it was reported that a second patient with a history of travel to Saudi Arabia who had been transferred from a hospital in Qatar to a hospital in London was infected with the same virus.²⁵ At present, links between the two infected patients or a potential common source of infection have not been identified. No additional cases have been identified, although several are still under investigation. Epidemiologic investigations, active case findings with the use of updated case definitions,²⁵ and syndrome surveillance in combination with sensitive diagnostic tests will be key to monitoring the present situation and — if necessary — to intervene in a potential outbreak. It will be equally important to test whether HCoV-EMC fulfills Koch’s postulates as the causative agent of severe respiratory disease.

This case is a reminder that although most infections with human coronaviruses are mild and associated with common colds, certain animal and human coronaviruses may cause severe and sometimes fatal infections in humans. Although HCoV-EMC does not have many of the worrisome characteristics of SARS-CoV, we should take notice of the valuable lessons learned during the 2003 SARS outbreak with respect to outbreak investigations and management.

https://www.nejm.org/doi/full/10.1056/nejmoa1211721

In Summary:

• According to Zaki, he had an indirect immunofluorescent assay that uses the sputum sample as an unknown antigen, which can then be tested against a panel of known antibodies induced by a group of commonly encountered respiratory “viral” pathogens (this uses the unproven theoretical antibodies to attempt to discover unknown theoretical “viruses”)
• All of them yielded negative results with sample #38
• Polymerase chain reaction (PCR) test results came back as negative for influenza H1N1
• Zaki claimed that he was almost certain that this sample had some sort of “virus” 
• Repeated inoculation of sample #38 into two types of cells, Vero and LLC-MK2, (monkey kidney cell lines) consistently yielded cytopathic effects (CPE) that are typical of “viral-induced” cellular damage
• Under the microscope, inoculated cells exhibited syncytium formations, which are large multinucleated cells that (are assumed to) result from fusion of multiple “viral” infected cells
• The patient was dead by that time and nobody was interested to carry on investigating the mysterious causative agent that killed patient zero
• It was around mid-July when Zaki had strong suspicions that this could have been a case of paramyxovirus infection due to the acute clinical picture of patient zero and his subsequent quick death
• Coupled with negative immunofluorescent and H1N1 PCR results and the syncytium formation, he began to suspect the deadly Nipah or Hendra “viruses,” both are said to be paramyxoviruses carried by bats
• Zaki went through the published literature and identified two sets of pan-paramyxovirus primers and one of them just so happened to be designed by the Dutch scientists at the Erasmus Medical Center (EMC) who later on confirmed the novel “coronavirus” in sample #38
• Zaki’s tests came back negative and fearing that his reaction conditions might have been suboptimal, he contacted the scientists at EMC asking for technical help
• Zaki went ahead and tested sample #38 for the presence of hantavirus, a fatal zoonotic disease carried by rodents, which also came back negative
• “Coronavirus” was another respiratory agent for which sample #38 had not been tested so Zaki ordered “pan-coronavirus” primers targeting conserved region within the “viral” polymerase gene from the same German company, and bingo, he got the expected 440 base-pair band that he expected to find
• Zaki thought he was dealing with “SARS” and could consistently reproduce the “pan-coronavirus” positive result, but it was not “SARS”
• This was the first evidence that he could be dealing with a novel human “coronavirus” that had not been described before
• Zaki wanted to confirm his findings but he was also concerned that he might lose his intellectual rights if he had kept circulating his sample around, yet fortunately for Zaki, the same Dutch company (EMC) decided to help him out
• EMC did “confirm” his initial findings and asked Zaki to send them a small portion of patient zero’s sample because they want to do some more testing and they were running out of RNA
• Zaki didn’t have any mechanism to ship a live “virus” sample while maintaining the cold chain during transit so he filtered the sputum sample and mixed the filtrate with Vero cells, packaged the tightly capped tube in appropriate biohazard containers and shipped it with a private carrier at room temperature as a diagnostic sample
• EMC received it in the Netherlands and managed to recover the live “virus,” the first genetic analysis of this novel “virus” published in New England Journal of Medicine
• Zaki claimed that his lab was subsequently shut down and that if he had not sent the sample to the Dutch lab, it would have been lost forever (an odd thing to say about a “virus” being lost)
• Zaki stated that his ProMED message served as an irrefutable record that protected his intellectual rights for this discovery
• He had kept all the clinical data of patient zero
• EMC scientists amplified the “virus” sample that Zaki sent them and managed to produce a full genome sequence for this novel “coronavirus” as well as the phylogenetic analysis that was published in the paper
• The paper was quickly accepted and published in early November

• A 60-year-old Saudi man was admitted to a private hospital in Jeddah, Saudi Arabia, on June 13, 2012, with a 7-day history of fever, cough, expectoration, and shortness of breath
• On day 1, treatment was started with:
  1. Oseltamivir
  2. Levofloxacin
  3. Piperacillin–tazobactam
  4. Micafungin
• On day 4, treatment with meropenem was started, since Klebsiella pneumoniae that was sensitive to meropenem was detected on bronchoscopy and tracheal lavage performed on day 2
• Staphylococcus aureus, which was sensitive to a wide range of antimicrobials, was collected from a sputum sample collected on admission
• Acinetobacter was detected in a tracheal aspirate sample collected on the day of death
• On the day after admission, the patient was transferred to an intensive care unit, where he underwent intubation for mechanical ventilation
• Levels of liver enzymes, blood urea nitrogen, and creatinine were within the normal ranges at admission but somewhat elevated liver enzymes were detected on day 7 and later (that couldn’t have had to do with the multiple antibiotics and antifungals he was given, now could it…?)
• The patient tested negative for the human immunodeficiency “virus” and testing was not performed for pneumocystis pneumonia
• On day 11 after admission (June 24, 2012), the patient died of progressive respiratory and renal failure and a postmortem examination was not performed
• How the samples were obtained/prepared:
  1. Blood samples were collected in vacutainers with and without EDTA (a chemical that binds certain metal ions, such as calcium, magnesium, lead, and iron; used in medicine to prevent blood samples from clotting and to remove calcium and lead from the body)
  2. Sputum samples were collected in sterile cups, after which “virus” transport medium was added
  3. Samples were stirred and centrifuged at 2000 rpm for 10 minutes
  4. Supernatant was transferred to a new sterile tube and used to inoculate Vero and LLC-MK2 cells by adsorption for 1 hour at room temperature, after which 2% fetal bovine serum in minimal essential medium Eagle was added
  5. Flasks were incubated in a carbon dioxide incubator at 37°C and observed daily for 15 days for cytopathic changes with change of medium every 3 days
• How “viral” diagnosis was made:
  1. Respiratory epithelial cells from sputum were washed three times in phosphate-buffered saline (PBS), resuspended in 1 ml of PBS, and spotted on Teflon-coated slides
  2. Slides were left to air-dry and then fixed for 10 minutes in chilled acetone
  3. Slides were tested by indirect immunofluorescence for for various “viruses” with the use of a Bartels Viral Respiratory Screening and Identification Kit
  4. Supernatant from sputum as well as from experimentally inoculated cell cultures that displayed cytopathic effects (and uninfected cultures as negative controls) was extracted with the use of a High Pure Viral Nucleic Acid Kit
  5. Extracted nucleic acids were tested by RT-PCR assay designed to detect all known “paramyxoviruses,” “coronaviruses,” and “enteroviruses” and by real-time PCR for “adenoviruses”
• During genome sequencing, supernatant was cleared from cellular debris by low-speed centrifugation, and “virus” was filtered through a 0.45-μm centrifugal filter unit (Millipore) to minimize bacterial background (minimize, not eliminate)
• They used OmniCleave endonuclease (Epicenter) to remove free DNA and RNA, according to the manufacturer’s protocol
• “Viral” RNA (assumed “viral” as no particles were purified/isolated) was extracted from supernatants in infected cell cultures with the use of a High Pure RNA Isolation Kit
• To remove mammalian ribosomal RNA, they used Ribo-Zero rRNA Removal Kit
• Using the 454 sequencing platform, they obtained approximately 90% of the “virus” genome sequence
• Subsequently, specific primers were designed to amplify overlapping fragments of approximately 800 bp by means of PCR
• For a sputum sample obtained on admission, inoculation in LLC-MK2 and Vero cells resulted in cytopathic changes suggestive of “virus” replication
• Cytopathic changes consisted of syncytium formation in LLC-MK2 cells at low pH and rounding and detachment of cells at neutral or alkaline pH in Vero and LLC-MK2 cells
• In other words, the CPE (signs of cell death) seen changed with the pH levels
• On passage of the culture supernatant to fresh cells, the same cytopathic effects were observed within 5 days
• “Virus” was not isolated from a blood sample collected on admission or from a tracheal aspirate sample collected 4 days after admission
• When these slides were incubated with serum samples collected from the patient 10 and 11 days after admission, the samples reacted strongly when dilutions of 1:20 were tested on immunofluorescence assay “specific” for IgG antibodies
• No attempts were made to detect “virus-specific” IgM antibodies
• These data suggested that antibodies to an unknown “virus” had developed in the patient, although such antibodies were not detectable in the general population over the previous 2 years
• Family-wide PCR assays for the detection of “coronaviruses” yielded PCR fragments of the expected sizes
• The PCR fragments of the “pan-coronavirus” PCR were sequenced
• This sequence corresponded with a conserved region of open reading frame 1b of the replicase gene of a “coronavirus”
• Reference “coronavirus” genome sequences were downloaded from GenBank and aligned with the amplified fragment of the newly discovered “virus,” hereafter called HCoV-EMC (for Erasmus Medical Center)
• A maximum-likelihood tree was constructed to infer the phylogenetic relationships
• In other words, Zaki and Co. came up with a small PCR fragment that matched a conserved region of one gene of all “coronaviruses” and then used the reference sequences of all “coronaviruses” to build and create their new one
• To further characterize the “virus,” approximately 90% of the “virus” genome sequence was obtained on sequence analysis with the use of the 454 platform
• Subsequently, specific primers were designed to amplify overlapping PCR fragments of approximately 800 bp each for conventional Sanger sequencing
• The nearly full-length sequence was obtained for which final annotation remained in progress at the time of this report
• The International Committee on Taxonomy of Viruses (ICTV) considers that “viruses” sharing more than 90% of sequence identity in the conserved replicase domains belong to the same species
• This 90% identity threshold serves as the sole species demarcation criterion
• Since the identity of amino acid sequences in these conserved domains of open reading frame 1ab between HCoV-EMC and HKU4 and HKU5 was less than 80%, Zaki concluded that HCoV-EMC represented a novel betacoronavirus species, although such classification requires formal ICTV approval
• Zaki states that the first decade of the 21st century has witnessed an increase in the number of “coronaviruses” that have been identified, along with a corresponding increase in the number of “coronavirus” genomes that have been sequenced (the more sequences obtained = the more “coronaviruses” they “discover”)
• Such increases were due to the discovery of the “SARS coronavirus” (why would the discovery of “SARS'” lead to the discovery of more “coronaviruses?”)
• On the basis of genetic data, the ICTV identified four “virus” clusters within the Coronavirinae subfamily, of which three represent approved genera; alphacoronavirus, betacoronavirus, and gammacoronavirus
• According to Zaki, as compared with other “coronaviruses,” HCoV-EMC was isolated and propagated relatively easily in Vero and LLC-MK2 cells
• The only other human “coronaviruses” that replicate well in these monkey-cell lines are “SARS-CoV” and HCoV-NL63,
• Zaki and Co. hypothesized that one or more species of animals, possibly bats, were the reservoir host of this new “coronavirus”
• The patient’s findings on chest radiography together with the clinical symptoms indicated acute respiratory distress syndrome (ARDS) with multiorgan dysfunction syndrome (MODS), similar to what has been described in severe cases of influenza and “SARS”
• These pneumonic changes did not respond to antibacterial treatment
• The patient was treated with oseltamivir for the possibility of infection with the H1N1 swine flu “virus”
• The patient also had progressive impairment of renal function, similar to what had been described in some patients with “SARS” and possibly attributed to direct infection of renal tissue by the “virus”
• The renal impairment in this case started on the 9th day of symptoms and progressed over the course of the patient’s illness
• Keep in mind that every drug given to the patient from day 1 alone can cause renal failure, and he was on at least 5 of them
• A second “case” was identified 3 months later leading Zaki to conclude that epidemiologic investigations, active case findings with the use of updated case definitions, and syndrome surveillance in combination with sensitive diagnostic tests will be key to monitoring the present situation and — if necessary — to intervene in a potential outbreak
• He also concluded that it will be equally important to test whether HCoV-EMC fulfills Koch’s postulates as the causative agent of severe respiratory disease
• This case was a reminder that although most infections with human “coronaviruses” are mild and associated with common colds, certain animal and human “coronaviruses” may cause severe and sometimes fatal infections in humans

Clearly all I had to do was highlight the sentence in the second to last paragraph where Zaki admits that Koch’s Postulates, the bare-minimum and absolutely essential criteria needed to be met in order to claim the discovery of any new pathogen, had remained unsatisfied for MERS. That is case-closed for MERS. He had no purified/isolated particles assumed to be a new “virus.” He had no electron microscope images of any “coronavirus-like” particles. He had no proof of pathogenicity resulting from experimentation with purified/isolated particles in animal studies. All Zaki had was a genome cobbled together from the supernatant of unpurified monkey kidney cell cultures aligned and created from reference genomes of other “coronaviruses” created in the same way. Oh, and his intellectual rights.

End of story.