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The story of the first MERS patient

نشرت بتاريخ 2 June 2014

How did a small laboratory attached to a private hospital in Saudi Arabia isolate and identify what later came to be known as the Middle East respiratory syndrome coronavirus (MERS-CoV)?

Islam Hussein

Ali Mohamed Zaki was the first person to isolate the coronavirus that causes MERS.
Ali Mohamed Zaki was the first person to isolate the coronavirus that causes MERS.
As he tries to unravel the untold story of Middle East respiratory syndrome coronavirus (MERS-CoV) discovery, MIT virologist Islam Hussein looks at the work of another prominent virologist, Ali Mohamed Zaki, whom he credits for the discovery of MERS-CoV. 

Zaki had diagnosed “patient zero” of the deadly virus at a Saudi Arabia virology diagnostic laboratory established in 1993. Besides coronavirus, Zaki also diagnosed Dengue fever for the first time in Saudi Arabia in 1994. And in 1997, he isolated a new tick-born flavivirus, known as Alkhurma, which causes severe hemorrhagic fever. 

Here he talks to Hussein about his experience with MERS-CoV, how he first identified the virus and the politics of science that saw him leave his work in Saudi and return to Egypt.  

Tell us about the laboratory where MERS-CoV was first diagnosed.

My laboratory was attached to a private Saudi hospital, named after its Egyptian-educated Saudi founder, Dr. Soliman Fakeeh. A case of a deadly viral disease called Crimean-Congo hemorrhagic fever was admitted to this hospital back in 1990 and was not diagnosed due to the lack of a virology diagnostic lab. Dr. Fakeeh realized the importance of having such lab and hired me to set it up. I transformed three empty rooms on the 6th floor into a fully equipped laboratory that had sterile cell culture room, serology and molecular biology units. 

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? 

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.

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.

Before sending the virus to the Dutch lab, I had to weigh up several risks. I now know that patient zero was infected with a novel virus that was not known to humans before. So, as a doctor and a scientist, I bear the responsibility of raising the alarm and making the information publicly available. However, based on my past experiences, I also knew that I would pay the price by losing my job. I had already made up my mind. My job is worth nothing compared to saving lives. 

So you decided to blow the whistle on MERS-CoV?

Yes. I submitted an alert message to ProMED mail on September 15, 2012. As soon as it appeared online on September 20, I was under attack by the Saudi Ministry of Health. My “crime” was breaching the rules by shipping sample #38, the very sample they have failed to diagnose, to the Dutch lab without permission. I have been working in Saudi Arabia for many years and I knew very well that this is a common procedure among Saudi hospitals and I had no other options. On September 23, the ProMED mail from a London hospital on a Qatari patient came online as well, and I felt that this should strengthen my case with the Saudi Ministry of Health. 

But the storm was unstoppable. On September 25, I took a flight back to Cairo leaving all my personal belongings behind.

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.

Saeed Al-Amoudi, the regional director of Jeddah’s labs and blood banks, blamed the recent MERS spread on you. He said that your lab was not an appropriate environment for handling this airborne virus and you might have unknowingly played a role in spreading it to the outside world. How do you respond to these allegations?

I saw patient zero dying, so I knew that this was something serious and that I had to take my precautions not to catch this mysterious disease. This was not the first time I had dealt with an unknown virus. I have a successful track record that demonstrates my competence in dealing with unknown infectious agents. 

As for my lab, I can comfortably classify it as at least a BSL2+ facility. My cell culture room was under negative pressure and was equipped with a double-door autoclave. I had all the necessary personal protective equipment and the filters of my biosafety cabinets were regularly inspected. My centrifuge cups were tightly capped. Strictly speaking, all lab procedures were virus aerosol-proof. My lab was an accredited diagnostic facility that, on a regular basis, received samples from the Saudi Ministry of Health itself. 

Now that you are back in Cairo, do you have any future plans to set up your own MERS lab?

I am back in my original academic position as a professor of microbiology at Ain Shams University. I helped the Egyptian Ministry of Health to set up their first diagnostic PCR assay for MERS-CoV. If I can secure enough funding, I will definitely establish my own lab. Otherwise, I would just live a peaceful quiet life side by side with my regular academic responsibilities.