From Anthrax to MERS: How Do We Identify New Pathogens?

Francesca Tomasi received her B.A. from the University of Chicago and is now a microbiologist.

​In 1876, the only methods a scientist could use in investigating the causative agent of an infectious disease were microscopy and serendipity. When Robert Koch set out to isolate Bacillus anthracis, the bacterium responsible for anthrax, he followed four key steps that later became known as Koch’s Postulates. If fulfilled, Koch’s Postulates prove the causative link between a microbe and disease. The postulates are (1) The organism can be cultured from an individual with the sickness; (2) This organism can be grown in pure culture; (3) This organism can be inoculated in a healthy individual, and the individual gets sick when this is done; (4) The same organism can be re-isolated from the newly sick patient. Koch performed these steps on infected and naïve animals to show that Bacillus anthracis was the causative agent of anthrax in animals; he was also able to observe the rod-shaped bacteria under a microscope and physically describe them. Thanks to his observations, he was able to visualize the fully living bacteria and an alternate spore form, which led him to hypothesize that B. anthracis exists both as a rod and as a spore. In fact, we now know that the “reservoir” form of B. anthracis is a highly environmentally-resistant spore that withstands the tests of time and turmoil; upon ingestion by an organism, the spores germinate into virulent rods that ultimately sicken and potentially kill the host. Quite a bit to learn using rudimentary technology, right? 

In 2013, scientists were faced with a similar challenge as Robert Koch; namely, to identify the causative agent of a new disease. When the index patient, an otherwise healthy 60-year-old man, presented to a hospital in Saudi Arabia with respiratory symptoms in June (an uncharacteristic time for flu-like symptoms) and failed to get better upon treatment with Tamiflu or other related medications, they started to think that something else was wrong with this man. They took X-rays of his lungs and found them to be white - unhealthy - and subsequently they took more cultures of his sputum. They identified several pathogenic bacteria such as Klebsiella pneumoniae, but the patient did not improve after different rounds of antibiotics. Doctors also ran viral cell cultures, which came out positive for the presence of a virus. As a result, they isolated the viral genetic material and ran PCR analysis, which amplifies a specific segment of DNA using primers, which bind to short, specific flanking sequences of a genetic region of interest. The primers used for the case in Saudi Arabia were specific for a wide array of common viruses. No amplicons (fragments of DNA that amplified using the primers) were observed, which led the diagnostic team to think that they could be looking at a new virus. They therefore performed PCR again but using pan-coronavirus primers, which would amplify a gene common to all coronaviruses, thus telling them the type of virus. Examples of illnesses caused by coronaviruses are SARS, some pneumonias, and the common cold. Indeed, these universal primers worked: this Saudi Arabian man's respiratory symptoms appeared to be caused by a coronavirus, but not one that had previously been described. 

In order to test if this was a brand-new virus (as opposed to one that might have been circulating for a long time but had never caused serious illness before), scientists obtained sera from thousands of individuals previously at that hospital and tested them for antibodies against this particular virus. No one had antibodies against it, meaning nobody had previously been exposed. The new virus was called Middle East Respiratory Syndrome Coronavirus, or MERS-CoV. 

In addition to microbiological techniques and observations that led to the isolation and description of a new pathogen, epidemiological studies were concurrently performed in order to further describe the virus. Epidemiologists contact traced the index patient to determine whether any secondary cases had occurred in people he interacted with. In fact, surveillance mechanisms from different hospitals around Saudi Arabia (and later on, other countries with patients who had recently traveled to Saudi Arabia) soon showed a pattern of sporadic outbreaks in different regions. This led researchers to hypothesize most likely that the virus was being introduced into the human population from an animal; it seemed that the virus was not easily spread from human to human, so there had to be some other explanation for the sporadic cases. Different samples were collected from MERS patients and their genetic sequences were compared using phylogeny-generating algorithms. The strains were different enough from each other for scientists to conclude that the outbreak did not come from one specific strain that proliferated to different humans but, rather, that the virus was being reintroduced in different strains of the same species to humans. This prompted an investigation for an animal reservoir. Scientists tested many individuals across Saudi Arabia for antibodies against this virus, and found the highest amounts in farmers, slaughterhouse workers, and people with similar professions. They also isolated the virus from the air in camel barns, and a lot of Saudi Arabian racing camels tested positive for the virus. These camels also had very mild symptoms, characteristic of an animal reservoir. Furthermore, direct observation of a camel to human transmission further underscored the idea that camels were a reservoir of MERS-CoV. Having identified the virus and its epidemiological patterns, scientists could now start to study the virus itself in order to come up with potential therapeutic targets and vaccinations, a project making significant headway today. 

Whereas Robert Koch was extremely limited in his laboratory techniques, his postulates were pivotal to the field of microbiology and infectious disease research. He was the first scientist to establish a definitive causative relationship between a microbe and a human illness. And while his postulates weren’t exactly implemented in the MERS-CoV investigation (especially because viruses cannot exactly be grown in pure culture), the same general investigation was accomplished using highly sophisticated, 21st century sequencing technology.