Lessons Learned From Serial Isolations Of Human Coronavirus 229E From Environmental Surfaces Of A Classroom During Influenza Season

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The word “infection” has different connotations in the fields of virology and medicine. Generally, physicians refer to a condition wherein a patient displays symptoms of illness due to an infectious agent when they use the word “infection.” In virology, the word infection is not synonymous with outward signs of illness, because people can have a “virus infection” without clinically apparent symptoms.  For example, up to 80% of those infected by Zika virus do not know they harbor the virus and never develop a rash, fever, and other outcomes of Zika fever.

It is not surprising, then, that some people unknowingly harbor respiratory viruses and act as spreaders of the viruses. Respiratory viruses include influenza and the various viruses that cause the common cold. Sometimes, mild respiratory infections are mistaken for common allergies, and no precautions to prevent spread of the causative agent are taken as a result. Yet symptomatic and asymptomatic individuals that harbor respiratory viruses release virus particles when they exhale, cough, talk, and sneeze.

The viruses released by these individuals are typically associated with salts and secretions, and the particles that contain these occur in a wide size range. The smaller particles can be “naked” virus particles not complexed with salt or other substances, or those complexed with other substances but with overall small dimensions such that they become airborne and can remain adrift for relatively long periods of time. The larger particles are droplets that settle (i.e., “fall-out”) quickly onto environmental surfaces (or persons close by). Apart from inhalation of airborne respiratory viruses, humans can unintentionally infect themselves with the viruses through self-inoculation by touching virus-contaminated environmental surfaces and then touching their mouth, nose, and, in some cases, eyes.

Areas wherein many people congregate (“mass-gathering” sites) pose high risks for contracting respiratory illnesses.  This includes classrooms, especially as people therein are typically seated in close proximity, increasing the chances of inhaling airborne viruses from nearby “virus emitters,” and by touching surfaces such as desk-tops contaminated with respiratory viruses followed by subsequent transfer of the virus by touching the nose, eyes, or mouth.

Bonny et al. (1) tested whether any viable (infectious) respiratory viruses would be detected at all on the surfaces of a classroom that was cleaned daily. The word “live” is considered inappropriate for viruses; instead, the word “viable” is used in reference to an infectious virus (i.e., capable of causing an infection), whereas non-viable indicates a “dead” virus.  The work was performed during “influenza season,” at a time many students were observed to be coughing and sneezing, yet felt well enough to attend classes. “High-touch” surfaces, such as a doorknob and desktops, were among the surfaces tested for presence of respiratory viruses; door knobs are often contaminated and selected for tests because hands often are used to cover the mouth during coughing  and sneezing, and the viruses deposited on the hands are transferred onto the door knob during entrance/exit from rooms.

As respiratory viruses cannot be seen using a standard microscope, sophisticated laboratory methods are required for their detection. Moreover, to determine whether a health risk may be present, it is necessary to show that the virus detected is viable; non-viable viruses cannot cause an infectious disease and thus pose no health risk(s).  Unlike many bacteria and fungi, viruses require a live host to replicate and complete their “life cycle.”  This necessitates “isolation” of the virus from specimens in either animals susceptible to the particular virus, or in specific living cells referred to as “cultured cells” that are grown in virology laboratories.

Bonny et al. (1) thus attempted to isolate respiratory viruses in various types of cultured cells grown under different temperatures and conditions.  They were “casting a wide net,” as each respiratory virus has its own specific requirements for isolation; there is no such thing as a one-size fits all simple process for the task. The team was surprised because over a test period of a few days, the same virus, Human coronavirus 229E (CoV-229E), was isolated from environmental surfaces, whereas they anticipated isolation of influenza A and B viruses.

Coronaviruses are widespread in nature, and human versions of these viruses are common causes of the common cold, but they can also cause more severe illnesses.  They are covered (“enveloped”) by a lipid membrane, and their genetic material is RNA, not DNA. The work of Bonny et al. (1) generated surprising results for various reasons: (a) In general, coronaviruses are considered “difficult” to isolate, yet they isolated CoV-229E repeatedly, (b) They confirmed CoV-229E survives on environmental surfaces longer than commonly assumed for viruses with lipid envelopes, and (c) Over a few days, the same virus was isolated from the surfaces of a classroom that is cleaned frequently.

Success at virus isolation was attributable to the wide net approach used by the authors, and their patience.  Many viruses require long periods of incubation time, and the lengthy process used for their isolation is considered expensive and time-consuming, and so cell cultures are often held and examined for only a few days after they are inoculated with material that may contain a virus. In contrast, the authors typically maintain their cultures and examined them daily for 30 days, resulting in better virus isolation success rates than achieved by many others.

After isolation of CoV-229E from various specimens, the virus’ complete genetic sequence (code) was determined, revealing that the same virus strain was isolated from the classroom surfaces over a few days.  These findings could mean that the virus strain that had been detected was causing a local outbreak, or that the same person was emitting the virus and contaminating surfaces over a few days.  The latter possibility is unlikely for many reasons, especially as courses are taught every other day so the virus emitter would not have attended classes in the same classroom at the same time every day.

Since there is sparse information regarding the environmental stability of CoV-229E, a well-studied laboratory strain was tested and shown to survive for at least seven days when dried on hard surfaces.  This finding is counter-intuitive to dogma, as it is usually considered a “fact” that enveloped viruses such as CoV-229E are easily inactivated (“killed”) upon drying. Studies performed after the report of Bonny et al. (1) was published revealed the same was true for the CoV-229E strain that had been isolated from the classroom.

Upon preparation of the manuscript, the authors were further surprised to find published literature that documented the following: (a) The cleaning agent used for the classroom, whose active ingredient consisted of alcohol ethoxylates, was reported ineffective for coronaviruses (2), and (b) The metal used for the doorknob has a direct effect on coronavirus stability; the classroom door knob was made of stainless steel, whereas brass is deleterious for coronaviruses (3) and would be a better choice in this case.

These findings are described in the article entitled Isolation and identification of human coronavirus 229E from frequently touched environmental surfaces of a university classroom that is cleaned daily, recently published in the journal American Journal of Infection Control. This work was conducted by Tania S. Bonny and John A. Lednicky from the University of Florida and Saber Yezli from the Ministry of Health, Riyadh, Saudi Arabia.

References:

  1. Bonny TS, Yezli S, Lednicky JA. Isolation and identification of human coronavirus 229E from frequently touched environmental surfaces of a university classroom that is cleaned daily. Am J Infect Control. 2018 Jan;46(1):105-107. doi: 10.1016/j.ajic.2017.07.014. Epub 2017 Oct 12.
  2. Ibfelt T, Engelund EH, Schultz AC, Andersen LP. Effect of cleaning and disinfection of toys on infectious diseases and micro-organisms in daycare nurseries. J Hosp Infect 2015;89:109-15.
  3. Warnes SL, Little ZR, Keevil CW.Human Coronavirus 229E Remains Infectious on Common Touch Surface Materials. MBio. 2015 Nov 10;6(6):e01697-15. doi: 10.1128/mBio.01697-15.
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