Mysterious Microbe in Water Challenges the Very Definition of a Virus

By Kelly A. Reynolds, MSPH, Ph.D.

Nicknamed ‘girus’ (gigantic virus), a new class of viruses called mimiviruses exhibit characteristics unlike other viruses. Originally mistaken for a Legionella-like bacterium, mimiviruses have a diameter of ~600 nm (0.6 µm), the largest known virus. In addition, they have complex genomes, with the greatest known genetic coding capacity of any virus. Like Legionella, their occurrence in finished water and association with pneumonia are a concern. Therefore, the need to properly classify mimiviruses and to determine their role in aquatic ecosystems and waterborne disease is evident.

Water origins
The original mimivirus isolate was collected in 1992 from amoeba within a cooling tower in Bradford, England. The organism was visible under the optical microscope, exhibited staining characteristics of bacteria, and thus was categorized as bacteria and given the name, Bradford coccus.¹ Researchers were sampling the cooling tower as part of an investigation of a pneumonia outbreak, with Legionella as a possible source. The Bradford coccus was identified as a new bacterial species at the time. Approximately a decade passed before La Scola et al. (2003) characterized the isolate as a new type of virus, mimivirus (mimicking microbe).² High-powered electron microscopy revealed an icosohedral shape and a capsid structure with extending protein filaments; in other words, a virus.

Like Legionella, mimiviruses are associated with the growth of free-living amoeba. Free-living amoeba are commonly isolated from drinking water sources, distribution systems and premise plumbing (plumbing inside buildings and residences; outside of US EPA’s regulatory jurisdiction). In the environment, amoeba serve as a host for mimiviruses and other microbes, including pathogens, to grow and be protected from environmental hazards and disinfectants. As with a number of other environmental isolates of bacteria, their ability to invade and persist in live amoeba cultures allowed for the development of a laboratory viability assay for the otherwise hard-to-grow microbes, and aided in their isolation from treated waters, such as humidifiers, cooling towers and hospital water systems.¹ Given that they grow in Acanthamoeba hosts, the viruses are often referred to as Acanthamoeba polyphaga mimivirus (APM). Environmental transmission routes and human pathogenicity factors of mimiviruses are currently unknown; however, genetic analysis suggests high concentrations are present in aquatic environments. A surprising number of large DNA viruses have been isolated from the marine environment, suggesting that viruses related to mimiviruses may be infecting marine microbes. Evidence of related viruses infecting marine sponges and corals is also growing.³ In any case, the large numbers of giruses in marine and freshwater environments suggest a prominent role in water ecology.

Novel discoveries
Genetic analysis of mimivirus isolates reveals still more surprising discoveries. Genetic similarities are noted between mimiviruses and the microbial communities of seawater and coral reef environments. In addition, various researchers found genetic relatedness to Entamoeba histolytica (a waterborne amoebic pathogen) and also a number of bacteria-like genes. Thus the genetic origins of mimiviruses, or other microbes, is debatable and has raised new evolutionary questions.

Viruses, by definition, are non-living, obligate intracellular parasites (microorganisms that cannot reproduce outside their host cell)—filterable agents, containing DNA or RNA but never both. These, and other common characteristics that previously defined viruses, are not consistent with mimiviruses. While they possess unique properties of viruses, they also have some of the cellular machinery associated with bacteria. Previously, viruses were recognized as being able to take over host-cell machinery, direct host cells to produce viral components and assemble into progeny viruses (new single infective viral particles called virions). Mimiviruses are not as dependent on host cell functions yet still do not possess the independent characteristics of a live bacterial cell. Newly published data suggest that the viruses are capable of replicating themselves but require other host- cell factors to initiate the process.⁴ Therefore, a new category of organisms has emerged—somewhere between a virus and a bacterium. Mimivirus has also been described as a “possible missing link between the cellular and the viral world”.⁵

Recently, more mysteries have unfolded regarding mimiviruses—they can be infected by other viruses, called Sputnik. Sputnik are 50-nm viruses that infect mimiviruses. They not only reduce the growth of APM in laboratory cultures, but change the morphology of the viruses; hence you have ’sick‘ viruses.⁵ A new strain of APM infected with Sputnik was isolated from a cooling tower in Paris in 2008. This new strain seemed even larger but similar to mimivirus and was differentiated via the name mamavirus.⁶

Emerging pathogens?
The discovery of mimiviruses has raised many questions across broad scientific disciplines. An obvious question from the water industry is: are mimiviruses, and related viruses, emerging waterborne pathogens? In addition, how did such large, prominent viruses, visible by optical microscopy, exist in aquatic organisms for so long without being noticed? The answer is probably that we were simply not looking for them.

In an attempt to lobby for more information, the American Society for Microbiology’s (ASM) Committee on Environmental Microbiology responded to requests from the US EPA for nominations of known or suspected waterborne pathogens that might be included in the third drinking water Contaminant Candidate List (CCL 3). Every five years, US EPA revises the CCL and targets specific chemical or microbial contaminants requiring increased data collection or consideration for national regulatory determination. The ASM proposed two candidates for the CCL 3: Naegleria fowleri, a highly fatal ameboflagellate, and mimivirus. CCL 3 was published in 2009 and included Naegleria fowleri as one of 12 priority microbial contaminants, but the mysterious mimivirus was not targeted for more research and evaluation.

Mimivirus may not be a regulatory priority due to inconclusive evidence of human pathogenicity. Several serology studies of humans with clinical cases of pneumonia have shown a detectable immune response to mimiviruses, suggesting current or previous infection. In one case, a 38-year-old laboratory technician working with the virus developed respiratory illness and pneumonia and tested positive for mimivirus antibodies. Antibody screening for mimiviruses was routinely performed with no positive results until symptoms occurred.⁷ The laboratory-acquired case added to the evidence of mimivirus infection previously suspected in a group of patients with community-acquired pneumonia in Canada; they tested positive for mimivirus antibodies but no other agents tested. Subsequent studies in France and among intensive-care-unit patients with pneumonia also documented a significant number of pneumonia patients with mimivirus antibodies compared to those without illness.⁷ Studies with experimentally inoculated mice provided further evidence of pneumonia in an animal model, with mimiviruses present in the lung but not proliferating.

Other studies have not found mimiviruses associated with pneumonia patients, including a survey of children (reviewed in Claverie and Abergel, 2009).⁴ Even in studies where antibody titers against mimivirus were high, the virus was never isolated from the mucosa of pneumonia patients. These mixed research results continue to raise doubt, despite the previously described associations of adverse effects of mimivirus infections in humans.

Final thoughts
Are mimiviruses an evolutionary missing link, providing clues about early life forms (’living viruses‘) or perhaps even a new form of life altogether? Do they cause disease in humans or contribute to the genetic exchange of information between larger organisms on earth? Why does the mimivirus have over 900 genes when other viruses are capable of replication with only five? Questions abound. Although mimiviruses are not bacteria, there may be some lessons to learn relative to the history of waterborne Legionella regrowth, infections and monitoring. Spread via the aerosol route, Legionella bacteria commonly grow in cooling towers, misters, water heaters and other aquatic environments, often under the protection of amoeba hosts. Legionella was added to the Center for Disease Prevention and Control’s (CDC) waterborne disease surveillance summary database of reportable diseases in 2001, and is now a regulated drinking water pathogen.

The most recent surveillance summary reports data from 2005-2006.⁸ Twenty outbreaks occurred during the two-year time frame with 10 outbreaks (50 percent) caused by Legionella growing in the plumbing system. Illnesses from Legionella resulted in four deaths, and is now the most common cause of waterborne outbreaks in the US, where an etiology is identified. Interestingly, the majority of contamination events occur in plumbing systems which are outside the jurisdiction of the regulatory agency. Chlorine residuals in premise plumbing are often very low, providing perfect conditions for growth of bacteria, free-living amoeba and their invading microbes like Legionella and mimiviruses.

Little is known about the ecology of mimiviruses, but they are present in natural waters along with the growth of free- living amoeba, like Legionella. Standard analytical methods for mimivirus have not been developed, and there are many data gaps, such as environmental occurrence, fate, transport, regrowth, treatment, exposure routes, health effects, etc. Maybe it’s time we start looking closer at this potential pathogen.

References

1. Raoult, D. et al., 2007. “The discovery and characterization of mimivirus, the largest known virus and putative pneumonia agent.” Clinical Infectious Diseases- Emerging Infections. 45:95-102.
2. La Scola, B., et al., 2003. “A giant virus in amoebae.” Science. 299:2033.
3. Claverie, J.M., et al., 2009. “Mimivirus and mimiviridae: giant viruses with an increasing number of potential hosts, including corals and sponges.” Journal of Invertebrate Pathology. 101: 172-180.
4. Claverie, J.M. and Abergel, C., 2009. “Mimivirus and its virophage.” Annual Reviews of Genetics. 43: 49-66.
5. Ogata, H. and Claverie, J.M., 2008. “How to infect a mimivirus.” Science. 321:1305-1306.
6. La Scola, B., et al., 2008. “A virophage is a unique parasite of the giant mimivirus.” Nature. 455: 100-105.
7. Raoult, D., et al., 2006. “Laboratory infection of a technician by mimivirus.” Annals of Internal Medicine. 144:702-703.
8. Yoder, et al., 2008. “Surveillance for waterborne diseases and outbreaks associated with drinking water and water not intended for drinking – United States,” 2005-2006. CDC-MMWR. 57(SS09): 39-62.

About the author

Dr. Kelly A. Reynolds is an Associate Professor at the University of Arizona College of Public Health. She holds a Master of Science Degree in public health (MSPH) from the University of South Florida and a doctorate in microbiology from the University of Arizona. Reynolds has been a member of the WC&P Technical Review Committee since 1997. She can be reached via email at [email protected]