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Viruses from extreme environments are now easier to detect in the lab

Culture techniques help detecting the infection of micro-organisms by viruses, but cannot be applied to some species living in the extreme environment of oceanic hydrothermal vents: another technique had to be designed to be able to study them.

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All living organisms are infected by viruses more or less specific of their species; this is also true for the Achaea, microorganisms belonging to the third domain of life. Studying the viruses infecting this group (called Archaeoviruses) is important because Archaea mainly live in extreme environmental conditions: high temperature, high pressure, absence of oxygen, presence of toxic compounds, etc. One environment where such conditions can be found is in the deep ocean, in hydrothermal vents located along mid-oceanic ridges.


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Purified fraction of virus TPV1 observed by transmission electronic microscopy (black bar: 150 nanometres)



A common and simple method for the detection and quantification of viruses is to inoculate a culture of host cells with a potentially infecting viral solution. Cells are grown on a culture medium. If they are sensitive, the virus multiplies inside them and eventually causes them to burst in order to disseminate into the medium. The lysis of infected cells around the point of virus inoculation produces a clear area known as a plaque, by which the viral infection can be detected.

However whereas virus reproduction is possible only within host cells, it does not always cause their lysis; therefore this technique is not applicable to all virus species. Indeed most Archaeviruses are not lytic and their infection does not form plaques on the culture medium. This is the case for PAV1 and TPV1, the only viruses known so far to infect marine hyperthermophilic Archaea (Thermococcales order). These Archaea are among the dominant groups in the oceanic hydrothermal vents, where they live under very high pressure, between 85°C and 110°C and in a water without oxygen but rich in sulfur. As PAV1 and TPV1 multiply in host cells and are released without causing their lysis, they cannot be detected by the classical plaque technique. In addition, not only are Thermococcales very difficult to cultivate in the laboratory on solid medium, but when they extract colloidal sulfur from the medium to meet their metabolic needs, the white milky aspect of the medium changes around them into a clear halo that is indistinguishable from a plaque caused by the infection by lytic viruses.

The aim of this study was to develop a technique based on the same principles but applicable to the case of Thermococcales and their non-lytic viruses. Strains of 15 Archaean species were incorporated into an appropriate solid culture medium, prepared with a hot gel and rapidly poured into Petri dishes before hardening. In each dish, three inoculations were made with pure fractions of PAV1 and TPV1 viruses. Dishes were incubated without oxygen and at temperatures of 85°C or 90 °C according to species. After six hours of incubation, observations were made every two hours for thirty hours.

Contrary to the traditional method, the spots appearing on the medium were not translucent but opaque. In the absence of viruses, cell growth causes dissolution of the colloidal sulfur of the medium and the clearing of its milky suspension; conversely, where viruses infect host cells and inhibits their growth, the culture medium is not transformed and remains turbid, thereby forming a trouble halo around the points of infection. If the interpretation of plaques is identical (detection of the viral infection), their origin is different as they are not formed by the modification of the medium appearance but on the contrary by the persistence of its original appearance.

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Before incubation (left), the medium is opaque; circles indicate the points of viral infection. After sixteen hours of incubation (right) the white plaques (arrows) in a medium which became translucent elsewhere shows the viral infection.

Some Thermococcales species were infected, others not. The presence of viruses within the plaques and their absence elsewhere were confirmed by the observation of culture extracts under epifluorescence microscopy: a large number of virions (free form of the virus) were observed in the white zones, whereas they were absent elsewhere. In addition, two controls introduced into the experiment confirmed that the inhibition of cell growth and thus the persistence of white spots was indeed caused by the virus. Firstly, the liquid used to purify the viruses does no cause the apparition of plaques when it is used alone, i.e. without viruses. Secondly, the presence of viruses alone (i.e. without host cells) in the culture medium has no action on its appearance.

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Epifluorescence microscopy confirms that viruses are very abundant in the white plaques (right) and absent in the clear zones (left)

The method thus developed makes it possible to remove the technical barriers specific to the study of Thermococcales Archaea. It enabled to identify a host range for these viruses, i.e. which species can be infected by them. It is an original, rapid and cost-effective method to determine their infectivity and to test several viral samples on a single culture; however its limitation is that, unlike the original method, it cannot be used to obtain a viral titer to quantify the amount of viruses. It also appears to be promising for other purposes, such as screening for new viruses or determining the specificity of the effect of diverse chemicals and biological agents against sulfur-metabolising and thermophilic prokaryotes.


The paper

Gorlas A. and Geslin C., 2013. A simple procedure to determine the infectivity and host range of viruses infecting anaerobic and hyperthermophilic microorganisms. Extremophiles 17:349–355.

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The authors

Both authors of this paper are members of the Laboratoire de microbiologie des environnements extrêmes (LMEE) of IUEM.


The journal

Extremophiles is a recent scientific journal (founded in 1997) of the international publisher Springer. Its interests are on the biology, structure, function and applications of microbial life at the limit conditions of survival: high or low temperature, pressure, acidity, alkalinity, salinity, or desiccation; or in the presence of organic solvents, heavy metals, normally toxic substances, or radiations.



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