New Giant Virus in Free-Living Amoeba
First Discovery of a Mimivirus-Like Giant Virus in Saccamoebae
- Fig. 1: Saccamoeba lacustris SL-5 18 h post infection with KLS-5. A large virus factory (vf) is surrounded by Platanovirus capsids (pc) at various assembly stages. Inset: KSL-5 virion with multilayer envelop (e) and fibrils (f) packing DNA (d). Between the virions are open precursor membranes (om).
- Fig. 2: Saccamoeba lacustris SL-5 24 h post infection with assembling Platanovirus capsids (pc) and a large number of small Comedo virus (cv) particles within in the virus factory (vf), fibrils (f) envelope (e), dense core (dc), vertex (v). Inset: Satellite virus Comedo, negative staining.
- Fig. 3 A: Negative staining image of KSL-5x virion with head (h) and very long stalk (s). B: Vertex (v) of KSL-5x with star gate (sg), negative staining.
Two similar Mimivirus-like giant viruses KLS-5 and KSL-5x were isolated from a sycamore tree. One isolate, KSL-5, was associated with a small satellite virus. The phylogenetic analysis of the host amoeba identified it as Saccamoeba lacustris. Only S. lacustris strains were susceptible to the new giant virus KLS-5 indicating a highly species-specific virus system in Saccamoeba lacustris. The name Platanovirus saccamoebae for the new giant virus is proposed.
Strains of free-living amoebae (FLA) are frequently used to trap and isolate giant viruses of different families. . Namely Acanthamoeba castellanii was shown to be susceptible to infections with a wide range of giant viruses . Pithovirus sibericum  and Mollivirus sibericum  were both isolated from permafrost soil using Acanthamoeba polyphaga as bait. In other cases, Vermamoeba vermiformis was successfully applied to capture isolates of Faustoviruses [5-7]. In most of these cases the identity of the natural host of the viruses or their host range remained however obscure.
In contrast, most of our isolates such as Pithovirus lacustris [8-10] and Pandoravirus inopinatum [11-13] were isolated within their natural hosts. In this study, we present a Mimivirus-like endoparasite (KSL-5) of Saccamoeba sp. (SL-5), that was isolated from the bark of a sycamore tree . Saccamoeba sp. (SL-5) could be identified as Saccamoeba lacustris by gene sequencing. Since this is the first Mimivirus-like organism that was isolated from a Saccamoeba we suggest the name “Platanovirus saccamoebae”. In addition to the giant virus, the Saccamoeba strain contained a small satellite virus . In order to study the developmental stages of the new giant virus (KSL-5), a continuous culture of the endoparasite in its original host (SL-5) was initiated. We also investigated whether FLA other than Saccamoebae were susceptible to an infection with the new isolate KSL-5. In a further experiment, a virus free strain of the host amoeba SL-5 was mixed with crushed material from the same sycamore tree resulting in the isolation of a second giant virus: KLS-5x.
The second isolate was to our surprise not associated with a satellite virus .
Material and Methods
Small pieces of loose fitting bark of a sycamore tree were transferred onto non nutrient (NN)-agar plates pre-seeded with Enterobacter cloacae as food bacteria. After liberation from various concomitant organisms, mass cultures for ultrastructural investigations were initiated. Plates with infected saccamoebae were incubated at 20°C and harvested at various time intervals. To study the infection cycle virus free SL-5 cells were reinfected with KSL-5 suspensions and samples were taken from 0 to 96 hours post infection. Transmission electron microscope (TEM) preparation was preceded as described before . Various other amoeba species were inoculated with equal aliquots of KSL-5 to study the host range of the new giant virus.
For the isolation of the second giant virus KSL-5x material from the bark of the same sycamore tree was crushed, suspended in amoeba saline ) and added to NN-agar plates pre-seeded with virus free cells of Saccamoeba sp. SL-5. The development of an infection was monitored daily by light microscopy. At the peak of the infection after 24 h cells were harvested for ultrastructural analysis. To study the infectious potential of the satellite virus it was purified in consequent filtration steps (5, 1.2 and 0.22 µm polycarbonate filters). Aliquots of filtered satellite virus were added to virus free cultures of SL-5 and monitored for signs of infection by light microscopy.
The phylogenetic identity of host amoeba Saccamoeba sp. was analysed by 18S rRNA gene sequencing.
Results and Discussion
18S rRNA gene analysis identified the host amoeba SL-5 with 99% similarities as Saccamoeba lacustris. Saccamoeba lacustris giant virus KSL-5 is a polyhedral virus with a vortex at one side and a neck or stem like protrusion at the opposite end. The average size of the capsids is 290 nm. The average length of the fibrils is 140 nm, resulting in an average virion size of 430-450 nm. Replication of the virus started after a long lag phase of at least 12 h. First virus particles and a developing virus factory were detected after 18 h with a peak at 30 h (fig. 1). There were still viable host cells after 96 h post infection. The virus factory was comparable to the virus factory seen in acanthamoebae . Virus assembly was mainly observed at the periphery of the virus factory starting with the formation and assembly of precursor membranes.
After 24 h, a small icosahedral virus of 50-60 nm became visible within the virus factory (fig. 2). The small virus seemed to interfere with the replication of KSL-5. Small virus capsids were detected within empty capsids of the giant virus resulting in defective giant virus particles.
We were able to infect only one other Saccamoeba lacustris strain (SL elo) with the new giant virus. Virus factories were produced but they were smaller in size (1-2 µm) than in the original host (3.5 – 6 µm). No accompanying satellite viruses could be detected. The infection was self-limiting and disappeared after a few transfer cycles.
All attempts to transfer KLS-5 to other amoeba including several Acanthamoeba species failed.
Preliminary results of a sequence analysis show the highest similarity of KSL-5 to Megavirus chilensis (~75%)
Regarding the satellite virus, we were unable to detect any signs of infection in Saccamoeba lacustris SL5-5 when the giant virus was missing .
The infection with the second isolate KSL-5x was more vigorous for the host cells. The virus factory presented itself star like with long projections and within two days the entire host cell was completely filled with virus particles causing the cells to finally burst. Virions showed very long necks or rather stalks (fig. 3, A) comparable to some bacteriophages .
A five-pointed-star structure similar to the star gate of other Mimiviridae  was visible opposite of the stalk (fig 3, B).
This is the first giant virus reported in Saccamoeba. The new giant virus showed many ultrastructural similarities with giant viruses of the Mimiviridae. However the average size of the new virus capsid is smaller than that of other Mimiviridae but their fibrils are longer. The development of the virus factory started after a much longer lag phase (12 – 18 h) compared to other Mimiviridae (4 - 5 h) and the size of the virus factory was smaller. Ultrastructural details and the association with a satellite virus suggest it to be a Mimivirus-like virus that was also supported by a preliminary sequence analysis. Except for one close related Saccamoeba lacustris strain, none of the other amoeba strains tested were susceptible to an infection with KLS-5. We therefore consider it to be a new giant virus within the Mimiviridae and suggest the name “Platanovirus saccamoebae”.
The accompanying satellite virus seems to affect the replication of the giant virus in favour of the host cell. It behaves like virophage Sputnik [20,21]. We therefore believe that it is a new virophage and suggest the name Comedo from the Latin word comedere (to eat, to devour).
We thank Dirk Bannert for excellent support in mounting the photographs.
Bärbel Hauröder1, Claudia Wylezich2, Liane Junglas1, Silke Loch1, Jenny Eisenkolb1,3, Rolf Michel1
1Central Hospital of the Deutsche Bundeswehr Medical Services, Koblenz, Germany
2Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany
3Institute for Integrated Natural Sciences, University Koblenz-Landau, Koblenz, Germany
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