Isolation and Quantification of Mimivirus-Like and Marseillevirus-Like Viruses from Soil Samples in An Aboriginal ( Orang asli) Village in Peninsular Malaysia

Giant amoebal viruses which were discovered over a decade ago comprised of 2 distinct families: Mimiviridae and Marseilleviridae. Viruses of the family Mimiviridae had been isolated and characterized with increasing frequency recently in Asian countries such as Japan, India, and Saudi Arabia [1-3]. However, at the time of writing, there were no reports of giant amoebal viruses detected in South East Asia. Due to the ubiquitous nature of giant amoebal viruses, both Mimivirus and Marseillevirus have been found in a wide range of environments. These include water samples from man-made structures like cooling tower and from natural sources like river, lake, or ocean [4-9]. They are also found in soil, on other animals like leech and oyster, and disturbingly, some on human samples [10-15]. In this paper, we describe the isolation and distribution both Mimivirus-like and Marseillevirus-like viruses from the Serendah Village, which is an aboriginal settlement in Peninsular Malaysia.

Nine sampling points (SR1-9) were tagged across Serendah village (Central Peninsular Malaysia; Fig. 1). For each sampling point, 9 samples were collected within a 500-m radius of the point. These samples were pooled, transported in cold chain, and subsequently stored at –80°C. Isolation of Mimivirus or Marseillevirus was performed through blind passages on Acanthamoeba castellanii (ATCC 30010) until cytopathic effects (CPE) were observed. Infected Acanthamoeba castellanii were then Giemsa-stained to detect the viral factories. DNA was extracted from the pooled soil samples using TIANamp soil DNA kit (TIAN, China) according to the manufacturer’s protocol. In those samples where Mimivirus-like or Marseillevirus-like viruses were successfully isolated, phylogeny was performed by using specific primers targeting the helicases of Mimivirus and Marseillevirus as previously described by Slimani et al. [16, 17]. DNA sequences were deposited in the Genbank (MG981284-7). Quantification of Mimivirus and Marseillevirus partial viral genome copies in the soil samples was performed as previously described by Ngounga et al. [18].

Out of the 9 sampling sites, 4 of them (SR1, 4, 5, and 9) were demonstrated to have CPE on the cultured Acanthamoeba castellanii after several rounds of blind passages. Viral inclusion factories were spotted in these cultures  with CPE that were stained with Giemsa stain, indicating Mimivirus-like infection (see online suppl. Fig. S1; for all online suppl. material, see www.karger.com/doi/10.1159/000491602). Further molecular identification through the construction of phylogenetic tree showed that Mimiviruses were detected on SR1, 4, and 9. The virus detected in SR5 belonged to Marseillevirus (Fig. 2). Quantification of the Mimivirus from all 9 sampling sites showed that these viruses were present in high levels in the respective soils where Mimiviruses were isolated (SR1, 4, and 9) and not in other sampling sites. Similarly, Marseillevirus was detected at a high level in SR5 (Fig. 3).

Although the impact of these viruses to the health of the Serendah village inhabitants is not known yet, there are of increasing evidences supporting that Mimiviruses and Marseillevirus are infectious agents in humans. These giant viruses had been isolated and detected from clinical specimens from patients as well as healthy individuals [15, 19]. In addition, Mimivirus was detected in domestic animals such as Bos taurus and evidences of neutralizing antibodies have been reported in farmers handling domestic animals, indicating the risk of exposure of Mimiviruses from the animals [20].

This study also showed that spatial distribution of Mimivirus-like and Marseillevirus-like viruses in the -Serendah village differ among the collection points especially where water source is within the vicinities. The data indicated that viral titers were higher in soils (SR1, SR4, SR5, and SR9) which are near the water source and lower in samples further away from the water sources. Marseillevirus-like virus was isolated from soils collected at the vicinity of the health clinic (SR5), located on top of the slope leading to the bank of a stream. Similarly, Mimivirus-like virus was isolated from soils collected at the water fall (SR1), midway to the water fall (SR4), and ex-agricultural land (SR9) situated next to a stream opening out to a lake. To conclude, viruses of Mimiviridae were discovered in Peninsular Malaysia further supporting the ubiquitous nature of the Mimivirus and Marseillevirus. This study warrants a further investigation on the circulation and epidemiology of Mimiviridae in aboriginal settlements in Malaysia.

We are grateful to Dr Chan Li Li for her generosity in providing Acanthamoeba castellanii culture.

All data generated or analyzed during this study are included in this published article and its online supplementary Figure and Table.

This article does not contain any studies with human participants or animals performed by any of the authors.

All authors consent for publication.

All authors declare non-competing interest.

This study was funded by International Medical University (BP I-01/11(37)2014 and BPI-01/12(13)2015).

K.V. and C.W.C. designed the experiments; Y.F.T. and C.Y.L. contributed to sample processing, viral DNA extraction, PCR and subcloning; K.V. performed the viral culture. PLKC provided the helminths egg counts data. P.P.L. and K.V. provided essential reagents and helped write the paper; C.W.C. provided valuable suggestion. Y.F.T. and K.V. revised the paper. All authors read and approved the final manuscript.