Isolation of new Brazilian giant viruses from environmental samples using a panel of protozoa

Abstract

The Megavirales are a newly described order capable of infecting different types of eukaryotic hosts. For the most part, the natural host is unknown. Several methods have been used to detect these viruses, with large discrepancies between molecular methods and co-cultures. To isolate giant viruses, we propose the use of different species of amoeba as a cellular support. The aim of this work was to isolate new Brazilian giant viruses by comparing the protozoa Acanthamoeba castellanii, A. polyphaga, A. griffini, and Vermamoeba vermiformis (VV) as a platform for cellular isolation using environmental samples. One hundred samples were collected from 3 different areas in September 2014 in the Pampulha lagoon of Belo Horizonte city, Minas Gerais, Brazil. PCR was used to identify the isolated viruses, along with hemacolor staining, labelling fluorescence and electron microscopy. A total of 69 viruses were isolated. The highest ratio of isolation was found in A. polyphaga (46.38%) and the lowest in VV (0%). Mimiviruses were the most frequently isolated. One Marseillevirus and one Pandoravirus were also isolated. With Brazilian environmental samples, we demonstrated the high rate of lineage A mimiviruses. This work demonstrates how these viruses survive and circulate in nature as well the differences between protozoa as a platform for cellular isolation.

Keywords: Marseillevirus; Megavirales; Mimivirus; Pandoravirus; acanthamoeba; giant virus; isolation.

FIGURE 1

Pictures from the three areas where the environmental samples were collected represented by letters (A–C). (A) Point 1, before the chemical treatment; (B) Point 2, after the chemical treatment; (C) Point 3, 3 is an isolated area of the lagoon (Google Earth).

FIGURE 2

Venn diagram, showing amoebas supports and isolated viruses. A total of 69 virus were isolated in the three different platform cellular AC Acanthamoeba polyphaga (AP), Acanthamoeba griffinii (AG) are represented. Viruses come from the same species and lineages detected by PCR are represented with the same color. The interception of the circle show viruses isolates coming from the same environmental samples in different Acanthamoeba platform of isolation. All the small graphics inside the circle represents the species and lineage detected by PCR. Mimivirus that were not detected by PCR is also represented. The number and the percentual of viruses isolated are also expressed.

FIGURE 3

Nucleotides sequence of a fragment of the mimivirus DNA polymerase B gene. Samples obtained in this study are underlined; boldface indicates polymorphic.