Isolation, identification, and biological characterization of extremely halophilic pleomorphic archaeal virus HRPV13

Abstract:

[Background] Compared with viruses infecting bacteria and eukaryotes, archaeal viruses are limited in number but morphologically diverse. Therefore, it is of great significance to isolate and identify new archaeal viruses. [Objective] To gain an in-depth understanding of the diversity of archaeal viruses, we isolated a new halophilic archaeal virus from water samples of Feicui Lake in Qinghai Province and analyzed its biological characteristics and taxonomic status. [Methods] Halophilic archaea were isolated by picking single colonies and the archaeal virus was obtained by plaque assay. Virus particles were concentrated and purified by two-step precipitation with PEG 6000 and CsCl density gradient centrifugation. After the negative staining with uranyl acetate, viruses were observed under a transmission electron microscope. Viral genome was extracted and sequenced and the bioinformation was analyzed. Virus samples were concentrated by trichloroacetic acid (TCA) and applied to SDS-PAGE gels for separation of viral proteins, followed by Coomassie brilliant blue staining and Sudan Black B staining, respectively, to observe the protein and lipid bands. [Results] A halophilic archaeal virus was isolated on a double-layer plate with Halorubrum sp. K2 as sensitive bacteria. The plaques were hazy with a polymorphic enveloped virus-like shape under transmission electron microscope, with a diameter of about 60 nm. It had the double-stranded circular DNA genome of 9 333 bp in size and shared about 75% identity to the reported HRPV11, HRPV12, and HRPV10 in Betapleolipovirus, which was a new viral species of this genus. According to morphology and genome characteristics, it was named Halorubrum pleomorphic virus 13 (HRPV13) in Betapleolipovirus. The virus can still infect bacteria at high salt concentration and had high viability at below 50 °C and pH 5.0–9.0. Moreover, it failed to cause significant lysis of host after infection. [Conclusion] HRPV13 is a new polymorphic species in Betapleolipovirus, as verified by electron microscopy, biological characterization, and genome sequencing. The result lays a foundation for research on the evolutionary relationship of polymorphic viruses in different regions.