Isolation And Biological Characterization Of Halobacterial Viruses From Salt Mines In Yunnan, China

Viruses are extraordinarily important for their roles in biological evolution and in ecological systems of material circulation, energy flow. With the rapid development of molecular biology, genomics and bioinformatics, an increasing numbers of evidences show that viruses play an essential part in horizontal gene transfer among different species. Halophiles and halophilic viruses lived in hypersaline environments are of great interest because of their special biological properties and evolutionary functions.Salt mines in Yunnan, China, as an important parts of hypersaline environments, have not been reported on their diversity of viruses until our research.Using electronic microscopy and double-layer plating method, virus particles were isolated and identified from the enriched water samples or soil samples of high-salt mines in Yunnan, China, and their morphological characteristics were further described and classified, i.e., three types of virus-like particles, head and tail-shaped virus, linear virus and spherical virus, were observed. The electron microscopy morphology of those virus-like particles were similar to those that isolated from the same kind of environments in Italy, Israel, Thailand, Spain and some other places, and most of them were head-tail viruses. In this research, two Halomonas viruses and two Chromohalobacter viruses were subsequently isolated from Qiaohou and Yipinglang salt mines in Yunnan, China. According to the sample source, electron microscopic morphology and characteristics of viruses, and genus names of the host strains, novel viruses were proposed as Qiaohou Halomonas Siphoviridae Virus1, YiPingLang Halomonas Siphoviridae Virus1, YiPingLang Chromohalobacter Pleomorphic Virus1, and YiPingLang Chromohalobacter Pleomorphic Virus2, and they were denoted as QHHSV-1, YPHSV-1, YPCPV-1, YPCPV-2, respectively. A bacteriovirus, named as QHHSV-1, infecting Halomonas sp. QH52-2were further studied. QHHSV-1was characterized by electron microscopy, biological, physical and chemical properties, SDS-PAGE analysis, enzyme digestion analysis, and genome sequencing and analysis..QHHSV-1is a typical siphovirus, which belongs to caudaevirus, with a47nm diameter icosahedral head and a75nm length tail. It can form clear plaques with a diameter of3mm on MLB with concentrations of2-25%NaCl, the optimum infecting concentration is5%NaCl. QHHSV-1has good thermal stability in neutral alkaline environment, and its optimal pH value is8.0. It is insensitive to chloroform and nonionic detergent Triton X-100, and less sensitive to the ionic detergent SDS. However, after treaed with proteinase K, its infection activity almost completely lost. It can be concluded that the proteins of QHHSV-1without lipid envelope and the capsid protein structure has certain specificity. According to the analysis of SDS-PAGE, four structural protein bands with preliminary estimated molecular size of48,42,34, and17kDa, respectively. Enzyme digestion analysis showed that QHHSV-1genome can be digested by BamHI, EcoRI, XhoI restriction enzymes, but HindⅢ doesn’t digest its genome.The genome of phage QHHSV-1, determined by genome annotation, had a contig length of37,270bp. Genomic analysis revealed that QHHSV-1genome consists of dsDNA with a GC content of66.80%. Analysis of the genome sequence suggests that QHHSV-1genome contains at least72coding genes with an average length of479bp and a single repetitive sequence (1,631bp), which with non-coding RNA. Thirty of the coding genes appear to be unique to QHHSV-1, as BLAST analysis didn’t reveal significant matches. Studying the diversity and distribution of halophilic viruses could make us obtain novel viruses resources and new functional genes, which also might help us to understand the halostability mechanism in halophiles and deserved for further study. The further analysis of haloviruses’ coding gene, protein and halophilic bacteria transcription mode and their corresponding proteins, supporting us to understand the complex interactions between specific virus and host under infection process of them in hypersaline environments.