极端嗜盐多形性古菌病毒HRPV13的分离鉴定及生物学特性

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

摘要:

【背景】与感染细菌和真核生物的病毒相比，目前发现的古菌病毒数量很少，但是却展现出形态多样性。因此，分离和鉴定新的古菌病毒具有重要意义。【目的】为了进一步了解古菌病毒的多样性，我们从青海省翡翠湖水样中分离到一株新的嗜盐古菌病毒株，研究其生物学特性并进行分类。【方法】首先通过挑取单菌落法分离嗜盐古菌，通过噬菌斑法获得嗜盐古菌病毒，PEG 6000两步沉淀法和CsCl密度梯度离心对病毒颗粒进行浓缩和纯化，用醋酸双氧铀对病毒负染染色，在透射电镜下观察病毒形态，提取病毒基因组后进行测序并进行生物信息学分析，以三氯乙酸法制备病毒蛋白样品并进行SDS-PAGE分析，分别用考马斯亮蓝和苏丹黑B染色并观察其蛋白和脂质条带。【结果】在以Halorubrum属极端嗜盐古菌K2菌株为敏感菌的双层平板上分离到了一株嗜盐古菌病毒，其噬菌斑为浊斑，透射电镜下呈多形性包膜病毒状，直径60 nm左右；含有9 333 bp大小的双链环状DNA基因组，与已报道的β多形包膜病毒属(Betapleolipovirus)的HRPV11、HRPV12和HRPV10具有约75%的一致性，是该属的一个病毒新种。根据形态及基因组特征，将其归属于β多形包膜病毒属，命名为Halorubrum pleomorphic virus 13(HRPV13)。该病毒在50℃以下及pH 5.0−9.0具有较好活性，高盐浓度下具有侵染能力，而且病毒侵染宿主后不会造成宿主的大量裂解。【结论】通过电镜观察和生物学特性研究以及基因组测序分析，证实HRPV13为β多形包膜病毒属的新种，其分离和鉴定丰富了多形性病毒种类，这为研究不同区域多形性病毒的进化奠定了基础。

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.