图1  嗜盐古菌K2与相近种的系统发育树   用分子进化遗传分析软件(MEGA 7.0)绘制菌株的进化树，用邻接法(neighbor-joining method)构树并设置bootstrap为1 000，分支上面的数字表示1 000次重复的引导支持值，标尺表示遗传距离，括号中的序号是每个菌的GenBank序列号

Figure 1  Phylogenetic tree of halophilic archaea K2 and related species. The phylogenetic tree was constructed by neighbor-joining method and bootstrap was set to 1 000, the number on the branch was the leading support value of 1 000 replications, and the scale was the genetic distance. The serial number in parentheses is the GenBank serial number of each archaea.

图2  Halorubrum sp. K2双层平板上HRPV13病毒的噬菌斑形态

Figure 2  Morphology of the plaques produced by HRPV13 on Halorubrum sp. K2 lawn.

图3  HRPV13病毒的纯化及电镜观察结果   A：PEG 6000沉淀浓缩后的HRPV13病毒液进行CsCl (1.3 g/mL)密度梯度离心后形成的光散射带(箭头所示)；B：横坐标为CsCl密度梯度离心后从上到下每次取1 mL病毒液的层数，线形图表示每一层的浮密度，条形图表示每层病毒液对应的效价；C：TEM观察1%醋酸双氧铀负染后HRPV13的形态

Figure 3　Purification and electron microscope observation of HRPV13 virus. A: Light scattering band formed after PEG 6000 precipitation of concentrated HRPV13 virion by CsCl (1.3 g/mL) density gradient centrifugation (shown by arrow); B: The horizontal axis is the number of layers of 1 mL of viral solution taken from top to bottom each time after centrifugation of the CsCl density gradient, the line graph indicates the density of each layer, and the bar graph indicates the titer corresponding to each layer of virus; C: TEM observation of HRPV13 after negative staining with 1% uranyl acetate.

图4  HRPV13基因组用不同酶消化处理的结果   A：分别用DNaseI (终浓度为1 U/μg-DNA)、RNaseA (终浓度为5 μg/μg-DNA)、EXO Ⅲ (终浓度为10 U/μg-DNA)消化HRPV13基因组；B：用不同种类的限制性内切酶处理HRPV13基因组；C：用不同浓度的MBN (10、5和0.5 U/μg-DNA)和NaOH (终浓度为0.1 mmol/L)处理HRPV13基因组。M：λ-Hind Ⅲ DNA单切marker；U：未处理的基因组

Figure 4　The HRPV13 genome was digested with different enzymes. A: HRPV13 genome was digested with DNaseI (final concentration of 1 U/μg-DNA), RNaseA (final concentration of 5 μg/μg-DNA), and EXO Ⅲ (final concentration of 10 U/μg-DNA), respectively; B: HRPV13 genome was treated with different kinds of restriction enzymes; C: HRPV13 genome was treated with different concentrations of MBN (10 U/μg-DNA, 5 U/μg-DNA and 0.5 U/μg-DNA) and NaOH (0.1 mmol/L). M: The λ-Hind Ⅲ DNA single cleavage marker; U: The untreated genome.

图5  HRPV13与其他β多形包膜病毒基因组比较   同源的基因用相同的颜色表示。HRPV13的各ORF与其他β属多形性病毒对应的ORF用EMBOSS Needle进行氨基酸比对得到的相似度标在对应的位置上

Figure 5  Genome comparison of HRPV13 with other Betapleolipoviruses. Homologous genes are indicated with the same color. The similarity between each ORF of HRPV13 and the ORFs corresponding to other Betapleolipoviruses was marked on the corresponding positions by the amino acid alignment obtained by EMBOSS Needle.

图6  HRPV13和多形性病毒科病毒GBDP进化树构建   在核酸水平(A)和氨基酸水平(B)用GBDP构建的进化树，以上的GBDP的假定bootstrap均为100次。图A和B用的GBDP距离公式分别是D0和D6。当前属于(或拟属于)多脂病毒科同一属的病毒用相同的颜色标记，蓝色区域代表α多形包膜病毒，绿色区域代表β多形包膜病毒，黄色区域表示代表γ多形包膜病毒

Figure 6  Phylogenomic GBDP trees for HRPV13 and other pleolipoviruses. The phylogenetic tree constructed with GBDP at the nucleic acid level (A) and amino acid level (B), the assumed bootstrap of GBDP above is 100 times. The GBDP distance formulas used in A and B are D0 and D6, respectively. Viruses currently belonging (or are proposed to belong) to the same genus of the Polylipoviridae family are marked with the same color. The blue regions represent Alphapleolipovirus, the green regions represent Betapleolipovirus, and the yellow regions represent Gammapleolipovirus.

图7  HRPV13的吸附和繁殖特性   A：37 ℃时Halorubrum sp. K2对HRPV13的吸附率；B：感染(实心圆圈)和未感染(空心圆圈) Halorubrum sp. K2培养物的OD₆₀₀值，红色条形图表示感染后不同时间采集的培养物的游离病毒的效价；C：侵染(实心圆圈)和未侵染(空心圆圈)病毒的活菌计数。A–C中误差线代表标准偏差(n=3)

Figure 7  Adsorption and life characteristics of HRPV13. A: The adsorption rate of HRPV13 by Halorubrum sp. K2 at 37 ℃; B: Turbidity of infected (closed circles) and uninfected (open circles) Halorubrum sp. K2 cultures; The red bars indicate the potency of the free virus in cultures collected at different times after infection; C: Viable counts of infected virus (filled circles) and uninfected (open circles). Error bars represent one standard deviation in A, B and C (n=3).

图8  HRPV13在不同条件处理时的稳定性   A：HRPV13在不同温度处理时的效价；B：HRPV13用不同浓度NaCl处理3 h和24 h的效价；C：HRPV13在缺少一种或两种盐离子时处理3 h和24 h的效价；D：HRPV13在不同pH时处理3 h和24 h的效价。误差线代表标准偏差(n=3)

Figure 8  Stability of HRPV13 under different conditions. A: The titer of HRPV13 when treated at different temperatures; B: The titers of HRPV13 treated with different concentrations of NaCl for 3 h and 24 h; C: The titers of HRPV13 in the absence of one or both salt ions for 3 h and 24 h; D: The titers of HRPV13 treated at different pH for 3 h and 24 h. Error bars represent one standard deviation (n=3).

图9  HRPV13病毒蛋白及脂质成分   A：病毒蛋白用考马斯亮蓝染色的SDS-PAGE图，1–9表示CsCl密度梯度离心后从上往下每1 mL为一层取的样，其中3为目的层；B：苏丹黑B检测病毒脂质成分，位于10 kDa条带下方。M：蛋白marker

Figure 9  HRPV13 viral protein and lipid components. A: SDS-PAGE of viral proteins stained with Coomassie brilliant blue, line 1–9 represent samples taken from top to bottom after CsCl density gradient centrifugation as one layer per 1 mL, of which line 3 is the target layer; B: Sudan Black B detects viral lipid components, located below the 10 kDa band. M: Protein marker.