体外传代对大口黑鲈蛙虹彩病毒毒株毒力的影响

doi:10.16535/j.cnki.dlhyxb.2022-323

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大连海洋大学学报 ›› 2023, Vol. 38 ›› Issue (4) : 623-629. DOI: 10.16535/j.cnki.dlhyxb.2022-323

体外传代对大口黑鲈蛙虹彩病毒毒株毒力的影响

王丛旭,潘晓艺,周可欣,穆雪娇,姚嘉赟,蔺凌云,赵建华,沈锦玉*

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Effect of in vitro passage on virulence of largemouth bass (Micropterus salmoides) ranavirus

WANG Congxu，PAN Xiaoyi，ZHOU Kexin，MU Xuejiao，YAO Jiayun，LIN Lingyun，ZHAO Jianhua，SHEN Jinyu*

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摘要

为研究不同代次大口黑鲈蛙虹彩病毒(Largemouth bass ranavirus，LMBV)的病毒特性变化及毒力差异，采用攻毒试验、qPCR、TCID50及二代测序的方法，对LMBV分离株(LMBV-ZJDSS)F5、F35和F65进行致病性、组织中病毒载量变化、病毒滴度和不同代次病毒基因组测序，在LMBV全基组中筛选出4个毒力基因，并对不同地区毒株的毒力基因进行分析。结果表明：攻毒浓度为108.5 TCID50/mL时，F5、F35和F65代毒株15 d内引起大口黑鲈(Micropterus salmoides)的死亡率分别为60%、25%和5%；F65代毒株最早产生细胞病变效应(cytopathic effect，CPE)；大口黑鲈肝脏、脾脏和肠的病毒载量较高，不同代次毒株在各组织中均有分布，96 h内F65代毒株在各器官组织的病毒载量呈上升趋势，48 h内F5代毒株在各器官组织的病毒载量呈上升趋势；对F5和F65代毒株的全基因组测序分析显示，E3、TNFR和ICP18基因编码区未发生突变，遗传稳定性较高，ICP46基因编码区有一处氨基酸由天冬氨酸突变为酪氨酸。研究表明，通过病毒的连续传代，病毒致细胞病变的速度加快，病毒对大口黑鲈的致病性减弱，病毒的毒力有所下降，毒力下降的原因可能是编码ICP46蛋白的氨基酸发生突变，且不同地区LMBV毒株毒力基因差异主要集中在TNFR上。

Abstract

The challenge test was conducted in largemouth bass (Micropterus salmoides) with body weight of (14±2)g to investigate the virulence of largemouth bass ranavirus (LMBV) in different generations. The growth kinetics of the LMBV virus were established with different titers by tissue culture infectious dose (TCID50) method, and the viral load was collected in tissue samples 1, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h after the challenge. The whole-genome sequences of F5 and F65 were obtained by next-generation sequencing and four virulence genes were screened out for the analysis of other genes from different strains. The results showed that lethality were found to be 60% in F5, 25% in F35 and 5% in F65 after challenge with 108.5 TCID50/mL. The higher virus load levels of the virus were detected in livers, spleens, and intestines, increase in virus loads in F65 and F35 at 96 h and 48 h post challenge. No mutations in E3, TNFR and ICP18 genes were observed among F5 and F65, and ICP46 had an amino acid mutation from aspartate to tyrosine, indicating that the stabilities of LMBV were observed during its successive passages. The mutation in ICP46 was hypothesized to be involved in the minimal virulence of F65. The findings were beneficial to verify the pathogenesis of LMBV and the application of its vaccine.

导出引用

王丛旭, 潘晓艺, 周可欣, 穆雪娇, 姚嘉赟, 蔺凌云, 赵建华, 沈锦玉. 体外传代对大口黑鲈蛙虹彩病毒毒株毒力的影响[J]. 大连海洋大学学报, 2023, 38(4): 623-629 https://doi.org/10.16535/j.cnki.dlhyxb.2022-323

WANG Congxu, PAN Xiaoyi, ZHOU Kexin, MU Xuejiao, YAO Jiayun, LIN Lingyun, ZHAO Jianhua, SHEN Jinyu. Effect of in vitro passage on virulence of largemouth bass (Micropterus salmoides) ranavirus[J]. Journal of Dalian Fisheries University, 2023, 38(4): 623-629 https://doi.org/10.16535/j.cnki.dlhyxb.2022-323

中图分类号： S 943