CAJ | 학술논문

利用RT－PCR从广东某猪场疑似猪瘟（ Classical swine fever， CSF）病例中检测出CSFV，并对扩增的E2全长基因进行了序列测定和系统进化分析。结果表明：引发该起猪瘟疫情的毒株属于CSFV 2?1b基因亚亚型，命名为GD176。为了进一步了解该毒株的复制特点，利用PK－15细胞对GD176进行了体外细胞适应性传代，并对获得的细胞适应毒GD176?F46进行了全基因组测序。结果表明：GD176通过在PK－15上不断传代，最终获得了高滴度的适应毒株，其滴度从第6代的102?61 TCID50／mL提高至第46代的108?06 TCID50／mL。病毒生长动力学结果显示，GD176?F46在感染后72 h病毒滴度达到最高值（108?17 TCID50／mL）。为进一步分析GD176在细胞传代适应过程中的稳定性，对不同代次细胞毒的E2全长基因进行扩增和序列测定，结果发现：GD176?F0与GD176?F6、F10、F15、F20、F25、F30、F35、F40和F46的E2基因序列完全一致，这表明囊膜蛋白E2在病毒适应PK－15细胞过程中非常稳定。通过比对GD176和其他2?1亚型毒株各蛋白的氨基酸序列发现，不同毒株之间同源性最小的病毒蛋白是NS5A，低于病毒囊膜糖蛋白E2。获得了高度适应PK－15细胞的GD176细胞毒，为进行病毒毒力评价、复制和致病特性研究奠定了基础。
이용RT－PCR종엄동모저장의사저온（ Classical swine fever， CSF）병례중검측출CSFV，병대확증적E2전장기인진행료서렬측정화계통진화분석。결과표명：인발해기저온역정적독주속우CSFV 2?1b기인아아형，명명위GD176。위료진일보료해해독주적복제특점，이용PK－15세포대GD176진행료체외세포괄응성전대，병대획득적세포괄응독GD176?F46진행료전기인조측서。결과표명：GD176통과재PK－15상불단전대，최종획득료고적도적괄응독주，기적도종제6대적102?61 TCID50／mL제고지제46대적108?06 TCID50／mL。병독생장동역학결과현시，GD176?F46재감염후72 h병독적도체도최고치（108?17 TCID50／mL）。위진일보분석GD176재세포전대괄응과정중적은정성，대불동대차세포독적E2전장기인진행확증화서렬측정，결과발현：GD176?F0여GD176?F6、F10、F15、F20、F25、F30、F35、F40화F46적E2기인서렬완전일치，저표명낭막단백E2재병독괄응PK－15세포과정중비상은정。통과비대GD176화기타2?1아형독주각단백적안기산서렬발현，불동독주지간동원성최소적병독단백시NS5A，저우병독낭막당단백E2。획득료고도괄응PK－15세포적GD176세포독，위진행병독독력평개、복제화치병특성연구전정료기출。
Classical swine fever virus ( CSFV) is the causative agent of highly contagious swine dis?ease CSF that has occurred in many countries over the world including China. Recently a CSF-sus?pected clinical sample from Guangdong province was collected and detected as CSFV positive by RT-PCR. Phylogenetic analysis based on the nucleotide sequence of full?length E2 gene showed that the CSFV isolate GD176 belongs to sub?subgenotype 2?1b of CSFV. To further understand the repli?cation characteristics of this sub?subgenotype 2?1 isolate, GD176 was isolated and adapted in PK-15 cells, whole?genome sequence of GD176cell?adapted virus was amplified and sequenced. Adapta?tion of GD176 in PK-15 cells started at F6 with 102?61 TCID50/mL and was increased to 108?06 TCID50/mL at F46. Growth dynamics of GD176?F46 with a MOI at 0?1 indicated that the peak titer reached at 72 h postinfection (108?17TCID50/mL). To further define the stability of GD176 during cellular adaptation, full?length E2 genes of different cell?adapted viruses of GD176 were sequenced. As a result, E2 gene of GD176?F0 was found to be identical with that of GD176?F6, F10, F15, F20, F25, F30, F35, F40 and F46, indicating the genetic stability of E2 gene during in vitro adap?tation of GD176 in PK-15 cells. In addition, comparison of individual viral protein showed that the most variable protein between GD176 and other 2?1 sub?subgenotype isolates is NS5A rather than vi?ral envelope protein E2. Overall, highly cell?adapted CSFV sub?subgenotype 2?1b strain GD176 was obtained in this study and it will be useful for future study on viral virulence, replication and patho?genesis.