利用口餵途徑感染人類SCARB2基因轉殖小鼠探討腸病毒A71型變異點在致病性上的影響

Pathogenesis study of Enterovirus-A71 variants in intragastric infection of human SCARB2 -Transgenic weaned mice

陳彥丞(Yen-Cheng Chen)

指導教授 ： 林靜宜

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202101991

摘要

病毒A71型是會造成手足口症的其中一種病毒，是屬於小RNA病毒科、腸病毒屬的正股RNA病毒，一般的感染症狀為嘔吐、發燒、紅疹、起水泡等，少數情況下會在幼童引起嚴重的無菌性腦膜炎及腦炎等中樞神經感染症狀，嚴重甚至會導致死亡。然而腸病毒A71型如何在人體內擴散感染致中樞神經系統的作用機轉仍不清楚。為了探討腸病毒在各組織中的基因變異以及其組織特異性，利用實驗室過去所建立的帶有冷光報導基因的腸病毒A71型重組病毒以口餵感徑感染21天大人類SCARB2基因轉殖小鼠，可以觀測到病毒在動物模式中擴散以及複製的情況。本研究利用此動物模式連續偵測感染腸病毒小鼠之冷光訊號，並於第三天出現較強訊號時收取12個組織器官，萃取RNA並利用次世代定序偵測病毒基因的變異。資料分析後發現變異佔百分之二十五以上之單核酸變異包括脊髓中的5’UTR -G597C、VP1-V69和VP1-E213血液中的VP1-A188K和3B-K8N。接著我們進一步針對單核酸變異，利用點突變的方式製造突變病毒，並於細胞模式及動物模式分析該點突變在病毒組織特異性上所扮演的腳色。結果發現於中樞神經系統發現的5’UTR-G597C和3B-K8N單點突變病毒感染橫紋肌瘤細胞株細胞(Rhabdomyosarcoma cell)以及膠質母細胞瘤 (SF268 cell)下，產生之RNA複製能力、病毒蛋白質表現亮以及病毒效價與野生型相比略微下降。然而在動物實驗方面5’UTR-G597C及3B-K8N單點突變腸病毒以口餵感染小鼠模式動物可以發現，相比於野生病毒株有較早產生神經致病性的現象。利用次世代定序發現到了腸病毒在各個組織的基因變異，在小鼠動物實驗中證實這些變異點會增強腸病毒致病性，其可能是經由病毒擴散的機轉造成致病性的差異，而詳細機轉還有待更多的實驗來驗證。

關鍵字

腸病毒A71型 ； 活體冷光顯影系統 ； 次世代定序 ； 單一核苷酸變異 ； 小鼠模式動物

並列摘要

Enterovirus A71 (EV-A71) is one of main causes of hand, foot and mouth disease (HFMD). Enterovirus A71 infection can also lead to severe neurological disease which can be the cause of mortality in children. However, the transmission of Enterovirus A71 from gastrointestinal tract to central nerve system still remained unclear. Here, we reported an Enterovirus A71 reporter construct containing nano-luciferase gene which can be used in in vivo imaging system and we inoculated this reporter virus in weaning aged human SCARB2-transgenic mice with intragastric gavage. With this natural infection route animal model, we can easily detect real time dynamics of viral replication and patterns of viral spreading in weaning aged mice. We next harvested the organs of infected mice detected with strong nano-luciferase signal in central nerve system and extract total RNA of each organ for high-throughput sequencing. We found several single nucleotide variants in Enterovirus A71 genome with over 25% portion of total reads, including 5’UTR G597C in spinal cord and VP1-A188K and 3B-K8N in blood. These results suggest that EV-A71 populations were quasispecies from different tissue or organs of virus-infected mice. In order to characterize the pathogenesis of these single point variants in EV-A71. We cloned these single point variants into EV-A71-MP4 infectious clone and analyzed two of these variant virus, 5’UTR G597C and 3B-K8N, in both cell based and animal model experiment. The RNA synthesis, viral protein expression and viral titer of EV-A71-MP4 5’UTR G597C and EV-A71-MP4 3B-K8N were lower than EV-A71-MP4 WT. To gain insight of the pathogenesis in these EV-A71-MP4 variants in mouse model. The EV-A71-MP4 variants were intragastric infected to 21-day-olds hSCARB2 transgenic mouse and the virulence of EV-A71-MP4 variants were significantly higher than EV-A71-MP4 WT. Moreover, EV-A71-MP4 with 5’UTR G597C variant has indication of improved transmission ability. Overall, we have identified several variants from EV-A71-MP4-Nluc5 with our intragastric infected 21-day-old human SCARB2-transgenic mice with NGS. The variants which identified from NGS data play important roles in EV-A71-MP4 virulence and to uncover the underlie mechanism more research is required.

並列關鍵字

Enterovirus A71 (EV-A71) ； in vivo imaging system (IVIS) ； next-generation sequence (NGS) ； single nucleotide variants (SNVs) ； mouse model

參考文獻

1. Huang, S.-W., D. Cheng, and J.-R. Wang, Enterovirus A71: virulence, antigenicity, and genetic evolution over the years. Journal of Biomedical Science, 2019. 26(1): p. 81.

2. Schmidt, N.J., E.H. Lennette, and H.H. Ho, An Apparently New Enterovirus Isolated from Patients with Disease of the Central Nervous System. The Journal of Infectious Diseases, 1974. 129(3): p. 304-309.

3. Chumakov, M., et al., Enterovirus 71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria. Arch Virol, 1979. 60(3-4): p. 329-40.

4. Nagy, G., et al., Virological diagnosis of enterovirus type 71 infections: experiences gained during an epidemic of acute CNS diseases in Hungary in 1978. Archives of virology, 1982. 71(3): p. 217-227.

5. Ho, M., et al., An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N Engl J Med, 1999. 341(13): p. 929-35.