2000-2007年間台灣柯沙奇A24型變異株分子流行病學研究

Molecular Epidemioloy of CA24 variant in Taiwan from 2000 to 2007

蔡延君(Yen-Chin Tsai)

指導教授 ： 林貴香

高雄醫學大學/醫學院/醫學研究所/碩士(2008年)

摘要

柯沙奇A24型變異株(Coxsackievirus A24 variant；CA24v)引起的急性出血性結膜炎(Acute hemorrhagic conjunctivitis；AHC)首次在1970年新加坡開始流行，之後分別在1975、1985-1986、1988-1989、1990-1994、2000-2001、2002、2006-2007流行於世界各地。為了瞭解CA24v在分子流行病學和基因在演化上之相關資訊，本研究將(1)建構自1970年至今全世界CA24v分離株之3C和VP1基因分子親緣分析樹，並(2)分析柯沙奇A24型變異株在演化上3C和VP1基因核苷酸與胺基酸基因序列取代情形，以及(3)台灣CA24v分離株其3C和VP1基因分子演化速率的比較，(4)最後，基因全長序列分析與基因重組研究將挑選台灣不同流行年代共四株分離株進行全長病毒基因體的定序。
在分子親緣樹分析方面，本研究挑選2000到2007年台灣CA24v分離株共18株，利用D1/U2引子經核酸增幅放大一個約674核苷酸大小產物片段(包含3C基因)，經定序後與來自全世界CA24v分離株共58株(包含原型株EH24/70)利用MEGA 4.0當中的Neighbor-joining method進行3C基因的親緣分析，得知親緣分析樹上可明顯區分為三個基因型，分別為Genotype I、II、III(GI-III)，Genotype III可再進一步細分為8個clusters(C1-C8)，其中2000-2001年台灣分離株屬cluster 7，而2002-2007年台灣與2005年新加坡分離株同屬cluster 8。本研究亦利用CA24v之部分VP1(235bp)建構親緣分析樹，將15株1985-2007年台灣CA24v分離株之VP1與世界各地共31株進行親緣分析。發現2002-2007年台灣分離株與2007年中國分離株屬於同一cluster。
在核苷酸與胺基酸變異方面，58株來自世界各地CA24v分離株之3C基因(510bp)核苷酸與胺基酸分別有86.5-98.5%和96.2-98.4%的相似度，而台灣分離株則分別有86.7-89.3%和96.7-98.9%的相似度。另外，流行於2000-2007年分離株共有23個變異特定發生在此群分離株，其中有4個為有意義突變造成胺基酸的變異，分別為98N→S、114V→I、151I→V、160M→I。在VP1基因(915bp)方面，本研究挑選14株台灣分離株和4株非台灣分離株(包含原型株)利用DNASTAR軟體進行分析，發現此群分離株VP1之核苷酸與胺基酸相似度分別為85.1-89.5%和92.8-96.4%。此外，流行於2000到2007年分離株VP1共有104個核苷酸發生變異，有34個變異特定發生在此分離株，其中有4個為有意義突變造成胺基酸的變異，分別為23K→R、25L→P/H、32S→L、100E→D。
在分子演化速率方面，分析過去22年(1985-2007)台灣CA24v分離株之3C和VP1基因核苷酸序列演化速率發現，3C基因(非結構基因)之核苷酸取代速率為1.39×10-3 nt/year，較VP1(結構基因)之1.96×10-3 nt/year的演化保守。最後，本研究挑選台灣不同流行年代CA24v分離株四株進行全長基因定序，此四株分離株全長基因序列與原型株之核苷酸相似性為85.6-88.5%。在結構基因方面，VP2與原型株相比有最高的相似性97-98.5%，而VP1則略低為93.1-94.8%。在非結構基因方面，以2C、3C、3D具有最高的相似性(皆大於95%)。
台灣自1985年流行CA24v以來，分離株皆屬Genotype III，其中1985-86年屬cluster 1、 1988-89年屬cluster 5、1990-94年屬cluster 6、2000-01年屬cluster 7、2002-07年屬cluster 8。分子演化速率的證據顯示VP1的演化速率略高於3C。在病毒株全長基因體研究方面，各功能區之核苷酸與胺基酸將與原型株做詳細比較。此外，insertion、deletion以及重組現象並沒有在本研究中發現。

關鍵字

柯沙奇A24型變異株

並列摘要

Acute hemorrhagic conjunctivitis caused by coxsackievirus A24 variant (CA24v) first appeared in Singapore in 1970, and was followed by several epidemics in 1975, 1985-1986, 1988-1989, 1990-1994, 2000-2001, 2002, and 2006-2007. In order to characterize information regarding the molecular epidemiology and genetic diversity, (1) phylogenetic trees were analyzed based on 3C and VP1 gene sequences of CA24v since 1970, (2) 3C and VP1 gene sequences of CA24v were used to analysis genetic/amino acid substitution, and (3) The evolutionary rate of 3C and VP1 gene will be re-estimated in Taiwanese isolates in 1985-2007. (4) To understand the homology of each gene region, full genome sequence of isolates from 1985 to 2007 in Taiwan and the prototype will be sequencing.
To analysis the 3C gene diversity of CA24v in Taiwan, D1/U2 primers were used to amplied a 674 nucleotides, which contains 3C gene. A total of 58 CA24v worldwide isolates (including prototype, EH24/70) were subjected to phylogenetic analysis based on the 3C gene (510bp) by Neighbor-joining method of MEGA 4.0, including the 18 strains identified in this study. Consistent with the results of previous studies, all 58 strains (including prototype) were divided into three genotypes (genotype I-III) on the phylogenetic tree. Genotype III can be further separated into eight clusters (C1-8). Molecular epidemiological analyses showed that isolates from 2000 to 2001 and from 2002 to 2007 in Taiwan were cluster 7 and 8 of genotype III separately, and the later was similar to the AHC strain isolated in Singapore in 2005.
The partial VP1 gene (235bp) was phylogenetically analyzed in 31 CA24v strains, including 15 strains identified in this study. Molecular epidemiological analyses showed that the cluster of isolates from 2002 to 2007 in Taiwan were similar to the those isolated in China in 2007.
Compare to prototype, 510 nucleotides of 3C gene of CA24v isolates, we found the nucleotide and amino acid identities of 3C region of 58 worldwide strains were 86.5-98.5% and 96.2-98.4%. However, the nucleotide and amino acid identities of 3C region of Taiwanese strains were 86.7-89.3% and 96.7-98.9% compare to prototype. Twenty-three nucleotides substitution and four amino acid change (98N→S、114V→I、151I→V、160M→I) were found at 3C gene of isolates from 2000 to 2007.
Fourteen strains from Taiwan and four non-Taiwanese strains were used to analysis nucleotide sequence of complete VP1 gene (915bp) of CA24v by using DNASTAR. The VP1 region showed nucleotide identities ranging from 85.1 to 89.5% and amino acid identities ranging from 92.8 to 96.4% compare to prototype. There are 34 nucleotide and 4 amino acid substitutions (23K→R、25L→P/H、32S→ L、100E→D) found at VP1 region of isolates from 2000 to 2007.
The evolutionary rate of CA24v was analyzed at the nucleotide level based on 3Cpro and VP1 capsid genes for Taiwanese strains, isolated during the past 22 years (from 1985 to 2007). The nucleotide substitution rate of the 3Cpro gene was estimated to be 1.39×10-3 nt/year, which was slightly more conserved than that of the VP1 capsid gene at 1.96×10-3 nt/year.
Finally, four virus strains from different epidemic (1986、2000、2001、2007) were randomly selected for full-length viral genome sequencing. In the structure region, VP2 has the most highest amino acid identities (97-98.5%) compare to prototype, and VP1 slighly lower than that of VP2. In the non-structure region, amino acid identities of 2C, 3C, 3D were higher than 95% compared to that of prototype .
Genotype III comprised of worldwide strains including Taiwanese strain since 1985. They were strains from 1985-1986(C1), 1988-1989(C5), 1990-1994(C6), 2000-2001(C7), and 2002-2007(C8). The VP1 gene had lower evolutionary rate than that of 3C gene. In the full genome sequencing of CA24v, pairwise nucleotide and amino acid comparisons of each region to prototype were showed in this study. However, neither insertion nor deletion was found, and recombination was also not found in this study.

並列關鍵字

CA24v

參考文獻

Alonso-Echanove J, García-Guadalupe Y, Rullán P, Pallansch MA, Alvarado-Ramy F, Cauthen B. 2003. Acute Hemorrhagic Conjunctivitis Outbreak Caused by Coxsackievirus A24 --- Puerto Rico. Morbidity and Mortality Weekly Report 53:632-634.

Andersson P, Edman K, Lindberg AM. 2002. Molecular analysis of the echovirus 18 prototype: evidence of interserotypic recombination with echovirus 9. Virus Res 85:71-83.

Andino R, Rieckhof GE, Baltimore D. 1990. A functional ribonucleoprotein complex forms around the 5′ end of poliovirus RNA. Cell 63:369-380.

Aoki K, Sawada H, Ishikawa H, Shimoji T, Kamada R, Chen CW, Huang WL, Sheu MM, Lin KH. 1987. An epidemic of hemorrhagic conjunctivitis due to coxsackievirus A24 in Okinawa Japan and Kaohsiung Taiwan. Jpn J Ophthalmol 41:755-758.

Arora RR, Gupta JP, Sabestian M. 1977. Epidemioligical and virological study of a conjunctivitis epidemic in Nem Delhi in 1975. Indian J Med Res 66:6-13.