豬流行性下痢病毒之親緣動態演化分析

Abstract

自2010年以來，屬於Genogroup 2的新變異PEDV在中國傳播，並進一步傳播到美國和其他亞洲國家包括台灣。為了詳細描述豬流行性下痢病毒 (PEDV) 的時間和地理關係，本研究系統地評估了整個PEDV基因組的每個基因的演化模式和親緣關係的解析，以確定哪些基因提供最大的解析力。從GenBank下載了48個全基因組序列，並分為三個數據集，即Worldwide，Genogroup 2和China，以涵蓋不同範圍的時間和空間趨勢。然後每個數據集被不同的基因區分成不同的序列比對，用於似然性映射和親緣關係分析。我們的研究顯示，NSP3和S基因都具有最高的親緣關係訊號，具有與從完整基因組獲得的相似的取代率和親緣關係拓撲結構。將來自三株PEDV感染豬的臨床檢體的NSP3基因序列與來自GenBank的其他病毒株進行比對，結果顯示導致台灣2014年爆發的PEDV與來自Genogroup 2的美國演化分枝I聚集在一起。此外，我們從唯一台灣的全序列PEDV發現此新變異PEDV是一經過重組 (斷點位置就在NSP3後面及S基因前面) 的病毒。因此，本研究的結果提供日後研究者需要瞭解新發生流行的病毒是否為重組病毒，可藉由定序NSP3 和S基因即可得知，而不需做全長定序。從PEDV全基因組所建構的PEDV族群演化史顯示PEDV起源於1956年，病毒以每年1.79×10-3核苷酸取代/位點的速度演化。全球的PEDV可分為G1及G2基因群；又分別區分G1a、G1b (Classical clade) 與G2a、G2b (Pandemic clade) 亞群。無論Worldwide、Genogroup2及China數據集中的S基因均有比同數據集中的NSP3及E基因較快的演化速率。PEDV的有效族群規模變化 (Bayesian skyline plot；BSP) 顯示從1978年至2010年保持穩定，然而PEDV S基因在2012年起的遺傳多樣性有比較明顯的變化，可能與2013年在美國爆發的新變異毒株有關。全基因組的平均dN/dS的比值均小於1，顯示處於負向選擇，但在S及NSP3基因仍可檢測出個別的陽性選擇位點。2013至2014年在美國、日本及台灣爆發的新變異PEDV起源地點均屬豬隻飼養密度較高地區，並經由豬隻、飼料及化製車的長距離運輸及農場間近距離相互傳播可能是造成快速散播的主因。應用BSP分析，配合mismatch distribution分析及Tajima's D 中性檢測partial S基因等族群變動分析結果顯示2013至2014年爆發的新變異PEDV是處於族群擴張狀態。其中以台灣爆發的新變異PEDV的partial S基因有最快的演化速率及最大的遺傳距離。儘管美國、日本及台灣的病毒株在遺傳上密切相關，但病毒的演化速率、遺傳距離與選擇壓力不同，這可能與飼養環境及生物安全等其他因素有關。

Since 2010, a new variant of porcine epidemic diarrhea virus (PEDV) belonging to Genogroup 2 has been transmitting in China and further spreading to the Unites States and other Asian countries including Taiwan. In order to characterize in detail the temporal and geographic relationships among PEDV strains, the present study systematically evaluated the evolutionary patterns and phylogenetic resolution in each gene of the whole PEDV genome in order to determine which regions provided the maximal interpretative power.Forty-eight full genome sequences were downloaded from GenBank and divided into three groups, namely, worldwide, Genogroup 2 and China, to cover different ranges of spatio-temporal trends. Each dataset was then divided into different alignments by different genes for likelihood mapping and phylogenetic analysis. Our study suggested that both NSP3 and S genes contained the highest phylogenetic signal with substitution rate and phylogenetic topology similar to those obtained from the complete genome.The NSP3 gene sequences from three clinical samples of swine with PEDV infections were aligned with other strains available from GenBank and the results suggested that the virus responsible for the 2014 PEDV outbreak in Taiwan clustered together with Clade I from the US within Genogroup 2.In addition, we found that the new variant PEDV from the only Taiwanese full genome sequence PEDV is a virus that has been recombined (the breakpoint is just behind NSP3 and in front of the S gene). Therefore, the results of this study provide a method for future researchers to reveal whether a newly occurring virus is a recombinant virus by sequencing the NSP3 and S genes without the need for full genome sequencing.The evolution history of the PEDV population constructed from the full genome of PEDV showed that PEDV originated in 1956,and the virus evolved at a rate of 1.79×10-3 nucleotide substitution per site per year.The global PEDV can be divided into G1 and G2 genogroups, which can be further divided into G1a, G1b (Classical clade) and G2a, G2b (Pandemic clade) subgroups.The S genes in the Worldwide, Genogroup2, and China datasets all have faster rates of evolution than the NSP3 and E genes in the same dataset.The effective population size (Bayesian skyline plot；BSP) change of PEDV showed that it remained stable from 1978 to 2010. However, the genetic diversity of PEDV S gene began to change significantly in 2012 and this may be correlated tothe occurrence ofthe new variant strains that caused the outbreak in the United States in 2013.The mean dN/dS ratio of the full genome was less than 1, indicating a negative selection; however the individual positive selection sites were still detected in the S and NSP3 genes.The new variant PEDV origins in the United States, Japan and Taiwan from 2013 to 2014 were in areas with high pig breeding density. Long-distance transportation through pigs, feed and rendering truck, and close communication between farms may be the main cause of rapid spread.Application BSP analysis, combined with mismatch distribution analysis and Tajima's D neutral test of partial S gene, showed that the new variant PEDV that led to the outbreak from 2013 to 2014 was in a state of population expansion.Among them, the partial S gene of the new variant PEDV that resulted in the outbreak in Taiwan has the fastest evolution rate and the largest genetic distance.Although the strains of the United States, Japan and Taiwan are genetically closely related, the evolution rate, genetic distance and selection pressure of the virus are different, indicating that other factors such as feeding environment and biosecurity are also relevant.