犬冠狀病毒之基因分型、病毒分離與序列分析

Abstract

冠狀病毒可以感染許多種動物並且造成不同的臨床症狀。犬冠狀病毒屬於第一群冠狀病毒中的A亞群，依據基因型別又可將之分成第一型犬冠狀病毒與第二型犬冠狀病毒。自從1970年代初期確診第一隻犬之下痢症狀為犬冠狀病毒所導致之後，此病毒一直被認為只會造成犬隻輕微下痢；但近年來發現具有強毒力的第二型犬冠狀病毒，會造成犬隻全身性臨床症狀且具有致命性。在台灣，本實驗室曾針對犬瘟熱疑似病例做犬冠狀病毒之調查，發現有47.5%之陽性率；本實驗將其中30 隻犬冠狀病毒陽性病例進行基因分型，結果發現其中有3隻為第一型犬冠狀病毒感染、8隻為第二型犬冠狀病毒感染與3隻混合兩型感染的病例，其它病例無法分型。 本實驗進一步使用犬纖維瘤細胞株(A-72)嘗試從兩隻病犬之糞材中分離第二型犬冠狀病毒，接種病毒後可以觀察到細胞皺縮與圓形化等細胞病變；其中一隻在盲目繼代七代後免疫螢光染色與RT-nPCR雖為陽性，但病毒仍無法大量的在此細胞株中被增殖出來。 由於冠狀病毒容易藉由重組交換大片段的基因形成新病毒株，造成新的疾病出現；而完整病毒基因體序列可提供重組病毒演化分析時之正確資訊。犬冠狀病毒發現至今已有將近40年歷史，全世界至今尚未有全長的序列被報告。因此本研究自下痢病犬之糞材利用反轉錄聚合酶鏈鎖反應增幅基因體核酸片段，同時使用末端快速增殖技術確立基因體末端，將產物經由反覆定序分析連接後確立其基因序列，結果得到全長為 29,226 nt （尚不包含 5’ end）之序列，將之命名為CCoVII/NTU336/F/2008，簡稱為NTU336。經由軟體分析後，發現NTU336與TGEV-like CCoV type II最為相似，且可在ORF 1b基因下游與S基因的上游發現TGEV與CCoV type II重組之位置。NTU336在S基因之後亦保留了ORF 3部份序列，此序列存在於CCoV type II、TGEV-like CCoV type II、FCoV type II與TGEV這些病毒基因中，此為上述這些病毒是由CCoV type I演化而來的證據；在比較第一群A亞群冠狀病毒，發現NTU336之M基因多了15-21 nt，為最大的M基因。另外 NTU336之ORF 1a到ORF 1b上游與ORF 1b下游到S基因上游與TGEV具有演化上共同祖先，而ORF 1b中游與S基因下游之後則與台灣CCoV type II有共同演化來源；此結果顯示NTU336與TGEV在演化上有相關性，而部份基因片段與台灣貓冠狀病毒NTU156之間亦存有地緣相關性。

Coronaviruses (CoVs) cause a variety of different clinical forms of disease in a wide range of animal species. Group 1a Canine coronaviruses (CCoVs) can be divided into two genotypes, CCoV type I and CCoV type II. The viruses are known to be mild enteropathogen of dogs since their first recognition in early 1970s. Recently, CCoV type II strains with uncommon virulence, including a pantropic variant causing systemic disease in puppies, have been reported. In this study, 80 samples collected from canine distemper suspected dogs were tested for CCoV, and 47.5% were found positive by RT-nPCR. Among 30 CCoV positive dogs, 3 dogs were positive for CCoV type I, 8 dogs for CCoV type II and 3 dogs for both genotypes. We further tried to use a canine fibroma (A-72) cell line to isolate CCoV type II from two fecal samples. After co-incubation, cytopathy effects with the characteristic of shrinking and rounding up of cells can be observed. One of the sample was immunofluorescence assay (IFA) and RT-nPCR tests positive after 7 passages. However, active multiplication of the viruses were unable to be observed on the cell line. High frequency of recombination of CoVs have been the reason of emerging of new disease. Full length of genomic sequence can provide the correct informations for the analysis of the evolution of recombined virus. Up to present, none of a whole genomic data concerning CCoV has been reported in the world. Here we reported the whole genomic RNA sequence of a field strain CCoV II. A consensus sequence of the feces-derived genomic RNA (CCoV II/NTU336/F/2008, NTU336) was determined from overlapping cDNA fragments produced by reverse transcriptase polymerase chain reaction (RT-PCR) amplification. RT-PCR products were sequenced by a reiterative sequencing strategy and the genomic RNA termini were determined using a rapid amplification of cDNA ends PCR strategy. The full length of NTU336 is 29226 nt, excluded poly-A tail and 5’ end. This local virus shows a higher genetic relatedness to TGEV-like CCoV type II bearing a recombination site in downstream of ORF 1b and upstream of S gene. Like other CCoV type II related viruses, CCoV type II, FCoV type II, TGEV and TGEV-like CCoV type II, which had ORF 3 residues after S gene. This is a evidence of CCoV type II related viruses are evolutionary from CCoV type I. Among group 1a CoVs, NTU336 has the largest M gene resulted from a 15-21 nt insertion. SimPlot and DNAstar analyses of the NTU336, TGEV, and FCoV type II, reveal NTU336 and TGEV sharing common ancestor on ORF 1a, downstream of ORF 1b, upstream of S gene. On the other parts, NTU336 is closely related to CCoV type II, and some parts are close related to NTU156, a FCoV type II virus of Taiwan.