Detection of new DNA polymerase genes of known and potentially novel herpesviruses by PCR with degenerate and deoxyinosine-substituted primers
Detection of new DNA polymerase genes of known and potentially novel herpesviruses by PCR with degenerate and deoxyinosine-substituted primers
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DOI：
10.1023/a:1008064118057
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发表时间：
1999-06-01
期刊：
影响因子：
1.6
通讯作者：
Buhk, HJ
中科院分区：
文献类型：
--
作者：
Ehlers, B;Borchers, K;Buhk, HJ
本研究采用聚合酶链式反应方法对野生马匹和动物园马匹(斑马、野驴、绒毛马)中疱疹病毒的存在情况进行了研究，并研究了鸽子疱疹病毒(疱疹病毒1型)与其他疱疹病毒物种的亲缘关系。基于针对疱疹病毒DNA聚合酶基因高度保守区域的简并引物，通过使用简并和脱氧肌苷取代的引物的混合物进行了修改。通过扩增已发表的16种疱疹病毒的DNA聚合酶基因以及马疱疹病毒3型(EHV-3)和马疱疹病毒5型(EHV-5)的DNA聚合酶基因，验证了该修饰的适用性。然后用改进的方法对柱状疱疹病毒1型的聚合酶进行部分扩增，目前根据生物学标准将其归类为β-疱疹病毒。对4株不同毒株的扩增产物序列分析表明，带状疱疹病毒1型与甲型疱疹病毒亚科成员关系密切，尤其是与马立克氏病疱疹病毒关系更为密切。另外1.6kb的柱状疱疹病毒1型聚合酶的特性证实了这一点。对斑马、一头野驴和一头美洲斑马的血液样本进行的共识PCR分析显示，扩增产物显示出与伽马疱疹病毒DNA聚合酶高度相同的序列百分比(>；50%)。特别是斑马与野驴的序列之间以及与马伽玛疱疹病毒EHV-2和EHV-5聚合酶的关系密切，序列同源性为80%。这是首次表明野生和动物园马匹中存在新型伽马疱疹病毒。
A consensus primer PCR approach was used to (i) investigate the presence of herpesviruses in wild and zoo equids (zebra, wild ass, tapir) and to (ii) study the genetic relationship of the herpesvirus of pigeons (columbid herpesvirus 1) to other herpesvirus species. The PCR assay, based on degenerate primers targeting highly conserved regions of the DNA polymerase gene of herpesviruses, was modified by using a mixture of degenerate and deoxyinosine-substituted primers. The applicability of the modification was validated by amplification of published DNA polymerase genes of 16 herpesvirus species and of the previously uncharacterized DNA polymerase genes of equine herpesvirus 3 (EHV-3) and equine herpesvirus 5 (EHV-5). The modified assay was then used for partial amplification of the polymerase of columbid herpesvirus 1 which is presently classified as a beta-herpesvirus based on biological criteria. Sequence analysis of amplicons obtained from four different viral strains revealed a close relationship of columbid herpesvirus 1 to members of the subfamily Alphaherpesvirinae, especially to Marek's disease herpesvirus. This was confirmed by characterization of additional 1.6 kb of the columbid herpesvirus 1 polymerase. Consensus PCR analysis of blood samples from zebras, a wild ass and a tapir revealed amplicons showing high percentages (> 50%) of sequence identity to DNA polymerases of gamma-herpesviruses. In particular, the zebra and the wild ass sequence were closely related to each other and to the polymerases of the equine gamma-herpesviruses EHV-2 and EHV-5 with sequence identities of > 80%. This is a first indication that novel gamma-herpesviruses are present in wild and zoo equids.