Rowley Daniel L, Farrar Robert R, Blackburn Michael B, Harrison Robert L
Invasive Insect Biocontrol and Behavior Laboratory, USDA Agricultural Research Service, Plant Sciences Institute, 10300 Baltimore Avenue, Beltsville, MD 20705, USA.
Virus Genes. 2010 Jun;40(3):458-68. doi: 10.1007/s11262-010-0462-y. Epub 2010 Mar 6.
A PCR-based method was used to identify and distinguish among 40 uncharacterized nucleopolyhedrovirus (NPV) isolates from larvae of the moth Spodoptera frugiperda that were part of an insect virus collection. Phylogenetic analysis was carried out with sequences amplified from two strongly conserved loci (polh and lef-8) from the 40 isolates in the collection and from eight previously studied S. frugiperda NPV (SfMNPV) isolates. To further distinguish these isolates, analysis was also carried out with sequences from two less-conserved loci, hr4 and hr5. Phylogenetic inference from the sequence data could distinguish among several of the individual isolates and between different groups of isolates from Georgia (USA) and Colombia, South America. A stronger degree of bootstrap support for the phylogenetic trees was obtained with the hr4 and hr5 homologous repeat sequences. Sequencing and phylogenetic analysis detected a relatively high degree of larva-to-larva sequence divergence occurring among isolates of SfMNPV collected from the same field in Missouri, USA. Restriction endonuclease analysis of viral DNA from larvae infected with five isolates from Georgia, Missouri, Louisiana, Florida (USA), and Colombia allowed for further comparison with other previously reported isolates of SfMNPV. Bioassays with these five geographically distinct isolates detected minor differences in virulence. This study highlights the use of PCR to rapidly distinguish and characterize large numbers of historical baculovirus isolates from the same host using minimal quantities of material, and the use of sequences from homologous repeat regions to distinguish closely related isolates of the same NPV species.
采用基于聚合酶链反应（PCR）的方法，对昆虫病毒库中40株未鉴定的来自草地贪夜蛾幼虫的核型多角体病毒（NPV）分离株进行鉴定和区分。利用从该病毒库中40株分离株以及8株先前研究过的草地贪夜蛾NPV（SfMNPV）分离株的两个高度保守位点（多角体蛋白基因polh和晚期表达因子8基因lef-8）扩增得到的序列进行系统发育分析。为进一步区分这些分离株，还利用两个保守性较低的位点hr4和hr5的序列进行了分析。从序列数据进行的系统发育推断能够区分部分单个分离株以及来自美国佐治亚州和南美洲哥伦比亚的不同分离株组。利用hr4和hr5同源重复序列对系统发育树获得了更强程度的自展支持。测序和系统发育分析检测到在美国密苏里州同一田间采集的SfMNPV分离株之间存在较高程度的幼虫与幼虫间序列差异。对来自美国佐治亚州、密苏里州、路易斯安那州、佛罗里达州以及哥伦比亚的5株分离株感染的幼虫的病毒DNA进行限制性内切酶分析，以便与其他先前报道的SfMNPV分离株作进一步比较。对这5个地理上不同的分离株进行生物测定，检测到毒力存在微小差异。本研究强调了利用PCR以极少量材料快速区分和鉴定来自同一宿主的大量历史杆状病毒分离株，以及利用同源重复区域的序列区分同一NPV物种的密切相关分离株。
