Li K, Chen Z, Plagemann P
Department of Microbiology, Medical School, Minneapolis, Minnesota, 55455, USA.
Virology. 1999 May 25;258(1):73-83. doi: 10.1006/viro.1999.9660.
On the basis of genome nucleotide differences between a nonneuropathogenic and a neuropathogenic lactate dehydrogenase-elevating virus (LDV) quasispecies (LDV-P and LDV-C, respectively), we have designed sets of primers for polymerase chain reaction (PCR) amplification that can detect recombinants between them in a 1276-nt-long segment ranging from ORF 5 to ORF 7. Mice were infected with large amounts of both LDVs and bled at various times postinfection (p.i.). RNA was extracted from plasma samples and reverse transcribed and the first-strand products were PCR amplified with four sets of sense and antisense primers that discriminate between parental (P/P and C/C) and recombinant (P/C and C/P) genomic segments. Both P/C and C/P recombinants were detected in plasma from six different mice at 1 day p.i. No recombinant products were generated with in vitro mixtures of LDV-P and LDV-C. End-point dilution experiments indicated that the generation of P/C and C/P recombinants varied between mice but that in some mice the frequency of recombination in the 1276-nt-long genome segment was as high as 5%. Sequence analyses of clones of some recombinants indicated that recombination had occurred at 26- to 43-nt-long stretches of homology between the LDV-P and the LDV-C genomes. Sequence analyses of the 3157-nt-long 3' end of the genomes of the neuropathogenic LDV-v and of a newly discovered nonneuropathogenic quasispecies, LDV-vx, showed that LDV-v is a natural recombinant of LDV-vx that has specifically acquired by a double recombination about 400 nt of the 5' end of ORF 5 of the neuropathogenic LDV-C and thereby the unique properties of LDV-C, neuropathogenicity and high sensitivity to antibody neutralization. In dual infections of mice with LDV-P and LDV-C all genetic recombinants, like the LDV-C parent itself, had been lost by 7 days p.i., and only LDV-P persisted. The results further support the view that LDV-P and LDV-vx have evolved to a highly stable relationship with their host, the mouse.
基于非神经致病性和神经致病性乳酸脱氢酶升高病毒（LDV）准种（分别为LDV-P和LDV-C）之间的基因组核苷酸差异，我们设计了多组聚合酶链反应（PCR）扩增引物，可在从开放阅读框5到开放阅读框7的1276个核苷酸长的片段中检测它们之间的重组体。用大量的两种LDV感染小鼠，并在感染后（p.i.）的不同时间采血。从血浆样本中提取RNA并进行逆转录，然后用四组有义引物和反义引物对第一链产物进行PCR扩增，这些引物可区分亲本（P/P和C/C）和重组（P/C和C/P）基因组片段。在感染后1天，在六只不同小鼠的血浆中检测到了P/C和C/P重组体。用LDV-P和LDV-C的体外混合物未产生重组产物。终点稀释实验表明，P/C和C/P重组体的产生在不同小鼠之间存在差异，但在一些小鼠中，1276个核苷酸长的基因组片段中的重组频率高达5%。对一些重组体克隆的序列分析表明，重组发生在LDV-P和LDV-C基因组之间26至43个核苷酸长的同源区段。对神经致病性LDV-v和新发现的非神经致病性准种LDV-vx的基因组3157个核苷酸长的3'末端进行序列分析表明，LDV-v是LDV-vx的天然重组体，它通过两次重组特异性获得了神经致病性LDV-C开放阅读框5 5'末端约400个核苷酸，从而具有LDV-C的独特特性，即神经致病性和对抗体中和的高敏感性。在用LDV-P和LDV-C双重感染小鼠时，所有遗传重组体，就像LDV-C亲本本身一样，在感染后7天就消失了，只有LDV-P持续存在。这些结果进一步支持了这样一种观点，即LDV-P和LDV-vx已经与其宿主小鼠进化到了一种高度稳定的关系。
