Wyatt L S, Carroll M W, Czerny C P, Merchlinsky M, Sisler J R, Moss B
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892, USA.
Virology. 1998 Nov 25;251(2):334-42. doi: 10.1006/viro.1998.9397.
The severely attenuated and host range (HR) restricted modified vaccinia virus Ankara (MVA) was derived by >500 passages in chick embryo fibroblasts, during which multiple deletions and mutations occurred. To determine the basis of the HR defect, we prepared cosmids from the parental vaccinia virus Ankara genome and transfected them into nonpermissive monkey BS-C-1 cells that had been infected with MVA. Recombinant viruses that formed macroscopic plaques were detected after transfections with DNA segments that mapped near the left end of the viral genome. Plaque-forming viruses, generated by transfections with four individual cosmids and one pair of minimally overlapping cosmids, were purified, and their HRs were evaluated in BS-C-1 cells, rabbit RK-13 cells, and human HeLa, MRC-5, and A549 cells. The acquisition of the K1L and SPI-1 HR genes and the repair of large deletions were determined by polymerase chain reaction or pulse-field gel electrophoresis of NotI restriction enzyme digests of genomic DNA. The following results indicated the presence of previously unrecognized vaccinia virus HR genes: (1) the major mutations that restrict HR are within the left end of the MVA genome because the phenotypes of some recombinants approached that of the parental virus, (2) acquisition of the K1L gene correlated with the ability of recombinant viruses to propagate in RK-13 cells but did not enhance replication in human or monkey cell lines, (3) acquisition of the SPI-1 gene correlated with virus propagation in A549 cells but not with the extent of virus spread in monkey or other human cell lines, (4) there are at least two impaired HR genes because rescue occurred with nonoverlapping or minimally overlapping cosmids and recombinant viruses with intermediate HRs were isolated, and (5) at least one of the new HR genes did not map within any of the major deletions because the size of the left terminal NotI fragment was not appreciably altered in some recombinant viruses.
严重减毒且宿主范围（HR）受限的改良痘苗病毒安卡拉株（MVA）是通过在鸡胚成纤维细胞中传代500多次获得的，在此过程中发生了多个缺失和突变。为了确定HR缺陷的基础，我们从亲本痘苗病毒安卡拉基因组制备了黏粒，并将其转染到已感染MVA的非允许性猴BS-C-1细胞中。在用定位在病毒基因组左端附近的DNA片段转染后，检测到形成宏观噬斑的重组病毒。用四个单独的黏粒和一对最小重叠黏粒转染产生的噬斑形成病毒被纯化，并在BS-C-1细胞、兔RK-13细胞以及人HeLa、MRC-5和A549细胞中评估其HR。通过聚合酶链反应或基因组DNA的NotI限制性酶切消化的脉冲场凝胶电泳来确定K1L和SPI-1 HR基因的获得以及大缺失的修复。以下结果表明存在先前未被认识的痘苗病毒HR基因：（1）限制HR的主要突变位于MVA基因组的左端，因为一些重组体的表型接近亲本病毒，（2）K1L基因的获得与重组病毒在RK-13细胞中的增殖能力相关，但不增强在人或猴细胞系中的复制，（3）SPI-1基因的获得与病毒在A549细胞中的增殖相关，但与病毒在猴或其他人细胞系中的传播程度无关，（4）至少有两个受损的HR基因，因为用非重叠或最小重叠黏粒进行了拯救，并且分离出了具有中等HR的重组病毒，（5）至少有一个新的HR基因不在任何主要缺失范围内，因为在一些重组病毒中左端NotI片段的大小没有明显改变。
