Moormann R J, van Gennip H G, Miedema G K, Hulst M M, van Rijn P A
Virology Department, Institute for Animal Science and Health, Lelystad, The Netherlands.
J Virol. 1996 Feb;70(2):763-70. doi: 10.1128/JVI.70.2.763-770.1996.
Infectious RNA was transcribed for the first time from a full-length cDNA template of the plus-strand RNA genome of a pestivirus. The genome of the C strain, which is a vaccine strain of classical swine fever virus, was sequenced and used to synthesize the template. The cDNA sequence of the C strain was found to be 12,311 nucleotides in length and contained one large open reading frame encoding a polyprotein of 3,898 amino acids. Although there were mostly only small differences between the sequence of the C strain and the published sequences of strains Alfort and Brescia, there was one notable insertion of 13 nucleotides, TTTTCTTTTTTTT, in the 3' noncoding region of the C strain. Furthermore, we showed that the sequences at the 5' and 3' termini of the C strain are highly conserved among pestiviruses. We found that the infectivity of the in vitro transcripts of DNA copies pPRKflc-113 and pPRKflc-133 depended on the correctness of the nucleotide sequence. The in vitro transcripts of pPRKflc-133 were infectious, whereas those of pPRKflc-113 were not. In fact, only 5 amino acids among the complete amino acid sequence determined this difference in infectivity. However, virus FLc-133, which was generated from pPRKflc-133, cannot be differentiated from native C-strain virus. Therefore, we exchanged the region encoding the antigenic N-terminal half of envelope protein E2 in pPRKflc-133 with the equivalent region of strain Brescia. The resulting hybrid virus, FLc-h6, could be differentiated from the C strain and from FLc-133 with monoclonal antibodies directed against envelope proteins Erns and E2 of strain Brescia and the C strain. To be suitable for further vaccine development, viruses generated from pPRKflc-133 should grow at least as well as native C-strain virus. In fact, we found that FLc-133, hybrid virus FLc-h6, and the C strain grew equally well. We concluded that pPRKflc-133 is an excellent tool for developing a classical swine fever marker vaccine and may prove valuable for studying the replication, virulence, cell and host tropism, and pathogenesis of classical swine fever virus.
传染性RNA首次从瘟病毒正链RNA基因组的全长cDNA模板转录而来。经典猪瘟病毒疫苗株C株的基因组被测序并用于合成模板。发现C株的cDNA序列长度为12311个核苷酸，包含一个大的开放阅读框，编码一个3898个氨基酸的多聚蛋白。虽然C株的序列与已发表的阿尔福特株和布雷西亚株的序列大多只有微小差异，但在C株的3'非编码区有一个13个核苷酸（TTTTCTTTTTTTT）的显著插入。此外，我们表明C株5'和3'末端的序列在瘟病毒中高度保守。我们发现DNA拷贝pPRKflc - 113和pPRKflc - 133的体外转录本的感染性取决于核苷酸序列的正确性。pPRKflc - 133的体外转录本具有感染性，而pPRKflc - 113的则没有。实际上，在完整氨基酸序列中只有5个氨基酸决定了这种感染性差异。然而，由pPRKflc - 133产生的病毒FLc - 133无法与天然C株病毒区分开来。因此，我们将pPRKflc - 133中编码包膜蛋白E2抗原性N端一半的区域与布雷西亚株的等效区域进行了交换。产生的杂交病毒FLc - h6可以用针对布雷西亚株和C株包膜蛋白Erns和E2的单克隆抗体与C株和FLc - 133区分开来。为了适合进一步的疫苗开发，由pPRKflc - 133产生的病毒生长情况应至少与天然C株病毒一样好。事实上，我们发现FLc - 133、杂交病毒FLc - h6和C株生长情况相同。我们得出结论，pPRKflc - 133是开发经典猪瘟标记疫苗的优秀工具，可能对研究经典猪瘟病毒的复制、毒力、细胞和宿主嗜性以及发病机制具有重要价值。
