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Genetic and biologic characterization of a porcine reproductive and respiratory syndrome virus isolate attenuated by cell culture passage

机译：通过细胞培养传代减弱的猪繁殖与呼吸综合征病毒分离株的遗传和生物学特性

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A highly virulent porcine reproductive and respiratory syndrome virus (PRRSV) isolate was serially passaged 84 times in a continuous cell line in order to produce an attenuated strain. Naive pigs were inoculated with VR-2385 passage 8 (p8) or passage 85 (p85) in order to determine if cell culture passage resulted in decreased virulence as compared to the parent isolate. The pigs inoculated with p8 VR-2385 showed moderate clinical respiratory disease and had an elevated temperature 5--11 days post-infection (DPI), whereas the pigs inoculated with p85 VR-2385 had only mild respiratory disease and no fever. Long term cell culture passage resulted in significantly (P ≤ 0.05) reduced gross lung lesions, microscopic lung lesions, and microscopic lymph node lesions at 10 and 31 DPI, as compared to the p8-infected group. At 42 DPI, however, interstitial pneumonia lesions were moderate in both the p8 and p85-infected groups. This suggests that there are temporal differences in the course of infection for the two passages of the virus. Serum virus titers were determined using selected pigs at 10, 28, and 35 DPI. The results of virus isolation and titration from sera indicated that p8 VR-2385 may replicate better in vivo than the p85 virus. Growth curves were conducted in both a continuous cell line and primary cells. VR-2385 p85 replicated to a higher titer in both cell types, indicating that replication in cell culture did not correlate with in vivo virulence. The complete genome of VR-2385 p85 was sequenced and compared to the sequence of the parental strain. The two strains of VR-2385 had greater than 99% nucleotide sequence identity. The structural proteins have amino acid identities of 95--98.3%, while the replicase proteins have identities of 99.0% for ORF1a and 99.5% for ORF1b. No clear genetic determinant of virulence was identified, indicating that the differences in virulence are due to small genetic changes. A model of high and low virulence strains from the same lineage was established to further investigate potential virulence markers.
机译：为了产生减毒株，将高毒力猪繁殖与呼吸综合征病毒（PRRSV）分离株在连续细胞系中连续传代84次。将未成熟的猪用VR-2385第8代（p8）或第85代（p85）接种，以确定与亲代分离株相比，细胞培养传代是否导致毒性降低。接种p8 VR-2385的猪表现出中度的临床呼吸道疾病，感染后5--11天（DPI）温度升高，而接种p85 VR-2385的猪仅出现轻度呼吸道疾病，无发热。与p8感染组相比，长期细胞培养传代可以在10 DPI和31 DPI显着（P≤0.05）减少总肺病变，微观肺病变和微观淋巴结病变。但是，在DPI为42 DPI时，p8和p85感染组的间质性肺炎病变均中等。这表明该病毒的两次传代在感染过程中存在时间差异。使用选定的猪在10、28和35 DPI下测定血清病毒滴度。从血清中分离和滴定病毒的结果表明，p8 VR-2385可能比p85病毒在体内复制更好。在连续细胞系和原代细胞中均进行了生长曲线。 VR-2385 p85在两种细胞类型中均复制至更高的滴度，表明细胞培养物中的复制与体内毒力无关。对VR-2385 p85的完整基因组进行测序，并与亲本菌株的序列进行比较。这两个VR-2385菌株具有大于99％的核苷酸序列同一性。结构蛋白的氨基酸同一性为95--98.3％，而复制酶蛋白的ORF1a同一性为99.0％，ORF1b为99.5％。没有确定毒性的明确遗传决定因素，表明毒性的差异是由于小的遗传变化所致。建立了来自同一谱系的高毒力和低毒力菌株模型，以进一步研究潜在的毒力标记。

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    Schommer, Susan Kay;

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  • 年度 2000
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  • 正文语种 en
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