Rodríguez Saint-Jean Sylvia, de Las Heras Ana I, Pérez Prieto Sara I
Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Departamento de Microbiología Molecular, C/Ramiro de Maeztu 9, 28040 Madrid, Spain.
Vet Immunol Immunopathol. 2010 Jul;136(1-2):81-91. doi: 10.1016/j.vetimm.2010.02.015. Epub 2010 Feb 26.
Persistent infection by IPNV was induced in RTG-2 and RTG-P1 cells in vitro and the influence of this phenomenon on viral infectivity, viral antigen expression and interference with homologous and heterologous viruses was characterized over successive passages. The induction of IFN was also assessed, as was the sequence of the VP2 viral capsid protein, the region believed to be responsible for virulence, attenuation or persistence. Viral antigen expression was recorded in cells with no evidence of cytopathic effects and in these conditions, flow cytometry was more sensitive than RT-PCR to demonstrate the presence of a non-lytic virus. Interference of homologous viral infection could be detected in cross-infection experiments and in RTG-P1 cells persistently infected with IPNV, the Mx1 promoter could still be activated for at least 5 successive passages. Indeed, although over-induction of luciferase was not observed by re-infection with homologous or heterologous viruses, a significant increase in luciferase was induced by poly I:C. IFN transcripts could be quantified by qRT-PCR in the persistent cells at several passages, suggesting that IFN plays a role in maintaining IPNV persistence. In addition, we observed the same determinants in the VP2 sequences from the persistent virus as those described previously for IPNV adaptation and persistence in cell culture.
在体外RTG - 2和RTG - P1细胞中诱导传染性胰腺坏死病毒（IPNV）的持续感染，并在连续传代过程中表征这种现象对病毒感染性、病毒抗原表达以及对同源和异源病毒干扰的影响。还评估了干扰素（IFN）的诱导情况，以及病毒衣壳蛋白VP2的序列，该区域被认为与毒力、减毒或持续性有关。在没有细胞病变效应证据的细胞中记录了病毒抗原表达，在这些条件下，流式细胞术比逆转录聚合酶链反应（RT - PCR）更敏感，可证明非裂解性病毒的存在。在交叉感染实验中可检测到同源病毒感染的干扰，在持续感染IPNV的RTG - P1细胞中，Mx1启动子至少在连续5代中仍可被激活。实际上，虽然再次感染同源或异源病毒未观察到荧光素酶的过度诱导，但聚肌胞苷酸（poly I:C）可诱导荧光素酶显著增加。通过实时定量逆转录聚合酶链反应（qRT - PCR）可在连续传代的持续细胞中定量IFN转录本，表明IFN在维持IPNV持续性中发挥作用。此外，我们在持续病毒的VP2序列中观察到与先前描述的IPNV在细胞培养中的适应性和持续性相同的决定因素。
