To overcome the lack of immortal shrimp cell lines for shrimp viral research, we constructed and tested DNA infectious clones of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) often found together in freshwater prawn (M. rosenbergii) exhibiting white tail disease (WTD). Full-length cDNAs of MrNV and XSV genomic RNA were individually inserted into the baculovirus pFastBacDUAL shuttle vector. Individual Sf9 (insect cell line) transfection resulted in production of RNA (RT-PCR) and capsid proteins (immunofluorescence) for both viruses. Presence of respective virions was confirmed by density gradient purification followed by RT-PCR and transmission electron microscopy. Infectivity was by tested in immersion-challenge tests with M. rosenbergii post-larvae (PL) using both semi-purified viruses, individually or combined, and confirmed by histological analysis (morphology and immunofluorescence) and quantitative RT-PCR. Mortality accompanied by WTD lesions occurred with MrNV alone or in combination with XSV but not with XSV alone, despite its replication.

中文翻译：

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在用罗氏沼虾诺达病毒（MrNV）和特小病毒（XSV）的感染性克隆进行实验性挑战时，仅MrNV会导致淡水虾（罗氏沼虾）死亡。

为了克服对虾病毒研究缺乏永生虾细胞系的不足，我们构建并测试了罗氏沼虾诺达病毒（MrNV）和特小病毒（XSV）的DNA感染性克隆，这些病毒通常在表现出白尾病的淡水虾（罗氏沼虾）中一起发现（WTD）。将MrNV和XSV基因组RNA的全长cDNA分别插入杆状病毒pFastBacDUAL穿梭载体中。单独的Sf9（昆虫细胞系）转染导致两种病毒均产生RNA（RT-PCR）和衣壳蛋白（免疫荧光）。通过密度梯度纯化，然后通过RT-PCR和透射电子显微镜确认各个病毒体的存在。感染性是通过使用单独或组合使用的两种半纯化病毒，在罗氏沼虾幼虫（PL）的浸没-攻击试验中进行的，并经组织学分析（形态学和免疫荧光）和定量RT-PCR证实。尽管有其复制，但单独使用MrNV或与XSV联合使用时，伴随WTD损伤的死亡率会发生，但单独使用XSV则不会发生。