Jasmeen Pagala, Gupta Priya, Kaur Charanpreet, Gauthami Sulgey, Pyasi Shruti, Nayak Debasis, Hegde Nagendra R
BRIC-National Institute of Animal Biotechnology, Hyderabad, India.
Regional Centre of Biotechnology, Faridabad, India.
Virusdisease. 2025 Mar;36(1):48-59. doi: 10.1007/s13337-024-00901-x. Epub 2024 Dec 23.
Bovine ephemeral fever (BEF) is caused by BEF virus (BEFV) belonging to the Genus under the Family . The BEFV carries a single-stranded, negative-sense RNA genome. Not much is known about the various aspects of BEFV replication, its interaction with cellular proteins or the cellular response to BEFV infection. Here, we report the rescue of BEFV through reverse genetics. A full-length cDNA copy of BEFV was assembled to be driven by the RNA polymerase I (PolI) promoter. Parallely, eukaryotic expression plasmids containing BEFV sequences encoding the helper proteins N, P and L, which form the replicase complex, were generated. The expression of N and P proteins were verified by using the in-house generated and purified polyclonal sera. Transfection of the full-length cDNA copy along with the helper plasmids rescued BEFV, as evaluated by transmission electron microscopy, reverse-transcription polymerase reaction, immunofluorescence and Western blotting. However, the virus did not produce a cytopathic effect and failed to be propagated beyond a certain number of passages. The results lay the foundation for establishment of reverse genetics for BEFV but also highlight the difficulties in studying this virus.
牛流行热（BEF）由属于 科 属的牛流行热病毒（BEFV）引起。BEFV携带单链负义RNA基因组。关于BEFV复制的各个方面、其与细胞蛋白的相互作用或细胞对BEFV感染的反应，人们了解得并不多。在此，我们报告了通过反向遗传学拯救BEFV的方法。组装了由RNA聚合酶I（PolI）启动子驱动的BEFV全长cDNA拷贝。同时，构建了真核表达质粒，其包含编码形成复制酶复合物的辅助蛋白N、P和L的BEFV序列。通过使用内部制备和纯化的多克隆血清验证了N和P蛋白的表达。通过透射电子显微镜、逆转录聚合酶反应、免疫荧光和蛋白质免疫印迹评估，全长cDNA拷贝与辅助质粒共转染拯救了BEFV。然而，该病毒未产生细胞病变效应，并且在传代一定次数后无法继续传代。这些结果为建立BEFV的反向遗传学奠定了基础，但也凸显了研究该病毒的困难。
