Lenhard Lea, Müller Martin, Diederich Sandra, Loerzer Lisa, Friedrichs Virginia, Köllner Bernd, Finke Stefan, Dorhoi Anca, Pei Gang
Institute of Immunology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, 17493 Greifswald, Germany.
Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, 17493 Greifswald, Germany.
Viruses. 2025 Apr 16;17(4):573. doi: 10.3390/v17040573.
Cedar virus (CedV), closely related to the Hendra and Nipah viruses, is a novel that was originally isolated from flying foxes in Australia in 2012. Although its glycoprotein G exhibits relatively low sequence similarity with its counterparts of the Hendra and Nipah viruses, CedV also uses ephrin receptors, i.e., ephrins B1, B2, A2 and A5, to enters human cells. Nevertheless, the entry mechanism of CedV into bat cells remains unexplored. Considering that (Egyptian Rousette bat, ERB) is postulated to be a reservoir host for henipaviruses, we aim to reveal the receptors utilized by CedV to enable its entry into ERB cells. To this end, we cloned the class A and B ephrins of ERB and generated CHO-K1 cells stably expressing individual ephrins. We also developed a lentivirus-based pseudovirus system containing the firefly luciferase reporter. Assessment of the luciferase activity in cells expressing single ephrins demonstrated that the ERB ephrin B1 and B2 mediated CedV pseudovirus entry. Further, we generated a recombinant CedV expressing the fluorescent protein TurboFP635 (rCedV-nTurbo635). By performing high-content microscopy and flow cytometry, we unveiled that, in addition to ephrin B1 and B2, ephrin A5 was also able to mediate rCedV-nTurbo635 entry, although to a much lesser extent. In contrast to human ephrin A2, ERB ephrin A2 failed to mediate rCedV-nTurbo635 entry. Finally, we generated ERB epithelial cells with ephrin B1 and/or ephrin B2 knockdown (KD). The entry of rCedV-nTurbo635 into ERB epithelial cells was drastically impaired by ephrin B1/B2 KD, validating the importance of ephrin B1 and B2 in its entry. Altogether, we conclude that CedV primarily employs ERB ephrin B1, B2 and, possibly, A5 for its entry into ERB cells.
雪松病毒（CedV）与亨德拉病毒和尼帕病毒密切相关，是一种新型病毒，于2012年最初从澳大利亚的狐蝠中分离出来。尽管其糖蛋白G与亨德拉病毒和尼帕病毒的对应物相比序列相似性相对较低，但CedV也利用 Ephrin 受体，即 Ephrins B1、B2、A2 和 A5 进入人类细胞。然而，CedV进入蝙蝠细胞的机制仍未被探索。鉴于埃及果蝠（Egyptian Rousette bat，ERB）被认为是亨尼帕病毒的储存宿主，我们旨在揭示CedV用于进入ERB细胞的受体。为此，我们克隆了ERB的A类和B类Ephrins，并生成了稳定表达单个Ephrins的CHO-K1细胞。我们还开发了一种基于慢病毒的含有萤火虫荧光素酶报告基因的假病毒系统。对表达单个Ephrins的细胞中荧光素酶活性的评估表明，ERB Ephrin B1和B2介导了CedV假病毒的进入。此外，我们生成了表达荧光蛋白TurboFP635的重组CedV（rCedV-nTurbo635）。通过进行高内涵显微镜和流式细胞术，我们发现，除了Ephrin B1和B2外，Ephrin A5也能够介导rCedV-nTurbo635的进入，尽管程度要小得多。与人类Ephrin A2不同，ERB Ephrin A2未能介导rCedV-nTurbo635的进入。最后，我们生成了Ephrin B1和/或Ephrin B2敲低（KD）的ERB上皮细胞。Ephrin B1/B2 KD极大地损害了rCedV-nTurbo635进入ERB上皮细胞的过程，证实了Ephrin B1和B2在其进入过程中的重要性。总之，我们得出结论，CedV主要利用ERB Ephrin B1、B2以及可能的A5进入ERB细胞。
