Abstract
The bank vole (Myodes glareolus) is a common small mammal in Europe and a natural host for several important emerging zoonotic viruses, e.g. Puumala hantavirus (PUUV) that causes hemorrhagic fever with renal syndrome (HFRS). Hantaviruses are known to interfere with several signaling pathways in infected human cells, and HFRS is considered an immune-mediated disease. There is no in vitro-model available for infectious experiments in bank vole cells, nor tools for analyses of bank vole immune activation and responses. Consequently, it is not known if there are any differences in the regulation of virus induced responses in humans compared to natural hosts during infection. We here present an in vitro-model for studies of bank vole borne viruses and their interactions with natural host cell innate immune responses. Bank vole embryonic fibroblasts (VEFs) were isolated and shown to be susceptible for PUUV-infection, including a wild-type PUUV strain (only passaged in bank voles). The significance of VEFs as a model system for bank vole associated viruses was further established by infection studies showing that these cells are also susceptible to tick borne encephalitis, cowpox and Ljungan virus. The genes encoding bank vole IFN-β and Mx2 were partially sequenced and protocols for semi-quantitative RT-PCR were developed. Interestingly, PUUV did not induce an increased IFN-β or Mx2 mRNA expression. Corresponding infections with CPXV and LV induced IFN-β but not Mx2, while TBEV induced both IFN-β and Mx2.In conclusion, VEFs together with protocols developed for detection of bank vole innate immune activation provide valuable tools for future studies of how PUUV and other zoonotic viruses affect cells derived from bank voles compared to human cells. Notably, wild-type PUUV which has been difficult to cultivate in vitro readily infected VEFs, suggesting that embryonic fibroblasts from natural hosts might be valuable for isolation of wild-type hantaviruses.
Highlights
Bank voles (Myodes glareolus) are found in most areas of Europe and in parts of Northern Asia [1]. This rodent is a natural host for several viruses with different characteristics, including the hantavirus Puumala virus (PUUV), the flavivirus tick borne encephalitis virus (TBEV), the orthopoxvirus cowpox virus (CPXV), and the parechovirus Ljungan virus (LV) [2,3,4,5]
To investigate if vole embryonic fibroblasts (VEFs), in addition to PUUV, are susceptible to other viruses for which bank voles are a known reservoir, cells were infected with CPXV, LV, and TBEV and subsequently stained for the presence of viral proteins at 17 hours post infection, 8 hpi and 24 hpi, respectively
For CPXV, viral titers in supernatants increased from 48 hours after infection (Fig. 2D), while LVinfected (Fig. 2E) and TBEV-infected (Fig. 2F) VEFs produced a stable amount of progeny virus over time
Summary
Bank voles (Myodes glareolus) are found in most areas of Europe and in parts of Northern Asia [1] This rodent is a natural host for several viruses with different characteristics, including the hantavirus Puumala virus (PUUV), the flavivirus tick borne encephalitis virus (TBEV), the orthopoxvirus cowpox virus (CPXV), and the parechovirus Ljungan virus (LV) [2,3,4,5]. As for many zoonotic agents, PUUV, TBEV and CPXV normally cause asymptomatic infections in their natural host. If LV infections cause disease in humans and/or in bank voles is controversial and remains to be clearly shown [6]. Rodent borne hantaviruses are the etiological agent of two zoonotic diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Due to the lack of specific reagents, it has not been possible to analyze if PUUV interferes with bank vole cell functions, and it is not known if PUUV affects infected cells in a species-specific manner
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