Abstract
Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Middle East, affecting both humans and ruminants. There are no licensed vaccines or antivirals available for humans, whereas research using RVF virus (RVFV) is strictly regulated in many countries with safety concerns. Nonpathogenic Arumowot virus (AMTV), a mosquito-borne phlebovirus in Africa, is likely useful for the screening of broad-acting antiviral candidates for phleboviruses including RVFV, as well as a potential vaccine vector for RVF. In this study, we aimed to generate T7 RNA polymerase-driven reverse genetics system for AMTV. We hypothesized that recombinant AMTV (rAMTV) is viable, and AMTV NSs protein is dispensable for efficient replication of rAMTV in type-I interferon (IFN)-incompetent cells, whereas AMTV NSs proteins support robust viral replication in type-I IFN-competent cells. The study demonstrated the rescue of rAMTV and that lacking the NSs gene (rAMTVΔNSs), that expressing green fluorescent protein (GFP) (rAMTV-GFP) or that expressing Renilla luciferase (rAMTV-rLuc) from cloned cDNA. The rAMTV-rLuc and the RVFV rMP12-rLuc showed a similar susceptibility to favipiravir or ribavirin. Interestingly, neither of rAMTV nor rAMTVΔNSs replicated efficiently in human MRC-5 or A549 cells, regardless of the presence of NSs gene. Little accumulation of AMTV NSs protein occurred in those cells, which was restored via treatment with proteasomal inhibitor MG132. In murine MEF or Hepa1-6 cells, rAMTV, but not rAMTVΔNSs, replicated efficiently, with an inhibition of IFN-β gene upregulation. This study showed an establishment of the first reverse genetics for AMTV, a lack of stability of AMTV NSs proteins in human cells, and an IFN-β gene antagonist function of AMTV NSs proteins in murine cells. The AMTV can be a nonpathogenic surrogate model for studying phleboviruses including RVFV.
Highlights
Rift Valley fever (RVF) is one of the most important zoonotic viral diseases for public health, which is classified as Category A Priority Pathogen by the National Institute of Allergy and Infectious Diseases in the United States (U.S.) and the Blueprint priority disease by the World Health Organization [1, 2]
The reverse genetics system for RVF virus (RVFV) has contributed to the virology, vaccinology, and antiviral screening for RVFV
We generated the first reverse genetics system for a mosquito-borne nonpathogenic phlebovirus (Arumowot virus; AMTV) endemic to Africa, which is phylogenetically related to RVFV
Summary
The use of RVFV is required for basic and translational research to develop countermeasures against RVF. The reverse genetics is the technology to rescue infectious recombinant RNA viruses from cloned cDNA [6]. It allows manipulation of RNA virus genome, which has contributed to virology and vaccinology since the discovery. Recombinant MP-12 (rMP-12), including that expressing reporter gene, has been generated for the use in BSL2 for basic virology, antiviral screening and vaccine sciences. A development of reverse genetics system for other phleboviruses has been reported for Uukuniemi virus (UUKV) or Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) [8,9,10]. SFTSV was renamed as Huaiyangshan banyangvirus and reclassified into the genus Banyangvirus
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