Establishment of Recombinant Trisegmented Mopeia Virus Expressing Two Reporter Genes for Screening of Mammarenavirus Inhibitors.

Highly pathogenic Arenaviruses, like the Lassa Virus (LASV), pose a serious public health threat in affected countries. Research and development of vaccines and therapeutics are urgently needed but hampered by the necessity to handle these pathogens under biosafety level 4 conditions. These containment restrictions make large-scale screens of antiviral compounds difficult. Therefore, the Mopeia virus (MOPV), closely related to LASV, is often used as an apathogenic surrogate virus. We established for the first time trisegmented MOPVs (r3MOPV) with duplicated S segments, in which one of the viral genes was replaced by the reporter genes ZsGreen (ZsG) or Renilla Luciferase (Rluc), respectively. In vitro characterization of the two trisegmented viruses (r3MOPV ZsG/Rluc and r3MOPV Rluc/ZsG), showed comparable growth behavior to the wild type virus and the expression of the reporter genes correlated well with viral titer. We used the reporter viruses in a proof-of-principle in vitro study to evaluate the antiviral activity of two well characterized drugs. IC50 values obtained by Rluc measurement were similar to those obtained by virus titers. ZsG expression was also suitable to evaluate antiviral effects. The trisegmented MOPVs described here provide a versatile and valuable basis for rapid high throughput screening of broadly reactive antiviral compounds against arenaviruses under BSL-2 conditions.

Non-invasive bioluminescence imaging of HCoV-OC43 infection and therapy in the central nervous system of live mice.

Human coronaviruses (HCoVs) are important pathogens that cause upper respiratory tract infections and have neuroinvasive abilities; however, little is known about the dynamic infection process of CoVs in vivo, and there are currently no specific antiviral drugs to prevent or treat HCoV infection. Here, we verified the replication ability and pathogenicity of a reporter HCoV-OC43 strain expressing Renilla luciferase (Rluc; rOC43-ns2DelRluc) in mice with different genetic backgrounds (C57BL/6 and BALB/c). Additionally, we monitored the spatial and temporal progression of HCoV-OC43 through the central nervous system (CNS) of live BALB/c mice after intranasal or intracerebral inoculation with rOC43-ns2DelRluc. We found that rOC43-ns2DelRluc was fatal to suckling mice after intranasal inoculation, and that viral titers and Rluc expression were detected in the brains and spinal cords of mice infected with rOC43-ns2DelRluc. Moreover, viral replication was initially observed in the brain by non-invasive bioluminescence imaging before the infection spread to the spinal cord of BALB/c mice, consistent with its tropism in the CNS. Furthermore, the Rluc readout correlated with the HCoV replication ability and protein expression, which allowed quantification of antiviral activity in live mice. Additionally, we validated that chloroquine strongly inhibited rOC43-ns2DelRluc replication in vivo. These results provide new insights into the temporal and spatial dissemination of HCoV-OC43 in the CNS, and our methods provide an extremely sensitive platform for evaluating the efficacy of antiviral therapies to treat neuroinvasive HCoVs in live mice.