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.

Full Genome of batCoV/MinFul/2018/SriLanka, a Novel Alpha-Coronavirus Detected in Miniopterus fuliginosus, Sri Lanka.

Coronaviruses (CoV) are divided into the genera α-CoVs, ß-CoVs, γ-CoVs and δ-CoVs. Of these, α-CoVs and ß-CoVs are solely capable of causing infections in humans, resulting in mild to severe respiratory symptoms. Bats have been identified as natural reservoir hosts for CoVs belonging to these two genera. Consequently, research on bat populations, CoV prevalence in bats and genetic characterization of bat CoVs is of special interest to investigate the potential transmission risks. We present the genome sequence of a novel α-CoV strain detected in rectal swab samples of Miniopterus fuliginosus bats from a colony in the Wavul Galge cave (Koslanda, Sri Lanka). The novel strain is highly similar to Miniopterus bat coronavirus 1, an α-CoV located in the subgenus of Minunacoviruses. Phylogenetic reconstruction revealed a high identity of the novel strain to other α-CoVs derived from Miniopterus bats, while human-pathogenic α-CoV strains like HCoV-229E and HCoV-NL63 were more distantly related. Comparison with selected bat-related and human-pathogenic strains of the ß-CoV genus showed low identities of ~40%. Analyses of the different genes on nucleotide and amino acid level revealed that the non-structural ORF1a/1b are more conserved among α-CoVs and ß-CoVs, while there are higher variations in the structural proteins known to be important for host specificity. The novel strain was named batCoV/MinFul/2018/SriLanka and had a prevalence of 50% (66/130) in rectal swab samples and 58% (61/104) in feces samples that were collected from Miniopterus bats in Wavul Galge cave. Based on the differences between strain batCoV/MinFul/2018/SriLanka and human-pathogenic α-CoVs and ß-CoVs, we conclude that there is a rather low transmission risk to humans. Further studies in the Wavul Galge cave and at other locations in Sri Lanka will give more detailed information about the prevalence of this virus.

Detection of Alpha- and Betacoronaviruses in Miniopterus fuliginosus and Rousettus leschenaultii, two species of Sri Lankan Bats.

Bats are known to be potential reservoirs of numerous human-pathogenic viruses. They have been identified as natural hosts for coronaviruses, causing Severe Acute Respiratory Syndrome (SARS) in humans. Since the emergence of SARS-CoV-2 in 2019 interest in the prevalence of coronaviruses in bats was newly raised. In this study we investigated different bat species living in a sympatric colony in the Wavul Galge cave (Koslanda, Sri Lanka). In three field sessions (in 2018 and 2019), 395 bats were captured (Miniopterus, Rousettus, Hipposideros and Rhinolophus spp.) and either rectal swabs or fecal samples were collected. From these overall 396 rectal swab and fecal samples, the screening for coronaviruses with nested PCR resulted in 33 positive samples, 31 of which originated from Miniopterus fuliginosus and two from Rousettus leschenaultii. Sanger sequencing and phylogenetic analysis of the obtained 384-nt fragment of the RNA-dependent RNA polymerase revealed that the examined M. fuliginosus bats excrete alphacoronaviruses and the examined R. leschenaultii bats excrete betacoronaviruses. Despite the sympatric roosting habitat, the coronaviruses showed host specificity and seemed to be limited to one species. Our results represent an important basis to better understand the prevalence of coronaviruses in Sri Lankan bats and may provide a basis for pursuing studies on particular bat species of interest.