A Mouse Model for Betacoronavirus Subgroup 2c Using a Bat Coronavirus Strain HKU5 Variant

Abstract

Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis.

Trypsin Treatment Unlocks Barrier for Zoonotic Bat Coronavirus Infection

“…Data from this study implicate the processing of the spike protein as a critical factor for CoV infection. In the absence of trypsin, the MERS-Uganda and HKU5-CoV spikes were unable to mediate infection and initially suggested a lack of receptor compatibility (6,16). However, exogenous trypsin treatment produced robust infection, indicating that despite binding to human cells, CoVs cannot overcome incomplete spike processing.…”

“…Previously, our group had generated a full-length infectious clone for HKU5-CoV, another group 2C coronavirus sequence isolated from bats. Similar to the MERS-Uganda chimera, the infectious clone of HKU5-CoV produced subgenomic transcripts but failed to achieve productive infection (6). Revisiting the full-length recombinant virus, we sought to determine if trypsin treatment could also rescue HKU5-CoV.…”

“…Despite their spontaneous emergence, several research approaches to rapidly respond and even predict outbreak strains already exist. During the MERS-CoV outbreak, our group and others were able to leverage reagents generated against related group 2C coronaviruses, namely, HKU4-and HKU5-CoV (6,7). These reagents, created independent of viable virus replication, provided valuable insights and models for testing serologic responses during the early stages of the MERS-CoV outbreak.…”

Structure of the S1 subunit C-terminal domain from bat-derived coronavirus HKU5 spike protein

“…An infectious clone of BatCoV HKU5 containing the ectodomain from the SARS-CoV S protein was constructed through reverse genetics and synthetic-genome design, and the recombinant virus replicates efficiently in cell culture and in young and aged mice (Agnihothram et al, 2014). In addition, the key proteins for virus replication, such as the 3C-like protease, polymerase, and exonuclease of BatCoV HKU5 display high amino acid sequence similarity to those in MERS-CoV, indicating that once the genome of BatCoV HKU5 is released into host cells, genome replication, virus particle assembly, and release can readily occur.…”

Computational modeling of the bat HKU4 coronavirus 3CLpro inhibitors as a tool for the development of antivirals against the emerging Middle East respiratory syndrome (MERS) coronavirus

“…MERS‐CoV 3CL pro purification was performed using consecutive steps of hydrophobic‐interaction chromatography, DEAE anion‐exchange chromatography, Mono S cation‐exchange chromatography, and size‐exclusion chromatography as described previously . HKU4‐CoV 3CL pro was produced and purified using a modified protocol from Agnihothram et al Final protein yield was calculated based on the measurement of total activity units (μM product/min), specific activity (units/mg), and milligrams of protein obtained (BioRad protein assay) after each chromatographic step.…”