Abstract
Flaviviruses such as yellow fever, dengue or Zika viruses are responsible for significant human and veterinary diseases worldwide. These viruses contain an RNA genome, prone to mutations, which enhances their potential to emerge as pathogens. Bamaga virus (BgV) is a mosquito-borne flavivirus in the yellow fever virus group that we have previously shown to be host-restricted in vertebrates and horizontally transmissible by Culex mosquitoes. Here, we aimed to characterise BgV host-restriction and to investigate the mechanisms involved. We showed that BgV could not replicate in a wide range of vertebrate cell lines and animal species. We determined that the mechanisms involved in BgV host-restriction were independent of the type-1 interferon response and RNAse L activity. Using a BgV infectious clone and two chimeric viruses generated as hybrids between BgV and West Nile virus, we demonstrated that BgV host-restriction occurred post-cell entry. Notably, BgV host-restriction was shown to be temperature-dependent, as BgV replicated in all vertebrate cell lines at 34°C but only in a subset at 37°C. Serial passaging of BgV in Vero cells resulted in adaptive mutants capable of efficient replication at 37°C. The identified mutations resulted in amino acid substitutions in NS4A-S124F, NS4B-N244K and NS5-G2C, all occurring close to a viral protease cleavage site (NS4A/2K and NS4B/NS5). These mutations were reverse engineered into infectious clones of BgV, which revealed that NS4B-N244K and NS5-G2C were sufficient to restore BgV replication in vertebrate cells at 37°C, while NS4A-S124F further increased replication efficiency. When these mutant viruses were injected into immunocompetent mice, alongside BgV and West Nile virus chimeras, infection and neurovirulence were enhanced as determined by clinical scores, seroconversion, micro-neutralisation, viremia, histopathology and immunohistochemistry, confirming the involvement of these residues in the attenuation of BgV. Our studies identify a new mechanism of host-restriction and attenuation of a mosquito-borne flavivirus.
Highlights
Flaviviruses have the capacity to emerge as major human pathogens and cause large scale epidemics
We investigated the host-restriction of a recently discovered flavivirus, Bamaga virus
We identified three amino acid substitutions located at two viral protease cleavage sites, which we have demonstrated are directly involved in Bamaga virus (BgV) host-restriction in vitro and attenuation in vivo
Summary
Flaviviruses have the capacity to emerge as major human pathogens and cause large scale epidemics. Most flaviviruses are transmitted through a classical arthropod-borne cycle between arthropod vectors and vertebrate hosts, and exhibit replication in cells from a wild range of invertebrate and vertebrate host species, some flaviviruses exhibit a more narrow host range These include flaviviruses that do not infect arthropod hosts (no known vector), fail to replicate in vertebrates (insect-specific) or exhibit host-restriction in vertebrate cells that is cell line- or temperature-dependent [1,2,3]. The mechanisms for emergence of pathogens from enzootic flaviviruses are not clear despite past examples of large-scale epidemics caused by under-studied flaviviruses such as Zika virus [4]. Flaviviruses are positive-sense, single-stranded RNA viruses with a genome of approximately 11 kilobases (kb), contained in an enveloped icosahedral nucleocapsid [6, 7]. Once replication complexes have been established, with the NS5-encoded RdRp at their core, the viral RNA is replicated using a newly generated genome-length negative-sense strand as a template for new positive strands [10]
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