Flavivirus genome recoding by codon optimisation confers genetically stable in vivo attenuation in both mice and mosquitoes.

Virus genome recoding is an attenuation method that confers genetically stable attenuation by rewriting a virus genome with numerous silent mutations. Prior flavivirus genome recoding attempts utilised codon deoptimisation approaches. However, these codon deoptimisation approaches act in a species dependent manner and were unable to confer flavivirus attenuation in mosquito cells or in mosquito animal models. To overcome these limitations, we performed flavivirus genome recoding using the contrary approach of codon optimisation. The genomes of flaviviruses such as dengue virus type 2 (DENV2) and Zika virus (ZIKV) contain functional RNA elements that regulate viral replication. We hypothesised that flavivirus genome recoding by codon optimisation would introduce silent mutations that disrupt these RNA elements, leading to decreased replication efficiency and attenuation. We chose DENV2 and ZIKV as representative flaviviruses and recoded them by codon optimising their genomes for human expression. Our study confirms that this recoding approach of codon optimisation does translate into reduced replication efficiency in mammalian, human, and mosquito cells as well as in vivo attenuation in both mice and mosquitoes. In silico modelling and RNA SHAPE analysis confirmed that DENV2 recoding resulted in the extensive disruption of genomic structural elements. Serial passaging of recoded DENV2 resulted in the emergence of rescue or adaptation mutations, but no reversion mutations. These rescue mutations were unable to rescue the delayed replication kinetics and in vivo attenuation of recoded DENV2, demonstrating that recoding confers genetically stable attenuation. Therefore, our recoding approach is a reliable attenuation method with potential applications for developing flavivirus vaccines.

Chelerythrine chloride inhibits Zika virus infection by targeting the viral NS4B protein.

Zika virus (ZIKV) is a mosquito-borne virus that has re-emerged as a significant threat to global health in the recent decade. Whilst infections are primarily asymptomatic, the virus has been associated with the manifestation of severe neurological complications. At present, there is still a lack of approved antivirals for ZIKV infections. In this study, chelerythrine chloride, a benzophenanthridine alkaloid, was identified from a mid-throughput screen conducted on a 502-compound natural products library to be a novel and potent inhibitor of ZIKV infection in both in-vitro and in-vivo assays. Subsequent downstream studies demonstrated that the compound inhibits a post-entry step of the viral replication cycle and is capable of disrupting viral RNA synthesis and protein expression. The successful generation and sequencing of a ZIKV resistant mutant revealed that a single S61T mutation on the viral NS4B allowed ZIKV to overcome chelerythrine chloride inhibition. Further investigation revealed that chelerythrine chloride could directly inhibit ZIKV protein synthesis, and that the NS4B-S61T mutation confers resistance to this inhibition. This study has established chelerythrine chloride as a potential candidate for further development as a therapeutic agent against ZIKV infection.

A single-dose live attenuated chimeric vaccine candidate against Zika virus.

The mosquito-borne Zika virus is an emerging pathogen from the Flavivirus genus for which there are no approved antivirals or vaccines. Using the clinically validated PDK-53 dengue virus vaccine strain as a backbone, we created a chimeric dengue/Zika virus, VacDZ, as a live attenuated vaccine candidate against Zika virus. VacDZ demonstrates key markers of attenuation: small plaque phenotype, temperature sensitivity, attenuation of neurovirulence in suckling mice, and attenuation of pathogenicity in interferon deficient adult AG129 mice. VacDZ may be administered as a traditional live virus vaccine, or as a DNA-launched vaccine that produces live VacDZ in vivo after delivery. Both vaccine formulations induce a protective immune response against Zika virus in AG129 mice, which includes neutralising antibodies and a strong Th1 response. This study demonstrates that VacDZ is a safe and effective vaccine candidate against Zika virus.

Betulinic acid exhibits antiviral effects against dengue virus infection.

Dengue virus (DENV) is an arthropod-borne virus that has developed into a prominent global health threat in recent decades. The main causative agent of dengue fever, the virus infects an estimated 390 million individuals across the globe each year. Despite the sharply increasing social and economic burden on global society caused by the disease, there is still a glaring lack of effective therapeutics against DENV. In this study, betulinic acid, a naturally occurring pentacyclic triterpenoid was established as an inhibitor of DENV infection in vitro. Time-course studies revealed that betulinic acid inhibits a post-entry stage of the DENV replication cycle and subsequent analyses also showed that the compound is able to inhibit viral RNA synthesis and protein production. Betulinic acid also demonstrated antiviral efficacy against other serotypes of DENV, as well as against other mosquito-borne RNA viruses such as Zika virus and Chikungunya virus, which are commonly found co-circulating together with DENV. As such, betulinic acid may serve as a valuable starting point for the development of antivirals to combat potential DENV outbreaks, particularly in tropical and subtropical regions which make up a large majority of documented infections.