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The ability of DENV2 to display different morphologies (hence different antigenic properties) complicates vaccine and therapeutics development. Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to “bumpy” surfaced morphology when the temperature is switched from 29°C at 37°C. Here we identified five envelope (E) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies. Several mutations performed on the smooth DENV2 infectious clone destabilized the surface, as observed by cryoEM. Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins. In contrast, three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C, and only at high fever temperature (40°C) did they become “bumpy”. These results imply vaccines should contain particles representing both morphologies. For prophylactic and therapeutic treatments, this study also informs on which types of antibodies should be used at different stages of an infection, i.e., those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is one of the most widespread arboviruses. The four DENV serotypes infect about 400 million people every year, causing 96 million clinical dengue cases, of which approximately 500’000 are severe and potentially life-threatening. The only licensed vaccine has a limited efficacy and is only recommended in regions with high endemicity. We previously reported that 2’-O-methyltransferase mutations in DENV-1 and DENV-2 block their capacity to inhibit type I IFNs and render the viruses attenuated in vivo, making them amenable as vaccine strains; here we apply this strategy to all four DENV serotypes to generate a tetravalent, non-chimeric live-attenuated dengue vaccine. 2’-O-methyltransferase mutants of all four serotypes are highly sensitive to type I IFN inhibition in human cells. The tetravalent formulation is attenuated and immunogenic in mice and cynomolgus macaques and elicits a response that protects from virus challenge. These results show the potential of 2’-O-methyltransferase mutant viruses as a safe, tetravalent, non-chimeric dengue vaccine. [ABSTRACT FROM AUTHOR]

The flavivirus NS5 harbors both a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Both enzyme activities of NS5 are critical for viral replication. Recently, the full-length NS5 crystal structure of Japanese encephalitis virus reveals a conserved MTase-RdRP interface that features two conserved components: a six-residue hydrophobic network and a GTR sequence. Here we showed for the first time that these key interface components are essential for flavivirus replication by various reverse genetics approaches. Interestingly, some replication-impaired variants generated a common compensatory NS5 mutation outside the interface (L322F), providing novel routes to further explore the crosstalk between MTase and RdRP. [ABSTRACT FROM AUTHOR]

Summary: Viral noncoding RNAs have been shown to play an important role in virus-host interplay to facilitate virus replication. We report that members of the genus Flavivirus, a large group of medically important encephalitic RNA viruses, produce a unique and highly structured noncoding RNA of 0.3–0.5 kb derived from the 3′ untranslated region of the viral genome. Using West Nile virus as a model, we show that this subgenomic RNA is a product of incomplete degradation of viral genomic RNA by cellular ribonucleases. Highly conserved RNA structures located at the beginning of the 3′ untranslated region render this RNA resistant to nucleases, and the resulting subgenomic RNA product is essential for virus-induced cytopathicity and pathogenicity. Thus, flaviviruses evolved a unique strategy to generate a noncoding RNA product that allows them to kill the host more efficiently. [Copyright &y& Elsevier]

Flaviviruses are significant human pathogens that cause frequent emerging and reemerging epidemics around the world. Better molecular tools for studying, diagnosing, and treating these diseases are needed. Reporter viruses represent potent tools to fill this gap but have been hindered by genetic instability. Recent advances have overcome these hurdles, opening the way for increased use of stable reporter flaviviruses to diagnose infections, screen and study antiviral compounds, and serve as potential vaccine vectors. [ABSTRACT FROM AUTHOR]

Abstract: Most RNA viruses exist in their hosts as a heterogeneous population of related variants. Due to error prone replication, mutants are constantly generated which may differ in individual fitness from the population as a whole. Here we characterize three WNV isolates that contain, along with full-length genomes, mutants with large internal deletions to structural and nonstructural protein-coding regions. The isolates were all obtained from lorikeets that died from WNV at the Rio Grande Zoo in Albuquerque, NM between 2005 and 2007. The deletions are approximately 2kb, in frame, and result in the elimination of the complete envelope, and portions of the prM and NS-1 proteins. In Vero cell culture, these internally deleted WNV genomes function as defective interfering particles, reducing the production of full-length virus when introduced at high multiplicities of infection. In mosquitoes, the shortened WNV genomes reduced infection and dissemination rates, and virus titers overall, and were not detected in legs or salivary secretions at 14 or 21days post-infection. In mice, inoculation with internally deleted genomes did not attenuate pathogenesis relative to full-length or infectious clone derived virus, and shortened genomes were not detected in mice at the time of death. These observations provide evidence that large deletions may occur within flavivirus populations more frequently than has generally been appreciated and suggest that they impact population phenotype minimally. Additionally, our findings suggest that highly similar mutants may frequently occur in particular vertebrate hosts. [Copyright &y& Elsevier]

Abstract: The West Nile virus (WNV) genome contains a single RNA-dependent RNA polymerase (RdRp) gene, which is responsible for replication of the viral genome and, as such, is an important target for antiviral therapy. Viral RdRps are known to lack proofreading capabilities and as a result viruses such as WNV exist as a mixture of viral genotypes within an infection, enabling the virus to readily emerge and adapt to new host environments. To test the consequences of subtle structural alterations remote from the RdRp active-site, the following single point mutations were engineered in the WNV NS5 RdRp coding region: T363N, A365N, and T537I; these mutations were selected in an effort to stabilize the secondary structural elements near the rNTP binding pocket of the RdRp. Mutant viruses were tested in vitro on Vero, C6/36, Culex tarsalis and DF-1 cell types and in vivo in one day old chickens and Culex pipiens mosquitoes. Plaque morphology was affected by each mutation and growth and RNA replication kinetics were altered as well. Our results demonstrate that subtle alteration of the RdRp protein away from the active site can have a significant overall biological effect on WNV fitness, and that this effect can be host-dependent. [Copyright &y& Elsevier]