Phylogenetics and New Approaches to Vaccine Development for West Nile

West Nile virus (WNV) was first isolated in 1937 from a febrile Ugandan woman. WNV now has a worldwide distribution from Australia and India in the east to Europe and the United States in the west. The first human cases of WNV in the United States were detected in New York in 1999. The North American isolates were also found to be highly neuroinvasive and neurovirulent and in many cases leading to mortality or permanent CNS sequelae among humans. WNV was first isolated in Louisiana in 2001. The genome of this virus named LSU-AR01was sequenced and a detailed genetic analysis revealed 26 amino acid changes in comparison to the prototypic New York-99 strain. Phylogenetic analysis using Neighbor-joining and the Bayesian approach showed that LSU-AR01 was closely related to a strain isolated from a mosquito in 1999 in Connecticut. This relationship was bolstered by a 58% bootstrap value and a 66% posterior probability by these algorithms respectively. Comparative pathology revealed that the LSU-AR01 was more neurovirulent and neuroinvasive especially at low doses indicating a virus with a competitive edge. Recombinant vesicular stomatitis virus (rVSV) based vaccines cleverly incorporate reverse genetics to recover a recombinant virus expressing a foreign antigen of interest. rVSV vectored vaccines expressing the LSU-AR01 envelope (E) glycoprotein were engineered and administered to mice in a prime-boost approach. The vaccines were able to confer high degree of protection in mice against lethal challenge with highly virulent WNV LSU-AR01. Detailed immunological analyses of immunized mice revealed the production of neutralizing antibody responses. In addition, vaccinated mice generated WNV E glycoprotein specific CD8+CD62Llow IFNã+ T cells response against WNV. Recombinant VSV expressing the Simian Retrovirus-2 (SRV-2) gag and Env gene constructs and the Herpes B virus glycoprotein D gene were constructed and characterized in cell culture experiments. The VSV-SRV-2 gag and env recombinants generated protective immune responses in non-human primates. The VSV Herpes B recombinant vaccine will be tested in non-human primates in the near future. Collectively, these experiments revealed that VSV-vectored vaccines are highly effective in generating humoral and cellular immune responses against viral infections

Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome

Outbreaks of Dengue impose a heavy economic burden on developing countries in terms of vector control and human morbidity. Effective vaccines against all four serotypes of Dengue are in development, but population replacement with transgenic vectors unable to transmit the virus might ultimately prove to be an effective approach to disease suppression, or even eradication. A key element of the refractory transgenic vector approach is the development of transgenes that effectively prohibit viral transmission. In this report we test the effectiveness of several hammerhead ribozymes for suppressing DENV in lentivirus-transduced mosquito cells in an attempt to mimic the transgenic use of these effector molecules in mosquitoes. A lentivirus vector that expresses these ribozymes as a fusion RNA molecule using an Ae. aegypti tRNAval promoter and terminating with a 60A tail insures optimal expression, localization, and activity of the hammerhead ribozyme against the DENV genome. Among the 14 hammerhead ribozymes we designed to attack the DENV-2 NGC genome, several appear to be relatively effective in reducing virus production from transduced cells by as much as 2 logs. Among the sequences targeted are 10 that are conserved among all DENV serotype 2 strains. Our results confirm that hammerhead ribozymes can be effective in suppressing DENV in a transgenic approach, and provide an alternative or supplementary approach to proposed siRNA strategies for DENV suppression in transgenic mosquitoes

Computer-Aided vaccine design for selected positive-sense single stranded RNA viruses

Spontaneous mutations and lack of replication fidelity in positive-sense single stranded RNA viruses (+ssRNA virus) result in emergence of genetic variants with diverse viral morphogenesis and surface proteins that affect its antigenicity. This high mutability in +ssRNA viruses has induced antiviral drug resistance and ability to overcome vaccines that subsequently resulted in rapid viral evolution and high mortality rate in human and livestock. Computer aided vaccine design and immunoinformatics play a crucial role in expediting the vaccine production protocols, antibody production and identifying suitable immunogenic regions or epitopes from the genome sequences of the pathogens. T cell and B cell epitopes can be identified in pathogens by immunoinformatics algorithms and methods that enhance the analysis of protective immunity, vaccine safety, immunity modelling and vaccine efficacy. This rapid and cost-effective computational vaccine design promotes development of potential vaccine that could induce immune response in host against rapidly mutating pathogens like +ssRNA viruses. Epitope-based vaccine is a striking concept that has been widely employed in recent years to construct vaccines targeting rapidly mutating +ssRNA viruses. Therefore, the present review provides an overview about the current progress and methodology in computer-aided vaccine design for the most notable +ssRNA viruses namely Hepatitis C virus, Dengue virus, Chikungunya virus and Coronaviruses. This review also highlights the applications of various immunoinformatics tools for vaccine design and for modelling immune response against +ssRNA viruses

The role of genetic diversity in the replication, pathogenicity and virulence of Murray Valley encephalitis virus

Genetic and phenotypic variation of genotype 1 (G1) and genotype 2 (G2) Murray valley encephalitis virus (MVEV) were characterised. G2 viruses were a minority, had lower levels of genetic diversity and an attenuated phenotype in the mouse model of MVE. G1 isolates were abundant with higher levels of genetic diversity and a virulent phenotype. The restricted evolution of MVEV was due to multiplication in mosquito cells. An RT-qPCR assay detecting all MVEV genotypes was developed

Flavivirus reverse genetic systems, construction techniques and applications: A historical perspective

AbstractThe study of flaviviruses, which cause some of the most important emerging tropical and sub-tropical human arbovirus diseases, has greatly benefited from the use of reverse genetic systems since its first development for yellow fever virus in 1989. Reverse genetics technology has completely revolutionized the study of these viruses, making it possible to manipulate their genomes and evaluate the direct effects of these changes on their biology and pathogenesis. The most commonly used reverse genetics system is the infectious clone technology. Whilst flavivirus infectious clones provide a powerful tool, their construction as full-length cDNA molecules in bacterial vectors can be problematic, laborious and time consuming, because they are often unstable, contain unwanted induced substitutions and may be toxic for bacteria due to viral protein expression. The incredible technological advances that have been made during the past 30years, such as the use of PCR or new sequencing methods, have allowed the development of new approaches to improve preexisting systems or elaborate new strategies that overcome these problems. This review summarizes the evolution and major technical breakthroughs in the development of flavivirus reverse genetics technologies and their application to the further understanding and control of these viruses and their diseases

Flavivirus control of lipid metabolism: implications for virion formation, function and pathogenesis

2018 Summer.Includes bibliographical references.Dengue viruses (DENV) are the most aggressive arthropod-born viruses worldwide with no currently available antivirals. There is a clear need to understand host viral interactions that can be exploited for therapeutic options. DENV are members of the Flaviviridae family with a positive sense single-stranded RNA genome surrounded by a virally encoded capsid protein, a host cell derived lipid envelope and an icosahedral shell of virally encoded glycoproteins. Its genome is replicated in virally–induced invaginations in the endoplasmic reticulum of the host cell that consistently develop in a time-dependent manner. These invaginations display a highly curved architecture and seem to increase the membrane contact sites within the ER and its vicinity. Functionally, these membranes condense the replication machinery, provide a scaffold to coordinate replication, and hide the viral double stranded RNA intermediate from the host cellular defenses. It has been shown that fatty acid synthesis is increased during infection to provide substrates for this membrane expansion. To identify further changes to cellular metabolism, we have profiled the metabolome of DENV serotype 2 (DENV2) infected Human Hepatoma cells (Huh7) cells at key time-points in virus replication. We have found time-dependent changes in cellular essential fatty acid metabolism. Furthermore, we have interrogated a library of siRNAs directed at the unsaturated fatty acid biosynthesis pathway to determine key enzymes involved in viral replication. We have identified that stearoyl Co-A desaturase 1 (SCD1), the rate-limiting enzyme responsible for converting stearic to oleic acid, is critical for viral replication, maturation and infectious particle formation. Finally, we have profiled the serum metabolome of acute-phase patients with dengue diseases, chikungunya virus infection, or an unknown febrile illness to identify metabolic changes with potential use as prognostic biomarkers. Hypothesis: Since dengue viruses are enveloped viruses, lipid metabolites in the human host are a critical resource hijacked by these viruses for their replicative advantage. Important metabolites will be altered during infection in a time dependent manner and can be quantified and correlated directly to their role in viral genome replication and infectious particle assembly and release. These metabolic changes could also be identified in human bio-fluids and could function as early biomarkers of disease manifestation