The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research)


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Detection, Diagnosis and Vaccine Development , the third volume of The Flaviviruses details the current status of technologies for detection and differentiation of these viruses, their use in surveillance and outbreak investigation, and also reviews the latest clinical research. Vaccines E-Book. Stanley A. Psoriatic Arthritis. Dafna D. Stiehm's Immune Deficiencies. Kathleen E Sullivan.

Paul Volberding. Infectious Causes of Cancer. Kenneth Campbell. Sami M. Lecture Notes: Oncology. Mark Bower. Dengue Virus. Alan L. Alan C. Histopathology of Preclinical Toxicity Studies. Peter Greaves. The Biology of Multiple Sclerosis. Professor Gregory Atkins. Alice Goepfert. Vaccines for Biodefense and Emerging and Neglected Diseases. Alan D. Mandy Elschner. Foodborne Viral Pathogens. Peter A. Fabrizio Bruschi. Pathogenesis of Bacterial Infections in Animals. Carlton L. Agnes Bloch-Zupan. Textbook of Influenza. Robert G. Molecular Typing in Bacterial Infections.

Marian L. Ross S. Challenges in Infectious Diseases. Research Advances in Rabies. Immunity to Parasitic Infection. Tracey Lamb. Placental-Fetal Growth Restriction. Christoph Lees. Nonhuman Primates in Biomedical Research. Christian R. George Saade. Rabies an Overview. Charan Kamal Singh. The Kidney. Peter D. Oxford Textbook of Medical Mycology.

Christopher C. Maternal Hemodynamics. William H. Human Emerging and Re-emerging Infections. Sunit Kumar Singh. Viral Hepatitis and Liver Disease. Kusuya Nishioka. Neurotropic Viral Infections. Carol Shoshkes Reiss. Emerging and Re-emerging Infectious Diseases of Livestock. Jagadeesh Bayry. Sunil Thomas. Epidemiological and Molecular Aspects on Cholera. Viral Infections of the Human Nervous System. Skin and Arthropod Vectors.

Nathalie Boulanger. Neglected Tropical Diseases - South Asia. Sunit K. Emerging Zoonoses. Infectious Diseases. Phyllis Kanki. Survival of the Sickest. Sharon Moalem. Viral Hepatitis. Steven Specter. Infectious Agents and Cancer. Anton G. The Folly of Fools. Robert Trivers. Mark Pagel. Zoonoses - Infections Affecting Humans and Animals.

Andreas Sing.


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Pale Rider. Laura Spinney. Nicholas Johnson. Current Progress in Medical Mycology. Leila M. Deadly Feasts. Richard Rhodes. Too Much of a Good Thing. Lee Goldman. Similarly, the desired fragment resulting from Acc65I-BstZ17I digestion of the pNonstructural plasmid was also gel-purified. However, when equal concentrations of the two fragments were used in a ligation reaction, the ligation product yield was inconsistent and often contained multiple ligation products of different sizes.


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  6. This mixed population of ligation templates yielded little to no RNA of the desired size when used as template in in-vitro transcription IVT reactions Fig 2. Only in the case of the YFD1construct, following several attempts of ligation-IVT, were we able to observe a visible band of the expected size on a RNA gel, and using this product, we were able to successfully recover infectious virus. Control lane indicates RNA transcript produced from the control DNA template included in the in vitro transcription kit. Due to the inconsistency of the ligation reactions and resulting failure to rescue recombinant viruses, we decided to perform LONG-PCR to amplify and enrich the template for the IVT reactions.

    Transfected cells were observed daily and they started showing dengue virus-like CPE around 4—6 days post transfection.

    Zika virus emergence, transmission and disease

    At around the same time point, plaques similar to those produced by dengue virus could be observed in RNA transfections with methyl cellulose overlay Fig 3A. These observations collectively indicated recovery of infectious recombinant CYD viruses from the transfections. In general, when cells were transfected with IVT RNA derived from a single plasmid template, recombinant virus was readily recovered. Additionally, this method required several empirical modifications to ligate the two fragments and to generate RNA transcript of the required quality through IVT.

    The additional work required for the successful recovery of this virus emphasizes the technical difficulty of the two-plasmid system. These challenges underscore the importance of a full-length DNA template for efficient RNA transcript production and virus recovery. Infectivity of RNA transcripts was determined in Vero cells.

    Virus recovery was also confirmed by immunostaining using pan-flavivirus antibody 4G2.

    Bioinformatics in New Generation Flavivirus Vaccines

    The supernatants from each of the transfections upon further passage onto fresh Vero cells showed DENV-like CPE and were also positive for dengue virus specific immunostaining when tested with a pan-flavivirus antibody 4G2 Fig 3B. Collectively, these findings confirm that the viruses recovered through these methods are authentic recombinant CYD viruses. All the recovered CYD viruses were grown in Vero cells for two additional passages.

    Table 2 provides the summary of sequence changes observed in the recovered recombinant CYD viruses. None of the prME regions in any of the CYD viruses contained sequence changes indicating that the dengue sequences in these chimeric viruses are intact, even in the context of a chimeric backbone. On the other hand, analysis of sequences of the YF virus genes revealed that 7 out of 8 CYDs contained 1—2 nucleotide nt changes Table 2.

    This mutation did not appear to be associated with the method of virus recovery. Furthermore, this same mutation has been observed in several other YF17D-based chimeric flaviviruses [ 23 , 24 ], indicating that it is probably a virus-associated change and not an artificially-induced one. Together, these results suggest that the LONG-PCR method in addition to offering a significant advantage to virus recovery does not result in the introduction of additional mutations into the viral genomes of the viruses generated.

    Although there is no validated disease model for dengue, the rhesus macaque model is well-recognized as the most relevant model for assessing dengue vaccine immunogenicity and efficacy. Therefore, we decided to evaluate the immunogenicity of the recovered CYDs as a tetravalent formulation in rhesus macaques. A group of 4 monkeys served as a negative control group and were administered saline. Another group of 4 monkeys were immunized with a tetravalent formulation of 10 5. Serum samples were collected at monthly intervals post vaccination and assessed for levels of neutralizing antibodies against each of the four dengue serotypes using a LiCor assay [ 21 ].

    In the case of animals immunized with the tetravalent CYDs, neutralizing antibodies to each of the dengue viruses could be detected one month post the first dose, as would be expected from a LAV vaccine Fig 4. These titers declined slightly at study month 2 but then remained steady until the 6 month boost. A second dose of the same tetravalent formulation at 6 months resulted in a slight boost in DENV3 titers but not for any of the other serotype titers, indicating sterilizing immunity resulting from the first dose. Furthermore, the tetravalent CYD formulation was also observed to induce a balanced and durable immune response against each serotype for the entire duration of the 9-month study period Fig 4 , S1 Table.

    These results indicate that the recovered recombinant CYDs are immunogenic and can induce a well-balanced, durable immune response against all four serotypes when given as a tetravalent combination without exhibiting any viral interference. Longitudinal geometric mean virus neutralization titers in rhesus monkeys immunized with a tetravalent formulation of recombinant CYD viruses. A negative control group of 4 monkeys was included that received saline via intramuscular route.

    Both groups were immunized at 0, and 6 months. Virus neutralizing antibody titers were measured over a period of 9 months using a LiCor-based neutralization assay. The use of reverse genetic systems has facilitated the basic discovery of and vaccine development for a number of RNA viruses. Full-length infectious clones are currently available for several flaviviruses [ 27 ]. A single full-length cDNA clone is preferred for efficient flavivirus engineering and recovery since it offers a consistent template for generating high quality viral RNA transcripts resulting in a significantly higher success rate for virus recovery.

    However, genetic instability of full-length cDNA in E.

    The Flaviviruses: Detection, Diagnosis and Vaccine Development

    There is even an example of having to use a three-fragment ligation strategy to overcome stability issues in E. The instability of full length cDNA clones in E. Two-plasmid or multi-plasmid systems offer both advantages and disadvantages. In an attempt to improve this step in virus recovery we have developed a novel LONG-PCR strategy to amplify and enrich the full-length templates of YF17D-dengue chimeric genomes prior to transcription. Our method combines the advantages of both the single and two-plasmid systems and hence resulted in rapid generation of recombinant flaviviruses with high efficiency.

    We recovered 4 of our 8 recombinant CYDs using this method on the first attempt without introducing any sequence changes in the recovered recombinant viruses. These chimeras behaved like authentic viruses and were immunogenic in rhesus macaques, indicating that vaccine virus candidates can be generated rapidly using the LONG-PCR method. Furthermore, with the availability of DNA polymerases with very high proof-reading abilities, the LONG-PCR step can be included to generate templates without concerns about unwanted sequence changes.

    This work is the first to demonstrate that infectious recombinant flaviviruses can be rescued through long range PCR in a rapid manner. In conclusion, we believe that the modified virus rescue strategy using LONG-PCR is a quick and simple method to efficiently generate flaviviruses for use in basic research studies or for development as vaccine candidates. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide.

    Funding: The authors received no specific funding for this work. Yellow-fever dengue chimeric constructs All constructs were synthesized as gene fragments using sequences available in the GenBank database. Download: PPT.

    How we grow flu inside an egg

    Fig 1. Outline of recombinant YFD-dengue chimeric virus recovery. Plaque assay Plaque assays to determine virus titers were performed on 6-well plates containing confluent Vero cells. Immunofluorescence Verification of virus recovery was performed using immunofluorescence on Vero cells grown in chamber slides. LiCor dengue neutralization assay The LiCor dengue neutralization assay is a sensitive, high-throughput Infra red dye IRD - based neutralization assay that was developed and utilized to determine all dengue neutralization titers [ 21 ]. Conclusion The use of reverse genetic systems has facilitated the basic discovery of and vaccine development for a number of RNA viruses.

    Supporting Information. S1 Table. Neutralization titers in immunized monkeys. References 1. Refining the global spatial limits of dengue virus transmission by evidence-based consensus. View Article Google Scholar 2. The global distribution and burden of dengue. Expert Rev Vaccines. View Article Google Scholar 4. Whitehead SS. View Article Google Scholar 5. The dengue vaccine pipeline: Implications for the future of dengue control.

    Dengue vaccines: progress and challenges. Curr Opin Immunol. Dengue vaccine candidates in development. Rothman A. Barrett AD. Current status of flavivirus vaccines. Ann N Y Acad Sci. New Biol. J Virol. Construction of a full length infectious clone for dengue-1 virus Western Pacific,74 strain. Virus Genes. Construction and characterisation of a complete reverse genetics system of dengue virus type 3. Mem Inst Oswaldo Cruz. J Gen Virol. Construction, safety, and immunogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent vaccine.

    ChimeriVax-West Nile virus live-attenuated vaccine: preclinical evaluation of safety, immunogenicity, and efficacy. Exchanging the yellow fever virus envelope proteins with Modoc virus prM and E proteins results in a chimeric virus that is neuroinvasive in SCID mice. Development of Sanofi Pasteur tetravalent dengue vaccine. Hum Vaccin. View Article Google Scholar Viral Immunol. Modification of dengue virus strains by passage in primary dog kidney cells: preparation of candidate vaccines and immunization of monkeys.

    Am J Trop Med Hyg. Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates. Dengue virus-specific and flavivirus group determinants identified with monoclonal antibodies by indirect immunofluorescence. Immunogenicity, genetic stability, and protective efficacy of a recombinant, chimeric yellow fever-Japanese encephalitis virus ChimeriVax-JE as a live, attenuated vaccine candidate against Japanese encephalitis.

    The Flaviviruses: Detection, Diagnosis and Vaccine Development - Google книги

    Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates. Prospects for a dengue virus vaccine. Nat Rev Microbiol.

    The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research) The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research)
    The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research) The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research)
    The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research) The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research)
    The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research) The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research)
    The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research) The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research)
    The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research) The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research)
    The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research) The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research)
    The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research) The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research)
    The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research) The Flaviviruses: Detection, Diagnosis and Vaccine Development: 61 (Advances in Virus Research)

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