Tickborne encephalitis virus

Outbred white mice (of various ages) were inoculated subcutaneously with the Skalica strain of TBEV. The appearance and severity of encephalitis and levels of virus in brain tissue decreased with increasing age of the animals (Rajcani & Gresikova 1982). TBEV (strain Neudorfl) caused a lethal infection following either subcutaneous or intraperitoneal infection of young adult BALB c mice. Viraemia became first detectable at 24 h post infection. The virus was first detected in the brain about 6...

Mechanisms of evolution in DENV

Like all RNA viruses, the fuel of evolutionary change in DENV is provided by high mutation rates, a by-product of an intrinsically error-prone RNA polymerase. Although mutation rates have not been measured directly in DENV, estimates of its substitution rate, at 1 X 10-3 substitutions per site, per year, are of the same order of magnitude as seen in other RNA viruses. When coupled to large population sizes and rapid replication rates, high mutation rates mean that populations of DENV viruses...

T cell responses and dengue haemorrhagic fever

Gavin Screaton and Juthathip Mongkolsapaya Department of Immunology, Hammersmith Hospital, Imperial College, Du Cane Road, London W12 0NN, UK Abstract. The enhancement of severe disease upon secondary infection makes dengue almost unique among infectious pathogens and presents a serious challenge to vaccine design. Several key observations have been made which shed light onto this phenomenon particularly that antibodies can enhance Fc receptor-dependent uptake of virus into macrophages thereby...

Barriers to vaccine delivery

Many concerns (e.g. reversion to virulence and recombination with other viruses) about the implementation of new vaccines, especially those against dengue, can be overcome by careful design, good quality pre-clinical research and careful monitoring during clinical trials. However, recent experience in many industrialized countries has shown that unpredictable societal issues can be a bigger barrier to successful vaccination strategies (Amanna & Slifka 2005). Political atrocities and...

Discussion

Canard How do you place the polymerase Lescar The only mapping that was done was before we got the structure. Vasudevan It was with the yeast two-hybrid system. Most likely it is in domain III. Canard So, if I understand correctly, the helicase is behind the polymerase, and the helicase is unwinding after the polymerase. The polymerase is synthesizing a double-stranded RNA, and the helicase is behind, unwinding. Kuhn Remember, the region is believed to be between the two domains of NS5. The...

Mechanism of membrane fusion

The fusion of flaviviruses with membranes is strictly dependent on low pH, consistent with the cell entry by receptor-mediated endocytosis. The pH threshold of fusion has been shown to be around pH 6.4, suggesting that this process already occurs in early endosomes. In vitro studies on the fusion of TBE virus with liposomes have revealed that this process is extremely efficient, does not require an interaction with specific receptors and proceeds significantly faster than that mediated by class...

Secretion of the NS1 protein in plasma is a hallmark of flavivirus infections

We investigated NS1 secretion in clinical flavivirus infections other than DEN, such as YF, and more recently WN. With the use of YF NSl-specific antibodies (generous gift from JJ Schlesinger), it was possible to capture the protein by ELISA in 7 out of l8 serum samples recovered from patients infected with YFV in Senegal (kindly provided by C. Mathiot) (Table 3). The NSl-positive sera clustered in the 0 5 1 0 15 20 25 30 FIG. 3. Status of the NS1-specific IgG response during the course of...

Structural and functional analysis of dengue virus RNA

Lodeiro, Claudia V. Filomatori, Silvana Fucito, Juan A. Mondotte and Andrea V. Gamarnik1 Fundaci n Instituto Leloir, Patricias Argentinas 435, Buenos Aires, Argentina Abstract. Sequences and structures present at the 5' and 3' UTRs of RNA viruses play crucial roles in the initiation and regulation of translation, RNA synthesis and viral assembly. In dengue virus, as well as in other mosquito-borne flaviviruses, the presence of complementary sequences at the ends of...

Prevention

Prevention and control of dengue fever DHF currently depends on controlling the mosquito vector, Ae. aegypti, in and around the home where most transmission occurs. Space sprays with insecticides to kill adult mosquitoes are usually ineffective, unless they are sprayed indoors where the mosquitoes are resting. The most effective way to control the mosquitoes that transmit dengue is larval control, including eliminating, cleaning or chemically treating water-holding containers that serve as the...

Mouse and hamster models for the study of therapy against flavivirus infections

Nathalie Charlier, Erik De Clercq and Johan Neyts Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium Abstract. Small animal models that are reminiscent of flaviviral disease in human will be instrumental in identifying therapeutic strategies against flavivirus infections. Here we review models in mice and hamsters for the most clinically important flavivi-ruses dengue virus, yellow fever virus, West Nile virus, Japanese...

Results and discussion

Subcellular localization of the NS5 protein during DENV infection in Vero cells To determine the subcellular location of the NS5 protein during dengue virus infection, Vero cells infected with DENV-2 NGC at an MOI of approximately 10 were examined at specific stages p.i., by indirect immunofluorescence using a poly-clonal anti-NS5 and an Alexa Fluor 488 green fluorescent dye-conjugated secondary antibody (Fig. 2A). Prior to 12 h p.i. (data not shown), NS5 was unable to be detected in either the...

Conclusion

We reported two mutations (Ile365Ala, Arg376Ala) for which the helicase activity is completely abolished despite an intact ATPase activity. Interestingly, a shallow FIG. 5. Surface representation of the Den NS3 helicase domain in the same orientation as in Fig. 1. The putative interaction between the enzyme and a nucleic acid substrate is shown. The model was generated in part by superposition with the HCV helicase in complex with a dU8 oligonucleotide (PDB code 1A1V Kim et al 1998). FIG. 5....

Antiviral compound screening

Based on assessment of this data, a comprehensive in silico screening of three small compound libraries from the National Cancer Institute against the BOG binding site in the pocket was performed (Zhou & Post, manuscript in preparation). A total of 200,000 ligands were screened by docking them into the BOG binding site. After a rigorous hierarchical and iterative process the lowest-energy conform-ers were selected. Ultimately, based on visual inspection and consideration of drug-like...

A role of viral variation in determining the host response

The role of dengue virus variation in determining host responses and clinical outcome to infection is largely unknown due to the paucity of data on the viral genome structure. Previous studies have concentrated on structural genes, often from an epidemiological perspective. With the development of high-throughput capillary sequencing for viral genomes, investigation of their functional aspects becomes possible, but only with large commitments of time and money. We are developing a rapid and...

Dengue virus

SCID (severe combined immune deficiency) mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID mice) and infected subsequently by the intraperitoneal route with DENV-1, developed viraemia, although virus production was highly variable (Wu et al 1995). DENV-4 replicates in SCID mice that TABLE 1 Mouse or models for dengue virus infection TABLE 1 Mouse or models for dengue virus infection SCID mice transplanted with human peripheral blood lymphocytes and infected with DENV-1...

Entry functions and antigenic structure of flavivirus envelope proteins

Karin Stiasny, Stefan Kiermayr and Franz X. Heinz1 Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A1095 Vienna, Austria Abstract. The envelope proteins (E) of flaviviruses form an icosahedral cage-like structure of homodimers that cover completely the surface of mature virions and are responsible for receptor-binding and membrane fusion. Fusion is triggered by the acidic pH in endosomes which induces dramatic conformational changes of E that drive the merger of the...

Results

Flavivirus replicase proteins are present behind a membrane barrier and are required in catalytic amounts As reported by us previously for JEV (Uchil & Satchidanandam, 2003b), trypsin treatment of heavy membrane fractions from WNV and DENV-infected PS cells did not compromise in vitro RdRp activity (Fig. 1, lanes 2-3 for WNV and 15-16 for DENV). This resistance to trypsin indicated the presence of a membrane barrier behind which not only the template RF as shown by us previously (Uchil &...

Multiple enzyme activities of flavivirus proteins

Reichert*, C. Yon*, T. Teramoto*, Y. Kono*, R. Takhampunya* , S. Ubol , N. Pattabiramanf, B. Falgout , V. K. Ganesh and K. Murthy * Department of Microbiology and Immunology and f Lombardi Cancer Center, Georgetown University School of Medicine, Washington DC, USA, CBER, FDA, Bethesda, MD, USA, University of Alabama-Birmingham, AL, USA and Department of Microbiology, Mahidol University, Bangkok, Thailand Abstract. Dengue viruses (DENV) have 5'-capped RNA genomes...

Summary and conclusions

The structural information of the flavivirus envelope organization as determined by X-ray crystallography and cryo-electron microscopy combined with the results of biochemical and immunological analyses have provided the basis for a molecular understanding of the mechanism of membrane fusion and lead to new insights into the molecular details of antibody-mediated neutralization and cross-reactivity. This knowledge opens new possibilities for the design of antiviral strategies and can contribute...

Analysis of regions of the 5 and 3 UTRs involved in DENV translation and RNA synthesis

Viral translation and RNA synthesis of positive-sense RNA viruses is often regulated by the 5' and 3' UTRs. Previously, we had demonstrated a role for the DENV-2 terminal 3'SL in stimulating viral translation via deletion analysis in RNA reporter constructs (Holden & Harris 2004). To examine other regions of the DENV-2 UTRs, we targeted conserved domains in the 5' and 3' UTRs by complementary peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMOs), as depicted in Fig. 3. The...

Modoc virus and Montana Myotis leukoencephalitis virus

The Modoc virus (MODV) and Montana Myotis leukoencephalitis virus (MMLV), were first isolated from the white footed deer mouse (Peromyscus maniculatus) (MODV) and bats (Myotis lucifugus). These viruses do not cause disease in human (Zarnke & Yuill 1985, Bell & Thomas 1964). Both viruses are neuroinvasive in immunodeficient mice. Although no major histopathological changes were observed in the brain of SCID mice that had obvious neurological symptoms following MODV (or MMLV) infection,...

Dengue Nsl Cost

Harris In your injection experiments did you use a control protein such as E or another dengue viral protein Flamand The difficult thing in having a pertinent control is to find a protein of similar size to the protein of interest but without any effect on the organism. This is difficult to find since NS1 is a hexamer of about 300 kDa. We used purified GFP as a negative control, injected it at a similar level to NS1 in mice and never found any signs of GFP anywhere. Canard How did you purify...

SsDNA High fluorescence

FRET-based helicase unwinding assay. The measured fluorescent signal depends on the interaction between the fluorescent tag and the quencher. Spatial separation of the two DNA strands through ATP driven unwinding increases the signal. An inhibitor would block the unwinding activity of the helicase. Dengue NS5 protein is the largest viral protein in the flavivirus genome and contains two enzymatic activities an S-adenosyl methionine transferase (SAM) and an RNA-dependent RNA polymerase...

Identification of host pathways regulating viral replication using in vitro analysis

In order to identify host factors involved in dengue replication, we have undertaken an analysis of the changes in gene expression in a human hepatocytic cell line following infection with dengue virus. The HepG2 cell line was infected with TSV01, a serotype 2 dengue virus (GenBank AY037116 McBride & Vasudevan 1995), and cells harvested over a succession of time points up to three days postinfection. Comparisons between live virus infection and heat-inactivated virus were used to provide...

References

Alcon S, Talarmin A, Debruyne M, Falconar A, Deubel V, Flamand M 2002 Enzyme-linked immunosorbent assay specific to dengue virus type 1 nonstructural protein reveals circulation of the antigen in blood during the acute phase of disease in patients experiencing primary or secondary infections. J Clin Microbiol 40 376-381 Alcon-LePoder S, Drouet MT, Roux P et al 2005 The secreted form of dengue virus nonstruc-tural protein NS1 is endocytosed by hepatocytes and accumulates in late endosomes...

Development of novel antivirals against flaviviruses

Kuhn1 Department of Biological Sciences, Purdue University, 915 West State Street, West Lafayette, IN 47907-2054, USA Abstract. Dengue virus is responsible for a significant amount of human disease in predominantly tropical areas of the world. Much effort has focused on the development of vaccines against the four serotypes of dengue, and within the next few years a vaccine is anticipated. Less progress has been made at developing antivirals that might reduce...

CRM1dependent nuclear export of dengue virus type 2 NS5

Rawlinson, Peter J. Wright* and David A. Jans1 Department of Biochemistry and Molecular Biology, and * Department of Microbiology Monash University, Victoria 3800, Australia Abstract. The dengue virus multidomain RNA polymerase NS5 has been observed in the nucleus in mammalian infected cell systems. We previously showed that NS5 nuclear localization is mediated by two nuclear targeting signals within the NS5 interdomain region that are recognized by distinct members...

NS3 protease

Dengue Virus Protease

An obvious drug target in this protein is the viral serine protease domain in the N-terminal part. The enzymatic activity of this protein, which is dependent on at least 40 amino acids of NS2B, is vital for the post-translational proteolytic processing of the polyprotein precursor and is essential for viral replication and maturation of infectious dengue virions Falgout et al 1991 . Leung et al 2001 have shown that the dengue protease domain fused to 40 amino acids of NS2B through a flexible...

A noncanonical mechanism of DENV translation initiation

While DENV and other flaviviruses containing capped RNA genomes undergo cap-dependent translation, DENV has been shown to translate efficiently under circumstances in which cap-dependent cellular translation is suppressed through the depletion of the cap-binding protein, eIF4E Edgil et al 2006 . To investigate this phenomenon, we used several approaches to inhibit cap-dependent translation. Initially, the compounds rapamycin, wortmannin and LY294002 were used to suppress cellular translation...

A genomics approach to understanding host response during dengue infection

Hibberd , Ling Ling , Thomas Tolfvenstam , Wayne Mitchell , Chris Wong , Vladimir A. Kuznetsov , Joshy George , Swee-Hoe Ong , Yijun Ruan , Chia L. Wei , Feng Guf, Joshua Finkf, Andy Yipf, Wei Liuf, Mark Schreiberf and Subhash G. Vasudevanf Genome Institute of Singapore, Biopolis, Singapore 138670 and fNovartis Institute for Tropical Diseases, 10 Biopolis Road 05-01 Chromos, Singapore 138670 Abstract. Dengue infection results in a wide clinical spectrum, ranging from asymptomatic,...

Natural history

Thomas Tolfvenstam

There are four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4. They belong to the genus Flavivirus, family Flaviviridae of which yellow fever is the type species , which contains approximately 56 viruses ICTV 2005 . Humans are infected with dengue viruses by the bite of an infective Ae. aegypti mosquito Gubler 1988 . Ae. aegypti is a small, black-and-white, highly domesticated urban mosquito that prefers to lay its eggs in artificial containers commonly found in and around homes in...

Replication of flaviviruses

As with other positive-sense RNA viruses Gamarnik amp Andino 1998 , the 5' terminal region and 3' UTR of flaviviruses play a key role in regulating viral translation and RNA synthesis Lindenbach amp Rice 2001 . Moreover, similar to poliovirus translation Novak amp Kirkegaard 1994 , flavivirus translation is coupled to RNA replication Khromykh et al 1999, 2000 . According to a model that has been proposed for the flavivirus Kunjin KUN , the replication complex is initiated during translation by...

Flavivirus E protein

Protein Structure Dengue

The atomic structures of the ectodomain residues 1-395 of TBEV, DENV-2 and DENV-3 E proteins have been solved using X-ray crystallographic methods and are structurally very similar Fig. 1 Rey et al 1995, Modis et al 2003, 2005 . The E protein ectodomain, minus the C-terminal 100 residues consisting of the stem-anchor regions, displays three domains domain I, the central domain that contains the N-terminus of the protein domain II, the extended, finger-like 'dimerization' domain that makes most...

History

The first reports of major epidemics of an illness thought to possibly be dengue occurred on three continents Asia, Africa and North America in 1779 and 1780 Rush 1789, Hirsch 1883, Pepper 1941, Howe 1977 . However, reports of illnesses compatible with dengue fever occurred even earlier. The earliest record found to date was in a Chinese 'encyclopaedia of disease symptoms and remedies,' first published during the Chin Dynasty AD 265 to 420 and formally edited in AD 610 Tang Dynasty and again in...

Contents

Novartis Foundation Symposium on New treatment strategies for dengue and other flaviviral diseases, held at the Novartis Institute for Tropical Diseases in Singapore, 26 27 September 2005 Editors Gregory Bock Organizer and Jamie Goode This symposium is based on a proposal made by Shubash Vasudevan, Thomas Keller, David Jans and Pad Padmanabhan Duane J. Gubler Dengue dengue haemorrhagic fever history and current status 3 Eva Harris, Katherine L. Holden, Dianna Edgil, Charlotta Polacek and Karen...

Participants

Bruno Canard AFMB UMR 6098 CNRS, 31 Chemin Joseph Aiguier, F-13402 Marseille cedex 20, France Nathalie Charlier University of Leuven, Rega Institute of Medical Research, Laboratory of Virology, Minderbroedersstraat 10, B-3000 Leuven, Belgium Thomas G. Evans Infectious Diseases Translational Medicine, Novartis Institute for Biomedical Research, 100 Technology Square 4153, Cambridge, MA 02139, USA David Fairlie Centre for Drug Design and Development, Institute for Molecular Bioscience, The...