Dengue viruses occur as four antigenically related but distinct serotypes transmitted to humans by Aedes aegypti mosquitoes. These viruses generally cause a benign syndrome, dengue fever, in the American and African tropics, and a severe syndrome, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), in Southeast Asian children. This severe syndrome, which recently has also been identified in children infected with the virus in Puerto Rico, is characterized by increased vascular permeability and abnormal hemostasis. It occurs in infants less than 1 year of age born to dengue-immune mothers and in children 1 year and older who are immune to one serotype of dengue virus and are experiencing infection with a second serotype. Dengue viruses replicate in cells of mononuclear phagocyte lineage, and subneutralizing concentrations of dengue antibody enhance dengue virus infection in these cells. This antibody-dependent enhancement of infection regulates dengue disease in human beings, although disease severity may also be controlled genetically, possibly by permitting and restricting the growth of virus in monocytes. Monoclonal antibodies show heterogeneous distribution of antigenic epitopes on dengue viruses. These epitopes serve to regulate disease: when antibodies to shared antigens partially neutralize heterotypic virus, infection and disease are dampened; enhancing antibodies alone result in heightened disease response. Further knowledge of the structure of dengue genomes should permit rapid advances in understanding the pathogenetic mechanisms of dengue.

https://science.sciencemag.org/content/sci/239/4839/476.full.pdf

Pathogenesis of dengue: challenges to molecular biology

Dengue, a major public health problem throughout subtropical and tropical regions, is an acute infectious disease characterized by biphasic fever, headache, pain in various parts of the body, prostration, rash, lymphadenopathy, and leukopenia. In more severe or complicated dengue, patients present with a severe febrile illness characterized by abnormalities of hemostasis and increased vascular permeability, which in some instances results in a hypovolemic shock. Four distinct serotypes of the dengue virus (dengue-1, dengue-2, dengue-3, and dengue-4) exist, with numerous virus strains found worldwide. Molecular cloning methods have led to a greater understanding of the structure of the RNA genome and definition of virus-specific structural and nonstructural proteins. Progress towards producing safe, effective dengue virus vaccines, a goal for over 45 years, has been made. Images

The dengue viruses.

The cleavages at the junctions of the flavivirus nonstructural (NS) proteins NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 share an amino acid sequence motif and are presumably catalyzed by a virus-encoded protease. We constructed recombinant vaccinia viruses expressing various portions of the NS region of the dengue virus type 4 polyprotein. By analyzing immune precipitates of 35S-labeled lysates of recombinant virus-infected cells, we could monitor the NS2A/NS2B, NS2B/NS3, and NS3/NS4A cleavages. A polyprotein composed of NS2A, NS2B, and the N-terminal 184 amino acids of NS3 was cleaved at the NS2A/NS2B and NS2B/NS3 junctions, whereas a similar polyprotein containing only the first 77 amino acids of NS3 was not cleaved. This finding is consistent with the proposal that the N-terminal 180 amino acids of NS3 constitute a protease domain. Polyproteins containing NS2A and NS3 with large in-frame deletions of NS2B were not cleaved at the NS2A/NS2B or NS2B/NS3 junctions. Coinfection with a recombinant expressing NS2B complemented these NS2B deletions for NS2B/NS3 cleavage and probably also for NS2A/NS2B cleavage. Thus, NS2B is also required for the NS2A/NS2B and NS2B/NS3 cleavages and can act in trans. Other experiments showed that NS2B was needed, apparently in cis, for NS3/NS4A cleavage and for a series of internal cleavages in NS3. Indirect evidence that NS3 can also act in trans was obtained. Models are discussed for a two-component protease activity requiring both NS2B and NS3.

Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins.

A new virus, Hepatitis C virus (HCV), which has proven to be the major etiologic agent of blood-borne NANBH, was discovered by Applicant. The initial work on this virus, which includes a partial genomic sequence of the prototype HCV isolate, is described in EPO Pub. No. 318,216, and PCT Pub. No. WO/89/04669. The present invention, which in part is based on new HCV sequences and polypeptides which are not disclosed in the above-cited publications, includes the application of these new sequences and polypeptides in immunoassays, probe diagnostics, anti-HCV antibody production, PCR technology, and recombinant DNA technology. Included within the invention also are novel immunogenic polypeptides encoded within clones containing HCV cDNA, novel methods for purifying an immunogenic HCV polypeptide, and antisense polynucleotides derived from HCV cDNA.

Karin Page

Papers

Partial sequence analysis of cloned dengue virus type 2 genome.