Dengue 2 Research Articles

Preparation of an Attenuated Dengue 4 (341750 Carib) Virus Vaccine

Dengue 4 (DEN-4) virus strain 341750 Carib was modified by serial passage in primary canine kidney (PCK) cell cultures. By the 15th PCK passage, this virus was less infectious for monkeys and resulted in a significantly reduced viremia as compared to the parent DEN-4 virus. The 30th PCK passage of DEN-4 341750 Carib was non-infectious for monkeys. A vaccine prepared at the 20th PCK passage in DBS-FRhL-2 cells stimulated the production of both neutralizing and hemagglutination inhibition antibodies in monkeys; these animals were also protected against challenge with the homologous strain as well as a heterologous strain of DEN-4. An ID50 titration in monkeys resulted in a titer of greater than 10(4) plaque-forming units (PFU) for the vaccine virus and 0.5 PFU for the parent virus. Reduced monkey infectivity of this magnitude has been correlated with human attenuation in previous dengue vaccine candidates. The DEN-4 strain 341750 Carib PCK-20/FRhL-4 vaccine has been characterized and sufficiently tested to be considered for safety and immunogenicity trials in humans.

Detection of Dengue 4 Virus Core Protein in the Nucleus. I. A Monoclonal Antibody to Dengue 4 Virus Reacts with the Antigen in the Nucleus and Cytoplasm

A mouse monoclonal antibody (MAb) to dengue 4 (DEN-4) virus reacted with the antigen in the nucleus as well as in the cytoplasm of DEN-4-infected mammalian and mosquito cells, as demonstrated by the peroxidase-antiperoxidase staining method. The intranuclear antigen appeared to accumulate at the nucleoli, forming spots, whereas the cytoplasmic antigen appeared to be localized mainly in large perinuclear foci in the infected cells. The MAb-reactive antigen was produced in the presence of actinomycin D, which caused the accumulation in the nucleus to be altered to a dispersed pattern. Radioimmunoprecipitation analysis of [35S]methionine-labelled purified virions and Western blot analysis of the antigens prepared from the infected mammalian and mosquito cells showed that the MAb was directed against the DEN-4 virus core protein (Mr 15.5K). These results indicated that the DEN-4 virus core protein was partially transported, soon after its synthesis in the cytoplasm, into the nucleus and accumulated at the nucleoli.

Identification of Continuous Epitopes of the Envelope Glycoprotein of Dengue Type 2 Virus

We reacted 490 hexapeptides homologous to the amino acid sequence of the dengue 2 (DEN-2) virus envelope glycoprotein with antisera from 7 patients with primary DEN-2 virus infections to identify the continuous epitopes recognized by human IgG. There were 124 peptides in 25 clusters (domains) that bound 2 or more antisera. Twenty-two peptides in 7 domains bound all 7 convalescent DEN-2 virus antisera tested, and thus appeared to represent immunodominant epitopes. The evidence that these domains represents continuous epitopes of the envelope glycoprotein is that peptide representing each domain bound multiple sera, peptide reactivity was highly ordered along the amino acid sequence, and in almost all cases, domains were regions of predicted hydrophilicity. Heterologous flavivirus antisera also exhibited binding to the majority of peptides reactive with anti-DEN-2 virus sera, though 4 candidate DEN-2 specific epitopes were identified along with an immunodominant epitope common to dengue, Japanese encephalitis and West Nile viruses. Synthetic peptides representing these epitopes may prove to be useful for a variety of purposes.

Antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever.

Serum specimens collected during a prospective study of dengue infections among schoolchildren in Bangkok were tested for their ability to enhance dengue 2 (DEN-2) virus growth in human monocytes in vitro. Two groups of dengue-immune sera were compared: 32 dengue antibody positive serum specimens from children who subsequently developed asymptomatic secondary dengue infections; and 9 dengue antibody positive serum specimens from children who subsequently developed severe symptomatic secondary dengue infections, 8 of which were clinically diagnosed as dengue hemorrhagic fever. Antibody-dependent enhancement of virus growth was quantitated by measurement of virus yields in supernatant fluids of normal human monocyte cultures that were infected with DEN-2 virus in the presence of undiluted test serum. Only 4 of 32 (12%) preinfection sera from asymptomatic children, but 6 of 9 (67%) preinfection sera from symptomatic children, had significant enhancing activity (P less than 0.001). High serum DEN-2 antibody dependent enhancing activity is a significant (relative risk = 6.2) risk factor for severe illness among children in a dengue hemorrhagic fever endemic region. Dengue antibodies can be neutralizing and therefore protective, or they can be enhancing and increase the risk of dengue hemorrhagic fever.

Flavivirus entry into cultured mosquito cells and human peripheral blood monocytes

The entry modes of Japanese encephalitis (JE) and dengue-2 (DEN-2) viruses into C6/36 mosquito cells and of DEN-2 virus into human peripheral blood monocytes in vitro were studied. Inoculation of either JE or DEN-2 virions into C6/36 cells resulted in direct penetration of the virions into the cytoplasm at the cell surface in 3 stages. At stage 1, virions attached to the plasma membrane of host cells by their envelope spikes; at stage 2, the virion envelopes approximated to and eventually overlapped the host plasma membrane, and in the process the plasma membrane at the attachment sites dissolved; and, at stage 3, virions penetrated into the cytoplasm through the plasma-membrane disruptions created at the adsorption sites. Virions themselves apparently disintegrated at or near the penetration sites, for no virions were seen in the deeper cytoplasm. Coated pits did not form at the virion attachment sites, and virion-containing vesicles were not found in the cytoplasm. In the entry of DEN-2 virus into human peripheral blood monocytes, virions were found, adsorbed onto the external surface of the plasma membrane and attached to the luminal surface of macropinocytic vacuolar membranes. The latter apparently occurred as the result of ruffling and macropinocytic activities of the cells. At both sites virions penetrated into the cytoplasm through the plasma or vacuolar membrane in the same manner as they did through the plasma membrane of C6/36 cells. No evidence of viral entry by receptor-mediated endocytosis was observed. Implications of the entry mode of the mosquito cell-generated DEN-2 virus into human peripheral blood monocytes to an early process of natural, mosquito-transmitted infection is discussed.

Disease severity‐related antigenic differences in dengue 2 strains detected by dengue 4 monoclonal antibodies

Low-passage epidemic Thai isolates of dengue 2 (DEN-2) were distinguished on the basis of associated disease severity by detection of virus determinants involved in enhanced macrophage infection when incubated with a panel of monoclonal antibodies derived from a DEN-4 strain associated with grade II dengue hemorrhagic fever (DHF). DEN-2 strains associated with DHF typically had multiple determinants that were involved in antibody-dependent enhancement of infection, whereas those associated with uncomplicated disease had few. These data provide further evidence that in epidemic areas where DHF is associated with prior circulation of low-level monotypic antibody, severe dengue disease could represent antibody-enhanced infection of human monocytes/macrophages.

Study of the distribution of antibody‐ dependent enhancement determinants on dengue 2 isolates using dengue 2‐derived monoclonal antibodies

Dengue 2 (DEN-2) strains isolated from children during the 1980 metropolitan Bangkok epidemic were shown to possess antigenic homogeneity when studied for determinants mediating antibody-dependent infection enhancement using DEN-2 monoclonal antibodies. All isolates possessed multiple enhancing determinants, but those associated with mild and severe dengue syndromes could not be distinguished. Either the basis of disease severity in dengue is more complex than the mere presence or absence of virus epitopes involved in enhanced infection or enhancing epitopes have differences not detected in this system with monoclonal antibodies raised to the same serotype.

Topological Mapping of Unique Epitopes on the Dengue-2 Virus NS1 Protein Using Monoclonal Antibodies

Monoclonal antibodies were produced against two distinct Thai dengue-2 (DEN-2) virus strains isolated in 1980 from dengue haemorrhagic fever patients. Nine of 36 hybridomas produced monoclonal IgG antibodies which reacted in radioimmune precipitation assays with the NS1 non-structural protein (42,000 mol. wt.) from DEN-2-infected C6/36 (Aedes albopictus) cells. The virus specificity of NS1-reactive monoclonal antibodies was determined by indirect immunofluorescence assays using LLC-MK2 cells infected with either the Thai 1980 DEN-2 isolates, prototype DEN viruses (four serotypes), Japanese encephalitis (JE), Murray Valley encephalitis, West Nile, Wesselbron or Tembusu viruses. Eight of the monoclonal antibody preparations were DEN-2-serotype specific. One preparation defined a special serological relationship between DEN-2 and JE viruses. Four preparations had detectable complement fixation titres using Thai DEN-2 virus antigen. Six spatially unique epitopes were identified using competitive binding assays.

Partial nucleotide sequence of St. Louis encephalitis virus RNA: Structural proteins, NS1, ns2a, and ns2b

cDNA clones of the St. Louis encephalitis (SLE) virus genome have been obtained and the nucleotide sequence of 4.7 kb corresponding to the 5′ terminal half of the genome determined. The genome contains a 5′ noncoding region of 98 nucleotides followed by a single continuous open reading frame that encodes three structural proteins in the order capsid (C), membrane precursor (prM)-membrane (M), and envelope (E). Immediately following the C-terminus of E are located nonstructural proteins NS1 through NS3. The SLE amino acid sequence homology with yellow fever (YF), Murray Valley encephalitis (MVE), West Nile (WN), and dengue-2 (DEN) viruses over the sequenced region is 39, 66, 64, and 43%, respectively. The start of each SLE protein has been assigned on the basis of N-terminal sequence data and potential proteolytic cleavage sites homologous with YF and MVE viruses. Flaviviruses have conserved glycosylation sites in prM and NS1 proteins, although only one of the two glycosylation sites in the SLE E protein is conserved in MVE and DEN viruses. An evolutionary tree showing relationships of SLE, MVE, WN, YF, and DEN-2 flaviviruses is proposed on the basis of the amino acid sequences of the C proteins.

The association of enhancing antibodies with seroconversion in humans receiving a dengue-2 live-virus vaccine.

A group of human subjects, some with yellow fever (YF) antibodies, volunteered for testing of a live-attenuated dengue-2 (DEN-2) vaccine. Serum samples taken before DEN-2 vaccination were tested for their ability to enhance infection of human monocytes by DEN-2 virus. A significantly greater proportion of enhancing antibodies (Eab) were found in YF-immune (YFI) individuals (50%) as compared to those with no evidence of flavivirus infection (9.5%). Geometric mean titers of neutralizing and hemagglutination inhibiting antibodies to DEN-2 virus in YFI subjects with Eab were fourfold to seven-fold higher than in the YFI subjects without Eab in prevaccine sera and 10- to 35-fold higher than in non-immune volunteers. Additionally, levels of Eab in prevaccine sera were directly related to antibody titers found in postvaccine sera. The presence of Eab in the serum of a human subject before DEN-2 vaccination was a good predictor of the immune response after vaccination, and may in part be responsible for the higher seroconversion rate in YF immunes (90%) as compared to nonimmunes (61%) receiving this vaccine. This is the first human study to demonstrate that circulating Eab in non-DEN-immune persons is associated with an augmented immune response to DEN virus infection. This finding supports the hypothesis that cross-reactive antibodies against one flavivirus enhance an infection with another closely related flavivirus.

Human antibody response to immunization with 17D yellow fever and inactivated TBE vaccine.

The antibody response against flaviviruses tick-borne encephalitis (TBE), Kyasanur Forest disease (KFD), Murray Valley encephalitis (MVE), West Nile fever (WNF), Japanese B encephalitis (JE), dengue 2 (DEN-2), and yellow fever (YF) was studied in humans after administration of an inactivated TBE virus vaccine. Individuals were either prevaccinated with 17D yellow fever (experimental group) or without any previous exposure to flaviviruses (control group). The appearance of serum titres of homologous and heterologous haemagglutination inhibition (HI) antibodies, heterotypic DEN-2 neutralizing antibodies, and TBE enzyme-linked immunosorbent assay (ELISA) antibodies were examined. Individuals prevaccinated with the 17D yellow fever developed an antibody pattern that contrasted with that of the control group. This pattern was characterized as follows: (1) Predominantly anti-TBE IgG antibodies appeared earlier and in higher titres than in the control group, (2) heterologous HI antibodies cross-reacting with the WN flavivirus subgroup preceded the appearance of homologous HI antibodies, (3) a broad spectrum HI response was observed against all flaviviruses tested, and (4) low titre heterotypic DEN-2 neutralizing antibodies were formed in about half of the cases. These observations are discussed in the context of cross-reactivity, cross-protection and virus infection enhancement.

A sero-epidemiological survey for certain arboviruses (Togaviridae) in Pakistan

Complement-fixation test reactions to eight viruses of the family Togaviridae were studied in 372 serum samples (157 rodents, 172 domestic animals, 43 humans) from Pakistan. Antibodies to each tested virus were detected. The highest over-all prevalence rates were for West Nile (WN) (7·8%), Japanese encephalitis (JE) (3·2%) and Zika (ZIKA) (2·4%) viruses, followed by Sindbis (SIN), Chikungunya (CHIK), Uganda S (UGS) and Royal Farm (RF) viruses (1·6 to 1·3%). One human serum (male, age 58 years) reacted with Dengue-1 (DEN) virus antigen (titre 1:32). Antibodies to each virus except RF were detected in human sera; antibodies to RF virus were detected only in rodent and domestic animal sera. The roles of rodents in the epidemiology of WN, JE and ZIKA viruses should be investigated. At least six of these eight viruses cause fevers in humans (fevers of unknown origin comprise about one third of the febrile episodes recorded in Pakistan).

Dengue outbreaks in Guánica-Ensenada and Villalba, Puerto Rico, 1972-1973.

Epidemics of dengue fever occurring in Puerto Rico in 1963 to 1964 and 1969 were caused by dengue-3 and dengue-2 (DN-2) viruses, respectively, but endemic dengue transmission has never been documented on the Island. Since the 1969 epidemic, a surveillance system has detected DN-2 activity on the Island during each of the years 1970 through 1973, which suggests endemic persistence of the virus. This report describes the investigation of localized outbreaks of DN-2 in Guanica-Ensenada (1972) and Villalba (1973), and presents epidemiological, serological, and virological data from the outbreaks. Analysis of geographic distribution of dengue activity in Puerto Rico in recent years indicates that the DN-2 transmission in 1970 to 1973 may represent a long tail-off of the 1969 epidemic rather than the emergence of a truly endemic situation.