Biomedical subjects

A K Falconar

Publications and source records attributed to A K Falconar.

7 recordsLinked to original sources

PubMed2000

The use of direct sequencing of dengue virus cDNA from individual field-collected Aedes aegypti for surveillance and epidemiological studies.

The relative efficiencies of four methods to extract viral RNA from individual dengue-2 virus (D-2V)-infected mosquitoes, Aedes aegypti (L.) (Diptera: Culicidae), were compared. The most efficient of these methods was then used to extract viral RNA for the preparation of cDNA from the abdomens of six engorged D-2V-infected mosquitoes and sera from three dengue fever (DF) patients collected in an isolated rural town in Colombia. Comparisons of viral envelope (E) gene sequences from each of these strongly suggested that the D-2V population which circulated in this study area was a homogeneous genotype which was unrelated to any of the D-2 viruses isolated from elsewhere in the world. When coupled with our rapid method to identify viruses in individual mosquitoes (Romero-Vivas et al. (1998) Medical and Veterinary Entomology, 12, 101-105), the methodology we describe should be useful for epidemiological and surveillance studies of dengue viruses and other arboviruses.

Abdomen↗

PubMed1999

Identification of an epitope on the dengue virus membrane (M) protein defined by cross-protective monoclonal antibodies: design of an improved epitope sequence based on common determinants present in both envelope (E and M) proteins.

The protective capacity of monoclonal antibodies (MAbs) generated to the dengue-2 virus envelope (E) and premembrane (prM) proteins was tested in vivo. Two anti-E MAbs, 2C5.1 and 4G2 and two anti-prM MAbs, 2A4.1 and 2H2 provided cross-protection against all four dengue virus serotypes. Overlapping sets of synthetic peptides spanning amino-acid sequence 301-401 (domain III) of the E protein and the entire prM protein were then used to locate their epitopes. The anti-E MAbs strongly reacted with the peptide sequence 349-GRLITVNPIVT-359 (E349-359) from domain III and the immunodominant epitope, 274-SGNLLFTGHL-283 (E274-283) from the hinge region between domains I and II. The anti-prM MAbs strongly reacted with the sequence, 40-PGFTVMAAIL-49 (M40-49) from the first membrane-spanning domain of the M protein. These anti-prM MAbs also reacted with peptides E274-283 and E349-359, while the anti-E MAbs reacted with a peptide sequence, 1-FHLTTRNGEP-10 from the prM protein and these cross-reactions with both proteins were confirmed using immunoblot assays. MAbs 2C5.1, 4G2 and 2H2 more strongly reacted with an MEH1 peptide GLFTPNLITI, which was designed as an antigenic hybrid between these E and prM peptide sequences, than with any of these natural peptide sequences. These peptide sequence will now be tested for their ability to generate cross-protective antibodies against each dengue virus serotype when delivered with appropriate T-helper epitopes.

Amino Acid Sequence↗

PubMed1998

Determination of dengue virus serotypes in individual Aedes aegypti mosquitoes in Colombia.

Adult Aedes aegypti mosquitoes were collected in Puerto Triunfo, central Colombia, where dengue is endemic, during a six month period. Viral infection within the head of each individual mosquito was identified by an immunofluorescent assay (IFA) using a flavivirus-specific monoclonal antibody. The dengue virus serotype, present in each flavivirus-positive specimen, was then determined in portions of the remaining thorax using IFAs with serotype-specific monoclonal antibodies. Among 2065 female Aedes aegypti collected and tested, twenty-four flavivirus-positive individuals were found (minimum infection rate 11.6%), three identified as dengue type-1 and twenty-one as dengue type-2 virus. This was consistent with the isolation of only these two serotypes of dengue virus from dengue fever patients within this town. No vertical transmission of dengue virus could be detected in 1552 male Aedes aegypti collected. This method is inexpensive, simple, rapid to perform and suitable for use in developing countries to identify and distinguish different serotypes of dengue virus in their vectors during eco-epidemiological investigations.

Aedes↗

PubMed1997

The dengue virus nonstructural-1 protein (NS1) generates antibodies to common epitopes on human blood clotting, integrin/adhesin proteins and binds to human endothelial cells: potential implications in haemorrhagic fever pathogenesis.

Antibody responses generated by mice to the dengue-2 virus NS1 protein (D-2V NS1) were influenced by MHC class II (I-A) haplotype but each antiserum cross-reacted with human fibrinogen, thrombocytes and endothelial cells. To investigate these findings, a highly avid subclone (MAb 1G5.4-A1-C3) was selected from a parent hybridoma that secreted a monoclonal antibody (MAb) specific for the native dimeric form of D-2V NS1. When MAb reactions were compared using a panel of overlapping synthetic peptides covering the entire protein sequence, dimer specificity was found to be a weak reaction with multiple ELK-type motifs present in either the positive (E/D-hydrophobic-K/R) or negative (K/R-hydrophobic-D/E) orientations. MAb 1G5.4-A1-C3 and highly avid anti-NS1 polyclonal antisera reacted with the NS1 proteins of the four dengue virus serotypes, but only weakly reacted with the NS1 proteins of the other flaviviruses. MAb 1G5.4-A1-C3 and several other anti-NS1 MAbs produced haemorrhage in mice, cross-reacted with human fibrinogen, thrombocytes and endothelial cells, with known epitopes or active sites on human clotting factors and integrin/adhesin proteins present on these cells. D-2V NS1 bound to human endothelial cells via a site within its N-terminal region, which led to significantly increased binding of avid anti-NS1 antibodies. These results identified a potential role of both 'antigenic' and 'biochemical' mimicry in dengue haemorrhagic fever pathogenesis, consistent with clinical data.

Animals↗

PubMed1994

Precise location of sequential dengue virus subcomplex and complex B cell epitopes on the nonstructural-1 glycoprotein.

The reactions of a panel of 34 mouse monoclonal antibodies (MAbs) specific for the dengue-2 virus nonstructural-1 glycoprotein (NS1), were analysed using 174 overlapping synthetic nonameric peptides covering the entire sequence. Using this methodology, four epitopes were identified. One pair of MAbs, which defined a dengue-2/4 virus subcomplex epitope (24C: amino acids 299-309) using native NS1 proteins, showed the same reaction pattern with synthetic peptides containing the corresponding NS1 sequences of each virus serotype. One amino acid substitution, present in the sequences from the dengue-1/3 virus subcomplex abrogated almost all reaction by these MAbs. A dengue complex epitope (LX1: amino acids 111-121) was also located and peptides containing the sequences of each serotype were shown to contain only antigenically silent amino-acid substitutions. In contrast, MAbs which defined a dengue type-specific epitope (LD2: amino acids 25-33) and another dengue subcomplex epitope (24A: amino acids 61-69) failed to show the same reaction profiles using peptides of each serotype, suggesting that these determinants were partially dependent upon conformation. The LX1 epitope is a good candidate for further trials aimed at generating cross-protective immune responses to these viruses without the risk of antibody-dependent enhancement.

Amino Acid Sequence↗

PubMed1991

Production of dimer-specific and dengue virus group cross-reactive mouse monoclonal antibodies to the dengue 2 virus non-structural glycoprotein NS1.

A panel of mouse monoclonal antibodies (MAbs) raised against the non-structural glycoprotein NS1 of dengue 2 virus (PR159) was studied for cross-reactivity with the NS1 protein of other dengue virus serotypes and other members of the Flaviviridae using immunoblotting. Most of the 35 anti-NS1 MAbs were found to be specific for dengue 2 virus NS1 (some of which were specific for the native, dimeric form of this protein), but others were found to cross-react within the dengue virus group. This latter group of MAbs, although dominated by MAbs defining a dengue 2 and 4 virus subgroup, also contained some MAbs that were shown to cross-react with both linear (sequential) and conformational epitopes common to the NS1 glycoproteins of all four dengue virus serotypes. Several of these MAbs were also able to cross-react with other flaviviruses, most notably viruses from the Japanese encephalitis antigenic complex. Although cross-reactive epitopes were previously demonstrated on this glycoprotein using polyclonal sera from dengue virus-infected animals and human, this is the first report of the isolation of MAbs which define these determinants and which will allow their further analysis.

Animals↗

PubMed1990

Immunoaffinity purification of native dimer forms of the flavivirus non-structural glycoprotein, NS1.

The flavivirus non-structural glycoprotein, NS1 has been shown to elicit an immune response in animals which may confer protection from subsequent virus challenge (Schlesinger et al., 1985 and 1987). While previous reports have outlined methods for obtaining cell-associated NS1 in monomeric form for these studies, we describe here an efficient method for the immunoaffinity purification of both cell-associated and secreted NS1 in their native dimeric configuration. These dimer preparations were shown to be both more antigenic and immunogenic than their monomeric counterparts, a finding which may in part explain the reported failure to obtain solid protection of mice from homologous dengue virus challenge. In moderately sized virus growth experiments, greater than 1 mg quantities of purified NS1 were obtained.

Animals↗