ZikaPLAN: Zika Preparedness Latin American Network.

The ongoing Zika virus (ZIKV) outbreak in Latin America, the Caribbean, and the Pacific Islands has underlined the need for a coordinated research network across the whole region that can respond rapidly to address the current knowledge gaps in Zika and enhance research preparedness beyond Zika. The European Union under its Horizon 2020 Research and Innovation Programme awarded three research consortia to respond to this need. Here we present the ZikaPLAN (Zika Preparedness Latin American Network) consortium. ZikaPLAN combines the strengths of 25 partners in Latin America, North America, Africa, Asia, and various centers in Europe. We will conduct clinical studies to estimate the risk and further define the full spectrum and risk factors of congenital Zika virus syndrome (including neurodevelopmental milestones in the first 3 years of life), delineate neurological complications associated with ZIKV due to direct neuroinvasion and immune-mediated responses in older children and adults, and strengthen surveillance for birth defects and Guillain-Barré Syndrome. Laboratory-based research to unravel neurotropism and investigate the role of sexual transmission, determinants of severe disease, and viral fitness will underpin the clinical studies. Social messaging and engagement with affected communities, as well as development of wearable repellent technologies against Aedes mosquitoes will enhance the impact. Burden of disease studies, data-driven vector control, and vaccine modeling as well as risk assessments on geographic spread of ZIKV will form the foundation for evidence-informed policies. While addressing the research gaps around ZIKV, we will engage in capacity building in laboratory and clinical research, collaborate with existing and new networks to share knowledge, and work with international organizations to tackle regulatory and other bottlenecks and refine research priorities. In this way, we can leverage the ZIKV response toward building a long-term emerging infectious diseases response capacity in the region to address future challenges.

The single water-surface sweep estimation method accurately estimates very low (n = 4) to low-moderate (n = 25-100) and high (n > 100) Aedes aegypti (Diptera: Culicidae) pupae numbers in large water containers up to 13 times faster than the exhaustive sweep and total count method and without any sediment contamination.

OBJECTIVE: To confirm that a single water-surface sweep-net collection coupled with three calibration factors (2.6, 3.0 and 3.5 for 1/3, 2/3 and 3/3 water levels, respectively) (WSCF) could accurately estimate very low to high Aedes aegypti pupae numbers in water containers more rapidly than the exhaustive 5-sweep and total count (ESTC) method recommended by WHO. METHODS: Both methods were compared in semi-field trials using low (n = 25) to moderate (n = 50-100) pupae numbers in a 250-l drum at 1/3, 2/3 and 3/3 water levels, and by their mean-time determinations using 200 pupae in three 220- to 1024-l water containers at these water levels. Accuracy was further assessed using 69.1% (393/569) of the field-based drums and tanks which contained <100 pupae. RESULTS: The WSCF method accurately estimated total populations in the semi-field trials up to 13.0 times faster than the ESTC method (all P < 0.001); no significant differences (all P-values ≥ 0.05) were obtained between the methods for very low (n = 4) to low-moderate (n = 25-100) and high (n > 100) pupae numbers/container and without sediment disturbance. CONCLUSION: The simple WSCF method sensitively, accurately and robustly estimated total pupae numbers in their principal breeding sites worldwide, containers with >20 l water volumes, significantly (2.7- to 13.0-fold: all P-values <0.001) faster than the ESTC method for very low to high pupae numbers/container without contaminating the clean water by sediment disturbance which is generated using the WHO-recommended ESTC method. The WSCF method seems ideal for global community-based surveillance and control programmes.

Three calibration factors, applied to a rapid sweeping method, can accurately estimate Aedes aegypti (Diptera: Culicidae) pupal numbers in large water-storage containers at all temperatures at which dengue virus transmission occurs.

The ability of a simple sweeping method, coupled to calibration factors, to accurately estimate the total numbers of Aedes aegypti (L.) (Diptera: Culicidae) pupae in water-storage containers (20-6412-liter capacities at different water levels) throughout their main dengue virus transmission temperature range was evaluated. Using this method, one set of three calibration factors were derived that could accurately estimate the total Ae. aegypti pupae in their principal breeding sites, large water-storage containers, found throughout the world. No significant differences were obtained using the method at different altitudes (14-1630 m above sea level) that included the range of temperatures (20-30 degrees C) at which dengue virus transmission occurs in the world. In addition, no significant differences were found in the results obtained between and within the 10 different teams that applied this method; therefore, this method was extremely robust. One person could estimate the Ae. aegypti pupae in each of the large water-storage containers in only 5 min by using this method, compared with two people requiring between 45 and 90 min to collect and count the total pupae population in each of them. Because the method was both rapid to perform and did not disturb the sediment layers in these domestic water-storage containers, it was more acceptable by the residents, and, therefore, ideally suited for routine surveillance purposes and to assess the efficacy of Ae. aegypti control programs in dengue virus-endemic areas throughout the world.

Pupal-productivity surveys to identify the key container habitats of Aedes aegypti (L.) in Barranquilla, the principal seaport of Colombia.

Surveys were conducted in three neighbourhoods of Barranquilla, the main seaport of Colombia, to identify, using counts of pupae in water containers during the wet and dry seasons, the most productive Aedes aegypti breeding sites. Overall, 3,433 premises were investigated in the wet season and 3,563 in the dry, representing, respectively, 82.3% and 84.6% of the total numbers of premises in the study areas. Despite a reasonably reliable supply of piped water, there were still some large storage containers for domestic water (cement ground tanks and plastic, metal and cement drums) in the area. Although such containers represented only 1.8%-16.3% of the total number of containers observed, they contributed 72.0%-78.2% and 65.0%-95.8% of the total Ae. aegypti pupal population in the three study neighbourhoods during the wet and dry seasons, respectively. In contrast, bottles represented 23.0%-88.9% of the total number of containers but produced no more than 0.1% of the total Ae aegypti pupal populations in these neighbourhoods. Other containers (tyres, vases, 'other discarded' and 'other used') generally produced only low numbers of pupae. In some settings, however, containers in the 'other discarded' category could contribute up to 19% of the total pupal population, and in one survey of one neighbourhood a single container in this category held 9.1% of all the pupae collected. These results, from a city where dengue fever is endemic, will help to focus local campaigns for Ae. aegypti source-reduction on the most productive categories of container.

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.

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.

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.

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.

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.

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.

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.