BackgroundSerological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1) has been identified as an early marker for acute dengue, and is typically present between days 1–9 post-onset of illness but following seroconversion it can be difficult to detect in serum.AimsTo evaluate the performance of a newly developed Panbio® Dengue Early Rapid test for NS1 and determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test.MethodologyThe clinical performance of the Dengue Early Rapid was evaluated in a retrospective study in Vietnam with 198 acute laboratory-confirmed positive and 100 negative samples. The performance of the Dengue Early Rapid in combination with the IgM/IgG Rapid test was also evaluated in Malaysia with 263 laboratory-confirmed positive and 30 negative samples.Key ResultsIn Vietnam the sensitivity and specificity of the test was 69.2% (95% CI: 62.8% to 75.6%) and 96% (95% CI: 92.2% to 99.8) respectively. In Malaysia the performance was similar with 68.9% sensitivity (95% CI: 61.8% to 76.1%) and 96.7% specificity (95% CI: 82.8% to 99.9%) compared to RT-PCR. Importantly, when the Dengue Early Rapid test was used in combination with the IgM/IgG test the sensitivity increased to 93.0%. When the two tests were compared at each day post-onset of illness there was clear differentiation between the antigen and antibody markers.ConclusionsThis study highlights that using dengue NS1 antigen detection in combination with anti-glycoprotein E IgM and IgG serology can significantly increase the sensitivity of acute dengue diagnosis and extends the possible window of detection to include very early acute samples and enhances the clinical utility of rapid immunochromatographic testing for dengue.

Understanding the molecular basis of the neutralizing antibody response to dengue virus (DENV) is an essential component in the design and development of effective vaccines and immunotherapeutics. Here we present the structure of a cross-reactive, neutralizing antibody, 3E31, in complex with domain III (DIII) of the DENV envelope (E) protein and reveal a conserved, temperature-sensitive, cryptic epitope on DIII that is not available in any of the known conformations of E on the dengue virion. We observed that 3E31 inhibits E-mediated membrane fusion, suggesting that the antibody is able to neutralize virus through binding an as-yet uncharacterized intermediate conformation of DENV E and sterically block trimer formation. Finally, we show that, unlike cross-reactive fusion peptide-specific antibodies, 3E31 does not promote antibody-dependent enhancement of infection at sub-neutralizing concentrations. Our results highlight the 3E31 epitope on the E protein DIII as a promising target for immunotherapeutics or vaccine design.

Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus.

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The immunogenicity of P97 adhesin repeat region R1 (P97R1) of Mycoplasma hyopneumoniae, an important pathogenesis-associated region of P97, was evaluated in mice as a mucosal vaccine. Mice were immunized orally with attenuated Salmonella typhimurium aroA strain CS332 harbouring a eukaryotic or prokaryotic expression vector encoding P97R1. Local and systemic immune responses were analysed by ELISA on mouse sera, lung washes and splenocyte supernatants following splenocyte stimulation with specific antigens in vitro. Although no P97R1-specific antibody responses were detected in serum and lung washes, significant gamma interferon was produced by P97R1-stimulated splenocytes from mice immunized orally with S. typhimurium aroA harbouring either expression system, indicating induction of a cell-mediated immune response. These results suggested that live bacterial vectors carrying DNA vaccines or expressing heterologous antigens preferentially induce a Th1 response. Surprisingly, however, mice immunized with the vaccine carrier S. typhimurium aroA CS332 induced serum IgG, but not mucosal IgA, against P97R1 or S. typhimurium aroA CS332 whole-cell lysate, emphasizing the importance of assessing the suitability of attenuated S. typhimurium antigen-carrier delivery vectors in the mouse model prior to their evaluation as potential vaccines in the target species, which in this instance was pigs. INTRODUCTIONPorcine enzootic pneumonia (PEP) caused by Mycoplasma hyopneumoniae is a chronic, non-fatal respiratory disease. It affects pigs of all ages, resulting in slow growth and reduced feed efficiency, and causes significant economical losses in the pig industry worldwide (Sheldrake et al., 1991;Thacker et al., 1998). PEP is often followed by secondary infections by Porcine reproductive and respiratory syndrome virus and opportunistic bacterial pathogens (Calsamiglia et al., 1999;Sheldrake et al., 1991;Thacker et al., 1998Thacker et al., , 1999. As current market vaccines (bacterins prepared by chemical inactivation of whole-cell M. hyopneumoniae) do not protect pigs completely from establishment of M. hyopneumoniae infection and/or secondary infection (Kristensen et al., 1981;Maes et al., 1999;Murphy et al., 1993; Pallares et al., 2000;Thacker et al., 1998), development of an improved vaccine is desirable. P97 adhesin has probably been the most-studied and bestdefined potential protective antigen of M. hyopneumoniae since it was identified as an important adhesin responsible for the adherence of M. hyopneumoniae to respiratory ciliated epithelial cells in swine (Zhang et al., 1995). Furthermore, a repeat region of P97 called R1 (P97R1) has been identified as containing both cilium-and antibodybinding sites (Hsu & Minion, 1998;Hsu et al., 1997) and has been reported to function independently from other P97 regions (Minion et al., 2000). P97R1 has been shown to induce significant P97R1-specific serum IgG titres in both mice and pigs (Chen et al., 2001), indicating that it is immunogenic. However, the immunogenicity of P97R1 has...