Prevalence and molecular characterization of Rickettsia spp. from wild small mammals in public parks and urban areas of Bangkok Metropolitan, Thailand

Rural areas usually show a higher prevalence of rickettsial infection than urban areas. However, information on the rickettsial infection status in urban settings (e.g., built-up areas and city parks) is still limited, particularly in the Bangkok metropolitan area. In this study, we performed a molecular rickettsial survey of spleen samples of small mammals caught in public parks and built-up areas of Bangkok. Out of 198 samples, the Rattus rattus complex was found to be most prevalent. The amplification of rickettsial gltA fragment gene (338 bp) by nested PCR assay revealed positive results in four samples, yielding a low prevalence of infection of 2.02%. DNA sequencing results confirmed that three samples were matched with Rickettsia typhi, and one was identified as R. felis. It is noteworthy that this is the first report of the occurrence of R. felis DNA in rodents in Southeast Asia

Antibodies against the Envelope Glycoprotein Promote Infectivity of Immature Dengue Virus Serotype 2

Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles

The Human Antibody Response to Dengue Virus Infection

Dengue viruses (DENV) are the causative agents of dengue fever (DF) and dengue hemorrhagic fever (DHF). Here we review the current state of knowledge about the human antibody response to dengue and identify important knowledge gaps. A large body of work has demonstrated that antibodies can neutralize or enhance DENV infection. Investigators have mainly used mouse monoclonal antibodies (MAbs) to study interactions between DENV and antibodies. These studies indicate that antibody neutralization of DENVs is a “multi-hit” phenomenon that requires the binding of multiple antibodies to neutralize a virion. The most potently neutralizing mouse MAbs bind to surface exposed epitopes on domain III of the dengue envelope (E) protein. One challenge facing the dengue field now is to extend these studies with mouse MAbs to better understand the human antibody response. The human antibody response is complex as it involves a polyclonal response to primary and secondary infections with 4 different DENV serotypes. Here we review studies conducted with immune sera and MAbs isolated from people exposed to dengue infections. Most dengue-specific antibodies in human immune sera are weakly neutralizing and bind to multiple DENV serotypes. The human antibodies that potently and type specifically neutralize DENV represent a small fraction of the total DENV-specific antibody response. Moreover, these neutralizing antibodies appear to bind to novel epitopes including complex, quaternary epitopes that are only preserved on the intact virion. These studies establish that human and mouse antibodies recognize distinct epitopes on the dengue virion. The leading theory proposed to explain the increased risk of severe disease in secondary cases is antibody dependent enhancement (ADE), which postulates that weakly neutralizing antibodies from the first infection bind to the second serotype and enhance infection of FcγR bearing myeloid cells such as monocytes and macrophages. Here we review results from human, animal and cell culture studies relevant to the ADE hypothesis. By understanding how human antibodies neutralize or enhance DENV, it will be possible to better evaluate existing vaccines and develop the next generation of novel vaccines

A Dynamic Landscape for Antibody Binding Modulates Antibody-Mediated Neutralization of West Nile Virus

Neutralizing antibodies are a significant component of the host's protective response against flavivirus infection. Neutralization of flaviviruses occurs when individual virions are engaged by antibodies with a stoichiometry that exceeds a required threshold. From this “multiple-hit” perspective, the neutralizing activity of antibodies is governed by the affinity with which it binds its epitope and the number of times this determinant is displayed on the surface of the virion. In this study, we investigated time-dependent changes in the fate of West Nile virus (WNV) decorated with antibody in solution. Experiments with the well-characterized neutralizing monoclonal antibody (MAb) E16 revealed a significant increase in neutralization activity over time that could not be explained by the kinetics of antibody binding, virion aggregation, or the action of complement. Additional kinetic experiments using the fusion-loop specific MAb E53, which has limited neutralizing activity because it recognizes a relatively inaccessible epitope on mature virions, identified a role of virus “breathing” in regulating neutralization activity. Remarkably, MAb E53 neutralized mature WNV in a time- and temperature-dependent manner. This phenomenon was confirmed in studies with a large panel of MAbs specific for epitopes in each domain of the WNV envelope protein, with sera from recipients of a live attenuated WNV vaccine, and in experiments with dengue virus. Given enough time, significant inhibition of infection was observed even for antibodies with very limited, or no neutralizing activity in standard neutralization assays. Together, our data suggests that the structural dynamics of flaviviruses impacts antibody-mediated neutralization via exposure of otherwise inaccessible epitopes, allowing for antibodies to dock on the virion with a stoichiometry sufficient for neutralization

In Vitro and In Vivo Studies Identify Important Features of Dengue Virus pr-E Protein Interactions

Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV) pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors

Antibodies against Immature Virions Are Not a Discriminating Factor for Dengue Disease Severity

Humoral immunity plays an important role in controlling dengue virus (DENV) infection. Antibodies (Abs) developed during primary infection protect against subsequent infection with the same dengue serotype, but can enhance disease following secondary infection with a heterologous serotype. A DENV virion has two surface proteins, envelope protein E and (pre)-membrane protein (pr)M, and inefficient cleavage of the prM protein during maturation of progeny virions leads to the secretion of immature and partially immature particles. Interestingly, we and others found that historically regarded non-infectious prM-containing DENV particles can become highly infectious in the presence of E- and prM-Abs. Accordingly, we hypothesized that these virions contribute to the exacerbation of disease during secondary infection. Here, we tested this hypothesis and investigated the ability of acute sera of 30 DENV2-infected patients with different grades of disease severity, to bind, neutralize and/or enhance immature DENV2. We found that a significant fraction of serum Abs bind to the prM protein and to immature virions, but we observed no significant difference between the disease severity groups. Furthermore, functional analysis of the Abs did not underscore any specific correlation between the neutralizing/enhancing activity towards immature DENV2 and the development of more severe disease. Based on our analysis of acute sera, we conclude that Abs binding to immature virions are not a discriminating factor in dengue pathogenesis

Prevalence and Molecular Characterization of Rickettsia spp. from Wild Small Mammals in Public Parks and Urban Areas of Bangkok Metropolitan, Thailand

Rural areas usually show a higher prevalence of rickettsial infection than urban areas. However, information on the rickettsial infection status in urban settings (e.g., built-up areas and city parks) is still limited, particularly in the Bangkok metropolitan area. In this study, we performed a molecular rickettsial survey of spleen samples of small mammals caught in public parks and built-up areas of Bangkok. Out of 198 samples, the Rattus rattus complex was found to be most prevalent. The amplification of rickettsial gltA fragment gene (338 bp) by nested PCR assay revealed positive results in four samples, yielding a low prevalence of infection of 2.02%. DNA sequencing results confirmed that three samples were matched with Rickettsia typhi, and one was identified as R. felis. It is noteworthy that this is the first report of the occurrence of R. felis DNA in rodents in Southeast Asia.</jats:p

Prevalence and Molecular Characterization of Rickettsia spp. from Wild Small Mammals in Public Parks and Urban Areas of Bangkok Metropolitan, Thailand

International audienceRural areas usually show a higher prevalence of rickettsial infection than urban areas. However, information on the rickettsial infection status in urban settings (e.g., built-up areas and city parks) is still limited, particularly in the Bangkok metropolitan area. In this study, we performed a molecular rickettsial survey of spleen samples of small mammals caught in public parks and built-up areas of Bangkok. Out of 198 samples, the Rattus rattus complex was found to be most prevalent. The amplification of rickettsial gltA fragment gene (338 bp) by nested PCR assay revealed positive results in four samples, yielding a low prevalence of infection of 2.02%. DNA sequencing results confirmed that three samples were matched with Rickettsia typhi, and one was identified as R. felis. It is noteworthy that this is the first report of the occurrence of R. felis DNA in rodents in Southeast Asia