Impact of flavivirus vaccine-induced immunity on primary Zika virus antibody response in humans.

BACKGROUND: Zika virus has recently spread to South- and Central America, causing congenital birth defects and neurological complications. Many people at risk are flavivirus pre-immune due to prior infections with other flaviviruses (e.g. dengue virus) or flavivirus vaccinations. Since pre-existing cross-reactive immunity can potentially modulate antibody responses to Zika virus infection and may affect the outcome of disease, we analyzed fine-specificity as well as virus-neutralizing and infection-enhancing activities of antibodies induced by a primary Zika virus infection in flavivirus-naïve as well as yellow fever- and/or tick-borne encephalitis-vaccinated individuals. METHODOLOGY: Antibodies in sera from convalescent Zika patients with and without vaccine-induced immunity were assessed by ELISA with respect to Zika virus-specificity and flavivirus cross-reactivity. Functional analyses included virus neutralization and infection-enhancement. The contribution of IgM and cross-reactive antibodies to these properties was determined by depletion experiments. PRINCIPAL FINDINGS: Pre-existing flavivirus immunity had a strong influence on the antibody response in primary Zika virus infections, resulting in higher titers of broadly flavivirus cross-reactive antibodies and slightly lower levels of Zika virus-specific IgM. Antibody-dependent enhancement (ADE) of Zika virus was mediated by sub-neutralizing concentrations of specific IgG but not by cross-reactive antibodies. This effect was potently counteracted by the presence of neutralizing IgM. Broadly cross-reactive antibodies were able to both neutralize and enhance infection of dengue virus but not Zika virus, indicating a different exposure of conserved sequence elements in the two viruses. CONCLUSIONS: Our data point to an important role of flavivirus-specific IgM during the transient early stages of infection, by contributing substantially to neutralization and by counteracting ADE. In addition, our results highlight structural differences between strains of Zika and dengue viruses that are used for analyzing infection-enhancement by cross-reactive antibodies. These findings underscore the possible impact of specific antibody patterns on flavivirus disease and vaccination efficacy.

Predictive value of clinical and laboratory features for the main febrile diseases in children living in Tanzania: A prospective observational study.

BACKGROUND: To construct evidence-based guidelines for management of febrile illness, it is essential to identify clinical predictors for the main causes of fever, either to diagnose the disease when no laboratory test is available or to better target testing when a test is available. The objective was to investigate clinical predictors of several diseases in a cohort of febrile children attending outpatient clinics in Tanzania, whose diagnoses have been established after extensive clinical and laboratory workup. METHOD: From April to December 2008, 1005 consecutive children aged 2 months to 10 years with temperature ≥38°C attending two outpatient clinics in Dar es Salaam were included. Demographic characteristics, symptoms and signs, comorbidities, full blood count and liver enzyme level were investigated by bi- and multi-variate analyses (Chan, et al., 2008). To evaluate accuracy of combined predictors to construct algorithms, classification and regression tree (CART) analyses were also performed. RESULTS: 62 variables were studied. Between 4 and 15 significant predictors to rule in (aLR+>1) or rule out (aLR+<1) the disease were found in the multivariate analysis for the 7 more frequent outcomes. For malaria, the strongest predictor was temperature ≥40°C (aLR+8.4, 95%CI 4.7-15), for typhoid abdominal tenderness (5.9,2.5-11), for urinary tract infection (UTI) age ≥3 years (0.20,0-0.50), for radiological pneumonia abnormal chest auscultation (4.3,2.8-6.1), for acute HHV6 infection dehydration (0.18,0-0.75), for bacterial disease (any type) chest indrawing (19,8.2-60) and for viral disease (any type) jaundice (0.28,0.16-0.41). Other clinically relevant and easy to assess predictors were also found: malaria could be ruled in by recent travel, typhoid by jaundice, radiological pneumonia by very fast breathing and UTI by fever duration of ≥4 days. The CART model for malaria included temperature, travel, jaundice and hepatomegaly (sensitivity 80%, specificity 64%); typhoid: age ≥2 years, jaundice, abdominal tenderness and adenopathy (46%,93%); UTI: age <2 years, temperature ≥40°C, low weight and pale nails (20%,96%); radiological pneumonia: very fast breathing, chest indrawing and leukocytosis (38%,97%); acute HHV6 infection: less than 2 years old, (no) dehydration, (no) jaundice and (no) rash (86%,51%); bacterial disease: chest indrawing, chronic condition, temperature ≥39.7°c and fever duration >3 days (45%,83%); viral disease: runny nose, cough and age <2 years (68%,76%). CONCLUSION: A better understanding of the relative performance of these predictors might be of great help for clinicians to be able to better decide when to test, treat, refer or simply observe a sick child, in order to decrease morbidity and mortality, but also to avoid unnecessary antimicrobial prescription. These predictors have been used to construct a new algorithm for the management of childhood illnesses called ALMANACH.

Synergistic inhibition in cell-cell fusion mediated by the matrix and nucleocapsid protein of canine distemper virus.

Canine distemper virus (CDV) causes a chronic, demyelinating, progressive or relapsing neurological disease in dogs, because CDV persists in the CNS. Persistence of virulent CDV, such as the A75/17 strain has been reproduced in cell cultures where it is associated with a non-cytolytic infection with very limited cell-cell fusion. This is in sharp contrast to attenuated CDV infection in cell cultures, such as the Onderstepoort (OP) CDV strain, which produces extensive fusion activity and cytolysis. Fusion efficiency may be determined by the structure of the viral fusion protein per se but also by its interaction with other structural proteins of CDV. This was studied by combining genes derived from persistent and non-persistent CDV strains in transient transfection experiments. It was found that fusion efficiency was markedly attenuated by the structure of the fusion protein of the neurovirulent A75/17-CDV. Moreover, we showed that the interaction of the surface glycoproteins with the M protein of the persistent strain greatly influenced fusion activity. Site directed mutagenesis showed that the c-terminus of the M protein is of particular importance in this respect. Interestingly, although the nucleocapsid protein alone did not affect F/H-induced cell-cell fusion, maximal inhibition occurred when the latter was added to combined glycoproteins with matrix protein. Thus, the present study suggests that very limited fusogenicity in virulent CDV infection, which favours persistence by limiting cell destruction involves complex interactions between all viral structural proteins.

Signal peptide and helical bundle domains of virulent canine distemper virus fusion protein restrict fusogenicity.

Persistence in canine distemper virus (CDV) infection is correlated with very limited cell-cell fusion and lack of cytolysis induced by the neurovirulent A75/17-CDV compared to that of the cytolytic Onderstepoort vaccine strain. We have previously shown that this difference was at least in part due to the amino acid sequence of the fusion (F) protein (P. Plattet, J. P. Rivals, B. Zuber, J. M. Brunner, A. Zurbriggen, and R. Wittek, Virology 337:312-326, 2005). Here, we investigated the molecular mechanisms of the neurovirulent CDV F protein underlying limited membrane fusion activity. By exchanging the signal peptide between both F CDV strains or replacing it with an exogenous signal peptide, we demonstrated that this domain controlled intracellular and consequently cell surface protein expression, thus indirectly modulating fusogenicity. In addition, by serially passaging a poorly fusogenic virus and selecting a syncytium-forming variant, we identified the mutation L372W as being responsible for this change of phenotype. Intriguingly, residue L372 potentially is located in the helical bundle domain of the F(1) subunit. We showed that this mutation drastically increased fusion activity of F proteins of both CDV strains in a signal peptide-independent manner. Due to its unique structure even among morbilliviruses, our findings with respect to the signal peptide are likely to be specifically relevant to CDV, whereas the results related to the helical bundle add new insights to our growing understanding of this class of F proteins. We conclude that different mechanisms involving multiple domains of the neurovirulent A75/17-CDV F protein act in concert to limit fusion activity, preventing lysis of infected cells, which ultimately may favor viral persistence.

Mechanism of reduction of virus release and cell-cell fusion in persistent canine distemper virus infection.

Canine distemper virus (CDV), a mobillivirus related to measles virus causes a chronic progressive demyelinating disease, associated with persistence of the virus in the central nervous system (CNS). CNS persistence of morbilliviruses has been associated with cell-to-cell spread, thereby limiting immune detection. The mechanism of cell-to-cell spread remains uncertain. In the present study we studied viral spread comparing a cytolytic (non-persistent) and a persistent CDV strain in cell cultures. Cytolytic CDV spread in a compact concentric manner with extensive cell fusion and destruction of the monolayer. Persistent CDV exhibited a heterogeneous cell-to-cell pattern of spread without cell fusion and 100-fold reduction of infectious viral titers in supernatants as compared to the cytolytic strain. Ultrastructurally, low infectious titers correlated with limited budding of persistent CDV as compared to the cytolytic strain, which shed large numbers of viral particles. The pattern of heterogeneous cell-to-cell viral spread can be explained by low production of infectious viral particles in only few areas of the cell membrane. In this way persistent CDV only spreads to a small proportion of the cells surrounding an infected one. Our studies suggest that both cell-to-cell spread and limited production of infectious virus are related to reduced expression of fusogenic complexes in the cell membrane. Such complexes consist of a synergistic configuration of the attachment (H) and fusion (F) proteins on the cell surface. F und H proteins exhibited a marked degree of colocalization in cytolytic CDV infection but not in persistent CDV as seen by confocal laser microscopy. In addition, analysis of CDV F protein expression using vaccinia constructs of both strains revealed an additional large fraction of uncleaved fusion protein in the persistent strain. This suggests that the paucity of active fusion complexes is due to restricted intracellular processing of the viral fusion protein.