Marie Flamand

Enzyme-Linked Immunosorbent Assay Specific to Dengue Virus Type 1 Nonstructural Protein NS1 Reveals Circulation of the Antigen in the Blood during the Acute Phase of Disease in Patients Experiencing Primary or Secondary Infections

ABSTRACT During flavivirus infection in vitro, nonstructural protein NS1 is released in a host-restricted fashion from infected mammalian cells but not vector-derived insect cells. In order to analyze the biological relevance of NS1 secretion in vivo, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) to detect the protein in the sera of dengue virus-infected patients. The assay was based on serotype 1 NS1-specific mouse and rabbit polyclonal antibody preparations for antigen immunocapture and detection, respectively. With purified dengue virus type 1 NS1 as a protein standard, the sensitivity of our capture ELISA was less than 1 ng/ml. When a panel of patient sera was analyzed, the NS1 antigen was found circulating from the first day after the onset of fever up to day 9, once the clinical phase of the disease is over. The NS1 protein could be detected even when viral RNA was negative in reverse transcriptase-PCR or in the presence of immunoglobulin M antibodies. NS1 circulation levels varied among individuals during the course of the disease, ranging from several nanograms per milliliter to several micrograms per milliliter, and peaked in one case at 50 μg/ml of serum. Interestingly, NS1 concentrations did not differ significantly in serum specimens obtained from patients experiencing primary or secondary dengue virus infections. These findings indicate that NS1 protein detection may allow early diagnosis of infection. Furthermore, NS1 circulation in the bloodstream of patients during the clinical phase of the disease suggests a contribution of the nonstructural protein to dengue virus pathogenesis.

Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein

Dengue virus (DENV) causes the major arboviral disease of the tropics, characterized in its severe forms by signs of hemorrhage and plasma leakage. DENV encodes a nonstructural glycoprotein, NS1, that associates with intracellular membranes and the cell surface. NS1 is eventually secreted as a soluble hexamer from DENV-infected cells and circulates in the bloodstream of infected patients. Extracellular NS1 has been shown to modulate the complement system and to enhance DENV infection, yet its structure and function remain essentially unknown. By combining cryoelectron microscopy analysis with a characterization of NS1 amphipathic properties, we show that the secreted NS1 hexamer forms a lipoprotein particle with an open-barrel protein shell and a prominent central channel rich in lipids. Biochemical and NMR analyses of the NS1 lipid cargo reveal the presence of triglycerides, bound at an equimolar ratio to the NS1 protomer, as well as cholesteryl esters and phospholipids, a composition evocative of the plasma lipoproteins involved in vascular homeostasis. This study suggests that DENV NS1, by mimicking or hijacking lipid metabolic pathways, contributes to endothelium dysfunction, a key feature of severe dengue disease.

Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia.

The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices, and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells as well as their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization, and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.

The Secreted Form of Dengue Virus Nonstructural Protein NS1 Is Endocytosed by Hepatocytes and Accumulates in Late Endosomes: Implications for Viral Infectivity

ABSTRACT The flavivirus nonstructural protein NS1 is expressed as three discrete species in infected mammalian cells: an intracellular, membrane-associated form essential for viral replication, a cell surface-associated form that may be involved in signal transduction, and a secreted form (sNS1), the biological properties of which remain elusive. To determine the distribution of the dengue virus (DEN) sNS1 protein in vivo, we have analyzed by immunohistological means the tissue tropism of purified DEN sNS1 injected intravenously into adult mice. The sNS1 protein was found predominantly associated with the liver, where hepatocytes appeared to represent a major target cell. We further showed that sNS1 could be efficiently endocytosed by human Huh7 and HepG2 hepatocytes in vitro. After its internalization, the protein was detected intracellularly for at least 48 h without being substantially degraded. Colocalization studies of sNS1 with markers of the endolysosomal compartments revealed that the protein was specifically targeted to lysobisphosphatidic acid-rich structures reminiscent of late endosomes, as confirmed by electron microscopy. Intracellular accumulation of sNS1 in Huh7 cells enhanced the fluid phase uptake of rhodamine-labeled dextran. Furthermore, preincubation of Huh7 cells with sNS1 increased dengue virus production after infection with the homologous strain of DEN-1 virus. Our results demonstrate that the accumulation of DEN sNS1 in the late endosomal compartment of hepatocytes potentializes subsequent dengue virus infection in vitro, raising the possibility that sNS1 may contribute to viral propagation in vivo.

Dengue virus activates polyreactive, natural IgG B cells after primary and secondary infection.

Background Dengue virus is transmitted by mosquitoes and has four serotypes. Cross-protection to other serotypes lasting for a few months is observed following infection with one serotype. There is evidence that low-affinity T and/or B cells from primary infections contribute to the severe syndromes often associated with secondary dengue infections. such pronounced immune-mediated enhancement suggests a dengue-specific pattern of immune cell activation. This study investigates the acute and early convalescent B cell response leading to the generation of cross-reactive and neutralizing antibodies following dengue infection. Methodology/Principal Findings We assayed blood samples taken from dengue patients with primary or secondary infection during acute disease and convalescence and compared them to samples from patients presenting with non-dengue related fever. Dengue induced massive early plasmablast formation, which correlated with the appearance of polyclonal, cross-reactive IgG for both primary and secondary infection. Surprisingly, the contribution of IgG to the neutralizing titer 4–7 days after fever onset was more than 50% even after primary infection. Conclusions/Significance Poly-reactive and virus serotype cross-reactive IgG are an important component of the innate response in humans during both primary and secondary dengue infection, and “innate specificities” seem to constitute part of the adaptive response in dengue. While of potential importance for protection during secondary infection, cross-reactive B cells will also compete with highly neutralizing B cells and possibly interfere with their development.

Apoptotic cell death in response to dengue virus infection: the pathogenesis of dengue haemorrhagic fever revisited.

BACKGROUND Dengue virus infection may be asymptomatic or lead to undifferentiated febrile illness or dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS). The major clinical manifestations of DHF/DSS are high fever, haemorrhage, hepatomegaly and circulatory failure. OBJECTIVES The relatively high level of viraemia only a few days after infection may reflect a large number of replication sites. However, the degree of cell injury in fatal cases of DHF/DSS is not sufficient to explain death and suggests metabolic disturbance rather than tissue destruction. This theory was investigated in this study. RESULTS We demonstrated that replication of dengue virus in infected cells induces stress leading to apoptotic cell death in vitro and in vivo. CONCLUSIONS The elimination of apoptotic bodies by phagocytic cells is a previously unsuspected pathway of dengue virus clearance from infected tissues. However, the mechanisms of host defence involving apoptosis and phagocytic cell activation may cause local tissue injury or transient homeostasis imbalance and may trigger further deleterious events.

Dengue nonstructural protein 1 antigen in the urine as a rapid and convenient diagnostic test during the febrile stage in patients with dengue infection.

A total of 136 matched serum and urine samples obtained from 55 patients with dengue infection and 30 other febrile illnesses were assayed for dengue nonstructural protein 1 (NS1) antigen. The urine NS1 ELISA was positive in patients with dengue fever (68.4%) and dengue hemorrhagic fever (63.9%), whereas the strip method showed a lower positive rate.

Value of Routine Dengue Diagnostic Tests in Urine and Saliva Specimens.

Background Dengue laboratory diagnosis is essentially based on detection of the virus, its components or antibodies directed against the virus in blood samples. Blood, however, may be difficult to draw in some patients, especially in children, and sampling during outbreak investigations or epidemiological studies may face logistical challenges or limited compliance to invasive procedures from subjects. The aim of this study was to assess the possibility of using saliva and urine samples instead of blood for dengue diagnosis. Methodology/Principal Findings Serial plasma, urine and saliva samples were collected at several time-points between the day of admission to hospital until three months after the onset of fever in children with confirmed dengue disease. Quantitative RT-PCR, NS1 antigen capture and ELISA serology for anti-DENV antibody (IgG, IgM and IgA) detection were performed in parallel on the three body fluids. RT-PCR and NS1 tests demonstrated an overall sensitivity of 85.4%/63.4%, 41.6%/14.5% and 39%/28.3%, in plasma, urine and saliva specimens, respectively. When urine and saliva samples were collected at the same time-points and tested concurrently, the diagnostic sensitivity of RNA and NS1 detection assays was 69.1% and 34.4%, respectively. IgG/IgA detection assays had an overall sensitivity of 54.4%/37.4%, 38.5%/26.8% and 52.9%/28.6% in plasma, urine and saliva specimens, respectively. IgM were detected in 38.1% and 36% of the plasma and saliva samples but never in urine. Conclusions Although the performances of the different diagnostic methods were not as good in saliva and urine as in plasma specimens, the results obtained by qRT-PCR and by anti-DENV antibody ELISA could well justify the use of these two body fluids to detect dengue infection in situations when the collection of blood specimens is not possible.

Expression and secretion of japanese encephalitis virus nonstructural protein NS1 by insect cells using a recombinant baculovirus

The nonstructural protein NS1 of Japanese encephalitis virus (JEV) was expressed at a high level under the control of the polyhedrin promoter in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus. Recombinant NS1 was designed to contain its natural signal sequence at its N-terminus and no C-terminal hydrophobic domain that could act as a membrane anchor. This recombinant protein exhibited similar size to native NS1 expressed in Aedes albopictus (C6/36) insect cells infected with wild-type JEV. The signal sequence of NS1 allowed translocation of the protein into the endoplasmic reticulum where it underwent glycosylation. A small fraction of synthesized NS1 was able, in the absence of any other viral protein, to associate as a homodimer, showing similar characteristics to the native dimer. Interestingly, this recombinant dimeric form seemed to be exported and released in the extracellular medium of infected cell culture. During its transport, one of the two N-linked oligosaccharides of the polymannose type was processed to an endoglycosidase H-resistant form, suggesting that the protein had passed through the Golgi compartment before reaching the cell surface. Moreover, Triton X-114 partitioning analysis showed that monomeric NS1 behaved essentially as a hydrophilic protein, whereas both intracellular and extracellular dimeric NS1 were either free of or associated to membraneous components.

The Rift Valley fever accessory proteins NSm and P78/NSm-GN are distinct determinants of virus propagation in vertebrate and invertebrate hosts

Rift Valley fever virus (RVFV) is an enzootic virus circulating in Africa that is transmitted to its vertebrate host by a mosquito vector and causes severe clinical manifestations in humans and ruminants. RVFV has a tripartite genome of negative or ambisense polarity. The M segment contains five in-frame AUG codons that are alternatively used for the synthesis of two major structural glycoproteins, GN and GC, and at least two accessory proteins, NSm, a 14-kDa cytosolic protein, and P78/NSm-GN, a 78-kDa glycoprotein. To determine the relative contribution of P78 and NSm to RVFV infectivity, AUG codons were knocked out to generate mutant viruses expressing various sets of the M-encoded proteins. We found that, in the absence of the second AUG codon used to express NSm, a 13-kDa protein corresponding to an N-terminally truncated form of NSm, named NSm′, was synthesized from AUG 3. None of the individual accessory proteins had any significant impact on RVFV virulence in mice. However, a mutant virus lacking both NSm and NSm′ was strongly attenuated in mice and grew to reduced titers in murine macrophages, a major target cell type of RVFV. In contrast, P78 was not associated with reduced viral virulence in mice, yet it appeared as a major determinant of virus dissemination in mosquitoes. This study demonstrates how related accessory proteins differentially contribute to RVFV propagation in mammalian and arthropod hosts.