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Sericin, a silk-derived non-immunogenic protein, has been used to improve cell culture performance by increasing viability, cell concentration, and promoting adherence of several cell lines. Here, we hypothesized that the properties of sericin can enhance the amplification of flaviviruses in cell cultures. The propagation of flavivirus is inefficient and limits scientific research. Zika virus (ZIKV) is an important human pathogen that has been widely studied because of its high impact on public health. There is a need to amplify Zika virus both for research and vaccine development. In this work, we show that sericin improves ZIKV amplification in insect (C6/36) and mammalian (Vero) cell cultures, and that it has a cryoprotectant capacity. Supplementation of cell culture media with sericin at 80 µg/mL resulted in a significant increase of 1 log in the concentration of ZIKV infectious particles produced from both cell lines. Furthermore, final virus yields increased between 5 and 10-fold in Vero cells and between 7 and 23-fold in C6/36 cells when sericin was supplemented, compared to control conditions. These results show that sericin is an effective supplement to increase ZIKV production by Vero and C6/36 cells. Additionally, sericin was a suitable cryoprotective agent, and hence an alternative to FBS and DMSO, for the cryopreservation of C6/36 cells but not for Vero cells.

The dengue virus NS1 is a multifunctional protein that forms part of replication complexes. NS1 is also secreted, as a hexamer, to the extracellular milieu. Circulating NS1 has been associated with dengue pathogenesis by several mechanisms. Cell binding and internalization of soluble NS1 result in endothelial hyperpermeability and in the downregulation of the innate immune response. In this work, we report that the HDL scavenger receptor B1 (SRB1) in human hepatic cells and a scavenger receptor B1-like in mosquito C6/36 cells act as cell surface binding receptors for dengue virus NS1. The presence of the SRB1 on the plasma membrane of C6/36 cells, as well as in Huh7 cells, was demonstrated by confocal microscopy. The internalization of NS1 can be efficiently blocked by anti-SRB1 antibodies, and previous incubation of the cells with HDL significantly reduces NS1 internalization. Significant reduction in NS1 internalization was observed in C6/36 cells transfected with siRNAs specific for SRB1. In addition, the transient expression of SRB1 in Vero cells, which lacks the receptor, allows NS1 internalization in these cells. Direct interaction between soluble NS1 and the SRB1 in Huh7 and C6/36 cells was demonstrated in situ by proximity ligation assays and in vitro by surface plasmon resonance. Finally, results are presented indicating that the SRB1 also acts as a cell receptor for Zika virus NS1. These results demonstrate that dengue virus NS1, a bona fide lipoprotein, usurps the HDL receptor for cell entry and offers explanations for the altered serum lipoprotein homeostasis observed in dengue patients. IMPORTANCE Dengue is the most common viral disease transmitted to humans by mosquitoes. The dengue virus NS1 is a multifunctional glycoprotein necessary for viral replication. NS1 is also secreted as a hexameric lipoprotein and circulates in high concentrations in the sera of patients. Circulating NS1 has been associated with dengue pathogenesis by several mechanisms, including favoring of virus replication in hepatocytes and dendritic cells and disruption of the endothelial glycocalyx leading to hyperpermeability. Those last actions require NS1 internalization. Here, we identify the scavenger cell receptor B1, as the cell-binding receptor for dengue and Zika virus NS1, in cultured liver and in mosquito cells. The results indicate that flavivirus NS1, a bona fide lipoprotein, usurps the human HDL receptor and may offer explanations for the alterations in serum lipoprotein homeostasis observed in dengue patients.

Dengue is the most common virus disease transmitted to humans by mosquitoes. The dengue virus NS1 is a multifunctional protein that form part of replication complexes. In addition, NS1 is also secreted, as a hexamer, to the extracellular milieu. Circulating NS1 has been associated with dengue pathogenesis by several different mechanisms. Cell binding and internalization of soluble NS1 result in the disruption of tight junctions and in down regulation of the innate immune response. In this work, we report that the HDL scavenger receptor B1 (SRB1) in human hepatic cells, and a scavenger receptor B1-like in mosquito C6/36 cells act as cell surface binding receptor for dengue virus NS1. The presence of the SRB1 on the plasma membrane of C6/36 cells, as well as in Huh-7 cells, was demonstrated by confocal microcopy. Internalization of NS1 can be efficiently blocked by anti-SRB1 antibodies and previous incubation of the cells with HDL significantly reduces NS1 internalization. In addition, the transient expression of SRB1 in Vero cells, which lack the receptor, renders these cells susceptible to NS1 entry. Direct interaction between soluble NS1 and the SRB1 in Huh7 and C6/36 cells was demonstrated in vivo by proximity ligation assays an in vitro by surface plasmon resonance. Finally, data is presented indicating that the SRB1 also act as cell receptor for zika virus NS1. These results demonstrate that dengue virus NS1, abona fidelipoprotein, usurps the HDL receptor for cell entry and offers explanations for the altered serum lipoprotein homeostasis observed in dengue patients.

Dengue virus nonstructural protein 1 (NS1) is a multifunctional glycoprotein. For decades, the notion in the field was that NS1 is secreted exclusively from vertebrate cells and not from mosquito cells. However, recent evidence shows that mosquito cells also secrete NS1 efficiently. In this review, we discuss the evidence for secretion of NS1 of dengue virus, and of other flaviviruses, from mosquito cells, differences between NS1 secreted from mosquito and NS1 secreted from vertebrate cells, and possible roles of soluble NS1 in the insect flavivirus vector.

Dengue virus NS1 is a glycoprotein of 46-50 kDa that is conserved among flaviviruses, associates as a dimer to cell membranes and is secreted as a hexamer to the extracellular milieu. Recent evidence showed that NS1 is secreted efficiently from infected mosquito cells. To explore the secretory route of NS1 in mosquito cells, infected cells were treated with brefeldin A (BFA) and methyl-beta-cyclodextrin (MβCD). The results showed that MβCD, but not BFA, significantly reduced the release of NS1. Moreover, silencing the expression of caveolin-1 (CAV1; a key component of the caveolar system that transports cholesterol inside the cell), but not SAR1 (a GTPase that participates in the classical secretory pathway), also results in a significant reduction of the secretion of NS1. These results indicate that NS1 is released from mosquito cells via an unconventional secretory route that bypasses the Golgi complex, with the participation of CAV1. In agreement with this notion, differences were observed in the glycosylation status between secreted NS1 and E proteins. Classical mechanics and docking simulations suggested highly favoured interactions between the caveolin-binding domain present in NS1 and the scaffolding domain of CAV1. Finally, proximity ligation assays showed direct interaction between NS1 and CAV1 in infected mosquito cells. These findings are in line with the lipoprotein nature of secreted NS1 and provide new insights into the biology of NS1.

Dengue is the most important mosquito-borne viral disease to humans. Bats are potential reservoirs for flaviviruses, including dengue virus (DENV). In this work, Artibeus jamaicensis bats were inoculated with two serotypes of DENV using different routes. For experimental inoculations (EI) 1 and 2, bats were inoculated subcutaneously or intraperitoneally with DENV-4; for EI-3 bats were inoculated intraperitoneally with DENV-1. Mock inoculated bats were kept as controls. In EI-4, bats were bitten by Aedes aegypti mosquitoes infected with DENV-1 or 4. Reverse transcription-polymerase chain reaction assays in plasma and spleen tissue collected from Day 1 to Days 9-17 after inoculation failed to reveal the presence of viral RNA in any of the samples. No evidence of circulating NS1 or specific anti-DENV IgG was detected in the plasma of the inoculated bats. These results indicate that A. jamaicensis bats are incapable of sustaining dengue virus replication and are unlikely to act as reservoirs for this virus.

Rotavirus infection induces an increase in [Ca2+]cyto, which in turn may affect the distribution of the cytoskeleton proteins in the infected cell. Changes in microfilaments, including the formation of stress fibers, were observed starting at 0.5 h.p.i. using fluorescent phalloidin. Western blot analysis indicated that RhoA is activated between 0.5 and 1 h.p.i. Neither the phosphorylation of RhoA nor the formation of stress fibers were observed in cells infected with virions pre-treated with an anti-VP5* non-neutralizing mAb, suggesting that RhoA activation is stimulated by the interaction of the virus with integrins forming the cell receptor complex. In addition, the structure of the tubulin cytoskeleton was also studied. Alterations of the microtubules were evident starting at 3 h.p.i. and by 7 h.p.i. when microtubules were markedly displaced toward the periphery of the cell cytoplasm. Loading of rotavirus-infected cells with either a Ca2+ chelator (BAPTA) or transfection with siRNAs to silence NSP4, reversed the changes observed in both the microfilaments and microtubules distribution, but not the appearance of stress fibers. These results indicate that alterations in the distribution of actin microfilaments are initiated early during infection by the activation of RhoA, and that latter changes in the Ca2+ homeostasis promoted by NSP4 during infection may be responsible for other alterations in the actin and tubulin cytoskeleton. publishedVersion

The circulation of Aichi virus in a major urban area was demonstrated using molecular detection with samples recovered from a major river polluted with sewage discharges in Caracas, Venezuela. Five out of 11 water samples studied were positive, being classified by phylogenetic analysis as genotype B. Analysis of sewage waters appears to be a useful methodology to uncover the presence of a hitherto undetected fecal pathogen in a given geographical area.

A primer pair (p289-p290) designed to detect both noroviruses and sapoviruses by reverse transcription-PCR (Jiang et al., J. Virol. Methods 83: 145, 1999) cross-reacts with rotaviruses. The rotavirus amplicon corresponds to genome segment 1. Furthermore, primer pair p289-p290 detected rotaviruses as efficiently as rotavirus-specific primers directed to rotavirus gene 4.

Dengue virus NS1 is a glycoprotein of 46–50kDa which associates as a dimer to internal and cytoplasmic membranes and is also secreted, as a hexamer, to the extracellular milieu. However, the notion exist that NS1 is secreted only from infected vertebrate and not mosquito cells. In this work, evidence is presented showing that NS1 is secreted efficiently by infected mosquito cells. NS1 was detected in cell supernatants starting at 6hpi with a continuous concentration increase up to 24hpi. Nevertheless, cell viability showed an average cell survival of 97%. At variance with observations with vertebrate cells, NS1 does not seems to associate with the cytoplasmic membrane of insect cells. Finally, evidence is presented indicating that NS1 is secreted from insect cells as a barrel-shaped hexamer. These findings provide new insights into the biology of NS1 and open questions about the role of secreted NS1 in the vector mosquito.