Studies of rotavirus morphogenesis, transport, and release have shown that although these viruses are released from the apical surface of polarized intestinal cells before cellular lysis, they do not follow the classic exocytic pathway. Furthermore, increasing evidence suggests that lipid rafts actively participate in the exit of rotavirus from the infected cell. In this study, we silenced the expression of VP4, VP7, and NSP4 by using small interfering RNAs (siRNAs) and evaluated the effect of shutting down the expression of these proteins on rotavirus-raft interactions. Silencing of VP4 and NSP4 reduced the association of rotavirus particles with rafts; in contrast, inhibition of VP7 synthesis slightly affected the migration of virions into rafts. We found that inhibition of rotavirus migration into lipid rafts, by either siRNAs or tunicamycin, also specifically blocked the targeting of VP4 to rafts, suggesting that the association of VP4 with rafts is mostly mediated by the formation of viral particles in the endoplasmic reticulum (ER). We showed that two populations of VP4 exist, one small population that is independently targeted to rafts and a second large pool of VP4 whose association with rafts is mediated by particle formation in the ER. We also present evidence to support the hypothesis that assembly of VP4 into mature virions takes place in the late stages of transit through the ER. Finally, we analyzed the progression of rotavirus proteins in the exocytic pathway and found that VP4 and virion-assembled VP7 colocalized with ERGIC-53, suggesting that rotavirus particles transit through the intermediate compartment between the ER and the Golgi complex.

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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.

Rotavirus infection of cells in culture induces major changes in Ca2؉ homeostasis. These changes include increases in plasma membrane Ca 2؉ permeability, cytosolic Ca 2؉ concentration, and total cell Ca 2؉ content and a reduction in the amount of Ca 2؉ released from intracellular pools sensitive to agonists. Various lines of evidence suggest that the nonstructural glycoprotein NSP4 and possibly the major outer capsid glycoprotein VP7 are responsible for these effects. In order to evaluate the functional roles of NSP4 and other rotavirus proteins in the changes in Ca 2؉ homeostasis observed in infected cells, the expressions of NSP4, VP7, and VP4 were silenced using the short interfering RNA (siRNA) technique. The transfection of specific siRNAs resulted in a strong and specific reduction of the expression of NSP4, VP7, and VP4 and decreased the yield of new viral progeny by more than 90%. Using fura-2 loaded cells, we observed that knocking down the expression of NSP4 totally prevented the increase in Ca 2؉ permeability of the plasma membrane and cytosolic Ca 2؉ concentration measured in infected cells. A reduction in the levels of VP7 expression partially reduced the effect of infection on plasma membrane Ca 2؉ permeability and Ca 2؉ pools released by agonist (ATP). In addition, the increase of total Ca 2؉ content (as measured by 45 Ca 2؉ uptake) observed in infected cells was reduced to the levels in mock-infected cells when NSP4 and VP7 were silenced. Finally, when the expression of VP4 was silenced, none of the disturbances of Ca 2؉ homeostasis caused by rotaviruses in infected cells were affected. These data altogether indicate that NSP4 is the main protein responsible for the changes in Ca 2؉ homeostasis observed in rotavirus-infected cultured cells. Nevertheless, VP7 may contribute to these effects. Viral-associated diarrhea remains one of the most common causes of morbidity and mortality among infants and young children. Worldwide estimations indicate that rotaviruses are the leading viral agent associated with severe diarrhea in children younger than 5 years old (20). In addition, rotavirus infections are also a main cause of diarrhea in calves, piglets, and the young of other animals of economic importance (20). Thus, further knowledge of the virus-cell interactions and the events leading to pathogenesis are necessary to improve or develop new strategies that may prevent or reduce the health and economic impact caused by rotavirus infections.Rotaviruses are members of the Reoviridae family. The rotavirus virion is icosahedral, nonenveloped, and composed of three concentric layers of proteins and a genome of 11 segments of double-stranded RNA. Each genomic segment, with the exception of segment 11, encodes one viral protein for a total of six structural (VP1 to VP7) and six nonstructural proteins (NSP1 to NSP6). The inner layer of the virion is formed by VP2 and also contains the RNA-dependent RNA polymerase VP1 and the guanylyltransferase/methylase VP3. The middle capsid is composed of the major virion...

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.