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Articular cartilage is a specialized tissue that provides a smooth surface for joint movement and load transmission. Unfortunately, it has limited regenerative capacity. Tissue engineering, combining different cell types, scaffolds, growth factors, and physical stimulation has become an alternative for repairing and regenerating articular cartilage. Dental Follicle Mesenchymal Stem Cells (DFMSCs) are attractive candidates for cartilage tissue engineering because of their ability to differentiate into chondrocytes, on the other hand, the polymers blend like Polycaprolactone (PCL) and Poly Lactic-co-Glycolic Acid (PLGA) have shown promise given their mechanical properties and biocompatibility. In this work, the physicochemical properties of polymer blends were evaluated by Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM) and were positive for both techniques. The DFMSCs demonstrated stemness by flow cytometry. The scaffold showed to be a non-toxic effect when we evaluated it with Alamar blue, and the samples were analyzed using SEM and phalloidin staining to evaluate cell adhesion to the scaffold. The synthesis of glycosaminoglycans was positive on the construct in vitro. Finally, the PCL/PLGA scaffold showed a better repair capacity than two commercial compounds, when tested in a chondral defect rat model. These results suggest that the PCL/PLGA (80:20) scaffold may be suitable for applications in the tissue engineering of articular hyaline cartilage.

BACKGROUNDZika virus (ZIKV) is highly teratogenic, in contrast with dengue virus (DENV) or the yellow fever virus vaccine (YFV-17D). The mechanisms employed by ZIKV to cross the placenta need to be elucidated.METHODSParallel infections with ZIKV, DENV and YFV-17D were compared in terms of efficiency, activation of mTOR pathways and cytokine secretion profile in human cytotrophoblastic HTR8 cells (CTB), and monocytic U937 cells, differentiated to M2 macrophages (M2-MØ).RESULTSIn CTB, ZIKV replication was significantly more efficient than DENV or YFV-17D. In M2-MØ, ZIKV replication continued to be more efficient, although differences between strains were reduced. Significantly greater activation of Phospho-S6r and Phospho-AKT/Ser473 fractions in CTB infected with ZIKV than with DENV or YFV-17D, was observed. CTB treated with the mTOR inhibitors rapamycin or AZD8055, showed a 20-fold-reduction in ZIKV yield, versus 5 and 3.5-fold for DENV and YFV-17D, respectively. Finally, we detected that ZIKV infection, but not DENV or YFV-17D, efficiently inhibited the interferon response of CTB cells.CONCLUSIONSThese results suggest that CTB cells are permissive and act favoring ZIKV entry into the placental stroma, over DENV and YFV-17D and that the mTOR complex is a switch that enhances the replication of ZIKV in CTB cells.

Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. A total of 817 mosquito targets were identified as protein–protein interacting with DENV NS1. Approximately 14% of them coincide with interactomes previously obtained in vertebrate cells, including the oligosaccharide transferase complex, the chaperonin containing TCP-1, vesicle localization, and ribosomal proteins. Notably, other protein pathways not previously reported in vertebrate cells, such as epigenetic regulation and RNA silencing, were also found in the NS1 interactome in mosquito cells. Due to the novel and strong interactions observed for NS1 and the epigenetic regulator DIDO1 (Death-Inducer Obliterator 1), the role of DIDO1 in viral replication was further explored. Interactions between NS1 and DIDO1 were corroborated in infected mosquito cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected cells silenced for DIDO1 revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD, and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response necessary for flavivirus replication in mosquito cells. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication, and add to the understanding of NS1 as a multifunctional protein. IMPORTANCE Dengue is the most important mosquito-borne viral disease to humans. Dengue virus NS1 is a multifunctional protein essential for replication and modulation of innate immunity. To gain insights into NS1 functions, the protein interactome of dengue virus NS1 in Aedes albopictus cells was investigated using a proximity biotinylation system and mass spectrometry. Several protein pathways, not previously observed in vertebrate cells, such as transcription and epigenetic regulation, were found as part of the NS1 interactome in mosquito cells. Among those, DIDO1 was found to be a necessary host factor for dengue and Zika virus replication in mosquito cells. Transcription analysis of infected mosquito cells silenced for DIDO1 revealed alterations of the IMD and Toll pathways, part of the antiviral response in mosquitoes. The results suggest that DIDO1 is a host factor involved in modulation of the antiviral response and necessary for flavivirus replication.

Zika virus (ZIKV) infection is a major public health threat, making the study of its biology a matter of great importance. By analyzing the viral-host protein interactions and proposing them as new drug targets, we would diminish the emergence of new resistant strains. In this work, we have shown that human cytoplasmic dynein-1 (Dyn) interacts with the ZIKV. We additionally demonstrate that the envelope protein of the ZIKV and the dimerization domain of the heavy chain of Dyn binds directly without dynactin or cargo adaptor. In addition, we have analyzed this interaction in Vero cells, where we are proposing that the interaction ZIKV-Dyn is finely tuned within the replication cycle. Altogether, our data suggest a new step in the previously described replication cycle of the ZIKV, introducing a suitable molecular target to modulate infection by ZIKV.

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.

Septins are a family of GTP-binding proteins identified in insects and mammals. Septins are components of the cytoskeleton and participate in cytokinesis, chromosomal segregation, intracellular vesicular traffic, and response to pathogens. Human septin 6 was identified as necessary for hepatitis C virus replication. Information about host factors necessary for flavivirus replication in mosquitoes is scarce. Thus, the role of septins in the replicative cycle of dengue virus in Aedes spp. derived cells was investigated. Through bioinformatic analysis, sequences of septin-like proteins were identified. Infected mosquito cells showed increased expression of Sep2. Colocalization analysis, proximity ligation and immunoprecipitation assays indicated that Sep2 interacts with proteins E, NS3 and NS5, but not NS1. Immunoelectron microscopy evidenced the presence of AalSep2 in replicative complexes. Finally, silencing of Sep2 expression resulted in a significant decrease in virus progeny, indicating that Sep2 is a host factor participating in dengue virus replication in mosquito cells.

Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. Approximately 14% of the 817 identified proteins coincide with interactomes obtained in vertebrate cells, including ontology groups of the oligosaccharide transferase complex, the chaperonin containing TCP-1, and nuclear import and export, vesicle localization and ribosomal proteins. Notably, other protein pathways such as epigenetic regulation and RNA silencing, not previously reported in vertebrate cells, were also found in the NS1 interactome in mosquito cells. Due to the novel interaction observed for NS1 and DIDO1 (Death Inducer-Obliterator 1), we further explored the role of DIDO1 in viral replication. Interactions between NS1 and DIDO1were corroborated in infected C6/36 and Aag2 cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression in C6/36 and Aag2 cells results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected C6/36 silenced for DIDO1, revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response and necessary for flavivirus replication. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication and add to the understanding of NS1 as a multifunctional protein.ABSTRACT IMPORTANCEDengue is the most important mosquito-borne viral disease to humans. Dengue virus NS1 is a multifunctional protein essential for replication and modulation of innate immunity. To gain insights into NS1 functions, the protein interactome of dengue virus NS1 in Aedes albopictus cells was investigated using a proximity biotinylation system and mass spectrometry. Several protein pathways, not previously observed in vertebrate cells, such as epigenetic regulation and RNA silencing, were found as part of the NS1 interactome in mosquito cells. Among those, DIDO1 was found to be a necessary host factor for dengue and Zika virus replication in vertebrate and mosquito cells. Transcription analysis of infected mosquito cells silenced for DIDO1, revealed alterations of the IMD and Toll pathways, part of the antiviral response in mosquitoes. The results suggest that DIDO1 is a host factor involved in modulation of the antiviral response and necessary for flavivirus replication.

The stress of the Golgi apparatus is an autoregulatory mechanism that is induced to compensate for greater demand in the Golgi functions. Few examples of Golgi stress responses due to physiological stimuli are known. Furthermore, the impact on this organelle of viral infections that occupy the vesicular transport during replication is unknown. In this work, we evaluated if a Golgi stress response is triggered during dengue and Zika viruses replication, two flaviviruses whose replicative cycle is heavily involved with the Golgi complex, in vertebrate and mosquito cells. Using GM-130 as a Golgi marker, and treatment with monensin as a positive control for the induction of the Golgi stress response, a significant expansion of the Golgi cisternae was observed in BHK-21, Vero E6 and mosquito cells infected with either virus. Activation of the TFE3 pathway was observed in the infected cells as indicated by the translocation from the cytoplasm to the nucleus of TFE3. Of note, no sign of activation of the stress response was observed in CRFK cells infected with Feline Calicivirus (FCV), a virus released by cell lysis, not requiring vesicular transport. Finally, dilatation of the Golgi complex and translocation of TFE3 was observed in vertebrate cells expressing dengue and Zika viruses NS1, but not NS3. These results indicated that infections by dengue and Zika viruses induce a Golgi stress response in vertebrate and mosquito cells due to the increased demand on Golgi complex imposed by virion and NS1 processing and secretion.

Background Dengue virus (DENV) infections pose one of the largest global barriers to human health. The four serotypes (DENV 1–4) present different symptoms and influence immune response to subsequent DENV infections, rendering surveillance, risk assessments, and disease control particularly challenging. Early diagnosis and appropriate clinical management is critical and can be achieved by detecting DENV nonstructural protein 1 (NS1) in serum during the acute phase. However, few NS1-based tests have been developed that are capable of differentiating DENV serotypes and none are currently commercially available. Methodology/Principle findings We developed an enzyme-linked immunosorbent assay (ELISA) to distinguish DENV-1-4 NS1 using serotype-specific pairs of monoclonal antibodies. A total of 1,046 antibodies were harvested from DENV-immunized mice and screened for antigen binding affinity. ELISA clinical performance was evaluated using 408 polymerase chain reaction-confirmed dengue samples obtained from patients in Brazil, Honduras, and India. The overall sensitivity of the test for pan-DENV was 79.66% (325/408), and the sensitivities for DENV-1-4 serotyping were 79.1% (38/48), 80.41% (78/97), 100% (45/45), and 79.6% (98/123), respectively. Specificity reached 94.07–100%. Significance Our study demonstrates a robust antibody screening strategy that enabled the development of a serotype NS1-based ELISA with maximized specific and sensitive antigen binding. This sensitive and specific assay also utilized the most expansive cohort to date, and of which about half are from Latin America, a geographic region severely underrepresented in previous similar studies. This ELISA test offers potential enhanced diagnostics during the acute phase of infection to help guide patient care and disease control. These results indicate that this ELISA is a promising aid in early DENV-1-4 diagnosis and surveillance in regions of endemicity in addition to offer convenient monitoring for future vaccine interventions., Author summary Dengue virus (DENV) infection is an increasingly significant threat to global health, with a yearly estimate of 390 million infections and an expected increasing burden with the rise of climate change and globalization. DENV is caused by one of the four serotypes (DENV-1-4), each of which have been associated with different immune responses and clinical manifestations. We developed a method to detect DENV serotypes by targeting the nonstructural 1 (NS1) antigen through an enzyme-linked immunosorbent-based assay (ELISA) with high sensitivity and specificity. We demonstrate that our high throughput mouse-derived antibody screening method selected for optimal test performance. The antibodies were integrated into an ELISA that can distinguish between the four different dengue serotypes by serotype-specific pairing. In addition, we provide a dengue universal antibody combination that enables pan-virus detection independently of the serotype. We use the ELISA in three different countries and calculate overall and site-specific sensitivities and specificities. The assay performs optimally when levels of viremia are high during the first five days of fever.

The role of Ca2+ during dengue virus (DENV) replication is unknown; thus, changes in Ca2+ homeostasis in DENV infected human hepatic HepG2 and Huh-7 cells were analyzed. Infected HepG2 cells, but not Huh-7 cells, showed a significant increase in plasma membrane permeability to Ca2+, while both cell lines showed marked reduced levels of Ca2+ stored in the endoplasmic reticulum. While the expression levels of STIM1 and ORAI1 showed no changes, STIM1 and ORAI1 were shown to co-localized in infected cells, indicating activation of the store-operated Ca2+ entry (SOCE) pathway. Finally, manipulation in the infected cells of the intra and extracellular Ca2+ levels by chelators (BAPTA-AM and EGTA), SOC inhibitor (SKF96365), IP3 Receptor antagonist (2APB) or increase of extracellular [Ca2+], significantly reduced DENV yield, but not vesicular stomatitis virus yield, used as a control. These results show that DENV infection alters cell Ca2+ homeostasis and that such changes favor viral replication.

Flaviviruses of major medical importance worldwide such as dengue (DENV), Zika (ZIKV), and yellow fever (YFV) viruses are transmitted by mosquitoesAedes sp. The non-structural protein 1 (NS1) of these flaviviruses is secreted from the infected cells using different secretion routes depending on the cell and virus nature. The NS1 of DENV and ZIKV contain in the hydrophobic region a conserved caveolin binding domain (CBD) (ΦXXΦXXXXΦ), which is not conserved in YFV NS1. To ascertain the role of the CBD in the secretory route followed by flavivirus NS1, expression vectors for the NS1 of DENV2, ZIKV and YFV were constructed. Using site-directed mutagenesis, substitutions were made in the aromatic residues within CBD; in addition, the full domain was replaced by those of other flaviviruses, creating chimeras in the CBD of NS1. Substitutions of the aromatic residues to Ala or Thr, or CBD chimeras, results in increased sensitivity of NS1 secretion to brefeldin A treatment, indicating a change to a classical secretion pathway. Likewise, the insertion of the DENV/ZIKV CBD into the recombinantGaussia-Luciferase results in a loss of sensitivity to BFA treatment, in luciferase secretion. These results suggest that the CBD sequence is a molecular determinant for the unconventional secretory route followed by DENV and ZIKV NS1 in mosquito cells. However, the cellular components that recognize the CBD in the NS1 of DENV and ZIKV and redirect them to an unconventional route and if this secretion route confers unique functions to NS1 within the vector mosquito are aspects currently unknown.ImportanceFlaviviruses are an important cause of mosquito borne diseases to humans. We have previously demonstrated that the non-structural protein 1 from dengue and zika virus are secreted efficiently from mosquito cells using an unconventional route, that depends on caveolin and molecular chaperones. In this work, we show evidence indicating that a caveolin binding domain, well conserved and exposed in dengue and Zika virus NS1, but absent in other flaviviruses such as yellow fever virus or West Nile virus, is important in determining the unconventional secretion pathway followed by dengue and zika virus NS1 in mosquito cells. The unique secretory pathway followed by NS1 in mosquito cells may result in distinctive viral-cellular protein associations required to facilitate viral infection in the mosquito vector. To identify viral and cellular elements that could disturb the traffic of dengue and Zika virus NS1 may be important to design of strategies for vector control.

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.

The cytotrophoblast of human placenta transitions into an outer multinucleated syncytiotrophoblast (STB) layer that covers chorionic villi which are in contact with maternal blood in the intervillous space. During pregnancy, the Zika virus (ZIKV) poses a serious prenatal threat. STB cells are resistant to ZIKV infections, yet placental cells within the mesenchyme of chorionic villi are targets of ZIKV infection. We seek to determine whether ZIKV can open the paracellular pathway of STB cells. This route is regulated by tight junctions (TJs) which are present in the uppermost portion of the lateral membranes of STB cells. We analyzed the paracellular permeability and expression of E-cadherin, occludin, JAMs &ndash, B and &ndash, C, claudins -1, -3, -4, -5 and -7, and ZO-1, and ZO-2 in the STB of placentae from ZIKV-infected and non-infected women. In ZIKV-infected placentae, the pattern of expression of TJ proteins was preserved, but the amount of claudin-4 diminished. Placentae from ZIKV-infected women were permeable to ruthenium red, and had chorionic villi with a higher mean diameter and Hofbauer hyperplasia. Finally, ZIKV added to the basolateral surface of a trophoblast cell line reduced the transepithelial electrical resistance. These results suggest that ZIKV can open the paracellular pathway of STB cells.

Bats are reservoirs for viruses with zoonotic potential in the Americas, and scattered evidence exists suggesting that bats may act as reservoirs for dengue virus (DENV). To explore further the role of bats as part of DENV sylvatic cycles, 240 bats of 18 species were captured in 2 states of Mexico with contrasting ecological characteristics but concurrent DENV activity in humans. RT-PCR analysis of RNA extracted from liver or spleen tissue from de bats failed to show evidence for the presence of DENV nucleic acids in these organs. In addition, plasma assayed by plaque reduction neutralization test showed no evidence of neutralizing anti-DENV antibodies. These results suggest that American bats may not be reservoirs or amplification host for DENV infection.

Dengue virus (DENV) is a mosquito-borne virus of the familyFlaviviridae.The RNA viral genome encodes for a polyprotein that is co-translationally processed into three structural proteins and seven non-structural proteins. The non-structural protein 1 (NS1) is a multifunctional viral protein actively secreted in vertebrate and mosquito cells during DENV infection. In mosquito cells, NS1 is secreted in a caveolin-1 (CAV-1) dependent manner by an unconventional pathway. The caveolin chaperone complex (CCC) is a cytoplasmic complex formed by caveolin-1 and the chaperones FKBP52, Cy40 and CyA which is responsible for cholesterol traffic inside the cell. In this work, we demonstrate that in infected mosquito cells, DENV NS1 is secreted by an early and unconventional route that bypasses the Golgi apparatus in close association with the CCC. Treatment of mosquito cells with classic secretion inhibitors such as brefeldin A, golgicide A and Fli-06 showed no effect on NS1 secretion, but significant reductions in recombinant luciferase secretion and virion release. Silencing the expression of CAV1, FKBP52 with siRNAs or the inhibition of CyA by cyclosporine A resulted in significant decrease in NS1 secretion without affecting virion release. Using co-localization, co-inmunoprecipitation and proximity ligation assays, NS1 was found to co-localize and interact with all the protein components of the CCC in mosquito infected cells. In addition, CAV-1 and FKBP52 expression was found augmented in DENV infected cells. Finally, the treatment of ZIKV infected mosquito cells with brefeldin A and golgicide A showed no effect on NS1 secretion, while affecting virion release. ZIKV NS1 was also found to co-localize with CAV-1 in infected mosquito cells. These results suggest that in mosquito cells, ZIKV NS1 follows the same secretory pathway observed for DENV NS1. The association of NS1 with the cholesterol transporter CCC agrees with the lipoprotein nature of secreted hexameric NS1.AUTHOR SUMMARYDengue protein NS1 is secreted in infected mosquito and vertebrate cells. In humans, secreted NS1 have been associated with pathogenesis. In mosquito cells, NS1 follows an unconventional secretion pathway that is dependent on Caveolin-1. This work shows that in mosquito cells, NS1 secretion is associated to the chaperone caveolin complex, a complex formed by caveolin-1 and several chaperones, in charge of cholesterol transport within the cells. Reduction of the expression or the activity of chaperone caveolin complex in mosquito infected cells, diminished the secretion of NS1 without affecting virion release. Direct interaction between NS1 and the chaperone caveolin complex proteins was demonstrated by several assays. Moreover, increased expression of the caveolin-1 and co-chaperone FKBP52 during dengue infection was found, presumably in response to the higher requirements of these proteins during dengue virus infection. Results obtained with ZIKV infected mosquito cells suggest that also ZIKV NS1 is released following an unconventional secretory route in association with the chaperone caveolin complex. The functions of secreted NS1 within mosquito are unclear. However, giving the importance of the soluble NS1 in the vertebrate host, manipulation of the NS1 secretory route may prove a valuable strategy for dengue mosquito control and patient treatment.

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.

The Cuatro Ciénegas Basin (CCB) is located in the Chihuahuan desert in the Mexican state of Coahuila; it has been characterized as a site with high biological diversity despite its extreme oligotrophic conditions. It has the greatest number of endemic species in North America, containing abundant living microbialites (including stromatolites and microbial mats) and diverse microbial communities. With the hypothesis that this high biodiversity and the geographic structure should be reflected in the virome, the viral communities in 11 different locations of three drainage systems, Churince, La Becerra, and Pozas Rojas, and in the intestinal contents of 3 different fish species, were analyzed for both eukaryotic and prokaryotic RNA and DNA viruses using next-generation sequencing methods. Double-stranded DNA (dsDNA) virus families were the most abundant (72.5% of reads), followed by single-stranded DNA (ssDNA) viruses (2.9%) and ssRNA and dsRNA virus families (0.5%). Thirteen families had dsDNA genomes, five had ssDNA, three had dsRNA, and 16 had ssRNA. A highly diverse viral community was found, with an ample range of hosts and a strong geographical structure, with very even distributions and signals of endemicity in the phylogenetic trees from several different virus families. The majority of viruses found were bacteriophages but eukaryotic viruses were also frequent, and the large diversity of viruses related to algae were a surprise, since algae are not evident in the previously analyzed aquatic systems of this ecosystem. Animal viruses were also frequently found, showing the large diversity of aquatic animals in this oasis, where plants, protozoa, and archaea are rare. IMPORTANCE In this study, we tested whether the high biodiversity and geographic structure of CCB is reflected in its virome. CCB is an extraordinarily biodiverse oasis in the Chihuahuan desert, where a previous virome study suggested that viruses had followed the marine ancestry of the marine bacteria and, as a result of their long isolation, became endemic to the site. In this study, which includes a larger sequencing coverage and water samples from other sites within the valley, we confirmed the high virus biodiversity and uniqueness as well as the strong biogeographical diversification of the CCB. In addition, we also analyzed fish intestinal contents, finding that each fish species eats different prey and, as a result, presents different viral compositions even if they coexist in the same pond. These facts highlight the high and novel virus diversity of CCB and its “lost world” status.

Background The role of rotavirus as main etiologic agent of diarrhea has been well documented worldwide, including in Venezuela. However, information about the prevalence of gastrointestinal viruses such as calicivirus, adenovirus and astrovirus is limited and the contribution of other agents as Aichi virus and klassevirus is largely unknown. To explore the etiological spectrum of diarrhea associated with agents other than rotaviruses, 227 stool samples from children under 5 years old with acute gastroenteritis, collected in Valencia (Venezuela) from 2001 to 2005, and previously tested as rotavirus-negative, were analyzed for caliciviruses, adenoviruses, astroviruses, Aichi viruses, klasseviruses, picobirnaviruses and enteroviruses by specific RT-PCRs. Results At least one viral agent was detected in 134 (59%) of the samples analyzed, mainly from children under 24 months of age and most of them belonging to the lowest socioeconomic status. Overall, enterovirus was identified as the most common viral agent (37.9%), followed by calicivirus (23.3%), adenovirus (11.5%), astrovirus (3.5%), klassevirus (1.3%) and Aichi virus (0.4%), while no picobirnavirus was detected. Klasseviruses were found during 2004 and 2005 and Aichi viruses only in 2005, indicating their circulation in Venezuela; meanwhile, the rest of the viruses were detected during the whole study period. Coinfections with two or more viruses were found in 39 (29.1%) of the infected children, most under 24 months of age. Adenovirus was involved as the coinfecting agent in at least 46.9% of the cases, but no differences concerning socio-demographic variables were observed between the coinfected and the single infected children. Conclusions The results show that various enteric viruses, including enteroviruses, caliciviruses and adenoviruses, accounted for a significant proportion of infantile diarrhea cases in Venezuela before rotavirus vaccine implementation. In addition, emerging viruses as Aichi virus and klassevirus were found, indicating the need to continue monitoring their spreading into the communities. Efforts are needed to develop more accurate methods to identify the major causes of diarrhea and to provide tools for more effective preventive measures.

Severe dengue pathogenesis is not fully understood, but high levels of proinflammatory cytokines have been associated with dengue disease severity. In this study, the cytokine levels in 171 sera from Mexican patients with primary dengue fever (DF) and dengue haemorrhagic fever (DHF) from dengue virus (DENV) 1 (n = 116) or 2 (n = 55) were compared. DF and DHF were defined according to the patient's clinical condition, the primary infections as indicated by IgG enzymatic immunoassay negative results, and the infecting serotype as assessed by real-time reverse transcription-polymerase chain reaction. Samples were analysed for circulating levels of interleukin (IL)-12p70, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IL-6, and IL-8 using a commercial cytometric bead array. Significantly higher IFN-γ levels were found in patients with DHF than those with DF. However, significantly higher IL-12p70, TNF-α, and IL-6 levels were associated with DHF only in patients who were infected with DENV2 but not with DENV1. Moreover, patients with DF who were infected with DENV1 showed higher levels of IL-12p70, TNF-α, and IL-6 than patients with DHF early after-fever onset. The IL-8 levels were similar in all cases regardless of the clinical condition or infection serotype. These results suggest that the association between high proinflammatory cytokine levels and dengue disease severity does not always stand, and it once again highlights the complex nature of DHF pathogenesis.

Given dengue virus (DENV) genome austerity, it uses cellular molecules and structures for virion entry, translation and replication of the genome. NS1 is a multifunctional protein key to viral replication and pathogenesis. Identification of cellular proteins that interact with NS1 may help in further understanding the functions of NS1. In this paper we isolated a total of 64 proteins from DENV infected human hepatic cells (Huh-7) that interact with NS1 by affinity chromatography and immunoprecipitation assays. The subcellular location and expression levels during infection of the ribosomal proteins RPS3a, RPL7, RPL18, RPL18a plus GAPDH were determined. None of these proteins changed their expression levels during infection; however, RPL-18 was redistributed to the perinuclear region after 48 hpi. Silencing of the RPL-18 does not affect cell translation efficiency or viability, but it reduces significantly viral translation, replication and viral yield, suggesting that the RPL-18 is required during DENV replicative cycle.

Dengue is the most important and widely distributed arthropod-borne viral disease affecting humans. The number of dengue virus infections has steadily grown and more than 100 countries survey dengue incidence every year. Due to the lack of an approved antiviral treatment or licensed preventative vaccine, accurate and opportune diagnosis is commended for efficient dengue epidemiological surveillance, to propose control measures in order to curtail outbreaks timely and treat patients satisfactorily. In this review, the basis, application and indications for different diagnostic tests are described, and their advantages and limitations considered. At the end of this piece, we speculate what the future may hold for the diagnosis of dengue infections.

The Cuatro Cienegas Basin (CCB) is a biologically exceptional place. In general, CCB aquatic systems are extremely oligotrophic, but paradoxically they are extraordinarily biodiverse, and viruses are not an exception. Previous studies had shown that two stable ecosystems within CCB had very different virus; one of them, Pozas Azules, presented divergent virus with a marine affiliation. In this chapter, we compare an earlier study with the virome of three sites at CCB, sampled in 2014, including fish guts from one of the sites. The idea was to test if the virus follows, like a mirror image, the diversity of a site, by showing us the predators vs their prey.

The role of Ca

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.

a b s t r a c t Background: Type 1 interferon (IFN/) has a significant role in establishing protection against virus infections. It has been well documented by in vitro studies that dengue virus (DENV) activates a robust IFN/ response. However, DENV also induces a down-regulation of the JAK/STAT pathway, inhibiting the induction of interferon regulated genes. As a consequence, the role played by the IFN type 1 response in the protection of dengue patients is not fully understood. Objective: To compare IFN- levels in dengue patients with dengue fever (DF) or dengue hemorrhagic fever (DHF) undergoing primary or secondary infections. Study design: Two hundred and four serum samples were analyzed for IFN- level by cytometric bead array. Patients' clinical condition was assigned following the WHO 1997 criteria and specific IgG and IgM antibodies were measured using commercial assays to determine primary and secondary infections. The infecting serotype was determined by qRT-PCR. Results and conclusion: The IFN- levels were found significantly higher in DF than DHF patients irrespec- tive of the infecting serotype (DENV1 or 2), and were found to decline rapidly at day 3 after fever onset. For DENV2 infections, higher IFN- level was found during primary than secondary infections. These results suggest that an early strong interferon response correlates with a better clinical condition.

Background The measurement and detection of viremia and antigenemia in sera have been used as a marker of risk for dengue disease severity and diagnosis. However, evidence exists suggesting that levels of viremia and antigenemia are affected by the presence of specific antibodies. Objective To compare viral load and circulating NS1 levels in sera from patients positive or negative for dengue specific IgM antibodies. Study design Three hundred and eighty serum samples were analyzed for viral load using qRT-PCR and for levels of circulating NS1 and the presence of specific antibodies using commercial EIAs. Results and conclusion Comparison of viremia levels in sera from patients positive or negative for dengue IgM antibodies showed that viral load was significantly higher ( p ≤ 0.0001) in patients negative for IgM antibodies. In contrast, levels of circulating NS1 were found unaffected by the presence of IgM ( p = 0.0038). Thus, dengue virus specific IgM antibodies in sera seem to be a strong negative modulator of viremia levels in patient's sera.

Several cellular molecules and components, specifically, cholesterol and lipid rafts have been described as necessary elements for dengue virus entry and signaling in several human cells. Thus, changes in lipid rafts formation and cholesterol levels were evaluated. Here we report that the amount of total cholesterol and lipid rafts formation increase early after infection of Huh-7 cells. This augment correlates with an increase in the amount of low density lipoprotein receptor (LDLr) on the surface of infected cells and also with a lower phosphorylation level of the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). None of the changes were observed in Huh 7 cells infected with VSV used as a control. These results suggest that dengue virus infection increases intracellular cholesterol levels at early times post infection by triggering the modulation of LDL particles uptake and the increase in the enzymatic activity of HMG-CoA reductase.

The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1-4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-μl serum sample, the sensitivity and specificity values of the DENV1-4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-μl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction-positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses.

Dengue is the most important mosquito-borne viral infection in humans. Recent evidence suggests that vitamin D influences virus replication. In this work, the effect of vitamin D treatment on dengue virus infection in human hepatic Huh-7 cells and on virus infection and cytokine production in the human monocytic U937 cells was evaluated. Exposure to 1α,25-dihydroxy-vitamin D3, resulted in a significant reduction in the number of infected cells, in conditions where cell viability was not affected. Viral replication in monocytic cells was more susceptible to vitamin D3 than replication in the hepatic cells. Moreover, vitamin D3 significantly reduced the levels of proinflammatory cytokines (TNF-α, IL-6, IL-12p70 and IL-1β) produced by infected U937 cells. These results suggest that vitamin D3 may represent a potentially useful antiviral compound.

The epidemiology and clinical symptoms in infants and young children with acute sporadic viral gastroenteritis due to viral etiologies other than rotaviruses have not been studied thoroughly in developing countries. Fecal specimens from 480 children5 years of age who were admitted to a large children's hospital in the city of Valencia, Venezuela, with acute diarrhea during January to December 2003 were collected and screened by ELISA and RT-PCR for rotavirus, adenovirus, norovirus, sapovirus, and astrovirus. Viral isolates were partially characterized by phylogenetic analysis. Norovirus viral load was determined by qRT-PCR. Viruses were identified in 205 (43%) of the 480 stool samples collected. Rotavirus was the virus detected most frequently (21%), followed by norovirus (13%), adenovirus (5%), sapovirus (3%), and astrovirus (2%). Viral infection rates were highest in the 6- to 11-month-old group (49%) and lowest in children24 months old. Norovirus GII was more prevalent (90%) than GI (10%). Enteric adenovirus (serotypes 40/41) was present in 43% of the adenovirus-positive samples. Rotavirus infection caused more severe clinical symptoms than the other viruses detected, with more vomiting (84%) and dehydration (11%) that led to hospital admission of 20% of the children with acute gastroenteritis. Rotavirus and norovirus showed marked and opposite seasonal patterns. No association was observed between disease severity and viral load in children infected with norovirus. These results not only confirm the impact of rotavirus infection in Venezuela but also indicate that other enteric viruses, especially noroviruses, contribute significantly to sporadic acute gastroenteritis and to the burden of disease.

Picobirnaviruses (PBV) are a group of small, nonenveloped viruses with bi-segmented dsRNA genomes. The large genomic segment encodes for the capsid protein while the small segment encodes for the RNA-dependent RNA polymerase. PBV are widely distributed and have been detected in the feces of a large variety of vertebrate species, including farm and captive animals and also in humans. Their etiological role as a cause of diarrhea remains elusive. Still, compelling evidence indicates that PBV may be opportunistic pathogens associated with diarrhea in immunocompromised individuals. The partial molecular characterization of human and animal strains revealed that PBVs are highly variable, and at least two distinct genogroups have been recognized. In addition, the possibility of interspecies transmission has been suggested. This article examines the molecular epidemiology and the biology of PBV and highlights the major challenges in the field.

Porcine enteric sapovirus (PES) has been shown to cause diarrhea under experimental conditions in gnotobiotic piglets. However, the role of PES as enteric pathogens in porcine farms remains unclear. To further understand the PES-host interactions under field conditions, a serological survey was carried out. To this end the capsid gene of a PES isolate was cloned in the baculovirus expression system and an ELISA was developed based on virus-like particles from the baculovirus-expressed PES capsid protein. A total of 85 serum samples collected from pigs ranging from 8 weeks to over 54 weeks of age were analyzed. An overall seroprevalence to PESs of 62% was found, with significant differences (p0.05) found between ages. Pigs younger than 10 weeks old and older than 12 weeks old showed high seroprevalences (70-100%), while pigs aged 10-12 weeks showed no detectable serum antibodies levels. Our results suggest that PES infections are common in pigs and that passively acquired maternal antibodies are soon replaced by actively acquired antibodies, whose titers increase gradually with age and that probably are maintained during lifetime.

A study aimed to further understand the biology of porcine picobirnaviruses (PBV) was conducted between November 2003 and January 2008, on a farm located in the outskirts of Córdoba City, Argentina. PBV prevalence was examined by polyacrylamide gel electrophoresis and silver staining (PAGE S/S) on a total of 265 samples collected from pigs divided into four groups, according to age and physiological status. PBV detection rate was highest in the group of sows sampled within the lactogenic period (38.02%; p0.05), followed by pregnant sows (15.09%), piglets aged 2-5 months of age (18.42%) and adult (or =50 weeks) male pigs (0%). In addition, 103 samples collected in 3 follow-up studies were analyzed by PAGE S/S and reverse transcription followed by PCR (RT-PCR). Two of these studies followed female pigs from weaning up to slaughter and a third one from weaning up to 4 pregnancy periods. The results provide evidence that PBV establishes a persistent infection in the host with periods of silence intermingled with periods of low and high viral excretion. High PBV excretion levels were detected by PAGE S/S and were conditioned by age (primary infection) and host physiological status. Low PBV excretion levels were detected by RT-PCR throughout the entire study period. Sequence analysis of selected amplicons indicated that the virus excreted through the follow-up study was the same. These results suggest that porcine PBV is maintained in nature by transmission from infected asymptomatic individuals to susceptible ones.

Infection with a broad diversity of viruses often activates host cell signaling pathways including the mitogen-activated protein kinase pathway. The present study established that dengue virus infection of human macrophages activates Jun NH(2)-terminal kinase (JNK) and the p38 MAPKs pathways. The activation was observed at early times after infection and occurs when either infectious or UV-inactivated dengue virus was used. The role of these activated kinases in dengue virus infection was evaluated using specific inhibitors. Inhibition of JNK and p38 kinases did result in a significant reduction in viral protein synthesis and in viral yield. Additionally, lipid rafts disruption induced a strong inhibition of JNK activation. These results suggest that, at early stages after dengue virus infection, MAPKs are activated and that activation of JNK and p38 is required for dengue virus infection.

Dengue virus (DENV) is the causative agent of dengue fever and the more severe forms of the infection known as dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS). Secondary infections with a serotype different from the primary infection are considered a risk factor for the development of DHF/DSS. One explanation for the increased risk of DHF/DSS development after heterologous secondary infections is the antibody-dependent enhancement (ADE) hypothesis. This hypothesis postulates that pre-existing non-neutralizing antibodies will form immune complexes with the new serotype-infecting virus that in turn will have enhanced capacity to infect macrophages and other Fcgamma receptor (FcgammaR)-bearing cells. Despite the evidence supporting the ADE hypothesis, the molecular mechanisms of ADE are not fully understood. In this work, we present evidence which indicates that intact lipid rafts are required for the ADE infection of U937 cells with DENV. Flow cytometry analysis to measure the percentage of infected cells showed that treatment of differentiated U937 cells with nystatin (30 microg ml(-1)), filipin (10 microg ml(-1)) or beta-methyl cyclodextrin (30 mM) significantly reduces (P

The endocytic pathway followed by dengue virus to infect the mosquito cells C6/36 HT was analyzed. Using DIL-labeled virions and real-time imaging it was determined that viral entry into C6/36 HT takes approximately 5 to 7 min. Pretreatment of C6/36 HT cells with sucrose and bafilomycin A, but not filipin, inhibited dengue virus infection up to 80%. Furthermore, the overexpression of dominant-negative mutants of Eps15, a molecule required for the formation of clathrin-coated vesicles, reduced dengue infection up to 50%, indicating that dengue virus entry is through clathrin-mediated endocytosis and is pH-dependent. By double-immunofluorescence assays, DIL-labeled particles were colocalized with early endosomes at 5 min and with lysosomes mainly at 30 min post-infection. Finally, disruption of the microtubule and microfilaments by nocodazole and by cytochalasin D reduced viral infection by more than 80%. Taken together these results indicate that dengue virions enter into C6/36 HT cells by clathrin-mediated endocytosis, using the endosomal pathway from early endosomes to acidic lysosomes before viral RNA is released into the cytoplasm.

The molecular characterization of partial- length genomic segment 2 of porcine picobirnavirus (PBV) strains and the development of a specific reverse transcription-PCR (RT-PCR) assay for detection of virus in feces are reported. Phylogenetic analysis indicated that the studied porcine isolates were more closely related (>85% identity) to human PBV belonging to genogroup I than to the other porcine PBV described so far. Analysis by RT-PCR and polyacrylamide gel electrophoresis of fecal samples collected in Venezuela and Argentina showed that PBV circulate at high frequencies in piglets.

Rotavirus infection is characterized by a number of Ca 2+ dependent virus–cell interactions. The structure of rotavirus triple-layered particles (TLP) is dependent on Ca 2+ concentration. Acquisition of the capsid outer layer requires a high Ca 2+ concentration inside the ER. Infection modifies Ca 2+ homeostasis of the cell, increasing ER Ca 2+ content, which may be advantageous to virus replication. We studied the role of sequestered Ca 2+ on the stabilization of already mature viral particles within the ER. Thapsigargin (TG), a SERCA pump inhibitor, added for 30 min at the end of infection depleted ER Ca 2+ and reduced the titer of already mature TLP accumulated in the cell. Another inhibitor, cyclopiazonic acid, and two Ca 2+ ionophores (A23187 and ionomycin) in the presence of EGTA had similar effects. TG eliminated the peak of radiolabeled TLP, increasing that of DLP in CsCl gradients. Electron microscopy revealed accumulation of clustered particles in the ER, which had lost their integrity. The [Ca 2+ ] in the ER of infected cells is important for virus maturation and for maintaining the integrity of mature TLP. Viral particles in this compartment may be potentially infectious, already containing VP7 and VP4.

The immune response elicited by the rotavirus nonstructural protein NSP4 and its potential role in protection against rotavirus disease are not well understood. We investigated the serological response to NSP4 and its correlation with disease protection in sera from 110 children suffering acute diarrhea, associated or not with rotavirus, and from 26 children who were recipients of the rhesus rotavirus tetravalent (RRV-TV) vaccine. We used, as antigens in an enzyme-linked immunosorbent assay (ELISA), affinity-purified recombinant NSP4 (residues 85 to 175) from strains SA11, Wa, and RRV (genotypes A, B, and C, respectively) fused to glutathione S -transferase. Seroconversion to NSP4 was observed in 54% (42/78) of the children who suffered from natural rotavirus infection and in 8% (2/26) of the RRV-TV vaccine recipients. Our findings indicate that NSP4 evokes significantly ( P < 0.05) higher seroconversion rates after natural infection than after RRV-TV vaccination. The serum antibody levels to NSP4 were modest (titers of ≤200) in most of the infected and vaccinated children. A heterotypic NSP4 response was detected in 48% of the naturally rotavirus-infected children with a detectable response to NSP4. Following natural infection or RRV-TV vaccination, NSP4 was significantly less immunogenic than the VP6 protein when these responses were independently measured by ELISA. A significant ( P < 0.05) proportion of children who did not develop diarrhea associated with rotavirus had antibodies to NSP4 in acute-phase serum, suggesting that serum antibodies against NSP4 might correlate with protection from rotavirus diarrhea. In addition, previous exposures to rotavirus did not affect the NSP4 seroconversion rate.

Tranfusion-transmitted virus (TTV), a single-stranded circular DNA virus that chronically infects humans and other animals, displays a high degree of genetic diversity and was originally thought to be associated with hepatitis. The prevalences of TTV infection among different populations of humans and non-human primates from Venezuela have now been evaluated, using serum samples and three different detection tests. All three tests were PCR-based, one involving a hemi-nested PCR and primers based on the N22 open-reading-frame-1 region (N22-PCR), another employing 55 cycles with primers from the more conserved untranslated region (UTR-PCR), and the other using a hemi-nested PCR with primers from the same region (HUTR-PCR). The overall prevalences of human infection appeared much higher with the HUTR-PCR (52%) than with the N22-PCR (13%) or the UTR-PCR (5%). When the products amplified by N22-PCR from 28 human isolates of TTV were sequenced, only two genotypes of the virus were detected. The non-human sera tested came from primates kept in a zoo in north-western Venezuela. TTV DNA was detected, by HUTR-PCR, in both of the chimpanzee sera tested but not in any of the sera from the 11 New-World primates or the other 12 Old-World primates that were investigated. The results, particularly those of the HUTR-PCR, indicate that TTV infection is common in Venezuela, especially in populations, such as many Amerindian groups, who live under poor sanitary conditions. Although TTV infection may be relatively rare among non-human primates from the New World, this will have to be investigated further, using many more samples collected throughout the Americas.

Background Despite vaccine availability, the infection rate and disease burden associated with rotavirus infection are still high. Thus, accurate diagnosis of rotavirus infection continues to be necessary for proper patient clinical management and disease control. Objective To evaluate the performance of a novel, second generation, commercial latex tests (Pastorex™ Rotavirus latex agglutination test, BIORAD, Marnes-La-Coquette, France), for the detection of rotavirus in human feces. Study design Using 166 fecal samples collected from children with acute diarrhea, and previously tested for rotavirus, calicivirus and astrovirus, the second generation Pastorex™ Rotavirus latex agglutination test was evaluated. Results and conclusion The test showed a sensitivity of 85.9% and a specificity of 97.7%. Positive and negative predicted values for the test were 97% and 88%, respectively. The results suggest that this commercial test is a good alternative for rotavirus diagnosis.

The gene encoding the VP8* trypsin-cleavage product of the VP4 protein of porcine rotavirus strain A34 was sequenced, and the predicted amino acid (aa) sequence was compared to the homologous region of all known P genotypes. The aa sequence of the VP8* of strain A34 shared low identity, ranging from 39% (bovine strain B223, P8[11]) to 76% (human strain 69M, P4[10]), with the homologous sequences of representative strains of the remaining 21 P genotypes. Phylogenetic relationships showed that the VP8* of strain A34 shares a common evolutionary lineage with those of human 69M (P4[10]) and equine H-2 (P4[12]) strains. Hyperimmune sera raised to strain A34 and to a genetic reassortant strain containing the VP4 gene from strain A34, both with high homologous neutralization titer via VP4, failed to neutralize strains representative of 15 different P genotypes. These results indicate that strain A34 should be considered as prototype of a new P genotype and serotype (P14[23]) and provide further evidence for the vast genetic and antigenic diversity of group A rotaviruses.

In Venezuela, the isolation of hantaviruses from rodents and the detection, in 1999, of a clinically confirmed human case of hantavirus infection led to increased interest in these viruses. In an attempt to estimate the problem posed by such viruses in Venezuela, ELISA based on purified, recombinant, nucleoprotein were used to check 1380 human serum samples for the presence of IgG antibodies to hantavirus. The ELISA results, as confirmed by indirect immunofluorescence and Western-blot assays, indicated that 23 (1.7%) of the serum samples contained antibodies to hantaviruses. Seroprevalences were similar among all age-groups and for both genders and were no higher among rural populations with a relatively high risk of exposure to rodents than among the overall study population. Although the numbers of samples involved were small, the seroprevalence among the subjects who were residents of Carabobo state was much higher than the overall value (10.3% v. 1.7%; P < 0.01). Human infection with hantavirus appears uncommon but widely distributed in Venezuela.

The rotavirus capsid is composed of three concentric protein layers. Proteins VP4 and VP7 comprise the outer layer. VP4 forms spikes, is the viral attachment protein, and is cleaved by trypsin into VP8* and VP5*. VP7 is a glycoprotein and the major constituent of the outer protein layer. Both VP4 and VP7 induce neutralizing and protective antibodies. To gain insight into the virus neutralization mechanisms, the effects of neutralizing monoclonal antibodies (MAbs) directed against VP8*, VP5*, and VP7 on the decapsidation process of purified OSU and RRV virions were studied. Changes in virion size were followed in real time by 90° light scattering. The transition from triple-layered particles to double-layered particles induced by controlled low calcium concentrations was completely inhibited by anti-VP7 MAbs but not by anti-VP8* or anti-VP5* MAbs. The inhibitory effect of the MAb directed against VP7 was concentration dependent and was abolished by papain digestion of virus-bound antibody under conditions that generated Fab fragments but not under conditions that generated F(ab′) 2 fragments. Electron microscopy showed that RRV virions reacted with an anti-VP7 MAb stayed as triple-layered particles in the presence of excess EDTA. Furthermore, the infectivity of rotavirus neutralized via VP8*, but not that of rotavirus neutralized via VP7, could be recovered by lipofection of neutralized particles into MA-104 cells. These data are consistent with the notion that antibodies directed at VP8* neutralize by inhibiting binding of virus to the cell. They also indicate that antibodies directed at VP7 neutralize by inhibiting virus decapsidation, in a manner that is dependent on the bivalent binding of the antibody.