Molecular characterization of human adenovirus causing infantile acute gastroenteritis in Venezuela before and after rotavirus vaccine implementation.

Human adenoviruses (HAdV) of species F are commonly involved in pediatric acute gastroenteritis (AGE). The real impact on Venezuelan health is unknown. To investigate the prevalence and molecular diversity of HAdV in Venezuela, 630 fecal samples collected from children with AGE in 3 cities, from 2001 to 2013, were tested by PCR. Species F and types F40/41 were identified by REA. HAdV was detected in 123 cases (19.5%), most from outpatient females under 24 months old. A progressive and substantial increase in the detection rate was observed over time, significantly higher in rotavirus vaccinated than unvaccinated children (28.4% vs. 9.5%, P = 0.00019). Phylogenetic analysis of 28 randomly selected genomes showed high similarity among HAdV-F40/41 and those worldwide. HAdV-F of type 41 prevailed (79.8%) and clustered into 2 intratypic major clades. The significant involvement of HAdV-F41 in AGE suggests the importance of actively monitoring viral agents other than rotavirus, especially after vaccine introduction.

Detection of rotavirus and norovirus among children with acute gastroenteritis in Merida and Chihuahua cities, Mexico.

INTRODUCTION: Infantile acute gastroenteritis (AGE) is a leading cause of morbidity and mortality, particularly in developing countries. The most frequent etiological agents of viral gastroenteritis in children are adenovirus, astrovirus, rotavirus, and norovirus, the last two, leading causes. Thus, the aim of this study was to identify the presence of these two viruses in children with AGE, from two cities located in the Southeast and the Northwest regions of México. METHODOLOGY: HuNoVs were detected and characterized by RT-PCR and sequencing, while RVs were detected by RNA electrophoresis. RESULTS: The presence of RV and HuNoV was evaluated in 81 stool samples; 37 were collected between April and July 2013 from patients with acute diarrhea in Merida, and 44 were collected between January and June 2017 in Chihuahua, who attended health services. Despite vaccination, RV resulted in the predominant viruses detected, with 30.8% (25/81) positivity, while HuNoV infection was present in 8.6% (7/81) of the stool samples; GII strains were identified circulating in the Southeast, while GI strains were identified in the Northwest. Moreover, co-infections with both viruses were detected at a prevalence rate of 2.4% (2/81). CONCLUSIONS: The circulation of RV and HuNoV in the country is continuous and should be constantly monitored due to their impact on public health.

Effect of sericin, a silk derived protein, on the amplification of Zika virus in insect and mammalian cell cultures.

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.

Dengue Virus NS1 Uses Scavenger Receptor B1 as a Cell Receptor in Cultured 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.

Secretion of Nonstructural Protein 1 of Dengue Virus from Infected Mosquito Cells: Facts and Speculations.

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.

Molecular detection of human enteric viruses circulating among children with acute gastroenteritis in Valencia, Venezuela, before rotavirus vaccine implementation.

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.

The dengue virus non-structural protein 1 (NS1) is secreted from infected mosquito cells via a non-classical caveolin-1-dependent pathway.

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 (MßCD). The results showed that MßCD, 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.

The dengue virus non-structural protein 1 (NS1) is secreted efficiently from infected mosquito cells.

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.

Experimental inoculation of Artibeus jamaicensis bats with dengue virus serotypes 1 or 4 showed no evidence of sustained replication.

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.

Seroepidemiology of porcine enteric sapovirus in pig farms in Venezuela.

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 (p<0.05) 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.

Estudio de la infección por rotavirus y calicivirus en neonatos de la Maternidad "Concepción Palacios" de Caracas / Study of rotavirus and calicivirus infections in neonates from the Maternity Hospital "Concepción Palacios", Caracas

En un estudio realizado en 1984 en la Maternidad "Concepción Palacios", Pérez-Schael y col. detectaron excreción asintomática de rotavirus en el 56% de los recién nacidos evaluados. El objetivo de este trabajo fue estudiar la infección por rotavirus en neonatos de la Maternidad y comparar datos con aquellos obtenidos anteriormente. Además, se investigó la presencia de calicivirus en dicha población. Entre agosto y diciembre de 2004, se recolectaron 307 muestras de heces provenientes de 215 neonatos sanos. Para la detección de los agentes virales se utilizaron ensayos tipo ELISA comerciales y no comerciales. Estos ensayos identificaron como positivas a rotavirus y calicivirus a un total de 14 y 58 muestras, respectivamente. Sin embargo, al realizar pruebas para corroborar la presencia de rotavirus por microscopia electrónica, EGPA y RT-PCR y para calicivirus por RT-PCR, ninguna de las muestras señaladas como positivas pudo ser confirmada. Estos resultados sugieren la no-circulación tanto de rotavirus como de calicivirus dentro de la Maternidad. Es posible que la interrupción de la transmisión de rotavirus dentro de la Institución pueda deberse a cambios en el manejo del par madre-neonato introducidos en la Maternidad desde 1995, promovidos por la OMS y UNICEF.

In a study carried out in 1984 at the "Concepción Palacios" Maternity Hospital, Perez-Schael et al (J Med Virol 1984, 14:127) detected asymptomatic excretion of rotavirus in 56% of the neonates evaluated. The purpose of this work was to study rotavirus infection in neonates at the Maternity Hospital and compare the new data with those previously obtained. We also studied calicivirus presence in said population. Between August and December 2004, 307 feces samples were collected from 215 healthy neonates. Commercial and non commercial ELISA type assays were used for detection of viral agents. These assays identified a total of 14 and 58 samples as rotavirus and calicivirus positive respectively. Nevertheless, when carrying out tests to corroborate rotavirus presence with electron microscopy, EGPA and RT-PCR, and for calicivirus with RT-PCR, none of the samples previously shown as positive could be confirmed. These results suggest the non-circulation of both rotavirus and calicivirus within the Maternity Hospital. It is possible that the interruption of rotavirus transmission within this Institution could be due to changes in the management of the mother-child unit promoted by WHO and UNICEF, and introduced at the Maternity Hospital since 1995.

Molecular detection of porcine enteric caliciviruses in Venezuelan farms.

Caliciviruses are a well-established cause of respiratory, vesicular and hemorrhagic diseases in animals. In addition, these viruses are an important cause of enteric diseases in humans. Recently, molecular analysis of several porcine enteric caliciviruses indicated that they are closely related to human enteric caliciviruses. The objective of this work was to determine the frequency, age distribution, and association with diarrhea of enteric calicivirus infections in piglets and to partially characterize the detected isolates. A total of 203 stool samples from animals 0 to 9 weeks of age, collected between 1993 and 2003 in seven porcine farms located in the central region of Venezuela were tested for enteric caliciviruses by reverse transcription-polymerase chain reaction (RT-PCR) amplification using primers designed to detect both norovirus and sapovirus. Selected amplicons were sequenced to establish phylogenetic relationships with reference strains. Calicivirus were detected in 18% (36/204) of the samples. Viruses were detected more frequently in animals between 3 and 4 weeks of age, and were detected in samples from animals with diarrhea and without diarrhea with equal frequencies (14 versus 19%, p>0.5). Phylogenetic studies based on partial RNA polymerase gene sequences indicated that the Venezuelan isolates were most closely related (75-95% identity) to the sapovirus Cowden reference strain. These results provide evidence that caliciviruses of the genus sapovirus circulate frequently in piglets but further studies are needed to clarify their importance as cause of diarrhea.

Primer pair p289-p290, designed to detect both noroviruses and sapoviruses by reverse transcription-PCR, also detects rotaviruses by cross-reactivity.

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.

[Molecular identification of bovine enteric calciviruses in Venezuela]. / Detección molecular de calicivirus entericos de bovino en Venezuela.

Caliciviruses are a well-established cause of respiratory, vesicular and hemorrhagic diseases in animals. In addition, these viruses are an important cause of enteric diseases in humans. Recently, molecular analysis of several bovine enteric calicivirus isolates indicated that they are genetically close to human enteric calicivirus. To investigate if bovine enteric caliciviruses circulate in Venezuela, 129 stool samples collected between 1994 and 2000 were assayed by reverse transcription-polymerase chain reaction amplification. The presence of calicivirus was confirmed in one of the samples analyzed, collected in the Lara State from a healthy calf, 2 months old. Phylogenetic studies based on partial RNA polymerase sequences indicated that the Venezuelan isolate (Bo/NV/Lara/2000/VE) is most closely related to the genogroup III, genus Noroviruses.

Detección molecular de calcivirus entericos de bovino en Venezuela / Molecular identification of bovine enteric calciviruses in Venezuela

Caliciviruses are a well-established cause of respiratory, vesicular and hemorrhagic diseases in animals. In addition, these viruses are an important cause of enteric diseases in humans. Recently, molecular analysis of several bovine enteric calicivirus isolates indicated that they are genetically close to human enteric calicivirus. To investigate if bovine enteric caliciviruses circulate in Venezuela, 129 stool samples collected between 1994 and 2000 were assayed by reverse transcription-polymerase chain reaction amplification. The presence of calicivirus was confirmed in one of the samples analyzed, collected in the Lara State from a healthy calf, 2 months old. Phylogenetic studies based on partial RNA polymerase sequences indicated that the Venezuelan isolate (Bo/NV/Lara/2000/VE) is most closely related to the genogroup III, genus Noroviruses.