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The COVID-19 pandemic was characterized by the emergence and succession of SARS-CoV-2 variants able to evade the antibody response induced by natural infection and vaccination. To evaluate the IgG reactivity and neutralizing capacity of the serum of individuals vaccinated with Sputnik V (105 volunteers vaccinated) against different viral variants. IgG reactivity to the Spike protein (S) was evaluated by ELISA. A plaque reduction neutralization test was performed using different viral variant isolates. At 42 days post-vaccination, the frequency of recognition and reactivity to the S protein of the Omicron variant was lower compared to that of the other variants. In general, a higher average neutralization titer was seen against the ancestral variant compared to the variants, especially Omicron. However, some sera exhibited a higher neutralization titer to the Gamma variant compared to the ancestral variant, suggesting unapparent exposure during the clinical trial. Antibodies induced by Sputnik V can recognize, persist, and neutralize SARS-CoV-2 variants, with Omicron being the one that best evades this response. These results represent a unique report on the humoral response induced by a globally lesser-studied vaccine in terms of efficacy and immune escape, offering insights into developing vaccines targeting unknown coronaviruses.

Objectives: To compare different methods to treat hydrosalpinx, based on both ablative and non-ablative approaches, in infertile patients before undergoing IVF-ET., Methods: Systematic review and network meta-analysis (NMA) of comparisons between different treatments of hydrosalpinx in infertile patients undergoing IVF., Data Sources: structured searches in common citation databases. Study inclusion criteria: peer-reviewed randomized trials (RCT) or cohort studies comparing effects of salpingectomy, laparoscopic proximal tubal occlusion (LTO), insertion of intratubal device (ITD), sclerotherapy, ultrasound-guided aspiration and no treatment, on live birth, ongoing pregnancy, clinical pregnancy as main outcomes, considering also miscarriage, ectopic pregnancy and complications as secondary outcomes. Principal NMA included RCT, and aggregated NMA of RCT and observational studies was carried out. Pooled effects have been estimated by Odds Ratio (OR) and its 95% confidence interval (CI) for direct and indirect-mixed comparisons, derived from random-effects models. Imprecision and heterogeneity of NMA estimations was assessed by comparison of its 95% CI with predefined intervals for clinically relevant size of effect (OR <0.9 or >1.1). Surface under the cumulative ranking curve (SUCRA) were used to predict treatment rankings for each outcome., Results: Nine RCT were included in main analysis, plus 17 additional observational studies in additional analysis. NMA of RCT did not identify significant differences in the effect of compared treatments on live birth rate, and LTO was the option with the highest value of SUCRA (0.92, mean rank: 1.2). Salpingectomy and US-aspiration associated to a significant increase of ongoing pregnancy rate compared to no treatment, according to NMA results (NMA OR: 4.35; 95% CI: 1.7, 11.14 and 2.8; 95% CI: 1.03, 7.58 respectively). Salpingectomy had the highest SUCRA value (0.88, mean rank: 1.4). NMA estimated significant increase of clinical pregnancy rate for salpingectomy compared with no treatment (NMA OR: 2.24; 95% CI: 1.3, 3.86) as well as for LTO versus no treatment (NMA OR: 2.55; 95% CI: 1.2, 5.41). Both comparisons were affected by a high grade of heterogeneity. For clinical pregnancy, LTO was the intervention with highest SUCRA (0.85; mean rank: 1.6). Regarding secondary outcomes, feasible NMA estimates did not support significant differences between treatments effects. According to aggregated NMA including randomized and observational studies, sclerotherapy showed significant beneficial effects on live birth rate compared to no treatment (NMA (OR: 4.6; 95% CI: 1.21, 17.46). Compared with untreated patients, the aggregated NMA estimates a higher ongoing pregnancy rate in patients treated with salpingectomy (NMA OR: 3.35; 95% CI: 2.12, 5.12), US-aspiration (NMA OR: 2.16; 95% CI: 1.28, 3.65) and LTO (NMA OR: 2.46; 95% CI: 1.11, 5.43). Salpingectomy and LTO produced a higher beneficial effect compared to ITD, based on both direct and indirect comparisons. Salpingectomy obtained the highest SUCRA value in rank of effects on ongoing pregnancy (0.94; mean rank: 1.2). NMA found significant effects on clinical pregnancy for comparisons between the different active management procedures compared with no treatments, with the exception of ITD insertion. LTO had more increasing effect on clinical pregnancy rate compared with US-aspiration (NMA OR: 2.04; 95% CI: 1.05, 3.97), while for the rest of the comparisons between procedures no significant differences were identified. NMA ranked LTO as the treatment with a highest SUCRA value (0.91; mean rank: 1.5). NMA prediction models identified LTO as best intervention to reduce miscarriage (SUCRA value: 0.84; mean rank: 1.8), as sclerotherapy as safer option in terms of ovarian response to IVF stimulation., Conclusions: The present NMA fails to support the effectiveness of any option to treat hydrosalpinx before IVF in order to improve live birth rates, although the beneficial effect of salpingectomy and US aspirations on ongoing pregnancy rates and of both salpingectomy and LTO on clinical pregnancy rates emerges from our analysis, which reinforces current recommendations. Based on the aggregated analyses, sclerotherapy could be a promising alternative to conventional laparoscopic techniques, combined with a favorable safety profile. This article is protected by copyright. All rights reserved., (This article is protected by copyright. All rights reserved.)

SARS-CoV-2 vaccines have contributed to attenuating the burden of the COVID-19 pandemic by promoting the development of effective immune responses, thus reducing the spread and severity of the pandemic. A clinical trial with the Sputnik-V vaccine was conducted in Venezuela from December 2020 to July 2021. The aim of this study was to explore the antibody reactivity of vaccinated individuals towards different regions of the spike protein (S). Neutralizing antibody (NAb) activity was assessed using a commercial surrogate assay, detecting NAbs against the receptor-binding domain (RBD), and a plaque reduction neutralization test. NAb levels were correlated with the reactivity of the antibodies to the spike regions over time. The presence of Abs against nucleoprotein was also determined to rule out the effect of exposure to the virus during the clinical trial in the serological response. A high serological reactivity was observed to S and specifically to S1 and the RBD. S2, although recognized with lower intensity by vaccinated individuals, was the subunit exhibiting the highest cross-reactivity in prepandemic sera. This study is in agreement with the high efficacy reported for the Sputnik V vaccine and shows that this vaccine is able to induce an immunity lasting for at least 180 days. The dissection of the Ab reactivity to different regions of S allowed us to identify the relevance of epitopes outside the RBD that are able to induce NAbs. This research may contribute to the understanding of vaccine immunity against SARS-CoV-2, which could contribute to the design of future vaccine strategies.

Background: Congenital uterine anomalies (CUA) can be associated with impairments of early and late pregnancy events., Objective: To assess the impact of CUA on reproductive outcomes in pregnancies conceived spontaneously or after assisted reproduction., Materials and Methods: Systematic review and meta-analysis of cohort studies comparing patients with CUA versus women with normal uterus. A structured literature search was performed in leading scientific databases to identify prospective and retrospective studies. The Newcastle-Ottawa scale, adapted to AHRQ standards, was used to assess the risk of bias. Pooled odds ratios (OR) were calculated. Publication bias and statistical heterogeneity were assessed, and meta-regression was used to analyse the heterogeneity., Main Outcome Measures: Miscarriage, ectopic pregnancy, placental abruption, term, and premature rupture of membranes (PROM), malpresentation at delivery, preterm delivery prior to 37, 34 and 32 weeks, caesarean delivery, intrauterine growth restriction/small for gestational age, foetal mortality and perinatal mortality., Results: 32 studies were included. CUAs increased significantly the risk of first/second trimester miscarriage (OR:1.54;95%CI:1.14-2.07), placental abruption (OR:5.04;3.60-7.04), PROM (OR:1.71;1.34-2.18), foetal malpresentation at delivery (OR:21.04;10.95-40.44), preterm birth (adjusted OR:4.34;3.59-5.21), a caesarean delivery (adjusted OR:7.69;4.17-14.29), intrauterine growth restriction/small for gestational age (adjusted OR:50;6.11-424), foetal mortality (OR:2.07;1.56-2.73) and perinatal mortality (OR:3.28;2.01-5.36)., Conclusions: CUA increases the risk of complications during pregnancy, delivery, and postpartum. Complications most frequent in CUA patients were preterm delivery, foetal malpresentation, and caesarean delivery., What Is New?: Bicornuate uterus was associated with the highest number of adverse outcomes, followed by didelphys, subseptate and septate uterus.

The emergence of the SARS-CoV-2 Variant of Concern (VOC), Omicron, has been characterized by an explosive number of cases in almost every part of the world. The dissemination of different sub-lineages and recombinant genomes also led to several posterior waves in many countries. The circulation of this VOC and its major sub-lineages (BA.1 to BA.5) was monitored in community cases and in international travelers returning to Venezuela by a rapid partial sequencing method. The specific sub-lineage assignment was performed by complete genome sequencing. Epidemic waves of SARS-CoV-2 cases were observed among international travelers during 2022, a situation not seen before December 2021. The succession of the Omicron VOC sub-lineages BA.1 to BA.5 occurred sequentially, except for BA.3, which was almost not detected. However, the sub-lineages generally circulated two months earlier in international travelers than in community cases. The diversity of Omicron sub-lineages found in international travelers was related to the one found in the USA, consistent with the most frequent destination of international travel from Venezuela this year. These differences are compatible with the delay observed sometimes in Latin American countries in the circulation of the different lineages of the Omicron VOC. Once the sub-lineages were introduced in the country, community transmission was responsible for generating a characteristic distribution of them, with a predominance of sub-lineages not necessarily similar to the one observed in travelers or neighboring countries.

The ongoing epidemic of monkeypox virus (MPXV) infection has already reached more than 50,000 persons worldwide until the end of August 2022. We report the first case detected in Venezuela. The patient reported traveling from Spain and contact with friends tested positive for MPXV after his return. Partial complete genome phylogenetic analysis allowed to group the isolate within the clade II of MPXV, the major one circulating worldwide. No other case of MPXV has been detected until the end of August 2022 in the country, although the presence of undiagnosed cases due to the fear of stigmatization cannot be ruled out.

Some of the lineages of SARS-CoV-2, the new coronavirus responsible for COVID-19, exhibit higher transmissibility or partial resistance to antibody-mediated neutralization and were designated by WHO as Variants of Interests (VOIs) or Concern (VOCs). The aim of this study was to monitor the dissemination of VOIs and VOCs in Venezuela from March 2021 to February 2022. A 614 nt genomic fragment was sequenced for the detection of some relevant mutations of these variants. Their presence was confirmed by complete genome sequencing, with a correlation higher than 99% between both methodologies. After the introduction of the Gamma VOC since the beginning of the year 2021, the variants Alpha VOC and Lambda VOI were detected as early as March 2021, at a very low frequency. In contrast, the Mu VOI, detected in May 2021, was able to circulate throughout the country. After the detection of the Delta VOC in June 2021, it became the predominant circulating variant. With the arrival of the Omicron VOC in December, this variant was able to displace the Delta one in less than one month.

The stress of the Golgi apparatus is an autoregulatory mechanism that is induced to compensate for greater demand in the Golgi functions. No 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 and increased expression of pathway targeted genes. 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 the Golgi complex imposed by virion and NS1 processing and secretion., (© 2021. The Author(s).)

SARS-CoV-2 is the new coronavirus responsible for COVID-19 disease. The first two cases of COVID-19 were detected in Venezuela on March 13, 2020. The aim of this study was the genetic characterization of Venezuelan SARS-CoV-2 isolates. A total of 7 full SARS-CoV-2 genome sequences were obtained by Sanger sequencing, from patients of different regions of Venezuela, mainly from the beginning of the epidemic. Ten out of 11 isolates (6 complete genomes and 4 partial spike genomic regions) belonged to lineage B, bearing the D614G mutation in the Spike protein. Isolates from the first outbreak that occurred in the Margarita Island harbored an in-frame deletion in its sequence, without amino acids 83-85 of the NSP1 of the ORF1. The search for deletions in 48,635 sequences showed that the NSP1 gene exhibit the highest frequency of deletions along the whole genome. Structural analysis suggests a change in the N-terminal domain with the presence of this deletion. In contrast, isolates circulating later in this island lacked the deletion, suggesting new introductions to the island after this first outbreak. In conclusion, a high diversity of SARS-CoV-2 isolates were found circulating in Venezuela, with predominance of the D614G mutation. The first small outbreak in Margarita Island seemed to be associated with a strain carrying a small deletion in the NSP1 protein, but these isolates do not seem to be responsible for the larger outbreak which started in July., Competing Interests: The authors have declared that no competing interests exist.

Pertussis vaccination of parents and household contacts ('cocooning') to protect newborn infants is an established strategy in many countries, although uptake may be low. Many aspects may influence such decision-making. We conducted a cross-sectional survey (NCT01890447) of households and other close contacts of newborns aged ≤6 months (or of expectant mothers in their last trimester) in Spain and Italy, using an adaptive discrete-choice experiment questionnaire. Aims were to assess the relative importance of attributes influencing vaccine adoption, and to estimate variation in vaccine adoption rates and the impact of cost on vaccination rates. Six hundred and fifteen participants (Spain, n = 313; Italy, n = 302) completed the survey. Of 144 available questionnaire scenarios, the most frequently selected (14% of respondents in both countries) were infant protection by household vaccination at vaccination center, recommendation by family physician and health authorities, with information available on leaflets and websites. The attribute with highest median relative importance was 'reduction in source of infection' in Spain (23.1%) and 'vaccination location' in Italy (18.8%). Differences between other attributes were low in both countries, with media attributes showing low importance. Over 80% of respondents indicated a definite or probable response to vaccine adoption (at no-cost) with estimated probability of adoption of 89-98%; applying vaccine costs (25€ per person) would reduce the probability of uptake by 7-20% in definite/probable respondents. Awareness of these determinants is helpful in informing Health Authorities and healthcare practitioners implementing a cocooning strategy for those populations where maternal immunization is not a preferred option.

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.

Background: Rotavirus (RV) is the most common cause of severe childhood diarrhea worldwide. Despite Venezuela was among the first developing countries to introduce RV vaccines into their national immunization schedules, RV is still contributing to the burden of diarrhea. Concerns exist about the selective pressure that RV vaccines could exert on the predominant types and/or emergence of new strains., Results: To assess the impact of RV vaccines on the genotype distribution 1 year after the vaccination was implemented, a total of 912 fecal specimens, collected from children with acute gastroenteritis in Caracas from February 2007 to April 2008, were screened, of which 169 (18.5%) were confirmed to be RV positive by PAGE. Rotavirus-associated diarrhea occurred all year-round, although prevailed during the coolest and driest months among unvaccinated children under 24 months old. Of 165 RV strains genotyped for G (VP7) and P (VP4) by seminested multiplex RT-PCR, 77 (46.7%) were G2P[4] and 63 (38.2%) G1P[8]. G9P[8], G3P[8] and G2P[6] were found in a lower proportion (7.3%). Remarkable was also the detection of <5% of uncommon combinations (G8P[14], G8P[4], G1P[4] and G4P[4]) and 3.6% of mixed infections. A changing pattern of G/P-type distribution was observed during the season studied, with complete predominance of G2P[4] from February to June 2007 followed by its gradual decline and the reemergence of G1P[8], predominant since January 2008. Phylogenetic analysis of VP7 and VP4 genes revealed a high similarity among G2P[4] and global strains belonging to G2-II and P[4]-V lineages. The amino acid substitution 96D → N, related with reemergence of the G2 genotype elsewhere, was observed. The G1P[8] strains from Caracas were grouped into the lineages G1-I and P[8]-III, along with geographically remote G1P[8] rotaviruses, but they were rather distant from Rotarix ® vaccine and pre-vaccine strains. Unique amino acid substitutions observed on neutralization domains of the VP7 sequence from Venezuelan post-vaccine G1P[8] could have conditioned their re-emergence and a more efficient dissemination into susceptible population., Conclusions: The results suggest that natural fluctuations of genotypes in combination with forces driving the genetic evolution could determine the spread of novel strains, whose long-term effect on the efficacy of available vaccines should be determined.

Two major maize viruses have been reported in the United States: Maize dwarf mosaic virus (MDMV) and Maize chlorotic dwarf virus (MCDV). These viruses co-occur in regions where maize is grown, such that co-infections are likely. Co-infection of different strains of MCDV is also observed, and a synergistic enhancement of symptoms in co-infected plants was previously reported. Here, we examined the impact of co-infections of two strains of MCDV (MCDV-S and MCDV-M1, severe and mild, respectively), and co-infections of MCDV and MDMV in the sweet corn hybrid 'Spirit' in greenhouse experiments. Quantitative plant growth and development parameters were measured and virus accumulation was measured by reverse-transcriptase quantitative polymerase chain reaction. Virus symptoms were enhanced and plants showed no recovery over time in co-infections of MDMV-OH and MCDV-S but virus titers and quantitative growth parameters did not indicate synergy in co-infected plants. MCDV-M1 co-infections with either MDMV-OH or MCDV-S did not show symptom enhancement or evidence of synergism.

Maize rayado fino virus (MRFV) causes one of the most important virus diseases of maize in America. Severe yield losses, ranging from 10 to 50% in landraces to nearly 100% in contemporary cultivars, have been reported. Resistance has been reported in maize populations, but few resistant inbred lines have been identified. Maize inbred lines representing the range of diversity in the cultivated types and selected lines known to be resistant to other viruses were evaluated to identify novel sources of resistance to MRFV. The virus was transmitted to maize seedlings using the vector Dalbulus maidis, and disease incidence and severity were evaluated beginning 7 days postinoculation. Most of the 36 lines tested were susceptible to MRFV, with mean disease incidence ranging from 21 to 96%, and severity from 1.0 to 4.3 (using a 0 to 5 severity scale). A few genotypes, including CML333 and Ki11, showed intermediate levels of resistance, with 14 and 10% incidence, respectively. Novel sources of resistance, with incidence of less than 5% and severity ratings of 0.4 or less, included the inbred lines Oh1VI, CML287, and Cuba. In Oh1VI, resistance appeared to be dominant, and segregation of resistance in F 2 plants was consistent with one or two resistance genes. The discovery of novel sources of resistance in maize inbred lines will facilitate the identification of virus resistance genes and their incorporation into breeding programs.

Background: To determine the degree of confidence of students regarding obstetric examination before and after training with simulators, and assess their satisfaction with the use of simulation as a tool for acquiring skills., Methods: Training groups involved 10 students per lecturer. Students learned how to perform Leopold's manoeuvres and measure uterine height with a simulator. The instructor supervised each physical exam. Surveys by those students who had previously done an internship at the Department of Obstetric & Gynecology were not taken into account., Results: Students' confidence in performing the procedures improved significantly (p≤0.001) after the intervention.

Rotavirus infection induces an increase in [Ca(2+)](cyto), which in turn may affect the distribution of the cytoskeleton proteins in the infected cell. Changes in microfilaments, including the formation of stress fibers, were observed starting at 0.5 h.p.i. using fluorescent phalloidin. Western blot analysis indicated that RhoA is activated between 0.5 and 1 h.p.i. Neither the phosphorylation of RhoA nor the formation of stress fibers were observed in cells infected with virions pre-treated with an anti-VP5* non-neutralizing mAb, suggesting that RhoA activation is stimulated by the interaction of the virus with integrins forming the cell receptor complex. In addition, the structure of the tubulin cytoskeleton was also studied. Alterations of the microtubules were evident starting at 3 h.p.i. and by 7 h.p.i. when microtubules were markedly displaced toward the periphery of the cell cytoplasm. Loading of rotavirus-infected cells with either a Ca(2+) chelator (BAPTA) or transfection with siRNAs to silence NSP4, reversed the changes observed in both the microfilaments and microtubules distribution, but not the appearance of stress fibers. These results indicate that alterations in the distribution of actin microfilaments are initiated early during infection by the activation of RhoA, and that latter changes in the Ca(2+) homeostasis promoted by NSP4 during infection may be responsible for other alterations in the actin and tubulin cytoskeleton.

Background: Rotaviruses are known to modulate the innate antiviral defense response driven by IFN. The purpose of this study was to identify changes in the cellular proteome in response to rotavirus infection in the context of the IFN response. We also sought to identify proteins outside the IFN induction and signaling pathway that were modulated by rotavirus infection., Methods: 2D-DIGE and image analysis were used to identify cellular proteins that changed in levels of expression in response to rotavirus infection, IFN treatment, or IFN treatment prior to infection. Immunofluorescence microscopy was used to determine the subcellular localization of proteins associated with the unfolded protein response (UPR)., Results: The data show changes in the levels of multiple proteins associated with cellular stress in infected cells, including levels of ER chaperones GRP78 and GRP94. Further investigations showed that GRP78, GRP94 and other proteins with roles in the ER-initiated UPR including PERK, CHOP and GADD34, were localized to viroplasms in infected cells., Conclusions: Together the results suggest rotavirus infection activates the UPR, but modulates its effects by sequestering sensor, transcription factor, and effector proteins in viroplasms. The data consequently also suggest that viroplasms may directly or indirectly play a fundamental role in regulating signaling pathways associated with cellular defense responses.

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.

Rotavirus infection modifies Ca(2+) homeostasis, provoking an increase in Ca(2+) permeation, the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyto)), and total Ca(2+) pools and a decrease in Ca(2+) response to agonists. A glycosylated viral protein(s), NSP4 and/or VP7, may be responsible for these effects. HT29 or Cos-7 cells were infected by the SA11 clone 28 strain, in which VP7 is not glycosylated, or transiently transfected with plasmids coding for NSP4-enhanced green fluorescent protein (EGFP) or NSP4. The permeability of the plasma membrane to Ca(2+) and the amount of Ca(2+) sequestered in the endoplasmic reticulum released by carbachol or ATP were measured in fura-2-loaded cells at the single-cell level under a fluorescence microscope or in cell suspensions in a fluorimeter. Total cell Ca(2+) pools were evaluated as (45)Ca(2+) uptake. Infection with SA11 clone 28 induced an increase in Ca(2+) permeability and (45)Ca(2+) uptake similar to that found with the normally glycosylated SA11 strain. These effects were inhibited by tunicamycin, indicating that inhibition of glycosylation of a viral protein other than VP7 affects the changes of Ca(2+) homeostasis induced by infection. Expression of NSP4-EGFP or NSP4 in transfected cells induced the same changes observed with rotavirus infection, whereas the expression of EGFP or EGFP-VP4 showed the behavior of uninfected and untransfected cells. Increased (45)Ca(2+) uptake was also observed in cells expressing NSP4-EGFP or NSP4, as evidenced in rotavirus infection. These results indicate that glycosylated NSP4 is primarily responsible for altering the Ca(2+) homeostasis of infected cells through an initial increase of cell membrane permeability to Ca(2+).

Rotavirus infection of cells in culture induces major changes in Ca(2+) 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.

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.

A highly regioselective palladium-catalyzed alpha-alkylation of allylic bromides 1a,c-e and chloride 1b with substituted and unsubstituted benzylic Grignard reagents is reported. The resulting all-trans polyenehomobenzene derivatives were obtained in excellent yields and regioselectivity. These products were easily converted to abietane-type diterpenes (10-12) and tetracyclic polyprenoid compounds (13, 14) through a Lewis acid-promoted cascade polyene cyclization reaction.