Use of a commercial enzyme immunoassay to monitor dengue virus replication in cultured cells

Current methods for dengue virus quantitation are either time consuming, technically demanding or costly. As an alternative, the commercial enzyme immunoassay Platelia™ Dengue NS1 AG (Bio-Rad Laboratories) was used to monitor semiquantitatively dengue virus replication in cultured cells. The presence of NS1 protein was evaluated in supernatants from Vero and C6/36 HT cells infected with dengue virus. The amount of NS1 detected in the supernatants of infected cells was proportional to the initial MOI used and to the time of post infection harvest. This immunoassay was also able to detect the presence of NS1 in the supernatants of infected human macrophages. Inhibition of dengue virus replication in C6/36 HT cells treated with lysosomotropic drugs was readily monitored with the use of this assay. These results suggest that the Platelia™ Dengue NS1 AG kit can be used as a fast and reliable surrogate method for the relative quantitation of dengue virus replication in cultured cells

Picobirnavirus causes persistent infection in pigs

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%; p<0.05), followed by pregnant sows (15.09%), piglets aged 2-5 months of age (18.42%) and adult (≥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.Fil: Martinez, Laura Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología Dr. J. M. Vanella; ArgentinaFil: Masachessi, Gisela. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología Dr. J. M. Vanella; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Carruyo, Gabriela. Universidad del Zulia; VenezuelaFil: Ferreyra, Leonardo Jesús. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología Dr. J. M. Vanella; ArgentinaFil: Barril, Patricia Angelica. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología Dr. J. M. Vanella; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Isa, Maria Beatriz. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología Dr. J. M. Vanella; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Giordano, Miguel Oscar. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología Dr. J. M. Vanella; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Ludert, Juan E.. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzados. Departamento de Física; MéxicoFil: Nates, Silvia Viviana. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología Dr. J. M. Vanella; Argentin

Dengue virus NS1 protein interacts with the ribosomal protein RPL18: This interaction is required for viral translation and replication in Huh-7 cells

AbstractGiven 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 48hpi. 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

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

Identification and Type Distribution of Astroviruses among Children with Gastroenteritis in Colombia and Venezuela

Astrovirus infections were detected by enzyme immunoassay in 12 (5%) of 251 stool samples from children with gastroenteritis from Bogota, Colombia. In addition, astroviruses were detected by reverse transcription-PCR in 3 (10%) of 29 stool samples negative for other enteric pathogens collected in Caracas, Venezuela, from children with gastroenteritis. Astrovirus type 1 was the most frequently detected virus

Rotavirus Infection of Cells in Culture Induces Activation of RhoA and Changes in the Actin and Tubulin Cytoskeleton

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

Dissecting Rotavirus Particle-Raft Interaction with Small Interfering RNAs: Insights into Rotavirus Transit through the Secretory Pathway

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