CITOTOXICIDAD Y ACTIVIDAD ANTIVIRAL DE EXTRACTOS DE CHILES (Capsicum spp), CYTOTOXICITY AND ANTIVIRAL ACTIVITY OF PEPPER EXTRACTS (Capsicum spp)

RESUMENEn este trabajo se evaluaron  la citotoxicidad y la actividad antiviral en contra del virus herpes simplex tipo 1 (VHS-1)  de extractos  de chile Jalapeño, Serrano, Guajillo, Ancho, Pimiento (Capsicum annuum L. var. annuum.) y Habanero (Capsicum chinense Jacq), así como de los fenilpropanoides, ácidos fenólicos y flavonoides puros que fueron identificados por HPLC-MS en los extractos  de chile. La concentración que causa el 50 % de toxicidad en células Vero fue determinada (CC50) así como el potencial antiviral de los extractos de chiles y de los compuestos puros,  expresado como la concentración inhibitoria del 50 %  (CI50) en contra de los efectos citopáticos en células Vero infectadas con el virus. El índice de selectividad fue calculado como la relación de CC50 entreCI50.  El extracto de Pimiento mostró las más baja citotoxicidad (CC50= 9.82 ± 0.06 mg/mL) y la más alta  actividad antiviral (IC50= 0.56 ± 0.02 mg/mL), con el mayor índice de selectividad de 17.5. Los extractos de chile Ancho y Guajillo mostraron también una alta capacidad antiviral. El extracto de chile Habanero presentó la mayor citotoxicidad y el menor índice de selectividad. Los compuestos fenólicos contenidos en los extractos también mostraron actividad antiviral, lo que sugiere que pueden ser estos compuestos los responsables de la actividad antiviral de los chiles.Palabras clave: Capsicum spp, citotoxicidad, actividad antiviral, compuestos fenólicos.

Evidence that the 45 kDa glycoprotein, part of a putative dengue virus receptor complex in the mosquito cell line C6/36, is a heat shock-related protein

Artículo científic

Characterization of Viral Interference in <i>Aedes albopictus</i> C6/36 Cells Persistently Infected with Dengue Virus 2

Arboviruses are an important group of pathogens that cause diseases of medical and veterinary concern worldwide. The interactions of these viruses with their host cells are complex, and frequently, the coexistence of two different viruses in the same cell results in the inhibition of replication in one of the viruses, which is a phenomenon called viral interference. This phenomenon can be exploited to develop antiviral strategies. Insect cell lines persistently infected with arboviruses are useful models with which to study viral interference. In this work, a model of C6/36-HT cells (from Aedes albopictus mosquitoes) persistently infected with Dengue virus, serotype 2, was used. Viral interference was evaluated via plaque and flow cytometry assays. The presence of heterotypic interference against the other serotypes of the same virus and homologous interference against yellow fever virus was determined; however, this cell line did not display heterologous viral interference against Sindbis virus. The mechanisms responsible for viral interference have not been fully elucidated, but small RNAs could be involved. However, the silencing of Ago3, a key protein in the genome-derived P-element-induced wimpy testis pathway, did not alter the viral interference process, suggesting that viral interference occurs independent of this pathway

Development of a multiplex PCR assay to detect gastroenteric pathogens in the feces of mexican children

Abstract Acute gastroenteritis (AGE) is a major cause of childhood morbidity and mortality worldwide; the etiology ofAGEincludes viruses, bacteria, and parasites.Amultiplex PCR assay to simultaneously identify human Astrovirus (HAstV), Calicivirus (HuCVs), Entamoeba histolytica (E. histolytica), and enteroinvasive Escherichia coli (EIEC) in stool samples is described. A total of 103 samples were individually analyzed by ELISA (enzyme-linked immunosorbent assays) and RT-PCR/PCR. HAstV and HuCVs were detected in four out of 103 samples (3.8 %) by RT-PCR, but ELISAs found only one sample as positive for HuCVs (2.5 %). E. histolytica was identified in two out of 19 samples (10.5 %) and EIEC in 13 out of 20 samples (70 %) by PCR, and all PCR products were sequenced to verify their identities. Our multiplex PCR results demonstrate the simultaneous amplification of different pathogens such as HAstV, EIEC, and E. histolytica in the same reaction, though the HuCVs signal was weak in every replicate. Regardless, this multiplex PCR protocol represents a novel tool for the identification of distinct pathogens and may provide support for the diagnosis of AGE in children

Differential Gene Expression Pattern of Importin &beta;3 and NS5 in C6/36 Cells Acutely and Persistently Infected with Dengue Virus 2

The establishment of persistent dengue virus infection within the cells of the mosquito vector is an essential requirement for viral transmission to a new human host. The mechanisms involved in the establishment and maintenance of persistent infection are not well understood, but it has been suggested that both viral and cellular factors might play an important role. In the present work, we evaluated differential gene expression in Aedes albopictus cells acutely (C6/36-HT) and persistently infected (C6-L) with Dengue virus 2 by cDNA-AFLP. We observed that importin &beta;3 was upregulated in noninfected cells compared with C6-L cells. Using RT-qPCR and plaque assays, we observed that Dengue virus levels in C6-L cells essentially do not vary over time, and peak viral titers in acutely infected cells are observed at 72 and 120 h postinfection. The expression level of importin &beta;3 was higher in acutely infected cells than in persistently infected cells; this correlates with higher levels of NS5 in the nucleus of the cell. The differential pattern of importin &beta;3 expression between acute and persistent infection with Dengue virus 2 could be a mechanism to maintain viral infection over time, reducing the antiviral response of the cell and the viral replicative rate

PTB Binds to the 3’ Untranslated Region of the Human Astrovirus Type 8: A Possible Role in Viral Replication

<div><p>The 3′ untranslated region (3′UTR) of human astroviruses (HAstV) consists of two hairpin structures (helix I and II) joined by a linker harboring a conserved PTB/hnRNP1 binding site. The identification and characterization of cellular proteins that interact with the 3′UTR of HAstV-8 virus will help to uncover cellular requirements for viral functions. To this end, mobility shift assays and UV cross-linking were performed with uninfected and HAstV-8-infected cell extracts and HAstV-8 3′UTR probes. Two RNA-protein complexes (CI and CII) were recruited into the 3′UTR. Complex CII formation was compromised with cold homologous RNA, and seven proteins of 35, 40, 45, 50, 52, 57/60 and 75 kDa were cross-linked to the 3′UTR. Supermobility shift assays indicated that PTB/hnRNP1 is part of this complex, and 3′UTR-crosslinked PTB/hnRNP1 was immunoprecipitated from HAstV-8 infected cell-membrane extracts. Also, immunofluorescence analyses revealed that PTB/hnRNP1 is distributed in the nucleus and cytoplasm of uninfected cells, but it is mainly localized perinuclearly in the cytoplasm of HAstV-8 infected cells. Furthermore, the minimal 3′UTR sequences recognized by recombinant PTB are those conforming helix I, and an intact PTB/hnRNP1-binding site. Finally, small interfering RNA-mediated PTB/hnRNP1 silencing reduced synthesis viral genome and virus yield in CaCo2 cells, suggesting that PTB/hnRNP1 is required for HAstV replication. In conclusion, PTB/hnRNP1 binds to the 3′UTR HAstV-8 and is required or participates in viral replication.</p></div

PTB bind to the HAstV8 virus 3′UTR.

<p><b>A)</b> Supermobility shift assay from uninfected or HAstV-8 infected CaCo2 S10 extracts were incubated without (lane 2) or with anti-hnRNP1 (lane 3) or with a non-related antibody (lane 4) after of the 3′UTR labeled RNA (lane 1). Supershifted complexes are indicated as CIII. <b>B)</b> Uninfected (U) and infected CaCo2 cells cytoplasmic and nuclear cell extracts (8 h p.i.) prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113113#pone-0113113-g002" target="_blank">Figure 2D</a> were probed with monoclonal anti-PTB and polyclonal anti-hnRNP1 antibodies by western blotting. <b>C)</b> Colocalization of hnRNP1 (green) in Mock infected (upper panel) or infected CaCo2 cells (lower panel). Cells were fixed at 12 h p.i. and were subjected to indirect immunofluorescence. The HAstV-8 infected positive cells were labeled with anti-HAstV CQFG sera (red). Nuclei are shown by DAPI (blue) staining. The images of cell were acquired with a confocal lasser scanning microscope LSM 710 (Carl Zeiss). <b>D)</b> 3′UTR RNA was cross-linked with 60 µg of HAstV-8-infected CaCo2 cells extracts (lanes 2 to 6 and 8) or mutant mPTB RNA labeled (lane 7) and immunoprecipitated with anti-hnRNP1 antibody (lanes 7 and 8) or with a non-related antibody (lane 5). The lanes 4 and 6 are internal controls. Probes are indicated with asterisks and the arrow indicates the position of the immunoprecipitated labeled PTB/hnRNP1.</p