Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

Modulatory effects of ammonia-N on the immune system of Penaeus japonicus to virulence of white spot syndrome virus

To study response to white spot syndrome virus (WSSV) under ammonia stress, Penaeus japonicus were exposed to 5 mg l(-1) ammonia-N and challenged orally with WSSV (NW). Controls consisted of an ammonia-N-exposed control group (N), a WSSV-challenged positive control group (W), and an untreated control group (control). Immune parameters measured were total haemocyte count (THC), haemocyte phagocytosis, plasma protein content and haemolymph enzymatic activities for prophenoloxidase (proPO), alkaline phosphatase (ALP), and nitric oxide synthase (NOS). THC and plasma protein had downward trends with time in all treatment groups (NW, N, and W) in contrast to the untreated control group (control). The percentage phagocytosis, NOS activity, and ALP and proPO activity of W and NW decreased initially then increased from 6 to 78 h (except for NOS and ALP, from 6 to 54 h) before declining thereafter until the end of the experiment. Compared with untreated controls (control), there was a downward trend for all measured parameters in the treatment groups (N, NW, and W), but the degree was W > NW > N. WSSV was detected at 78 h postchallenge in both W and NW. In conclusion, 5 mg l(-1) ammonia-N reduced the immunocompetence of P japonicus and may have decreased the virulence of WSSV (C) 2004 Elsevier B.V. All rights reserved.

Studies on the transmission of WSSV (white spot syndrome virus) in juvenile Marsupenaeus japonicus via marine microalgae

We studied the possible role that marine microalgae may play during the outbreaks of WSS (white spot syndrome). In order to elucidate the possibility of marine microalgae carrying WSSV (white spot syndrome virus), six marine microallgae (Isochr.vsis galbana, Skeletonema costatum, Chlorella sp., Heterosigma akashiwo, Scrippsiella trochoidea, Dunaliella salina) were co-cultured with adult Marsupenaeus japollicus infected with WSSV and were assayed daily by nested-PCR to study whether they could carry WSSV. Further experiments were conducted to investigate whether the virus carried by microalgae could re-infect juvenile M. japonicus. Results showed that all of the experimental microalgae, except H. akashiwo could carry WSSV, and among them, Chlorella sp. and S. trochoidea had the strongest WSSV-carrying ability. Unlike other invertebrate carriers of WSSV, the WSSV detections in microalgae, which were positive after I and 3 days, were negative after 10 days of incubation. WSSV detection results in juvenile M. japonicus showed that the juvenile shrimp were re-infected by co-cultured Chlorella sp., although the juvenile M. japonicus carried so small an amount of WSSV that it could only be detected by nested-PCR. The results of this experiment suggest that microalgae might be one possible horizontal transmission pathway for WSSV. Further research, however, is required to better understand the factors behind the different carrying abilities and virus-carrying mechanisms of different microalgae. (c) 2007 Elsevier Inc. All rights reserved.

Studies on nitric oxide synthase activity in haemocytes of shrimps Fenneropenaeus chinensis and Marsupenaeus japonicus after white spot syndrome virus infection

The nitric oxide synthase (NOS) activity in the haemocytes of shrimps Fenneropenaeus chinensis (Osbeck) and Marsupenaeus japonicus (Bate) was Studied after white spot syndrome virus (WSSV) infection to determine its characteristics in response to virus infection. First, the NOS activity in haemocytes of shrimps was determined by the means of NBT reduction and changes in cell conformation. And the variations of NOS activity in shrimps after challenge with WSSV intramuscularly were evaluated through the analysis Of L-citrulline and total nitrite/nitrate (both as NO derivates) concentrations. The result showed that NOS activity in the haemocytes of F chinensis increased slightly from 0 to 12 h postchallenge, indicated by the variations Of L-Citrulline (from 11.15 +/- 0.10 to 12.08 +/- 0.64 mu M) and total nitrite/nitrate concentrations (from 10.45 +/- 0.65 to 12.67 +/- 0.52 mu M). Then it decreased sharply till the end of the experiment (84 h postchallenge), the concentrations Of L-Citrulline and total nitrite/nitrate at 84 It were 1.58 +/- 0.24 and 2.69 +/- 0.70 mu M, respectively. The LPS-stimulated NOS activity kept constant during the experiment. However, in M. japonicus, the NOS activity kept increasing during the first 72 It postchallenge, the concentrations Of L-Citrulline and total nitrite/nitrate increased from 7.82 +/- 0.77 at 0 h to 10.79 +/- 0.50 mu M at 72 h, and from 8.98 +/- 0.43 at 0 h to 11.20 +/- 0.37 mu M at 72 h, respectively. Then it decreased till the end of the experiment (216 h postchallenge), and the concentrations of L-Citrulline and total nitrite/nitrate at 216 h were 5.66 +/- 0.27 and 4.68 +/- 0.16 mu M, respectively. More importantly, an apparent increase of I-PS-stimulated NOS activity was observed in M japonicus at 48 h postchallenge, which was about 4 times higher than that in the control group of health shrimps. In correspondence with the difference of NOS activity between the two species of shrimps, the Cumulative mortalities of the shrimps were also different. All shrimps of F. chinensis in the mortality experiment died in 66 h, much more quickly than M. japonicus, Whose accumulative mortality reached 100% after 240 h. Data here reported let us hypothesize that NOS activity in the haemocytes of shrimps F chinensis and M. japonicus responses to WSSV infection differently, and this might be one of the reasons for the different susceptibility of F chinensis and M. japonicus to WSSV infection. (c) 2005 Elsevier Inc. All rights reserved.

H5N1 avian influenza re-emergence of Lake Communication Qinghai: phylogenetic and antigenic analyses of the newly isolated viruses and roles of migratory birds in virus circulation

Highly pathogenic avian influenza H5N1 virus has swept west across the globe and caused serious debates on the roles of migratory birds in virus circulation since the first large-scale outbreak in migratory birds of Lake Qinghai, 2005. In May 2006, another outbreak struck Lake Qinghai and six novel strains were isolated. To elucidate these QH06 viruses, the six isolates were subjected to whole-genome sequencing. Phylogenetic analyses show that QH06 viruses are derived from the lineages of Lake Qinghai, 2005. Five of the six novel isolates are adjacent to the strain A/Cygnus olor/Croatia/1/05, and the last one is related to the strain A/duck/Novosibirsk/ 02/05, an isolate of the flyway. Antigenic analyses suggest that QH06 and QH05 viruses are similar to each other. These findings implicate that QH06 viruses of Lake Qinghai may travel back via migratory birds, though not ruling out the possibility of local circulation of viruses of Lake Qinghai.

Detecção do melon yellowing associated virus (MYaV) em áreas produtoras de melão na Região Nordeste.

2009

Ocorrência do Barley/Cereal yellow dwarf virus e seus vetores em cereais de inverno no Rio Grande do Sul, Santa Catarina, Paraná e Mato Grosso do Sul em 2008.

2009

Ocorrência do Barley/Cereal yellow dwarf virus e seus vetores em cereais de inverno no Rio Grande do Sul em 2007.

2008

Identificação do vírus do mosqueado do feijoeiro ("bean pod mottle virus") em soja no Brasil (2000).

O virus do mosqueado do feijoeiro ("bean pod mottle virus" BPMV) foi identificado em plantas da soja, cultivar Itiquira, com sintomas de mosqueado, em Planaltina, Distrito Federal. Uma preparacao purificada do virus examinada em microcospio eletronico revelou a presenca de particulas isometricas em torno de 30 nm de diametro. Em testes de SDS-PAGE, foram detectadas tres proteinas com massas moleculares estimadas de 39,7, 22,9 e 21,2 kDa. A eficiencia media de transmissao do isolado do BPMV em estudo pelo crisomelideo Cerotoma arcuata Oliv. Em tres experimentos foi de 66,7%. Em experimentos de campo, o BPMV reduziu a producao de graos nas cultivares de soja Garca Branca, Garimpo, Doko, Itiquira e Pioneira em 17,1% , 17,1%, 20,4%, 20,9% e 21,4%, respectivamente.

Incidencia e distribuicao do virus do endurecimento dos frutos do maracujazeiro no cerrado do Brasil Central.

2001

Altered of apoptotic markers of both extrinsic and intrinsic pathways induced by hepatitis C virus infection in peripheral blood mononuclear cells

Background: Chronic hepatitis C (CHC) has emerged as a leading cause of cirrhosis in the U. S. and across the world. To understand the role of apoptotic pathways in hepatitis C virus (HCV) infection, we studied the mRNA and protein expression patterns of apoptosis-related genes in peripheral blood mononuclear cells (PBMC) obtained from patients with HCV infection.Methods: the present study included 50 subjects which plasma samples were positive for HCV, but negative for human immunodeficiency virus (HIV) or hepatitis B virus (HBV). These cases were divided into four groups according to METAVIR, a score-based analysis which helps to interpret a liver biopsy according to the degree of inflammation and fibrosis. mRNA expression of the studied genes were analyzed by reverse transcription of quantitative polymerase chain reaction (RT-qPCR) and protein levels, analyzed by ELISA, was also conducted. HCV genotyping was also determined.Results: HCV infection increased mRNA expression and protein synthesis of caspase 8 in group 1 by 3 fold and 4 fold, respectively (p < 0.05). in group 4 HCV infection increased mRNA expression and protein synthesis of caspase 9 by 2 fold and 1,5 fold, respectively (p < 0.05). Also, caspase 3 mRNA expression and protein synthesis had level augumented by HCV infection in group 1 by 4 fold and 5 fold, respectively, and in group 4 by 6 fold and 7 fold, respectively (p < 0.05).Conclusions: HCV induces alteration at both genomic and protein levels of apoptosis markers involved with extrinsic and intrinsic pathways.

Exploitation of Ebola Virus VP35 Protein to identify new drugs counteracting its type I IFN antagonism

Ebolaviruses (EBOVs) are among the most virulent and deadly pathogens ever known, causing fulminant haemorrhagic fevers in humans and non-human primates. The 2014 outbreak of Ebola virus disease (EVD) in West Africa has claimed more lives than all previous EVD outbreaks combined. The EBOV high mortality rates have been related to the virus-induced impairment of the host innate immunity reaction due to two virus-coded proteins, VP24 and VP35. EBOV VP35 is a multifunctional protein, it is essential for viral replication as a component of the viral RNA polymerase and it also participates in nucleocapsid assembly. Early during EBOV infection, alpha-beta interferon (IFN-α/β) production would be triggered upon recognition of viral dsRNA products by cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). However, this recognition is efficiently prevented by the double-stranded RNA (dsRNA) binding activity of the EBOV VP35 protein, which hides RLRs binding sites on the dsRNA phosphate backbone as well the 5’-triphosphate (5’-ppp) dsRNA ends to RIG-I recognition. In addition to dsRNA binding and sequestration, EBOV VP35 inhibits IFN-α/β production preventing the activation of the IFN regulatory factor 3 (IRF-3) by direct interaction with cellular proteins. Previous studies demonstrated that single amino acid changes in the VP35 dsRNA binding domain reduce EBOV virulence, indicating that VP35 is an attractive target for antiviral drugs development. Within this context, here we report the establishment of a novel method to characterize the EBOV VP35 inhibitory function of the dsRNA-dependent RIG-I-mediated IFN-β signaling pathway in a BLS2 cell culture setting. In such system, a plasmid containing the promoter region of IFN-β gene linked with a luciferase reporter gene was transfected, together with a EBOV VP35 mammalian expression plasmid, into the IFN-sensitive A549 cell line, and the IFN-induction was stimulated through dsRNA transfection. Through alanine scanning mutational studies with biochemical, cellular and computational methods we highlighted the importance of some VP35 residues involved in dsRNA end-capping binding, such as R312, K282 and R322, that may serve as target for the development of small-molecule inhibitors against EBOV. Furthermore, we identified a synthetic compound that increased IFN-induction only under antiviral response stimulation and subverted VP35 inhibition, proving to be very attractive for the development of an antiviral drug. In conclusion, our results provide the establishment of a new assay as a straightforward tool for the screening of antiviral compounds that target i) dsRNA-VP35 or cellular protein-VP35 interaction and ii) dsRNA-dependent RIG-I-mediated IFN signaling pathway, in order to potentiate the IFN response against VP35 inhibition, setting the bases for further drug development.

Cobertura de la vacuna del virus del papiloma humano en la zona básica de Barañáin

El cáncer de cuello de útero o cáncer de cérvix es el segundo tumor más frecuente en las mujeres del mundo después del de mama y el quinto de todos los canceres. En España, es el sexto más frecuente, constituyendo el 4,8% de los canceres en la mujer. Este tipo de cáncer es el resultado final de una infección no resuelta por el virus del papiloma humano (VPH). Hoy en día, el VPH representa una de las infecciones de trasmisión sexual más común. El 70-80 % de hombres y mujeres sexualmente activos están o han estado expuestos al VPH. El cambio en la conducta sexual de las mujeres ha llevado a que las infecciones de trasmisión sexual sean ahora más frecuentes que en las generaciones anteriores. Hay un inicio más precoz de las primeras relaciones sexuales y un número más elevado de compañeros sexuales. Estos cambios han hecho aumentar la prevalencia del VPH en los últimos años en las mujeres jóvenes de España. La combinación de estrategias de prevención primaria (vacunación contra el VHP) y secundaria (cribado) permitiría reducir la incidencia y la mortalidad del cáncer de cuello de útero. Existen dos vacunas frente al VPH que son Gardasil ® y Cervarix ®. Al tratarse de dos vacunas nuevas, el éxito de la introducción dependerá de la comprensión apropiada de los riesgos y beneficios de la vacuna para prevenir la infección del VPH. Esto se consigue mediante programas efectivos de educación y formación sobre el VPH.

The Terrorism Virus: Contemporary Traits and Mechanisms

In order to present and understand the nature of modern terrorism it is important to realize its key properties as well the mechanisms that shape terrorism. Selected properties and mechanisms shaping modern terrorism which can be exemplified by the following: evolutionary nature of terrorism, asymmetry of terrorism, interferentiality of terrorism, multitude of components of terrorism, diffusion of terrorism, duality of terrorism, positive dimension of terrorism, terrorist as the system, diversity of terrorist activity goals, changeability of terrorist threat, the broad and narrow dimension of terrorism, counter-anti-terrorism, the confrontational and cooperational character of relations, calculation and operational strategy, disintegrational nature of terrorism, multidisciplinarity of terrorism, horizontal and vertical dimension of terrorism and a the few other traits or mechanisms.

Human papillomavirus detection in women with and without human immunodeficiency virus infection in Colombia

Background: HIV infection leads to a decreasing immune response, thereby facilitating the appearance of other infections, one of the most important ones being HPV. However, studies are needed for determining associations between immunodeficiency caused by HIV and/or the presence of HPV during the course of cervical lesions and their degree of malignancy. This study describes the cytological findings revealed by the Papanicolaou test, laboratory characteristics and HPV molecular profile in women with and without HIV infection. Methods: A total of 216 HIV-positive and 1,159 HIV-negative women were invited to participate in the study; PCR was used for the molecular detection of HPV in cervical samples. Statistical analysis (such as percentages, Chi-square test and Fisher's exact test when applicable) determined human papillomavirus (HPV) infection frequency (single and multiple) and the distribution of six types of high-risk-HPV in women with and without HIV infection. Likewise, a logistic regression model was run to evaluate the relationship between HIV-HPV infection and different risk factors. Results: An association was found between the frequency of HPV infection and infection involving 2 or more HPV types (also known as multiple HPV infection) in HIV-positive women (69.0% and 54.2%, respectively); such frequency was greater than that found in HIV-negative women (44.3% and 22.7%, respectively). Statistically significant differences were observed between both groups (p = 0.001) regarding HPV presence (both in infection and multiple HPV infection). HPV-16 was the most prevalent type in the population being studied (p = 0.001); other viral types had variable distribution in both groups (HIV-positive and HIV-negative). HPV detection was associated with <500 cell/mm(3) CD4-count (p = 0.004) and higher HIV-viral-load (p = 0.001). HPV-DNA detection, <200 cell/mm(3) CD4-count (p = 0.001), and higher HIV-viral-load (p = 0.001) were associated with abnormal cytological findings. Conclusions: The HIV-1 positive population in this study had high multiple HPV infection prevalence. The results for this population group also suggested a greater association between HPV-DNA presence and cytological findings. HPV detection, together with low CD4 count, could represent useful tools for identifying HIV-positive women at risk of developing cervical lesions.