Comparative Flavivirus-Host Protein Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis.

Mosquito-borne flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), are a growing public health concern. Systems-level analysis of how flaviviruses hijack cellular processes through virus-host protein-protein interactions (PPIs) provides information about their replication and pathogenic mechanisms. We used affinity purification-mass spectrometry (AP-MS) to compare flavivirus-host interactions for two viruses (DENV and ZIKV) in two hosts (human and mosquito). Conserved virus-host PPIs revealed that the flavivirus NS5 protein suppresses interferon stimulated genes by inhibiting recruitment of the transcription complex PAF1C and that chemical modulation of SEC61 inhibits DENV and ZIKV replication in human and mosquito cells. Finally, we identified a ZIKV-specific interaction between NS4A and ANKLE2, a gene linked to hereditary microcephaly, and showed that ZIKV NS4A causes microcephaly in Drosophila in an ANKLE2-dependent manner. Thus, comparative flavivirus-host PPI mapping provides biological insights and, when coupled with in vivo models, can be used to unravel pathogenic mechanisms.

Targeting Viral Proteostasis Limits Influenza Virus, HIV, and Dengue Virus Infection.

Viruses are obligate parasites and thus require the machinery of the host cell to replicate. Inhibition of host factors co-opted during active infection is a strategy hosts use to suppress viral replication and a potential pan-antiviral therapy. To define the cellular proteins and processes required for a virus during infection is thus crucial to understanding the mechanisms of virally induced disease. In this report, we generated fully infectious tagged influenza viruses and used infection-based proteomics to identify pivotal arms of cellular signaling required for influenza virus growth and infectivity. Using mathematical modeling and genetic and pharmacologic approaches, we revealed that modulation of Sec61-mediated cotranslational translocation selectively impaired glycoprotein proteostasis of influenza as well as HIV and dengue viruses and led to inhibition of viral growth and infectivity. Thus, by studying virus-human protein-protein interactions in the context of active replication, we have identified targetable host factors for broad-spectrum antiviral therapies.

Dengue virus genomic variation associated with mosquito adaptation defines the pattern of viral non-coding RNAs and fitness in human cells.

The Flavivirus genus includes a large number of medically relevant pathogens that cycle between humans and arthropods. This host alternation imposes a selective pressure on the viral population. Here, we found that dengue virus, the most important viral human pathogen transmitted by insects, evolved a mechanism to differentially regulate the production of viral non-coding RNAs in mosquitos and humans, with a significant impact on viral fitness in each host. Flavivirus infections accumulate non-coding RNAs derived from the viral 3'UTRs (known as sfRNAs), relevant in viral pathogenesis and immune evasion. We found that dengue virus host adaptation leads to the accumulation of different species of sfRNAs in vertebrate and invertebrate cells. This process does not depend on differences in the host machinery; but it was found to be dependent on the selection of specific mutations in the viral 3'UTR. Dissecting the viral population and studying phenotypes of cloned variants, the molecular determinants for the switch in the sfRNA pattern during host change were mapped to a single RNA structure. Point mutations selected in mosquito cells were sufficient to change the pattern of sfRNAs, induce higher type I interferon responses and reduce viral fitness in human cells, explaining the rapid clearance of certain viral variants after host change. In addition, using epidemic and pre-epidemic Zika viruses, similar patterns of sfRNAs were observed in mosquito and human infected cells, but they were different from those observed during dengue virus infections, indicating that distinct selective pressures act on the 3'UTR of these closely related viruses. In summary, we present a novel mechanism by which dengue virus evolved an RNA structure that is under strong selective pressure in the two hosts, as regulator of non-coding RNA accumulation and viral fitness. This work provides new ideas about the impact of host adaptation on the variability and evolution of flavivirus 3'UTRs with possible implications in virulence and viral transmission.

Collateral Damage during Dengue Virus Infection: Making Sense of DNA by cGAS.

Early sensing of viral components or infection-induced tissue damage is a prerequisite for the successful control of pathogenic viruses by the host innate immune system. Recent results from our laboratory show how immune cells use the DNA-sensing machinery to detect intracellular damage generated early during infection by an RNA virus, namely, dengue virus (DENV). Conversely, we found that DENV can efficiently dismantle this sensing mechanism by targeting the cyclic GMP-AMP synthase (cGAS) and the stimulator of interferon (IFN) genes (STING), two crucial host factors involved in DNA detection and type I IFN production. These findings highlight the relevance of the DNA-sensing mechanism in the detection and control of infections by RNA viruses. In this review, we discuss how DENV modulates the innate immune DNA-sensing pathway, activated in the context of cellular damage during infection.

DENV inhibits type I IFN production in infected cells by cleaving human STING.

Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.

Generation and Characterization of Human-Mouse STING Chimeras That Allow DENV Replication in Mouse Cells.

Our group was the first to describe direct antagonism of the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway by dengue virus (DENV) in human cells, and here, we report new findings on the characterization of the interaction between the DENV nonstructural protein 2B (NS2B)-NS3 (NS2B3) protease complex and STING. We demonstrate interactions between NS2B and the transmembrane domains of human STING and between NS3 and a portion of the cytoplasmic C-terminal domain of human STING. One significant obstacle we face today in the DENV field is the lack of small animal models available that can effectively recapitulate DENV pathogenesis in the early events of infection. The existing mouse models are either immunocompromised mice lacking interferon (IFN) receptors or "humanized" mice reconstituted with human stem cells. However, both approaches fail to capture important aspects of human pathogenesis because they lack critical innate immunity components or have deficiencies in immune cell development or maintenance. As an important step toward developing an immunocompetent mouse model for DENV, we have generated two chimeric human-mouse STING constructs that have promise in retaining both cleavability by NS2B3 and signaling capacity in the mouse. IMPORTANCE This article characterizes the interaction between human STING and DENV viral protease complex NS2B3 by constructing serial deletion mutants of STING. Our findings suggest that DENV nonstructural protein NS2B interacts with the transmembrane domains and NS3 with the C-terminal cyclic dinucleotide binding domain of human STING. Furthermore, as there exists no ideal immunocompetent murine model that can simultaneously support robust DENV replication and recapitulate the clinical manifestation of dengue disease observed in humans, we expressed and characterized two promising human-mouse chimeric STING constructs that can be used for developing a relevant transgenic mouse model to study dengue in the future. Both constructs can activate normal IFN responses in the overexpression system and be cleaved under infection conditions. We believe our findings offer a roadmap to the further development of a murine model that can greatly facilitate antiviral discoveries and vaccine research for DENV.

Molecular Characterization of Embryos with Different Buoyancy Levels in the Yellowtail Kingfish (Seriola lalandi).

The buoyancy of eggs and embryos is associated with successful development in pelagic fish. Buoyancy is the result of oocyte hydration, which depends on the osmotic force exerted by free amino acids (FAA) generated by yolk proteolysis, and cathepsins are the main enzymes involved in this process. Seriola lalandi is a pelagic fish whose farming has been hampered by development failure that have been partially attributed to decreased buoyancy of embryos. Therefore, the aim of this study was to compare the mRNA expression and activity of cathepsins B, D, and L, as well as the FAA content in floating and low-floating embryos at different developmental stages. The chosen stages were eggs, morula, blastula, gastrula and 24 h embryos. Complementary assessments showed that there were no differences attributed to buoyancy status in embryo and oil droplet diameters, as well as the transcriptional status at any developmental stage. Cathepsin B did not show differences in mRNA expression or activity related to buoyancy at any stage. Cathepsin D displayed higher transcript and activity levels only in low-floating eggs compared with those floating. Cathepsin L showed higher expression in floating eggs and 24 h embryos compared with that of low-floating, but the activity of this enzyme was higher in floating eggs and morula. Total FAA content constantly decreased throughout development in floating embryos, but it was always higher than low-floating embryos until gastrula stage. In 24 h embryos floating and low-floating embryos share similar quantities of FAA. In summary, differences in the expression and activity of cathepsins between floating and low-floating embryos could be revealed at specific embryonic stages, suggesting different functions of these enzymes throughout development. Besides 24 h embryos, FAA content seems to be a decisive factor for buoyancy of embryos during early development of S. lalandi. Overall, considering the main role of cathepsins and FAA in buoyancy acquisition process and therefore in both embryo quality and viability, our study identifies good marker candidates to evaluate embryo quality in the farming of this species.

Educative Hybrid Intervention as a Strategy for Reintegration to the Clinical Courses of Undergraduate Students in COVID-19 Pandemic.

The SARS-CoV-2 pandemic generated the need to modify the current clinical educational model with the challenge of promoting safety and the continuity of clinical education through the use of virtual platforms. Since clinical training in hospital institutions cannot be substituted, a strategic training plan was developed to guarantee protection, safety, and academic continuity for students upon returning to clinical clerkships. The objective of this project was to develop and evaluate the impact of a massive hybrid training plan as an educative strategy to give the theoretical and practical knowledge required for the safe return of undergraduate students to their respective clinical activities in the context of this pandemic. An academic program was designed through a massive hybrid strategy to train 616 undergraduate students studying clinical cycles by presential, virtual, synchronous, and asynchronous activities. To know the program's impact, a study based on an initial evaluation and a final evaluation was carried out to evaluate the acquisition of the critical knowledge and skills of the program. A significant difference was found between the means of the initial and final evaluations (p <0.001), as well as a high impact of the intervention (d 1.6). Significant improvements in the areas of COVID-19 initial management (p <0.001) and personal protective equipment use (p <0.001) were seen in the post-test when compared to the initial evaluation. Both a quantitative and a qualitative analysis were carried out, finding positive results on the course design, quality of didactic resources, and instructors' performance. Massive hybrid training is an effective strategy to facilitate the reintegration of undergraduate students into their face-to-face clinical rotations.

Association of serum vitamin D levels with chronic disease and mortality.

INTRODUCTION: Introduction: whether hypovitaminosis D is an overarching cause of increased mortality or a prognostic marker of poor health has not been well elucidated. Objectives: we sought to determine the association of serum 25-hydroxyvitamin D [25-(OH)-D3] levels with the clinical biochemical parameters and mortality risk in chronic diseases. Methods: we reviewed the clinical charts and collected the clinical biochemical parameters of patients diagnosed with chronic conditions who had at least one 25-(OH)-D3 determination, with or without calcium and vitamin D supplementation, and who were selected using a cluster random sampling design (n = 1,705). The analysis was focused on metabolic disorders (type-2 diabetes mellitus [T2DM] and obesity), autoimmune disorders, and mortality. Multivariate logistic regression analyses were performed. Results: low 25-(OH)-D3 levels were reported in 1,433 (84.0%) patients, of which 774 (45.4%) had insufficiency (20-29 ng/mL) and 659 (38.6%) patients had deficiency (< 20 ng/mL). Lower 25-(OH)-D3 levels in T2DM patients were associated with higher glycosylated hemoglobin levels (p < 0.001). Patients with 25-(OH)-D3 levels < 12.5 ng/mL had a higher mortality risk than those with levels ≥ 12.5 ng/mL (HR: 3.339; 95% CI: 1.342-8.308). We observed lower 25-(OH)-D3 levels in patients with grade-III obesity (p = 0.01). We found a higher risk of 25-(OH)-D3 deficiency in rheumatoid arthritis, type-1 diabetes, and systemic lupus erythematosus (p = 0.032, p = 0.002, p = 0.049, respectively). Conclusions: we found a significant relationship between 25-(OH)-D3 levels and glycemic control, body mass index, autoimmune disease, and mortality risk. Nevertheless, whether hypovitaminosis D plays a causal role or is a consequence of chronic disease remains controversial.

INTRODUCCIÓN: Introducción: si la hipovitaminosis D constituye una causa general de mayor mortalidad o un marcador de mal pronóstico para la salud no se ha dilucidado por completo. Objetivos: determinar la asociación de los niveles séricos de 25-hidroxivitamina D [25-(OH)-D3] con los parámetros clínico-bioquímicos y el riesgo de mortalidad en la enfermedad crónica. Métodos: se revisaron los expedientes clínicos y recopilamos los parámetros clínico-bioquímicos de pacientes diagnosticados de enfermedades crónicas que tenían al menos una determinación de 25-(OH)-D3, con o sin suplemento de calcio y vitamina D, y que se seleccionaron mediante muestreo aleatorio por grupos (n = 1705). El análisis se centró en los trastornos metabólicos (diabetes mellitus de tipo 2 [DM2] y obesidad), los trastornos autoinmunes y la mortalidad. Se realizaron análisis multivariados de regresión logística. Resultados: se encontraron niveles bajos de 25-(OH)-D3 en 1433 (84,0%) pacientes, de los cuales 774 (45,4%) tenían insuficiencia (20-29 ng/mL) y 659 (38,6%) tenían deficiencia (< 20 ng/mL) de esta vitamina. Los niveles más bajos de 25-(OH)-D3 en los pacientes con DM2 se asociaron a niveles más altos de hemoglobina glucosilada (p < 0,001). Los pacientes con niveles de 25-(OH)-D3 < 12,5 ng/mL tenían mayor riesgo de mortalidad que aquellos con niveles ≥ 12,5 ng/mL (HR: 3,339; IC del 95%: 1,342-8,308). Apreciamos niveles más bajos de 25-(OH)-D3 en los pacientes con obesidad de grado III (p = 0,01). Se encontró un mayor riesgo de deficiencia de 25-(OH)-D3 en la artritis reumatoide, la diabetes de tipo 1 y el lupus eritematoso sistémico (p = 0,032, p = 0,002, p = 0,049, respectivamente). Conclusiones: apreciamos una relación significativa entre los niveles de 25-(OH)-D3 y el control glucémico, el índice de masa corporal, la enfermedad autoinmune y el riesgo de mortalidad. Sin embargo, sigue siendo controvertido si la hipovitaminosis D desempeña un papel causal o constituye una consecuencia de las enfermedades crónicas.

Evaluation of the mesenteric arterial vasculature by computed tomography angiography and its implications for colorectal cancer surgery / Avaliação da vasculatura arterial mesentérica pela angiotomografia computadorizada e suas implicações na cirurgia do câncer colorretal

Abstract Objective: To determine the branching patterns of the inferior mesenteric artery (IMA) and to describe the clinical applicability of computed tomography (CT) angiography in the evaluation of these vessels to facilitate the planning of colorectal cancer surgery. Materials and Methods: We included 100 patients who underwent CT angiography of the abdomen and pelvis. The branching patterns of the IMA were examined and classified as type 1 (bifurcated), including 1A (sigmoid and left colic arteries arising from a common trunk), 1B (sigmoid and superior rectal arteries arising from a common trunk) and 1C (sigmoid arteries arising from both trunks); type 2 (trifurcated); and type 3 (no left colic branch). Results: Among the 100 patients evaluated, we found the variant to be type 1A in 9%, type 1B in 47%, type 1C in 24%, type 2 in 16%, and type 3 in 4%. Conclusion: Preoperative CT angiography for evaluating the IMA branching pattern could inform decisions regarding the surgical approach to colorectal cancer.

Resumo Objetivo: Determinar os padrões de ramificação da artéria mesentérica inferior (AMI) e descrever a aplicabilidade clínica da angiografia por tomografia computadorizada na avaliação desses vasos na elaboração das estratégias pré-operatórias de cirurgia de câncer colorretal. Materiais e Métodos: Foram incluídos 100 pacientes submetidos a angiografia por tomografia computadorizada abdominal e pélvica. Os padrões de ramificação da AMI foram examinados e classificados como tipo 1 (bifurcado), incluindo 1A (artérias sigmoide e cólica esquerda originando-se de um tronco comum), 1B (artérias sigmoide e retal superior originando-se de um tronco comum) e 1C (artérias sigmoide originando-se de ambos os troncos); tipo 2 (trifurcado); e tipo 3 (sem ramo cólico esquerdo). Resultados: Do total de participantes incluídos no estudo, a variante do tipo 1A foi observada em 9%, a do tipo 1B em 47%, e a do tipo 1C em 24%. Com relação à variante tipo 2, esta foi observada em 16% dos pacientes, e a do tipo 3, em 4% dos casos. Conclusão O uso da angiografia por tomografia computadorizada pré-operatória para avaliar o padrão de ramificação da AMI pode ajudar a escolher a abordagem cirúrgica no câncer colorretal.

Can Responsible Ownership Practices Influence Hunting Behavior of Owned Cats?: Results from a Survey of Cat Owners in Chile.

The domestic cat (Felis catus) has become a worldwide threat to wildlife. The potential impact of owned cats on wildlife in Chile has not been documented at a large scale. The purpose of this study was to investigate the number and type of prey that owned cats bring back in Chile and its relation with responsible ownership practices. An online survey was distributed to 5216 households that included questions about the type of pet, responsible ownership practices, and in the case of cats, the type of prey they brought home. Descriptive statistics as well as univariate and multivariate logistic regression analysis were applied. The results showed that 94.3% of respondents had a pet, and from these, 49.9% had at least one cat. A total of 84.1% of owners reported that their cats had brought back prey. Birds were the most common type of prey, followed by mammals and insects. Not being registered with a microchip, not having a litter box, living in a house with access to a garden, not having a hiding place for the cats, and having free access to the outdoors significantly increased the odds of cats bringing back prey. Body condition score or providing ad libitum food to cats did not have an effect on bringing prey.

Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely used in animals as an efficient genome editing tool. In fish cells, the technique has been difficult to implement due to the lack of proper vectors that use active promoters to drive the expression of both small guide RNA (sgRNA) and the S. pyogenes Cas9 (spCas9) protein within a single expression platform. Until now, fish cells have been modified using co-transfection of the mRNA of both the sgRNA and the spCas9. In the present study, we describe the optimization of a new vector for the expression of a CRISPR/Cas9 system, designed to edit the genome of fish cell lines, that combines a gene reporter (mCherry), sgRNA, and spCas9 in a single vector, facilitating the study of the efficiency of piscine and non-piscine promoters. A cassette containing the zebrafish U6 RNA III polymerase (U6ZF) promoter was used for the expression of the sgRNA. The new plasmid displayed the expression of spCas9, mCherry, and sgRNA in CHSE/F fish cells. The results demonstrate the functionality of the mammalian promoter and the U6ZF promoter in fish cell lines. This is the first approach aimed at developing a unified genome editing system in fish cells using bicistronic vectors, thus creating a powerful biotechnological platform to study gene function.

Transcriptomic response of rainbow trout (Oncorhynchus mykiss) skeletal muscle to Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the etiologic agent of rainbow trout fry syndrome (RTFS). This pathogen infects a wide variety of salmonid species during freshwater stages, causing significant losses in the aquaculture industry. Rainbow trout (Oncorhynchus mykiss) infected with F. psychrophilum, presents as the main external clinical sign ulcerative lesions and necrotic myositis in skeletal muscle. We previously reported the in vitro cytotoxic activity of F. psychrophilum on rainbow trout myoblast, however little is known about the molecular mechanisms underlying the in vivo pathogenesis in skeletal muscle. In this study, we examined the transcriptomic profiles of skeletal muscle tissue of rainbow trout intraperitoneally challenged with low infection dose of F. psychrophilum. Using high-throughput RNA-seq, we found that 233 transcripts were up-regulated, mostly associated to ubiquitin mediated proteolysis and apoptosis. Conversely, 189 transcripts were down-regulated, associated to skeletal muscle contraction. This molecular signature was consistent with creatine kinase activity in plasma and oxidative damage in skeletal muscle. Moreover, the increased caspase activity suggests as a whole skeletal muscle atrophy induced by F. psychrophilum. This study offers an integrative analysis of the skeletal muscle response to F. psychrophilum infection and reveals unknown aspects of its pathogenesis in rainbow trout.

Stocking density induces differential expression of immune-related genes in skeletal muscle and head kidney of fine flounder (Paralichthys adspersus).

Immunity can be modulated by different internal and external factors, being stress one of the most important. However, the stress effects on the immunocompetence of the skeletal muscle has not been studied in detail in earlier vertebrates. Here, we examine the effect of chronic (4 and 7 weeks) crowding stress on the immunocompetence of skeletal muscle and head kidney in the fine flounder (Paralichthys adspersus). Corticosteroid receptor transcript levels and their target genes; pro-inflammatory cytokines, and Toll-, NOD-, and RIG-like receptors were quantified by qPCR. The results indicate that chronic stress down-regulates the expression of these genes in muscle, compromising skeletal muscle immunocompetence, while the expression of these genes is upregulated in head kidney after seven weeks of crowding stress. The data suggests that chronic stress modulates the expression of these immune-related genes in a tissue-specific manner.

Topoisomerase II Inhibitors Induce DNA Damage-Dependent Interferon Responses Circumventing Ebola Virus Immune Evasion.

Ebola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication in vitro and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication.IMPORTANCE Ebola virus and other emerging RNA viruses are significant but unpredictable public health threats. Therapeutic approaches with broad-spectrum activity could provide an attractive response to such infections. We describe a novel assay that can identify small molecules that overcome Ebola virus-encoded innate immune evasion mechanisms. This assay identified as hits cancer chemotherapeutic drugs, including doxorubicin. Follow-up studies provide new insight into how doxorubicin induces interferon (IFN) responses, revealing activation of both the DNA damage response kinase ATM and the DNA sensor cGAS and its partner signaling protein STING. The studies further demonstrate that the ATM and cGAS-STING pathways of IFN induction are a point of vulnerability not only for Ebola virus but for other RNA viruses as well, because viral innate immune antagonists consistently fail to block these signals. These studies thereby define a novel avenue for therapeutic intervention against emerging RNA viruses.

Dengue virus NS2B protein targets cGAS for degradation and prevents mitochondrial DNA sensing during infection.

During the last few decades, the global incidence of dengue virus (DENV) has increased dramatically, and it is now endemic in more than 100 countries. To establish a productive infection in humans, DENV uses different strategies to inhibit or avoid the host innate immune system. Several DENV proteins have been shown to strategically target crucial components of the type I interferon system. Here, we report that the DENV NS2B protease cofactor targets the DNA sensor cyclic GMP-AMP synthase (cGAS) for lysosomal degradation to avoid the detection of mitochondrial DNA during infection. Such degradation subsequently results in the inhibition of type I interferon production in the infected cell. Our data demonstrate a mechanism by which cGAS senses cellular damage upon DENV infection.

Characterization of infectious bursal disease viruses from Argentina.

Infectious bursal disease (IBD) viruses detected in commercial flocks of different regions of Argentina were analyzed by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RFLP) of a VP2 gene fragment, followed by sequence analysis. Two out of eight IBD viruses presented an SspI restriction site, typical of the very virulent phenotype. Three IBD viruses presented a SacI restriction site, typical of classic virulent strains, and one isolate presented restriction sites for both enzymes. The Argentine IBD viruses showed three different molecular patterns by RFLP with the restriction endonuclease BstNI and five different patterns with MboI. By comparison of nucleotide and deduced amino acid sequences of the hypervariable region of the VP2 protein, four Argentine viruses were found to be closely related to Brazilian subclinical strains and two isolates were found to be related to vaccine IBDV strains in use in Argentina. Strain LD9569 was genetically characterized as a very virulent strain and was found to be closely related to international and regional vvIBDV strains. This is the first report on variability of IBDV strains circulating in Argentina.

Innate immunity evasion by Dengue virus.

For viruses to productively infect their hosts, they must evade or inhibit important elements of the innate immune system, namely the type I interferon (IFN) response, which negatively influences the subsequent development of antigen-specific adaptive immunity against those viruses. Dengue virus (DENV) can inhibit both type I IFN production and signaling in susceptible human cells, including dendritic cells (DCs). The NS2B3 protease complex of DENV functions as an antagonist of type I IFN production, and its proteolytic activity is necessary for this function. DENV also encodes proteins that antagonize type I IFN signaling, including NS2A, NS4A, NS4B and NS5 by targeting different components of this signaling pathway, such as STATs. Importantly, the ability of the NS5 protein to bind and degrade STAT2 contributes to the limited host tropism of DENV to humans and non-human primates. In this review, we will evaluate the contribution of innate immunity evasion by DENV to the pathogenesis and host tropism of this virus.

Development and validation of a TaqMan-MGB real-time RT-PCR assay for simultaneous detection and characterization of infectious bursal disease virus.

Rapid and reliable detection and classification of infectious bursal disease viruses (IBDVs) is of crucial importance for disease surveillance and control. This study presents the development and validation of a real-time RT-PCR assay to detect and discriminate very virulent (vv) from non-vv (classic and variant) IBDV strains. The assay uses two fluorogenic, minor groove-binding (MGB) TaqMan probes targeted to a single nucleotide polymorphism (SNP) embedded in a highly conserved genomic region. The analytical sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA. The assay demonstrated a wide dynamic range between 10(2) and 10(8) standard RNA copies per reaction. Good reproducibility was also detected, with intra- and inter-assay coefficients of variation ranging from 0.13% to 2.23% and 0.26% to 1.92%, respectively. The assay detected successfully all the assessed vv, classical, and variant field and vaccine strains and correctly discriminated all vvIBDV strains from non-vvIBDV strains. Other common avian RNA viruses tested negative, indicating high specificity of the assay. The high sensitivity, rapidity, reproducibility, and specificity of the real-time RT-PCR assay make this method suitable for general and genotype-specific detection and quantitation.