Dengue virus non-structural protein 3 inhibits mitochondrial respiration by impairing complex I function.

Dengue virus (DENV) infection is known to affect host cell metabolism, but the molecular players involved are still poorly known. Using a proteomics approach, we identified six DENV proteins associated with mitochondria isolated from infected hepatocytes, and most of the peptides identified were from NS3. We also found an at least twofold decrease of several electron transport system (ETS) host proteins. Thus, we investigated whether NS3 could modulate the ETS function by incubating recombinant DENV NS3 constructs in mitochondria isolated from mouse liver. We found that NS3pro (NS3 protease domain), but not the correspondent catalytically inactive mutant (NS3proS135A), impairs complex I (CI)-dependent NADH:ubiquinone oxidoreductase activity, but not the activities of complexes II, III, IV, or V. Accordingly, using high-resolution respirometry, we found that both NS3pro and full-length NS3 decrease the respiratory rates associated with malate/pyruvate oxidation in mitochondria. The NS3-induced impairment in mitochondrial respiration occurs without altering either leak respiration or mitochondria's capacity to maintain membrane potential, suggesting that NS3 does not deeply affect mitochondrial integrity. Remarkably, CI activity is also inhibited in DENV-infected cells, supporting that the NS3 effects observed in isolated mitochondria may be relevant in the context of the infection. Finally, in silico analyses revealed the presence of potential NS3 cleavage sites in 17 subunits of mouse CI and 16 subunits of human CI, most of them located on the CI surface, suggesting that CI is prone to undergo proteolysis by NS3. Our findings suggest that DENV NS3 can modulate mitochondrial bioenergetics by directly affecting CI function. IMPORTANCE: Dengue virus (DENV) infection is a major public health problem worldwide, affecting about 400 million people yearly. Despite its importance, many molecular aspects of dengue pathogenesis remain poorly known. For several years, our group has been investigating DENV-induced metabolic alterations in the host cells, focusing on the bioenergetics of mitochondrial respiration. The results of the present study reveal that the DENV non-structural protein 3 (NS3) is found in the mitochondria of infected cells, impairing mitochondrial respiration by directly targeting one of the components of the electron transport system, the respiratory complex I (CI). NS3 acts as the viral protease during the DENV replication cycle, and its proteolytic activity seems necessary for inhibiting CI function. Our findings uncover new nuances of DENV-induced metabolic alterations, highlighting NS3 as an important player in the modulation of mitochondria function during infection.

Mutations in the non-structural protein coding region regulate gene expression from replicon RNAs derived from Venezuelan equine encephalitis virus.

Self-replicating RNA (repRNA) derived from Venezuelan equine encephalitis (VEE) virus is a promising platform for gene therapy and confers prolonged gene expression due to its self-replicating capability, but repRNA suffers from a suboptimal transgene expression level due to its induction of intracellular innate response which may result in inhibition of translation. To improve transgene expression of repRNA, we introduced point mutations in the non-structural protein 1-4 (nsP1-4) coding region of VEE replicon vectors. As a proof of concept, inflammatory cytokines served as genes of interest and were cloned in their wild type and several mutant replicon vectors, followed by transfection in mammalian cells. Our data show that VEE replicons bearing nsP1GGAC-nsP2T or nsP1GGAC-nsP2AT mutations in the nsP1-4 coding region could significantly reduce the recognition by innate immunity as evidenced by the decreased production of type I interferon, and enhance transgene expression in host cells. Thus, the newly discovered mutant VEE replicon vectors could serve as promising gene expression platforms to advance VEE-derived repRNA-based gene therapies.

Expression of recombinant dengue virus type 1 non-structural protein 1 in mammalian cells and preliminary assessment of its suitability to detect human IgG antibodies elicited by viral infection.

In recent years dengue has become a rapidly growing public health problem worldwide, however, the availability of accurate and affordable diagnostic immunoassays is limited, partly due to the difficulty of producing large quantities of purified antigen. Non-structural protein 1 (NS1) has shown to be a good candidate for inclusion in diagnostic assays and for serosurveys, particularly in endemic countries as a prerequisite for vaccination. In this work the NS1 antigen derived from dengue virus type-1 (DENV1) was expressed in HEK293-T cells and purified by affinity chromatography. The recombinant protein was recovered properly folded as dimers, highly purified and with good yield (1.5 mg/L). It was applied as a serological probe in an indirect ELISA developed in this work to detect human IgG antibodies. Preliminary comparative performance values of 81.1% sensitivity and 83.0% specificity of the developed and preliminary validated iELISA, relative to a commercial kit were obtained, suggesting that the purified recombinant DENV1 NS1 antigen is suitable to detect IgG antibodies, indicative of past DENV infection.

In silico evaluation of flavonoids as potential inhibitors of SARS-CoV-2 main nonstructural proteins (Nsps)-amentoflavone as a multitarget candidate.

Despite the development of vaccines against COVID-19 disease and the multiple efforts to find efficient drugs as treatment for this virus, there are too many social, political, economic, and health inconveniences to incorporate a fully accessible plan of prevention and therapy against SARS-CoV-2. In this sense, it is necessary to find nutraceutical/pharmaceutical drugs as possible COVID-19 preventives/treatments. Based on their beneficial effects, flavonoids are one of the most promising compounds. Therefore, using virtual screening, 478 flavonoids obtained from the KEGG database were evaluated against non-structural proteins Nsp1, Nsp3, Nsp5, Nsp12, and Nsp15, which are essential for the virus-host cell infection, searching for possible multitarget flavonoids. Amentoflavone, a biflavonoid found mainly in Ginkgo biloba, Lobelia chinensis, and Byrsonima intermedia, can interact and bind with the five proteins, suggesting its potential as a multitarget inhibitor. Molecular docking calculations and structural analysis (RMSD, number of H bonds, and clustering) performed from molecular dynamics simulations of the amentoflavone-protein complex support this potential. The results shown here are theoretical evidence of the probable multitarget inhibition of non-structural proteins of SARS-CoV-2 by amentoflavone, which has wide availability, low cost, no side effects, and long history of use. These results are solid evidence for future in vitro and in vivo experiments aiming to validate amentoflavone as an inhibitor of the Nsp1, 3, 5, 12, and 15 of SARS-CoV-2.

Self-association features of NS1 proteins from different flaviviruses.

Flaviviruses comprise a large group of arboviral species that are distributed in several countries of the tropics, neotropics, and some temperate zones. Since they can produce neurological pathologies or vascular damage, there has been intense research seeking better diagnosis and treatments for their infections in the last decades. The flavivirus NS1 protein is a relevant clinical target because it is involved in viral replication, immune evasion, and virulence. Being a key factor in endothelial and tissue-specific modulation, NS1 has been largely studied to understand the molecular mechanisms exploited by the virus to reprogram host cells. A central part of the viral maturation processes is the NS1 oligomerization because many stages rely on these protein-protein assemblies. In the present study, the self-associations of NS1 proteins from Zika, Dengue, and West Nile viruses are examined through constant-pH coarse-grained biophysical simulations. Free energies of interactions were estimated for different oligomeric states and pH conditions. Our results show that these proteins can form both dimers and tetramers under conditions near physiological pH even without the presence of lipids. Moreover, pH plays an important role mainly controlling the regimes where van der Waals interactions govern their association. Finally, despite the similarity at the sequence level, we found that each flavivirus has a well-characteristic protein-protein interaction profile. These specific features can provide new hints for the development of binders both for better diagnostic tools and the formulation of new therapeutic drugs.

Inhibitors of Venezuelan Equine Encephalitis Virus Identified Based on Host Interaction Partners of Viral Non-Structural Protein 3.

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus and a category B select agent. Currently, no FDA-approved vaccines or therapeutics are available to treat VEEV exposure and resultant disease manifestations. The C-terminus of the VEEV non-structural protein 3 (nsP3) facilitates cell-specific and virus-specific host factor binding preferences among alphaviruses, thereby providing targets of interest when designing novel antiviral therapeutics. In this study, we utilized an overexpression construct encoding HA-tagged nsP3 to identify host proteins that interact with VEEV nsP3 by mass spectrometry. Bioinformatic analyses of the putative interactors identified 42 small molecules with the potential to inhibit the host interaction targets, and thus potentially inhibit VEEV. Three inhibitors, tomatidine, citalopram HBr, and Z-VEID-FMK, reduced replication of both the TC-83 strain and the Trinidad donkey (TrD) strain of VEEV by at least 10-fold in astrocytoma, astroglial, and microglial cells. Further, these inhibitors reduced replication of the related New World (NW) alphavirus Eastern equine encephalitis virus (EEEV) in multiple cell types, thus demonstrating broad-spectrum antiviral activity. Time-course assays revealed all three inhibitors reduced both infectious particle production and positive-sense RNA levels post-infection. Further evaluation of the putative host targets for the three inhibitors revealed an interaction of VEEV nsP3 with TFAP2A, but not eIF2S2. Mechanistic studies utilizing siRNA knockdowns demonstrated that eIF2S2, but not TFAP2A, supports both efficient TC-83 replication and genomic RNA synthesis, but not subgenomic RNA translation. Overall, this work reveals the composition of the VEEV nsP3 proteome and the potential to identify host-based, broad spectrum therapeutic approaches to treat new world alphavirus infections.

Dynamics and allostery of Zika virus non-structural protein 5 methyltransferase.

The methyltransferase (MTase) domain of non-structural protein 5 (NS5) plays a fundamental role in flaviviruses replication, and its inhibition is a strategy for antiviral development. MTase methylates viral RNA cap at guanine N-7 and the ribose 2'OH of the first adenosine. Many structures of Zika virus (ZIKV) and other flaviviruses MTases bound to cofactors, substrates and inhibitor candidates have been solved. Still, the dynamical modulation of MTase binding and catalytic activity yet needs to be clarified. Here, we investigated the structural dynamics of ZIKV NS5 MTase domain free and bound to Guanosine-5'-triphosphate (GTP) and S-Adenosyl-Lmethionine (SAM), to identify the molecular dynamics changes related to ligand binding and methyl transfer reaction. We have observed that the binding of the GTP and SAM individually and GTP-SAM in the ternary complex has induced allostery in the RNA binding site, showing the complexity of ZIKV MTase conformational equilibrium and its impact on viral RNA recognition. We also mapped in molecular dynamics trajectories, conformations of GTP guanine moiety that mimics guanine orientations visualized in human-specific N-methyltransferase structures solved by X-ray crystallography. It is the first time that N-7 methylation-prone guanine orientation has been proposed and modeled in flavivirus MTase. Communicated by Ramaswamy H. Sarma.

Identification of potential drugs against SARS-CoV-2 non-structural protein 1 (nsp1).

Non-structural protein 1 (nsp1) is found in all Betacoronavirus genus, an important viral group that causes severe respiratory human diseases. This protein has significant role in pathogenesis and it is considered a probably major virulence factor. As it is absent in humans, it becomes an interesting target of study, especially when it comes to the rational search for drugs, since it increases the specificity of the target and reduces possible adverse effects that may be caused to the patient. Using approaches in silico we seek to study the behavior of nsp1 in solution to obtain its most stable conformation and find possible drugs with affinity to all of them. For this purpose, complete model of nsp1 of SARS-CoV-2 were predicted and its stability analyzed by molecular dynamics simulations in five different replicas. After main pocket validation using two control drugs and the main conformations of nsp1, molecular docking based on virtual screening were performed to identify novel potential inhibitors from DrugBank database. It has been found 16 molecules in common to all five nsp1 replica conformations. Three of them was ranked as the best compounds among them and showed better energy score than control molecules that have in vitro activity against nsp1 from SARS-CoV-2. The results pointed out here suggest new potential drugs for therapy to aid the rational drug search against COVID-19. Communicated by Ramaswamy H. Sarma.

Specificity Studies of the Venezuelan Equine Encephalitis Virus Non-Structural Protein 2 Protease Using Recombinant Fluorescent Substrates.

The non-structural protein 2 (nsP2) of alphavirus Venezuelan equine encephalitis virus (VEEV) is a cysteine protease that is responsible for processing of the viral non-structural polyprotein and is an important drug target owing to the clinical relevance of VEEV. In this study we designed two recombinant VEEV nsP2 constructs to study the effects of an N-terminal extension on the protease activity and to investigate the specificity of the elongated enzyme in vitro. The N-terminal extension was found to have no substantial effect on the protease activity. The amino acid preferences of the VEEV nsP2 protease were investigated on substrates representing wild-type and P5, P4, P2, P1, P1', and P2' variants of Semliki forest virus nsP1/nsP2 cleavage site, using a His6-MBP-mEYFP recombinant substrate-based protease assay which has been adapted for a 96-well plate-based format. The structural basis of enzyme specificity was also investigated in silico by analyzing a modeled structure of VEEV nsP2 complexed with oligopeptide substrate. To our knowledge, in vitro screening of P1' amino acid preferences of VEEV nsP2 protease remains undetermined to date, thus, our results may provide valuable information for studies and inhibitor design of different alphaviruses or other Group IV viruses.

Venezuelan Equine Encephalitis Virus nsP3 Phosphorylation Can Be Mediated by IKKß Kinase Activity and Abrogation of Phosphorylation Inhibits Negative-Strand Synthesis.

Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-κB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKKß inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKß and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKß kinase activity can directly phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5. Alanine substitution mutations at sites 204/5, 142, or 134/5 reduced VEEV replication by >30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKKß can phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204/5, but may be important for infectious particle production at site 134/5.

Dengue Non-structural Protein 5 Polymerase Complexes With Promyelocytic Leukemia Protein (PML) Isoforms III and IV to Disrupt PML-Nuclear Bodies in Infected Cells.

Dengue virus (DENV) threatens almost 70% of the world's population, with no therapeutic currently available. The severe, potentially lethal forms of DENV disease (dengue hemorrhagic fever/dengue shock syndrome) are associated with the production of high level of cytokines, elicited as part of the host antiviral response, although the molecular mechanisms have not been fully elucidated. We previously showed that infection by DENV serotype 2 (DENV2) disrupts promyelocytic leukemia (PML) gene product nuclear bodies (PML-NBs) after viral protein translation in infected cells. Apart from playing a key role as the nucleating agent in forming PML-NBs, PML has antiviral activity against various viruses, including DENV. The present study builds on this work, showing for the first time that all four DENV serotypes elicit PML-NB breakdown. Importantly, we show for the first time that of the nuclear localizing proteins of DENV, DENV non-structural protein (NS) 5 polymerase alone is sufficient to elicit PML-NB disassembly, in part through complexing with PML isoforms III and IV, but not other PML isoforms or other PML-NB components. The results raise the possibility that PML-NB disruption by nuclear localized NS5 contributes to DENV's suppression of the host antiviral response.

Transient Expression of Dengue Virus NS1 Antigen in Nicotiana benthamiana for Use as a Diagnostic Antigen.

Dengue is a viral disease that represents a significant threat to global public health since billions of people are now at risk of infection by this mosquito-borne virus. The implementation of extensive screening tests is indispensable to control this disease, and the Dengue virus non-structural protein 1 (NS1) is a promising antigen for the serological diagnosis of dengue fever. Plant-based systems can be a safe and cost-effective alternative for the production of dengue virus antigens. In this work, two strategies to produce the dengue NS1 protein in Nicotiana benthamiana leaves were evaluated: Targeting NS1 to five different subcellular compartments to assess the best subcellular organelle for the expression and accumulation of NS1, and the addition of elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags to NS1. The transiently expressed proteins in N. benthamiana were quantified by Western blot analysis. The NS1 fused to ELP and targeted to the ER (NS1 ELP-ER) showed the highest yield (445 mg/kg), approximately a forty-fold increase in accumulation levels compared to the non-fused protein (NS1-ER), representing the first example of transient expression of DENV NS1 in plant. We also demonstrated that NS1 ELP-ER was successfully recognized by a monoclonal anti-dengue virus NS1 glycoprotein antibody, and by sera from dengue virus-infected patients. Interestingly, it was found that transient production of NS1-ER and NS1 ELP-ER using vacuum infiltration of whole plants, which is easier to scale up, rather than syringe infiltration of leaves, greatly improved the accumulation of NS1 proteins. The generated plant made NS1, even without extensive purification, showed potential to be used for the development of the NS1 diagnostic tests in resource-limited areas where dengue is endemic.

Dengue perinatal: Reporte de caso / Perinatal Dengue: A case Report

Resumen Introducción: El dengue perinatal es una patología de la que poco se sabe, los reportes disponibles describen riesgo de resultados perinatales adversos. Objetivo: Reportar un caso de dengue perinatal, como diagnóstico diferencial de sepsis neonatal, que debe tenerse en cuenta en zonas endémicas. Caso clínico: Recién nacido de una mujer de 23 años quien a las 36 semanas de gestación presentó cuadro de dengue con antígeno Non-Structural Protein 1 (NS1) positivo y anticuerpos anti-dengue negativos. Al sexto día de enfermedad dio a luz a un recién nacido sano, quien, al segundo día de vida, presentó fiebre sin otros hallazgos patológicos al examen físico, asociado a trombocitopenia severa (17.900 plaquetas/uL) y aumento de la proteína C reactiva, antígeno viral NS1 positivo e in-munoglobulina G (IgG) anti dengue positiva. Fue manejado con antibióticoterpia con ampicilina y gentamicina por protocolo de la institución para sepsis neonatal probable. El neonato mostró me joría clínica, con estabilidad hemodinámica y aumento significativo de plaquetas, siendo dado de alta. Conclusiones: El dengue en el embarazo trae consigo el riesgo de resultados perinatales adver sos, particularmente bajo peso al nacer y parto pre-término. Los hijos de madres diagnosticadas con dengue al final del embarazo deberían ser observados estrechamente con realización de hemograma seriado en los primeros días de vida, debido al riesgo de transmisión vertical.

Abstract Introduction: Few reports are available about perinatal dengue, with controversial results in regards the risk of perinatal outcome. Objective: To report a case of perinatal dengue as a differential diagno sis with neonatal sepsis, which must be considered in endemic areas. Clinical case: Male newborn of a 23 year-old female, who presented a Non-Structural Protein 1 (NS1) antigen positive to dengue at 36 weeks of gestation and negative anti-dengue antibodies. At day six of the illness a healthy newborn was born. On the second day of life the neonate presented fever with no other pathological findings on the physical exam, associated with severe thrombocytopenia (17,900 platelets/uL), increased C-reactive protein, a positive NS1 antigen, and positive anti-dengue immunoglobulin G (IgG). He was treated with ampicillin and gentamicin according the Institution protocol of neonatal sepsis. The newborn showed clinical improvement, with hemodynamic stability and significant increase of platelets, receiving the medical discharge. Conclusions: Dengue in pregnancy produces the risk of adverse perinatal outcomes, particularly low birth weight and preterm delivery. Children of mothers diagnosed with dengue at the end of pregnancy should be observed closely with serial hemograms during child's first days of life, due to the high risk of vertical transmission.

The New High Resolution Crystal Structure of NS2B-NS3 Protease of Zika Virus.

Zika virus (ZIKV) is the cause of a significant viral disease affecting humans, which has spread throughout many South American countries and has also become a threat to Southeastern Asia. This commentary discusses the article "Crystal structure of unlinked NS2B-NS3 protease from Zika virus" published recently in the journal Science by Zhang et al. of Nanyang Technological University, Singapore. They resolved a 1.58 Å resolution structure of the NS2B-NS3 protease of ZIKV and demonstrated how peptide and non-peptide inhibitors interact with this structure, along with the different conformational states that were observed. This protease crystal structure offers new opportunities for the design and development of novel antiviral drugs used for the treatment and control of ZIKV.

Structural and biophysical analysis of sequence insertions in the Venezuelan Equine Encephalitis Virus macro domain.

Random transposon insertions in viral genomes can be used to reveal genomic regions important for virus replication. We used these genomic data to evaluate at the protein level the effect of such insertions on the Venezuelan Equine Encephalitis Virus nsP3 macro domain. The structural analysis showed that transposon insertions occur mainly in loops connecting the secondary structure elements. Some of the insertions leading to a temperature sensitive viral phenotype (ts) are close to the cleavage site between nsP2 and nsP3 or the ADP-ribose binding site, two important functions of the macro domain. Using four mutants mimicking the transposon insertions, we confirmed that these insertions can affect the macro domain properties without disrupting the overall structure of the protein.

Molecular analysis of NSP4 coding gene of porcine rotavirus in Brazil / Análise molecular do gene codificador da NSP4 de rotavírus suínos no Brasil

The non-structural protein 4 (NSP4) has different roles in rotaviral replication, morphogenesis, and enterotoxin-like activity causing secretory diarrhea. A total of 11 partial nucleotide sequences of NSP4 coding gene were defined from group A rotavirus circulating in Brazilian swine herds. On comparing the viral sequences of diarrheagenic peptide area (amino acid 114-135), there was a single point mutation at amino acid 135 presented by two strains with amino acid alanine, and valine in the others. The NSP4 gene phylogeny showed that all strains clustered into E1 genotype, and the nucleotide identity between Brazilian strains ranged from 92.4% and 100%, while the putative amino acid identity, between 95.8% and 100%. Only one site (138aa) was positively selected and at least 119 were negatively selected. As a conclusion, these data demonstrate the occurrence of a common NSP4 genotype described elsewhere in pigs and low diversity between the samples from the surveyed areas(AU)

A proteína não estrutural 4 (NSP4) desempenha diferentes funções na replicação e na morfogênese dos rotavírus, apresentando, ainda, uma atividade de enterotoxina, causando diarreia do tipo secretória. Um total de 11 sequências parciais de nucleotídeos do gene codificador da NSP4 de rotavírus suínos de criações brasileiras foram definidas como pertencentes ao grupo A. Comparando-se as sequências virais da área do peptídeo toxigênico, que compreende a porção entre os aminoácidos de 114 a 135, constatou-se uma única mutação pontual no aminoácido 135, sendo que duas amostras apresentaram alanina, e as demais, valina. A análise filogenética do gene demonstrou que todas as amostras pertencem ao genotipo E1, e que a identidade nucleotídica das amostras brasileiras variou de 92,4% a 100%, enquanto que a identidade de aminoácidos, de 95,8% a 100%. Apenas um resíduo (aa 138) sofreu seleção positiva enquanto que pelo menos outros 119 apresentam seleção negativa. Assim, esses dados mostram a ocorrência de um genotipo comum da NSP4 já descrito anteriormente em suínos, com uma baixa diversidade entre as amostras encontradas(AU)

Molecular analysis of NSP4 coding gene of porcine rotavirus in Brazil / Análise molecular do gene codificador da NSP4 de rotavírus suínos no Brasil

The non-structural protein 4 (NSP4) has different roles in rotaviral replication, morphogenesis, and enterotoxin-like activity causing secretory diarrhea. A total of 11 partial nucleotide sequences of NSP4 coding gene were defined from group A rotavirus circulating in Brazilian swine herds. On comparing the viral sequences of diarrheagenic peptide area (amino acid 114-135), there was a single point mutation at amino acid 135 presented by two strains with amino acid alanine, and valine in the others. The NSP4 gene phylogeny showed that all strains clustered into E1 genotype, and the nucleotide identity between Brazilian strains ranged from 92.4% and 100%, while the putative amino acid identity, between 95.8% and 100%. Only one site (138aa) was positively selected and at least 119 were negatively selected. As a conclusion, these data demonstrate the occurrence of a common NSP4 genotype described elsewhere in pigs and low diversity between the samples from the surveyed areas

A proteína não estrutural 4 (NSP4) desempenha diferentes funções na replicação e na morfogênese dos rotavírus, apresentando, ainda, uma atividade de enterotoxina, causando diarreia do tipo secretória. Um total de 11 sequências parciais de nucleotídeos do gene codificador da NSP4 de rotavírus suínos de criações brasileiras foram definidas como pertencentes ao grupo A. Comparando-se as sequências virais da área do peptídeo toxigênico, que compreende a porção entre os aminoácidos de 114 a 135, constatou-se uma única mutação pontual no aminoácido 135, sendo que duas amostras apresentaram alanina, e as demais, valina. A análise filogenética do gene demonstrou que todas as amostras pertencem ao genotipo E1, e que a identidade nucleotídica das amostras brasileiras variou de 92,4% a 100%, enquanto que a identidade de aminoácidos, de 95,8% a 100%. Apenas um resíduo (aa 138) sofreu seleção positiva enquanto que pelo menos outros 119 apresentam seleção negativa. Assim, esses dados mostram a ocorrência de um genotipo comum da NSP4 já descrito anteriormente em suínos, com uma baixa diversidade entre as amostras encontradas

The relevance of dengue virus genotypes surveillance at country level before vaccine approval.

Dengue is a major threat for public health in tropical and subtropical countries around the world. In the absence of a licensed vaccine and effective antiviral therapies, control measures have been based on education activities and vector elimination. Current efforts for developing a vaccine are both promising and troubling. At the advent of the introduction of a tetravalent dengue vaccine, molecular surveillance of the circulating genotypes in different geographical regions has gained considerable importance. A growing body of in vitro, preclinical, and clinical phase studies suggest that vaccine conferred protection in a geographical area could depends on the coincidence of the dengue virus genotypes included in the vaccine and those circulating. In this review we present the state-of-the-art in this field, highlighting the need of deeper knowledge on neutralizing immune response for making decisions about future vaccine approval and the potential need for different vaccine composition for regional administration.