Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus / 生物医学与环境科学（英文）

OBJECTIVE@#To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene.@*METHODS@#A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay.@*RESULTS@#The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses.@*CONCLUSION@#A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.

Caracterización molecular de la nucleoproteína del virus de la rabia en canes procedentes de Arequipa, Perú / Molecular characterization of the rabies virus' nucleoprotein in dogs from Arequipa, Peru

RESUMEN Objetivos. Caracterizar la nucleoproteína (N) y establecer el origen del virus de la rabia en canes procedentes de Arequipa. Materiales y métodos. Se analizaron 30 muestras de tejido nervioso procedentes de los departamentos de Arequipa y Puno. Se extrajo el ARN total de las muestras y se sintetizó ADNc para amplificar el gen de la nucleoproteína, secuenciarlo y realizar el análisis bioinformático. Resultados. Se obtuvo la formación de un grupo definido con respecto al grupo externo (European bat lyssavirus). Este grupo fue clasificado en dos subgrupos, uno constituido por muestras procedentes de Puno y Arequipa (subgrupo A), y otro por muestras de Puno (subgrupo B), observándose una identidad nucleotídica de 99,9% en el subgrupo A. Conclusiones. Los agrupamientos de las secuencias virales muestran que los casos de rabia canina notificados en Arequipa son el resultado de la expansión de rabia canina procedente de la región endémica de Puno.

ABSTRACT Objective . To characterize the nucleoprotein (N) and establish the origin of the rabies virus in dogs coming from Arequipa. Materials and Methods. Thirty samples of nervous tissue from the departments of Arequipa and Puno were analyzed. Total RNA was extracted from the samples and cDNA was synthesized to amplify the nucleoprotein gene, sequence it, and perform bioinformatics analysis. Results . A defined group was formed with respect to the external group (European bat lyssavirus). This group was classified into two subgroups, one constituted by samples coming from Puno and Arequipa (subgroup A), and another one by samples from Puno (subgroup B), exhibiting a nucleotide identity of 99.9% in subgroup A. This group was classified in two subgroups, one constituted by samples coming from Puno and Arequipa (subgroup A), and another one by samples from Puno (subgroup B), observing a nucleotide identity of 99.9% in subgroup A. Conclusions. The groupings of viral sequences show that the cases of canine rabies reported in Arequipa are the result of the expansion of canine rabies from the endemic region of Puno.

Evolution of equine influenza viruses (H3N8) during a Brazilian outbreak, 2015

Abstract Equine influenza is one of the major respiratory infectious diseases in horses. An equine influenza virus outbreak was identified in vaccinated and unvaccinated horses in a veterinary school hospital in São Paulo, SP, Brazil, in September 2015. The twelve equine influenza viruses isolated belonged to Florida Clade 1. The hemagglutinin and neuraminidase amino acid sequences were compared with the recent isolates from North and South America and the World Organisation for Animal Health recommended Florida Clade 1 vaccine strain. The hemagglutinin amino acid sequences had nine substitutions, compared with the vaccine strain. Two of them were in antigenic site A (A138S and G142R), one in antigenic site E (R62K) and another not in antigenic site (K304E). The four substitutions changed the hydrophobicity of hemagglutinin. Three distinct genetic variants were identified during the outbreak. Eleven variants were found in four quasispecies, which suggests the equine influenza virus evolved during the outbreak. The use of an out of date vaccine strain or updated vaccines without the production of protective antibody titers might be the major contributing factors on virus dissemination during this outbreak.

In silico analysis of amino acid variation in human respiratory syncytial virus: insights into immunodiagnostics

BACKGROUND The highly contagious nature of human respiratory syncytial virus (HRSV) and the gravity of its infection in newborns and vulnerable adults pose a serious public health problem. Thus, a rapid and sensitive diagnostic test for viral detection that can be implemented upon the first appearance of symptoms is needed. The genetic variation of the virus must be considered for immunodiagnostic purposes. OBJECTIVES To analyse HRSV genetic variation and discuss the possible consequences for capture immunoassay development. METHODS We performed a wide analysis of N, F and G protein variation based on the HRSV sequences currently available in the GenBank database. We also evaluated their similarity with homologous proteins from other viruses. FINDINGS The mean amino acid divergences for the N, F, and G proteins between HRSV-A and HRSV-B were determined to be approximately 4%, 10% and 47%, respectively. Due to their high conservation, assays based on the full-length N and F proteins may not distinguish HRSV from human metapneumovirus and other Mononegavirales viruses, and the full-length G protein would most likely produce false negative results due to its high divergence. MAIN CONCLUSIONS We have identified specific regions in each of these three proteins that have higher potential to produce specific results, and their combined utilisation should be considered for immunoassay development.

Short interfering RNAs targeting a vampire-bat related rabies virus phosphoprotein mRNA

Abstract The aim of this study was to assess the in vitro and in vivo effects of short-interfering RNAs (siRNAs) against rabies virus phosphoprotein (P) mRNA in a post-infection treatment for rabies as an extension of a previous report (Braz J Microbiol. 2013 Nov 15;44(3):879-82). To this end, rabies virus strain RABV-4005 (related to the Desmodus rotundus vampire bat) were used to inoculate BHK-21 cells and mice, and the transfection with each of the siRNAs was made with Lipofectamine-2000™. In vitro results showed that siRNA 360 was able to inhibit the replication of strain RABV-4005 with a 1 log decrease in virus titter and 5.16-fold reduction in P mRNA, 24 h post-inoculation when compared to non-treated cells. In vivo, siRNA 360 was able to induce partial protection, but with no significant difference when compared to non-treated mice. These results indicate that, despite the need for improvement for in vivo applications, P mRNA might be a target for an RNAi-based treatment for rabies.

Características de la estructura molecular de las proteínas E del virus del Zika y E1 del virus de la rubéola y posibles implicaciones en el neurotropismo y en las alteraciones del sistema nervioso / E-ZIKV and E1-RV proteins molecular structure and their potential implications in neurotropism and nervous system disorders

Resumen Introducción. El virus del Zika (ZIKV) es un flavivirus con envoltura, transmitido a los seres humanos principalmente por el vector Aedes aegypti. La infección por ZIKV se ha asociado con un gran neurotropismo y con efectos neuropáticos, como el síndrome de Guillain-Barré en el adulto y la microcefalia fetal y posnatal, así como con un síndrome de infección congénita similar al producido por el virus de la rubéola (RV). Objetivo. Comparar las estructuras moleculares de la proteína de envoltura E del virus del Zika (E-ZIKV) y de la E1 del virus de la rubéola (E1-RV), y plantear posibles implicaciones en el neurotropismo y en las alteraciones del sistema nervioso asociadas con el ZIKV. Materiales y métodos. La secuencia de aminoácidos de la proteína E-ZIKV (PDB: 5iZ7) se alineó con la de la glucopreteína E1 del virus de la rubéola (PDB: 4ADG). Los elementos de la estructura secundaria se determinaron usando los programas Vector NTI Advance®, DSSP y POSA, así como herramientas de gestión de datos (AlignX®). Uno de los criterios principales de comparación y alineación fue la asignación de residuos estructuralmente equivalentes, con más de 70 % de identidad. Resultados. La organización estructural de la proteína E-ZIKV (PDB: 5iZ7) fue similar a la de E1-RV (PDB: 4ADG) (70 a 80 % de identidad), y se observó una correspondencia con la estructura definida para las glucoproteínas de fusión de membrana de clase II de los virus con envoltura. E-ZIKV y E1-RV exhibieron elementos estructurales de fusión muy conservados en la región distal del dominio II, asociados con la unión a los receptores celulares de entrada del virus de la rubéola (glucoproteína de mielina del oligodendrocito, Myelin Oligodendrocyte Glycoprotein, MOG), y con los receptores celulares Axl del ZIKV y de otros flavivirus. Conclusión. La comparación de las proteínas E-ZIKV y E1-RV es un paso necesario hacia la definición de otros factores moleculares determinantes del neurotropismo y la patogenia del ZIKV, el cual puede contribuir a generar estrategias de diagnóstico, prevención y tratamiento de las complicaciones neurológicas inducidas por el ZIKV.

Abstract Introduction: Zika virus (ZIKV) is an enveloped flavivirus transmitted to humans mainly by Aedes aegypti. ZIKV infection has been associated with high neurotropism and neuropathic effects such as the Guillain-Barré syndrome in adults, and fetal and postnatal microcephaly and the congenital Zika virus syndrome similar to that produced by rubella virus (VR). Objective: To compare Zika virus membrane protein E (E-ZIKV) and rubella virus membrane protein E1 (E1-RV), and to propose possible implications for neurotropism and nervous system disorders associated with ZIKV infections. Materials and methods: The amino acid sequence of E-ZIKV protein (PDB: 5iZ7) was aligned to that of rubella virus glycoprotein E1 (PDB: 4ADG). The secondary structure elements were determined using the programs Vector NTI Advance®, DSSP, and POSA, and integrated data management tools (AlignX®). One of the main comparison and alignment criteria was the allocation of structurally equivalent residues with more than 70% identity. Results: E-ZIKV structural organization (PDB: 5iZ7) was similar to that of E1-RV (PDB: 4ADG) (70%-80% identity), and it was consistent with relevant structural features of viral membrane class II fusion glycoproteins. E-ZIKV and E1-RV exhibited highly conserved fusion structural elements at the distal region of domain II, which has been associated with the RV myelin oligodendrocyte glycoprotein and Axl cell receptors in ZIKV and other flaviviruses. Conclusion: The comparison of E-ZIKV and E1-RV proteins constitutes an essential step towards the definition of ZIKV neurotropism and pathogenesis molecular determinants, and for the adoption of diagnosis, prevention and treatment strategies against neurological complications induced by ZIKV infection.

Surveillance of antiviral resistance markers in Argentina: detection of E119V neuraminidase mutation in a post-treatment immunocompromised patient

Although vaccines are the best means of protection against influenza, neuraminidase inhibitors are currently the main antiviral treatment available to control severe influenza cases. One of the most frequent substitutions in the neuraminidase (NA) protein of influenza A(H3N2) viruses during or soon after oseltamivir administration is E119V mutation. We describe the emergence of a mixed viral population with the E119E/V mutation in the NA protein sequence in a post-treatment influenza sample collected from an immunocompromised patient in Argentina. This substitution was identified by a real-time reverse transcriptase polymerase chain reaction (RT-PCR) protocol and was confirmed by direct Sanger sequencing of the original sample. In 2014, out of 1140 influenza samples received at the National Influenza Centre, 888 samples (78%) were A(H3N2) strains, 244 (21.3%) were type B strains, and 8 (0.7%) were A(H1N1)pdm09 strains. Out of 888 A(H3N2) samples, 842 were tested for the E119V substitution by quantitative RT-PCR: 841 A(H3N2) samples had the wild-type E119 genotype and in one sample, a mixture of viral E119/ V119 subpopulations was detected. Influenza virus surveillance and antiviral resistance studies can lead to better decisions in health policies and help in medical treatment planning, especially for severe cases and immunocompromised patients.

Sequence similarity between thyroid self-protein and hepatitis c virus polyprotein: possible triggering mechanism of autoimmune thyroiditis / Similaridade de sequências entre auto-proteínas da tireoide e poliproteínas do vírus da hepatite C: possível mecanismo de desencadeamento da tireoidite auto-imune

ABSTRACT Background - Exposure to viral antigens that share amino acid sequence similar with self- antigens might trigger autoimmune diseases in genetically predisposed individuals, and the molecular mimicry theory suggests that epitope mimicry between the virus and human proteins can activate autoimmune disease. Objective - The purpose of this study is to explore the possible sequence similarity between the amino acid sequences of thyroid self-protein and hepatitis C virus proteins, using databanks of proteins and immunogenic peptides, to explain autoimmune thyroid disease. Methods - Were performed the comparisons between the amino acid sequence of the hepatitis C virus polyprotein and thyroid self-protein human, available in the database of National Center for Biotechnology Information on Basic Local Alignment Search Tool. Results - The sequence similarity was related each hepatitis C virus genotype to each thyroid antigen. The similarities between the thyroid and the viral peptides ranged from 21.0 % (31 identical residues out of 147 amino acid in the sequence) to 71.0% (5 identical residues out of 7 amino acid in the sequence). Conclusion - Bioinformatics data, suggest a possible pathogenic link between hepatitis C virus and autoimmune thyroid disease. Through of molecular mimicry is observed that sequences similarities between viral polyproteins and self-proteins thyroid could be a mechanism of induction of crossover immune response to self-antigens, with a breakdown of self-tolerance, resulting in autoimmune thyroid disease.

RESUMO Contexto - A exposição a antígenos virais que compartilham sequência de aminoácidos semelhantes a auto-antígenos pode provocar doenças auto-imunes em indivíduos predispostos geneticamente, e a teoria do mimetismo molecular sugere que o mimetismo entre epítopos de vírus e proteínas humanas pode ativar doenças auto-imunes. Objetivo - O objetivo deste estudo foi explorar a possível semelhança entre as sequências de aminoácidos de auto-proteinas da tireóide e proteínas do vírus da hepatite C, utilizando bancos de dados de proteínas e peptídeos imunogênicos, para explicar a doença auto-imune da tireóide. Métodos - Foram realizadas comparações entre as sequências de aminoácidos de poliproteínas do vírus da hepatite C e auto-proteinas da tireóide humana, disponível na base de dados do National Center for Biotechnology Information no Basic Local Alignment Search Tool. Resultados - A semelhança de sequências foi relacionada para cada genótipo de vírus da hepatite C e proteínas da tireóide. As semelhanças entre proteínas da tireóide e os peptídeos virais variaram de 21,0% (31 resíduos idênticos da sequência de 147 aminoácidos) a 71,0% (cinco resíduos idênticos da sequência de 7 aminoácidos). Conclusão - Dados de bioinformática sugerem uma possível ligação entre vírus da hepatite C e doença auto-imune da tireóide. Através de mimetismo molecular observa-se que as semelhanças entre as sequências de poliproteínas virais e auto-proteínas da tireóide pode ser um mecanismo de indução de resposta imune resultando em doença auto-imune da tireóide.

Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system

The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.

Genotyping Influenza Virus by Next-Generation Deep Sequencing in Clinical Specimens

Rapid and accurate identification of an influenza outbreak is essential for patient care and treatment. We describe a next-generation sequencing (NGS)-based, unbiased deep sequencing method in clinical specimens to investigate an influenza outbreak. Nasopharyngeal swabs from patients were collected for molecular epidemiological analysis. Total RNA was sequenced by using the NGS technology as paired-end 250 bp reads. Total of 7 to 12 million reads were obtained. After mapping to the human reference genome, we analyzed the 3-4% of reads that originated from a non-human source. A BLAST search of the contigs reconstructed de novo revealed high sequence similarity with that of the pandemic H1N1 virus. In the phylogenetic analysis, the HA gene of our samples clustered closely with that of A/Senegal/VR785/2010(H1N1), A/Wisconsin/11/2013(H1N1), and A/Korea/01/2009(H1N1), and the NA gene of our samples clustered closely with A/Wisconsin/11/2013(H1N1). This study suggests that NGS-based unbiased sequencing can be effectively applied to investigate molecular characteristics of nosocomial influenza outbreak by using clinical specimens such as nasopharyngeal swabs.

Possible Transfusion-Related Acute Lung Injury Following Convalescent Plasma Transfusion in a Patient With Middle East Respiratory Syndrome

No abstract available.

Adeno-Associated Virus 2-Mediated Hepatocellular Carcinoma is Very Rare in Korean Patients

BACKGROUND: The incidence and etiology of hepatocellular carcinoma (HCC) vary widely according to race and geographic regions. The insertional mutagenesis of adeno-associated virus 2 (AAV2) has recently been considered a new viral etiology of HCC. The aim of this study was to investigate the frequency and clinical characteristics of AAV2 in Korean patients with HCC. METHODS: A total of 289 unrelated Korean patients with HCC, including 159 Hepatitis-B-related cases, 16 Hepatitis-C-related cases, and 114 viral serology-negative cases, who underwent surgery at the Samsung Medical Center in Korea from 2009 to 2014 were enrolled in this study. The presence of AAV2 in fresh-frozen tumor tissues was investigated by DNA PCR and Sanger sequencing. The clinical and pathological characteristics of AAV2-associated HCC in these patients were compared with previous findings in French patients. RESULTS: The AAV2 detection rate in Korean patients (2/289) was very low compared with that in French patients (11/193). Similar to the French patients, the Korean patients with AAV2-related HCC showed no signs of liver cirrhosis. The Korean patients were younger than the French patients with the same AAV2-associated HCC; the ages at diagnosis of the two Korean patients were 47 and 39 yr, while the median age of the 11 French patients was 55 yr (range 43-90 yr). CONCLUSIONS: AAV2-associated HCC was very rare in Korean patients with HCC. Despite a limited number of cases, this study is the first to report the clinical characteristics of Korean patients with AAV2-associated HCC. These findings suggest epidemiologic differences in viral hepatocarcinogenesis between Korean and European patients.

Human Cytomegalovirus DNA Quantification and Gene Expression in Gliomas of Different Grades

Gliomas are the most common type of primary brain tumors. The most aggressive type, Glioblastoma multiforme (GBM), is one of the deadliest human diseases, with an average survival at diagnosis of about 1 year. Previous evidence suggests a link between human cytomegalovirus (HCMV) and gliomas. HCMV has been shown to be present in these tumors and several viral proteins can have oncogenic properties in glioma cells. Here we have investigated the presence of HCMV DNA, RNA and proteins in fifty-two gliomas of different grades of malignancy. The UL83 viral region, the early beta 2.7 RNA and viral protein were detected in 73%, 36% and 57% by qPCR, ISH and IHC, respectively. Positivity of the viral targets and viral load was independent of tumor type or grade suggesting no correlation between viral presence and tumor progression. Our results demonstrate high prevalence of the virus in gliomas from Brazilian patients, contributing to a better understanding of the association between HCMV infection and gliomas worldwide and supporting further investigations of the virus oncomodulatory properties.

A glycoprotein E gene-deleted bovine herpesvirus 1 as a candidate vaccine strain

A bovine herpesvirus 1 (BoHV-1) defective in glycoprotein E (gE) was constructed from a Brazilian genital BoHV-1 isolate, by replacing the full gE coding region with the green fluorescent protein (GFP) gene for selection. Upon co-transfection of MDBK cells with genomic viral DNA plus the GFP-bearing gE-deletion plasmid, three fluorescent recombinant clones were obtained out of approximately 5000 viral plaques. Deletion of the gE gene and the presence of the GFP marker in the genome of recombinant viruses were confirmed by PCR. Despite forming smaller plaques, the BoHV-1△gE recombinants replicated in MDBK cells with similar kinetics and to similar titers to that of the parental virus (SV56/90), demonstrating that the gE deletion had no deleterious effects on replication efficacy in vitro. Thirteen calves inoculated intramuscularly with BoHV-1△gE developed virus neutralizing antibodies at day 42 post-infection (titers from 2 to 16), demonstrating the ability of the recombinant to replicate and to induce a serological response in vivo. Furthermore, the serological response induced by recombinant BoHV-1△gE could be differentiated from that induced by wild-type BoHV-1 by the use of an anti-gE antibody ELISA kit. Taken together, these results indicated the potential application of recombinant BoHV-1 △gE in vaccine formulations to prevent the losses caused by BoHV-1 infections while allowing for differentiation of vaccinated from naturally infected animals.

Epilepsy-induced electrocardiographic alterations following cardiac ischemia and reperfusion in rats

The present study evaluated electrocardiographic alterations in rats with epilepsy submitted to an acute myocardial infarction (AMI) model induced by cardiac ischemia and reperfusion. Rats were randomly divided into two groups: control (n=12) and epilepsy (n=14). It was found that rats with epilepsy presented a significant reduction in atrioventricular block incidence following the ischemia and reperfusion procedure. In addition, significant alterations were observed in electrocardiogram intervals during the stabilization, ischemia, and reperfusion periods of rats with epilepsy compared to control rats. It was noted that rats with epilepsy presented a significant increase in the QRS interval during the stabilization period in relation to control rats (P<0.01). During the ischemia period, there was an increase in the QRS interval (P<0.05) and a reduction in the P wave and QT intervals (P<0.05 for both) in rats with epilepsy compared to control rats. During the reperfusion period, a significant reduction in the QT interval (P<0.01) was verified in the epilepsy group in relation to the control group. Our results indicate that rats submitted to an epilepsy model induced by pilocarpine presented electrical conductivity alterations of cardiac tissue, mainly during an AMI episode.

Rotavirus genotypes and the indigenous children of Brazilian midwest in the vaccine era, 2008-2012: footprints of animal genome

World group A rotavirus (RVA) surveillance data provides useful estimates of the disease burden, however, indigenous population might require special consideration. The aim of this study was to describe the results of G­ and P­types from Brazilian native children ≤3 years. Furthermore, selected strains have been analyzed for the VP7, VP6, VP4, and NSP4 encoding genes in order to gain insight into genetic variability of Brazilian strains. A total of 149 samples, collected during 2008­2012, were tested for RVA using ELISA and PAGE, following by RT­PCR and sequencing. RVA infection was detected in 8.7% of samples (13/149). Genotype G2P[4] was detected in 2008 and 2010, G8P[6] in 2009, and G3P[8] in 2011. The phylogenetic analysis of the VP7 and VP4 genes grouped the Brazilian G2P[4] and G3P[8] strains within the lineages currently circulating in humans worldwide. However, the phylogenetic analysis of the VP6 and NSP4 from the Brazilian G2P[4] strains, and the VP7 and NSP4 from the Brazilian G3P[8] strains suggest a distant common ancestor with different animal strains (bovine, caprine, and porcine). The epidemiological and genetic information obtained in the present study is expected to provide an updated understanding of RVA genotypes circulating in the native infant population, and to formulate policies for the use of RVA vaccines in indigenous Brazilian people. Moreover, these results highlight the great diversity of human RVA strains circulating in Brazil, and an in­depth surveillance of human and animal RVA will lead to a better understanding of the complex dynamics of RVA evolution

Determinants of emergency contraception non-use among women in unplanned or ambivalent pregnancies / Determinantes del no uso de la anticoncepción de emergencia entre mujeres con embarazo no planeado u ambivalente / Determinantes do não uso da anticoncepção de emergência entre mulheres com gravidez não planejada ou ambivalente

Objective To analyze the determinants of emergency contraception non-use among women in unplanned and ambivalent pregnancies. Method Cross-sectional study with a probabilistic sample of 366 pregnant women from 12 primary health care units in the city of São Paulo, Brazil. A multinomial logistic regression was performed, comparing three groups: women who used emergency contraception to prevent ongoing pregnancies (reference); women who made no use of emergency contraception, but used other contraceptive methods; and women who made no use of any contraceptive methods at all. Results Cohabitation with a partner was the common determinant of emergency contraception non-use. No pregnancy risk awareness, ambivalent pregnancies and no previous use of emergency contraception also contributed to emergency contraception non-use. Conclusion Apart from what is pointed out in the literature, knowledge of emergency contraception and the fertile period were not associated to its use. .

Objetivo Analizar los determinantes del no uso de la anticoncepción de emergencia entre las mujeres con embarazo no planeado o ambivalente. Método Estudio transversal en una muestra probabilística de 366 mujeres embarazadas de 12 Unidades Básicas de Salud de São Paulo. Mediante regresión logística multinomial, se comparó tres grupos de mujeres: aquellas que usaron la anticoncepción de emergencia para prevenir el embarazo en curso (referencia), aquellas que usaron algún método anticonceptivo, pero no la anticoncepción de emergência; y aquellas que no usaron ningún método. Resultados Los hallazgos mostraron que vivir com la pareja fue el determinante común del no uso de la anticoncepción de emergencia. No tener conciencia del riesgo de embarazo, estar en un embarazo ambivalente y nunca tener utilizado la anticoncepción de emergencia también fueron associados con su no uso para prevenir el embarazo en curso. Conclusión Contrariamente a lo que reporta la literatura, el conocimiento de la anticoncepción de emergencia y el período fértil no mostró asociación con el no uso. .

Objetivo Analisar os determinantes do não uso da anticoncepção de emergência entre mulheres com gravidez não planejada ou ambivalente. Método Estudo transversal com amostra probabilística de 366 gestantes de 12 Unidades Básicas de Saúde da cidade de São Paulo. Por meio de regressão logística multinomial, compararam-se três grupos de mulheres: as que usaram anticoncepção de emergência para prevenir a gravidez em curso (referência); as que usaram algum método contraceptivo, mas não anticoncepção de emergência; e as que não usaram nenhum método. Resultados Os achados mostraram que morar com o parceiro foi o determinante comum do não uso da anticoncepção de emergência. Não ter consciência do risco de engravidar, estar em uma gravidez ambivalente e nunca ter usado anticoncepção de emergência também foram associados ao seu não uso para prevenir a gravidez em curso. Conclusão Diferentemente do que relata a literatura, o conhecimento sobre anticoncepção de emergência e sobre o período fértil não mostrou qualquer associação ao não uso. .

Molecular cloning and characterization of Pseudorabies virus EP0 gene.

The pseudorabies virus (PRV) early protein EP0 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP0, which is a multifunctional protein and important for HSV-1 infection. However, the exact function of EP0 is not clear. In this study, using polymerase chain reaction, a 1,104 base-pair sequence of the EP0 gene was amplified from the PRV Becker strain genome and identification of the EP0 gene was confirmed by further cloning and sequencing. Bioinformatics analysis indicated that the PRV EP0 gene encoded a putative polypeptide with 367 amino acids. The encoded protein, designated as EP0 contained a conserved RING-finger superfamily domain and was found to be closely related with the herpes virus RING-finger superfamily and was highly conserved among the counterparts encoded by RING-finger genes. Multiple nucleic acid sequence and amino-acid sequence alignments suggested that PRV EP0 showed a relatively higher similarity with EP0-like proteins of genus Varicellovirus than with those of other genera of Alphaherpesvirinae. In addition, phylogenetic analysis showed that PRV EP0 had a close evolutionary relationship with members of genus Varicellovirus, especially bovine herpesvirus 1 (BoHV-1) and BoHV-5. Antigen prediction indicated that several potential B-cell epitopes were located in EP0. Also, subcellular localization analysis demonstrated that EP0 was predominantly localized in the nucleus, suggesting that it might function as a nuclear-targeted protein.

Production and immunogenicity of chimeric virus-like particles containing the spike glycoprotein of infectious bronchitis virus

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.

Cloning and expression analysis of multiple proteins encoding P gene of Newcastle disease virus.

Viral gene oncotherapy is emerging as a biotherapeutic cancer treatment modality based on targeted killing of cancer cells by viral genes. Newcastle disease virus (NDV) has the property to cause selective oncolysis of tumor cells sparing normal cells. NDV has a single stranded negative sense RNA genome, which is 15,186 nucleotide long and consists of six genes, which codes for eight proteins. NDV like other paramyxoviruses has the ability to generate multiple proteins from the P gene. P protein is encoded by an unedited transcript of the P gene, whereas the V and W protein are the results of RNA editing event in which one and two G residues are inserted at a conserved editing site within the P gene mRNA resulting in V and W transcripts, respectively. Although NDV is known to cause oncolysis by triggering apoptosis, the role of different viral proteins in selective oncolysis is still unclear. P gene edited products are known for its anti-apoptotic property in homologous host. In the present study, NDV P gene and its RNA edited products were amplified, cloned, sequenced and in vitro expression was done in HeLa cells. Further constructs were assayed for their apoptosis inducing ability in HeLa cells. Preliminary study suggested that P, V and W proteins are not apoptotic to HeLa cells.