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Chinese Journal of Biotechnology ; (12): 4809-4823, 2023.
Article in Chinese | WPRIM | ID: wpr-1008060


In order to understand the prevalence and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) in China and to develop subunit vaccine against the epidemic lineage, the genetic evolution analysis of PRRSV strains isolated in China from 2001 to 2021 was performed. The representative strains of the dominant epidemic lineage were selected to optimize the membrane protein GP5 and M nucleotide sequences, which were used, with the interferon and the Fc region of immunoglobulin, to construct the eukaryotic expression plasmids pCDNA3.4-IFNα-GP5-Fc and pCDNA3.4-IFNα-M-Fc. Subsequently, the recombinant proteins IFNα-GP5-Fc and IFNα-M-Fc were expressed by HEK293T eukaryotic expression system. The two recombinant proteins were mixed with ISA206VG adjuvant to immunize weaned piglets. The humoral immunity level was evaluated by ELISA and neutralization test, and the cellular immunity level was detected by ELISPOT test. The results showed that the NADC30-like lineage was the main epidemic lineage in China in recent years, and the combination of IFNα-GP5-Fc and IFNα-M-Fc could induce high levels of antibody and cellular immunity in piglets. This study may facilitate the preparation of a safer and more effective new PRRSV subunit vaccine.

Humans , Animals , Swine , Porcine respiratory and reproductive syndrome virus/genetics , Porcine Reproductive and Respiratory Syndrome/prevention & control , HEK293 Cells , Viral Envelope Proteins/genetics , Antibodies, Viral , Viral Vaccines/genetics , Recombinant Proteins , Vaccines, Subunit
Braz. j. infect. dis ; 20(1): 1-7, Jan.-Feb. 2016. tab, graf
Article in English | LILACS | ID: lil-776471


Abstract Background The mechanism underlying the coexistence of hepatitis B surface antigen and antibodies to HBsAg in chronic hepatitis B patients remains unknown. Aims This research aimed to determine the clinical and virological features of the rare pattern. Methods A total of 32 chronic hepatitis B patients infected by HBV genotype C were included: 15 carrying both HBsAg and anti-HBs (group I) and 17 solely positive for HBsAg (group II). S gene and reverse transcriptase region sequences were amplified, sequenced and compared with the reference sequences. Results The amino acid variability within major hydrophilic region, especially the “a” determinant region, and within reverse transcriptase for regions overlapping the major hydrophilic region in group I is significantly higher than those in group II. Mutation sI126S/T within the “a” determinant was the most frequent change, and only patients from group I had the sQ129R, sG130N, sF134I, sG145R amino acid changes, which are known to alter immunogenicity. Conclusions In chronic patients, the concurrent HBsAg/anti-HBs serological profile is associated with an increased aa variability in several key areas of HBV genome. Additional research on these genetic mutants are needed to clarify their biological significance for viral persistence.

Adult , Female , Humans , Male , Young Adult , Hepatitis B Antibodies/blood , Hepatitis B Surface Antigens/blood , Hepatitis B virus/genetics , Hepatitis B, Chronic/genetics , Hepatitis B, Chronic/immunology , RNA-Directed DNA Polymerase/genetics , Viral Envelope Proteins/genetics , China , DNA, Viral , Genotype , Hepatitis B virus/immunology , Mutation , Polymerase Chain Reaction , Sequence Analysis, DNA
Annals of Laboratory Medicine ; : 450-456, 2016.
Article in English | WPRIM | ID: wpr-59850


BACKGROUND: During the 2015 outbreak of Middle East Respiratory Syndrome coronavirus (MERS-CoV), six different commercial MERS-CoV RNA detection kits based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) were available in Korea. We performed analytical and clinical validations of these kits. METHODS: PowerChek (Kogene Biotech, Korea), DiaPlexQ (SolGent, Korea), Anyplex (Seegene, Korea), AccuPower (Bioneer, Korea), LightMix (Roche Molecular Diagnostics, Switzerland), and UltraFast kits (Nanobiosys, Korea) were evaluated. Limits of detection (LOD) with 95% probability values were estimated by testing 16 replicates of upstream of the envelope gene (upE) and open reading frame 1a (ORF1a) RNA transcripts. Specificity was estimated by using 28 nasopharyngeal swabs that were positive for other respiratory viruses. Clinical sensitivity was evaluated by using 18 lower respiratory specimens. The sensitivity test panel and the high inhibition panel were composed of nine specimens each, including eight and six specimens that were positive for MERS-CoV, respectively. RESULTS: The LODs for upE ranged from 21.88 to 263.03 copies/reaction, and those for ORF1a ranged from 6.92 to 128.82 copies/reaction. No cross-reactivity with other respiratory viruses was found. All six kits correctly identified 8 of 8 (100%) positive clinical specimens. Based on results from the high inhibition panel, PowerChek and AccuPower were the least sensitive to the presence of PCR inhibition. CONCLUSIONS: The overall sensitivity and specificity of all six assay systems were sufficient for diagnosing MERS-CoV infection. However, the analytical sensitivity and detection ability in specimens with PCR inhibition could be improved with the use of appropriate internal controls.

Humans , Coronavirus Infections/diagnosis , Middle East Respiratory Syndrome Coronavirus/genetics , Nasopharynx/virology , Open Reading Frames/genetics , RNA, Viral/analysis , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction , Viral Envelope Proteins/genetics
Mem. Inst. Oswaldo Cruz ; 110(6): 719-725, Sept. 2015. tab, graf
Article in English | LILACS | ID: lil-763100


Saint Louis encephalitis virus (SLEV) is a member of the Japanese-encephalitis virus serocomplex of the genus Flavivirus. SLEV is broadly distributed in the Americas and the Caribbean Islands, where it is usually transmitted by mosquitoes of the genus Culex and primarily to birds and mammalian-hosts. Humans are occasionally infected by the virus and are dead-end hosts. SLEV causes encephalitis in temperate regions, while in tropical regions of the Americas, several human cases and a wide biological diversity of SLEV-strains have been reported. The phylogenetic analysis of the envelope (E) protein genes indicated eight-genotypes of SLEV with geographic overlap. The present paper describes the genotyping of two SLEV viruses detected in mosquito-pools collected in northern Colombia (department of Cordoba). We used reverse transcription-polymerase chain reaction to amplify a fragment of theE-gene to confirm the virus identity and completeE-gene sequencing for phylogenetic analysis and genotyping of the two-SLEV viruses found circulating in Córdoba. This is the first report of SLEV genotype IV in Colombia (Córdoba) in mosquitoes from a region of human inhabitation, implicating the risk of human disease due to SLEV infection. Physicians should consider SLEV as a possible aetiology for undiagnosed febrile and neurologic syndromes among their patients who report exposure to mosquito-bites.

Animals , Humans , Culicidae/virology , Encephalitis Virus, St. Louis/genetics , Viral Envelope Proteins/genetics , Colombia , Consensus Sequence , DNA Barcoding, Taxonomic , Epidemiological Monitoring , Encephalitis Virus, St. Louis/classification , Genotype , Phylogeny , Polymorphism, Genetic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment
Indian J Exp Biol ; 2015 Apr; 53(4): 236-240
Article in English | IMSEAR | ID: sea-158433


Envelope gene is of great evolutionary significance and had been targeted as the vaccine candidate for dengue virus. We analyzed partial sequences of this gene to understand its genetic variability among viral isolates from Kerala state, India, if any. The current study focused on the evolutionary trends of this phylogenetically important gene among DENV-3 isolates through 2008 to 2010 outbreaks. The results gave an insight into the microevolutionary trends of the dengue viral genome. A unique mutation was recorded in the Domain II of the Envelope gene (EDII) of the viral genome at the amino acid position 219 (A219T). The evolutionary implication of this non-synonymous mutation near the EDI/EDII hinge remains to be explored. The study also provided knowledge on the genetic ancestral history of the viral isolates. Two variants of different phylogenetic origin were recorded in Kerala State. The findings in the study have significant implications on the development of dengue vaccines based on the Envelope gene of the virus.

Dengue Virus/classification , Dengue Virus/genetics , Evolution, Molecular , Genes, Viral , India , Phylogeny , Viral Envelope Proteins/genetics
Rev. bras. enferm ; 67(5): 679-683, Sep-Oct/2014.
Article in Portuguese | LILACS, BDENF | ID: lil-731223


Estudo Histórico Social que tem como objeto notícias sobre o Levantamento de Recursos e Necessidades de Enfermagem no Brasil, publicadas na Revista Brasileira de Enfermagem entre 1955 e 1958. A fonte primária foi constituída pelos exemplares da Revista Brasileira de Enfermagem, publicados dentro do recorte temporal do estudo. As fontes secundárias foram constituídas por livros, artigos, dissertações e teses relativas à história da Enfermagem. A análise dos dados teve apoio das fontes secundárias e do pensamento do sociólogo Pierre Bourdieu. Os dados evidenciaram que a Revista Brasileira de Enfermagem, além de oportunizar a divulgação de notícias acerca do Levantamento, proporcionou visibilidade ao mesmo mediante a veiculação dessas notícias e, por fim, teve o efeito simbólico de conferir poder e prestígio à Enfermagem Brasileira.

Social historical study that has as object news related to the Assessment of the Resources and Needs of Nursing in Brazil published in the Revista Brasileira de Enfermagem between 1955 and 1958. The primary source is constituted of copies of Revista Brasileira de Enfermagem published within the selected period of the study. The secondary sources are constituted of books, papers, dissertations and thesis related to the Nursing history. The data analysis was supported by the secondary sources and the thought of the sociologist Pierre Bourdieu. The results evidenced that Revista Brasileira de Enfermagem, in addition to making possible the dissemination of news about the Assessment provided visibility to it and, at last, had the symbolic effect of giving power and prestige to the Brazilian Nursing.

Estudio Histórico Social que tiene como objeto noticias referentes al Levantamiento de Recursos y Necesidades de Enfermería en Brasil publicadas en la Revista Brasileira de Enfermagem entre 1955 y 1958. La fuente primaria se constituye de los ejemplares de la Revista Brasileira de Enfermagem publicados dentro del recorte temporal do estudio. Las fuentes secundarias están constituidas de libros, artículos disertaciones y tesis relativas a la historia de la Enfermería. El análisis de los datos tuvo apoyo de las fuentes secundarias y del pensamiento del Sociólogo Pierre Bourdieu. Los resultados evidencian que la Revista Brasileira de Enfermagem, además de posibilitar la divulgación de noticias acerca del Levantamiento proporcionó visibilidad al mismo mediante la divulgación de esas noticias y, por fin, tuve el efecto simbólico de conferir poder y prestigio a la Enfermería Brasileña.

Animals , Female , Mice , Hepatitis B Surface Antigens/genetics , Hepatitis B Vaccines/genetics , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Viral Hepatitis Vaccines/genetics , Artificial Gene Fusion , Base Sequence , Hepacivirus/genetics , Hepacivirus/immunology , Hepatitis B virus/genetics , Hepatitis B virus/immunology , Immunization , In Vitro Techniques , Molecular Sequence Data , Vaccines, DNA/genetics
Invest. clín ; 55(2): 155-167, jun. 2014. ilus, tab
Article in Spanish | LILACS | ID: lil-749973


Estudios previos han demostrado que la adaptación de diversos virus a crecer en líneas celulares de vertebrados, conduce a la selección de variantes virales que unen al heparán sulfato (HS) con alta afinidad. En el presente trabajo se determinó la susceptibilidad de cepas del virus dengue (DENV) a la heparina hipersulfatada un análogo al HS, después de pases seriados en células BHK-21. A aislados de campo de los cuatro serotipos de DENV, se les realizaron ocho pases seriados en células BHK-21. La adaptación de los DENV al cultivo celular seleccionó variantes virales con una aumentada capacidad replicativa en células BHK-21 y una incrementada susceptibilidad a la heparina, en relación a las respectivas cepas no adaptadas, obteniéndose una inhibición de la infectividad más significativa en DENV-2 y DENV-4. Las cepas de DENV adaptadas presentaron cambios en la secuencia de aminoácidos de la proteína de envoltura (E), en particular una substitución K204R para DENV-1, N67K para DENV-2, K308R y V452A para DENV-3 y E327G para DENV-4. Estas sustituciones implicaron ganancia de residuos básicos que incrementaron la carga neta positiva de la proteína. Los resultados sugieren, que la adaptación de cepas de DENV a células BHK-21 selecciona variantes virales sensibles a la heparina y que la efectividad de este compuesto varía dependiendo de la cepa viral. Además sugieren que el HS puede jugar un papel importante en la infectividad de las cepas de DENV adaptadas al cultivo celular, a diferencia de los aislados de DENV no adaptados.

Several studies have shown that adaptation of various viruses to grow in certain cell lines of vertebrates, leads to the selection of virus variants that bind heparan sulfate (HS) with high affinity. In this study we investigated the susceptibility of strains of dengue virus (DENV) to oversulfated heparin an analogue of HS after passages in BHK-21 cells. Field isolates of the four serotypes of DENV with a limited number of passes in mosquito cells C6/36HT were serially passaged eight times in BHK-21 cells. The adaptation of the DENV to the cell culture selected viral variants with an increased replicative capacity in BHK-21 cells and an increased susceptibility to heparin compared with the original not adapted strains, with a more significant inhibition of the infectivity in DENV-2 and DENV-4.The E protein of the adapted strains showed changes in the amino acid sequence, particularly at the position K204R to DENV-1, N67K to DENV-2, K308R and V452A for DENV-3 and E327G to DENV-4. These substitutions implicated a gain of basic residues that increased the net positive charge of the protein. These results suggest that adaptation of DENV strains to BHK-21 cells implies changes in the envelope protein, changes associated to the protein reactivity with heparin, the inhibitory effectiveness of this compound varying depending on the viral strain. In addition, these results suggest that the HS can play an important role in the infectivity of the DENV strains adapted to vertebrate cell culture, but not in the infectivity of non-adapted DENV isolates.

Animals , Cricetinae , Dengue Virus/drug effects , Heparin/pharmacology , Selection, Genetic , Viral Envelope Proteins/genetics , Aedes/cytology , Cell Line , Chlorocebus aethiops , Dengue Virus/growth & development , Kidney/cytology , Mesocricetus , Models, Molecular , Mutation , Mutation, Missense , Protein Binding , Protein Conformation , RNA, Viral/genetics , Sequence Analysis, RNA , Vero Cells , Viral Plaque Assay , Virus Cultivation , Virus Replication , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/physiology
Journal of Veterinary Science ; : 389-398, 2014.
Article in English | WPRIM | ID: wpr-194859


The UL49.5 gene of most herpesviruses is conserved and encodes glycoprotein N. However, the UL49.5 protein of duck enteritis virus (DEV) (pUL49.5) has not been reported. In the current study, the DEV pUL49.5 gene was first subjected to molecular characterization. To verify the predicted intracellular localization of gene expression, the recombinant plasmid pEGFP-C1/pUL49.5 was constructed and used to transfect duck embryo fibroblasts. Next, the recombinant plasmid pDsRed1-N1/glycoprotein M (gM) was produced and used for co-transfection with the pEGFP-C1/pUL49.5 plasmid to determine whether DEV pUL49.5 and gM (a conserved protein in herpesviruses) colocalize. DEV pUL49.5 was thought to be an envelope glycoprotein with a signal peptide and two transmembrane domains. This protein was also predicted to localize in the cytoplasm and endoplasmic reticulum with a probability of 66.7%. Images taken by a fluorescence microscope at different time points revealed that the DEV pUL49.5 and gM proteins were both expressed in the cytoplasm. Overlap of the two different fluorescence signals appeared 12 h after transfection and continued to persist until the end of the experiment. These data indicate a possible interaction between DEV pUL49.5 and gM.

Animals , Ducks/virology , Genes, Viral/genetics , Mardivirus/genetics , Membrane Glycoproteins/genetics , Microscopy, Fluorescence , Phylogeny , Polymerase Chain Reaction/veterinary , Viral Envelope Proteins/genetics
Journal of Veterinary Science ; : 199-208, 2014.
Article in English | WPRIM | ID: wpr-191849


Currently, killed-virus and modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccines are used to control porcine reproductive and respiratory syndrome. However, both types of vaccines have inherent drawbacks; accordingly, the development of novel PRRSV vaccines is urgently needed. Previous studies have suggested that yeast possesses adjuvant activities, and it has been used as an expression vehicle to elicit immune responses to foreign antigens. In this report, recombinant Kluyveromyces lactis expressing GP5 of HP-PRRSV (Yeast-GP5) was generated and immune responses to this construct were analyzed in mice. Intestinal mucosal PRRSV-specific sIgA antibody and higher levels of IFN-gamma in spleen CD4+ and CD8+ T cells were induced by oral administration of Yeast-GP5. Additionally, Yeast-GP5 administered subcutaneously evoked vigorous cell-mediated immunity, and PRRSV-specific lymphocyte proliferation and IFN-gamma secretion were detected in the splenocytes of mice. These results suggest that Yeast-GP5 has the potential for use as a vaccine for PRRSV in the future.

Animals , Mice , Administration, Oral , Antibodies, Viral/immunology , B-Lymphocytes/immunology , Enzyme-Linked Immunosorbent Assay , Immunity, Cellular , Immunity, Mucosal , Injections, Subcutaneous , Kluyveromyces/genetics , Mice, Inbred BALB C , Porcine respiratory and reproductive syndrome virus/immunology , Recombinant Proteins/genetics , T-Lymphocytes/immunology , Viral Envelope Proteins/genetics , Viral Vaccines/administration & dosage
Journal of Veterinary Science ; : 99-109, 2014.
Article in English | WPRIM | ID: wpr-56428


The purpose of this study was to investigate the effects of porcine interleukin (IL)-2 and IL-4 genes on enhancing the immunogenicity of a porcine reproductive and respiratory syndrome virus ORF5 DNA vaccine in piglets. Eukaryotic expression plasmids pcDNA-ORF5, pcDNA-IL-2, and pcDNA-IL-4 were constructed and then expressed in Marc-145 cells. The effects of these genes were detected using an indirect immunofluorescent assay and reverse transcription polymerase chain reaction (RT-PCR). Characteristic fluorescence was observed at different times after pcDNA-ORF5 was expressed in the Marc-145 cells, and PCR products corresponding to ORF5, IL-2, and IL-4 genes were detected at 48 h. Based on these data, healthy piglets were injected intramuscularly with different combinations of the purified plasmids: pcDNA-ORF5 alone, pcDNA-ORF5 + pcDNA-IL-2, pcDNA-ORF5 + pcDNA-IL-4, and pcDNA-ORF5 + pcDNAIL-4 + pcDNA-IL-2. The ensuing humoral immune responses, percentages of CD4+ and CD8+ T lymphocytes, proliferation indices, and interferon-gamma expression were analyzed. Results revealed that the piglets co-immunized with pcDNA-ORF5 + pcDNA-IL-4 + pcDNA-IL-2 plasmids developed significantly higher antibody titers and neutralizing antibody levels, had significantly increased levels of specific T lymphocyte proliferation, elevated percentages of CD4+ and CD8+ T lymphocytes, and significantly higher IFN-gamma production than the other inoculated pigs (p < 0.05).

Animals , Cell Line , Escherichia coli/genetics , Haplorhini , Immunity, Cellular , Interleukin-2/genetics , Interleukin-4/genetics , Neutralization Tests/veterinary , Plasmids , Porcine Reproductive and Respiratory Syndrome/prevention & control , Porcine respiratory and reproductive syndrome virus/immunology , Recombinant Proteins/genetics , Swine , Vaccines, DNA/immunology , Viral Envelope Proteins/genetics , Viral Vaccines/immunology
Biomédica (Bogotá) ; 33(supl.1): 22-30, set. 2013. graf, tab
Article in Spanish | LILACS | ID: lil-695793


Introducción. Los cuatro serotipos del virus del dengue circularon en el departamento de Santander entre 1998 y 2008. No existe información sobre el papel del serotipo 1 (DENV-1) en la epidemiología de la enfermedad. Objetivo. Analizar la relación entre el cambio de predominancia del (DENV-1) con su diversificación genética, predominancia de los otros serotipos y presentación del dengue grave. Materiales y métodos. La diversificación genética se estudió por análisis filogenético usando la secuencia del gen E de 12 cepas del virus. Para el análisis se utilizaron datos sobre predominancia de los serotipos obtenidos en estudios previos y datos oficiales de incidencia del dengue. Resultados. Los virus seleccionados se agruparon en el genotipo V junto a (DENV-1) de países de Latinoamérica y se evidenció segregación en cuatro linajes. Los cambios en la predominancia del virus coincidieron con el reemplazo de linaje y esto, a su vez, con incremento en la prevalencia de DENV-2 y DENV-3, e incremento del dengue grave. Conclusión. La diversificación genética podría contribuir a cambios de predominancia de (DENV-1), y la relación del virus con el DENV-2 y DENV-3 en situaciones que favorecen la presentación de casos graves. Se necesitan más estudios para precisar el papel de los serotipos en la epidemiología del dengue.

Introduction: Between 1998 and 2008 all dengue virus serotypes circulated in the Departamento de Santander, an endemic region in northeastern Colombia. No information is available as to the role of serotype 1 (DENV-1) with respect to epidemiology of dengue. Objective: To analyze the relationship between changes in DENV-1 predominance with respect to genetic diversity, prevalence of others serotypes and occurrence of severe dengue. Methods: Virus genetic diversity was studied by phylogenetic analysis comparing E gene sequences from 12 viral strains. Data about serotypes predominance obtained in previous studies and official data about dengue incidence were used for analysis. Results: Selected viruses grouped into genotype V together DENV-1 from Latin America countries, and segregation in four lineages was evidenced. Changes in virus predominance coincided with replacement of lineage, increase in prevalence of DENV-2 and DENV-3 and increase of severe dengue. Conclusion: Genetic divergence could have contributed to changes in DENV-1 predominance. The relationship of the virus with DENV-2 and DENV-3 could create scenarios that promote occurrence of severe cases. More studies are required to ascertain the precise role of serotypes in the epidemiology of dengue.

Humans , Dengue Virus/isolation & purification , Dengue/virology , Colombia/epidemiology , Disease Outbreaks , Dengue Virus/classification , Dengue Virus/genetics , Dengue Virus/pathogenicity , Dengue/epidemiology , Genetic Variation , Genotype , Incidence , Phylogeny , Prevalence , RNA, Viral/genetics , Sequence Alignment , Sequence Homology, Nucleic Acid , Serogroup , Serotyping , Severe Dengue/epidemiology , Severe Dengue/virology , Virulence , Viral Envelope Proteins/genetics , Viral Envelope Proteins/physiology
Rev. argent. microbiol ; 44(2): 75-84, jun. 2012. ilus, graf, tab
Article in Spanish | LILACS | ID: lil-657615


En la Argentina, la rabia está circunscripta a algunas provincias del norte. La disponibilidad de nuevas vacunas que eliminen la manipulación del virus rábico y que permitan el control de la enfermedad es de importancia estratégica nacional y regional. Las vacunas basadas en poxvirus recombinantes se han utilizado con éxito como vacunas antirrábicas a nivel mundial. SI bien estos sistemas no están disponibles comercialmente, la plataforma de obtención de virus canarypox (CNPV) recombinantes ya ha sido implementada en nuestro laboratorio. El objetivo de este trabajo fue obtener y evaluar un candidato a vacuna antirrábica basado en CNPV recombinantes que expresan la glicoproteína G (RG) del virus rábico (RV). Se construyó un virus recombinante que expresa la secuencia codificante de RG (CNPV-RG). La inoculación de ratones con este virus indujo altos títulos de anticuerpos seroneutralizantes de RV (3,58 y 9,76 Ul/ml después de una o dos inmunizaciones, respectivamente) y protegió al 78 % de los animales desafiados intracerebralmente con RV. Además, se determinó que el CNPV-RG posee una potencia relativa de 3,5 Ul/ml. Los resultados obtenidos constituyen la primera etapa en la evaluación del CNPV-RG como candidato a vacuna antirrábica. Se requerirán nuevos ensayos para confirmar su utilidad en especies de interés veterinario.

In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 lU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 lU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary Interest.

Animals , Chick Embryo , Cricetinae , Mice , Antigens, Viral/immunology , Canarypox virus/immunology , Glycoproteins/immunology , Rabies Vaccines , Viral Envelope Proteins/immunology , Antibodies, Viral/biosynthesis , Antibodies, Viral/immunology , Antigens, Viral/genetics , Chlorocebus aethiops , Canarypox virus/genetics , Canarypox virus/growth & development , Canarypox virus/isolation & purification , Cell Line/virology , Fibroblasts/virology , Glycoproteins/genetics , Kidney , Mesocricetus , Peptide Fragments/genetics , Peptide Fragments/immunology , Rabies Vaccines/immunology , Rabies/prevention & control , Specific Pathogen-Free Organisms , Virus Cultivation , Vaccines, Synthetic/immunology , Vero Cells/virology , Viral Envelope Proteins/genetics
Mem. Inst. Oswaldo Cruz ; 106(7): 785-793, Nov. 2011. ilus
Article in English | LILACS | ID: lil-606640


Genome sequences for Schistosoma japonicum and Schistosoma mansoni are now available. The schistosome genome encodes ~13,000 protein encoding genes for which the function of only a minority is understood. There is a valuable role for transgenesis in functional genomic investigations of these new schistosome gene sequences. In gain-of-function approaches, transgenesis can lead to integration of transgenes into the schistosome genome which can facilitate insertional mutagenesis screens. By contrast, transgene driven, vector-based RNA interference (RNAi) offers powerful loss-of-function manipulations. Our laboratory has focused on development of tools to facilitate schistosome transgenesis. We have investigated the utility of retroviruses and transposons to transduce schistosomes. Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) can transduce developmental stages of S. mansoni including eggs. We have also observed that the piggyBac transposon is transpositionally active in schistosomes. Approaches with both VSVG-MLV and piggyBac have resulted in somatic transgenesis and have lead to integration of active reporter transgenes into schistosome chromosomes. These findings provided the first reports of integration of reporter transgenes into schistosome chromosomes. Experience with these systems is reviewed herewith, along with findings with transgene mediated RNAi and germ line transgenesis, in addition to pioneering and earlier reports of gene manipulation for schistosomes.

Animals , Humans , Mice , Gene Transfer Techniques , Genome, Helminth/genetics , Schistosoma japonicum/genetics , Schistosoma mansoni/genetics , Animals, Genetically Modified , Chromosomes/genetics , Chromosomes/virology , DNA Transposable Elements , DNA, Helminth/genetics , DNA, Viral/genetics , DNA, Viral/isolation & purification , Genetic Vectors , Leukemia Virus, Murine/genetics , Leukemia Virus, Murine/isolation & purification , Membrane Glycoproteins/genetics , Membrane Glycoproteins/isolation & purification , RNA Interference , Schistosoma japonicum/virology , Schistosoma mansoni/virology , Viral Envelope Proteins/genetics , Viral Envelope Proteins/isolation & purification
Braz. j. med. biol. res ; 44(5): 421-427, May 2011. ilus
Article in English | LILACS | ID: lil-586516


Anti-cancer DNA vaccines have attracted growing interest as a simple and non-invasive method for both the treatment and prevention of tumors induced by human papillomaviruses. Nonetheless, the low immunogenicity of parenterally administered vaccines, particularly regarding the activation of cytotoxic CD8+ T cell responses, suggests that further improvements in both vaccine composition and administration routes are still required. In the present study, we report the immune responses and anti-tumor effects of a DNA vaccine (pgD-E7E6E5) expressing three proteins (E7, E6, and E5) of the human papillomavirus type 16 genetically fused to the glycoprotein D of the human herpes simplex virus type 1, which was administered to mice by the intradermal (id) route using a gene gun. A single id dose of pgD-E7E6E5 (2 µg/dose) induced a strong activation of E7-specific interferon-γ (INF-γ)-producing CD8+ T cells and full prophylactic anti-tumor effects in the vaccinated mice. Three vaccine doses inhibited tumor growth in 70 percent of the mice with established tumors. In addition, a single vaccine dose consisting of the co-administration of pgD-E7E6E5 and the vector encoding interleukin-12 or granulocyte-macrophage colony-stimulating factor further enhanced the therapeutic anti-tumor effects and conferred protection to 60 and 50 percent of the vaccinated mice, respectively. In conclusion, id administration of pgD-E7E6E5 significantly enhanced the immunogenicity and anti-tumor effects of the DNA vaccine, representing a promising administration route for future clinical trials.

Animals , Female , Mice , Cancer Vaccines/administration & dosage , /immunology , Oncogene Proteins, Viral/immunology , Simplexvirus/immunology , Vaccines, DNA/administration & dosage , Viral Envelope Proteins/immunology , /immunology , Cancer Vaccines/genetics , Cancer Vaccines/immunology , /genetics , Injections, Intradermal , Neoplasms, Experimental/immunology , Neoplasms, Experimental/prevention & control , Oncogene Proteins, Viral/genetics , Simplexvirus/genetics , Vaccines, DNA/genetics , Vaccines, DNA/immunology , Viral Envelope Proteins/genetics
Rev. Inst. Med. Trop. Säo Paulo ; 53(2): 82-88, Mar.-Apr. 2011. tab
Article in English | LILACS | ID: lil-584138


BACKGROUND: Cytomegalovirus (CMV) remains an important pathogen to immunocompromised patients even in the era of HAART. The present study aimed at evaluating the influence of CMV viral load and its gB genotypes on AIDS patients' outcome. METHODS: Blood samples of 101 AIDS patients were collected and tested for HIV load, CD4 - cell count and opportunistic pathogens, including CMV. Semi-nested PCRs were run to detect CMV genome and in the positive samples, gB genotyping and CMV load were established using enzymatic restriction and real time PCR, respectively. All patients were clinically followed for four years. RESULTS: In thirty patients (31 percent) CMV was detected and all fatal cases (n = 5) occurred in this group of patients (p = 0.007), but only two patients had CMV disease (1.9 percent). However, viral load was not statistically associated with any analyzed parameter. The most frequently observed CMV genotype was gB2 (45.16 percent) followed by gB3 (35.48 percent). gB2 genotype was more frequently found in patients with CD4-cell counts under 200 cells/mm³ (p = 0.0017), and almost all fatal cases (80 percent) had gB2 genotype. CONCLUSIONS: Our study suggests that CMV and its polymorphisms in biologically relevant genes, such as the gB encoding ORF, may still influence the prognosis and outcome of AIDS patients. The gB2 genotype was associated to patient's bad outcome.

ANTECEDENTES: O citomegalovírus (CMV) permanece um importante patógeno para pacientes imunocomprometidos, mesmo na era da HAART. O presente estudo teve como objetivo avaliar a influência da carga viral do CMV e seu genótipo gB sobre a evolução de pacientes com AIDS. MÉTODOS: Amostras de sangue de 101 pacientes com AIDS foram coletadas e testadas para carga viral de HIV, a contagem de células CD4 e patógenos oportunistas, incluindo o CMV. Um sistema de PCRs seminested foi utilizado para detectar o genoma do CMV e em amostras positivas a carga viral de CMV e genotipagem foram estabelecidos por restrição enzimática e PCR em tempo real, respectivamente. Todos os pacientes foram acompanhados clinicamente durante quatro anos. RESULTADOS: Trinta pacientes (31 por cento) tiveram CMV detectado e todos os casos fatais (n = 5) ocorreram em pacientes deste grupo (p = 0,007), porém apenas dois pacientes tinham doença por CMV (1,9 por cento). No entanto, a carga viral não foi associada estatisticamente a nenhum dos parâmetros analisados. O genótipo de CMV mais freqüentemente observado foi gB2 (45,16 por cento), seguido por gB3 (35,48 por cento). O genótipo gB2 foi mais freqüente em pacientes com contagens abaixo de 200 células/mm³ CD4cell (p = 0,0017), e quase todos os casos fatais (80 por cento) tinham o genótipo gB2. CONCLUSÃO: Nosso estudo sugere que CMV e seu polimorfismo em genes relevantes biologicamentes, como a gB, pode ainda influenciar no prognóstico e evolução de pacientes com AIDS. O genótipo gB2 foi associado ao mau prognóstico do paciente.

Adult , Female , Humans , Male , AIDS-Related Opportunistic Infections/virology , Cytomegalovirus Infections/virology , Cytomegalovirus/genetics , Leukocytes/virology , Viral Envelope Proteins/genetics , Genotype , Polymerase Chain Reaction , Prognosis , Viral Load
Braz. j. med. biol. res ; 43(2): 217-224, Feb. 2010. ilus, graf
Article in English | LILACS | ID: lil-538233


Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE∆) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric â-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK∆) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE∆) and TK + gE (BoHV-5 gE/TK∆) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

Animals , Cattle , Gene Deletion , /genetics , Thymidine Kinase/genetics , Viral Envelope Proteins/genetics , Defective Viruses/genetics , Electrophoresis, Polyacrylamide Gel , Green Fluorescent Proteins/genetics , /immunology , /pathogenicity , Immunoblotting , Polymerase Chain Reaction , Recombination, Genetic/genetics , Thymidine Kinase/immunology , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Viral Envelope Proteins/immunology , Viral Vaccines/genetics , Viral Vaccines/immunology , Virulence/genetics
Rev. argent. microbiol ; 42(1): 11-17, feb. 2010. ilus, tab
Article in English | LILACS | ID: lil-634646


Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.

La arteritis viral equina (AVE) ocasiona infecciones, en su mayoría subclínicas, pero puede causar abortos y enfermedad respiratoria. Si bien se ha descrito un solo serotipo de AVE, existen diferencias en cuanto a la antigenicidad, patogenicidad y patrones de neutralización en las cepas de campo. Los ORF5 y ORF6 del virus codifican las proteínas de envoltura GP5 y M; la interacción entre estas proteínas es crítica para la infectividad. Los cambios en las secuencias de aminoácidos en la proteína GP5, especialmente en la región V1, afectan el fenotipo neutralizante, sin tener en cuenta variaciones aminoacídicas de otras proteínas virales. En este estudio evaluamos los fenotipos neutralizantes de las 5 únicas cepas de arteritis viral equina aisladas en Argentina y los comparamos con los de la cepa de referencia EAV-UCD por virus neutralización cruzada y análisis de secuencias aminoacídicas de las proteínas M y GP5. Las cepas argentinas presentaron un patrón de neutralización similar cuando se utilizaron sueros positivos del banco de sueros, mientras que fueron neutralizadas en menor medida por los sueros policlonales de referencia anti-AVE. A excepción de la cepa LP01, las cepas argentinas tienen casi las mismas sustituciones aminoacídicas en la primera región variable V1 de la proteína GP5, específicamente en los sitios neutralizantes B y C, pero difieren en gran medida respecto de la cepa de referencia EAV-UCD. Las diferencias encontradas en los aislamientos LP02/R, LP02/C, LP02/P y LT-LP-ARG no se reflejaron en variaciones en el fenotipo neutralizante.

Animals , Antigens, Viral/immunology , Equartevirus/immunology , Arterivirus Infections/virology , Horse Diseases/virology , Viral Envelope Proteins/immunology , Viral Matrix Proteins/immunology , Amino Acid Sequence , Argentina , Antigens, Viral/genetics , Equartevirus/classification , Equartevirus/genetics , Equartevirus/isolation & purification , DNA, Complementary/genetics , DNA, Viral/genetics , Genetic Variation , Horses , Molecular Sequence Data , Neutralization Tests , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity , Viral Envelope Proteins/genetics , Viral Matrix Proteins/genetics
Mem. Inst. Oswaldo Cruz ; 105(1): 92-98, Feb. 2010. tab, ilus
Article in English | LILACS | ID: lil-539301


Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The NS5A and E2 proteins of HCV genotype 1 were reported to inhibit the double-stranded (ds) RNA-dependent protein kinase (PKR), which is involved in the cellular antiviral response induced by interferon (IFN). The response to IFN therapy is quite different between genotypes, with response rates among patients infected with types 2 and 3 that are two-three-fold higher than in patients infected with type 1. Interestingly, a significant percentage of HCV genotype 3-infected patients do not respond to treatment at all. The aim of this paper was to analyse the sequences of fragments of the E2 and NS5A regions from 33 outpatients infected with genotype 3a, including patients that have responded (SVR) or not responded (NR) to treatment. HCV RNA was extracted and amplified with specific primers for the NS5A and E2 regions and the PCR products were then sequenced. The sequences obtained covered amino acids (aa) 636-708 in E2 and in NS5A [including the IFN sensitivity determining region (ISDR), PKR-binding domain and extended V3 region)]. In the E2 and NS5A regions, we did observe aa changes among patients, but these changes were not statistically significant between the SVR and NR groups. In conclusion, our results suggest that the ISDR domain is not predictive of treatment success in patients infected with HCV genotype 3a.

Adult , Female , Humans , Male , Hepacivirus/genetics , Hepatitis C, Chronic/virology , RNA, Viral/genetics , Viral Envelope Proteins/genetics , Viral Nonstructural Proteins/genetics , Amino Acid Sequence , Antiviral Agents/therapeutic use , Genotype , Hepatitis C, Chronic/drug therapy , Interferon-alpha/therapeutic use , Molecular Sequence Data , Polymerase Chain Reaction , Ribavirin/therapeutic use