Seroprevalence of Hepatitis E virus (HEV) in domestic non-commercial pigs reared in small-scale farms and wild boar in South of Brazil

Hepatitis E is a zoonotic emerging disease distributed worldwide. The domestic swine and wild boars (Sus scrofa) are known as important reservoirs of HEV although HEV infections have been detected in other animal species. The southern region of Brazil has the largest swine productions in the country, ranging from highly-specialized commercial swine productions to small-scale non-commercial pig farms. The small-scale farms allow interactions between wild boars and domestic pigs, when occasionally pathogens transmission can occur between these populations. The aim of this study was to determine HEV seroprevalence in non-commercial domestic pigs and wild boars from two southern Brazilian states (RS: Rio Grande do Sul; SC: Santa Catarina), and discuss if the consumption of raw or undercooked meat from these animals is a potential risk to public health. Animals from RS and SC States were sampled. Serum was harvested from wild boar hunted between 2012 and 2016, and from non-commercial small-scale pig farms in 2014. Overall 249 wild boars (56 from RS and 193 from SC) and 382 pigs (261 from RS and 121 from SC) were tested to detect anti-HEV IgG antibodies using a commercial HEV antibody ELISA kit (Thermo fisher), specific for swine. Overall difference was observed (P\u3c0.0001) regarding HEV seroprevalence between wild boar 4.42% (n=249) and non-commercial domestic pigs 46.60% (n=382). In relation to wild boars samples, higher seroprevalence for Hepatitis E was observed in RS (14.29%; n=56) and lower in SC (1.55%; n=193; P\u3c0.0004). In relation to pigs, RS had also higher seroprevalence (53.26%; n=261) than SC (32.23%; n=121; P\u3c0.0002). Although interactions between wild boar and non-commercial domestic pigs are known to occur, the lowest antibody detection in wild boar suggest that these contact may not be sufficient to explain seroprevalence in studied populations. Our results indicate that non-commercial pigs are a more likely source of infection for the human population than wild boar

Gene expression analysis in anterior pituitary to investigate genetics of swine fertility

In the present study we investigated the effect of selection for fertility traits on gene expression in anterior pituitary (AP) of NE selection lines. The objective of this study was to identify differentially expressed genes in the anterior pituitary of sows with different fertility phenotype. A White Composite base population originated two selection lines, one control line and another selected line for prolificacy. Selection was conducted based on an index, in which ovulation rate and embryo survival were included as parameters. Sows from both lines were slaughtered during follicular development phase. Differential display PCR (DD-PCR) was used to search for differences between lines in AP gene expression. Northern analysis and microarray were used to confirm DD-PCR results. Microarray was constructed using swine anterior pituitary clones (from the present study) and follicle clones from a concurrent DD-PCR project. In the expression study, three genes were identified as differentially expressed in the same direction of DD-PCR using Northern hybridizations. Using microarray analysis, 13 genes were differently expressed (P \u3c 0.05) for the two lines. At D2 nine genes were differently expressed, and at D4, the expression of six genes differed between lines. In general, genes differentially expressed are involved in cell growth, iron pool regulation, and energy production. In conclusion, long-term selection for fertility plays an important role in changing gene expression patterns in the anterior pituitary. Selected transcripts isolated from DD-PCR were mapped. Eleven markers were mapped using somatic cell hybrid panel (SCHP). Eight markers were mapped using a radiation hybrid panel (RH). SCHP mapping allows regional assignment of markers to chromosomes, and RH mapping refines mapping location and defines marker order not resolved in the linkage maps of the pig, which is important in finding genes in QTL region, in applying marker-assisted selection, and in increasing gene density in the porcine genomic map. Furthermore, they are useful for development of the porcine transcript map and the high-resolution BAC contig map

Gene expression analysis in anterior pituitary to investigate genetics of swine fertility

In the present study we investigated the effect of selection for fertility traits on gene expression in anterior pituitary (AP) of NE selection lines. The objective of this study was to identify differentially expressed genes in the anterior pituitary of sows with different fertility phenotype. A White Composite base population originated two selection lines, one control line and another selected line for prolificacy. Selection was conducted based on an index, in which ovulation rate and embryo survival were included as parameters. Sows from both lines were slaughtered during follicular development phase. Differential display PCR (DD-PCR) was used to search for differences between lines in AP gene expression. Northern analysis and microarray were used to confirm DD-PCR results. Microarray was constructed using swine anterior pituitary clones (from the present study) and follicle clones from a concurrent DD-PCR project. In the expression study, three genes were identified as differentially expressed in the same direction of DD-PCR using Northern hybridizations. Using microarray analysis, 13 genes were differently expressed (P \u3c 0.05) for the two lines. At D2 nine genes were differently expressed, and at D4, the expression of six genes differed between lines. In general, genes differentially expressed are involved in cell growth, iron pool regulation, and energy production. In conclusion, long-term selection for fertility plays an important role in changing gene expression patterns in the anterior pituitary. Selected transcripts isolated from DD-PCR were mapped. Eleven markers were mapped using somatic cell hybrid panel (SCHP). Eight markers were mapped using a radiation hybrid panel (RH). SCHP mapping allows regional assignment of markers to chromosomes, and RH mapping refines mapping location and defines marker order not resolved in the linkage maps of the pig, which is important in finding genes in QTL region, in applying marker-assisted selection, and in increasing gene density in the porcine genomic map. Furthermore, they are useful for development of the porcine transcript map and the high-resolution BAC contig map

A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate

Dengue infections represent one of the most prevalent arthropod-borne diseases worldwide, causing a wide spectrum of clinical outcomes. Engineered infectious clone is an important tool to study Dengue virus (DENV) biology. Functional full-length cDNA clones have been constructed for many positive-strand RNA viruses and have provided valuable tools for studying the molecular mechanisms involved in viral genome replication, virion assembly, virus pathogenesis and vaccine development. We report herein the successful development of an infectious clone from a primary Brazilian isolate of dengue virus 3 (DENV3) of the genotype III. Using a two-plasmid strategy, DENV3 genome was divided in two parts and cloned separately into a yeast-bacteria shuttle vector. All plasmids were assembled in yeast by homologous recombination technique and a full-length template for transcription was obtained by in vitro ligation of the two parts of the genome. Transcript-derived DENV3 is infectious upon transfection into BHK-21 cells and in vitro characterization confirmed its identity. Growth kinetics of transcript-derived DENV3 was indistinguishable from wild type DENV3. This system is a powerful tool that will help shed light on molecular features of DENV biology, as the relationship of specific mutations and DENV pathogenesis

Construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique

Submitted by Kamylla Nascimento ([email protected]) on 2017-12-14T13:56:10Z No. of bitstreams: 1 art. Construction of yellow - queiroz.pdf: 525676 bytes, checksum: cb44d51971a226b1f1a734f0831cdeb8 (MD5)Approved for entry into archive by Kamylla Nascimento ([email protected]) on 2017-12-14T14:05:56Z (GMT) No. of bitstreams: 1 art. Construction of yellow - queiroz.pdf: 525676 bytes, checksum: cb44d51971a226b1f1a734f0831cdeb8 (MD5)Made available in DSpace on 2017-12-14T14:05:56Z (GMT). No. of bitstreams: 1 art. Construction of yellow - queiroz.pdf: 525676 bytes, checksum: cb44d51971a226b1f1a734f0831cdeb8 (MD5) Previous issue date: 2013Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brasil.Universidade Federal de Pernambuco. Departamento de Bioquímica. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brasil.O replicon de RNA derivado do genoma de Flavivirus é uma ferramenta valiosa para o estudo de replicação viral independente da montagem e da maturação do virion, além de possuir um grande potencial para expressão de genes heterólogos. Neste estudo nós descrevemos a construção de replicons subgenômicos do vírus da febre amarela utilizando a técnica de recombinação homóloga em levedura. O plasmídeo contendo o replicon do vírus febre amarela cepa 17D (pBSC-repYFV-17D), caracterizado anteriormente, foi manipulado para a expressão heteróloga dos genes repórteres green fluorescent protein (repYFV-17D-GFP) e firelly luciferase (repYFV-17D-Luc). Ambos os replicons foram construídos por recombinação homóloga entre o vetor pBSC-repYFV-17D linearizado e o produto de PCR contendo 25 nucleotídeos terminais homólogos incorporados aos oligonucleotídeos iniciadores. A organização genômica dos construídos é semelhante ao repYFV-17D, com inserção de um gene repórter entre os restantes 63 nucleotídeos N-terminais da proteína do capsídeo e 72 nucleotídeos C-terminais da proteína E. Os replicons repYFV-17D-GFP e repYFV-17D-Luc mostraram uma eficiente replicação e expressão dos genes repórteres. A técnica de recombinação homóloga em levedura usada neste estudo demonstrou ser aplicável à manipulação do genoma do vírus da febre amarela para a construção de replicons subgenômicos.RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potential for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid containing the yellow fever 17D strain replicon (pBSC-repYFV-17D), previously characterized, was handled to heterologous expression of the green fluorescent protein (repYFV-17D-GFP) and firefly luciferase (repYFV-17D-Luc) reporter genes. Both replicons were constructed by homologous recombination between the linearized vector pBSC-repYFV-17D and the PCR product containing homologous 25 nucleotides ends incorporated into PCR primers. The genomic organization of these constructs is similar to repYFV-17D, but with insertion of the reporter gene between the remaining 63 N-terminal nucleotides of the capsid protein and 72 C-terminal nucleotides of the E protein. The replicons repYFV-17D-GFP and repYFV-17D-Luc showed efficient replication and expression of the reporter genes. The yeast-based homologous recombination technique used in this study proved to be applicable for manipulation of the yellow fever virus genome in order to construct subgenomic replicons

Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

Submitted by Kamylla Nascimento ([email protected]) on 2018-01-17T14:14:38Z No. of bitstreams: 1 IAM - Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase.pdf: 1744508 bytes, checksum: da44e9a7e3b2c7e91aa652ad0b067eea (MD5)Approved for entry into archive by Kamylla Nascimento ([email protected]) on 2018-01-17T14:47:40Z (GMT) No. of bitstreams: 1 IAM - Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase.pdf: 1744508 bytes, checksum: da44e9a7e3b2c7e91aa652ad0b067eea (MD5)Made available in DSpace on 2018-01-17T14:47:40Z (GMT). No. of bitstreams: 1 IAM - Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase.pdf: 1744508 bytes, checksum: da44e9a7e3b2c7e91aa652ad0b067eea (MD5) Previous issue date: 2017Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-470453 / 2012-5).Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brasil.Universidade Federal de Pernambuco. Departamento de Bioquímica. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brasil.Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV) expressing Gaussia luciferase (GLuc) (YFV-GLuc). We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967), indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct

Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

ABSTRACT Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV) expressing Gaussia luciferase (GLuc) (YFV-GLuc). We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967), indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct