Effect of encephalomyocarditis virus 3'Untranslated region on GFP transient expression: implications in recombinant protein production / Efecto de la Región 3'-no traducida del virus de la encefalomiocarditis sobre la expresión transitoria de GFP: implicaciones en la producción de proteínas recombinantes

The enrichment of therapeutic protein production yield in mammalian cell cultures by modulating mRNA stability is a fairly new strategy in biotechnological applications. Here, we describe the application of 3'-untranslated region (3'UTR) from RNA viral genome to modulate mRNA stability.The data obtained showed that the use of the 3 'UTR sequence of the encephalomyocarditis virus (EMCV 3'UTR) downstream of the target gene was not able to significantly modulate the free energy density indicators of the RNA. However, the sequence influenced the stability of the mRNA (and, therefore, the amount of protein production) in a cell type and time-dependent manner, indicating a central role of mRNA-stabilizing binding sites/cellular factors in this process. Our data might be of interest for the biotechnology community to improve recombinant protein production in mammalian cell cultures and RNA-based therapy/vaccination approaches.

El enriquecimiento de la producción terapéutica de proteínas en cultivos de células de mamíferos mediante la modulación de la estabilidad del ARNm es una estrategia nueva en aplicaciones biotecnológicas. Se describe la aplicación de la región 3'-no traducida (3'UTR) del genoma viral ARN para modular la estabilidad del ARNm. Los datos obtenidos mostraron que el uso de la secuencia 3'UTR del virus de la encefalomiocarditis (EMCV 3'UTR) aguas abajo del gen objetivo no pudo modular significativamente los indicadores de densidad de energía libre del ARN. Sin embargo, la secuencia influyó en la estabilidad del ARNm (y, por lo tanto, en la cantidad de producción de proteínas) dependiente de la célula y del tiempo, lo que indica un papel central de los sitios de unión estabilizadores de ARNm/factores celulares en este proceso. Nuestros datos podrían ser de interés para la comunidad biotecnológica para mejorar la producción de proteínas recombinantes en cultivos de células de mamíferos y en enfoques de terapia/vacunación basados en ARN.

Isolation, identification and full-length genome sequence analysis of encephalomyocarditis virus from local aardvarks / 病毒学报

Encephalomyocarditis virus (EMCV) is a natural epidemic zoonotic pathogen. However, no reports have been published regarding the isolation, identification and full-length genome of EMCV from a local aardvark population. In present study, an EMCV isolate HNXX13 was isolated from aardvarks named Huainan-pig in Henan Province. The systematic identification, full-length genome sequencing and molecular characteristic analysis of the isolate HNXX13 were conducted. The result showed that the isolate was spherical with a diameter of 24-30 nm, neither heat- nor acid-resistant, sensitive to trypsin, insensitive to chloroform, not protected by bivalent cationic, and the specific fluorescence was observed in the cytoplasm of BHK-21 cells infected with the isolate by using indirect fluorescence assay. The full-length genome of EMCV HNXX13 generated a 7 725bp sequence (GenBank: F771002), with 81.0%-99.9% nucleotide identity to reference strains from different animals, and 99.5% with a Chinese reference strain isolated earlier from a commercial pig herd. The phylogenetic tree based on the full-length genome and ORF sequences identified that all EMCV strains were divided into three groups G1, G2 and G3, and strain HNXX13 belonging to the G1 group with other Chinese reference strains. The result also identified that this EMCV infection could cause severe clinical signs in a local aardvark population, and enriches the molecular epidemiological data of EMCV in China. Regional differences exist in EMCV genome and transmission is limited within a certain area. However, the cross-infection and transmission of EMCV between aardvark and mice appears most likely. Mutations have occurred in some amino acids of EMCV strain HNXX13 during the transmission in local aardvark herd and these mutations might make the virus easier to infect the aardvark.

Sequencing and analysis of the complete genome of encephalomyocarditis virus strain GXLC isolated from swine / 病毒学报

The complete genome of encephalomyocarditis virus (EMCV)strain GXLC isolated from swine was sequenced and analyzed. Five overlapped gene fragments covering the entire open reading frame (ORF) were amplified by RT-PCR, and the 3'-untranslated region (UTR) and 5'-UTR were amplified by the 3'-rapid amplification of cDNA ends (RACE) and 5'-RACE method, respectively. The genome sequences of strain GXLC were obtained by assembling the sequences of RT-PCR-generated cDNA fragments. The length of the complete genome was 7 725 nucleotides (nt). The homology comparison and phylogenetic analysis of the nucleotide and deduced amino acid sequences between strain GXLC and other EMCV strains available in GenBank were performed. The results showed that the complete genome identity between GXLC strain and the strains from China, i.e. GX0601, GX0602, BJC3 and HB1 and the strains from other countries, i.e. CBNU, K3, K11, TEL-2887A, EMCV-R and PV21 was over 99%. The phylogenetic trees based on the complete genome, the structural protein or the non-structural protein gene sequences revealed that the tree topology was similar. All the EMCV strains could be divided into two groups: group I and group II, and group I could be subdivided into subgroup Ia and subgroup Ib. The strains from swine belonged to subgroup Ia or Ib, and the strains from mice belonged to subgroup Ia, while the strains from Sus scro fa belonged to group II. Strain GXLC, together with other EMCV isolates from China, belonged to subgroup Ia.

A RT-PCR assay for the detection of encephalomycarditis virus infections in pigs

Encephalomyocarditis virus (EMCV) infections can cause losses in pig farms all over the world. Rapid, sensitive and unequivocal detection of this virus is therefore essential for the diagnosis and control of the disease. An RT-PCR assay was developed, optimized and evaluated for encephalomyocarditis virus detection in organ based on a pair of primers that amplifies a 165 bp DNA fragment from a highly conserved nucleotide region of the viral 3D glycoprotein. PCR products of the expected size were obtained from Cuban EMCV 744/03 strain. Non-specific reactions were not observed when other porcine RNA genome viruses and uninfected cells were used. The analytical sensitivity of the test was estimated to be 2 TCID50/50 mL. The analysis of tissue homogenate samples from naturally infected animals proved the potential usefulness of the method for a rapid disease diagnosis from field cases.

Antiviral profile of Nyctanthes arbortristis L. against encephalitis causing viruses.

The ethanolic extracts, various fractions and two pure compounds isolated from the plant N. arbortris were tested against Encephalomyocarditis Virus (EMCV) and Semliki Forest Virus (SFV). Pronounced in vitro virus inhibitory activity was observed with the ethanolic and n-butanol fractions as well as with the pure compounds arbortristoside A and arbortristoside C. In addition, ethanolic extracts and n-butanol fraction protected EMCV infected mice to the extent of 40 and 60% respectively against SFV at a daily dose of 125 mg/kg body weight.

Dual Expression of Two Transgenes Introduced by Lentiviral Vectors

Lentiviruses can infect mitotic and non-dividing cells owing to the karyophilic properties of their pre-integrating complex, which allow its active import through the nucleopore. Thus lentiviral vectors derived from human immunodeficiency virus type 1 can mediate an efficient transfer integration, and stable expression of transgenes into proliferating and stationary cells both in vivo and in vitro. By adopting the internal ribosome entry site of encephalomyocarditis virus for bicistronic expression or two promoters of EF-1alpha and SV40 for separate expression of two genes of interest, we developed two lentiviral vectors that express two genes. On FACS analysis, RT-PCR, and immunofluorescence assay, it was shown that the target cells expressed two genes of interest at different levels as the transducing vectors designed for. This vector system is useful especially for a stable, dual-gene expression and two transgene deliveries to non-dividing cells.

Factors influencing induction and reversal of virus induced diabetes.

Injection of encephalomyocarditis virus -D strain in SJL/J mice leads to development of diabetes. In order to ascertain various factors involved in this process, effect of age of the host, dose of virus and glucose pretreatment on incidence of diabetes and its possible reversal were studied. Blood and urine glucose levels of control and experimental mice were followed for 6-8 weeks to reveal diabetic and reversal from diabetic state. It is observed that incidence of diabetes is directly proportional to the age of the host and dose of the virus, leading to maximum destruction of beta cells and minimum chances of recovery from the diabetic state. Glucose injection prior to low dose virus inoculation reduced the incidence of diabetes and enhanced the process of reversal of diabetes. The data reveal the importance of age of host, dose of virus, metabolic state of beta cells and residual beta cell mass in recovery and reversal of virus induced.

Encephalomyocarditis virus (EMCV) in swine in the State of Rio Grande do Sul, Brazil

Foi observada mortalidade em nove leitöes de uma leitegada de dez em uma granja no Estado do Rio Grande do Sul, Brasil. A enfermidade se estendeu por um período de 15 dias, afetando animais em torno de 30 dias de idade, com morte súbita, sem prévios sinais clíncios evidentes. Leitöes de outras leitegadas permaneceram sadios. Material coletado de leitöes mortos pela doença permitiram o isolamento de um vírus, posteriormente caracterizado como Vírus da Encefalomiocardite (VEMC) por testes de índice de neutralizaçäo. Exames histopatológicos revelaram lesöes compatíveis com a enfermidade provocada pelo VEMC