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.

Serological survey of veterinarians to assess the zoonotic potential of three emerging swine diseases in Mexico.

We conducted an immunological assay of blood samples taken from 85 swine-specialist veterinarians attending the Congress of the Mexican Association of Swine Specialist Veterinarians in Mexico in 2011. Serum samples were assayed for Porcine rubulavirus (PorPV), Encephalomyocarditis virus (EMCV) and Leptospira spp. antibodies. Using a hemagglutination inhibition test, we registered 2.3% and 27% seropositivity for PorPV and EMCV, respectively. Using viral neutralization tests, we registered 5.8% and 47% seropositivity for PorPV and EMCV, respectively. For Leptospira spp., we registered a seropositivity of 38.8%. The variables (sex, age, years of exposure, number of visited farms, biosecurity level and region) showed no significant effect (P > 0.05) on the seropositivity for EMCV, PorPV and Leptospira spp. except for number of visited farms on HI seropositivity for EMCV (P < 0.05; odds ratio: 1.38). The data obtained provide information on the epidemiology of emerging diseases with zoonotic potential in occupational risk groups.

IRES-based Venezuelan equine encephalitis vaccine candidate elicits protective immunity in mice.

Venezuelan equine encephalitis virus (VEEV) is an arbovirus that causes periodic outbreaks that impact equine and human populations in the Americas. One of the VEEV subtypes located in Mexico and Central America (IE) has recently been recognized as an important cause of equine disease and death, and human exposure also appears to be widespread. Here, we describe the use of an Internal Ribosome Entry Site (IRES) from encephalomyocarditis virus to stably attenuate VEEV, creating a vaccine candidate independent of unstable point mutations. Mice infected with this virus produced antibodies and were protected against lethal VEEV challenge. This IRES-based vaccine was unable to establish productive infection in mosquito cell cultures or in intrathoracically injected Aedes taeniorhynchus, demonstrating that it cannot be transmitted from a vaccinee. These attenuation, efficacy and safety results justify further development for humans or equids of this new VEEV vaccine candidate.

Prevalence and risk factors for encephalomyocarditis virus infection in Peru.

Although encephalomyocarditis virus (EMCV) infection has been commonly documented among domestic animals, less is known about EMCV transmission among humans. Recently, we described the isolation of EMCV from two febrile patients in Peru. To further investigate EMCV transmission in Peru, we screened febrile patients reporting to health clinics in Peru for serological evidence of recent EMCV infection. We also conducted a serological survey for EMCV-neutralizing antibodies in the city of Iquitos, located in the Amazon basin department of Loreto, Peru. Additionally, we screened serum from rodents collected from 10 departments in Peru for evidence of EMCV exposure. EMCV infection was found to be only rarely associated with acute febrile disease in Peru, accounting for <1% of febrile episodes analyzed. Despite the low acute disease burden associated with the virus, human exposure was quite common, as prevalence of EMCV-neutralizing antibodies ranged between 6.0% in the coastal city of Tumbes and >17% in cities in the tropical rainforest of northeastern Peru (Iquitos and Yurimaguas). On the basis of the serological survey conducted in Iquitos, risk factors for past infection include increased age, socioeconomic indicators such as residence construction materials and neighborhood, and swine ownership. Evidence from the rodent survey indicates that EMCV exposure is common among Murinae subfamily rodents in Peru (9.4% EMCV IgG positive), but less common among Sigmodontinae rodents (1.0% positive). Further studies are necessary to more precisely delineate the mode of EMCV transmission to humans, other potential disease manifestations, and the economic impact of EMCV transmission among swine in Peru.

Antiviral activity of Bignoniaceae species occurring in the State of Minas Gerais (Brazil): part 1.

AIMS: To evaluate the antiviral activity of Bignoniaceae species occurring in the state of Minas Gerais, Brazil. METHODS AND RESULTS: Ethanol extracts of different anatomical parts of bignoniaceous plant species have been evaluated in vitro against human herpesvirus type 1 (HSV-1), vaccinia virus (VACV) and murine encephalomyocarditis virus (EMCV) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A total of 34 extracts from 18 plant species selected according to ethnopharmacological and taxonomic criteria were screened. Fifteen of the 34 extracts (44.1%) have disclosed antiviral activity against one or more of the viruses assayed with EC(50) values in the range of 23.2 ± 2.5-422.7 ± 10.9 µg ml(-1). CONCLUSIONS: Twelve of the 34 extracts (35.3%) might be considered promising sources of antiviral natural products, as they have shown EC50 ≤ 100 µg ml(-1). The present screening discloses the high potential of the Bignoniaceae family as source of antiviral agents. SIGNIFICANCE AND IMPACT OF THE STUDY: Active extracts were identified and deserve bioguided studies for the isolation of antiviral compounds and studies on mechanism of action.

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.

Human febrile illness caused by encephalomyocarditis virus infection, Peru.

Etiologic studies of acute febrile disease were conducted in sites across South America, including Cusco and Iquitos, Peru. Patients' clinical signs and symptoms were recorded, and acute- and convalescent-phase serum samples were obtained for serologic examination and virus isolation in Vero E6 and C6/36 cells. Virus isolated in Vero E6 cells was identified as encephalomyocarditis virus (EMCV) by electron microscopy and by subsequent molecular diagnostic testing of samples from 2 febrile patients with nausea, headache, and dyspnea. The virus was recovered from acute-phase serum samples from both case-patients and identified with cardiovirus-specific reverse transcription-PCR and sequencing. Serum samples from case-patient 1 showed cardiovirus antibody by immunoglobulin M ELISA (acute phase <8, convalescent phase >1,024) and by neutralization assay (acute phase <10, convalescent phase >1,280). Serum samples from case-patient 2 did not contain antibodies detectable by either assay. Detection of virus in serum strongly supports a role for EMCV in human infection and febrile illness.

Rotavirus protein NSP3 shuts off host cell protein synthesis.

A recombinant vaccinia virus encoding rotavirus protein NSP3 driven by an internal ribosome entry site (IRES) from the encephalomyocarditis (EMC) virus was able to abate protein synthesis in BSC1 cells by 25-fold, with as much as 30% of the remaining protein synthesis being NSP3. Hence NSP3 shuts off host cell protein synthesis down to the level seen during rotavirus infection but is unable to prevent translation from EMC IRES-driven genes. This effect was abolished by deletions in the eIF4G-binding (aa 274-313) and the dimerization (aa 150-206) but not the viral mRNA-binding (aa 83-149) domains, supporting that NSP3 functions in vivo as a dimer. Binding of eIF4G by NSP3 has been implicated in interfering with mRNA 5'-3' circularization, hence such circularization is essential for translation in mammalian cells.

Serological survey for viral diseases in the Cayo Santiago rhesus macaque population.

The free-ranging population of rhesus monkeys (Macaca mulatta) on Cayo Santiago was sero-surveyed for human measles, simian virus 40, B virus (Herpes simiae), rhesus cytomegalovirus, human and simian retroviruses and encephalomyocarditis virus to determine the prevalence of these viruses in the colony. The results of this study indicate that the colony is free of SV40, HTLVIII (HIV-1), STLVIII (SIV) and SRV1; has a low prevalence of measles and EMCV; and high prevalence rates for B virus, CMV and HTLVI.

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

Comparative studies of plaque variantes derived from a Florida strain of Venezuelan equine encephlomyelitis virus.

Small- and large-plaque variants of a Florida strain (Fe 3-7c) of Venezuelan equine encephalomyelitis virus were studied in vivo and in vitro. The small-plaque variant was less virulent in mice, hamsters, and guinea pigs than the large-plaque variant. The variants could be distinguished by calcium phosphate chromatography. The implications of plaque variants within a mixed virus population are discussed.