Recombination Located over 2A-2B Junction Ribosome Frameshifting Region of Saffold Cardiovirus.

Here we report the nearly full-length genome of a recombinant Saffold virus strain (SAFV-BR-193) isolated from a child with acute gastroenteritis. Evolutionary analysis performed using all available near-full length Saffold picornavirus genomes showed that the breakpoint found in the Brazilian strain (SAFV-BR-193) is indeed a recombination hotspot. Notably, this hotspot is located just one nucleotide after the ribosomal frameshift GGUUUUU motif in the SAFV genome. Empirical studies will be necessary to determine if this motif also affects the binding affinity of RNA-dependent RNA-polymerase (RdRp) and therefore increases the changes of RdRp swap between molecules during the synthesis of viral genomes.

Kir 4.1 inward rectifier potassium channel is upregulated in astrocytes in a murine multiple sclerosis model.

Multiple sclerosis (MS) is a high prevalence degenerative disease characterized at the cellular level by glial and neuronal cell death. The causes of cell death during the disease course are not fully understood. In this work we demonstrate that in a MS model induced by Theiler's murine encephalomyelitis virus (TMEV) infection, the inward rectifier (Kir) 4.1 potassium channel subunit is overexpressed in astrocytes. In voltage clamp experiments the inward current density from TMEV-infected astrocytes was significantly larger than in mock-infected ones. The cRNA hybridization analysis from mock- and TMEV-infected cells showed an upregulation of a potassium transport channel coding sequence. We validated this mRNA increase by RT-PCR and quantitative PCR using Kir 4.1 specific primers. Western blotting experiments confirmed the upregulation of Kir 4.1, and alignment between sequences provided the demonstration that the over-expressed gene encodes for a Kir family member. Flow cytometry showed that the Kir 4.1 protein is located mainly in the cell membrane in mock and TMEV-infected astrocytes. Our results demonstrate an increase in K+ inward current in TMEV-infected glial cells, this increment may reduce the neuronal depolarization, contributing to cell resilience mechanisms.

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.

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.

Genomic features and evolutionary constraints in Saffold-like cardioviruses.

This study identified the complete genomic sequence of four type 2 and type 3 human Saffold-like cardioviruses (SLCVs) isolated in Germany and Brazil. The secondary structures of the SLCV internal ribosome entry sites (IRESs) were deduced based on RNA base-pairing conservation and co-variation, using an established Theiler's murine encephalomyelitis virus (TMEV) IRES structure as a reference. The SLCV IRES was highly similar to that of TMEV, but motifs critical in TMEV for binding of the polypyrimidine tract-binding protein (PTB) were disrupted. In TMEV, corresponding alterations have been associated with reduced neurovirulence in mice. In the non-structural genome region, there was evidence of multiple intertypic recombination events between different SLCV types. Between viruses of the same type, recombination also occurred in the capsid-encoding genome region. There were apparently no recombination events between mouse TMEV and human SLCV. In another genus of the family Picornaviridae, Enterovirus, natural recombination occurs strictly within species and can serve as an additional criterion for delimiting species. Accordingly, the results of this study suggest that SLCV and TMEV may represent distinct species within the genus Cardiovirus.

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.

Evaluation of an indirect enzyme-linked immunosorbent assay for routine screening of Theiler's murine encephalomyelitis virus antibodies in mice colonies.

The current study demonstrates the ability of an indirect enzyme-linked immunosorbent assay (iELISA) to detect antibodies against Theiler's murine encephalomyelitis virus in mice colonies. The antigen was produced from infected baby hamster kidney (BHK)-21 cells and treated with 1% Nonidet P40 in saline buffer. Control antigen was prepared following the same procedure using uninfected BHK-21 cells. The optimal antigen and serum dilutions were established. The reaction was revealed using an anti-mouse-horseradish peroxidase conjugate and 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Optimized iELISA was validated by detection of antibodies in known positive and negative serum samples before testing the samples of unknown status. Performance of the iELISA was compared with the indirect fluorescent antibody test, and the cutoff value was determined by receiver operating curve. Indirect ELISA showed 100% sensitivity, 99.38% specificity, and 97.78% predictive positive value. The antigen used is easy to produce, and no special equipment is required. The iELISA developed is simple and provides a rapid and less costly tool for diagnosis and research.

Circulation of 3 lineages of a novel Saffold cardiovirus in humans.

Cardioviruses cause serious disease, mainly in rodents, including diabetes, myocarditis, encephalomyelitis, and multiple sclerosis-like disseminated encephalomyelitis. Recently, a human virus isolate obtained 25 years ago, termed Saffold virus, was sequenced and classified as a cardiovirus. We conducted systematic molecular screening for Saffold-like viruses in 844 fecal samples from patients with gastroenteritis from Germany and Brazil, across all age groups. Six cardioviruses were identified in patients <6 years of age. Viral loads were 283,305-5,044,412,175 copies/g of stool. Co-infections occurred in 4 of 6 children. No evidence for outbreak-like epidemic patterns was found. Phylogenetic analysis identified 3 distinct genetic lineages. Viral protein 1 amino acids were 67.9%-77.7% identical and had a distance of at least 39.4% from known cardioviruses. Because closely related strains were found on 2 continents, global distribution in humans is suspected. Saffold-like viruses may be the first human cardiovirus species to be identified.