Sônia de Avila Botton

The Leader Proteinase of Foot-and-Mouth Disease Virus Inhibits the Induction of Beta Interferon mRNA and Blocks the Host Innate Immune Response

ABSTRACT We have previously shown that the leader proteinase (Lpro) of foot-and-mouth disease virus (FMDV) blocks cap-dependent mRNA translation and that a genetically engineered FMDV lacking the leader proteinase coding region (A12-LLV2) is attenuated in cell culture and susceptible animals. The attenuated phenotype apparently is a consequence of the inability of A12-LLV2 to block the expression of type I interferon (IFN-α/β) protein, resulting in IFN-induced inhibition of FMDV replication. Here we show that in addition to preventing IFN-α/β protein synthesis, Lpro reduces the level of immediate-early induction of IFN-β mRNA and IFN-stimulated gene products such as double-stranded RNA-dependent protein kinase R (PKR), 2′,5′-oligoadenylate synthetase, and Mx1 mRNAs in swine cells. Down-regulation of cellular PKR by RNA interference did not affect wild-type virus yield but resulted in a higher yield of A12-LLV2, indicating a direct role of PKR in controlling FMDV replication in the natural host. The observation that Lpro controls the transcription of genes involved in innate immunity reveals a novel role of this protein in antagonizing the cellular response to viral infection.

Identification of Pythium insidiosum by nested PCR in cutaneous lesions of Brazilian horses and rabbits.

Pythium insidiosum is a fungus-like organism present in subtropical and tropical areas, such as Brazil, known to infect humans and various animal species. P. insidiosum is the etiological agent of pythiosis, an emerging and granulomatous disease characterized mainly by cutaneous and subcutaneous lesions in horses, the principal species affected. Accurate diagnosis of pythiosis and identification of its causal agent by microbiological and serological tests can be often difficult and inconclusive principally for horses and humans. The aim of this study was to evaluate the application of the previously described P. insidiosum-specific nested polymerase chain reaction (PCR) assay to directly detect P. insidiosum DNA in clinical and experimental lesions. Universal fungal primers (ITS1 and ITS4) were used during the first-round of PCR to amplify ITS1, 5.8s, and ITS2. A second-round of PCR was conducted with P. insidiosum-specific primers (PI1 and PI2) to amplify a variable region within this ITS1. In this study, a total of 21 equine clinical samples (kunkers) and 28 specimens from experimentally infected rabbits were analyzed by nested PCR. The first-round of PCR generated 800-base pair products, and the second-round produced 105-base pair amplicons for each P. insidiosum-specific sample; no amplicons were generated in negative control samples. Our results suggest that nested PCR is an important and efficient tool for diagnosis of both endemic (horse samples) and experimental (rabbit samples) pythiosis.

Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV) were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs) (Corapi WV, Donis RO and Dubovi EJ (1990) American Journal of Veterinary Research, 55: 1388-1394). Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs--one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48--recognized 100% of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3%) reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R) was calculated, 49 of 66 comparisons (74.24%) between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R value suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.

In vitro susceptibility of fluconazole-susceptible and -resistant isolates of Malassezia pachydermatis against azoles

OBJECTIVES The first aim of this study was to evaluate the in vitro efficacies of fluconazole, ketoconazole, itraconazole and voriconazole on M. pachydermatis growth inhibition. This study also evaluated M. pachydermatis azole cross-resistance, comparing wild clinical isolates and the same isolates with in vitro-induced fluconazole resistance. METHODS Two techniques were used: (1) a broth microdilution method based on protocol M27-A3 from the Clinical and Laboratory Standards Institute to determine the minimum inhibitory concentration (MIC) and (2) the Fekete-Forgács method to induce fluconazole resistance in vitro. The isolates were divided into two groups: group 1 included fluconazole-susceptible clinical isolates (n=30) and group 2 contained the same isolates with in vitro-induced fluconazole resistance (n=30). RESULTS The two groups exhibited differences in susceptibility (p<0.001). Group 1 isolates were susceptible to azoles: ketoconazole (MIC 0.01-1.0 μg/mL), itraconazole (MIC 0.01-1.0 μg/mL), voriconazole (MIC 0.01-4.0 μg/mL), and fluconazole (MIC 0.01-4.0 μg/mL). Group 2 isolates demonstrated a wider range of MICs to azoles: ITZ (MIC 0.06-64.0 μg/mL), KTZ (MIC 0.25-32.0 μg/mL), VRZ (MIC 2.0-128.0 μg/mL), and FLZ (MIC 64.0-128.0 μg/mL). CONCLUSIONS It was shown that FLZ-resistant M. pachydermatis isolates exhibit cross-resistance to other azoles, reinforcing the importance of susceptibility tests as a guide for the therapeutic prescription of antifungals in medical and veterinary mycology.

In vitro activity of terbinafine associated to amphotericin B, fluvastatin, rifampicin, metronidazole and ibuprofen against Pythium insidiosum.

We evaluated the in vitro activities of terbinafine alone and in combination with amphotericin B, fluvastatin, rifampicin, metronidazole or ibuprofen against 17 clinical isolates of Pythium insidiosum. The assays were based on technique M38-A2, as well as the checkerboard microdilution method. The main synergism observed was by combination of terbinafine plus amphotericin B (41.18%). Antagonisms were observed in combinations of terbinafine with fluvastatin (35.30%) or rifampicin (5.88%).

Phylogenetic relationships of Brazilian isolates of Pythium insidiosum based on ITS rDNA and cytochrome oxidase II gene sequences

Pythium insidiosum is an aquatic oomycete that is the causative agent of pythiosis. Advances in molecular methods have enabled increased accuracy in the diagnosis of pythiosis, and in studies of the phylogenetic relationships of this oomycete. To evaluate the phylogenetic relationships among isolates of P. insidiosum from different regions of Brazil, and also regarding to other American and Thai isolates, in this study a total of thirty isolates of P. insidiosum from different regions of Brazil was used and had their ITS1, 5.8S rRNA and ITS2 rDNA (ITS) region and the partial sequence of cytochrome oxidase II (COX II) gene sequenced and analyzed. The outgroup consisted of six isolates of other Pythium species and one of Lagenidium giganteum. Phylogenetic analyses of ITS and COX II genes were conducted, both individually and in combination, using four different methods: Maximum parsimony (MP); Neighbor-joining (NJ); Maximum likelihood (ML); and Bayesian analysis (BA). Our data supported P. insidiosum as monophyletic in relation to the other Pythium species, and COX II showed that P. insidiosum appears to be subdivided into three major polytomous groups, whose arrangement provides the Thai isolates as paraphyletic in relation to the Brazilian ones. The molecular analyses performed in this study suggest an evolutionary proximity among all American isolates, including the Brazilian and the Central and North America isolates, which were grouped together in a single entirely polytomous clade. The COX II network results presented signals of a recent expansion for the American isolates, probably originated from an Asian invasion source. Here, COX II showed higher levels bias, although it was the source of higher levels of phylogenetic information when compared to ITS. Nevertheless, the two markers chosen for this study proved to be entirely congruent, at least with respect to phylogenetic relationships between different isolates of P. insidiosum.

Echinococcus canadensis (G7) and Echinococcus granulosus sensu stricto (G1) in swine of southern Brazil

The cystic echinococcosis (CE) is an important zoonotic disease caused by the parasite Echinococcus spp. In Brazil, this parasite is present in Rio Grande do Sul (RS) state, border with Argentina and Uruguay, causing several damages to human and animal health. This study aimed to identify Echinococcus spp. in hydatid cysts of swine and evaluate the similarity of the genotypes through the phylogenetic analysis. A total of 3,101,992 swine were slaughtered in the central/northern region of RS/Brazil, during 2008-2012. Five isolates were characterized as hydatid cyst by molecular analysis, based on the mitochondrial gene cytochrome c oxidase subunit I (cox-I). The genotypes E. granulosus sensu stricto (G1) (n=2) and E. canadensis (G7) (n=3) were identified in the hydatid cysts. The swine represents a potential intermediate host for different genotypes of Echinococcus spp., besides it can contribute to the perpetuation of the parasites life cycle in rural areas.

Synergysm of voriconazole or itraconazole with other antifungal agents against species of Fusarium.

BACKGROUND Infections caused by Fusarium are difficult to treat because these fungi show in vitro and in vivo resistance to practically all the antifungal agents available, which explains the high mortality rates. An attempt to overcome fungal resistance is the combination of antifungal agents, especially those with different mechanisms of action. AIMS Evaluate the in vitro interactions of combinations of voriconazole or itraconazole with other antifungal agents against 32 isolates of Fusarium spp.: Fusarium chlamydosporum, Fusarium oxysporum, Fusarium proliferatum and Fusarium solani. METHODS Drug interactions were assessed by a checkerboard microdilution method that also included the determination of the MIC of each drug alone according to CLSI (Clinical and Laboratory Standards Institute) document M38-A2, 2008. RESULTS The best combinations were voriconazole+terbinafine which showed synergism against 84% of Fusarium strains. Other synergistic combinations were voriconazole+itraconazole (50%), voriconazole+fluconazole (50%), voriconazole+miconazole (38%), voriconazole+flucytosine (22%) and voriconazole+ketoconazole (25%). The synergisms observed with itraconazole combinations were itraconazole+terbinafine (25%) and itraconazole+flucytosine (9.37%). The antagonisms observed were: voriconazole+fluconazole (3%) and itraconazole+flucytosine (12.5%). CONCLUSIONS The synergism showed by voriconazole+terbinafine was remarkable. To better elucidate the potential usefulness of our findings, new in vivo and in vitro studies deserve be performed.

Diversidade antigênica de amostras do vírus da diarréia viral bovina isoladas no Brasil: implicações para o diagnóstico e estratégias de imunização

Nucleotide sequencing and phylogenetic analysis of 17 Brazilian isolates of bovine viral diarrhea virus (BVDV) identified four isolates (23.5%) belonging to genotype 1a (BVDV-1a), nine isolates (52.9%) of the genotype 1b (BVDV-1b) and four isolates (23.5%) belonging to genotype 2 (BVDV-2). The Brazilian BVDV type 2 isolates were shown to be genotypically different from the BVDV type 2 identified so far in North America and Europe, suggesting they might belong to a new subgenotype. Antigenic characterization of these isolates by cross-neutralization revealed a very low serologic cross-reactivity with vaccine BVDV strains. The immune serum raised in lambs to three vaccine strains presented a very low and even negligible neutralizing activity against some Brazilian BVDV type 1 and 2 isolates. Up to 128-fold differences in serum neutralizing antibody titers were observed between the vaccine strains and some Brazilian BVDV isolates. The potential impact of the antigenic variability of BVDV on serologic diagnosis was investigated by testing field serum samples by serum-neutralization (SN) against viruses of both genotypes. Two hundred and eighty samples (24.7%) had neutralizing antibodies against BVDV. Out of these, 215 (76.8%) had neutralizing activity against viruses of both genotypes, 37 samples (13.2%) reacted only against BVDV type 2 and 28 (10%) were positive only to BVDV type 1. These results demonstrate that SN tests using only one virus may result in a significant number of false-negative results. The reduced neutralizing activity of the antisera to vaccine strains against some Brazilian isolates raises the question about the degree of protection conferred by these vaccines and indicates the need of producing vaccines based on local viruses and/or including viruses of both genotypes.

A retrospective search for bovine respiratory syncytial virus (BRSV) antigens in histological specimens by immunofluorescence and immunohistochemistry

Bovine respiratory syncytial virus (BRSV) has been only sporadically identified as a causative agent of respiratory disease in Brazil. This contrasts with frequent reports of clinical and histopathological findings suggestive of BRSV-associated disease. In order to examine a possible involvement of BRSV in cases of calf pneumonia, a retrospective search was performed for BRSV antigens in histological specimens submitted to veterinary diagnostic services from the states of Rio Grande do Sul and Minas Gerais. Ten out of 41 cases examined (24.4%) were positive for BRSV antigens by immunohistochemistry (IPX). Eight of these cases (19.5%) were also positive by indirect immunofluorescence (IFA), and 31 cases (75.6%) were negative in both assays. In the lungs, BRSV antigens were predominantly observed in epithelial cells of bronchioles and less frequently found in alveoli. In one case, antigens were detected only in the epithelium of the alveolar septae. The presence of antigen-positive cells was largely restricted to epithelial cells of these airways. In two cases, positive staining was also observed in cells and cellular debris in the exudate within the pulmonary airways. The clinical cases positive for BRSV antigens were observed mainly in young animals (2 to 12 month-old) from dairy herds. The main microscopic changes included bronchointerstitial pneumonia characterized by thickening of alveolar septae adjacent to airways by mononuclear cell infiltrates, and the presence of alveolar syncytial giant cells. In summary, the results demonstrate the suitability of the immunodetection of viral antigens in routinely fixed tissue specimens as a diagnostic tool for BRSV infection. Moreover, the findings provide further evidence of the importance of BRSV as a respiratory pathogen of young cattle in southeastern and southern Brazil.