Gene Expression Profile Induced by Two Different Variants of Street Rabies Virus in Mice.

Pathogenicity and pathology of rabies virus (RABV) varies according to the variant, but the mechanisms are not completely known. In this study, gene expression profile in brains of mice experimentally infected with RABV isolated from a human case of dog rabies (V2) or vampire bat-acquired rabies (V3) were analyzed. In total, 138 array probes associated with 120 genes were expressed differentially between mice inoculated with V2 and sham-inoculated control mice at day 10 post-inoculation. A single probe corresponding to an unannotated gene was identified in V3 versus control mice. Gene ontology (GO) analysis revealed that all of the genes upregulated in mice inoculated with V2 RABV were involved in the biological process of immune defense against pathogens. Although both variants are considered pathogenic, inoculation by the same conditions generated different gene expression results, which is likely due to differences in pathogenesis between the dog and bat RABV variants. This study demonstrated the global gene expression in experimental infection due to V3 wild-type RABV, from the vampire bat Desmodus rotundus, an important source of infection for humans, domestic animals and wildlife in Latin America.

Vaccinia virus in Feces and Urine of Wild Rodents from São Paulo State, Brazil.

The origin of Vaccinia virus (VACV) outbreaks in Brazil remains unknown, but since the isolation of VACV in Mus musculus mice during a zoonotic outbreak affecting cattle and milkers, peridomestic rodents have been suggested to be a link between cows and wild animals. Considering that experimentally infected mice eliminate viral particles in their feces, we investigated the presence of VACV in the feces and urine of wild rodents that were captured in the forest areas surrounding milking farms in the central west region of São Paulo State. For the first time, this work reports the detection of VACV by PCR in the feces of naturally infected Oligoryzomys flavescens, Oligoryzomys nigripes, and Sooretamys angouya, and in the urine of Oligorizomys flavescens, which raises important questions about the spread of VACV by rodent feces and its potential to induce clinical infections in cows.

Vaccinia Virus in Blood Samples of Humans, Domestic and Wild Mammals in Brazil.

Outbreaks of Vaccinia virus (VACV) affecting cattle and humans have been reported in Brazil in the last 15 years, but the origin of outbreaks remains unknown. Although VACV DNA have been already detected in mice (Mus musculus), opossums (Didelphis albiventris) and dogs during VACV zoonotic outbreaks, no transmission to cattle or humans from any of these were reported during Brazilian outbreaks. In this work, we assessed the PCR positivity to VACV in blood samples of cows and other domestic mammals, wild rodents and other wild mammals, and humans from areas with or without VACV infection reports. Our results show the detection of VACV DNA in blood samples of cows, horse and opossums, raising important questions about VACV spread.

Viral type characterization and clinical aspects of canine parvovirus in naturally infected dogs in São Paulo State, Brazil / Caracterização viral e aspectos clínicos de parvovirose em cães naturalmente infectados no Estado de São Paulo

Since the first isolation of canine parvovirus type 2 (CPV-2) in late 70's new virus types as CPV-2a and CPV-2b have been emerged and becoming prevalent in natural canine population and more recently, a third subtype was identified , CPV-2c. The main purpose of this study was to detect and characterize canine parvovirus currently present in Central-West region of São Paulo state, in Brazil. Fecal samples were collected of vaccinated and non-vaccinated dogs, clinically suspected of having CPV infection brought to the Infectious Diseases Service, Veterinary Hospital of FMVZ-UNESP. All samples (n=30) were screening for canine parvovirus through hemagglutination test and those resulting as positive (n=20) were submitted to PCR and the products were subsequently sequenced for subtype characterization. Results were tested for association with age, hematological values, viral hemagglutination titers in the feces, vaccination status and survival. Leukopenia was found in all animals, death occurred in 30% of unvaccinated dogs and in 42% of vaccinated ones. In a total of 20 positive sequenced samples, 18 were classified as CPV-2b, one as CPV-2c, and one as CPV-2a, being CPV2a and CPV2c detected in unvaccinated puppies. Compared to the reference samples amino acid change at position 426 in those circling virus was identified. The study results demonstrate the predominance of CPV-2b and the presence of CPV-2a and CPV-2c in naturally infected, vaccinated and unvaccinated dogs in in São Paulo region.(AU)

Desde o primeiro isolamento do parvovirus canino tipo 2 (CPV-2) no final dos anos 70 novos subtipos virais como CPV-2a e CPV-2b surgiram e foram se tornando prevalentes na população canina; posteriormente um terceiro subtipo foi identificado, CPV- 2-C. O principal objetivo deste estudo foi detectar e caracterizar os subtipos de parvovírus canino atualmente presente na região Centro-Oeste do Estado de São Paulo-Brasil. Amostras de fezes foram coletadas de cães vacinados e não vacinados, atendidos no Serviço de Enfermidades Infecciosas dos Animais, Hospital Veterinário da FMVZ-UNESP, com suspeita clínica parvovirose . Todas as amostras (n = 30) foram submetidas teste de hemaglutinação para parvovirus canino e as positivas (n = 20) submetidas a PCR; os produtos amplificados foram subsequentemente sequenciados para caracterização do subtipo viral. Os resultados foram associados com a idade, os valores hematológicos, os títulos de hemaglutinação viral nas fezes, estado de vacinação e sobrevivência. A leucopenia foi encontrada em todos os animais; Obito foi observado em 30% dos cães não vacinados e 42% dos vacinados. Em um total de 20 amostras positivas sequenciadas, 18 foram classificadas como CPV-2b, uma como CPV-2c, e uma como CPV-2a. CPV 2a e CPV2c foram detectados em filhotes não vacinados. Em comparação com a amostra de referência foi evidenciada uma mudança de aminoácido na posição 426 nas amostras virais circulantes. Os resultados do estudo demonstram a predominância de CPV-2b e a presença de CPV-2a e CPV-2c em cães naturalmente infectados, vacinados e não vacinados na região de São Paulo.(AU)

Dogs and Opossums Positive for Vaccinia Virus during Outbreak Affecting Cattle and Humans, São Paulo State, Brazil.

During a vaccinia virus (VACV) outbreak in São Paulo State, Brazil, blood samples were collected from cows, humans, other domestic animals, and wild mammals. Samples from 3 dogs and 3 opossums were positive for VACV by PCR. Results of gene sequencing yielded major questions regarding other mammalian species acting as reservoirs of VACV.

Rabies virus phosphoprotein interacts with mitochondrial Complex I and induces mitochondrial dysfunction and oxidative stress.

Our previous studies in an experimental model of rabies showed neuronal process degeneration in association with severe clinical disease. Cultured adult rodent dorsal root ganglion neurons infected with challenge virus standard (CVS)-11 strain of rabies virus (RABV) showed axonal swellings and reduced axonal growth with evidence of oxidative stress. We have shown that CVS infection alters a variety of mitochondrial parameters and increases reactive oxygen species (ROS) production and mitochondrial Complex I activity vs. mock infection. We have hypothesized that a RABV protein targets mitochondria and triggers dysfunction. Mitochondrial extracts of mouse neuroblastoma cells were analyzed with a proteomics approach. We have identified peptides belonging to the RABV nucleocapsid protein (N), phosphoprotein (P), and glycoprotein (G), and our data indicate that the extract was most highly enriched with P. P was also detected by immunoblotting in RABV-infected purified mitochondrial extracts and also in Complex I immunoprecipitates from the extracts but not in mock-infected extracts. A plasmid expressing P in cells increased Complex I activity and increased ROS generation, whereas expression of other RABV proteins did not. We have analyzed recombinant plasmids encoding various P gene segments. Expression of a peptide from amino acid 139-172 increased Complex I activity and ROS generation similar to expression of the entire P protein, whereas peptides that did not contain this region did not increase Complex I activity or induce ROS generation. These results indicate that a region of the RABV P interacts with Complex I in mitochondria causing mitochondrial dysfunction, increased generation of ROS, and oxidative stress.

Antiviral therapy for human rabies.

Human rabies is virtually always fatal despite numerous attempts at aggressive therapy. Most survivors received one or more doses of rabies vaccine prior to the onset of the disease. The Milwaukee Protocol has proved to be ineffective for rabies and should no longer be used. New approaches are needed and an improved understanding of basic mechanisms responsible for the clinical disease in rabies may prove to be useful for the development of novel therapeutic approaches. Antiviral therapy is thought to be an important component of combination therapy for the management of human rabies, and immunotherapy and neuroprotective therapy should also be strongly considered. There are many important issues for consideration regarding drug delivery to the central nervous system in rabies, which are in part related to the presence of the blood-brain barrier and also the blood-spinal cord barrier. Ribavirin and interferon-α have proved to be disappointing agents for the therapy of rabies. There is insufficient evidence to support the continued use of ketamine or amantadine for the therapy of rabies. Minocycline or corticosteroids should not be used because of concerns about aggravating the disease. A variety of new antiviral agents are under development and evaluation, including favipiravir, RNA interference (for example, small interfering [si]RNAs) and novel targeted approaches, including interference with viral capsid assembly and viral egress.

Evaluation of three formulations of culture media for isolation of Brucella spp. regarding their ability to inhibit the growth of contaminating organisms.

Three culture media (Brucella agar, Farrell medium, and CITA) were compared for their effectiveness in inhibiting contamination and for isolating Brucella spp. One hundred lymph nodes from pigs (n = 50) and wild boars (n = 50) with lymphadenitis were collected in slaughterhouses in the State of São Paulo and were assessed on these three selective media for Brucella spp. All of the samples were negative for Brucella spp. on the three culture media. On the agar medium, fungal (70 plates) and Gram-positive bacterial (59 plates) contaminants were observed; in the CITA medium, the absence of fungal and Gram-positive bacteria on 15 plates was observed; no bacterial or fungal growth was observed on the Farrell media. The results demonstrated that the CITA and Farrell media inhibited the growth of contaminants better than the Brucella agar.