Establishment and characterization of a novel lung cell line derived from the common bottlenose dolphin.

Cetaceans are specialized marine mammals with a unique respiratory system adapted for diving behavior. Furthermore, respiratory diseases are commonly observed in these mammals. Nevertheless, much of their respiratory physiology remains unknown due to the limited supply and poor quality of their biological samples for research. In this study, we established a novel lung cell line, dLu, derived from the common bottlenose dolphin (Tursiops truncatus), which can prove useful in cetacean research, including for understanding the pathogenesis of respiratory diseases in cetaceans. The cells were cultured in a simple medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The morphology of the cells was fibroblast-like. dLu was produced by transfecting the simian virus 40 large T antigen into primary cultured cells. Although dLu exhibited approximately 80 cell divisions, it was unable to achieve complete immortalization, as the cells stopped proliferating beyond this number. dLu cells expressed toll-like receptor 3 but not toll-like receptor 4. Immunostimulation with poly(I:C) altered the gene expressions of interferon beta 1 and tumor necrosis factor alpha in dLu cells. In summary, dLu established in this study is a novel cetacean cell resource that can be easily cultured and is a useful in vitro tool in cetacean research, particularly for studying host immune responses in the lungs.

Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus.

RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3' untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections.

An endogenous adeno-associated virus element in elephants.

An endogenous viral element derived from adeno-associated virus containing a nearly intact open reading frame (ORF) of the rep gene (enAAV-rep) has been identified in the genomes of various mammals including degu and African elephant. Particularly, in degu, mRNA expression of enAAV-rep has been observed specifically in the liver. Here we newly identified enAAV-rep in Asian elephant and rock hyrax, both of which are afrotherians. The enAAV-rep of African and Asian elephants appeared to be orthologous and originated from an integration event of the entire genome of AAV into the ancestral genome of elephants more than 6 million years ago, whereas that of rock hyrax appeared to have originated independently. Negative selection operating at the amino acid sequence level was detected for the ORF of enAAV-rep in elephants. As in degu, mRNA expression of enAAV-rep was specifically observed in the liver in Asian elephant. Integrations of enAAV-rep appeared to have occurred independently on the evolutionary lineages of elephants and degu, suggesting that the AAV Rep protein has been co-opted repeatedly in the mammalian liver.

[Urachal Actinomycosis : A Case Report].

The patient was a 66-year-old woman who was examined by a local physician for the chief complaint of a mass palpable in the left lower abdomen. Abdominal plain computed tomography (CT) indicated a subcutaneous mass extending continuously from the apex of the bladder to the retropubic space, and she was referred to our medical department. Tumor markers were normal, and cystoscopic examination indicated no clear findings. Abdominal contrast-enhanced CT and plain abdominal magnetic resonance imaging results led to suspicion of actinomycosis. An open biopsy was performed on the subcutaneous mass, and subsequent histopathological testing led to a definitive diagnosis of actinomycosis. After 2 weeks of antibiotic therapy, the mass had diminished on CT. There has been no relapse approximately 24 weeks after discontinuation of the antibiotic therapy.

Cloning and characterization of bottlenose dolphin (Tursiops truncatus) interleukin-10.

The function of cytokines in cetaceans has so far only been determined for the proinflammatory cytokines. In this study, we cloned bottlenose dolphin (Tursiops truncatus) interleukin-10 (IL-10) cDNA from concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC), and investigated the mRNA expression levels in various tissues and the bioactivity of recombinant dolphin (rd) IL-10. The gene encodes a polypeptide of 178 amino acids which encompasses the mature protein sequence of 158 amino acids. Quantitative expression analysis of dolphin IL-10 revealed that the highest mRNA levels are found in the spleen. To assess its function, rdIL-10 was produced in human embryonic kidney 293 cells and its bioactivity was demonstrated through IL-10-induced inhibition of proinflammatory cytokine mRNA expression IFN-γ, TNF-α, and IL-2 of Con A-stimulated PBMC. These results indicated that the structure and function of bottlenose dolphin IL-10 is similar to that of other animals. This is the first report of the characterization of an anti-inflammatory cytokine in cetaceans.

Astrocytoma in an African hedgehog (Atelerix albiventris) suspected wobbly hedgehog syndrome.

A 28-month-old African hedgehog was referred to our hospital with progressive tetraparesis. On the first presentation, the hedgehog was suspected as having wobbly hedgehog syndrome (WHS) and the animal was treated with medication and rehabilitation. The animal died 22 days after onset. Pathological examination revealed that the animal was involved in astrocytoma between the medulla oblongata and the spinal cord (C1). This report indicates that a primary central nervous system tumor should be considered as one of the differential diagnoses for hedgehogs presenting with progressive paresis, together with WHS.

Molecular cloning and expression of bottlenose dolphin CD34.

In terrestrial mammals, the surface molecule CD34 is used as a marker to identify hematopoietic progenitor cells. To clarify whether CD34 expression can be used to confirm the undifferentiated state of hematopoietic-like cells isolated from the bone marrow of bottlenose dolphin, Tursiops truncates, we determined in this study the sequence of dolphin CD34 cDNA and analyzed its mRNA expression. Dolphin CD34 cDNA can be expressed as two forms, one that encodes a full-length version and a variant, truncated version of the gene. Both forms were detected in bone marrow mononuclear cells and in various tissues using RT-PCR. The truncated form was not detected in peripheral blood mononuclear cells, and neither form was detected in polymorphonuclear leukocytes. This is the first report on CD34 in marine mammals and our results suggest that dolphin CD34 may be a useful marker to identify hematopoietic progenitor cells.

Hematopoietic cell populations in dolphin bone marrow: Analysis of colony formation and differentiation.

Bone marrow biopsy is useful for diagnosis of hematopoietic diseases. We have recently reported that bone marrow biopsy from the flipper might be useful for diagnosis of hematopoietic diseases in dolphins. In this study, to demonstrate whether biopsy from the flipper is useful for clinical diagnosis, we investigated the gene expression profiles and proliferation and differentiation of bone marrow mononuclear cell (BMMC) isolated from the humeral bone marrow of bottlenose dolphins. BMMC exhibited gene expression profiles considered to be characteristic of hematopoietic cells. Similarly, a colony forming unit assay showed that dolphin BMMC possessed vigorous colony forming ability. The proliferation of hematopoietic progenitor cells resulted in the formation of three types of colonies, containing neutrophils, monocytes/macrophages and eosinophils with or without megakaryocytes, all of which could be identified based on the morphological characteristics and gene expression profiles typically associated with hematopoietic markers. Thus, dolphin BMMCs from humeral bone marrow contain many hematopoietic progenitor cells, and bone marrow biopsy from the flipper is suggested useful for clinical diagnosis for the dolphins.

Magnetic resonance imaging features and clinical signs associated with presumptive and confirmed progressive myelomalacia in dogs: 12 cases (1997-2008).

OBJECTIVE: To characterize clinical signs and findings from magnetic resonance imaging (MRI) and CSF analysis for dogs with progressive myelomalacia (PM) diagnosed on the basis of clinical and histologic features. DESIGN: Retrospective case series. ANIMALS: 5 dogs with confirmed PM and 7 with presumptive PM evaluated from 1997 through 2008. PROCEDURES: Medical records of study dogs were reviewed, and clinical signs and MRI and CSF findings were evaluated. A clinical diagnosis of PM was made on the basis of detection of disk-associated spinal cord compression via MRI and progression of clinical signs from initial paraparesis or paraplegia to thoracic limb lower motor neuron paresis to tetraplegia associated with cranial migration of the extent of cutaneous trunci reflex extinction and analgesia, terminating in death due to respiratory paralysis. RESULTS: All dogs were paraplegic and had signs of lower motor neuron lesions. As revealed by MRI of the vertebral column, the length of abnormal signal intensity was more than 6-fold, compared with the body length of L2. In some dogs, these abnormal MRI findings were observed before PM was clinically diagnosed. The CSF examination revealed hemorrhagic xanthochromia. CONCLUSIONS AND CLINICAL RELEVANCE: A hyperintense region of the spinal cord > 6 times the length of L2 on T2-weighted imaging might be a characteristic MRI finding of PM. In some dogs, such MRI findings were observed before PM was clinically diagnosed. Progressive myelomalacia may thus be diagnosable at the early stage through MRI and CSF examination before signs of PM develop.

Pathogenicity of different rabies virus isolates and protection test in vaccinated mice / Patogenicidade de diferentes isolados do vírus da raiva e teste de proteção em camundongos vacinados

This study was aimed to evaluate and compare the pathogenicity of rabies virus isolated from bats and dogs, and to verify the efficacy of a commercial rabies vaccine against these isolates. For evaluation of pathogenicity, mice were inoculated by the intramuscular route (IM) with 500MICLD50/0.03mL of the viruses. The cross-protection test was performed by vaccinating groups of mice by the subcutaneous route and challenged through the intracerebral (IC) route. Isolates were fully pathogenic when inoculated by the IC route. When inoculated intramuscularly, the pathogenicity observed showed different death rates: 60.0 percent for the Desmodus rotundus isolate; 50.0 percent for dog and Nyctinomops laticaudatus isolates; 40.0 percent for Artibeus lituratus isolate; 9.5 percent Molossus molossus isolate; and 5.2 percent for the Eptesicus furinalis isolate. Mice receiving two doses of the vaccine and challenged by the IC route with the isolates were fully protected. Mice receiving only one dose of vaccine were partially protected against the dog isolate. The isolates from bats were pathogenic by the IC route in mice. However, when inoculated through the intramuscular route, the same isolates were found with different degrees of pathogenicity. The results of this work suggest that a commercial vaccine protects mice from infection with bat rabies virus isolates, in addition to a canine rabies virus isolate.

O estudo avaliou e comparou as propriedades patogênicas de cinco isolados do vírus da raiva de morcegos e um isolado do vírus da raiva de cão e analisou a eficácia de vacina comercial contra estes isolados, em camundongos. Para o estudo de patogenicidade camundongos foram inoculados pela via IM com 0,1 mL contendo 500MICLD50/0,03mL das amostras de vírus. Quando inoculados pela via IC, os isolados do vírus da raiva provocaram a morte de 100 por cento dos camundongos. No entanto, 500MICLD50/0,03mL das mesmas amostras, inoculadas pela via IM, ocasionaram mortalidade de: 60,0 por cento quando a amostra era de Desmodus rotundus; 50,0 por cento de cão e de Nyctinomops laticaudatus; 40,0 por cento de Artibeus lituratus; 9,5 por cento de Molossus molossus; e 5,2 por cento de Eptesicus furinalis. Camundongos que receberam duas doses de vacina foram protegidos quando desafiados pela via IC, com todas as amostras testadas. Quando os camundongos receberam uma dose da mesma vacina, houve proteção parcial daqueles desafiados com a amostra de cão. Todos os isolados do vírus da raiva testados foram patogênicos para camundongos, inoculados pela IC. No entanto, pela via IM, os mesmos isolados mostraram diferentes graus de patogenicidade. Concluiu-se também que a vacina comercial contra raiva protegeu os camundongos desafiados com amostras de vírus isolados de morcegos e de cão.

Low genetic diversities of rabies virus populations within different hosts in Brazil.

The low rates of nonsynonymous evolution observed in natural rabies virus (RABV) isolates are suggested to have arisen in association with the structural and functional constraints operating on the virus protein and the infection strategies employed by RABV within infected hosts to avoid strong selection by the immune response. In order to investigate the relationship between the genetic characteristics of RABV populations within hosts and the virus evolution, the present study examined the genetic heterogeneities of RABV populations within naturally infected dogs and foxes in Brazil, as well as those of bat RABV populations that were passaged once in suckling mice. Sequence analyses of complete RABV glycoprotein (G) genes showed that RABV populations within infected hosts were genetically highly homogeneous whether they were infected naturally or experimentally (nucleotide diversities of 0-0.95x10(-3)). In addition, amino acid mutations were randomly distributed over the entire region of the G protein, and the nonsynonymous/synonymous rate ratios (d(N)/d(S)) for the G protein gene were less than 1. These findings suggest that the low genetic diversities of RABV populations within hosts reflect the stabilizing selection operating on the virus, the infection strategies of the virus, and eventually, the evolutionary patterns of the virus.

Bone marrow biopsy from the flipper of a dolphin.

To find macroscopically palpable bone marrow cavities in dolphins is difficult because of their extremely retrogressive limbs and pelvis and because they do not contain abundant modular cavities (as in terrestrial mammals) that can serve as sites for bone marrow biopsies. Three-dimensional computed tomography analysis of dolphin skeletons suggests that bone marrow could be harvested from the humerus and radius. In this report, post-mortem paracentesis of the humerus from a captive rough-toothed dolphin using a biopsy needle provided a marrow preparation containing myelocytes, erythroblasts and megakaryocytes. This type of bone marrow collection from the flipper might be useful for clinical diagnostic work in cetaceans.

Estudo imunológico e genético de 10 isolados do vírus da raiva de morcegos do Rio de Janeiro, Sudeste do Brasil / Immunological and genetic study of 10 bat rabies virus isolates from Rio de Janeiro State, Southeast

O presente trabalho visou estudar 10 isolados de vírus da raiva de morcegos hematófagos e não-hematófagos do Estado do Rio de Janeiro em suas características genéticas quanto aos genes N e G, além da resposta de camundongos vacinados com a vacina anti-rábica produzida pela replicação da amostra Pitman-Moore em cultivo celular, frente ao desafio com estes isolados virais, utilizando-se um ensaio imunológico baseado no teste de potência NIH. A vacina anti-rábica utilizada na imunização dos camundongos ofereceu proteção em mais de 80% dos camundongos vacinados com a diluição 1:5 da vacina, frente à maioria dos isolados. A análise filogenética do gene da proteína N apresentou um padrão de agregação dividido em variante de morcego hematófago e variante de morcego insetívoro, com todos os isolados de morcegos frugívoros Artibeus sp. tendo sido segregados com a variante característica de morcegos Desmodus rotundus. Foram observadas diferenças filogenéticas entre as variantes do vírus da raiva de morcego hematófago isoladas na Região Noroeste do Estado do Rio de Janeiro e aquelas isoladas nas Regiões Metropolitana e Sul do Estado. A substituição do resíduo ácido aspártico por ácido glutâmico na posição 118, encontradas na caracterização genética da proteína G dos isolados 704/97BR-DR e 151/98BR-DR, permite inferir que esta posição esteja relacionada à antigenicidade viral. Não foram observadas diferenças genéticas temporais entre os isolados estudados. A vacina anti-rábica utilizada ofereceu proteção satisfatória contra a maioria dos isolados estudados.

In the present study we analyzed 10 bats rabies viruses isolated from Rio de Janeiro State, focusing on its genetic characteristics from genes N and G, and also in the response of mice vaccinated with cell-culture rabies vaccine, produced with the Pitman-Moore strain, after viral challenge with bat rabies isolates, using an immunologic essay based on NIH vaccine potency test. The vaccine used conferred protection in more than 80% of the mice vaccinated with 1:15 vaccine dilution, after viral challenge. N gene genetic analysis divided the rabies virus isolates into haematophagous and insectivorous bat variants, with all isolates from Artibeus sp. frugivorous bats being clustered with the variant characteristic of the Desmodus rotundus vampire bat. Phylogenetic differences between isolates from Northeast Region and those from the Metropolitan and South Regions of Rio de Janeiro State were observed. The substitution of an aspartic acid to a glutamic acid found in the position 118 of G gene genetic characterization from samples 704/97BR-DR and 151/98BR-DR seems to be related to viral antigenicity. There were no time-related genetic differences between the studied samples. The vaccine employed was found with satisfactory protection against the majority of the isolates used.

Magnetic resonance imaging and computed tomography findings of vertebral osteosarcoma in a cat.

An 8-year-old female Persian cat with a gait disorder was brought to our hospital. Pelvic limb mobility had gradually reduced over the preceding 3 months, then rapidly deteriorated 2 weeks before consultation. Signs also occurred in the thoracic limbs. With a tentative diagnosis of neural disease, magnetic resonance imaging and computed tomography were performed. T1-weighted imaging showed isointensity in the seventh cervical vertebra, while T2-weighted imaging revealed hypointensity. Contrast-enhanced T1-weighted imaging revealed a uniformly enhancing mass. Extirpation of the mass relieved the clinical signs, leading to disappearance of the neurological signs. The histopathological examination suggested osteosarcoma.

Quantitative analysis of inflammatory cytokines expression in peripheral blood mononuclear cells of the ferret (Mustela putorius furo) using real-time PCR.

This study describes the expression pattern of cytokines, interferon-gamma (IFN-gamma), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and IL-10, produced by LPS stimulation in peripheral blood mononuclear cells (PBMCs) of the ferret (Mustela putorius furo). Real-time PCR was used with TaqMan probes, which were modified by dual-labeled probes (TAMRA/FAM), quantitative analysis of cytokine mRNA comparing the cytokine with the housekeeping gene, ferret GAPDH, as the relative C(t) value. Expression peaks of IFN-gamma, TNF-alpha, IL-6 mRNA occurred 2 h after LPS stimulation, whereas the IL-10 peak was 8 h post-LPS. In the present study, peak cytokine expression was detected within 8 h, similar to several other mammalian studies. This current study provides baseline information on inflammatory cytokines of ferret PBMCs.

Production of monoclonal antibody specific for bottlenose dolphin neutrophils and its application to cell separation.

The authors produced a monoclonal antibody (mAb) against dolphin neutrophils by fusing mouse myeloma cells with lymph node cells from a Wistar rat immunized with bottlenose dolphin (Tursiops truncatus) polymorphonuclear leukocytes (PMNs). This mAb (DN1) was reactive against 77.1 +/- 8.6% of dolphin peripheral blood PMN by flow cytometric analysis; furthermore, there was no cross-reactivity with human or bovine leukocytes. The DN1-positive cells isolated with a sorting cytometer were almost all (99.7%) neutrophils. By using DN1 in conjunction with magnetic-activated cell sorting (MACS), the authors isolated neutrophils and eosinophils from density gradient-fractionated PMN with 100% and 95.6 +/- 4.8% purities, respectively. These results suggest that this mAb specific for bottlenose dolphin neutrophils is useful as a potential reagent to study bottlenose dolphin neutrophils and eosinophils.

Experimental infection of vampire bats Desmodus rotundus (E. Geoffroy) maintained in captivity byfeeding defibrinated blood added with rabies vírus / Infecção experimental de morcegos hematófagos Desmodus rotundus(E. Geoffroy) mantidos em cativeiro e alimentados com sangue desfibrinado adicionado de vírus da raiva

In vampire bats, food sharing behavior would contribute for the oral transmission of rabies virus among the roostmates. To test this hypothesis, 10 captive Desmodus rotundus bats were fed defibrinateds wine blood containing mice brain suspension of PV-strain of rabies virus. Other 10 bats were fed blood mixed with a mice brain suspension of T-9/95 vampire-bat-field isolate of rabies virus. Another group of 10 bats was inoculated intramuscularly with a mice brain suspension of the T-9/95 isolate. Other 20 bats were maintained without treatment and fed defibrinated swine blood for 158 days. All animals found dead during the observation period or those sacrificed at the end of the experiment were necropsied and specimens such as the brain and non-nervous tissues were collected for rabies examination. Four bats inoculated intramuscularly developed clinical rabies, with signs lasting 1-2 days, and the survival periods ranged from 11-14 days. The initial rabies diagnosis was based on direct fluorescent antibody (dFA) and mouse inoculation test (MIT) performed only on brain specimens, and subsequently, brains andthe non-nervous materials were further reexamined by means of dFA, MIT and heminested-polymerase chain reaction (ht-PCR)technique. The intake of the PV-strain caused rabies in 2 bats, with survival period of 25 and 32 days, while the three bats ingesting theT-9/95 isolate presented periods of 26-31 days. Although discrepant results were found among the diagnostic tests, viruses have disseminated to the central nervous system and other organs, as seenin bats inoculated intramuscularly

Em morcegos hematófagos, o hábito de compartilhar alimentopoderia contribuir na transmissão oral do vírus da raiva. Para verificar esta hipótese, 10 morcegos Desmodus rotundus em cativeiro foram alimentados com sangue suíno desfibrinado, contendo suspensão de cérebros de camundongos infectados com vírus rábico PV. Outros 10 camundongos receberam sangue contendo suspensão cerebral de camundongos infectados com vírus de morcego hematófago (T-9/95). Um grupo de 10 camundongos foi inoculado intramuscularmente com suspensão de vírus T-9/95. Outros 20 morcegos foram mantidossem tratamento e alimentados com sangue desfibrinado por 158 dias. Todos os animais encontrados mortos durante o período de observação ou sacrificados no final do experimento foram necropsiados e os cérebros e órgãos não-nervosos foram colhidos para a confirmação da raiva. Quatro morcegos inoculados intramuscularmente apresentaram raiva clínica, com sinais persistind opor 1-2 dias e os períodos de sobrevivência variaram de 11-14 dias. O diagnóstico da raiva inicialmente foi realizado somente com os fragmentos do cérebro, submetendo-os às provas de imunoflurescência direta (IFD) e inoculação em camundongos (IC).Subseqüentemente, os cérebros e os órgãos não-nervosos foram reexaminados com as técnicas de IFD, IC e heminested-polymerase chain reaction (ht-PCR). A ingestão do vírus PV causou raiva em dois morcegos, com período de sobrevivência de 25 e 32 dias, enquanto que os três morcegos que ingeriram o isolado T-9/95 apresentaram períodos de 26-31 dias. Embora encontrando resultados discrepantes entre as técnicas diagnósticas utilizadas, os vírus ingeridos pelos morcegos foram detectados no sistema nervoso central e outros órgãos não-nervosos, como nos morcegos inoculados intramuscularmente

Molecular cloning and phylogenetic analysis of inflammatory cytokines of the ferret (Mustela putorius furo).

The present study determined the cDNA and deduced amino acid sequences of ferret (Mustela putorius furo) inflammatory cytokines, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor (TNF)-alpha. The homologies of the nucleotide sequences of IFN-gamma, IL-1beta, IL-6, IL-8 and TNF-alpha of the ferret to those from other mammalian species ranged from 64.3-92.9%, 73.0-83.9, 58.1-84.8%, 58.1-89.7% and 79.0-95.0%, respectively. As distinctive amino acid residues constituting various motifs and ligand-binding sites and cysteine residues were highly conserved in ferret inflammatory cytokine proteins, ferret cytokines may have fundamentally similar functions to those of other mammals. Phylogenetic analyses based on the deduced amino acid sequences revealed that all ferret inflammatory cytokines were more closely related to those of the Carnivora order, specifically dog and cat, than to other species.

Genetic analysis of phosphoprotein and matrix protein of rabies viruses isolated in Brazil.

To investigate the genetic characteristics of phosphoprotein (P) and matrix protein (M) genes of variable rabies virus (RV) prevalent in Brazil, the authors genetically characterized the P and M genes from 30 Brazilian RV field isolates. Phylogenetic analysis based on the P and M genes revealed the presence of six RV variants that consisted primarily of three insectivorous bats, the vampire bat, dog and fox in Brazil. Specific amino acid substitutions corresponding to these phylogenetic lineages were observed, with Asp(42) and Glu(62) in the P protein found to be characteristic of Brazilian chiroptera- and carnivora-related RVs, respectively. Amino acid sequence motifs predicted to associate with a viral function in the P and M proteins were conserved among Brazilian RV variants.

Experiments on intramuscular inoculation and feeding domestic cats (Felis catus) with brains of mice previously infected by rabies viruses

Nineteen kittens divided into four groups were fed with brains of mice infected with rabies viruses. Each four kittens (group I) received four brains infected with the PV fixed strain; nine kittens (group II) ingested 4-5 brains infected with the field isolate T-9/95, isolated from the Desmodus rotundus vampire bat; two kittens (group III) fedten T-9/95-infected brains, and four cats consumed 32-37 PV strain infected brains. One adult male, inoculated into masseter muscle with a 20% T-9/95-infected brain suspension, presented rabies after an incubation period of six days, followed with 8 days of clinical evolution, and died there after and this cat was considered as the rabies “positive standard”. After observing for 20-230 days, all the cats feeding the rabid brains were submitted to euthanasia, by using Acepran®, Zoletil®,and T-61®. At necropsy, samples of brain, heart, lung, kidney, submaxillary salivary gland, and cervical medulla were collected from all the cats and further submitted to the direct fluorescence antibodytest (dFA), mouse inoculation test (MIT) and to the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Brain, cervical medulla, and the submaxillary salivary gland of the positive standard cat were dFA-positive, and brain and cervical medulla were positive for MIT. All specimens of this cat tested by the RT-PCR were found positive. No animals ingesting PV or T-9/95 virus-infectedbrains developed clinical signs and all materials tested were negativeby dFA and MIT. Several specimens, however, showed positive reactions by the RT-PCR technique, but cats were resistant to rabies through the viruses administered orally.

Dezenove gatos, divididos em quatro grupos, foram alimentadoscom cérebros de camundongos infectados com vírus de raiva. Cada um dos quatro gatos (grupo I) receberam quatro cérebros infectados com vírus fixo PV; nove gatos (grupo II) ingeriram 4-5 cérebros infectados com uma amostra de campo T-9/95, isolada do morcego Desmodus rotundus; dois gatos (grupo III) ingeriram 10 cérebros infectados com T-9/95 e quatro gatos (grupo IV) ingeriram 32-37 cérebros infectados com vírus PV. Um macho adulto, inoculado no músculo masséter, com uma suspensão cerebral a 20% da amostra T-9/95, desenvolveu raiva após período de incubação de seis dias,seguidos por oito dias de evolução clínica, morrendo em seguida. Este gato foi denominado de “padrão positivo”. Após observação por um período de 20-230 dias, todos os gatos que receberam cérebros foram submetidos à eutanásia, utilizando Acepran®, Zoletil® e T-61®. À necropsia, foram colhidas amostras do cérebro, coração, pulmão,rim, glândula salivar submaxilar e medula cervical e submetidas à prova de imunofluorescência direta (IFD), inoculação em camundongos (IC), e reação em cadeia pela polimerase-transcriptase reversa (RT-PCR). No “padrão positivo”, cérebro, medula cervical eglândula salivar foram positivos à IFD e à IC, cérebro e medula cervical foram os positivos. Todos os espécimes do “padrão positivo” foram positivos à RT-PCR. Nenhum animal que ingeriu cérebros contendo amostras de vírus PV ou T-9/95 apresentou sinais clínicos e todos osespécimes testados foram negativos à IFD e IC, no entanto, alguns espécimes reagiram positivamente à RT-PCR, porém, os gatos foram resistentes à raiva com vírus administrados oralmente. Master thesis submitted to the Faculty of Veterinary Medicine and Zootechny of the University of São Paulo, on June 24th, 2003. Fellowship from the Fundação de Amparo à Pesquisa do Estado de São Paulo-FAPESP, process No. 01/07188-8.