Youko Shoji

An improved method for recovering rabies virus from cloned cDNA.

A new system for recovery of rabies virus from cDNA plasmid, the transcription of which was driven by cellular RNA polymerase II, was developed. The plasmid contains full-length viral cDNA flanked by hammerhead ribozyme and hepatitis delta ribozyme sequences, arranged downstream of the cytomegalovirus (CMV) promotor. Transfection with the full-length cDNA plasmid together with helper plasmids encoding viral N, P, and L proteins without supply of T7 RNA polymerase produced a recombinant rabies virus in several cell lines. The efficiency of recovery between the conventional T7 promotor system and the new CMV promotor system was compared using these plasmid constructs. The newly established system is applicable to various cell lines and allows rapid and efficient generation of recombinant rabies virus.

Discrimination between dog-related and vampire bat-related rabies viruses in Brazil by strain-specific reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism analysis

BACKGROUND There is a geographical overlap between the two main rabies epidemiological cycles maintained by dogs and vampire bats in Latin America. The geographical and temporal coincidence of rabies outbreaks of respective origins is not unusual in rural areas of Latin America. These circumstances make it difficult to discriminate the intraspecies and interspecies transmission pathways of rabies. OBJECTIVE This study was conducted to develop techniques to discriminate dog-related and vampire bat-related rabies virus isolates (DRRV and VRRV, respectively) in Brazil. STUDY DESIGN The 1396 nucleotides of the nucleoprotein gene of a total of 27 DRRV and VRRV were sequenced. Strain-specific (SS) primers were developed based on these sequences. Forty-nine rabies virus strains isolated from animals and humans in several parts of Brazil were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) with SS primers. These rabies viruses were also amplified by RT-PCR with general rabies primers and the PCR products were cut by three restriction enzymes, Blp I, Bsu36 I and BspE I. RESULTS All the DRRV and VRRV were distinguished by RT-PCR with SS primers. The PCR products obtained from DRRV were cut at one site by Blp I, but not by Bsu36 I. The PCR products obtained from VRRV were cut at one or two sites by Bsu36 I, but not by Blp I. Blp I and Bsu36 I clearly discriminated DRRV and VRRV in restriction fragment length polymorphysim (RFLP) assays. The results of SS RT-PCR and RFLP were consistent. CONCLUSION SS RT-PCR and RFLP assays have been developed for determining the origins of rabies virus isolates in Brazil. These assays are simple and rapid, and will be useful for identifying the rabies virus reservoirs of field isolates in Brazil, especially when used together.

Molecular epidemiology of rabies from Maranhão and surrounding states in the northeastern region of Brazil.

Summary.Although many outbreaks of rabies have been reported in northern Brazil, few epidemiological studies of these outbreaks have been undertaken. In this study, molecular epidemiological analyses were performed using 41 rabies virus samples isolated in the Maranhão (MA), Pará (PA), and Tocantins (TO) states of northeastern Brazil. A 599-bp region of the glycoprotein (G) gene was first amplified from each sample by RT-PCR, then sequenced and subjected to phylogenetic analysis. A phylogenetic tree divided the 41 isolates into two clades: Clade I was associated with terrestrial carnivores and Clade II was associated with vampire bats. The Clade I isolates were further sub-divided into two groups. The first group was closer to carnivore isolates that predominate in central Brazil, whereas the second group more closely resembled wild fox isolates from the northeastern coastal state of Paraíba (PB). MA isolates of Clade II formed an entirely separate group. These results demonstrate that bat- and dog-transmitted rabies occur in northwestern Brazil.

Molecular cloning and functional characterization of bottlenose dolphin (Tursiops truncatus) tumor necrosis factor alpha

Bottlenose dolphin tumor necrosis factor alpha (doTNF-alpha) cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) and the nucleic and deduced amino acid sequences were determined. The sequence of the cDNA clones shows that doTNF-alpha has an open reading frame of 699bp encoding 233 amino acids. The nucleic acid sequence of doTNF-alpha indicates 90, 88, 87, and 79% similarity with the cattle, pig, human, and mouse TNF-alpha gene, respectively. Based on the analysis of human and mouse TNF-alpha molecules, doTNF-alpha is processed to a mature protein with 157 amino acids. The 233 amino acids precursor has a hydrophobic region that could serve as a transmembrane domain. The recombinant doTNF-alpha expressed in Escherichia coli as a glutathione S-transferase fusion protein reacted with anti-human TNF-alpha antibody and exerted cytotoxity to the TNF-alpha sensitive murine cell line L929.

Pathogenicity of different rabies virus isolates and protection test in vaccinated mice

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 500MICLD₅₀/0.03 mL 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% for the Desmodus rotundus isolate; 50.0% for dog and Nyctinomops laticaudatus isolates; 40.0% for Artibeus lituratus isolate; 9.5% Molossus molossus isolate; and 5.2% 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.

Priming of dolphin neutrophil respiratory burst by recombinant tumor necrosis factor alpha

We studied the effects of recombinant dolphin tumor necrosis factor alpha (rdoTNFalpha) on the respiratory burst activity of dolphin neutrophils. rdoTNFalpha enhanced the luminol-dependent chemiluminescence response of dolphin neutrophils induced by concanavalin-A, opsonized zymosan, and heated plasma, but not that induced by phorbol myristate acetate. The TNF-associated priming activity was concentration- and preincubation time-dependent, and heat-instable. These data suggest that, as in human neutrophils, TNFalpha enhances the respiratory burst in dolphin neutrophils that follows short-term incubation with various receptor-mediated agonists.