[Epidemiology of rabies virus and other lyssaviruses].

Rabies is a zoonosis that infects domestic and wild animals through close contacts with saliva from infected animals. The annual number of deaths worldwide caused by rabies is estimated approximately 55,000 by World Health Organization (WHO). There has been no indigenous rabies case in Japan since 1957; however, there was only one imported case, a traveler who was bitten by a stray dog in Nepal and died in 1970. Dogs in Asia and Africa remain the main reservoir and transmitter of rabies to humans. The others are mainly coyotes, foxes, jackals, mongooses, raccoons, skunks, wolves and bats. The efficacy of the current human and veterinary vaccines against emergent lyssaviruses should be evaluated because the newly discovered rabies-related viruses have been isolated from bats.

Bat lyssaviruses (Aravan and Khujand) from Central Asia: phylogenetic relationships according to N, P and G gene sequences.

Bat lyssaviruses Aravan and Khujand were isolated in southern Kyrgyzstan in 1991 and in northern Tajikistan in 2001, respectively. Preliminary studies with anti-nucleocapsid monoclonal antibodies suggested that the viruses were distinct from other lyssavirus serotypes. These data were supported by sequencing of the N gene of Aravan virus. In the present study, we sequenced the entire N, P and G genes of both Aravan and Khujand viruses and compared them with respective sequences of other lyssaviruses available from GenBank. The results suggested that each virus should be considered as a newly recognized genotype according to the current approaches for genotype definition (amount of nucleotide identity of the N gene and bootstrap support of joining to certain phylogenetic groups). Use of different phylogenetic methods and comparison of different parts of the genomes generally suggested that Khujand virus was mainly related to genotype 6, while Aravan virus, on the one hand, was related to Khujand virus, and, on the other hand, demonstrated moderate similarity to genotypes 4, 5 and 6. The potential significance of these new lyssaviruses for veterinary and public health should not be underestimated.

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.

[Phylogenetic analysis of two rabies viruses, Takamen and Komatsugawa strains isolated in Japan in the 1940's].

The entire coding region of the nucleoprotein (N) gene of Takamen and Komatsugawa strains of rabies virus isolated in Japan in 1940's were determined. Phylogenetic analysis was performed on 140 lyssaviruses (128 viruses of genotype 1 and 12 lyssaviruses of other genotypes) isolated in various parts of the world, including the two Japanese rabies strains, based on the sequences of 1,350 nucleotides of the N gene. The rabies viruses were divided into 12 distinct clusters at least, reflecting geographical areas and hosts as reservoirs. The Takamen, Nishigahara, and RC-HL strains derived from the Nishigahara strain were grouped into the same cluster as the Chinese strain (3aG) in the worldwide distribution group. The Komatsugawa strain was grouped into the same cluster as the viruses from a raccoon dog from Khabarovsk, and from a steppe fox in area of Lake Baikal in Russia in a group consisting of Canada, Greenland, and the Arctic. These data along with the historical evidence suggest that Japanese rabies viruses, the Takamen and Komatsugawa strains, belong to two different clusters and moved into Japan from China and Russia, respectively.

New lyssavirus genotype from the Lesser Mouse-eared Bat (Myotis blythi), Kyrghyzstan.

The Aravan virus was isolated from a Lesser Mouse-eared Bat (Myotis blythi) in the Osh region of Kyrghyzstan, central Asia, in 1991. We determined the complete sequence of the nucleoprotein (N) gene and compared it with those of 26 representative lyssaviruses obtained from databases. The Aravan virus was distinguished from seven distinct genotypes on the basis of nucleotide and amino acid identity. Phylogenetic analysis based on both nucleotide and amino acid sequences showed that the Aravan virus was more closely related to genotypes 4, 5, and--to a lesser extent--6, which circulates among insectivorus bats in Europe and Africa. The Aravan virus does not belong to any of the seven known genotypes of lyssaviruses, namely, rabies, Lagos bat, Mokola, and Duvenhage viruses and European bat lyssavirus 1, European bat lyssavirus 2, and Australian bat lyssavirus. Based on these data, we propose a new genotype for the Lyssavirus genus.

Antibody responses induced by immunization with a Japanese rabies vaccine determined by neutralization test and enzyme-linked immunosorbent assay.

The immunogenicity of a Japanese purified chick embryo cell culture rabies vaccine (PCECV) was examined. Serum samples were obtained from 86 subjects after pre-exposure or post-exposure prophylaxis. Rabies antibody titres were determined by neutralization test and enzyme-linked immunosorbent assay (ELISA). Titres higher than 0.5 international units (IU)/ml were demonstrated by neutralization test in all the 19 subjects after three-time pre-exposure immunization on days 0, 30 and 180. Titres higher than 0.5IU/ml were also demonstrated by neutralization test in all the 23 subjects after four-time post-exposure immunization on days 0, 3, 7 and 14. There was a correlation between neutralization and ELISA antibody titres (r=0.697); however, neutralization titers were higher than ELISA titres for most of the samples. The results suggest that current Japanese rabies vaccine induces recommended levels of neutralizing antibodies after pre- and post-exposure prophylaxes.