Genetic characterization and geographic distribution of rabies virus isolates in Brazil: identification of two reservoirs, dogs and vampire bats.

We analyzed 50 rabies virus samples isolated in Brazil from 12 dogs, 11 cats, 5 vampire bats, 15 cattle, 2 horses, 1 pig, 1 sheep, and 3 humans to investigate the molecular epidemiology of rabies viruses. We sequenced 203 nucleotides on the nucleoprotein gene by direct sequencing of the PCR-amplified products. All the isolates belonged to the genotype 1 and homology of the 203 nucleotides was at least 83.7% among isolates. The main reservoirs were estimated based on the homology of nucleotide sequences. Brazilian rabies virus isolates were clustered into two reservoir groups: dogs and vampire bats. All the dog-related rabies virus isolates showed nucleotide homology greater than 99.0%. Vampire bat-related rabies virus isolates showed nucleotide homology greater than 96.6% and could be further divided into subgroups corresponding to areas where viruses were isolated. These data suggest that circulating rabies variants belong to at least two different genotype clusters in Brazil and that these two clusters are maintained independently among vampire bats and dogs.

Detection of rabies virus RNA isolated from several species of animals in Brazil by RT-PCR.

Brain samples from different animal species including humans: five vampire bats, 14 cattle, 12 dogs, 11 cats, two horses, one pig, one sheep and three humans collected from various geographical regions of Brazil were found to be positive for rabies by means of the fluorescent antibody test (FAT) and the mouse inoculation test (MIT). The brain samples were retested for rabies by means of the reverse transcription and polymerase chain reaction (RT-PCR) with 2 primer sets (P1/P2 and RHNI/RHNS3), which amplified full or partial regions on the nucleoprotein (N) gene of the rabies virus, respectively. Brain samples from five vampire bats, 13 cattle, one horse and one sheep failed to yield PCR products when the RHN1/RHNS3 primer pair was used, but all brain samples successfully yielded the products when the P1/P2 primer pair was used. These results suggest that Brazilian rabies virus isolates could be principally divided into two populations according to genetic difference.

Characterization of Sri Lanka rabies virus isolates using nucleotide sequence analysis of nucleoprotein gene.

Thirty-four suspected rabid brain samples from 2 humans, 24 dogs, 4 cats, 2 mongooses, I jackal and I water buffalo were collected in 1995-1996 in Sri Lanka. Total RNA was extracted directly from brain suspensions and examined using a one-step reverse transcription-polymerase chain reaction (RT-PCR) for the rabies virus nucleoprotein (N) gene. Twenty-eight samples were found positive for the virus N gene by RT-PCR and also for the virus antigens by fluorescent antibody (FA) test. Rabies virus isolates obtained from different animal species in different regions of Sri Lanka were genetically homogenous. Sequences of 203 nucleotides (nt)-long RT-PCR products obtained from 16 of 27 samples were found identical. Sequences of 1350 nt of N genes of 14 RT-PCR products were determined. The Sri Lanka isolates under study formed a specific cluster that included also an earlier isolate from India but did not include the known isolates from China, Thailand, Malaysia, Israel, Iran, Oman, Saudi Arabia, Russia, Nepal, Philippines, Japan and from several other countries. These results suggest that one type of rabies virus is circulating among human, dog, cat, mongoose, jackal and water buffalo living near Colombo City and in other five remote regions in Sri Lanka.

Molecular epidemiology of rabies in Thailand.

For the purpose of making clear the dynamics of rabies viruses that are prevalent among dogs in Asia, especially Thailand, nucleoprotein (N) genes of isolates derived from Thailand were partially sequenced, and a phylogenetic analysis was performed on the basis of the sequencing data. Firstly, all 27 isolates from Thailand belonged to one group that was distantly related to an isolate from China and was separated into at least six lineages. On the other hand, the isolate from Japan was related to viruses from the Arctic. Secondly, in order to analyze the diversity of the N gene more conveniently, restriction fragment length polymorphism (RFLP) analysis was performed on the N gene of 27 isolates from Thailand. The RFLP analysis could distinguish the lineages of each isolate, and the lineages of additional 34 isolates were deduced by this method. On examination of the geographical distribution of the six lineages, based on the results of phylogenetic and RFLP analyses, it was clear that infection cycles of the rabies virus in Thailand have tended to be maintained endemically.

Nucleoprotein gene analysis of fixed and street rabies virus variants using RT-PCR.

A simple and rapid single-step reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the nucleoprotein (N) gene of 11 rabies viruses. A conserved set of RT-PCR primers was designed to amplify the most variable region in the N gene. N gene regions were amplified from 6 fixed laboratory viruses, 4 street viruses from dogs in Thailand, and a horse in Zambia. Sequences of the amplified products, together with the database of 91 additional sequences, were analyzed by using PILEUP program of the GCG package. The rabies viruses grouped into at least 9 distinct clusters by < 90% nucleotide similarity of the N gene region: I (4 isolates, USA), II (2 isolates, South America), III (3 isolates, Africa), IV (52 strains, Europe, Middle East, Africa and South America), V (16 isolates, North America and Arctic), VI (17 isolates, Africa), VII (1 isolate, Africa), VIII (6 isolates, Thailand and Malaysia) and IX (1 isolate, Sri Lanka). A unique group of rabies viruses from Thailand and clusters of isolates corresponding to their geographic origin also were determined. The simple and rapid single-step RT-PCR proved to be useful for identifying rabies viruses, and for grouping the viruses into clades by sequence analysis.

Comparative studies on chick embryo cell rabies vaccine and human diploid cell vaccine: pre-exposure use and purity of the vaccines.

Neutralizing antibody responses to commercial Japanese-produced chick embryo cell (CEC) vaccines in Japanese and to commercial human diploid cell rabies vaccines (HDCV) in Filipinos were compared by the rapid fluorescent focus inhibition test (RFFIT). Neutralizing antibody titers of the Japanese after subcutaneous (sc) immunization with two doses of the CEC vaccines were not lower than those of the Filipinos with two or three doses of HDCV by intramuscular (i.m.) or intradermal (i.d.) route, respectively. Protein nitrogen content of the CEC vaccine was about 1/300th that of the HDCV. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis showed that non-viral protein content in the CEC vaccine was much lower than that in the HDCV. The CEC rabies vaccine seems to be as safe and effective as the HDCV for human pre-exposure immunization.

Studies on Japanese-produced chick embryo cell culture rabies vaccines.

We studied the potency, antibody response, and side reactions of commercial Japanese chick embryo cell (CEC) rabies vaccines for humans. The CEC rabies vaccines had indexes of 10(5.1) and 10(6.0) in Habel tests, and have had potencies higher than those of the International Reference Vaccine II by National Institutes of Health (NIH) tests. Thirty healthy adults received 1 ml of the CEC rabies vaccines subcutaneously as primary immunization on days 0 and 7. Between six and 12 months after the primary immunization, 22 of the 30 subjects showed neutralizing antibody levels greater than or equal to 1:40 by the rapid fluorescent focus inhibition test (RFFIT). The 30 subjects had been given booster immunizations of the CEC rabies vaccines at 8-14 months in addition to the primary immunization. Six to 12 months after the booster immunizations, 27 of the 30 subjects showed antibody levels greater than or equal to 1:40. No severe side reactions were reported during the course of vaccination. Thus we conclude that CEC rabies vaccine is effective and safe for pre-exposure immunization.

Inhibitory effect of a new antibiotic, guanine 7-N-oxide, on the replication of several RNA viruses.

Guanine 7-N-oxide (G-7-Ox) was examined for its antiviral activity against 9 viruses based on plaque reduction, neuraminidase activity reduction, a fluorescent antibody technique or ELISA. The following viruses were included in the tests: influenza, Sendai, simian virus 5 (SV5), respiratory syncytial, western equine encephalitis, Japanese encephalitis, vesicular stomatitis, rabies and polio. G-7-Ox showed broad anti-RNA viral activity against all viruses tested, except for poliovirus. Inhibition of persistent SV5 infection by G-7-Ox indicates that its antiviral activity is independent of cytotoxicity.

Further studies on an improved haemagglutination inhibition test with higher sensitivity for rabies virus antibody.

The efficiency of the removal of non-specific inhibitors of rabies virus haemagglutinin by treatment with colloidal silicic acid, which was proposed in an earlier study, was examined in a number of test samples. The non-specific inhibitors were removed in 289 out of 296 normal human sera (97.6%) by this treatment to a level that was undetectable at the 1:4 starting dilution in the haemagglutination inhibition test. Antigenic differences among three strains of rabies virus were detected in the haemagglutination inhibition test and the antibody titres to the homologous antigens were apparently higher than those to heterologous antigens.

Virulence of chick embryo fibroblast-passaged flury HEP rabies virus and its revertants in mice.

Chick embryo fibroblast-passaged Flury high egg passage (HEP) rabies virus failed to kill nude mice or cyclophosphamide-treated mice when inoculated intracerebrally. The virus regained neurovirulence for adult mice after three passages in mouse neuroblastoma C1300 cells (NA cells). However, even after 20 passages in NA cells, the virulence could not be increased to the level shown by the virus passaged several times in suckling mice. Some physiological and biological properties of the virus showing and not showing mouse virulence after five serial passages and after one single passage in NA cells, respectively, were compared.

Structural proteins of fixed rabies virus in the suckling mouse brain.

Structural proteins of fixed rabies virus grown in the suckling mouse brain were analysed by SDS-polyacrylamide gel electrophoresis (PAGE). The relative content of G protein to L, N and M1 proteins of brain-grown virus was much lower when compared with that of the virus grown in chick embryo fibroblasts. The results indicate that nucleocapsid complexes, consisting of L, N and M1 proteins were the predominant antigens accumulating in the brain.

Influence of cell type and virus upon lysis of rabies virus-infected cells by antibody and complement.

Mouse neuroblastoma (MNB) cells infected with ERA virus were specifically lysed in the presence of rabbit complement by antisera produced in mice to challenge virus standard (CVS), ERA, Flury HEP or street virus (SV) strains of rabies. MNB or EL-4 cells persistently infected with ERA, MNB cells infected with CVS, and BHK-21/S13 cells infected with ERA or Flury HEP also were suitable targets. CER cells infected with either ERA, CVS or Flury HEP, BHK-21/S13 cells infected with CVS and MNB cells infected with Flury HEP were not suitable targets. Two unusual findings indicated that 1. some cells which were greater than 80 percent positive for rabies viral membrane antigen(s) were poorly lysed, and 2. some cells that expressed cytoplasmic antigen lacked detectable membrane antigens.

Demonstration of non-infectious hemagglutinating particles of rabies virus and isolation of the hemagglutinin by disruption of the virion with Nonidet P-40.

Non-infectious hemagglutinating particles of rabies virus accumulated in the fluid phase of chick embryo cell cultures at 6 days post-infection, though they were undetectable at 4 days. They were characterized as looped filaments resembling viral envelope as revealed by electron microscopy. Another form of hemagglutinin (HAnin) was obtained by solubilization of partially purified virions with Nonidet P-40 (NP-40) followed by successive high speed and CsCl density gradient centrifugations. The density of the isolated HAnin averaged 1.28 g/cm3. SDS-polyacrylamide gel electrophoresis of the HAnin demonstrated that it was mainly composed of a glycoprotein (G) with a molecular weight of 83,000. Electron microscopically, it differed from the above non-infectious hemagglutinating particles, being much smaller in size and showing a star- or rosette-like appearance with a diameter of about 25 nm, composed of a central particle surrounded by particles resembling envelope-spikes. Virus-neutralizing (VN) and hemagglutination inhibiting (HI) antibodies were produced in rabbits immunized with the HAnin isolated from virions.