The emergence of wildlife species as a source of human rabies infection in Brazil.

Forty-five human rabies virus isolates from a wide geographical area of Brazil were characterized using an anti-nucleoprotein monoclonal antibody panel and by partial nucleotide sequencing analysis of the nucleoprotein gene. Three major antigenic groups related to the antigenic variants maintained in domestic dogs, vampire bats and marmosets were identified. Phylogenetic analyses revealed that the viruses from dog-related cases segregated into four sister clades: three associated with dog-endemic cycles in Brazil and one with the crab-eating fox cycle in the northeastern region of the country. The vampire bat- and marmoset-related viruses formed two independent groups. The topology of these clades was conserved when these samples were compared to virus representatives of the currently reported rabies endemic cycles in the Americas. These results indicated the presence of multiple endemic transmission cycles maintained in four different reservoirs, domestic dogs, crab-eating foxes, vampire bats and marmosets, which are being transmitted directly to humans and should be considered as a high-risk for rabies infection.

Rabies in marmosets (Callithrix jacchus), Ceará, Brazil.

A new Rabies virus variant, with no close antigenic or genetic relationship to any known rabies variants found in bats or terrestrial mammals in the Americas, was identified in association with human rabies cases reported from the state of Ceará, Brazil, from 1991 to 1998. The marmoset, Callithrix jacchus acchus, was determined to be the source of exposure.

[Molecular epidemiological characterization of rabies virus isolated from Guangxi].

BACKGROUND: To analyze the genotype of rabies virus isolated from different areas in Guangxi at different time. METHODS: mRNA of 8 Guangxi isolates, 1 Anhui isolates and 10 isolates from different countries of rabies virus were amplified by reverse transcription polymerse chain reaction (TR-PCR),and the products were sequenced. Phylogenetic analysis was carried out by phylip program. RESULTS: Phylogenetic analysis showed that 8 Guangxi isolates were related regionally, the homologic difference was lower than 2% among the isolates of the same area; higher than 15% among the isolates of two different areas.8 Guangxi isolates belonged to genotype A1, A2 a and A2 b; 10 chb of Guangxi isolates and thbkrb 2123 and thdg 2933 of Thailand isolates belonged to genotype A2 b, while Guangxi isolates were different from isolates of USA, Mexico and South Africa, which all belonging to genotype B. CONCLUSIONS: There are at least three groups of rabies virus based on limited sequencing of N gene in Guangxi.

Bat rabies in urban centers in Chile.

One hundred and five rabies isolates obtained from domestic animals and insectivorous bats in Chile between 1977 and 1998 were molecularly characterized by limited sequence analysis of their nucleoprotein genes. These isolates were compared with viruses isolated from known domestic and wildlife rabies reservoirs in the Americas to identify potential reservoirs of rabies in Chile. The phylogenetic analyses showed that none of the Chilean isolates segregated with viruses from the terrestrial reservoirs. No non-rabies lyssaviruses were found in this study. The Chilean samples were not related to viruses of the sylvatic cycle maintained by the common vampire bat (Desmodus rotundus) in Latin America. Five genetic variants were identified from insectivorous bats in Chile. The Brazilian free-tailed bat (Tadarida brasiliensis) was identified as the reservoir for the rabies genetic variant most frequently isolated in the country between 1977 and 1998. The close association of a group of viruses obtained from a domestic dog (Canis familiaris), Brazilian free-tailed bats, and a red bat (Lasiurus borealis) with viruses maintained by Lasiurus spp. in North America implicated species of this genus as the possible reservoirs of this particular genetic variant in Chile. Reservoirs for the other three variants remain unknown.

Molecular characterization of rabies virus isolates from Mexico: implications for transmission dynamics and human risk.

Twenty-eight samples from humans and domestic and wild animals collected in Mexico between 1990 and 1995 were characterized by using anti-nucleoprotein monoclonal antibodies and limited sequence analysis of the nucleoprotein gene. The variants of rabies viruses identified in these samples were compared with other isolates from Mexico and the rest of the Americas to establish epidemiologic links between cases and outbreaks and to increase the understanding of rabies epidemiology in the Western Hemisphere. Antigenic and genetic diversity was found in all samples from dogs and dog-related cases, suggesting a long-term endemic situation with multiple, independent cycles of virus transmission. Two isolates from bobcats were antigenically and genetically homologous to the rabies variant circulating in the Arizona gray fox population, indicating a wider distribution of this variant than previously reported. Rabies isolates from skunks were unrelated to any variant analyzed in this study and represent a previously unrecognized cycle of rabies transmission in skunks in Baja California Sur. Two antigenic and genetic variants co-circulating in southern and eastern Mexico were found in viruses obtained from cases epidemiologically related to vampire bats. These results serve as a baseline for the better understanding of the molecular epidemiology of rabies in Mexico.

Phylogenetic comparison of the S3 gene of United States prototype strains of bluetongue virus with that of field isolates from California.

To better define the molecular epidemiology of bluetongue virus (BTV) infection, the genetic characteristics and phylogenetic relationships of the S3 genes of the five U.S. prototype strains of BTV, the commercially available serotype 10 modified live virus vaccine, and 18 field isolates of BTV serotypes 10, 11, 13, and 17 obtained in California during 1980, 1981, 1989, and 1990 were determined. With the exception of the S3 gene of the U.S. prototype strain of BTV serotype 2 (BTV 2), these viruses had an overall sequence homology of between 95 and 100%. Phylogenetic analyses segregated the prototype U.S. BTV 2 strain to a unique branch (100% bootstrap value), whereas the rest of the viruses clustered in two main monophyletic groups that were not correlated with their serotype, year of isolation, or geographical origin. The lack of consistent association between S3 gene sequence and virus serotype likely is a consequence of reassortment of BTV gene segments during natural mixed infections of vertebrate and invertebrate hosts. The prototype strain of BTV 13, which is considered an introduction to the U.S. like BTV 2, presents an S3 gene which is highly homologous to those of some isolates of BTV 10 and especially to that of the vaccine strain. This finding strongly suggests that the U.S. prototype strain of BTV 13 is a natural reassortant. The different topologies of the phylogenetic trees of the L2 and S3 genes of the various viruses indicate that these two genome segments evolve independently. We conclude that the S3 gene segment of populations of BTV in California is formed by different consensus sequences which cocirculate and which cannot be grouped by serotype.

Bluetongue disease and the molecular epidemiology of viruses from the western United States.

Prototype and field isolates of United States bluetongue viruses were evaluated for genetic heterogeneity by sequence analyses. Prototype viruses BTV 2, 10, 11, 13 and 17 from the United States, BTV 10 vaccine virus, and field isolates of BTV 10 and 17 from California were analyzed. Gene segment 2 from BTV 10 and 17 isolated in 1980-81 and 1990 was sequenced along with gene segment 9 from BTV 10 isolates. The Wisconsin Package was used to analyze nucleotide sequences and to predict amino acid composition of the putative proteins. Phylogenetic analyses were done using DNADIST and FITCH programs of the PHYLIP Package, v. 3.4. Gene segment 2 segregated into two monophyletic groups of BTV 2 and 13 and BTV 10, 11 and 17. BTV prototype 10, isolated in 1953 and field isolates through 1980 were similar, whereas the 1990 isolates differed by 4.5%, indicating two BTV 10 monophyletic groups over 37 years. Gene segment 2 of BTV 17 prototype virus differed from the California isolates. Gene segment 9 of BTV 10 field isolates formed into two monophyletic groups. This gene segment reassorted with all serotypes. Gene segment 9 of the 1953 BTV 10 vaccine virus was essentially the same as gene segment 9 of the BTV 13 prototype virus, isolated in Idaho in 1967. This suggested that prototype BTV 13 is a reassortant virus with gene segment 9 derived from a vaccine virus parent.

Genetic characterization of rabies field isolates from Venezuela.

Twenty samples from cases of rabies in humans and domestic animals diagnosed in Venezuela between 1990 and 1994 and one sample from a vampire bat collected in 1976 were characterized by reactivity to monoclonal antibodies against the viral nucleoprotein and by patterns of nucleotide substitution in the nucleoprotein gene. Three antigenic variants were found: 1, 3, and 5. Antigenic variant 1 included all samples from dogs and humans infected by contact with rabid dogs. Unique substitutions permitted identification of two separate outbreaks of dog rabies in the Maracaibo Depression and Los Llanos region and in the Andean region of Venezuela. Samples from the vampire bat and two head of cattle were characterized as antigenic variant 3 and showed a nucleotide sequence homology of 96 to 98% to each other and to samples of vampire bat-associated rabies throughout Latin America. Ten of the remaining 12 samples were characterized as antigenic variant 5. Genetic studies indicated that 11 of these samples formed a highly homologous and distinctive group but were closely related to samples of vampire bat-associated rabies. The 12th sample of variant 5 (from a cat) showed only 78 to 80% genetic homology to samples of rabies associated with vampire bats. The application of antigenic and genetic typing to rabies surveillance in Latin America is essential to improve control programs. Recognition of the source of outbreaks of dog rabies and identification of wildlife species maintaining sylvatic cycles of rabies transmission permit better utilization of public health resources.

Evolution of the L2 gene of strains of bluetongue virus serotype 10 isolated in California.

The genetic characteristics and phylogenetic relationships between the U.S. prototype strain of bluetongue virus serotype 10 (BTV 10), the modified live virus vaccine currently used in California, and three field isolates of BTV 10 obtained in 1980 and three in 1990 in California were determined by comparison of their L2 gene sequences. The L2 genes of the 1980 field isolates were very closely related to the L2 genes of the prototype strain and the vaccine strain, differing by only 0.1 to 0.5%. The 1990 field isolates diverged from all the other viruses by an average of 4.8%. They showed a high degree of genetic similarity that ranged from 98.2 to 99.7% and formed a separate group. All BTV 10 viruses derived from a common ancestor (bootstrap value 100%) from which two different lineages have diverged giving rise to two monophyletic groups, one including all the 1990 viruses and the other the prototype, the vaccine, and all 1980 field strains. The bootstrap analyses placed a 100% confidence value at each of these two nodes. These results indicate that two different lineages of BTV 10 circulated in California between 1953 and 1990. The effect of the vaccine on the evolutionary pathways of the BTV 10 population present in California in 1980 was not clearly established, but it did not influence the evolution of the BTV 10 field isolates obtained in 1990.

Association of bluetongue virus gene segment 5 with neuroinvasiveness.

Two strains (UC-2 and UC-8) of bluetongue virus were used to determine genetic factors influencing neuroinvasiveness. Reassortants were produced in vitro, and the parental origins of their genes were determined by polyacrylamide gel electrophoresis profiles and restriction endonuclease digestion. Gene segment 5 of UC-8 correlated with neuroinvasiveness of reassortants when inoculated subcutaneously into newborn mice.

Heterogeneity of the L2 gene of field isolates of bluetongue virus serotype 17 from the San Joaquin Valley of California.

Genome segment 2 (L2) from six field isolates of bluetongue virus (BTV) serotype 17 was sequenced by cycling sequencing after the amplification of the viral cDNA by the polymerase chain reaction. The viruses were isolated from sheep, cattle and a goat in the San Joaquin Valley of California during the years 1981 and 1990. These viruses exhibit divergent patterns of neutralization with BTV 17-specific monoclonal antibodies. The six L2 genes of the BTV 17 field isolates all encode a protein of 955 amino acids. Similarity of the nucleotide sequences of the L2 genes with respect to the prototype strain ranges between 93.8% and 95.1%, whereas the similarity between the field isolates ranges from 96.8% to 99.1%. Although very closely related, the L2 gene of each virus is distinct. Furthermore, mutations in the L2 gene of field isolates of BTV do not consistently follow a linear pattern of accumulation over time. Some amino acid changes in the VP2 protein of field strains were conserved over time, whereas others were not correlated with the year of isolation and some substitutions were unique to individual viruses. The predicted VP2s constitute a group of non-identical, but closely related proteins. Phylogenetic analyses suggest that the viral variants which co-circulate in the San Joaquin Valley could evolve by different evolutionary pathways.

Comparison of slot blot nucleic acid hybridization, immunofluorescence, and virus isolation techniques to detect bluetongue virus in blood mononuclear cells from cattle with experimentally induced infection.

A slot blot hybridization technique was applied for detection of bluetongue virus (BTV) in blood mononuclear cells (BMNC) obtained from cattle with experimentally induced infection. This technique lacked sensitivity to detect the viral nucleic acid directly in clinical specimens. When aliquots of mononuclear cells from these cattle were cultivated in vitro for 10 days to amplify virus titer, only 33.3% of the samples collected during viremia gave a positive signal in the slot blot hybridization format. By contrast, results for 34.3% of noncultured and 63.3% of cultured mononuclear cell samples collected during viremia were positive by immunofluorescence. The average number of infected cells, as detected by immunofluorescence in the noncultured mononuclear cell samples, was 1 to 5/300,000, and was usually > 10/300,000 in the cultured cell samples. Virus was isolated from all postinoculation blood samples obtained from 4 heifers that were seronegative at the time of inoculation, but was not isolated from any of the preinoculation samples, or from any of the postinoculation samples obtained from 2 heifers that were seropositive at the time of inoculation. When virus isolation was attempted from separated mononuclear cells in 2 heifers, 43.7% of the noncultured and 87.5% of the cultured samples had positive results.

Hybridization relatedness of Israeli and U.S. bluetongue (BLU) serotypes using cDNA probes from BLU virus strain 11-UC8.

Partial cDNA clones representing 47%, 96%, and 98% of genome segments 7, 9, and 10, respectively, of a US bluetongue virus (BLU) 11 virulent strain were used to study, for the first time, the genetic relationships between Israeli BLU proto-serotypes and field isolates, and US BLU proto-serotypes. Their usefulness as group-specific identification probes was also determined. The viral nucleic acid was extracted from the infected cells and the purified dsRNA genome segments were fractionated by polyacrylamide gel electrophoresis, transferred to a nylon membrane and hybridized to the 32P labeled DNA probes. The three probes recognized all the samples tested. Genome segment 7, that code for the mayor inner capsid protein VP7, showed the most variation in the hybridization signal with the US proto-serotypes and all the Israeli samples studied. The genome segments 9 and 10 that code for the minor inner capsid protein VP6 and the nonstructural protein NS3, respectively, were highly conserved in all the samples tested despite their distant geographical regions of origin. The last two mentioned clones showed to be good group-specific probes for the identification of BLU samples from Israel and United States. The obtained cloned genetic probes were also tested against US epizootic haemorrhagic disease virus (EHDV) serotype 1 and 2 viral dsRNA, a distantly related orbivirus. None of them hybridized with the viral dsRNA of these two viruses.

Evidence of genome segment 5 reassortment in bluetongue virus field isolates.

A recombinant cDNA probe from genome segment 5 obtained from a virulent US bluetongue virus strain (BTV-11 strain UC8) was hybridized to US and Israeli BTV prototypes and field isolates. The cloned genetic probe hybridized with US BTV prototype 10, but not with US prototypes 2, 11, 13, and 17; with the avirulent BTV-11 strain UC2; and with the Israeli prototype 10. When the probe was hybridized to field isolates from the US serotypes, it hybridized to 12 of 14 BTV-10 isolates and 4 of 17 BTV-11 samples, but not to the BTV-13 and BTV-17 samples tested. Hybridization was not observed with the Israeli field isolates studied. Results indicate that a reassortant event occurred between a strain of US BTV-10 and US BTV-11 that originated the BTV-11 strain UC8.

Identifying bluetongue virus ribonucleic acid sequences by the polymerase chain reaction.

A primer-directed nucleic acid amplification reaction, commonly known as the polymerase chain reaction (PCR), has been adapted to the identification of bluetongue virus (BTV) RNA. The protocol described in this article is designed for the detection of a purported globally-conserved, serogroup specific nucleic acid sequence within the BTV genome. Due to the double-stranded RNA composition of the BTV genome, the original polymerase chain reaction protocol has been modified to include chemical denaturation and reverse transcription steps that allow selective amplification from this unique template molecule. The amplification procedure yields a 210 base pair product when tested on samples of RNA from the prototypic strains of U.S. BTV serotypes 2, 10, 11, 13, 17. The amplification product is tentatively identified by agarose gel electrophoresis of the PCR samples. Final positive identification of the amplified BTV-specific product is determined by Southern blot hybridization of the PCR samples. RNA samples from uninfected cell culture controls and from cell cultures infected with two serotypes of epizootic hemorrhagic disease of deer (EHDV) yielded negative results. Preliminary experiments to quantify the threshold of sensitivity of this protocol, as described here, indicate positive detection of the BTV target RNA sequence at a level of less than 2 fg, or the amount of target sequence derived from less than 7500 viral particles. While this falls short of the theoretical limit of sensitivity of the PCR, the enhanced threshold of sensitivity of the PCR reaction compared to standard nucleic acid hybridization methodology suggests that rapid detection of BTV RNA directly within clinical specimens may be feasible with further refinements. Potential methods of enhancing this reaction are discussed.

Recombinant cDNA probe from bluetongue virus genome segment 10 for identification of bluetongue virus.

Genome segment 10 of bluetongue virus (BTV) serotype 11 UC8 strain was cloned and subsequently hybridized to viral double-stranded RNA extracted from 90 field isolates of BTV serotypes 10, 11, 13, and 17; the prototype strains of BTV 2, 10, 11, 13, and 17; the prototype strain epizootic hemorrhagic disease virus (EHDV) serotype 1; and 4 field isolates of EHDV serotype 2. The 90 field isolates were obtained from different counties in California, Louisiana, and Idaho during the years 1979, 1980, and 1981. The cloned genetic probe hybridized with all the BTV samples tested, showing different degrees of cross-hybridization at the stringency conditions used in this study. This indicated that BTV genome segment 10 has conserved nucleotide sequences among the BTV serotypes 2, 10, 11, 13, and 17. No cross-hybridization signals were detected between the cloned genome segment 10 of BTV 11 UC8 strain and the prototype strain of EHDV serotype 1 and the field isolates of serotype 2. This probe recognized a wide variety of BTV isolates.

Recombinant DNA probe for serotype-specific identification of bluetongue virus 17.

The double-stranded RNA genome from 117 field isolates of bluetongue virus (BTV) serotypes 10, 11, 13, and 17 was blotted onto nitrocellulose paper and hybridized with a radioactively labeled cloned copy of DNA genome segment 2 of BTV-17. Viral RNA from BTV prototype strains 2, 10, 11, 13, and 17 were used as controls. The probe hybridized only with the viral RNA from prototype BTV-17 virus and field isolates of BTV-17. There was no cross hybridization with field isolates of BTV serotypes 10, 11, and 13. A complementary DNA probe developed from genes coding for BTV serotype specificity was effectively used in a slot-blot hybridization system for efficiently characterizing the viral serotype.