Molecular epidemiology of rabies epizootics in Colombia: evidence for human and dog rabies associated with bats

Rabies is caused by highly neurotropic viruses, most belonging to a single serotype of the genus Lyssavirus, family Rhabdoviridae (Beran & Steele, 1994). The virion contains a single-stranded, non-segmented RNA genotype of 1112 kb that encodes five structural proteins (Wunner et al., 1988; Tordo & Kouknetzoff, 1994). Rabies virus vectors belong mainly to Carnivora and Chiroptera species, which may transmit the disease to cul-de-sac mammals, including humans. Rabies occurs in two different epidemiological forms: urban rabies, with the domestic dog as the main reservoir and transmitter, and sylvatic rabies, with different wildlife species acting as reservoirs and/or transmitters, such as the fox, the bat and the jackal among others. Both urban and sylvatic rabies are endemic in most South American countries, including Colombia. Of 1088 cases of urban rabies registered to the Ministry of Health between January 1992 and December 2002, 1038 have been dog rabies and 42 have been human rabies. Of these cases, 95 % can be grouped into three main geographical regions: Arauca (61 total cases), the Central Region (246 total cases) and the Caribbean Region (719 total cases). The additional 62 cases are distributed throughout the rest of the country. The rabies outbreaks in Arauca and the Central Region ended in 1997, whereas the outbreak in the Caribbean Region is still ongoing. The aim of this study is to derive phylogenetic relationships between rabies viruses that were isolated in Arauca, the Central region and the Caribbean Region, as well as two strains isolated from insectivorous bats in the city of Cali (southwest of Colombia) in 1995 and 2000. For this, two regions of the rabies virus genome were sequenced and used to draw phylogenetic trees. These are region G, 147 nt long encoding aa 467514 of the G protein, and region Psi-L, which consists of 174 nt of the non-coding pseudogene Psi (Tordo et al., 1986) and 84 nt encoding aa 128 of the L protein. Study samples.
This study includes 68 urban rabies virus strains (62 isolated from dogs and six from humans) and two strains isolated from insectivorous bats (Eptesicus brasiliensis and Molossus molossus) in the city of Cali (southwest of Colombia). All 68 urban viruses were isolated between the period August 1994 and April 2002 from rabies outbreaks in Arauca (eight isolates), the Central Region (15 isolates) and the Caribbean Region (45 isolates). Rabies diagnosis was achieved by virus isolation in ICR mice and immunofluorescence assays using antibodies. Rabies virus isolates were stored at -80 °C in the form of frozen mouse brain material.

Total RNA extraction.
This process was achieved as described previously (Páez et al., 2002). Briefly, passaged material from 100 mg frozen mouse brain was dissolved in 0·75 ml TRIzol (Gibco) and extracted once with 0·25 ml chloroform. Total RNA was precipitated with 1 vol. 100 % isopropyl alcohol, washed with 70 % ethanol and made up to 50 µl with 1 % DEPC in double-distilled water.

Primers.
An oligonucleotide primer pair (designated G/L) (Sacramento et al., 1991, 1992) was used for PCR to amplify a 902 nt fragment containing the region encoding the cytoplasmic domain of the glycoprotein (aa 442495 of the native glycoprotein), the adjacent pseudogene Psi and 84 nt of the L gene encoding aa 128 of protein L. The positive-strand primer G (5'-GACTTGGGTCTCCCAACTGGGG-3') primes the PCR at position 46654687 of the rabies virus genome and the negative-strand primer L (5'-CAAAGGAGAGTTGAGATTGTAGTC-3') primes at position 55435566, according to the numbering of the published Pasteur virus (PV) sequence (Tordo et al., 1988; Sacramento et al., 1991; von Teichman et al., 1995).

RT-PCR.
This process was achieved as described previously (Páez et al., 2002). Briefly, total brain RNA was hybridized with primer G (150 ng) at 65 °C for 2 min and reverse-transcribed at 42 °C for 90 min in a total volume of 10 µl (Sacramento et al., 1991). Amplification using the G/L primer set was carried out using 2 µl of the cDNA reaction mixture in a total volume of 50 µl using a Thermal Cycler (Perkin Elmer) and conditions similar to those described by von Teichman et al. (1995).

DNA sequence and phylogenetic analysis.
Direct sequencing of gel-purified PCR products was performed using the G/L primer set in an automatic sequencing apparatus (ABI Prism 310, Applied Biosystems). DNA sequence analysis and the construction of phylogenetic trees were performed using the PHYLIP package, version 3.52 (Felsenstein, 1993), and the TREEVIEW program. For construction of phylogenetic trees, the neighbour-joining method of Saitou & Nei (1987) was used combined with bootstrapping 500 resampling, a statistical method that calculates confidence limits with respect to the phylogenetic tree (Nei, 1992).

A total of 70 rabies virus isolates were included in this study; their geographical origin is shown in Fig. 1 and epidemiological information is given in Table 1. According to the alignment of G and Psi-L nucleotide sequences (Figs 2 and 3) and phylogenetic analysis (Fig. 4), the sample of urban rabies virus isolates used in this paper can be distributed into three different groups (Fig. 1). In this study, Colombian genetic variant I consists of seven rabies virus strains isolated from domestic dogs in Arauca during the period between February 1995 and March 1997 and 14 rabies virus strains isolated from domestic dogs in the Central Region during the period between September 1994 and July 1996. The average nucleotide similarity among Colombian genetic variant I viruses is approximately 98 %, which indicates that these viruses are closely related. Our results also show a high level of nucleotide similarity (average 97·5 %) among rabies viruses isolated in the Caribbean Region (Colombian genetic variant II rabies viruses). In this study, Colombian genetic variant II consists of 43 strains isolated from domestic dogs (38 isolates) and humans (five isolates). All 43 strains were isolated during the period between August 1994 and April 2002. In the raw sequence data, genetic variant I and II viruses were defined and distinguished by nucleotide substitutions at nine of the 147 bp of sequence in the G region and at eight of the 258 bp of sequence in the Psi-L region (Figs 2 and 3). The average nucleotide similarity between genetic variant I and II viruses was approximately 92·5 %. In the amino acid sequence, genetic variants I and II were distinguished by substitutions at four of the 49 positions encoded in region G (aa 442495 of the native G protein). In contrast, genetic variant I and II viruses have no amino acid differences (residues 128 of the L protein) (Figs 2 and 3). A third group of rabies viruses consisted of six strains isolated from two insectivorous bats (M. molossus and E. brasiliensis) in the city of Cali in 1995 and 2000, three domestic dogs in Arauca, the Caribbean Region and the Central Region in 1997 and a human in the Caribbean Region in 1997. According to phylogenetic analyses, our data suggest that these are bat rabies virus variants and must have been transmitted by bats to dogs and humans; however, the number of passages of infection between the bat reservoir and the final host from which these isolates were obtained is unknown. The average nucleotide similarity for regions G and Psi-L between the bat rabies virus variants in this study was 83 %, which suggests a greater diversity in this group. The average nucleotide similarity between the group of bat rabies virus variants and genetic variants I or II was 73 and 72 %, respectively.

(34K): Fig. 1. Map of Colombia showing the geographical location of places where rabies virus strains were isolated. A, Caribbean Region; B, Central Region; C, Department of Arauca. Symbols and colours identify host species and rabies virus variants. For clarity, the prefixes C (canid), H (human) and B (bat) were added to the isolate numbers. Numbers next to the back slash indicate the year of isolation.

Table 1 Epidemiological information for the rabies virus isolates For each of the 70 rabies virus isolates studied in this paper, the geographical origin (town and department in Colombia), vertebrate host and date of isolation (month and year) are shown, as well as the GenBank accession numbers for the nucleotide sequences of regions G and Psi-L.

(65K): Fig. 2. Alignment of nt 47214867 (region G) of the genome of rabies viruses and of aa 467514 of the native G protein. (A) Prototype sequence of genetic variant I viruses. (B) Prototype sequence of genetic variant II viruses. (C) Bat rabies variants, isolates H03/97, B01/95, B01/00, C23/97, C27/97, C31/97 and a strain isolated from a silver-haired bat in North America (Morimoto et al., 1996).

(49K): Fig. 3 Alignment of nt 52435500 (region Psi-L) of the genome of rabies viruses and of aa 128 of the L protein. (A) Prototype sequences of genetic variant I viruses. (B) Prototype sequence of genetic variant II viruses. (C) Bat rabies virus variants, isolates H03/97, B01/95, B01/00, C23/97, C27/97 and C31/97.

(17K): Fig. 4. Phylogenetic relationships among 70 rabies virus isolates from three outbreaks in Colombia based on combined sequences of the gene encoding the G protein cytoplasmatic domain, the GL intergenic region and a region encoding 28 amino-terminal amino acids of the L protein. Genetic distances were measured by the Kimura two-parameter method and neighbour-joining analysis (500 bootstrap trails) using the PHYLIP software, version 3.52. PV was used as the outgroup. Numbers at nodes indicate confidence limits greater than 70 %.

During the 1980s, urban rabies spread over approximately 19 000 and 8 000 km² in the Central Region and Arauca, respectively. Our results indicate that the rabies viruses transmitted in these two areas were genetically related, with 98 % nucleotide similarity; these were grouped into a single genetic variant (Colombian genetic variant I) in this study. In 1997, the urban rabies outbreaks in Arauca and the Central Region ended. Colombian genetic variant I rabies viruses have no longer been found in Colombia, indicating that these are extinct in the country. Rabies control campaigns that include vaccination of dogs may have been crucial for the extinction of genetic variant I viruses in Colombia. In the 1990s, urban rabies spread over approximately 40 000 km² in the Caribbean Region. Our results indicate that the viruses transmitted in this region are genetically related, with an average nucleotide similarity of 97·5 %; these were grouped into a single genetic variant (Colombian genetic variant II) in this study. Despite the rabies control campaigns, urban rabies is still being transmitted in the Caribbean Region and is a major health problem that could eventually spread to other areas of Colombia or to other countries.

It has been reported widely that rabies virus is transmitted from bats to humans causing several deaths (Morimoto et al., 1996; Crawford-Miksza et al., 1999; Warner et al., 1999; Madsen, 2000). In our study sample, we found four rabies virus isolates (H03/97, C23/97, C27/97 and C31/97) that are genetically related to three strains isolated from bats (B01/95, isolated from M. molossus, B01/00, isolated from E. brasiliensis, and a strain isolated from a silver-haired bat in the United States) (Morimoto et al., 1996; GenBank accession no. U52946). All seven of these isolates are located in a clade with a bootstrap value of 99 %, which underlines the significance of the genetic relationships among isolates in this clade. This suggests that isolates H03/97, C23/97, C27/97 and C31/97 could be of bat origin. These four isolates form a subclade from which bat isolates are excluded, suggesting that rabies virus mutation rates in bats may be different from those in dogs or humans. In fact, the number of passage infections that occurred between the bat reservoir and the final host remains unknown. Strains C23/97, C27/97 and C31/97 were isolated from dogs, indicating that in Colombia bat rabies could eventually spread in dog populations, thereby becoming a public health problem. Isolate H03/97 was isolated in Central Colombia in 1997 from a human (young woman) who had no apparent contact with bats, which suggests that in Colombia bat rabies can be transmitted from bats to other vertebrates and from these to humans. In our study, bat rabies variants were isolated in places that are free of urban rabies outbreaks and which are hundreds of kilometres away from each other (Fig. 1), indicating that in Colombia bat rabies is a threat for human health even in places that are far away from outbreaks of urban rabies.

This work was supported by Instituto Nacional de Salud of Colombia, the Ministry of Health of Colombia, Saldarriaga Concha Fundation and Banco de la Republica of Colombia. The authors of this paper wish to thank Dr Vladimir Corredor and Dr Luis Murillo at Instituto de Immunología, Bogotá, Dr Jaime Castellanos at Instituto Nacional de Salud in Bogotá, and Dr Ignacio Sarante at Universidad Javeriana, Bogotá for their useful help in the PCR, sequencing and phylogenetic analyses. The authors wish to thank Drs Noël Tordo, Hassan Badrane and Herve Bourhy at Pasteur Institute in Paris for their useful comments on the manuscript.