Phylogenetic relationships of Irkut and West Caucasian bat viruses within the Lyssavirus genus and suggested quantitative criteria based on the N gene sequence for lyssavirus genotype definition

Abstract

The nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) genes of Irkut and West Caucasian bat viruses (WCBV) were sequenced and compared with those of other lyssaviruses. N gene nucleotide identities provided unequivocal separation of all lyssavirus genotypes with an identity threshold of 82%. On this basis, Irkut virus should be considered as a new genotype with particular relatedness to genotypes 4 and 5 (78.0–78.6% identity for N gene nucleotides and 90.4–92.6% for amino acids). Furthermore, genotypes 4–6, together with Aravan, Khujand and Irkut viruses, present a solid phylogroup of Old World bat lyssaviruses. This relationship is apparent using all three viral genes, and causes overlap between intragenotype and intergenotype identities for the P gene (Aravan, Khujand viruses and genotype 6) and for the G gene (Aravan, Khujand, genotypes 5 and 6). WCBV is the most divergent of known lyssaviruses with only limited relatedness to genotypes 2 and 3.

Introduction

Until recently, the Lyssavirus genus, family Rhabdoviridae, was believed to consist of seven distinct genotypes (GTs). Rabies virus (RABV; GT 1), is distributed worldwide among terrestrial mammals and bats, presents the most comprehensive collection of isolates, and has been extensively studied, due to its health and economic significance. Lagos bat virus (LBV; GT 2) was first isolated in Nigeria from the frugivorous bat (Eidolon helvum) in 1956 (Boulger and Porterfield, 1958). This virus was also isolated from the bat Micropteropus pussilus in the Central Africa Republic (Sureau et al., 1980), from the bat Epomophorus wahlbergi in South Africa (Meredith, 1980), cats in South Africa (King and Crick, 1988) and Zimbabwe (Foggin, 1988), and from a dog in Ethiopia (Mebatsion et al., 1992). Mokola virus (MOKV; GT 3) was first isolated from shrews in Nigeria in 1968 (Shope et al., 1970). Thereafter, MOKV was detected in shrews from Nigeria and Cameroon, humans in Nigeria, domestic cats in Zimbabwe, Ethiopia and South Africa, a domestic dog in Zimbabwe and a rodent (Lophuromys sikapusi) from the Central Africa Republic (reviewed by Nel et al., 2000). Duvenhage virus (DUVV, GT 4) was isolated from a human, who died after a bat bite in 1970 in South Africa (Meredith et al., 1971). It has also been identified in the insectivorous bat (Miniopterus sp.) in South Africa and another bat species (Nycteris thebaica) in Zimbabwe (Van der Merwe, 1982, King and Crick, 1988). Since its discovery in 1968, European bat lyssavirus, type 1 (EBLV-1; GT 5) has been isolated from a number of European countries, where it was suspected since the 1950s (Schneider et al., 1985). The primary host species for the virus is considered the serotine bat (Eptesicus serotinus) (Amengual et al., 1997, Serra-Cobo et al., 2002). One human case of EBLV-1 infection occurred following a bat bite in Russia in 1985 (Selimov et al., 1989). European bat lyssavirus, type 2 (EBLV-2; GT 6) was isolated in Finland from a biologist, who died of rabies (Lumio et al., 1986). Later, it was isolated from bats (primarily from the genus Myotis) in northwestern Europe (Amengual et al., 1997), and more recently caused a second recorded human death, this time in the United Kingdom (Fooks et al., 2002). Phylogenetic analysis has demonstrated that EBLV-1 and EBLV-2 form relatively homogenous clusters, but each may be additionally subdivided into two lineages, “a” and “b” (Amengual et al., 1997). Australian bat lyssavirus (ABLV; GT 7) was discovered in 1996. Initially it was isolated from pteropid bats, and two humans that died of rabies following bat exposure. Later ABLV was identified from five different bat species with at least two separate lineages originating from frugivorous and insectivorous species (Fraser et al., 1996, Gould et al., 2002, Guyatt et al., 2003).

Clearly, most non-rabies lyssaviruses are associated with bats. The ecology of MOKV has not been sufficiently studied, so it is not possible to determine the principal host or suggest inferences regarding its epidemiology. Shortly after the discovery of LBV and MOKV, Africa was considered as the probable birth place of the Lyssavirus genus, and the Chiroptera were suggested as the first affected order on the route of adaptation from plant and arthropod rhabdoviruses to mammalian hosts (Shope, 1982). Furthermore, since EBLV-1 had been demonstrated to be related to DUVV, an introduction of the virus from Africa to Europe with migrating bats was hypothesised (Schneider et al., 1985, Amengual et al., 1997, Serra-Cobo et al., 2002).

Very limited information on bat lyssaviruses is available for Asia. Only a few records reported presumable rabies virus isolates of Chiropteran origin in India (Pal et al., 1980) and Thailand (Smith et al., 1968), but these have never been confirmed by further identification or other observations. The presence of antibodies to ABLV was demonstrated in sera from bats in the Philippines, however, no isolates were obtained (Arguin et al., 2002).

Two lyssaviruses have been isolated from bats in Central Asia. Aravan virus was isolated in southern Kyrgyzstan in 1991 (Kuzmin et al., 1992). Nucleotide sequencing of the entire nucleoprotein (N) gene has provided an indication that Aravan virus might be regarded as a new lyssavirus genotype with distant relatedness to GT 4 and GT 5 (Arai et al., 2003). A second example, Khujand virus, was isolated in Northern Tajikistan during 2001 (Kuzmin et al., 2002). Further phylogenetic analysis of the N, phosphoprotein (P) and glycoprotein (G) genes of these viruses suggests that Khujand virus is most related to genotype 6, while Aravan virus is related to Khujand virus, and demonstrated moderate similarity to GTs 4–6 (Kuzmin et al., 2003).

During 2002, two new lyssaviruses have been isolated from Eurasian bats. According to preliminary identification with anti-nucleocapsid monoclonal antibodies and comparison of limited N gene sequences, both may be considered as new genotypes of the Lyssavirus genus (Botvinkin et al., 2003). These viruses have been named West Caucasian bat virus (WCBV) and Irkut virus.

In this study we describe the genetic properties of Irkut virus and WCBV, determine their phylogenetic relationships according to the entire N, P and G gene sequences, and discuss quantitative approaches for lyssavirus genotype definition considering the bat lyssaviruses discovered during recent years.