Genetic variation of foot-and-mouth disease virus isolates recovered from persistently infected water buffalo (Bubalus bubalis)

Abstract

Genetic variation of foot-and-mouth disease virus (FMDV) isolates, serotype O, recovered serially over a 1-year period from persistently infected buffalos was assessed. The persistent state was established experimentally with plaque-purified FMDV, strain O₁Campos, in five buffalos (Bubalus bubalis). Viral isolates collected from esophageal–pharyngeal (EP) fluids for up to 71 weeks after infection were analyzed at different times by nucleotide sequencing and T₁ RNase oligonucleotide fingerprinting to assess variability in the VP1-coding region and in the complete genome, respectively. Genetic variation increased, although irregularly, with time after infection. The highest values observed for the VP1-coding region and for the whole genome were 2.5% and 1.8%, respectively. High rates of fixation of mutations were observed using both methodologies, reaching values of 0.65 substitutions per nucleotide per year (s/nt/y) and 0.44 s/nt/y for nucleotide sequencing and oligonucleotide fingerprinting, respectively, when selected samples recovered at close time periods were analyzed.

The data herein indicate that complex mixtures of genotypes may arise during FMDV type O persistent infection in water buffalos, which can act as viral reservoirs and also represent a potential source of viral variants. These results fit within the quasi-species dynamics described for FMDV, in which viral populations are constituted by related, non-identical genomes that evolve independently from each other, and may predominate at a given time.

Introduction

Foot-and-mouth disease (FMD) is an acute, febrile highly contagious disease of domestic cloven-hoofed animals like cattle, buffalo, sheep and pig, among others (Bachrach, 1968, Pereira, 1981). Though mortality due to the disease is very low and mostly restricted to young animals, drastic reduction in productivity and working capacity of the recovered animals causes considerable losses to the livestock industry (Astudillo et al., 1990).

The causative agent of FMD virus (FMDV) is distinguished immunologically in seven serotypes (O, A, C, SAT-1, SAT-2, SAT-3, and Asia-1), which can be further subdivided into more than 65 subtypes. Immunization with inactivated whole virus of a given vaccine strain does not confer complete protection against other serotypes, nor, even in some cases, between subtypes (Pereira, 1981). The viral particle contains a single-stranded positive RNA of about 8200 nucleotides, within an icosahedric non-enveloped capsid consisting of 60 copies of each of the four proteins VP1, VP2, VP3 and VP4 (Acharya et al., 1989).

RNA viruses exhibit a high mutation rate, which together with reassortment and recombination is probably the basis of the remarkable genetic and phenotypic variability and rapid evolution of these genomes (Holland et al., 1982). In FMDV one of the consequences of these high mutation rates is that populations are composed of ensembles of closely related non-identical genomes that are known as viral quasi-species (Holland et al., 1992, Domingo et al., 2001).

An important aspect of FMDV is its capacity to establish persistent infection in both vaccinated and non-vaccinated ruminants exposed to the virus (van Bekkum et al., 1959, Sutmöller and Gaggero, 1965, Augé de Mello et al., 1970). The mechanisms that mediate this persistence are unclear, but are likely to result from a dynamic equilibrium between the host immune response and the selection of viral antigenic variants at mucosae of upper respiratory tract (Gebauer et al., 1988, Salt, 1993). Currently, virus isolation from esophageal–pharyngeal (EP) fluid is the recommended method to recognize persistently infected animals, although detection of viral sequences in other tissues and fluids has been reported during FMDV persistence in cattle (Bergmann et al., 1996).

The emergence of genetic and antigenic variants of FMDV during cytolytic and persistent infections, and their dominance through positive selection and random drift, has been extensively documented in cell cultures (Sobrino et al., 1983, de la Torre et al., 1985, Borrego et al., 1993). Genetic and antigenic heterogeneity has also been described in virus populations recovered from persistently infected cattle (Gebauer et al., 1988, Malirat et al., 1994), and genetic characterization of FMDV during persistent infection in buffalos has also been illustrated in southern Africa (Vosloo et al., 1996), where the central role played by the African buffalo (Syncerus caffer) in the epidemiological situation has been studied (Dawe et al., 1994a, Dawe et al., 1994b, Bastos et al., 2000, Bastos et al., 2001). Although previous work has been published on FMDV infection in water buffalo (Bubalus bubalis) in the early and persistent stages (Gomes et al., 1997, Samara and Pinto, 1983), no reports are recorded describing the genetic variability of FMDV recovered during persistent infection in this host.

In the present study we describe the extent of genetic variation of sequential FMDV isolates, type O, recovered from five persistently infected buffalos by nucleotide sequencing of the VP1-coding region and T₁ RNase oligonucleotide fingerprinting, and raise discussions about the putative viral evolutionary pathways under controlled conditions.