Downloaded from www.microbiologyresearch.org by IP: 23.22.50.124 On: Tue, 03 May 2016 15:46:41 Journal of General Virology (1999), 80, 639–651. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Molecular evolution of swine vesicular disease virus Gang Zhang, 1, 3 Daniel T. Haydon, 1, 2 † Nick J. Knowles 1 and John W. McCauley 1, 3 1 Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking GU24 0NF, UK 2 Wellcome Centre for the Epidemiology of Infectious Diseases, Department of Zoology, University of Oxford, Oxford OX1 3PS, UK 3 Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, Compton, Newbury RG20 7NN, UK Phylogenetic analysis was used to examine the evolutionary relationships within a group of coxsackie B viruses that contained representatives of the major serotypes of this group and 45 isolates of swine vesicular disease virus (SVDV) from Asia and Europe. Separate analyses of sequence data from two regions of the viral genomes encoding the VP1 and 3BC genes both revealed that the SVDV belonged to a single monophyletic group which could be clearly distinguished from all other sampled coxsackieviruses. Regression analysis revealed that within the SVDV clade at least 80 % of the synonymous variation in evolutionary divergence between isolates was explained by time, indicating the existence of an approximate molecular clock. Calibration of this clock according to synonymous substitutions per year indicated the date of occurrence of a common ancestor for the SVDV clade to be between 1945 and 1965. Introduction Swine vesicular disease (SVD) is a highly contagious disease of pigs which was first recorded in Italy in October 1966 (Nardelli et al., 1968). Subsequent outbreaks have been reported in Hong Kong (1970–1991), Japan (1973–1975) and Europe (1972–1997) (Knowles & Sellers, 1994 ; Brocchi et al., 1997). SVD is caused by a virus belonging to the genus Enterovirus within the family Picornaviridae that is antigenically closely related to the human pathogen coxsackievirus B5 (CV- B5; Graves, 1973; Brown et al., 1973). The complete genome sequences of three SVD viruses (SVDV) and a CV-B5 isolate have been described (Inoue et al., 1989, 1993 ; Seechurn et al., 1990 ; Zhang et al., 1993) and the phylogenetic relationship between the two viruses has been confirmed. Zhang et al. (1993) have previously speculated that SVDV may have arisen from human CV-B5 by recombination since the part of the genome that encodes the 3C protease showed a close relationship to the corresponding nucleotide sequence of another human enterovirus, echovirus 9 (EV-9 strain Barty). Author for correspondence : John McCauley (at the Compton Laboratory). Fax 44 1635 577263. e-mail john.mccauleybbsrc.ac.uk † Present address : Centre for Tropical Veterinary Medicine, Easter Bush, Roslin EH25 9RG, UK. In this paper, phylogenetic analysis of SVDV has been carried out on virus isolates from Asia, where SVDV strains have been isolated almost continually since 1970, and from countries in Europe, where no long-term continuous series of stored isolates was available because either the virus was eliminated or virus isolates were not retained. Phylogenetic analysis of the sequences of two separate genes obtained from these isolates together with those from a panel of coxsackie B (CB) and CB-like viruses, was used to show that all sampled SVDV isolates form a single monophyletic group that is clearly distinct from the other CB viruses included in the analysis. This evidence is consistent with the hypothesis that SVDV has most likely arisen from a single transfer of CV-B5 into pigs. This hypothesis of SVDV monophyly is statistically supported using parametric bootstrap methods. SVDV monophyly was exploited to establish and compare rates of nucleotide substitution in these two separate genes ; these rates were then in turn used to estimate the date of the earliest occurrence of SVDV. Methods  Selection of virus isolates examined. Forty-two SVDV isolates were selected to be representative of viruses isolated throughout the history of the disease (Table 1). The panel comprised 25 virus isolates from Asia and 17 from Europe. In addition, three strains that have been sequenced previously were included : one from Europe (UKG2772; 0001-5729  1999 SGM GDJ