Virzzs Research, 18 (1990) 3-8 Elsevier
VIRUS
00613
Foot-and-mouth disease virus strains isolated from persistently infected cell cultures are attenuated for mice and cattle J. Diez 2, M. Hofner
‘, E. Domingo
2 and A.I. Donaldson’
’ AFRC Institute for Animal Health, Pirbright Laboratory, Firbright, Waking, U.K. and ,’ Centro de Biologia Molecular (CSIC- UA MJ, Universidad Autonoma, (Accepted
8 August
Canto Blanco, Madrid, Spain
1990)
Summary
It was previously reported that during serial passage of a BHK-derived cell line persistently infected with fit-Ed-mouth disease virus, (termed C,-BHK-Rcl) the virus became increasingly more virulent for BHK-21 cells (de la Torre et al 1988). Virus strains isolated from different cell passage levels were tested for virulence in mice and cattle. The results showed that in the course of persistence in BHK cells, FMDV became progressively less virulent for mice and cattle. Foot-and-mouth
disease virus; Persistently infected cultures
Persistent, inapparent infection of ruminant animals with foot-and-mouth disease virus (FMDV) was first recognised and described by van Bekkum et al. (1959). Persistently infected animals are termed “carriers”. The carrier state may occur both in animals which have recovered from disease and in immune (vaccinated) animals exposed to virus (McVicar and Sutmoller, 1969). The carrier state is recognised as a means whereby disease may recrudesce or be spread over distance when carriers are moved (Sutmoller and Gaggero, 1965; Sutmoller et al., 1968; AugC de Mello et al., 1970). The carrier is a potential source of new antigeni~ variants of FMDV (Gebauer et al., 1988). Despite its epidemiological importance, studies of the carrier state at the cellular level have been few and the mechanisms which underlie FMDV persistence remain
Correspondence to: A.I. Donaldson, Woking GU24 ONF, U.K. 0168-1702/90/$03.50
AFRC
Institute
for Animal
0 1990 Elsevier Science Publishers
Health,
B.V. (Biomedical
Pirbright
Laboratory,
Division)
P&bright,
4
largely unknown. Cell cultures persistently infected with FMDV have been established and characterised (de la Torre et al.$ 1985, 1988). These cells are useful models for investigating virus persistence and may serve to shed light on the mechanisms of virus persistence in vivo. Both in cell culture and in animals, persisting FMDV undergoes a number of genetic modifications such as those which result in reduced replication at elevated temperatures and a greater tendency towards production of small-plaques in cell monolayers (compare Sutmoller and Gaggero, 1965; Sutmoller et al., 1968; de la Torre et al., 1985). A persistently infected cell line, termed C,-BHK-Rcl, has been established by infection of cloned BHK-21 cells with plaque-purified FMDV strain C-S8cl (serotype C,, isolated in Santa Pau, Spain, 1970). The method used was as described by de la Torre et al. (1985). During the serial passage of C,-BHK-Rcl a co-evolution of the host cells and of the persistent virus took place, as reflected by an increased resistance of the cells to infection when challenged with the parental C-S8cl virus and hyper-virulence of the virus for BHK-21 cells (de la Torre et al., 1988, 1989a,b). The finding of an increased virulence of the persistent virus was unexpected as attenuation is a phenotypic trait which is often selected upon extended passage of virus in cell culture (or in an atypical host animal). Attenuation favours the survival of a virus in nature. Thus, it was of interest to investigate if the in vitro hyper-virulence of FMDV rescued from C,-BHK-Rcl was also manifested in host animals exposed to strains of virus at varying passage. We report the results of virulence assays in mice and in cattle according to the assay methods of Skinner (1960) and Henderson (19491, respectively. The virus strains used were the parental FMDV C-S8cl strain and strains isolated from the persistently infected cell line C,-BHK-Rcl at passage level 19, 58 and 100. These strains are referred to as VR 19, VR 58 and VR 100, respectively. Table 1 shows the result obtained. It can be seen that VR 58 and VR 100 were considerably attenuated for mice when compared to the parental strain FMDV C-S8cl. Strain VR 19 was also attenuated but to a much lesser extent. De la Torre et al. (1985, 1988) also noted a progressive modification of these strains in vitro. Assays in cattle showed that strains VR 19, VR 58 and VR 100 were considerably attenuated for that species. The parental strain FMDV C-S8cl only produced localised lesions at a few sites following tongue inoculation showing that it had been partially selected by cell passage. The three other strains all failed to elicit any clinical reaction indicating that further selection towards a less virulent population had taken place. The manifestations of attenuation were, however, less marked in cattle than in mice. The main differences elicited by the strains in cattle were the varying extent to which they replicated - as judged by the direct evidence of virus recovery in samples of oesophageal-pharyngeal fluid and indirectly by serum antibody responses. On the basis of these parameters, the parental strain FMDV C-S8cl replicated extensively but strains VR 58 and VR 100 failed to replicate. Strain VR 19 was intermediate in behaviour between the other strains in that it did not produce vesicular lesions on the tongue but replicated sufficiently to be recovered from the pharyngeal area which is a predilection site for FMD virus (Burrows et al., 1981).
TABLE 1 Comparison virus strain
C-S8cl VR 19 VR 58 VR 100
of virulence
of FMD virus for mice, cattle and BHK-21
Titre a
Serum titre
(ID&d) Mouse
Cattle
1.2 6.1 1.85 2.1
6.6 cl.0 cl.0 cl.0
r=O
f=lS
VIRUS
00613
Foot-and-mouth disease virus strains isolated from persistently infected cell cultures are attenuated for mice and cattle J. Diez 2, M. Hofner
‘, E. Domingo
2 and A.I. Donaldson’
’ AFRC Institute for Animal Health, Pirbright Laboratory, Firbright, Waking, U.K. and ,’ Centro de Biologia Molecular (CSIC- UA MJ, Universidad Autonoma, (Accepted
8 August
Canto Blanco, Madrid, Spain
1990)
Summary
It was previously reported that during serial passage of a BHK-derived cell line persistently infected with fit-Ed-mouth disease virus, (termed C,-BHK-Rcl) the virus became increasingly more virulent for BHK-21 cells (de la Torre et al 1988). Virus strains isolated from different cell passage levels were tested for virulence in mice and cattle. The results showed that in the course of persistence in BHK cells, FMDV became progressively less virulent for mice and cattle. Foot-and-mouth
disease virus; Persistently infected cultures
Persistent, inapparent infection of ruminant animals with foot-and-mouth disease virus (FMDV) was first recognised and described by van Bekkum et al. (1959). Persistently infected animals are termed “carriers”. The carrier state may occur both in animals which have recovered from disease and in immune (vaccinated) animals exposed to virus (McVicar and Sutmoller, 1969). The carrier state is recognised as a means whereby disease may recrudesce or be spread over distance when carriers are moved (Sutmoller and Gaggero, 1965; Sutmoller et al., 1968; AugC de Mello et al., 1970). The carrier is a potential source of new antigeni~ variants of FMDV (Gebauer et al., 1988). Despite its epidemiological importance, studies of the carrier state at the cellular level have been few and the mechanisms which underlie FMDV persistence remain
Correspondence to: A.I. Donaldson, Woking GU24 ONF, U.K. 0168-1702/90/$03.50
AFRC
Institute
for Animal
0 1990 Elsevier Science Publishers
Health,
B.V. (Biomedical
Pirbright
Laboratory,
Division)
P&bright,
4
largely unknown. Cell cultures persistently infected with FMDV have been established and characterised (de la Torre et al.$ 1985, 1988). These cells are useful models for investigating virus persistence and may serve to shed light on the mechanisms of virus persistence in vivo. Both in cell culture and in animals, persisting FMDV undergoes a number of genetic modifications such as those which result in reduced replication at elevated temperatures and a greater tendency towards production of small-plaques in cell monolayers (compare Sutmoller and Gaggero, 1965; Sutmoller et al., 1968; de la Torre et al., 1985). A persistently infected cell line, termed C,-BHK-Rcl, has been established by infection of cloned BHK-21 cells with plaque-purified FMDV strain C-S8cl (serotype C,, isolated in Santa Pau, Spain, 1970). The method used was as described by de la Torre et al. (1985). During the serial passage of C,-BHK-Rcl a co-evolution of the host cells and of the persistent virus took place, as reflected by an increased resistance of the cells to infection when challenged with the parental C-S8cl virus and hyper-virulence of the virus for BHK-21 cells (de la Torre et al., 1988, 1989a,b). The finding of an increased virulence of the persistent virus was unexpected as attenuation is a phenotypic trait which is often selected upon extended passage of virus in cell culture (or in an atypical host animal). Attenuation favours the survival of a virus in nature. Thus, it was of interest to investigate if the in vitro hyper-virulence of FMDV rescued from C,-BHK-Rcl was also manifested in host animals exposed to strains of virus at varying passage. We report the results of virulence assays in mice and in cattle according to the assay methods of Skinner (1960) and Henderson (19491, respectively. The virus strains used were the parental FMDV C-S8cl strain and strains isolated from the persistently infected cell line C,-BHK-Rcl at passage level 19, 58 and 100. These strains are referred to as VR 19, VR 58 and VR 100, respectively. Table 1 shows the result obtained. It can be seen that VR 58 and VR 100 were considerably attenuated for mice when compared to the parental strain FMDV C-S8cl. Strain VR 19 was also attenuated but to a much lesser extent. De la Torre et al. (1985, 1988) also noted a progressive modification of these strains in vitro. Assays in cattle showed that strains VR 19, VR 58 and VR 100 were considerably attenuated for that species. The parental strain FMDV C-S8cl only produced localised lesions at a few sites following tongue inoculation showing that it had been partially selected by cell passage. The three other strains all failed to elicit any clinical reaction indicating that further selection towards a less virulent population had taken place. The manifestations of attenuation were, however, less marked in cattle than in mice. The main differences elicited by the strains in cattle were the varying extent to which they replicated - as judged by the direct evidence of virus recovery in samples of oesophageal-pharyngeal fluid and indirectly by serum antibody responses. On the basis of these parameters, the parental strain FMDV C-S8cl replicated extensively but strains VR 58 and VR 100 failed to replicate. Strain VR 19 was intermediate in behaviour between the other strains in that it did not produce vesicular lesions on the tongue but replicated sufficiently to be recovered from the pharyngeal area which is a predilection site for FMD virus (Burrows et al., 1981).
TABLE 1 Comparison virus strain
C-S8cl VR 19 VR 58 VR 100
of virulence
of FMD virus for mice, cattle and BHK-21
Titre a
Serum titre
(ID&d) Mouse
Cattle
1.2 6.1 1.85 2.1
6.6 cl.0 cl.0 cl.0
r=O
f=lS
