Microbial Pathogenesis 1992 ; 12: 39-46
A murine model of pseudorabies virus latency F . A . Osorio and D . L . Rock* Department of Veterinary Sciences, 126 VDC-East Campus, University of Nebraska-Lincoln, Lincoln, NE 68583-0905, U.S .A . (Received June 26, 1991 ; accepted September 10, 1yy ; )
Osorio, F . A . (Dept of Veterinary Sciences, 126 VDC-East Campus, University of NebraskaLincoln, Lincoln, NE 68583-0905, U .S .A .) and D . L . Rock . A murine model of pseudorabies virus latency . Microbial Pathogenesis 1992 ; 12 : 39-46 . The mouse is a useful laboratory animal for studying various aspects of pseudorabies virus (PRV) virulence . Mice are highly susceptible hosts for PRV infection and are unable to survive acute viral infection . Because of this, mouse models have not been useful for studying PRV latent infections . Here, we report an efficient strategy for establishing latent PRV infections in laboratory mice . Passive transfer of high titered neutralizing antibodies to mice prior to inoculation with highly lethal doses of PRV (Bartha) resulted in survival rates of at least 60% with establishment of latent infections in survivors . Latent PRV infection in mice was demonstrated by : (1) recovery of infectious PRV-Bartha from explants of trigeminal ganglion (TG), and (2) detection of PRV nucleic acids in latently infected TGs by in situ hybridization and polymerase chain reaction (PCR), between 2-8 months post-infection . This PRV latency model indicates that attenuated PRV strains, those currently used extensively in vaccination programs worldwide, can establish a reactivatable latent infection in an experimental host . The mouse model may be particularly useful for examining the molecular bases of PRV latency and reactivation . Key words : pseudorabies virus ; Aujeszky's disease virus ; herpes virus latency; in situ hybridization ; polymerase chain reaction .
Introduction Pseudorabies (Aujeszky's disease) is a disease of worldwide economic significance that is caused by an alpha-herpes virus, pseudorabies virus (PRV) . The natural reservoir for PRV is swine, a species in which the disease is contagious and can become endemic .' Pseudorabies is perpetuated in swine herds by the virus's ability to establish alternating cycles of latency and reactivation in the host, which result in shedding of S . 2,3 infectious PRV and transmission to uninfected anima I Pseudorabies virus latency is beginning to be investigated at the cellular and molecular level .'- ' Although the mouse has been useful to develop practical, economical laboratory models of latency for other alpha-herpes viruses, 7-9 its potential use in establishing a laboratory species model for PRV latency has not been pursued . The mouse is a sensitive experimental host for
PRV, thus mouse virulence has been
frequently used as a marker to characterize PRV strains ." , " As is true for other secondary host species for PRV, laboratory mice as well as naturally infected rodents * Present address : Plum Island Animal Disease Center USDA/ARS, Greenport, New York, 11944-0848, U .S .A . 0882-4010/92/010039+08 $03 .00/0
© 1992 Academic Press Limited
40
F . A. Osorio and D . L . Rock
Table 1 Block titration of PRV (Bartha) challenge and PRV convalescent swine serum to produce latently infected mice Dilution of PRV convalescent serum PRV (Bartha) dose 10 4 10 3 10 2 10 a
LD 50 LD 50 LD 50 LD 50
Undiluted
1 :2
9/10a 9/10 10/10 10/10
6/10 7/10 7/10 10/10
1 :4
1 :8
1/10 0/10 0/10 8/10
0/10 0/10 0/10 3/10
No serum 0/10 0/10 0/10 3/10
Ratios indicate number of surviving mice/number of infected mice .
have always been considered 'terminal' hosts, unable to survive acute viral replication to establish latency .' 12 Here we report the successful establishment of PRV latent infection in laboratory mice . For these experiments we used the PRV strain Bartha . This is a gl - , non-virulent PRV strain which is extensively used for vaccination and which was produced by conventional attenuation/selection in alternative hosts and cell cultures .' The PRV Bartha strain was selected for these experiments because it has been demonstrated to be less virulent than other field and attenuated strains in laboratory mice .'" i Results Effect of passive transfer of PRV-specific antibodies on survival rate upon PRV challenge
Table 1 shows the survival rate of mice passively receiving decreasing amounts of swine PRV antibodies after challenge with decreasing doses of PRV (Bartha) . Sixty to 90% of the 'serum-protected' mice survived infection with the highest dose of PRV Bartha, which killed 100% of the untreated mice (Table 1) . Typically, the unprotected control mice died at 6-7 days p .i . showing signs of PRV infection which included alternating periods of prostration and hyperexcitability, tremors, and pruritus . Pseudorabies virus was isolated in cell cultures and identified by immunofluorescence from the brains of dead animals . The surviving mice exhibited positive PRV serologic status by latex agglutination at 61 days p .i . These antibodies were interpreted as actively induced (i .e . produced by the mice) as evaluated by indirect immunofluorescence (IFA) using fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin (Table 2) . Control Table 2
Serologic status of latently infected mice
Virus dose 10 ° 10 3 10 2 10
LD 50 LD 50 LD50 LD 50
Serum dilution
Mice with persistence of PRV antibodies'
Undiluted Undiluted Undiluted Undiluted
8/10 5/10 0/10 0/10
Pre-immunized mice were challenged with PRV (Bartha) as described in Materials and methods. The evaluation of murine antibodies against PRV was conducted by IFA . Serum samples were collected at 61 days p .i . by tail bleeding . a Ratios : no . of seropositive mice/no . of mice surviving virulent challenge .
Pseudorabies virus latency model
Table 3 mice Mouse no .
41
Explantation/co-cultivation of TGs from latently infected
Time p .i . (days)'
Recovery of PRV from TGs explants
Experiment I 1 2 3 4 5 6 7 8 9
45 56 56 98 98 98 98 98 98
+ (5 days post-explant) °
Experiment II 1 2 3 4 5
65 65 65 65 65
PRV DNA PCR
+ (7 days post-explant)
ND ND ND
+ (5 days post-explant)
ND ND
+ (5 days post-explant)
+
+ (5 days post-explant) + (5 days post-explant)
ND ND ND ND ND
ND : not done. In all cases, the latent infection was established using an inoculum of 10 ° LD50 PRV (Bartha)/mouse and a pre-immunizing dose of 0 .25 ml of convalescent swine serum (SN : 1 :64) per mouse . b Number of days required for detection of the reactivated PRV . a
mice that had been pre-immunized with PRV swine serum at the same time as the principals, but not challenged with PRV, were serologically negative at 61 days p .i . I n vitro reactivation of latent PRV from surviving mice Infectious PRV (Bartha) was recovered from ganglionic explant cultures of 6/14 mice performed at different times ranging from 45 to 98 days p .i . (Table 3) . No excretion of infectious PRV was detected in nasal or conjunctival samples collected at the time of necropsy and explantation . Similarly, infectious PRV was not detected in homogenized samples of the latently infected TGs . Assessment of PRV nucleic acids in trigeminal ganglia (TG) of latently infected mice By in situ hybridization, PRV DNA and/or RNA transcripts in TG neurons were evident at different times throughout the (late p .i .) period of study (Fig . 1) . Viral RNA/DNA containing neurons, present in all animals examined (n = 8; ranging from 45 to 240 days p .i .), exhibited a dense accumulation of autoradiographic grains that, generally, were restricted to the cell nucleus as previously described for TGs of latently infected swine . 4,13 These cells were histologically normal in appearance . Although no detailed quantitation of the positive neurons was performed, their frequency ranged from 1 to several (
A murine model of pseudorabies virus latency F . A . Osorio and D . L . Rock* Department of Veterinary Sciences, 126 VDC-East Campus, University of Nebraska-Lincoln, Lincoln, NE 68583-0905, U.S .A . (Received June 26, 1991 ; accepted September 10, 1yy ; )
Osorio, F . A . (Dept of Veterinary Sciences, 126 VDC-East Campus, University of NebraskaLincoln, Lincoln, NE 68583-0905, U .S .A .) and D . L . Rock . A murine model of pseudorabies virus latency . Microbial Pathogenesis 1992 ; 12 : 39-46 . The mouse is a useful laboratory animal for studying various aspects of pseudorabies virus (PRV) virulence . Mice are highly susceptible hosts for PRV infection and are unable to survive acute viral infection . Because of this, mouse models have not been useful for studying PRV latent infections . Here, we report an efficient strategy for establishing latent PRV infections in laboratory mice . Passive transfer of high titered neutralizing antibodies to mice prior to inoculation with highly lethal doses of PRV (Bartha) resulted in survival rates of at least 60% with establishment of latent infections in survivors . Latent PRV infection in mice was demonstrated by : (1) recovery of infectious PRV-Bartha from explants of trigeminal ganglion (TG), and (2) detection of PRV nucleic acids in latently infected TGs by in situ hybridization and polymerase chain reaction (PCR), between 2-8 months post-infection . This PRV latency model indicates that attenuated PRV strains, those currently used extensively in vaccination programs worldwide, can establish a reactivatable latent infection in an experimental host . The mouse model may be particularly useful for examining the molecular bases of PRV latency and reactivation . Key words : pseudorabies virus ; Aujeszky's disease virus ; herpes virus latency; in situ hybridization ; polymerase chain reaction .
Introduction Pseudorabies (Aujeszky's disease) is a disease of worldwide economic significance that is caused by an alpha-herpes virus, pseudorabies virus (PRV) . The natural reservoir for PRV is swine, a species in which the disease is contagious and can become endemic .' Pseudorabies is perpetuated in swine herds by the virus's ability to establish alternating cycles of latency and reactivation in the host, which result in shedding of S . 2,3 infectious PRV and transmission to uninfected anima I Pseudorabies virus latency is beginning to be investigated at the cellular and molecular level .'- ' Although the mouse has been useful to develop practical, economical laboratory models of latency for other alpha-herpes viruses, 7-9 its potential use in establishing a laboratory species model for PRV latency has not been pursued . The mouse is a sensitive experimental host for
PRV, thus mouse virulence has been
frequently used as a marker to characterize PRV strains ." , " As is true for other secondary host species for PRV, laboratory mice as well as naturally infected rodents * Present address : Plum Island Animal Disease Center USDA/ARS, Greenport, New York, 11944-0848, U .S .A . 0882-4010/92/010039+08 $03 .00/0
© 1992 Academic Press Limited
40
F . A. Osorio and D . L . Rock
Table 1 Block titration of PRV (Bartha) challenge and PRV convalescent swine serum to produce latently infected mice Dilution of PRV convalescent serum PRV (Bartha) dose 10 4 10 3 10 2 10 a
LD 50 LD 50 LD 50 LD 50
Undiluted
1 :2
9/10a 9/10 10/10 10/10
6/10 7/10 7/10 10/10
1 :4
1 :8
1/10 0/10 0/10 8/10
0/10 0/10 0/10 3/10
No serum 0/10 0/10 0/10 3/10
Ratios indicate number of surviving mice/number of infected mice .
have always been considered 'terminal' hosts, unable to survive acute viral replication to establish latency .' 12 Here we report the successful establishment of PRV latent infection in laboratory mice . For these experiments we used the PRV strain Bartha . This is a gl - , non-virulent PRV strain which is extensively used for vaccination and which was produced by conventional attenuation/selection in alternative hosts and cell cultures .' The PRV Bartha strain was selected for these experiments because it has been demonstrated to be less virulent than other field and attenuated strains in laboratory mice .'" i Results Effect of passive transfer of PRV-specific antibodies on survival rate upon PRV challenge
Table 1 shows the survival rate of mice passively receiving decreasing amounts of swine PRV antibodies after challenge with decreasing doses of PRV (Bartha) . Sixty to 90% of the 'serum-protected' mice survived infection with the highest dose of PRV Bartha, which killed 100% of the untreated mice (Table 1) . Typically, the unprotected control mice died at 6-7 days p .i . showing signs of PRV infection which included alternating periods of prostration and hyperexcitability, tremors, and pruritus . Pseudorabies virus was isolated in cell cultures and identified by immunofluorescence from the brains of dead animals . The surviving mice exhibited positive PRV serologic status by latex agglutination at 61 days p .i . These antibodies were interpreted as actively induced (i .e . produced by the mice) as evaluated by indirect immunofluorescence (IFA) using fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin (Table 2) . Control Table 2
Serologic status of latently infected mice
Virus dose 10 ° 10 3 10 2 10
LD 50 LD 50 LD50 LD 50
Serum dilution
Mice with persistence of PRV antibodies'
Undiluted Undiluted Undiluted Undiluted
8/10 5/10 0/10 0/10
Pre-immunized mice were challenged with PRV (Bartha) as described in Materials and methods. The evaluation of murine antibodies against PRV was conducted by IFA . Serum samples were collected at 61 days p .i . by tail bleeding . a Ratios : no . of seropositive mice/no . of mice surviving virulent challenge .
Pseudorabies virus latency model
Table 3 mice Mouse no .
41
Explantation/co-cultivation of TGs from latently infected
Time p .i . (days)'
Recovery of PRV from TGs explants
Experiment I 1 2 3 4 5 6 7 8 9
45 56 56 98 98 98 98 98 98
+ (5 days post-explant) °
Experiment II 1 2 3 4 5
65 65 65 65 65
PRV DNA PCR
+ (7 days post-explant)
ND ND ND
+ (5 days post-explant)
ND ND
+ (5 days post-explant)
+
+ (5 days post-explant) + (5 days post-explant)
ND ND ND ND ND
ND : not done. In all cases, the latent infection was established using an inoculum of 10 ° LD50 PRV (Bartha)/mouse and a pre-immunizing dose of 0 .25 ml of convalescent swine serum (SN : 1 :64) per mouse . b Number of days required for detection of the reactivated PRV . a
mice that had been pre-immunized with PRV swine serum at the same time as the principals, but not challenged with PRV, were serologically negative at 61 days p .i . I n vitro reactivation of latent PRV from surviving mice Infectious PRV (Bartha) was recovered from ganglionic explant cultures of 6/14 mice performed at different times ranging from 45 to 98 days p .i . (Table 3) . No excretion of infectious PRV was detected in nasal or conjunctival samples collected at the time of necropsy and explantation . Similarly, infectious PRV was not detected in homogenized samples of the latently infected TGs . Assessment of PRV nucleic acids in trigeminal ganglia (TG) of latently infected mice By in situ hybridization, PRV DNA and/or RNA transcripts in TG neurons were evident at different times throughout the (late p .i .) period of study (Fig . 1) . Viral RNA/DNA containing neurons, present in all animals examined (n = 8; ranging from 45 to 240 days p .i .), exhibited a dense accumulation of autoradiographic grains that, generally, were restricted to the cell nucleus as previously described for TGs of latently infected swine . 4,13 These cells were histologically normal in appearance . Although no detailed quantitation of the positive neurons was performed, their frequency ranged from 1 to several (
