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Animal Models of Herpesvirus Genital Infection
Chapter 108
Animal Models of Herpesvirus Genital Infection: Guinea-Pig N. Bourne and L. R. Stanberry
Background of human infection Genital herpes is a common sexually transmitted disease caused by herpes simplex virus (HSV) either type 1 or 2. It occurs worldwide and is recognized as a major public health problem in many developed countries (Stanberry et aL, 1997). Recent seroprevalence data indicate that more than one in five adult Americans have been infected with HSV-2, the predominant cause of genital herpes in the United States (Fleming et al., 1997). The natural history of genital herpes includes the initial or primary infection, the establishment of a latent infection in paraspinal sensory ganglia, and the periodic reactivation of latent infection which causes recurrent infections (Wald and Corey, 1996). Initial and recurrent infections in humans may be either symptomatic or asymptomatic. Symptomatic infection is characterized by the development of vesicles and/or ulcers at mucocutaneous sites in the anogenital area. Initial infection is generally more severe than recurrent infections and may be associated with systemic findings such as fever, urinary retention or dysuria, and meningitis. In humans, HSV-1 and HSV-2 produce indistinguishable initial genital infections but the type 2 virus typically causes more frequent symptomatic recurrent infections than HSV-1. Anecdotal reports suggest that a variety of stimuli, including stress and onset of menses, can trigger recurrent infections. To date only experimental ultraviolet radiation exposure has been proven to trigger symptomatic recurrent infections. Asymptomatic initial or recurrent infections are common and probably play a major role in the transmission of virus since people experiencing asymptomatic infections shed virus from anogenital sites but are unaware they are contagious. Intravenous acyclovir and oral acyclovir, valacyclovir, and famciclovir have been proven effective in the treatment of initial genital herpes and impact both the clinical and virological course of the infection (CDC, 1998). Oral acyclovir, valacyclovir, and famciclovir have a modest effect on recurrent infections when used episodically to treat recurrences but all are highly effective at preventing symptomatic and asymptomatic recurrent infections when taken daily as suppressive therapy (CDC, 1998). While there are animal data to suggest that treatment with anti-HSV nucleHandbook of Animal Models of Infection
ISBN 0-12-775390-7
oside analogs early in the course of the initial infection can impact latent infection, the clinical trials conducted to date suggest that antiviral drugs like acyclovir cannot prevent the establishment of the latent infection that occurs during the initial infection, nor can they eliminate the latent infection once it is established.
Background of model Originally described in the 1970s by Austrian scientists, the female guinea-pig model of genital herpes was extensively characterized in the 1980s and has found wide use in exploring the viral pathogenesis and in the evaluation of antiviral drugs and experimental HSV vaccines. The model mimicks many of the clinical, virologic and immunologic features of human infection (Stanberry et aL, 1982, 1985; Stanberry, 1991). Intravaginal inoculation of guinea-pigs with HSV-1 or 2 produces either symptomatic or asymptomatic infection depending on the inoculum titer. Infection results in high titer replication in the lower genital tract and intraneural spread to lumbosacral dorsal root ganglia and spinal cord. Symptomatic infection is characterized by vesiculoulcerative lesions on and about the perineum and some animals exhibit urologic (urinary retention) and neurologic (hind limb paralysis) findings. Initial infection results in the establishment of latent HSV infection in lumbosacral dorsal root ganglia. After recovery from the initial infection, guinea-pigs experience spontaneous recurrent infections manifested as either shedding of virus in vaginal secretions in the absence of recognizable lesions (subclinical viral shedding) or discrete erythematous or vesicular lesions on the perineal skin. The frequency of recurrent infections declines over time and animals infected with HSV-2 exhibit significantly more recurrences than HSV-l-infected guinea-pigs. In addition to spontaneous recurrences, recurrent infections can be induced by exposure of the perineal skin to ultraviolet radiation (Stanberry, 1989). Local and systemic immune responses to the genital infection have been reported. A male model of genital herpes involving intraurethral HSV inoculation has also been described (Stephanopoulos Copyright 9 1999Academic Press All rights of reproduction in any form reserved
908
N. Bourne and R. Stanberry
et aL, 1989). The male model has been used in the evaluation of candidate antiviral drugs (Stanberry et aL, 1992), but
compared to the female model has a higher mortality rate, is difficult to assess recurrent infections, and lacks the ease of repetitive sampling for virology specimens afforded by vaginal swabbing.
Animal species Symptomatic genital infection can be produced in either inbred or outbred female animals but outbred female Hartley guinea-pigs (250-350 g) are typically used. Fowler et aL (1992) showed that the viral inoculum required to produce infection in 50% of inbred (strain 2) and outbred (Hartley) animals was similar but that the inoculum required to produce clinical disease in 50% of the animals (CD50) was 10 times greater for the strain 2 animals. In addition, while the lethal dose for 50% of the animals was 100fold greater than the CD50 in Hartley animals, the two values were equivalent in strain 2 animals.
Preparation of animals No specialized housing, care nor pretreatment is required.
Storage, preparation of inocula Viral inocula can be prepared on any susceptible cell monolayer, it is not necessary to grow the virus in guinea-pig cells. Virus stocks are titrated, aliquoted and stored frozen at
-80~ When ready for use the virus is quickly thawed, diluted to the appropriate titer and kept on ice until used. Both HSV-1 and HSV-2 strains have been used in genital herpes studies in guinea-pigs. The characteristics of primary and recurrent disease produced by a number of laboratoryadapted strains are shown in Table 108.1. Both virus types produce comparable primary infections. However, nearly all HSV-2-infected animals subsequently manifest recurrent disease with recurrences developing frequently, while many HSV- 1-infected animals experience no recurrent disease and those that do have only infrequent recurrences. In general, HSV-2 strain MS and strain 333 both cause reproducible primary and recurrent infections and are widely used for the testing of antiviral drugs. Low passage clinical virus isolates can also be used; however, care should be taken since they are frequently more virulent than laboratory-adapted strains and can produce high mortality during primary infection.
Infection process Prior to virus inoculation, the vaginal closure membrane is ruptured using a moistened calcium alginate-tipped swab. The vaginal vault is then swabbed gently with the moistened swab and subsequently with a dry swab. The viral inoculum (typically -5.7 log,0 pfu for HSV-2 strain MS or strain 333) is instilled into the vagina in a volume of 0.1-0.2ml using a 1 cc tuberculin syringe and plastic catheter or a micropipettor. To optimize the number of animals that become infected, virus instillation can be performed twice within a short time interval. Some investigators anesthetize the animals prior to inoculation and some plug the vaginal opening with gelfoam after inoculation in an attempt to minimize leakage of the inoculum. Neither of these procedures is necessary.
Table 108.1
Genital herpes simplex virus (HSV) infection in female guinea-pigs
Virus*
Number infected~r
Number with skin diseases$
11/12 10/12 7/12
11/11 8/10 5/7
11/12 11/12 11/12 11/12
10/11 11/11 11/11 11/11
Severity{}
Number with recurrences**
Frequency of recurrencestt
HSV-1 17 syn + KOS(W) MP
8.0 + 0.4 1.4 + 0.3 4.3 + 1.6
7/10 2/6 2/5
1.4 + 0.4 0.5 + 0.3 0.2 + 0.2
HSV-2 HG52 333 MS 186
2.6 15.7 11.7 14.3
+ + + +
0.5 1.4 1.5 1.7
919 8/8 9/9 2/2
7.8 6.8 11.6 17.5
+ + + +
1.7 1.2 1.7 7.5
* Animals received 5 x 10s plaque-forming units (pfu)intravaginally. 1 Defined as infected if virus was recovered from genital tract (vaginal specimens) 24 hours post-infection. :1: Symptonl[atic animals/infected animals. s Area under the lesion score day curve for all animals infected (mean + SE). ** Number of animals with recurrences/number of symptomatic animals that could be evaluated. 11 Lesion days for valuable animals -- days 15-63 (mean + SE).
ANIMAL MODELS OF HERPESVIRUS GENITAL INFECTION: GUINEA-PIG
Key parameters to monitor infection and evaluate antimicrobial therapy Virus replication in the genital tract and neural tissue
The magnitude and duration of acute virus replication in the genital tract can be quantified by plaque titration of vaginal swab samples. Peak virus titers (usually 6.0-7.0 log,0 pfu/ml) are typically seen on day 1 or 2 post-inoculation (p.i.) and then gradually decline until approximately day 10 p.i. (Stanberry et aL, 1982). Following intravaginal inoculation the virus travels via the genitofemoral nerve, reaching the lumbosacral dorsal root ganglia by day 2, the spinal cord by day 3 and the brain stem and cerebral cortex by day 5. Virus replication in the lumbrosacral dorsal root ganglia and spinal cord continues until about day 10 p.i. and can be quantified by plaque titration of tissue homogenates. Measurement of the effects of treatment on the incidence, magnitude and duration of virus replication in the genital tract and neural tissues can provide a quantitative assessment of the efficacy of antiviral treatment.
Primary disease
Following virus inoculation animals are examined daily during acute infection for evidence of primary disease. Genital skin disease typically develops by day 4 p.i. beginning as discrete lesions with an erythematous base and progressing to multiple vesiculoulcerative lesions by days 5-8p.i. Hemorrhagic crusts cover the lesions by days 8-10 p.i. with complete healing of the external genitalia by days 13-15 p.i. The severity of the primary genital skin disease can be quantified using a lesion score-scale ranging from 0 for no disease to 4 for severe vesiculoulcerative disease of the perineum (Stanberry et aL, 1982). The time to appearance of lesions, severity of lesions, peak lesion score and time to healing are all assessed and can be used as indices of therapeutic efficacy. During the primary infection some animals may experience urinary retention and/or hind limb paralysis, both of which are generally transient. Urinary retention can be detected by palpation of the bladder and must be relieved by manual expression of the retained urine.
Recurrent disease
Following recovery from acute genital infection, guineapigs typically develop periodic spontaneous recurrent lesions on the perineum (Stanberry et aL, 1985). Individual lesions may be erythematous and/or vesicular and generally last 1-2 days. The frequency of spontaneous recurrent disease declines over time. By 100 days after HSV-2 inocula-
909
tion spontaneous recurrences are rare, although they may be induced by exposure to ultraviolet radiation (Stanberry, 1989). In addition to recurrent lesions animals experience cervicovaginal shedding of virus which can occur independently of recognizable recurrences (Stanberry et aL, 1985). During the initial week after recovery from primary infection, virus can be isolated by culture in the cervicovaginal secretions of 70-90% of animals with the frequency of isolation decreasing with time after virus inoculation. The incidence and frequency of both recurrent lesions and asymptomatic shedding provide indices to measure the efficacy of antiviral treatment. Animals treated during primary infection can be evaluated to determine whether treatment results in any subsequent effect on recurrent disease. In addition, latently infected animals can be treated to evaluate the effects of therapy both during treatment and once treatment has ceased.
Antimicrobial therapy Guinea-pigs can be administered antiviral compounds by a variety of routes including orally (in drinking water or by gavage), topically to the perineal skin, intravaginally, or by intraperitoneal injection.
Pitfalls (advantages/disadvantages) of the model Advantages
Guinea-pigs are docile and widely available. The pathogenesis of genital herpes in the guinea-pig appears very similar to the infection in humans and involves virus replication in genital and neural tissues. Unlike with the mouse, viremia does not occur and infection is generally self-limiting. A unique feature of the model is the ability of guinea-pigs to experience spontaneous and induced recurrent infections. The model can be used to test experimental therapies for the treatment of either primary or recurrent genital herpes and there are easily quantifiable clinical and virological parameters that can be used to assess efficacy. The model offers an advantage over less sophisticated lethal challenge models in that the numerous efficacy parameters can be used to distinguish between different antiviral compounds, thus providing a basis for identifying a lead compound for further clinical evaluation. The effects of acyclovir in the guinea-pig model of genital herpes parallels what has been observed in human clinical trials, proving strong validation for the model.
910
Disadvantages
Guinea-pigs are expensive and the number of immunological reagents available for the guinea-pig is limited. Occasionally infected animals can develop a secondary bacterial or fungal infection of the perineal skin, making evaluation of recurrent infections impossible. Metabolism of nucleoside analogs can be more rapid in guinea-pigs than in humans, making evaluation of systemically administered drugs more complex.
Contributions of the model to infectious disease therapy Much of our understanding of the pathogenesis of genital herpes comes from studies using the guinea-pig model (Stanberry, 1996). The model has also proven valuable in the assessment of antiviral drugs, vaccines and immunomodulators. Studies in this model accurately predicted the effectiveness of acyclovir in treating HSV-1 and HSV-2 primary genital infection and the usefulness of suppressive acyclovir therapy in controlling recurrent disease (Kern, 1982). The model also demonstrated that treatment of the primary infection did not prevent the establishment of latent infection and hence did not prevent the subsequent development of recurrent infections (Bernstein et aL, 1986). Studies using the guinea-pig model of recurrent genital herpes established that therapeutic vaccines administered to latently infected animals could reduce the frequency of recurrent infections (Stanberry et aL, 1988). This important observation was subsequently confirmed in a clinical trial of patients with frequently recurring genital herpes (Straus et al., 1994). The effectiveness of imiquimod, a potent immunomodulator, in the treatment of a viral sexually transmitted disease was first shown in the guinea-pig model of genital herpes (Bernstein and Harrison, 1989). Imiquimod was subsequently shown to be effective in the treatment of genital warts (human papillomavirus infection) for which it is currently licensed. The model has also shown the effectiveness of several novel prophylactic vaccine strategies, including genetically attenuated and replication-incompetent vaccine viruses and nucleic acid-based (DNA) vaccines (Meignier et aL, 1988;' Bourne et aL, 1996; Da Costa et aL , 1997).
References Bernstein, D. I., Harrison, C. J. (1989). Effects of the immunomodality agent R837 on acute and latent herpes simplex virus type 2 infection. Antimicrob. Agents Chemother., 33, 1511-1515. Bernstein, D. I., Stanberry, L. R., Harrison, C. J., Kappes, J., Myers, M. G. (1986). Effects of oral acyclovir treatment of ini-
N. Bourne and R. Stanberry
tial genital HSV-2 infection upon antibody response, recurrence patterns and subsequent HSV-2 reinfection in guinea pigs.J. Gen. ViroL, 67, 1601-1612. Bourne, N., Stanberry, L. R., Bernstein, D. I., Lew, D. (1996). DNA immunization against experimental genital herpes simplex virus infection. J. Infect. Dis., 173, 800-807. Centers for Disease Control and Prevention (1998). Guidelines for treatment of sexually transmitted diseases. MMWR, 47, 20-26. Da Costa, X. J., Bourne, N., Stanberry, L. R., Knipe, D. M. (1997). Construction and characterization of a replication-defective herpes simplex virus 2 ICP8 mutant strain and its use in immunization studies in a guinea pig model of genital disease. Virology, 232, 1-12. Fleming, D. T., McQuillan, G. M., Johnson, R. E. et al. (1997). Herpes simplex virus type 2 in the United States, 1976 to 1994. N. Engl. J. Med., 337; 1105-1111. Fowler, S. L., Harrison, C. J., Myers, M. G., Stanberry, L. R. (1992). Outcome of herpes simplex virus type 2 infection in guinea pigs. J. Med. Virol., 36, 303-308. Kern, E. R. (1982). Acyclovir treatment of experimental genital herpes simplex virus infections. Am. J. Med., 73, 100-107. Meignier, B., Longnecker, R., Roizman, R. (1988). In vivo behavior of genetically engineered herpes simplex viruses R7017 and R7020: construction and evaluation in rodents. J. Infect. Dis., 58, 602-614. Stanberry, L. R. (1989). An animal model of ultraviolet radiationinduced recurrent herpes simplex virus infection. J. Med. Virol., 28, 125-128. Stanberry, L. R. (1991). Herpes simplex virus vaccine evaluation in animals: the guinea pig model. Rev. Infect. Dis., 13 (suppl. 11), $920-$923. Stanberry, L. R. (1996). The pathogenesis of herpes simplex virus infections. In Genital and Neonatal Herpes (ed Stanberry, L. R.), pp. 31-48. John Wiley, London. Stanberry, L. R., Kern, E. R., Richards, J. T., Abbott, T. M., Overall, J. C. Jr. (1982). Genital herpes in guinea pigs: pathogenesis of the primary infection and description of recurrent disease. J. Infect. Dis., 146, 397-404. Stanberry, L. R., Kern, E. R., Richards. J. T., Overall, J. C., Jr. (1985). Recurrent genital herpes simplex virus infection in guinea pigs. Intervirol., 24, 226-231. Stanberry, L. R. Burke, R. L., Myers, M. G. (1988). Herpes simplex virus glycoprotein treatment of recurrent genital herpes. J. Infect. Dis., 157, 156-163. Stanberry, L. R., Bourne, N., Bravo, F. J., Bernstein, D. I. (1992). Capsaicin sensitive peptidergic neurons are involved in the zosteriform spread of herpes simplex virus infection. J. Med. Virol., 38, 142-146. Stanberry, L. R., Jorgensen, D. M., Nahmias, A. J. (1997). Herpes simplex viruses 1 and 2. In Viral Infection of Humans: Epidemiology and Control, 4th edn (eds Evans, A. S., Kaslow, R. A.), pp. 419-454. Plenum Press, New York. Stephanopoulos, D. E., Myers, M. G., Bernstein, D. I. (1989). Genital infections due to herpes simplex virus type 2 in male guinea pigs. J. Infect. Dis., 159, 89-95. Straus, S. E., Corey, L., Burke, R. L. et al. (1994). Placebo-controlled trial of vaccination with glycoprotein D of herpes simplex virus type 2 immunotherapy of genital herpes. Lancet, 343, 1460-1463. Wald, A., Corey, L. (1996). The clinical features and diagnostic evaluation of genital herpes. In Genital and Neonatal Herpes (ed Stanberry, L. R.), pp. 109-137. John Wiley, London.
Animal Models of Herpesvirus Genital Infection: Guinea-Pig N. Bourne and L. R. Stanberry
Background of human infection Genital herpes is a common sexually transmitted disease caused by herpes simplex virus (HSV) either type 1 or 2. It occurs worldwide and is recognized as a major public health problem in many developed countries (Stanberry et aL, 1997). Recent seroprevalence data indicate that more than one in five adult Americans have been infected with HSV-2, the predominant cause of genital herpes in the United States (Fleming et al., 1997). The natural history of genital herpes includes the initial or primary infection, the establishment of a latent infection in paraspinal sensory ganglia, and the periodic reactivation of latent infection which causes recurrent infections (Wald and Corey, 1996). Initial and recurrent infections in humans may be either symptomatic or asymptomatic. Symptomatic infection is characterized by the development of vesicles and/or ulcers at mucocutaneous sites in the anogenital area. Initial infection is generally more severe than recurrent infections and may be associated with systemic findings such as fever, urinary retention or dysuria, and meningitis. In humans, HSV-1 and HSV-2 produce indistinguishable initial genital infections but the type 2 virus typically causes more frequent symptomatic recurrent infections than HSV-1. Anecdotal reports suggest that a variety of stimuli, including stress and onset of menses, can trigger recurrent infections. To date only experimental ultraviolet radiation exposure has been proven to trigger symptomatic recurrent infections. Asymptomatic initial or recurrent infections are common and probably play a major role in the transmission of virus since people experiencing asymptomatic infections shed virus from anogenital sites but are unaware they are contagious. Intravenous acyclovir and oral acyclovir, valacyclovir, and famciclovir have been proven effective in the treatment of initial genital herpes and impact both the clinical and virological course of the infection (CDC, 1998). Oral acyclovir, valacyclovir, and famciclovir have a modest effect on recurrent infections when used episodically to treat recurrences but all are highly effective at preventing symptomatic and asymptomatic recurrent infections when taken daily as suppressive therapy (CDC, 1998). While there are animal data to suggest that treatment with anti-HSV nucleHandbook of Animal Models of Infection
ISBN 0-12-775390-7
oside analogs early in the course of the initial infection can impact latent infection, the clinical trials conducted to date suggest that antiviral drugs like acyclovir cannot prevent the establishment of the latent infection that occurs during the initial infection, nor can they eliminate the latent infection once it is established.
Background of model Originally described in the 1970s by Austrian scientists, the female guinea-pig model of genital herpes was extensively characterized in the 1980s and has found wide use in exploring the viral pathogenesis and in the evaluation of antiviral drugs and experimental HSV vaccines. The model mimicks many of the clinical, virologic and immunologic features of human infection (Stanberry et aL, 1982, 1985; Stanberry, 1991). Intravaginal inoculation of guinea-pigs with HSV-1 or 2 produces either symptomatic or asymptomatic infection depending on the inoculum titer. Infection results in high titer replication in the lower genital tract and intraneural spread to lumbosacral dorsal root ganglia and spinal cord. Symptomatic infection is characterized by vesiculoulcerative lesions on and about the perineum and some animals exhibit urologic (urinary retention) and neurologic (hind limb paralysis) findings. Initial infection results in the establishment of latent HSV infection in lumbosacral dorsal root ganglia. After recovery from the initial infection, guinea-pigs experience spontaneous recurrent infections manifested as either shedding of virus in vaginal secretions in the absence of recognizable lesions (subclinical viral shedding) or discrete erythematous or vesicular lesions on the perineal skin. The frequency of recurrent infections declines over time and animals infected with HSV-2 exhibit significantly more recurrences than HSV-l-infected guinea-pigs. In addition to spontaneous recurrences, recurrent infections can be induced by exposure of the perineal skin to ultraviolet radiation (Stanberry, 1989). Local and systemic immune responses to the genital infection have been reported. A male model of genital herpes involving intraurethral HSV inoculation has also been described (Stephanopoulos Copyright 9 1999Academic Press All rights of reproduction in any form reserved
908
N. Bourne and R. Stanberry
et aL, 1989). The male model has been used in the evaluation of candidate antiviral drugs (Stanberry et aL, 1992), but
compared to the female model has a higher mortality rate, is difficult to assess recurrent infections, and lacks the ease of repetitive sampling for virology specimens afforded by vaginal swabbing.
Animal species Symptomatic genital infection can be produced in either inbred or outbred female animals but outbred female Hartley guinea-pigs (250-350 g) are typically used. Fowler et aL (1992) showed that the viral inoculum required to produce infection in 50% of inbred (strain 2) and outbred (Hartley) animals was similar but that the inoculum required to produce clinical disease in 50% of the animals (CD50) was 10 times greater for the strain 2 animals. In addition, while the lethal dose for 50% of the animals was 100fold greater than the CD50 in Hartley animals, the two values were equivalent in strain 2 animals.
Preparation of animals No specialized housing, care nor pretreatment is required.
Storage, preparation of inocula Viral inocula can be prepared on any susceptible cell monolayer, it is not necessary to grow the virus in guinea-pig cells. Virus stocks are titrated, aliquoted and stored frozen at
-80~ When ready for use the virus is quickly thawed, diluted to the appropriate titer and kept on ice until used. Both HSV-1 and HSV-2 strains have been used in genital herpes studies in guinea-pigs. The characteristics of primary and recurrent disease produced by a number of laboratoryadapted strains are shown in Table 108.1. Both virus types produce comparable primary infections. However, nearly all HSV-2-infected animals subsequently manifest recurrent disease with recurrences developing frequently, while many HSV- 1-infected animals experience no recurrent disease and those that do have only infrequent recurrences. In general, HSV-2 strain MS and strain 333 both cause reproducible primary and recurrent infections and are widely used for the testing of antiviral drugs. Low passage clinical virus isolates can also be used; however, care should be taken since they are frequently more virulent than laboratory-adapted strains and can produce high mortality during primary infection.
Infection process Prior to virus inoculation, the vaginal closure membrane is ruptured using a moistened calcium alginate-tipped swab. The vaginal vault is then swabbed gently with the moistened swab and subsequently with a dry swab. The viral inoculum (typically -5.7 log,0 pfu for HSV-2 strain MS or strain 333) is instilled into the vagina in a volume of 0.1-0.2ml using a 1 cc tuberculin syringe and plastic catheter or a micropipettor. To optimize the number of animals that become infected, virus instillation can be performed twice within a short time interval. Some investigators anesthetize the animals prior to inoculation and some plug the vaginal opening with gelfoam after inoculation in an attempt to minimize leakage of the inoculum. Neither of these procedures is necessary.
Table 108.1
Genital herpes simplex virus (HSV) infection in female guinea-pigs
Virus*
Number infected~r
Number with skin diseases$
11/12 10/12 7/12
11/11 8/10 5/7
11/12 11/12 11/12 11/12
10/11 11/11 11/11 11/11
Severity{}
Number with recurrences**
Frequency of recurrencestt
HSV-1 17 syn + KOS(W) MP
8.0 + 0.4 1.4 + 0.3 4.3 + 1.6
7/10 2/6 2/5
1.4 + 0.4 0.5 + 0.3 0.2 + 0.2
HSV-2 HG52 333 MS 186
2.6 15.7 11.7 14.3
+ + + +
0.5 1.4 1.5 1.7
919 8/8 9/9 2/2
7.8 6.8 11.6 17.5
+ + + +
1.7 1.2 1.7 7.5
* Animals received 5 x 10s plaque-forming units (pfu)intravaginally. 1 Defined as infected if virus was recovered from genital tract (vaginal specimens) 24 hours post-infection. :1: Symptonl[atic animals/infected animals. s Area under the lesion score day curve for all animals infected (mean + SE). ** Number of animals with recurrences/number of symptomatic animals that could be evaluated. 11 Lesion days for valuable animals -- days 15-63 (mean + SE).
ANIMAL MODELS OF HERPESVIRUS GENITAL INFECTION: GUINEA-PIG
Key parameters to monitor infection and evaluate antimicrobial therapy Virus replication in the genital tract and neural tissue
The magnitude and duration of acute virus replication in the genital tract can be quantified by plaque titration of vaginal swab samples. Peak virus titers (usually 6.0-7.0 log,0 pfu/ml) are typically seen on day 1 or 2 post-inoculation (p.i.) and then gradually decline until approximately day 10 p.i. (Stanberry et aL, 1982). Following intravaginal inoculation the virus travels via the genitofemoral nerve, reaching the lumbosacral dorsal root ganglia by day 2, the spinal cord by day 3 and the brain stem and cerebral cortex by day 5. Virus replication in the lumbrosacral dorsal root ganglia and spinal cord continues until about day 10 p.i. and can be quantified by plaque titration of tissue homogenates. Measurement of the effects of treatment on the incidence, magnitude and duration of virus replication in the genital tract and neural tissues can provide a quantitative assessment of the efficacy of antiviral treatment.
Primary disease
Following virus inoculation animals are examined daily during acute infection for evidence of primary disease. Genital skin disease typically develops by day 4 p.i. beginning as discrete lesions with an erythematous base and progressing to multiple vesiculoulcerative lesions by days 5-8p.i. Hemorrhagic crusts cover the lesions by days 8-10 p.i. with complete healing of the external genitalia by days 13-15 p.i. The severity of the primary genital skin disease can be quantified using a lesion score-scale ranging from 0 for no disease to 4 for severe vesiculoulcerative disease of the perineum (Stanberry et aL, 1982). The time to appearance of lesions, severity of lesions, peak lesion score and time to healing are all assessed and can be used as indices of therapeutic efficacy. During the primary infection some animals may experience urinary retention and/or hind limb paralysis, both of which are generally transient. Urinary retention can be detected by palpation of the bladder and must be relieved by manual expression of the retained urine.
Recurrent disease
Following recovery from acute genital infection, guineapigs typically develop periodic spontaneous recurrent lesions on the perineum (Stanberry et aL, 1985). Individual lesions may be erythematous and/or vesicular and generally last 1-2 days. The frequency of spontaneous recurrent disease declines over time. By 100 days after HSV-2 inocula-
909
tion spontaneous recurrences are rare, although they may be induced by exposure to ultraviolet radiation (Stanberry, 1989). In addition to recurrent lesions animals experience cervicovaginal shedding of virus which can occur independently of recognizable recurrences (Stanberry et aL, 1985). During the initial week after recovery from primary infection, virus can be isolated by culture in the cervicovaginal secretions of 70-90% of animals with the frequency of isolation decreasing with time after virus inoculation. The incidence and frequency of both recurrent lesions and asymptomatic shedding provide indices to measure the efficacy of antiviral treatment. Animals treated during primary infection can be evaluated to determine whether treatment results in any subsequent effect on recurrent disease. In addition, latently infected animals can be treated to evaluate the effects of therapy both during treatment and once treatment has ceased.
Antimicrobial therapy Guinea-pigs can be administered antiviral compounds by a variety of routes including orally (in drinking water or by gavage), topically to the perineal skin, intravaginally, or by intraperitoneal injection.
Pitfalls (advantages/disadvantages) of the model Advantages
Guinea-pigs are docile and widely available. The pathogenesis of genital herpes in the guinea-pig appears very similar to the infection in humans and involves virus replication in genital and neural tissues. Unlike with the mouse, viremia does not occur and infection is generally self-limiting. A unique feature of the model is the ability of guinea-pigs to experience spontaneous and induced recurrent infections. The model can be used to test experimental therapies for the treatment of either primary or recurrent genital herpes and there are easily quantifiable clinical and virological parameters that can be used to assess efficacy. The model offers an advantage over less sophisticated lethal challenge models in that the numerous efficacy parameters can be used to distinguish between different antiviral compounds, thus providing a basis for identifying a lead compound for further clinical evaluation. The effects of acyclovir in the guinea-pig model of genital herpes parallels what has been observed in human clinical trials, proving strong validation for the model.
910
Disadvantages
Guinea-pigs are expensive and the number of immunological reagents available for the guinea-pig is limited. Occasionally infected animals can develop a secondary bacterial or fungal infection of the perineal skin, making evaluation of recurrent infections impossible. Metabolism of nucleoside analogs can be more rapid in guinea-pigs than in humans, making evaluation of systemically administered drugs more complex.
Contributions of the model to infectious disease therapy Much of our understanding of the pathogenesis of genital herpes comes from studies using the guinea-pig model (Stanberry, 1996). The model has also proven valuable in the assessment of antiviral drugs, vaccines and immunomodulators. Studies in this model accurately predicted the effectiveness of acyclovir in treating HSV-1 and HSV-2 primary genital infection and the usefulness of suppressive acyclovir therapy in controlling recurrent disease (Kern, 1982). The model also demonstrated that treatment of the primary infection did not prevent the establishment of latent infection and hence did not prevent the subsequent development of recurrent infections (Bernstein et aL, 1986). Studies using the guinea-pig model of recurrent genital herpes established that therapeutic vaccines administered to latently infected animals could reduce the frequency of recurrent infections (Stanberry et aL, 1988). This important observation was subsequently confirmed in a clinical trial of patients with frequently recurring genital herpes (Straus et al., 1994). The effectiveness of imiquimod, a potent immunomodulator, in the treatment of a viral sexually transmitted disease was first shown in the guinea-pig model of genital herpes (Bernstein and Harrison, 1989). Imiquimod was subsequently shown to be effective in the treatment of genital warts (human papillomavirus infection) for which it is currently licensed. The model has also shown the effectiveness of several novel prophylactic vaccine strategies, including genetically attenuated and replication-incompetent vaccine viruses and nucleic acid-based (DNA) vaccines (Meignier et aL, 1988;' Bourne et aL, 1996; Da Costa et aL , 1997).
References Bernstein, D. I., Harrison, C. J. (1989). Effects of the immunomodality agent R837 on acute and latent herpes simplex virus type 2 infection. Antimicrob. Agents Chemother., 33, 1511-1515. Bernstein, D. I., Stanberry, L. R., Harrison, C. J., Kappes, J., Myers, M. G. (1986). Effects of oral acyclovir treatment of ini-
N. Bourne and R. Stanberry
tial genital HSV-2 infection upon antibody response, recurrence patterns and subsequent HSV-2 reinfection in guinea pigs.J. Gen. ViroL, 67, 1601-1612. Bourne, N., Stanberry, L. R., Bernstein, D. I., Lew, D. (1996). DNA immunization against experimental genital herpes simplex virus infection. J. Infect. Dis., 173, 800-807. Centers for Disease Control and Prevention (1998). Guidelines for treatment of sexually transmitted diseases. MMWR, 47, 20-26. Da Costa, X. J., Bourne, N., Stanberry, L. R., Knipe, D. M. (1997). Construction and characterization of a replication-defective herpes simplex virus 2 ICP8 mutant strain and its use in immunization studies in a guinea pig model of genital disease. Virology, 232, 1-12. Fleming, D. T., McQuillan, G. M., Johnson, R. E. et al. (1997). Herpes simplex virus type 2 in the United States, 1976 to 1994. N. Engl. J. Med., 337; 1105-1111. Fowler, S. L., Harrison, C. J., Myers, M. G., Stanberry, L. R. (1992). Outcome of herpes simplex virus type 2 infection in guinea pigs. J. Med. Virol., 36, 303-308. Kern, E. R. (1982). Acyclovir treatment of experimental genital herpes simplex virus infections. Am. J. Med., 73, 100-107. Meignier, B., Longnecker, R., Roizman, R. (1988). In vivo behavior of genetically engineered herpes simplex viruses R7017 and R7020: construction and evaluation in rodents. J. Infect. Dis., 58, 602-614. Stanberry, L. R. (1989). An animal model of ultraviolet radiationinduced recurrent herpes simplex virus infection. J. Med. Virol., 28, 125-128. Stanberry, L. R. (1991). Herpes simplex virus vaccine evaluation in animals: the guinea pig model. Rev. Infect. Dis., 13 (suppl. 11), $920-$923. Stanberry, L. R. (1996). The pathogenesis of herpes simplex virus infections. In Genital and Neonatal Herpes (ed Stanberry, L. R.), pp. 31-48. John Wiley, London. Stanberry, L. R., Kern, E. R., Richards, J. T., Abbott, T. M., Overall, J. C. Jr. (1982). Genital herpes in guinea pigs: pathogenesis of the primary infection and description of recurrent disease. J. Infect. Dis., 146, 397-404. Stanberry, L. R., Kern, E. R., Richards. J. T., Overall, J. C., Jr. (1985). Recurrent genital herpes simplex virus infection in guinea pigs. Intervirol., 24, 226-231. Stanberry, L. R. Burke, R. L., Myers, M. G. (1988). Herpes simplex virus glycoprotein treatment of recurrent genital herpes. J. Infect. Dis., 157, 156-163. Stanberry, L. R., Bourne, N., Bravo, F. J., Bernstein, D. I. (1992). Capsaicin sensitive peptidergic neurons are involved in the zosteriform spread of herpes simplex virus infection. J. Med. Virol., 38, 142-146. Stanberry, L. R., Jorgensen, D. M., Nahmias, A. J. (1997). Herpes simplex viruses 1 and 2. In Viral Infection of Humans: Epidemiology and Control, 4th edn (eds Evans, A. S., Kaslow, R. A.), pp. 419-454. Plenum Press, New York. Stephanopoulos, D. E., Myers, M. G., Bernstein, D. I. (1989). Genital infections due to herpes simplex virus type 2 in male guinea pigs. J. Infect. Dis., 159, 89-95. Straus, S. E., Corey, L., Burke, R. L. et al. (1994). Placebo-controlled trial of vaccination with glycoprotein D of herpes simplex virus type 2 immunotherapy of genital herpes. Lancet, 343, 1460-1463. Wald, A., Corey, L. (1996). The clinical features and diagnostic evaluation of genital herpes. In Genital and Neonatal Herpes (ed Stanberry, L. R.), pp. 109-137. John Wiley, London.