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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hantaviruses (family Bunyaviridae, genus Hantavirus) are negative-sense RNA viruses that comprise more than 20 virus species, each maintained by a specific rodent host.4,5 Field and laboratory studies illustrate that rodents infected with hantaviruses are stable reservoirs, shedding the virus in saliva, urine, and feces, but showing no overt signs of disease.6 Unlike other Bunyaviridae viruses, which are transmitted by arthropods, hantaviruses are transmitted directly among rodents through inhalation of aerosolized virus shed in excreta and through passage of virus in saliva during aggressive encounters.7–9
Among several rodent reservoirs, including deer mice (Peromyscus maniculatus), western harvest mice (Reithrodontomys megalotis), brush mice (Peromyscus boylii), bank voles (Clethrionomys glareolus), cotton rats (Sigmodon hispidus), and to a lesser extent Norway rats (Rattus norvegicus), males are more frequently infected with hantaviruses than are females.10–14 Males also are more likely to engage in aggression than are females.7,15,16 Because infection as well as the incidence and severity of wounding are higher among adult males than among conspecific females or juvenile males, hantavirus infection of adult male rodents likely occurs during aggressive encounters.7,17
Hantaviruses are associated with increased aggression in their rodent hosts.7,17,18 Longitudinal studies with marked and released Norway rats illustrate that animals wounded between captures are more likely to develop antibody against Seoul virus (i.e., the naturally occurring hantavirus in Norway rats) upon recapture, and seroconversion is more prevalent among adult males than among adult females or juvenile males.7 Thus, increased aggressive behavior at the onset of sexual maturity may increase Seoul virus transmission among adult Norway rats. Whether aggression (as measured by wounding) correlates with shedding of Seoul virus or the presence of Seoul virus RNA in tissue has not been reported.
Norway rats infected with Seoul virus remain persistently infected, despite the presence of effector immune responses.8,19 Antibody responses are detectable 15–20 days post-inoculation and persist for a lifetime.8,20–22 Only during a limited period of time, however, are animals shedding virus in saliva, urine, and feces and capable of infecting uninfected individuals.8,20 Virus shedding typically occurs immediately after infection and is followed by a chronic period of infection that is characterized by active immune responses in the absence of virus shedding.19,20 During this time of chronic infection, animals often fail to transmit virus to uninfected cage mates.8 Therefore, because virus shedding occurs during the acute phase of infection,8,23 shedding may be a better indicator of a recent infection caused by wounding than antibody, which only becomes detectable two weeks after infection.
The present study characterized the relationship between Seoul virus infection and aggression among natural populations of male and female Norway rats and sought to uncover the mechanisms mediating this relationship. We hypothesized that adult male rats with wounds, an indicator of aggressive behavior, would be more likely to be infected with Seoul virus than would males with no wounds. Also, because adult males are more likely to engage in aggression than either adult females or juvenile males, the relationship between wounding and infection may be more pronounced among adult males than among either females or juvenile males. If wounding is the primary mode of infection, then animals with wounds may be more likely to shed virus, an indicator of an acute infection, than rats with no wounds. We further hypothesized that Seoul virus may cause increased aggressive behavior by infecting cells in areas of the brain associated with aggression (e.g., hippocampus, hypothalamus, amygdala, and cingulate cortex). Alternatively, if Seoul virus does not cross the blood-brain barrier in adult rodents,24–26 then infection may alter aggression through effects on endocrine glands and hormones (e.g., testosterone and corticosterone) that relay chemical signals to the central nervous system.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Procedure. Following trapping, male and female rats were brought to the laboratory where they were anaesthetized with a cocktail of ketamine HCl (80 mg/kg) and xylazine (6 mg/kg) (Phoenix Pharmaceutical, St. Joseph, MO), sexed, weighed, and bled from the retro-orbital sinus. Plasma was used to measure concentrations of IgG antibody to Seoul virus, testosterone, and corticosterone. Age was determined based on body mass.7,28 Of the 49 adult females that were trapped, 17 females were pregnant and were assigned to a mass class based on their body mass with fetuses. Based on the average birth weight of Norway rats, we determined that the additional fetal mass did not significantly alter assignment of females to the mass classes. Each rat was examined for wounds by combing the fur to uncover scars, abscesses, or scabs that corresponded with wounding. Wounds originally were scored into five classes: 0 = no wounds; 1 = minor tail wounds; 2 = larger tail wounds and small wounds (< 0.25 cm) on the rump; 3 = larger wounds (0.25–0.5 cm) on the body; and 4 = many extensive wounds on the body and tail.7 For subsequent analyses, wounding categories were partitioned into high (score = 3–4), low (score = 1–2), or no wounds. Saliva samples were collected from anaesthetized rats after injecting them intraperitoneally with 2.5 mg/kg of pilocarpine HC1 (Sigma, St. Louis, MO) suspended in 0.9% sterile saline. Pilocarpine is a naturally occurring, nonselective muscarinic acetylcholine receptor agonist that, at low doses, stimulates salivary secretion.29 After saliva was collected, animals were killed and urine, feces, lung, kidney, spleen, adrenal glands, gonads, secondary sex organs (e.g., epididymis in males and uterine horns in females), and brain samples were collected and stored at -80°C or in 10% formaldehyde.
Enzyme-linked immunosorbent assay.
Microtiter plates were coated overnight at 4°C with gamma-irradiated Vero E6 cells infected with Seoul virus or gamma-irradiated uninfected Vero E6 cells diluted in carbonate buffer. Thawed plasma samples, as well as positive and negative control samples, were diluted 1:100 in phosphate-buffered saline-Tween (PBS-T) with 2% fetal bovine serum (FBS) and added in duplicate to antigen-coated wells containing either infected or uninfected Vero E6 cells. The plates were incubated at 37°C for one hour and secondary antibody (alkaline phosphatase-conjugated anti-rat IgG (heavy plus light chain; Kirkegaard and Perry Laboratories, Gaithersburg, MD) diluted in PBS with 2% FBS was added. The plates were incubated for one hour at 37°C and substrate buffer (0.5 mg/ mL of p-nitrophenylphosphate diluted in diethanolamine substrate buffer) was added. The enzyme-substrate reaction was terminated, the optical density (OD) was measured at 405 nm, and the average OD for each set of uninfected Vero E6 duplicates was subtracted from the average OD for each set of infected Vero E6 duplicates. Samples were considered positive if the average adjusted OD was 
0.100 nm.20,21 To minimize intra-plate and inter-plate variability, the average adjusted OD for each sample was expressed as a percentage of its plate positive control OD for statistical analyses.
Radioimmunoassays (RIAs). Plasma testosterone concentrations in males and corticosterone concentrations in both sexes were assayed using double antibody RIA kits and the manufacturer’s protocols (ICN Biochemicals, Inc., Carson, CA).
Isolation of RNA. Virus RNA was isolated using a guanidine isothiocyanate procedure.30 RNA was isolated from saliva, urine, feces, and organs using Trizol LS and the manufacturer’s protocol as described previously (Invitrogen, Carlsbad, CA).20,21
Nested reverse transcriptase-polymerase chain reaction (RT-PCR). First-strand synthesis cDNA was prepared using TaqMan RT reagents and the manufacturer’s protocol (Applied Biosystems, Foster City, CA). The positive control was Seoul virus RNA isolated from our virus stock (strain SR–11) and the negative control was diethylpyrocarbonate-treated water included in the cDNA syntheses and in both primary and secondary amplifications. Outer primers were used to amplify a 280-basepair sequence of the Seoul virus genome in a 100-µL reaction mixture containing 20 µL of the cDNA.30 The nested 176-basepair sequence was amplified in a 100-µL reaction mixture containing the nested primers and 2 µL of the first DNA amplification product as described previously.20,21 Primary and secondary reactions each were amplified for one cycle at 94°C for three minutes and 40 cycles at 94°C for 30 seconds, 55°C for 45 seconds, and 72°C for 60 seconds, followed by incubation for 10 minutes at 72°C. The nested PCR products were subjected to electrophoresis on a 4% gel (3% NuSieve plus 1% agarose; FMC Bioproducts, Rockland, ME), stained with ethidium bromide, and examined for bands of 176 basepairs.
Immunohistochemistry. Brains, testes, epididymides, ovaries, uterine horns, and adrenal glands were fixed in 10% buffered formaldehyde, embedded in paraffin, cut into 5–µm sections, and mounted on glass slides. Sections were deparaffinized and post-fixed in Streck tissue fixative, and antigen retrieval was performed using Vector Antigen Retrieval (1:100; Vector Laboratories, Burlingame, CA). Tissue sections were incubated with monoclonal antibody against the Seoul virus nucleocapsid protein (1:250; EC01–AB08, gift of Connie Schmaljohn, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD),31 labeled, and stained using the manufacture’s protocol for the super sensitive immunodetection system and an automated processor (Biogenex, San Ramon, CA). Immune complexes were visualized using an alkaline phosphatase label (Vector Red; Vector Laboratories) and counterstained with hematoxylin. After counterstaining, sections were dehydrated and mounted with Permount.
Statistical analyses.
Body-mass classes were partitioned into juveniles (
199 grams), young adults (200–399 grams), and adults ( 400 grams), and wounding categories were partitioned into high (score = 3–4), low (score = 1–2), or no wounds. Antibody and virus prevalence (i.e., the proportion of animals with detectable antibody and virus, respectively) were compared among groups using chi-square analyses. Differences in hormone concentrations were assessed using one-way analysis of variance. In cases where the data violated the assumptions of normality, nonparametric statistics were used. Significant interactions were further analyzed using the Tukey or Dunn method for pairwise multiple comparisons. Mean differences were considered statistically significant if P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mass class.
IgG antibody to Seoul virus.
Forty-seven percent (55 of 116) of wild-caught Norway rats trapped in Baltimore, Maryland had detectable IgG antibody to Seoul virus. For all Norway rats trapped, 15% (3 of 20) of juvenile rats had detectable IgG antibody to Seoul virus, 23% (12 of 52) of young adults were antibody positive, and 91% (40 of 44) of adult rats had IgG antibody to Seoul virus in circulation. For both male (
2 = 26.27, degrees of freedom [df] = 2, P < 0.001) and female (2 = 28.26, df = 2, P < 0.001) Norway rats, adults were more likely to have detectable IgG antibody to Seoul virus than were young adults or juveniles (Figure 1
). A similar proportion of males and females had detectable IgG antibody to Seoul virus in each of the three mass classes (P > 0.05, in each case).
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2 = 14.40, df = 2, P < 0.001; Figure 1A
). In contrast, there was no significant difference in Seoul virus shedding among adult, young adult, and juvenile female rats (P = 0.113; Figure 1B).
Eighty-five percent (99 of 117) of the male and female rats had Seoul virus RNA in target tissues (i.e., in at least one of the tissues examined). Adult male Norway rats were more likely to have Seoul virus RNA in saliva (2 = 8.57, df = 2, P =0.014) and adrenal glands (2 =14.66, df =2, P < 0.001) than were young adult or juvenile males (Table 1). Adult males were also more likely to have detectable Seoul virus RNA in the kidneys than were juveniles (2 = 8.30, df = 2, P = 0.016; Table 1). Conversely, the percentage of adult, young adult, and juvenile males that had viral RNA present in the lung, spleen, testes, epididymis, urine, and feces did not differ (P > 0.05 in each case; Table 1).
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2 =10.83, df =2, P =0.004), lungs (2 = 16.25, df = 2, P < 0.001), and adrenal glands (2 = 14.25, df = 2, P < 0.001) than did juvenile or young adult females (Table 1). Adult females also were more likely to have Seoul virus RNA in kidneys than were young adults (2 = 8.65, df = 2, P = 0.013; Table 1). The proportion of adult, young adult, and juvenile females that had viral RNA in the spleen, ovaries, uterine horns, urine, and feces did not differ (P > 0.05 in each case; Table 1). A similar percentage of males and females, regardless of mass class, had detectable Seoul virus RNA in tissues, saliva, urine, and feces (P > 0.05 in each case).
Seoul virus nucleocapsid protein.
Immunohistochemistry was used to localize viral nucleocapsid protein in cells of the brain and adrenal glands of both sexes, the testes and epididymides of males, and the ovaries and uterine horns of females. Seoul virus antigen was not detectable in the brains of male or female Norway rats (n = 33; juveniles = 6, young adults = 15, and adults = 12). Seoul virus antigen was detected in the testes of adult male rats with viral protein identified both in the seminiferous tubules and interstitial regions of the testes (21 of 45; Figure 2A–C). Adult males (13 of 19) were more likely to have Seoul virus antigen in the testes than were either young adult (8 of 26) or juvenile (0 of 10) males (2 = 14.14, df = 2, P < 0.001). Seoul virus nucleocapsid protein also was localized in the heads of elongated spermatids in the seminiferous tubules of adult (5 of 19) and young adult males (1 of 26; Figure 2D). No Seoul virus antigen was detected in the epididymides of either adult (0 of 28) or juvenile (0 of 6) males. Seoul virus protein was identified in the adrenal glands of adult (7 of 19) and young adult (5 of 26), but not juvenile (0 of 10) males (P = 0.067). Among females, Seoul virus antigen was not detected in either the ovaries or uterine horns (0 of 27 in each case). Seoul virus nucleocapsid protein also was not detected in the adrenal glands of adult (0 of 24) or juvenile (0 of 3) female rats.
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Wounding.
Wounding frequency.
For both males and females, wounding frequency increased with age (Figure 3). Adult rats were more likely to have severe wounds (i.e., high-grade wounds) than were juveniles or young adults (Figure 3). The relationship between wounding grade and age was more pronounced among male than among female rats. If non-wounded animals were omitted from the analyses, older males were still more likely to have severe wounds (r =0.675, P < 0.001). Conversely, there was no significant correlation between wounding and age among females if non-wounded animals were removed from the analyses (r = 0.195, P = 0.284). Of the trapped rats, 86% (18 of 21) of adult males had severe wounds (i.e., high wounding grade) at the time of trapping; whereas only 39% (9 of 23) of adult females had severe wounds (2 = 8.18, df = 2, P = 0.004).
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2 =28.04, df =2, P < 0.001; Figure 4A). Females with both high- and low-grade wounds were more likely to have IgG antibody to Seoul virus than were females with no wounds (2 = 12.78, df = 2, P = 0.002; Figure 4B).
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2 = 7.73, df = 2, P = 0.021; Figure 4A). In contrast, the relationship between virus shedding and wounding was not statistically significant among female rats (P = 0.162; Figure 4B).
Males with high-grade wounds had a higher incidence of Seoul virus RNA in target organs than did males with no wounds (2 = 6.32, df = 2, P = 0.042; Table 2). The relationship between wounding and viral RNA in tissues was not present among females (P = 0.232; Table 2). Severely wounded males were more likely to have Seoul virus RNA in the lungs (2 = 6.28, df = 2, P = 0.043) and adrenal glands (2 = 14.42, df = 2, P < 0.001) than were males with no or low-grade wounds (Table 2). Males with high-grade wounds were more likely to have Seoul virus RNA in kidneys than were males with no wounds (2 = 6.47, df = 2, P = 0.039; Table 2). The percentage of males with high-grade, low-grade, or no wounds that had virus present in the spleen, testes, epididymis, saliva, urine, and feces did not differ (P > 0.05 in each case).
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2 = 6.94, df = 2, P = 0.031; Table 2). Conversely, there was no correlation between wounding grade and Seoul virus RNA in the lungs, kidneys, spleen, ovaries, uterine horns, saliva, urine, or feces of female rats (P > 0.05 in each case).
Seoul virus nucleocapsid protein.
Males with high-grade wounds (14 of 21) were more likely to have Seoul virus nucleocapsid protein in their testes than were males with either low-grade wounds (4 of 18) or no wounds (3 of 16; 2 = 11.72, df =2, P =0.003). Males with severe wounds (9 of 21) also were more likely to have Seoul virus antigen detected in their adrenal glands than were males with either no (1 of 16) or low-grade (2 of 18) wounds (2 =8.93, df =2, P =0.011).
Hormone concentrations.
Males with high-grade wounds had higher testosterone concentrations than did males with low-grade or no wounds (H =9.51, df =2, P =0.009; Figure 5). Corticosterone concentrations did not differ among males with high-grade (mean ± SEM = 384.10 ± 47.30 ng/mL), low-grade (493.47 ± 65.60 ng/mL), or no wounds (492.33 ± 71.00 ng/mL; P = 0.314). Females with no wounds (707.35 ± 80.47 ng/mL) had higher corticosterone concentrations than did females with high-grade wounds (429.33 ± 71.59 ng/mL), but not than females with low-grade wounds (468.14 ± 76.69 ng/mL; F (2, 40) = 3.93, P < 0.028).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, Seoul virus was not detected in the brains of wild-caught rats. Other hantaviruses, including Sin Nombre virus, do not cross the blood-brain barrier of adult rodents.24,26 In contrast, viral RNA and viral protein were detected in the testes of males. Seoul virus nucleocapsid protein was identified in sperm and sperm progenitors as well as in steroid hormone-producing cells (i.e., Leydig cells) in the testes. Additionally, wounded males had significantly higher testosterone concentrations than did non-wounded males (although age-related effects on hormone concentrations could contribute to this difference). Taken together, these data suggest that Seoul virus may alter host behavior by infecting cells in the testes and altering production of testosterone. Black Creek Canal virus and Sin Nombre virus RNA have been reported in the gonads of deer mice and cotton rats, respectively.23,24 Whether hantavirus infection of the gonads directly causes increased production of androgens requires further investigation. Laboratory studies of mice infected with tick-borne encephalitis virus (family Flaviviridae) illustrate that testosterone concentrations and aggression are higher among infected than uninfected mice.32
In addition to testosterone, glucocorticoids, produced by the adrenal glands, are associated with the expression of aggression.33 Among male rats, the prevalence of Seoul virus RNA in the adrenal glands was higher among wounded than among non-wounded animals; 80% of the males with high-grade wounds had Seoul virus RNA in the adrenal glands. In the present study, a lower percentage of animals had detectable viral protein than viral RNA in the adrenal glands. The discrepancy between the presence of viral RNA and viral protein in the adrenal glands, as well as in the epididymides, ovaries, and uterine horns, may suggest that production of Seoul virus protein is below our limit of detection. Alternately, infection of cells in some organs may result in production of early gene products but not late viral proteins, suggesting that virus may not replicate in these organs. Future studies should use in situ hybridization to localize viral RNA in specific cells.
Although wounding grade predicted the presence of viral RNA in the adrenal glands, wounding grade was not related to corticosterone concentrations in either male or female rats. In contrast, age-related differences in corticosterone concentrations were observed in which juveniles had higher concentrations than adults. Elevated corticosterone concentrations among juvenile animals, however, may be an artifact of the trapping procedure used in the present study, in which juvenile rats may respond to the trapping with an elevated stress response.34
Whether engaging in aggressive behavior increases exposure to hantaviruses (i.e., host-mediated hypothesis) or whether infection increases the propensity to engage in aggression (i.e., parasite-mediated hypothesis) remains unclear. We currently are investigating whether dominant males that engage in elevated levels of aggression and have increased testosterone concentrations are more susceptible to Seoul virus than subordinant males. Recent laboratory studies of male Norway rats inoculated with Seoul virus reveal that males tested 30 days after inoculation with Seoul virus spend more time engaged in aggression during resident-intruder tests than either uninfected males or males tested 15 days after inoculation.17 Males that engage in aggression for a longer duration of time have more virus present in lung, kidney, and testis than do males that engage in less aggression.17 Seoul virus is not present in the brains of infected adult male laboratory rats. Thus, the changes in host aggressive behavior may be a consequence of elevated virus replication in peripheral tissues, including the gonads.
Adult males were more likely to engage in aggression than were adult females. Also, the relationship between wounding and infection status (as measured by viral shedding and the presence of viral RNA in tissues) was more pronounced among male than among female Norway rats, suggesting that the behavioral mode of infection may differ between the sexes. In contrast, the relationship between wounding and IgG antibody to Seoul virus was present in both sexes. Because circulating antibody persists for a lifetime, the prevalence of IgG antibody to Seoul virus among wounded animals is more likely to be confounded by age than viral RNA in saliva, excrement, and tissues, which is transient. Thus, viral RNA may be a better indicator of a recent infection caused by wounding.
Although males and females may differ in how they contract Seoul virus infection, a comparable number of males and females were infected with Seoul virus in the present study. Specifically, the prevalence of circulating IgG antibody to Seoul virus and Seoul virus RNA in target organs was similar between the sexes. A lack of sex differences in the prevalence of infection among natural populations of Norway rats has been previously reported.7,27 Unlike other rodents (e.g., deer mice, western harvest mice, cotton rats, and bank voles), for which sex differences in hantaviral infection are reported, Norway rats are opportunistic breeders and live in mixed-sex communal groups instead of in solitary territories.28 Thus, there are sufficient opportunities for both males and females to be exposed to Seoul virus by engaging in social behaviors, such as grooming, mating, and/or aggression. The presence of viral RNA in elongated spermatids in the testes and in the epididymides of males as well as in the ovaries and uterine horns of females suggests that mating may be one mode of Seoul virus transmission. Hantaviruses are not transmitted vertically and there is no evidence of congenital infection.35,36 Whether infected spermatids are viable also requires investigation.
The present study demonstrates that a relationship exists between aggression and Seoul virus infection and this relationship may be mediated by the effects of infection on the endocrine system. Future studies should examine the relationship between hantaviral infection and aggression in other rodent reservoirs, such as deer mice, western harvest mice, cotton rats, and bank voles, which differ from Norway rats in their breeding and social behaviors. Investigating the relationship between hantaviral infection and social behavior is important to fully understand how hantaviruses are transmitted and maintained in rodent reservoir populations.
Received September 16, 2003. Accepted for publication November 1, 2003.
Acknowledgments: We thank Connie Schmaljohn, Cindy Rossi, and Kristen Spik, (United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD) for providing hantavirus reagents. We also thank Jennifer Uhrlaub (Johns Hopkins School of Medicine) for assistance with developing the immunohistochemistry protocol, Ann Lawler (Johns Hopkins School of Medicine) for assistance with the radioimmunoassay, and Joshua Fine, Mike Johnson, Nikhil Joshi, Rebekah Kent, Marilyn Klein, and John Pisciotta (Johns Hopkins Bloomberg School of Public Health) for assistance with rat trapping.
Financial support: This work was supported by National Aeronautics and Space Administration grant NCC5–305 (Gregory E. Glass), National Institutes of Health (NIH) grant F32 AI–10324 (Sabra L. Klein), and in part by NIH grant P30 HD 06268.
Authors’ addresses: Ella R. Hinson, Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06520, E-mail: ella.hinson{at}yale.edu. Scott M. Shone, Gregory E. Glass, and Sabra L. Klein, Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205, E-mails: sshone{at}jhsph.edu, ggurrigl{at}jhsph.edu, and saklein{at}jhsph.edu M. Christine Zink, Department of Comparative Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205, E-mail: mczink{at}jhmi.edu.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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