Experimental Infection with Puumala Virus,the Etiologic Agent of. Nephropathia Epidemica, in Bank Voles (Clethrionomys glareolus). RICHARD YANAGIHARA ...
JOURNAL OF VIROLOGY, JUlY 1985, p. 34-38 0022-538X/85/070034-05$02.00/0 Copyright ©3 1985, American Society for Microbiology
Vol. 55, No. 1
Experimental Infection with Puumala Virus, the Etiologic Agent of Nephropathia Epidemica, in Bank Voles (Clethrionomys glareolus) RICHARD YANAGIHARA,* HERBERT L. AMYX, AND D. CARLETON GAJDUSEK Laboratory of Central Nervous System Studies, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland 20205 Received 19 December 1984/Accepted 11 March 1985
Subclinical chronic infections characterized by transient viremia, prolonged virus shedding in oropharyngeal secretions and feces, and virus persistence in tissues (particularly lung) developed in laboratory-bred weanling bank voles (Clethrionomys glareolus) inoculated intramuscularly with Puumala virus (strain Hallnas), the etiologic agent of nephropathia epidemica. Viral antigen, as evidence by granular fluorescence, was detected in the lungs, liver, spleen, pancreas, salivary glands, and small intestine. Infectious virus was found in the lungs from 14 to 270 days postinoculation, and feces and urine collected 35 to 130 days postinoculation were regularly and sporadically infectious, respectively. Horizontal transmission coincided with virus shedding in oropharyngeal secretions. Suckling voles also developed asymptomatic persistent infections after intracerebral inoculation, and histopathological changes were absent deqpite widespread infection. Our data resemble findings in Apodemus agrarius experimentally infected with Hantaan virus, the prototype virus of hemorrhagic fever with renal syndrome, suggesting that the mechanisms of maintenance and transmission of Puumala and Hantaan viruses are similar in their respective wild-rodent hosts.
Although the zoonotic nature of nephropathia epidemica (NE), the Fennoscandian form of hemorrhagic fever with renal syndrome (HFRS) (5, 7, 13, 14, 23), has long been known (6, 7, 15, 16), the wild-rodent reservoirs have only recently been identified (2, 12, 19). Much is currently known about the epidemiology and ecology of NE (1, 6, 7, 16) and about the principal rodent host, bank voles (Clethrionomys glareolus) (2, 15, 16, 19), but the duration of virus shedding and persistence and the modes of virus transmission in bank voles remain unclear. The etiologic agent of NE, Puumala virus (2, 18), has been isolated and propagated in laboratorybred C. glareolus (22). We now present data which indicate that experimental Puumala virus infections in bank voles resemble experimental infections of striped field mice (Apodemus agrarius) with Hantaan virus (10, 11), the etiologic agent of Korean hemorrhagic fever and the prototype virus of HFRS, suggesting that similar mechanisms are probably operative in virus maintenance and in the transmission of disease to humans in different geographical foci of HFRS. (This work was presented in part at the joint meeting of the Royal and American Societies of Tropical Medicine and Hygiene in Baltimore, Md., in December 1984.)
old) voles were kept in cages that were covered with polyester filter bonnets and housed in Horsfall isolators. Virus. Puumala virus (strain Hallnas) isolated from the lungs of a bank vole trapped in November 1981 in Vasterbotten County, Sweden, was passaged twice in laboratory-bred C. glareolus (22). Animals were sacrificed 42 days postinoculation, and lung tissues were triturated in Eagle minimal essential medium (HEM Research, Inc., Rockville, Md.) supplemented with 2% heat-inactivated fetal bovine serum and gentamicin (50 Fjg/ml); the homogenate was then centrifuged at 900 x g for 10 min. The resulting 5% suspension had an infectivity titer of 1045 50% infective doses (ID50) per ml in bank voles, as calculated by the method of Reed and Muench (17). Virus distribution and persistence. To determine the duration of viremia and of virus persistence and the distribution of viral antigen in tissues, we sacrificed weanling voles at different times from 7 to 270 days after intramuscular (i.m.) inoculation with 103-5 ID50 of virus. Sections of lung, liver, spleen, kidney, pancreas, and salivary gland and occasionally of lymph node, small intestine, adrenal gland, anJ brain were examined for viral antigen by the indirect immunofluorescent-antibody (IFA) technique (22). Heparinized blood and lungs were assayed for virus infectivity in bank voles. Representative portions of selected tissues were fixed for 1 week in buffered Formalin, and paraffin-embedded sections were examined by light microscopy after hematoxylin and eosin staining. To assess the influence of age on experimental infections, we inoculated suckling voles intracerebrally with 750 ID50 Of virus. Animals were examined daily for the first 5 weeks and biweekly thereafter for signs of clinical illness and were sacrificed 35 or 270 days postinoculation, at which time the brain, lungs, kidneys, liver, heart, spleen, and salivary glands were tested for antigen. Virus secretion and excretion. Virus shedding was studied by testing secretions and excretions from weanling voles inoculated i.m. with 103.5 ID50 of virus. Oropharyngeal secretions were obtained at weekly intervals from 7 to 42
MATERIALS AND METHODS Bank voles. Mating pairs of C. glareolus (Schreber) derived from a colony established in 1959 with animals captured near Rhein-Main-Airbase in Frankfurt, Federal Republic of Germany, were received in December 1981 from Heinz E. Krampitz (Institute of Infection and Tropical Medicine, University of Munich, Munich, Federal Republic of Germany) and were bred in our laboratory. Animals lacked serological evidence of infection with reovirus type 3, Sendai virus, mouse hepatitis virus, ectromelia virus, polyomavirus, papovavirus K, and Puumala virus. After inoculation, suckling (5- to 8-day-old) and weanling (4- to 8-week*
Corresponding author. 34
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EXPERIMENTAL PUUMALA VIRUS INFECTION IN BANK VOLES
suspensions. The infectivities of secretions and excretions determined by inoculating two or three weanling voles with 0.3 ml of oropharyngeal secretions, 0.3 ml of undiluted urine, or 0.3 ml of clarified fecal suspensions and examining the lungs for viral antigen 35 days postinoculation. Horizontal transmission. Two or three noninoculated
TABLE 1. Infectivity of blood, oropharyngeal secretions, urine, and feces from bank voles experimentally infected with Puumala virus" Days postinoculation
7 10 14 21 28 35' 42 49 56 63 99C 103 191 270
were
No. of voles with infectious virus/total no. of voles tested Blood Oropharyngeal Urine Feces secretions
0/3 3/3 3/3 0/3 NT NT NT NT NT NT NT NT NT NT
NT"
0/3 NT 3/3 3/3
NT NT NT
NT 0/3 0/3 0/3 0/5 2/3 0/3 0/3 2/3 0/5
NT NT 0/2 0/3 0/3 3/3 3/3 1/3 1/3 1/3 3/3
NT NT NT
0/3 0/3 0/3
3/3 0/3
3/3 0/3
0/3 NT
35
weanling voles were exposed in the same cage to four voles which had been inoculated with 103.5 ID50 of virus. Exposed voles were removed after 2 to 3 weeks of exposure and replaced with other noninoculated animals. After exposure, voles were kept an additional 5 weeks in individual cages before being sacrificed, and lung tissues were examined for viral
antigen.
Detection of viral antigen. Cryostat-cut frozen sections (6 pm thick) of tissues from inoculated animals and from animals exposed to infected voles were examined for viral antigen by the IFA method (22) by using 32 fluorescent-antibody units of convalescent-phase sera from patients with NE (kindly supplied by Arne Svedmyr, Central Microbiology Laboratory, Stockholm, Sweden) and 8 antiglobulin units of fluorescein isothiocyanate-labeled goat antibodies to human immunoglobulins (Cappel Laboratories, Cochranville, Pa.). The presence of viral antigen in the lungs was interpreted as evidence of infection. Multiple sections of tissues were also stained with both control (seronegative) human sera and rabbit anti-reovirus type 3 serum. Slides were viewed with a Leitz epifluorescence microscope equipped with a 111-RS condenser. Measurement of fluorescent antibody. Serum samples collected at various times after virus inoculation were tested for antibody to Puumala virus by the IFA method (21). A 1:16 dilution of each serum sample prepared in 0.01 M phosphatebuffered saline (pH 7.4) was initially tested by using frozen lung sections from virus-infected C. glareolits or virusinfected Vero-E6 cells (CRL 1586) (American Type Culture Collection, Rockville, Md.) (21) and fluorescein isothiocyanate-conjugated goat antibodies to rat immunoglobulin G (Cappel Laboratories). Sera exhibiting characteristic fluorescence were then titrated in twofold increments against infected and noninfected Vero-E6 cells, and IFA titers were expressed as the reciprocal of the highest serum dilution giving specific fluorescence. The specificity of the antibody in bank vole sera was verified by a two-step immunofluorescence inhibition method (11, 21).
0/3 a The infectivity of blood, secretions, and excretions was determined after i.m. inoculation of two or three weanling bank voles with 0.15 ml of heparinized whole blood, 0.3 ml of undiluted urine, 0.3 ml of a 10% fecal suspension, or 0.3 ml of oropharyngeal secretions (estimated dilution, 1:30) collected from voles on the indicated days after inoculation with Puumala virus (strain Hallnas). Animals were sacrificed 35 days later, and the presence of viral antigen in lung tissue, as determined by the IFA technique (22), was interpreted as evidence of infectivity. b NT, Not tested. ' On these days, urine samples were collected both by direct bladder puncture and passively in metabolic cages. Samples were assayed separately.
days postinoculation by using moistened cotton-tipped swabs (Hardwood Products, Guilford, Maine). Swab contents from each of three animals were expressed into 1.0 ml of Eagle minimal essential medium supplemented with 2% heat-inactivated fetal bovine serum and gentamicin (50 ,ug/ml) (estimated dilution, 1:30). At weekly intervals from 14 to 63 days postinoculation and at ca. 30- to 60-day intervals thereafter for 7 months, urine and feces were collected by placing three or four inoculated animals in metabolic cages (Nalgene Labware Div., Nalge/Sybron Corp., Rochester, N.Y.) for 3 h. On several occasions, urine was also collected by direct bladder puncture at the time of sacrifice. Fecal samples were homogenized in Eagle minimal essential medium supplemented with 2% heat-inactivated fetal bovine serum and gentamicin (50 ,ug/ml); the mixture was then centrifuged at 900 x g for 10 min to yield 10%
RESULTS Viremia and virus shedding. Viremia occurred transiently from 10 to 14 days after i.m. inoculation. Infectious virus
TABLE 2. Distribution of Puumala virus antigen in tissues of experimentally infected bank voles" Tissue
Lung Liver Spleen Pancreas
Kidney Salivary gland Lymph node Intestine Adrenal gland Brain
7
14
21
0/2 0/2 0/2 0/2 0/2 0/2 0/2
1/4 1/4 1/4 0/2 0/4 1/2 0/2 NT NT NT
4/4 2/2 4/4 1/2 0/4 1/2 0/2 NT NT
NT NT NT
NT
No. of voles with antigen/total no. of voles tested on 28 35 42 50 63
2/2 2/2 2/2 2/2 0/2 1/2 0/2 1/2 NT NT
6/8 3/4 3/4 0/2 0/6 0/2 0/2 1/2 0/2 0/2
3/3 2/2 3/3 1/2 0/3 NT 0/2
1/2 NT NT
a On the indicated days postinoculation, weanling bank voles inoculated i.m. with examined for virus-specific fluorescence (22). NT, Not tested.
3/4 2/2 2/4 1/2 0/2 0/2 /1 1/2 NT NT
indicated day postinoculation 95
152
191
225
270
2/4
2/2
0/2
0/2 0/2 0/1 1/2 0/2
0/2 0/4 0/2 0/3 0/1 0/2 1/2 0/2
0/2
NT
1/4 1/4 1/2 0/1 0/4 NT NT 0/2 NT NT
3/4 1/4 0/4 0/2 0/2 0/1 0/1 0/2 NT NT
2/2 2/2 0/2 0/2 0/2 0/2 0/2 0/2 NT NT
3/4 1/2 1/2 0/2
0/2
NT") 0/2 NT NT NT NT NT
103i0' ID,(, of Puumala virus (strain Hiillnds) were sacrificed, and tissues were
36
YANAGIHARA, AMYX, AND GAJDUSEK
J. VIROL.
FIG. 1. Puumala virus antigen in tissues of weanling (A through D) and suckling (E and F) bank voles 28 and 35 days, respectively, postinoculation. Cryostat-cut frozen tissue sections were stained by the IFA technique (22). Antigen is shown in epithelial cells and alveolar macrophages of the lung (A and E), acinar cells of the salivary glands (B), Kupffer and endothelial cells of the liver (C), cells of the lamina propria of the small intestine (D), and neurons and glial cells of the brain (F). Original magnification, x250.
was detected in oropharyngeal secretions and in feces collected 14 to 28 days and 35 to 130 days postinoculation, respectively (Table 1). In comparison, urine was irregularly infectious, whether collected by direct bladder puncture or passively in metabolic cages (Table 1). Distribution of virus in tissues. Puumala virus antigen, as evidenced by granular fluorescence, was detected initially in epithelial cells and alveolar macrophages of lung tissues 14 days after i.m. inoculation. Virus-specific fluorescence was most intense 35 to 50 days postinoculation (Fig. 1), and although it was diminished, it was still evident 270 days postinoculation (Table 2). Virus infectivity was demonstrated in the lungs throughout this period on 14, 42, 95, 152, 191, and 270 days postinoculation. Viral antigen was also found in Kupffer and endothelial cells of the liver and in macrophages of the spleen 21 to 50 days postinoculation. In addition, antigen was detected in the pancreas, salivary glands, and small intestine (Fig. 1) but was absent from the lymph nodes, adrenal glands, kidneys,
and brain of weanling voles (Table 2). The distribution of viral antigen was similar in the tissues of suckling voles 35 and 270 days postinoculation, except that fluorescence was more intense and antigen was found in the neurons and glial cells of the brain (Fig. 1). Overt signs of infection were not apparent at any time in either suckling or weanling animals, and histopathological changes were absent in antigen-bearing tissues despite widespread infection. Also, specific staining was not observed in tissues of infected animals when control human sera or rabbit anti-reovirus serum was used. Antibody response. Fluorescent antibodies against Puumala virus appeared 18 days after i.m. inoculation in weanling voles. IFA titers were maximal 4 to 5 weeks postinoculation (Fig. 2) and declined gradually thereafter but persisted at moderate levels (IFA titer, 512) for the entire 270-day observation period. Some seropositive animals lacked viral antigen in tissues at 191 and 225 days postinoculation. Horizontal intracage transmission. Weanling voles exposed
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for 2 weeks to inoculated animals became infected beginning 14 days after inoculation of index animals (Table 3). The period of greatest communicability occurred between 14 and 28 days postinoculation and coincided with the period of virus shedding in saliva. The distribution of viral antigen in tissues of animals infected horizontally was indistinguishable from that in inoculated animals. DISCUSSION Although small litter sizes and occasional difficulties in breeding C. glareolus in captivity restricted the number of animals available for assaying and ultimately the magnitude of this study, our data adequately document that bank voles inoculated with Puumala virus develop clinically inapparent, chronic infections characterized by transient viremia, prolonged shedding of virus in oropharyngeal secretions and feces, virus persistence in tissues (particularly lung), and no histopathological changes in infected tissues. In general, our data resemble findings in A. agrarius experimentally infected with Hantaan virus (10), suggesting that the mechanisms of virus maintenance and transmission in HFRS-endemic areas are similar in the respective wild-rodent hosts. In a case control study of NE, small rodents were more often reported in the dwellings and cupboards of affected patients than in those of control individuals, implicating a close contact with rodents or their excrement in disease transmission (6). The finding of virus in oropharyngeal secretions, feces, and urine of experimentally infected voles supports the contention that direct contamination of food or other household articles with secretions or excretions or that aerosolization of contaminated fomites constitutes the likely mode of disease transmission to humans (6, 7). The recent demonstration of Hanta virus antigen in the salivary glands, feces, and urine of C. glareolus trapped in endemic regions in the European USSR (4) further confirms the importance of virus-containing secretions and excretions in the transmission of HFRS. Prolonged viruria occurs in Hantaan virus-infected Apodemus mice (10). In comparison, urine specimens from infected voles were only sporadically infectious, indicating intermittent virus shedding, low-titered viruria, or virus inactivation during the collection process. Our data do not
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