key: cord-329061-1xut73dq authors: Bhatt, Pravin N.; Percy, Dean H.; Jonas, Albert M. title: Characterization of the Virus of Sialodacryoadenitis of Rats: A Member of the Coronavirus Group date: 1972-08-17 journal: J Infect Dis DOI: 10.1093/infdis/126.2.123 sha: doc_id: 329061 cord_uid: 1xut73dq The virus that causes sialodacryoadenitis in rats has been isolated in mice and in primary cultures of rat-kidney cells and has been characterized as a heat-labile RNA virus that is sensitive to lipid solvents and is relatively stable at pH 3.0. This virus is antigenically related to the virus of hepatitis in mice and to coronavirus of rats. The range of hosts of this agent appears to be narrow. On the basis of available biologic characteristics, it has been placed in the coronavirus group. cated that neither source had detectable serum antibodies to SDA virus and that both were susceptible to infection with this agent. Rats were inoculated intranasally with 0.1 ml of virus-infected salivary-gland suspension, observed daily for evidence of overt illness, and sacrificed at various intervals. All rats were maintained in rigid plastic isolators with high-efficiency air filters. Tissue culture. Three cell lines, baby-hamster kidney (BHK-21), VERa, and Hep-2, and primary monolayer cultures of rat embryo, rabbit kidney, rhesus-monkey kidney, guinea-pig-embryo skin, muscle, and kidney were used as previously described [l ] . Monolayers obtained from explant cultures of submaxillary, parotid, Harderian, and exorbital glands of germfree rats, monolayers of trypsin-dispersed brain cells of infant mice, and a line of polyoma-transformed mouse cells (Py-AL/N) [6J were also tested. At a later stage in the study, primary rat-kidney (PRK) cultures prepared from kidneys of weanling Charles River CD germfree or conventional inbred dark Agouti fDA) rats were used. Inoculated tubes were kept in a roller drum at 37 C and observed for cytopathic effect (CPE) at intervals of two to four days for at least 21 days. The fluid medium was changed when necessary. In the absence of CPE, a blind passage was made between the eighth and 16th day after inoculation. Cultures for passage were observed for one to two weeks for development of CPE, and, in the absence of CPE, VERa, BHK-21, and PMK cultures were challenged on the 12th day after inoculation with Chandipura [8J virus, an arbovirus of the vesicular stomatitis viral group, for determination of interference. In addition, the fluid from each of the second-passage cultures was inoculated ic into infant mice, and the mice were observed for 21 days. In some instances tissueculture fluid from inoculated tubes was passaged into infant mice without further passages in tissue culture. Monolayer cultures of PRK, infant-mouse brain, and Py-AL/N cells were also examined by indirect immunofluorescence for the presence of viral antigen [5] . Characterization of the virus. For determination of the effect of 5-bromodeoxyuridine (5-BUDR), lipid solvent, low pH, and various temperatures, methods described by Bhatt et al. [9J were used. The hemagglutination method will be Bhatt, Percy, and Jonas described under Results. Staining with acridine orange was done according to the method of Hsiung [10] . Preparation of immune sera. Hyperimmune sera were prepared in rats and mice by repeated inoculation of a suspension of salivary glands from infected rats and of brains from infected mice, respectively. Complement-fixation test. Complement-fixing antigen was prepared by sucrose-acetone extraction from infected brains of two-to four-day-old mice. Polyvalent mouse-hepatitis CF antigen prepared in tissue culture was obtained from Microbiological Associates. The CF test was performed by the micromethod [l l ] using two units of complement and four to eight units of antigen. Serum of mice immune to SDA strain 681 was tested against 118 viral antigens by the CF test. Neutralization test. The neutralization (N) test with sera immune to murine viruses was performed in infant mice, and these animals were observed for 14 days after inoculation. PRK cultures were used for cross-N tests, using SDA strain 681 and Parker's RCV. Sera were inactivated at 56 C for 30 min. Cultures were examined on the third and fifth days after inoculation. The titers of antibody and virus were calculated by the method of Reed and Muench [12J. Fluorescent-antibody method. Pieces of mouse brain 2-4 mm thick were quick-frozen in a dry ice-alcohol bath and stored at -83 C. Sections 6-8f!m thick were cut in a cryostat, two sections were mounted per slide, and then the slides were fixed in acetone at 25 C for 15-20 min and dried at 37 C for 15 min. Sections were stained immediately or stored at -25 C for 1-30 days before use. Tissue-culture cover slips were similarly prepared but at times were kept in chilled acetone at -25 C for 18 hr. The section and cover-slip preparations were reacted with sera immune to virus for 20 min at 25 C and then exposed to mouse or rat antiglobulin conjugate for 20 min. Phosphate-buffered saline (PBS) was used for washing. Preparations were examined with a Carl Zeiss microscope fitted with an HBO 200 W/4 supermercury lamp, a UG-5 exciter filter, and a 47/65 barrier filter. Histopathology. Histologic examination of tissue from inoculated rats included Harderian, exorbital, parotid, and submaxillary glands. In suckling mice, coronal sections of brain and serial transverse sections of the thoracic and abdominal regions were examined. Attempts to induce disease in weanling mice. Sixty female mice, three-to four-weeks old, were given 2.5 mg of cortisone im twice a week beginning a week before inoculation and continuing until the end of the experiment. Twenty mice each were inoculated ic and ip with 0.03 ml and 0.1 ml of viral suspension containing 2 X 1()3·9 and 6.3 X 10 3 . 9 infant mouse LD50 (IMLD50), respectively. Twenty control mice were inoculated with diluent, 10 by the ic route and 10 by the ip route. Another group of controls was neither inoculated nor given cortisone. Six mice from each group were killed for histologic studies on the seventh day after inoculation and two were killed on the 14th day after inoculation. Remaining mice were observed until the 21st day after inoculation, when the experiment was terminated. A complete necropsy was done on each mouse. Induction of sialodacryoadenitis in susceptible rats by mouse-brain-adapted virus. Eleven rats, weighing 250 g and from a colony known to be susceptible to SDA virus, were inoculated by the intranasal route with fourth passage, infectedmouse-brain material. The inoculum contained approximately 6.6 X 10 3 . 8 IMLD 50 of virus. Rats were sacrificed on the fifth, sixth, and eighth days after inoculation. Harderian and submaxillary glands were processed for isolation of virus and histologic examination, whereas parotid gland was collected for histologic examination only. Adaptation to mice and related observations. A 10% suspension of infected salivary glands was inoculated ic into one-day-old mice. One mouse was sick on the fifth day after inoculation, eight more were sick on the seventh day after inoculation, and six on the eighth day after inoculation. Some of these animals were killed, and tissues were harvested for passages and histologic study, but mice that were sick but not killed died on the 10th day after inoculation. One mouse was unaffected and survived until the 21st day, when it was discarded. The disease was characterized by ataxia and uncoordination, followed by paresis, paralysis, and death. The same pattern of illness was observed on further passages. By the fifth mouse-brain passage, the incubation period was 125 shortened to two to three days. There was usually a random pattern of illness and death from two to eight days and occasionally up to 10 days after inoculation. The pattern has remained unchanged for 29 passages with this strain of virus. One other observation made during the first passage in mice and amply confirmed during subsequent work was emaciation of sick mice as compared to uninoculated control mice of the same age. These differences were more marked in mice that were two to four days old or older when inoculated. Other significant observations can be summarized as follows: (l) The agent of SDA does not cause detectable illness in weanling (three-to four-week-old) mice when inoculated ic or ip or in infant mice inoculated ip. (2) A comparative titration was done in mice two days old, 13 days old, and 22 days old that were inoculated ic with viral stock passaged 12 times in mouse brain. Titers were 10 4 . 00, 10 4 . 25 , and <10 2 . 0 IMLD50/0.015 mI, respectively. (3) Virus has undergone 29 serial ic passages in zero-to six-day-old mice, the cumulative dilution of which exceeds 10-100. (4) The titer of virus between the fifth and 29th passage in mouse brain has remained relatively stable at 10 3 . 5_105.0 IMLD50/0.015 ml (usually around 1Q3·7 IMLD50/0.015 ml). (5) The original salivary-gland suspension was titrated in one-day-old mice and had a titer of 1()3·6 IMLD 50/O.015 ml. (6) When inoculated intranasally into susceptible rats, mouse-brain-adapted virus produced sialodacryoadenitis. (7) Brains from two uninoculated mice (two days old) were harvested as controls; seven serial ic passages of this material were made at intervals of six to seven days in mice three to four days old. No agent pathogenic for mice was isolated from these control animals. Histopathologic and immunofluorescent observations in inoculated mice. In general, histologic changes observed in the central nervous system of inoculated mice were characterized by diffuse and focal neuronal degeneration with minimal inflammatory cell response. Regions of brain most frequently involved were the cortices of the occipital and parietal lobes. Other foci of neuronal destruction were scattered elsewhere in the central nervous system; there was relatively little destruction 0 Some important observations are summarized as follows. (l) Cultures were most sensitive when used within a week after seeding; then sensitivity decreased. The CPE was delayed and less extensive in older cultures. (2) Development of virus in PRK cells was monitored by CPE, detection of viral antigen by indirect immunofluorescence, and quantitation of infectious virus in PRK tubes. Results are presented in table 1. Significantly, detectable viral antigen developed by 12 hr and was followed by release of infectious virus into the medium. CPE was detected at 24 hr. Beyond 24 hr, quantitation of viral antigen was difficult due to lysis of cell sheets, and after 36 hr, titer of infectious virus decreased. (3) The sensitivity of inoculation of mice ic with strain 681 virus was compared with that of inoculation of PRK cultures. Titers obtained with a mouse-brain-adapted virus were 2.5 X 10 4 IMLD50 in mice and lOX 10 4 . 3 TCID50 in PRK cultures. Similar differences were also noted in other experiments. Characterization of the agent. The effect of 5-BUDR on viral multiplication was determined by the method of Bhatt et al. [9] . Chandipura virus was used as RNA control (P. N. Bhatt, unpub-Hour in the cerebellum. Affected neurons were pyknotic and densely eosinophilic. In addition, there was a scattering of shrunken, densely staining astrocytes in these areas. Occasionally there was hypertrophy and hyperplasia of capillary endothelial cells and minimal perivascular cuffing with mononuclear cells. Sometimes a few polymorphonuclear leukocytes were scattered in areas of destruction. Spinal cord, salivary glands, lung, heart, liver, kidney, spleen, and intestine were histologically normal. Immunofluorescence procedures detected viral antigen in regions where frank cellular necrosis was seen by standard histologic techniques. In addition, intense staining was observed in neuronal cytoplasm of scattered cells that were intact and not associated with frank necrosis. Serial coronal sections had immunofluorescence staining in dorsal cortical areas, the ventral portion of Ammon's horn, the hypothalamus, and the brain stem, but fluorescence was rarely found in cerebellar folia and white matter. A ttempts to adapt the agent to monolayer cell cultures. The original salivary-gland suspension inoculated onto various monolayer cell cultures produced no detectable CPE up to 21 days after inoculation. When blind passages were made and cultures were challenged with Chandipura virus [8] , interference was not observed. Fluid from the second passage in tissue culture was inoculated ic into infant mice; the results were negative. Attempts were made to propagate mouse-brainadapted virus to cell-culture systems, such as monolayers obtained from explant cultures of parotid, Harderian, exorbital, and submaxillary glands of germfree rats and monolayers of trypsindispersed infant-mouse-brain cultures. There was no detectable CPE. Similar results were obtained with the Py-AL/N cell line. Infectious virus or viral antigen was not detected when tissue-culture fluids from infected-mouse-brain and Py-AL/N cultures were inoculated ic into infant mice or when monolayers were examined by indirect immunofluorescence. However, PRK cultures showed CPE characterized by formation of multinucleated giant cells, which were seen as highly reflective masses. These cells fell off the glass wall a few hours later and were seen floating in medium. Tissue-culture fluids of these cultures contained virus as detected in infant mice, and cultures were positive for viral antigen by indirect immunofluorescence. Sensitivity of SDA virus to a lipid solvent was also tested. The titer of virus was 10 3 . 8 and < 10 2 . 0 IMLD50/0.015 ml for controls and chloroformtreated samples, respectively. The test was repeated with similar results, and it was concluded that the agent is sensitive to lipid solvents. Effect of low pH on infectivity. The test was performed as described by Leibhaber [13] . Tenfold serial dilutions of infected-mouse-brain suspension kept at different pH values were made in Eagle's minimal essential medium in Earle's base with 3% fetal bovine serum (FBS). The pH of each dilution was adjusted to approximately 7.0 by addition of Tris, and this solution was inoculated into mice. End points of infectivity were calculated by the method of Reed and Muench [12] . The titers of infectious virus detected in PBS after incubation for 3.0 hr at 25 C was 103. 6 IMLD50/0.015 ml, whereas at pH 7.0 and pH 3.0 it was 10 3 . 9 IMLD50/0.015 m1 and lQ2·8 1MLD50/ 0.015 ml, respectively. Thus infectious virus was relatively stable at low pH. Effect of temperature on infectivity. The effect of a temperature of 37 C on infectious virus was determined as outlined by Bhatt et al. [9] . To determine the effect of a temperature of 56 C, infectivity was determined at intervals of 0, 5, and 10 min. An aliquot of viral stock was kept at 4 C, and infectivity was determined on days 0, 7, and 28. Infectivity of viral strain 681 was stable in PBS plus 3% FBS at 37 C for 3 hr; the titer then decreased by 1.1 log., by 5 hr. At 56 C the infectivity decreased from 10 3 . 8 1MLD50 at zero time to trace levels by 5 The size of infectious viral particles. The approximate size of infectious viral particles was determined by the method of Atoynatan and Hsiung [14] and Casals [15] as modified by Bhatt et al. [9] . A fresh, 10% suspension of mouse brain was made in PBS plus FBS, clarified by centrifugation at 1,000 g for 20 min, and filtered through MilIipore filters (Millipore Corp., Bedford, Mass.) of various pore sizes. Viral titers obtained were 103. 5, 104.2, 103. 8 , 10 3 . 0, and < lQ2 1MLD50/0.015 m1 for unfiltered virus and after filtration through pore sizes 1,200 urn, 450/lm, 220/lm, and 100 urn, respectively. Results indicate that the size of the virus is less than 220 urn but greater than 100 urn. The particles without membranes measured by electron microscopy were previously reported to be 6