key: cord-317617-snrumw3x authors: Sugiyama, K.; Amano, Y. title: Morphological and biological properties of a new coronavirus associated with diarrhea in infant mice date: 1981 journal: Arch Virol DOI: 10.1007/bf01318134 sha: doc_id: 317617 cord_uid: snrumw3x Biological and morphological properties of a virus, isolated from the intestine of infant mice with clinical signs of diarrhea and designated as diarrhea virus of infant mice (DVIM), were examined. The first infective virus was detected on the cells 4 hours post infection, followed by rapid release of the virus into the culture fluids. Virus-induced syncytia in BALB/c-3T3 cell cultures caused hemadsorption at 4° C and viral antigens were shown to be located in the cytoplasm of the syncytia by immunofluorescent techniques. By scanning electron-microscopy, budding virus-like particles were detected on the surface of virus-induced syncytia. Morphologically the virus was shown to be enveloped and approximately 100 nm in diameter. Two types of projections were demonstrated, one type of projection was club-shaped, 20 nm in length and the other type was small, granular, 5 nm in length. The latter type of projection might be the basal part of the clubshaped type and related to the hemagglutinating activity. Among the coronaviruses, there are strains that cause intestinal infections leading to enteritis and diarrhea in swine (11, 25) , calves (4, 19, 23) , rodents (21) and possibly man (9) . Recently, a new viral agent (DVIM) was isolated from an infant mouse with diarrhea, by the utilization of cultured cells. It has also been shown by complement fixation tests that this new isolate is antigenically related to known coronaviruses, avian infectious bronchitis virus (IBV) and mouse hepatitis virus strain-2 (MHV-2). The viral agent was propagated in BALB/c-3T3 cells with the formation of sync)~ia and caused hemadsorption and hemagglutination of mouse or rat erythrocytes (24), 17" 0304-8608/81/0067/0241 / $ 02.20 (18, 26) . A virus isolated from t h e intestine of a n i n f a n t mouse w i t h clinical signs of diarrhea, d e s i g n a t e d DVIM, was generously p r o v i d e d b y Dr. K o z a b u r o Sato (Central L a b o r a t o r y of Shionogi P h a r m . Co., Osaka, J a p a n ) . G r o w t h a n d purification of D V I M h a v e been previously described (24) . H e m a d s o r p t i o n a n d h e m a g g l u t i n a t i o n (HA) assays were p e r f o r m e d as previously described (24) Cells were cultured on glass cover-slips (6 × 23 ram) infected with D V I M as described above. A t designated intervals cover-slips t r e a t e d or u n t r e a t e d w i t h erythrocytes were rinsed five t i m e s w i t h eold-PBS, fixed w i t h g l u t a r a l d e h y d e (4 p e r cent in PBS), dried in a criticaLpoint dryer a n d coated w i t h gold-palladium alloy in v a c u u m (10 -6 tort) a n d e x a m i n e d b y SEM. tubes were collected r a n d o m l y a n d e x a m i n e d for extracellular virus (ECV). The same tubes were w a s h e d five times with PBS, a n d t h e cells were d i s r u p t e d w i t h t ml of m a i n t e n a n c e m e d i u m b y two cycles of freezing a n d t h a w i n g . The s u p e r n a t e s of t h e d i s r u p t e d cells were collected after c e n t r i f u g a t i o n a t 2000 x g for 10 m i n u t e s a n d t h e virus c o n t a i n e d in t h e s u p e r n a t e was designated cell-associated virus (CAV). The i n f e c t i v i t y of E C V a n d CAV was a s s a y e d as described above, a n d expressed as a n u m b e r of m e d i a n tissue culture infectious doses (TCIDs0) per 0.1 ml. Hyperimmune Serum H y p e r i m m u n e serum a g a i n s t purified D V I M was p r e p a r e d in a r a b b i t as follows: F o r t h e first a n d second inoculation, virus was injected i n t r a m u s c u l a r l y w i t h a n equal v o l u m e of complete F r e u n d ' s a d j u v a n t within a three-week interval, a n d a booster injection was given w i t h o u t a d j u v a n t t h r e e weeks a f t e r t h e second-injection. A n t ib o d y t i t e r for t h e i m m u n e serum was d e t e r m i n e d as 2,048 a n d 1,024 in h e m a g g l u t i n ation a n d n e u t r a l i z a t i o n test respectively. I n f e c t e d cell cultures on coverslips were fixed w i t h acetone at 4 ° C for t5 m i n u t e s a n d tile indirect m e t h o d using r a b b i t a n t i -D V I M s e r u m a n d fluoresceine isothioc y a n a t e -e o n j u g a t e d g o a t a n t i -r a b b i t globulin was applied. Virus samples were negatively stained with 2 per cent potassium phosphotungstate (pH 6.5) and examined in JEM-100B electron microscope. The eytopathic effect of DVIM was characterized by the formation of syncytia in BALB/e-3 T 3 cell cultures. When cells were infected at low multiplicities of infection (approximately, M O I = 0 . 1 ) , small syneytia were detectable at 8 hours p.i. At 18 to 24 hours p.i., all areas of the cell sheet consisted of syncytia. Subsequently, syneytial lysis began from the center of each syncytium and progressed toward the periphery. The relationships between virus production and formation of syncytium were examined by a comparison of HA titers of the culture fluid with the cell-fusion ratio during the replicative cycle oi the virus (Fig. 1) . As can be seen from Fig. 1 , HA activity was detected at 8 hours p.i. but development of syncytium lagged behind. Maximum syneytium development occurred between 12 and 18 p.i. HA titers reached a peak by 11 hours p.i. and remained constant. The surface of uninfected cells were covered with microvilli (Fig. 3A) , as previously shown for many celt lines, but following infection with DVIM (t0 hours p.i.) the surface area of syney¢ia were shown to be covered with a large number of spherical buds (Fig. 3B) . The buds were moderately pleomorphic in shape, approximately 100--130 nm in diameter. Particles of a similar size were not detected on the surface of uninfected cells. Therefore we conclude those particles are budding virions. This conclusion is further substa.ntiated by the observation that mouse erythrocytes were exclusively adsorbed to syncy~ia. Moreover, as shown in Fig. 4A the size of the syneytia could be delineated by the location of adsorbed erythrocytes. At a high magnification, a large number of particles were etearly visible in the area of hemadsorption (Fig. 4B ). Fig. 5 shows the growth curve of DVIM in BALB/c-3T3 cells. The production of CAV was detected within 4 hours p.i., followed by a rapid exponential increase. At 6 hours p.i., CAV titer reached a maximum level (105.3 TCIDs0/0.1 ml). The production of ECV was later than that of CAV, but the titer was 105.3 TCID50/ 0.1 ml at 10 hours p.i. Although syncytium formation was very rarely detected at 6 hours p.i., approximately 80 per cent of the cells were fused by 18 hour and detachment from the glass surface followed. H A titers of CAV increased rapidly from 4 to 6 hours p.i. corresponding to a rapid increase in virus production from 10 °.s to 105.a TCIDs0/0.i ml, and ~he titer continued to increase until 8 hours p.i. HA titers of ECV were delayed by approximately 2 hours compared to those of CAV. Infectivity of CAV and ECV decreased from 12 hours p.i., corresponding to rapid degeneration of infected cells and possibly virions, while the HA titer of ECV remained constant until 24 hours p.i. at 37 ° C. The estimated time required for one step growth of DVIM was approximately 6 hours. An eleetron-mierograph of partially purified DVIM is shown in Fig. 6 . The diameter of virions varied from 100 to 110 nm, excluding the surface projections. The projections formed a fringe radiating from the viral envelope and they appeared to be club-shaped, 20 nm in length and t0 nm in width at the distal end of the projections. In addition, noticeable structures which consisted of apparent additional small granular projections were observed. These small projections were 5 nm in length and firmly fixed on the viral envelope. Purified virus partieles, in mcsg of the preparations, were only partially covered by club-shaped projections but always covered with small granular projections, even after a considerable period of sonieation. Fig. 7 shows the negatively stained DVIM particles after sonication at 20 l(Hz for 10 minutes. Although club-shaped projections had completely disappeared, small projections were still present on the surface of partially disrupted virions. Furthermore, the inner structure of the virion was clearly observed after sonieation suggesting penetration of the stain. The thickness of the envelope was measured as 10 nm and a fine filamentous structure, irregularly tangled, was observed in broken virions. Recently, some viral agents, which are responsible for diarrhea of new-borne mice, have been classified, geovirus is a major cause of routine diarrhea. (8) . Epizootic diarrhea of infant mice (EDIM) virus is now classified as a rotavirus by HOLMES st al. (15) . Mouse hepatitis virus (MHV), a member of coronaviruses, has been described as a common cause of enteric infection in baby mice (8, 21) . On the other hand, lethal intestinal virus of infant mice (LIVIM), first described by K~AST, is probably the most important viral agent of diarrhea in new-born mice (3, 17) , but there was no viral agent detected. However, the epidemiological and pathological characterization of diarrhea of new-borne mice is consistent with the recorded pathology of LIVIM, and suggests that a MHV could be the cause of this disease (5, 7, 14, 16) . Previous results reported by SATO st al. (22) indicated that the infectious agent (DVIM) isolated from the intestine of mice with diarrhea possesses I~NA and lipid. The agent passes through a 100 nm filter but not a 50 nm filter. Serologically, DVIM was shown to be related to IBV and MHV-2 by a complement fixation test but they were clearly distinguishable from each other by neutralization assays. These observations suggested that this new isolate, DVIM, is a member of coronavirus. However, it was important to establish the morphological characteristics of purified DVIM, since eoronavirus classification, is established mainly by means of characteristic morphology at present. The results presented in this report showed that diameter of virion was approximately 100 nm, excluding surface projections. Characteristic coronavirus projections attached to the virion were demonstrable. However morphologically DVIM appears to be slightly different in that additional small granular projections were observed. A similar small projection on enteric bovine coronavirus has been reported recently (4) . But no additional projection has been detected on MHV-2, propagated and purified in the same manner as DVIM (data not shown). The morphological relationships of the two projections, club-shaped and granular, are at present not clear. However, it is possible that the club-shaped projection might be attached to the granular projection. Moreover, the HA activity of DVIM was only slightly affected by sonication, which suggests that the HA activity of ])VIM might be associated with the small granular projection. We showed by SEN that a large number of particles were present on the DVIM-induced syncytia but not on uninfected cells. The size of the particle was similar to that of negatively stained DVIM, while hemadsorption was restricted to the cells which possess surface particles. The surface profile of DVIMinfected cells has a prominent resemblance to the cells infected with ortho-or para-myxoviruses (1, 12, 27). As described previously (240, it is clear that the hemadsorption of DVIM was not due to the "pseudohemadsorption" (6). These observations suggest that DVIM particles are assembled and bud through the plasma membrane of syncytium. In addition to these morphological properties, the existence of HA and receptor destroying activities as described previously (24), have not been reported for any of the other routine coronaviruses. Several studies of coronaviruses by the thin sections and transmissible electron microscopy show unequivocally that coronaviruses are formed by budding from the surface of intracellular cistelme (2, 10, 13, 20) . The more detailed morphogenesis of DVIM remains to be investigated in future. Electron microscope study of hemadsorption in measles virus infection Morphogenesis of avian infectious bronchitis virus and a related h u m a n virus (strain 229E) Lethal intestinal virus infection of mice (LIVIM). Amer Replication of an enteric bovine eoronavirus in intestinal organ cultures Lethal enteritis in infant mice caused by mouse hepatitis virus Intracellular development and mechanism of hemadsorption of h u m a n coronavirus Lethal intestinal virus of infant mice is mouse hepatitis virus Mouse hepatitis, reo-3, and the Theiler viruses Coronavirus particles in faeces from patients with gastroenteritis Morphogenesis of avian infectious bronchitis virus in primary chick kidney cells A transmissible gastroenteritis in pigs Itemadsorption of m u m p s virus examined by light and eleetron microscopy Growth and intracellular development of a new respiratory virus New strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice Infantile enteritis viruses. Morphogenesis and Morphology Isolation of mouse hepatitis virus from infant mice with fetal diarrhea An apparently new lethal virus disease of infant mice Coronavirus; A comparative review. C u r l Top Pathology of neonatal calf diarrhea induced by a coronavirus-like agent Electron microscopic studies of coronavirus Mouse hepatitis virus infection as a highly contagious, prevalent, enteric infection of mice Some characteristics of corona-like virus isolated from infant mice with diarrhea and inf]arnentory submaxillary gland of rats Characterization of a calf diarrhea coronavirus Hemagglutination and structural polypeptides of a new coronavirus associated with diarrhea in infant mice Morphology of transmissible gastro-enteritis virus of pigs. A possible member of coronaviruses Adsorption-hemagglutination test for influenza virus in monkey kidney tissue cultures We wish to thank Dr. Kozaburo Sato, Central Laboratory, Shionogi Pharma. Co., Osaka, for his useful advices during this work.