Archives of Virology 67, 241--251 (1981) Archives of Virology © by Springer-Verlag 1981.

Morphological and Bioloflieal Properties of a New Coronavirus Assoeiated

with Diarrhea in Infant Miee

By

K. SUGIYAM:A and Y. A~A~o Department of Microbiology and Central Laboratory,

Akita University School of Medicine, Akita, Japan

With 7 Figures

Accepted September 26, 1980

Summary Biological and morphological properties of ~ 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 hem- adsorption at 4 ° C and viral antigens were shown to be located in the cytoplasm of the syncy~ia by immunofluorescent techniques. By scanning electron-micro- scopy, 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 par t of the club- shaped type and related to the hemagglutinating activity.

Introduction Among the coronaviruses, there are strains tha t 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. I t has also been shown by complement fixation tests tha t 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 ra t erythrocytes (24),

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242 K. SVGIYAMA a n d Y. A~IA~¢O:

T h e p r e s e n t r e p o r t d e s c r i b e s t h e m o r p h o l o g y a n d s o m e b io log ica l c h a r a c t e r i s t i c s

of t h e n e w e o r o n a v i r u s - l i k e a g e n t ( D V I M ) , a n d s h o w s t h a t s o m e of t h e p r o p e r t i e s

of t h i s v i r u s a re u n i q u e c o m p a r e d to t h e p r e v i o u s l y s t u d i e d c o r o n a v i r u s e s (18, 26).

Materials and Methods

Virus and Cell Cultures A vi rus i sola ted f rom t h e i n t e s t i ne of a n i n f a n t mouse w i t h cl inical signs of d ia r rhea ,

d e s i g n a t e d DVIM, was generous ly p r o v i d e d b y Dr. K o z a b u r o Sa to (Centra l 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 pur i f i ca t ion of D V I M h a v e been p rev ious ly desc r ibed (24).

Hemadsorption and Hemagglutination 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 p rev ious ly

desc r ibed (24), us ing 0.5 pe r cen t mouse e r y t h r o c y t e s in P B S c o n t a i n i n g 0.3 per cen t b o v i n e s e r u m a l b u m i n .

Scanning Electron Microscopy (SEM) Cells were cu l t u r ed on glass cover-s l ips (6 × 23 ram) in fec ted w i th D V I M as de-

sc r ibed above . A t de s igna t ed i n t e rva l s cover-s l ips t r e a t e d or u n t r e a t e d w i t h e ry th ro - cy tes were r insed f ive t i m e s w i t h eo ld-PBS, f ixed w i t h g l u t a r a l d e h y d e (4 p e r cen t in PBS) , d r ied in a c r i t i caLpo in t d rye r a n d coa ted w i t h go ld -pa l l ad ium al loy in v a c u u m (10 -6 to r t ) a n d e x a m i n e d b y SEM.

Virus Growth and Assay Cul tu re t ubes c o n t a i n i n g a p p r o x i m a t e l y 106 of B A L B / e - 3 T 3 cells were i n o c u l a t e d

w i t h 107 TCIDs0 o f / ) V I M . Af te r one h o u r of v i rus a d s o r p t i o n a t 4 ° C, t h e tubes were w a s h e d f ive t i m e s w i t h P B S a n d fed w i t h 1 ml of m a i n t e n a n c e m e d i u m , t h e n i n c u b a t e d on rol ler d r u m a t 37 ° C. A t var ious t ime i n t e rva l s of i n cu b a t i o n , t h e cu l tu re m e d i a of f ive t ubes were col lec ted r a n d o m l y a n d e x a m i n e d for ex t r ace l lu l a r v i rus (ECV). The s ame tubes were w a s h e d f ive t imes w i th 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 f reezing 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 col lected a f t e r c en t r i f uga t i o n a t 2000 x g for 10 m i n u t e s a n d t h e v i rus c o n t a i n e d in t he s u p e r n a t e was des igna t ed cel l -associa ted v i rus (CAV). The i n f ec t i v i t y of E C V a n d CAV was a s sayed as desc r ibed above , a n d expressed as a n u m b e r of m e d i a n t i ssue cu l tu re infec t ious doses (TCIDs0) pe r 0.1 ml .

"lleasurement o/the Cell-Fusion Ratio I n f e c t e d cell cu l tu res on coversl ips, were h a r v e s t e d a t va r ious t ime i n t e rva l s

pos t in fec t ion (p.i.), f ixed w i t h 70 pe r cent. e t h a n o l a n d s t a i n ed w i t h G iemsa ' s so lut ion. The cell-fusion ra t io was ca lcu la ted , based on the t o t a l nucle i pe r fused cells aga ins t t o t a l nuclei c o u n t e d in t he same microscopic v iewing area.

Hyperimmune Serum H y p e r i m m u n e se rum a g a i n s t pur i f ied D V I M was p r e p a r e d in a r a b b i t as fol lows:

F o r t he f i rs t a n d second inocu la t ion , v i rus was in jec ted i n t r a m u s c u l a r l y w i t h a n equa l v o l u m e of comple t e F r e u n d ' s a d j u v a n t w i th in a t h ree -week in te rva l , a n d a boos te r i n j ec t i on was g iven 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- in jec t ion . A n t i - b o d y t i t e r for t he i m m u n e se rum 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 a - t ion a n d n e u t r a l i z a t i o n t e s t respec t ive ly .

Immuno]luorescent Staining I n f e c t e d cell cu l tu res on covers l ips were f ixed w i t h ace tone a t 4 ° C for t5 m i n u t e s

a n d ti le i nd i r ec t m e t h o d us ing r a b b i t a n t i - D V I M s e r u m a n d f luoresceine isothio- c 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 g lobu l in was appl ied .

Morphology of DVIM 243

Electron Microscopy Virus samples were negatively stained with 2 per cent potassium phosphotungstate

(pH 6.5) and examined in JEM-100B electron microscope.

Results

Cytopathic E//ect o/DVL~f

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.

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Fig° 1. Comparison of cell fusion ratio with HA development in the BALB/e-3T3 cell cultures infected with DVIM. The cell fusion ratio (~; o--- o average) was expressed as a percentage of total nuclei counted in fused cells against total nuclei counted in the same microscopic viewing area. HA (z~--- A) was titrated by

conventional method

DVIM specific antigen was first detectable in the cytoplasm of syneytia at

8 hours p.i. From Fig. 2 it can be seen that the antigens are restricted to the

cytoplasm of the syncytium. Antigens were not detected in the nucleus at any

time of viral infection.

244 K. SVGIYAMA and Y. A~ANo:

Fig. 2. Fluorescent-antibody staining of DVIM infected cells. At 10 hours post infec- tion, the infected monolayer on coverslips was stained with fluorescent antibody by

the indirect method

Scanning Electron Microscopy (SEM)

Surface details of BALB/c-3T3 cell cultures, pre- and post-infection with DVIM, were investigated by SEM.

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).

Growth Curve

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.

Morphology of DVIM 245

L

Fig. 3. Surface profile of DVIM uninfected (A) and infected (B) BALB/c-3T3 cells, by scanning electron microscopy. Bar, I000 nm

246 K. SLrGIYAS~A a,nd Y. AMANO:

Fig. 4. Hemadsorbed syneytium, by scanning electron microscopy. (A) An entire surface profile of syneytium. Bar, 20 ~xm. (B) Higher magnification view of the center

area of (A), Bar, 1000 nm

Morphology of ])VIM 247

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 ap- proximately 2 hours compared to those of CAV. Infect ivi ty of CAV and ECV decreased from 12 hours p.i., corresponding to rapid degeneration of infected cells and possibly virions, while the HA ti ter 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.

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Fig. 5. Growth of DVIM in BALB/c-3T3 cells. Cell associated virus (o - - . ) and extraeetlular virus (o- - - - - -o) were assayed as described in Materials and Methods. I-IA activity of cell associated virus (A --A) and extracel!ular virus ( ~ - - - ~)

were also assayed, dg cells degenerated

Morphology o / the Virus Particles

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 appear- ed 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 addi- tional 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 projec- tions but always covered with small granular projections, even after a considerable period of sonieation.

248 K. SUGIYAMA and Y, A~IANO:

Fig. 6. A t ransmiss ion electron mierograph of DVIM negat ive ly s tained with 2 per cent PTA. Arrow shows small project ion. Bar, 100 n m

Fig. 7. A transmission electron mierograpb of DVIM a~ter sonieation at 20 KI~Iz for I0 minutes. Virions were negatively stained with 2 per cent PTA. Bar, i00 nm

Morphology of DVIM 249

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 soniea- tion 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.

Diseussion

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 tha t 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. Serol- ogically, 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 neutraliza- tion 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 tha t additional small granular projec- tions 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 tha t the club-shaped projection might be attached to the granular projection. Moreover, the HA act ivi ty of DVIM was only slightly affected by sonication, which suggests tha t the HA activi ty of ])VIM might be associated with the small granular projection.

We showed by SEN tha t 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 tha t of negatively stained DVIM, while hemadsorption was restrict- ed to the cells which possess surface particles. The surface profile of DVIM- infected cells has a prominent resemblance to the cells infected with ortho- or para-myxoviruses (1, 12, 27). As described previously (240, it is clear tha t the

250 K. SUGIYA~A and Y. AMANO:

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 corona- viruses 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.

Acknowledgments

We wish to thank Dr. Kozaburo Sato, Central Laboratory, Shionogi Pharma. Co., Osaka, for his useful advices during this work.

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Morphology of DVIM 251

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Authors' address: Dr. K. SUGIYAMA, Department of Microbiology, University of Alabama in Birmingham, University Station, Birmingham, AL 35294, U.S.A.

Received August 20, 1980