key: cord-0005006-ble8gn1l authors: Daniel, C.; Talbot, P. J. title: Physico-chemical properties of murine hepatitis virus, strain A59 date: 1987 journal: Arch Virol DOI: 10.1007/bf01320963 sha: 9f2a87c32a20383bff96d910aaf2c686f28e34ec doc_id: 5006 cord_uid: ble8gn1l The infectivity of murine hepatitis virus (MHV-A59) was optimally stable at pH 6.0 and was unaffected by ionic strength or at least 15 cycles of freezing and thawing. It was completely inactivated within 25 minutes at 56° C, but was protected by 1m magnesium chloride or magnesium sulphate. It was completely inactivated within 14 days at 37 and 22°C, but was relatively stable for as long as 72 days at 4°C and optimal pH. The infectivity of marine hepatitis virus (MHV-A 59) was optimally stable at pH 6.0 and was unaffected by ionic strength or at least 15 cycles of freezing and thawing. It was completely inactivated within 25 minutes at 56 ° C, but was protected by 1 M magnesium chloride or magnesium sulphate. It was completely inactivated within 14 days at 37 and 22°C, but was relatively stable for as long as 72 days at 4 ° C and optimal pH. $ Murine hepatitis viruses (MHV), important members of the Coronaviridae family (25) , are responsible for several diseases of rodents, in particular respiratory, neurological and gastrointestinal disorders (24, 34) . They are characterized by club-shaped surface projections, about 20 nm in length and form pleomorphic virions of 80 to 120 nm in diameter; they possess single stranded I~NA of positive polarity and a lipid containing envelope (23, 25, 28) . Physico-chemicM properties of some coronaviruses have been described by several authors. These studies showed that pH and temperature stabilities were variable among different members of the coronavirus group, according to the strains used. For instance, some strains of the avian int~ctious bronchitis virus (IBV) were stable to acid pH whereas others were labile (1, 6, 7, 8, 26) . On the other hand, transmissible gastroenteritis virus (TGEV) has been reported to be stable for at least one hour at 37°C between pH 4 and 8 (18) . However, this virus showed marked instability at pH above its optimum pH of 6.5, when incubated for 24 hours (21) . Similar to IBV, TGEV strains showed great variability in their stabilities to an acidic environment (12) . Moreover, TGEV strains were generally heat-labile since they were inactivated in a few days at 37°C and in 10 minutes at 56°C (3, 15, 18) . Interestingly, Coria (5) reported that 1 ~ magnesium sulphate could protect IBV from thermal inactivation at pH values ranging from ¢ to 10 whereas Cowen et al. (7) found that this virus was inactivated at high temperature (50-56 ° C) with or without 1 5~ magnesium chloride. Finally, sialodaeryoadenitis virus (SDAV), a possible member of the coronavirus group (17, 24) , was characterized by Bhatt et al. (2) , who found it to be relatively acidresistant and heat-labile. Some physico-chemieal properties of strains JHM, 2 and A 59 of tile murine hepatitis virus group have been described. Cheever etM. (4) observed rapid inaetivation of MHV-JHM at 56 ° C, persistence of infectivity at. 37 ° C for 3 hours and a slow inactivation at 4 ° C. Manaker et M. (16) first isolated the A 59 strain and reported properties similar to those of JHM. Sturman (27) reported on the pH dependence of MHV-A 59 infectivity. At 37 ° C, virus infectivity was most stable between pH 6.0 and 6.5 whereas at 4 ° C, virus was stable over the range of pH 4 to 8. Hirano et M. (14) determined that MHV-2 was heat-labile but was protected from inactivation at 56 ° C by 1 M magnesium chloride or 1 M magnesium sulphate. This virus was also stable at pH 3 to 9 at 37 ° C for 60 minutes. In summary, it appears that physieo-chemieal properties differ within the eoronavirus group according to the experimental conditions and the intrinsic properties of the virions. In the present study, we report on some of the physieo-ehemieal properties of MHV-A 59 grown on DBT cells. Murine hepatitis virus (MHV), strain A 59, was obtained t~om the American Type Culture Collection (l~ockville, MD, U.S.A.), plaque purified twice and passaged four times on DBT cells prior to use us inoeulum in all experiments described. The DBT murine cell line (13) was kindly supplied by Dr. Michael J. Buehmeier (Scripps Clinic and l~eseareh Foundation, La Jolla, CA, U.S.A.) and grown at 37 ° C in Earle's minimum essential medium/ Hunk's M 199 (1:1, v/v) supplemented with 5 percent (v/v) foetal calf serum (FCS) and 0.13 percent (w/v) sodium bicarbonate (Gibco Canada, Burlington, Ontario). Plaque assay of MHV-A 59 was carried out by a modification of the method reported by Hirano et al. (13) in 35 mm diameter tissue culture 6-well cluster plates (Flow Laboratories, Mississauga, Ontario, Canada). All samples were homogenized by vortexing and ultra-sonieation for 5 to 10 seconds in order to eliminate possible virus aggregates before dilution and inoeula¢ion. After one hour adsol~)tion at 37 ° C on a rocker platform, the virus inoeula were removed, the monolayers overlaid with medium containing 1.5 percent (w/v) bucto-agar (Difco Laboratories, Detroit, MI, U.S.A.), 0.05 mg/ml gentamycin and 10 percent (v/v) FCS. Plates were incubated for 48 to 72 hours at 37 ° C, fixed with 9.25 percent (v/v) formMdehyde and stained with erystM violet. Virus was incubated in buffers of various pHs for 6 hours at 37 or 4 ° C and residual infectivity was titrated. The results are shown in Fig. 1 . We found t h a t MHV-A 59 was stable over a wide pH range (3.0-10.0) at lower t e m p e r a t u r e (4 ° C), whereas at 37 ° C, it was only stable b e t w e e n pH 5.0 and 7.4. Thus c o m p a r e d with MHV-2 (14) and MHV-3 (20) , MHV-A 59 was more sensitive to pH, particularly at 37 ° C. A similar relationship shown between t~m p e r a m r e and pH sensitivity was also observed with other eoronaviruses such as T G E V (21) and I B V (1) and it has been suggested t h a t p H -d e p e n d e n t thermolability is due to aggregation of the E 2 peplomerie glyeoprotein (27) . MHV-A 59 was m o s t stable at pH 6.0, as described for IBV and T G E V (29) and in extension of a r e p o r t by S t u r m a n (27) . The pH sensitivity test described in Fig. 1 was also perIbrmed in buffers without sodium chloride, with similar results (data not shown). The sensitivity of MHV-A 59 to ionic strength was further investigated with sodium chloride concentrations of 0 to 500 mM added to solutions of 10 mM Tris acid maleate pH 6.0, containing 5 percent (v/v) FCS. Ten-fold dilutions of virus in these buffers were incubated at 4 or 37 ° C for 6 hours and stored a t -7 0 ° C On the other hand, Wallis and Melnick (30, 31) found t h a t 2 M sodium ions (Na +) e n h a n c e d inactivation of enteroviruses (non-enveloped RNA viruses) at 37 ° C whereas it p r o t e c t e d t h e m from inactivation at 50 ° C. F u r t h e r m o r e , Na ÷ conferred some protection to vaccinia and adenoviruses from heat, inactivation (32) . As shown in Fig. 2 , the A 59 strain was rapidly (25 minutes) inactivated at 56 ° C. It was significantly protected by 1 • magnesium chloride for at least 30 minutes, and slightly m o r e by 1 M magnesium sulphate. H e a t stability has not been associated with morphological characteristics such as the presence of an envelope or nucleic acid type. Indeed, poxviruses, papovaviruses and reoviruses were relatively stable when incubated at 50 ° C for 30 minutes whereas herpes simplex viruses, adenoviruses, enteroviruses and m3~xoviruses were labile in similar conditions (10). Nevertheless, m o s t corona-viruses were reported to be inactivated within l0 to 30 minutes at 56 ° C as we have shown for MHV-A 59 (19, 20, 29) . The effect of divalent cations, such as magnesium, has been shown to vary according to the virus and the salt used. Wallis et al. (33) showed that stabilization of polioviruses by salts could not be generalized to cations or anions. Indeed, 1 M magnesium chloride stabilized entero-and reoviruses whereas 1 M magnesium sulphate did not stabilize enteroviruses and enhanced inactivation of reoviruses (31, 33) . In addition, l M magnesium chloride enhanced inactivation of adeno-, papova-, herpes-, myxo-and poxviruses whereas myxoviruses were the only one stabilized by 1 M magnesium sulphate (22, 33) . In the coronaviruses family, TGEV was not protected by 1 M magnesium chloride at 50 ° C for 60 minutes (18) whereas IBV was protected for 80 minutes at 50°C by 1 M magnesium sulphate (5) . On the other hand, Hirano et al. (14) found that MHV-2 in 1 M magnesium chloride or sulphate was not inactivated after heating at 50 ° C for 15 minutes, results similar to those obtained in our studies on MHV-A 59. The kinetics of virus inactivation at 4, 22 and 3 7 ° C were studied and the results are presented in Fig. 3 . The virus was relatively stable for 3 months at 4 ° C whereas infectivity was unmeasurable after 14 days at 22 and 37 ° C (<200 PFU/ml). The 37 and 4°C samples were kept in the dark but t~he 22°C sample was in room light and might have increased the inactivation rate at 22 ° C above that at 37 ° C. Manaker et al. (16) observed that the infectivity titre of the A 59 strain was not altered after 7 months at 4 ° C. Other authors described coronaviruses as slowly inactivated at 37 ° C and moderately stable at 4°C in optim~l suspending medium (19, 29) . On the other hand, some TGEV strains were inactivated in less than 4 days at 37 ° C (II, 18) and MHV-2 showed a 400-fold decrease in infectivity after 24 hours at 37 ° C (14) . However, it is not clear whether the other components of the suspending fluid were comparable in these experiments. FinMly, one ml aliquots of virus in growth medium were subjected to cycles of thawing in a 37 ° C water bath, followed immediately by freezing for at least 2 hours at -70 ° C. The infectious titre was stable for at least 15 such cycles (data not shown) as was found for several murine hepatitis viruses (19) . For instance, MHV-3 resisted six cycles (20) , MHV-2 resisted ten (14) and MHV-A 59 six (16) . The physico-ehemical properties of MHV-A 59 reported in this study show that these varied between strains though they were morphologically similar and confirm and extend previous reports on the subject. The stabilization of A 59 by magnesium ions had not been reported previously. These results may also be of practical value in selecting conditions Ibr viral production or purification. Effect of pH on the growth and cytopathogenicity of avian infectious bronchitis virus in chick kidney cells Characterization of the virus of sialodacryoadenitis of rats A cytopathic virus causing a transmissible gastroenteritis in swine A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin. I. Isolation and biologic properties of the virus Stabilizing effect of magnesium sulfate on avian infectious bronchitis virus propagated in chicken embryo kidney cells pH stability studies with avian infectious bronchitis virus (coronavirus) strains Characterization of a new infectious bronchitis virus isolate. II. Some chemieM and physical properties of Clark 333 The pH stability of the virus ofini~ctious bronchitis of chickens Buffer solutions. In: Fasman GD (ed) Handbook of biochemistry and molecular biology Contributions to characterization and classification of animal viruses Studies on transmissible gastroenteritis in pigs. IV. Physicochemical and biological properties of TGE virus In vitro differentiation and pH sensitivity of field and cell culture-attenuated strains of transmissible gastroenteritis virus Replication and plaque formation of mouse hepatitis virus (MHV-2) in mouse cell line DBT culture Physieo-ehemical properties of mouse hepatitis virus (MHV-2) grown on DBT cell culture Thermal inactivation studies of coronavirus, transmissible gastroenteritis virus A hepatitis virus complicating studies with mouse leukemia Characterization of a coronavirus isolated from rats with sialoadenitis Studies on transmissible gastroenteritis of swine. II. Selected characteristics of a cytopathogenic virus common to five isolates from transmissible gastroenteriti~ Coronaviruses: a comparative review Experimental viral hepatitis The influence ofpH on the growth and stability oftransmissible gastroenteritis virus in vitro Protection of measles virus by sulfate ions against thermal inactivation The structure and replication of coronaviruses The biology of coronaviruses Neutralizing antibody complex of infectious bronchitis virus The structure and behavior of coronavirus A 59 glycoproteins The molecular biology of corenaviruses Stabilization of polioviruses by cations Cationic stabilization-a new property of enteroviruses Effect of cations on thermal inactivation of vaceinia, herpes simplex and adenoviruses Different effects of MgCl~ and MgS04 on the thermostabflity of viruses The biology and pathogenesis of coronaviruses We thank Francine Lambert and Francine Allard for excellent technical assistance and Lucie Summerside and Lise Brady for typing the manuscript. The work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (U 0387) and the Fonds de la recherche en sant6 du Qu6bee (840491). A summer studentship award to C.D. from Employment and Immigration Canada is gratefully acknowledged. P.J.T. is recipient of a scholarship from the Natural Sciences and Engineering Igeseareh Council of Canada.