key: cord-0004834-1hn83tyo
authors: Hasony, H. J.; Macnaughton, M. R.
title: Antigenicity of mouse hepatitis virus strain 3 subcomponents in C57 strain mice
date: 1981
journal: Arch Virol
DOI: 10.1007/bf01315263
sha: ccc0b2eeb7696486c1a4d77a1c46bf58e1fff054
doc_id: 4834
cord_uid: 1hn83tyo

C57 strain mice were inoculated intraperitoneally with denatured mouse hepatitis virus strain 3 particles and virus surface projection, membrane and ribonucleoprotein subcomponents, obtained from detergent treated purified virus preparations. All immunised animals developed high levels of serum antibody directed against the respective antigens, detectable by enzyme-linked immunosorbent assay. Mice that had been immunised with denatured virus particles or surface projections were protected against infection with mouse hepatitis virus strain 3, whereas immunisation with virus membrane or ribonucleoprotein subcomponents failed to protect mice against virus challenge.

Mouse hepatitis virus (MHV) is a member of the Coronaviridae group of viruses which are all lipid-containing, enveloped, positive-stranded viruses that bud from endoplasmic reticular membranes (17, 23) . The structural polypeptides of a number of MHV strains, including strains A59 (2, 21) , J H M (1, 2, 25) and 3 (1, 12) have been described, and consist of 4 to 6 polypeptides of similar size and composition. These polypeptides are of 3 main types, with up to 3 high tool. wt. glycopolypeptides comprising the surface projections, up to 2 low tool. wt. polypeptides forming membrane proteins, and a single polypeptide of about 50,000 mol. ~.. comprising the ribonucleoprotein (]~NP) (12, 21, 25) . Several reports have described the separation of some or all of the subviral components of MHV A59 (22) and other coronaviruses (4, 6, 14, 18) by disruption of virus particles with ~Nonidet P40 or Triton X 100.

Most strains of mice can be infected with MHV 3 with the development of fulminant hepatitis, although numerous other organs are also infecte~ (17) . The 

M H V 3 was grown in confluent secondary m o u s e e m b r y o n i c fibroblasts. Monolayers were infected a t a n i n p u t m u l t i p l i c i t y of 0.1 infectious particles per ceil a n d following a n adsorption period of 1.5 h o u r s a t 37 ° C, were i n c u b a t e d for 72 h o u r s a t 37 ° C in Eagle's M E M w i t h 2 per cent foetal calf s e r u m (13) . Aliquots of this virus suspension were stored a t --7 0°C a n d used for t h e p r e p a r a t i o n of purified virus particles a n d s u b c o m p o n e n t s .

Virus was purified at 0 ° to 4 ° C as described previously (13) . The virus was pelleted at 75,000 × g for 1 h o u r a n d t h e n resuspended in 1 ml Dulbecco's p h o s p h a t e buffered saline " A " (PBSA). T h e resuspended virus was overlaid on to a linear 25 to 55 p e r cent (w/w) sucrose g r a d i e n t in P B S A a n d centrifuged for 16 hours at 90,000 × g. T h e virus peak a t 1.18 g/ml was collected.

P r e p a r a t i o n s of purified virus part, icier were dialysed against, P B S A for 16 hours a t 4 ° C, a n d t h e n d i s r u p t e d w i t h i per cent N o n i d e t :P40 in P B S A at 21 ° C in order t h a t all virus c o m p o n e n t s were available for iodination. T h e i o d i n a t i o n procedure used was based on t h a t described b y GREENWOOD et al. (7) . 

Sucrose-gra~tient-purified virus particles were d i s r u p t e d at, 21 ° C with 1 p e r cent N o n i d e t P 4 0 in P B S A a n d layered on to either 10 to 55 per cent (w/w) or 25 to 65 per cent (w/w) sucrose gradients in P B S A a n d centrifuged for 16 hours a t 90,000 × g at 4 ° C. S u b c o m p o n e n t peaks were located at 1.13 g/ml in 10 to 55 per cent (w/w) sucrose gradients a n d at 1.23 a n d 1.27 g/ml in 25 to 65 per cent (w/w) sucrose gradients as described previously for H C V 229E (14) .

I o d i n a t e d virus a n d virus s u b e o m p o n e n t fractions were t r e a t e d w i t h 5 per cent sodium dodecyl sulphate, 2 per cent 2 -m e r c a p t o e t h a n o l a t 100°C for 1.5 minutes. A trace of b r o m o p h e n y l blue was a d d e d to t h e reduced preparations, a n d t h e polypeptides were eleetrophoresed t h r o u g h 7.5 per cent p o l y a c r y l a m i d e gels as described previously (15) . After electrophoresis t h e gels were e x t r u d e d a n d sliced into t m m discs a n d their r a d i o a c t i v i t y d e t e r m i n e d .

Immunisation Procedure 4 --6 weeks old C57 BL/10 s t r a i n mice were o b t a i n e d from t h e specific-pathogenfree (SPF) u n i t of this Centre. Groups of 10 mice were i m m u n i s e d w i t h dilutions of purified d e n a t u r e d M H V 3 particle p r e p a r a t i o n s of titres a b o u t 107 IDa0, virus sub-components derived from them, or PBSA. MHV3 particles were denatured in i : 1000 formalin diluted in PBSA for 7 days at 4 ° C (11) . The antigens were suspended in an equal volume of Freund's Complete Adjuvant and 0.1 ml volumes were injected intraperitoneally into the mice in two doses at 10 day intervals.

At 20 days after immunisation, mice were challenged intraperitoneally with 0.1 ml of different titres of infectious MttV 3. Control mice were challenged with PBSA.

Flat-bottomed wells in polystyrene microtitre plates (Dynatech) were coated with dupIicatc 0.2 ml amounts of antigen diluted in 0.1 M carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at room temperature. After incubation the plates ware washed 4 times with phosphate-buffered saline containing 0.05 per cent Tween 20 and 0.02 per cent sodium azide (PBST) and shaken dry. Portions of 0.2 ml of sara diluted in PBST were added to the wells and incubated for 4 hours at room temperature. After 4 additional washes with PBST, 0.2 ml quantities of rabbit anti-mouse IgG antiserum (Miles Laboratories) at a dilution of l : i 0 a was added and left for 3 hours at room temperature. The plates were then washed a further 4 times in PBST. After washing, goat anti-rabbit IgG antiserum labelled with alkaline phosphatase conjugate (Miles Laboratories) at a dilution of 1 : 800 was added in 0.2 ml quantities at room temperature overnight. After 4 additional washes with PBST, 0.2 ml of phosphatasc substrate, consisting of a 0.1 per cent solution of p-nitrophenylphosphate in 10 per cent (w/v) diethanolamine buffer (pH 9.8) with 0.02 per cent sodium azide and 0.01 per cent MgC12 6 I-I20, was added to each well. Absorbance values were read after 30 minutes at 405 nm in a Flow Titertek Multiscan photometer.

Purified M H V 3 particles were obtained from sucrose density gradients as described previously (12) . Subviral components were obtained b y centrifuging N o n i d e t P 4 0 disrupted M H V 3 particles on sucrose density gradients in a similar w a y to t h a t described previously for t h e p r e p a r a t i o n of HCV 229E subcomponents (14) . Surface projections were isolated on 10--55 per cent (w/w) sucrose d e n s i t y gradients at 1.13 g/ml, a n d m e m b r a n e a n d R N P fractions were isolated on 25--65 per cent (w/w) sucrose gradients a t 1.23 and 1.27 g/ml respectively. The p u r i t y of these subcomponents was d e t e r m i n e d b y iodinating t h e m and t h e n analysing their polypeptides on p o l y a c r y l a m i d e gets. Fig. 1 shows a p o l y a c r y l a m i d e gel of iodinated polypeptides from N o n i d e t 1)40 d i s r u p t e d particles, which were similar to those obtained previously for unlabelled or 3H-leucine labelled M H V 3 polypeptides (12) . The polypeptides have m e a n mol. wt. of 170,000, 50,000, 22,000 and 20,000, and have been called V G P 170, V P 50, V G P 22 and V P 20 (VP, virus p o l y p e p t i d e ; VGP, virus glycopolypeptide) (12) . No p o l y p e p t i d e of m e a n mot. wt. 90,000, observed previously (12) , was resolved, although it m a y be present b u t m a s k e d b y the high b a c k g r o u n d radioactivity.

Similar polypeptides have been obtained for other M H V strains (1, 19, 21, 25) , and the polypeptidcs comprising the surface projections, m e m b r a n e and R N P have been shown to correspond to V G P 170, V G P 22 and V P 20, and VP 50, respectively (12, 19, 21, 25) . Fig. 2 a --e are polyacrylamide gels of MHV 3 subcomponent preparations isolated on sucrose density gradients from Nonidet P40 disrupted particles. The gels revealed a good separation of the structural polypeptides with the surface projection preparation containing VGP 170 (Fig. 2a) , the membrane preparation VGP 22 and VP 20 (Fig. 2 b) , and the RNP preparation VP 50 (Fig. 2 c) . There was no evidence, from a number of polyaerylamide gels, of contamination of any of the subcomponents with other subcomponents, although there was considerable background radioactivity in all the gels of the iodinated subeomponents. 

Groups of 10 mice were each immunised with one of a number of dilutions of sucrose-gradient-purified inactivated virus particle or with subcomponent preparations. The corresponding dilutions of the MIIV 3 subcomponent fractions contained comparable amounts of antigen as determined by ~25I labelling (Fig. 2) , The mice were then observed for 20 days, and during this period no mice died or showed any symptoms of disease. Furthermore, sera were taken from them before inoculation, and 8, 14 and 20 days after inoculation, and antibody rises in the postinoculation sera were measured by ELISA using homologous antigens. Table 1 shows typical ELISA absorbance values obtained for sera taken 14 days after immunisation. For all these sera the ratios of postinoculation to preinoculation serum absorbance values at the same antigen and serum dilutions were sigTfficantly over 2, indicating that specific antibodies had developed (9, 10, 14) . Thus, both virus particles and subcomponents elicited a significant antibody response. ELISAs using heterologous combinations of subcomponents and sera produced no antigenic reactions. All readings were taken at 405 nm after 20 minutes b Antigen dilution 1 : 50 c Sera from mice given 10-~ dilution of virus particles or virus subeomponents obtained 14 days after immunisation. Sera dilutions 1:200 Table 2 shows ELISA ratios, obtained for sera from animals immunised for 8, t4 and 20 days with a number of dilutions of denatured virus particles or virus subeomponents, tested against, homologous antigens. Significant antibody rises, as measured by ELISA ratios, were observed in all sera from mice inoculated with denatured virus particles, although not in those from mice inoculated with subcomponent fractions. The antibody rises detected against purified subviral components were dose dependent and in all eases tested the membrane and R N P components were less immunogenic in mice than the surface projection components, even after repeated immunisation.

Dilutions of 10 -1 of denatured virus particles and subcomponents and an immunisation period of 20 days were selected from Table 2 as suitable for producing high levels of antibody in immunised mice. After immunisation, mice were H . J . ItASONY a n d M. R. 5'][ACNAVG~TON: challenged with different titres of infectious MHV 3 or PBSA (Table 3 ). The mice were then observed daily for 20 days and the numbers surviving on each day noted. Between 5 and 8 out of 10 mice immunised with denatured virus particles, and between 5 and 7 out of 10 mice immunised with surface projections survived up to 20 days after challenge with MHV 3. All non-immunised mice were killed by 7 days after virus challenge by the MHV 3 dilutions tested, with the first deaths occurring by day 5. However, non-immunised mice challenged with PBSA showed no signs of illness. Mice immunised with purified membrane and R N P fractions were not protected against MHV 3 challenge --all of them died within 6 days. Diseussion In this paper we report the isolation and purification of MHV 3 subviral components and have shown the role of each subcomponent in the protection of immnnised mice against challenge with infectious MHV 3. I t was difficult to ensure that mice immunised with different virus subcomponent preparations all produced comparable amounts of antibody, as there was considerable variation in the immunogenieity of the subeomponents. The highest antibody rises detected by ELISA were directed against surface projections, while lower antibody rises were observed against membrane and RNP, suggesting that the most immunogenic part of the virus is an antigen(s) associated with the surface projections. Similar results have been obtained previously with human eoronaviruses (14) and the porcine coronavirus transmissible gastroenteritis virus (TGEV) (5) .

We have shown a close correlation between the protection of mice against infection and antibody rises in inactivated virus particles and surface projections. Fm%hermore, experiments have been reported showing that rabbits and sows inoculated with whole TGEV particles or surface projections may be protected against virus challenge by the stimulation of neutralising antibody (5). Similar properties have been observed for the surface projections of other lipid-containing, enveloped RNA viruses (3, 8, 24) . Our results with MHV 3 membrane and R N P subcomponents suggest, that although antibody was induced by these subeomponents in mice, this antibody was produced in relatively low amounts and had no deteetable protective effect against infection. Repeated immunisation of mice with membrane and R N P subeomponents did not lead to increased amounts of antibody or to any protection against MHV 3 infection. Thus, it is unlikely that these subeomponents were protective and this protection was missed due to the poor immunogenieity of these components.

There was no decline in the antigenicity of subeomponents after Nonidet P40 treatment, dialysis or eentrifugation in sucrose gradients (HAsoNY, unpublished data) as has been shown for other viruses, including herpesviruses (20) . In our ELISAs, only IgG antibodies were measured in the postinoculation mouse sera, although other immunoglobulin elasses may have important roles in the protection of mice against, infection. However, by the 20th day after immunisation, IgG antibodies should be the most common antibodies present and have the predominant role in protection against infection.

Further studies are in progress to extend these experiments to other coronaviruses and to determine the antigenic and structural relationships between coronavirus subcomponents.

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Received February 10, 1981