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Haemagglutination by mouse hepatitis virus type 3
Ann. Virol. (Inst. Pasteur) 1(.180, 131 B, 517-520
PRELIMINARY NOTE
HAEMAGGLUTINATION BY
MOUSE
HEPATITIS
VIRUS
TYPE
3
by D. P. Walker and G. M. Cleator
Department o/Bacteriology and Virolofly, Universil!l o/Jianchesler, Manchester ( Em.lland)
BESU.~IE L ' H I ~ M A G G L U T I N A T I O N PAR LE V I R U S T Y P E D E L ~ H E P A T 1 T E I)E LA S O U R I S
3
Une suspension partiellement purifi6e tie virus (le I'h6patite de la souris (MHV3) agglutine les 6rythrocytes tie poule, 1)oussin d'uu jour, furet, hamster, et les 6rythrocytes humains du groupe 0, aux temp6ratures de 4, 20 et 37 ° C, tandis que les 6rythrocytes de souris ne sont agglutinOs qu'fi 4 ° C. .'~'IoTs-CL~S : H6patite (le la souris, MHV.~ ; H6magglutination, Coronavirus.
INTF~ODUCTION Heretofore, mouse hepatitis virus type 3 (MHV3), a coronavirus causing a fatal infection of mice, has not been observed to agghltinate erythrocytes [4, 7, 9]. However, other coronaviruses do cause agglutination of red cells from several species, including rat, chicken, hamster and mouse, but some, such as avian infectious bronchitis virus (IBV) must tirst be purified in sucrose gradients [6, 8, 1, 5]. Previous t)relinfinary work in this laboratory had shown that untreated extracts from mouse livers containing MHV3 gave inconsistant llacmagglutination an(I thus, in the present study, partially purified preparations of the virus were tested for haemagglutinating properties.
Manuserit re(:u le 28 juin 1980, acceptO le 26 n o v e m b r e 1980.
518
!). P. WAI,KEI~ AND G. M. CLEATOB.
MATEI~IAI~S AND METHODS
Culture and purification o/ oirtts. MIIVa (obtained from Dr .I.S. Porterlleld, Sir Williams Dunn School of Pathology, Oxford, l:ngland) was propagated by th- intraperitoneal inoculation of 21-~lay old Manchester strain mice. Livers were removed 72 h after infection. macerated in I ml of l)ulb:~eco lal phosphate buffered saline ,, A ,, (PBS-A) per liver, and centrifugated at 1,500 .q for 10 rain. Supernatant fluids (3 ml) were overlaid onto 20 to 60 % (v/v) m~crose gradients (55 ml) prepared in distilled water and centrifuged at 70,000 .q for 48 h. The gradients were fractionated in 3-ml aliqnots which were diluted four fold in PBS-A and centrifuged for 2 h at 30,000 g. Sedimented material from each fraction was resuspended in PBS-A ((1.5 nil}, and screened for infectivity in mice. Control preparations of uninfected mouse livers were processed as above.
Erylhrocyles. Ervthrocvtes were collected into Alsever's solution, washed in PBS-A and resusp'ended'to 8 "i, (v/v) by haematocrit.
ltaemayffiulinalion lesls. Haemagglutination tests were carried out as described except that each well (WHO plates) contained 0.2 and 0.2 ml of a 0.2 % (v/v) erythrocytc suspension. Fractions virus were pooled and tested at 4, 20 and 37° C, using pH 6.4, 6.8 and 7.2. s a l s 12],
by Clarke and Casml of virus dilution containing infectious values 5.75, 6.0, 6.2,
R E S U L T S AViD DISCUSSION Coronavirus-like particles and ,;mall amounts of amorphous material were seen by electron microscopy in pooled materials recovered from virus-containing fractions detectc(! towards the middle of the sucrose gradients. Haemagglutination was detected only in those fractions which contained infectious virus (titre of pooled material 10s.s LDs0/ml in mice). Optimal haemagglutination titres using mouse and ferret erythrocytes were obtained at pH values between 5.75 and 6.4, and erythrocytes from all species were thence tested at pH 6.2 (table I). Haemagglutination was not observed with erythrocytes from the following species: sheep, rabbit, rhesus monkey, rat, gerbil, pigeon, cat and guinea-pig. Haemagglutination was not detected in control material from fractions of sucrose gradients used to ,, partially purify )) uninfected liver material. In common with other coronaviruses, MHV3 agglutinates erythrocytes from several species and, as with IBV, it is necessary to partially purify the preparations of virus to obtain consistent results. Haemagglutination IBV = infectious bronchitis virus. M H V a = m o u s e h e p a t i t i s v i r u s t y p e 3. P B S - A = p h o s p h a t e - b u f f e r e d s a l i n e ,, A ,.
I lAI';MAGGIAVI'INATION BY MHV3
519
]'ABIA~ 1. --- Haemagglutination (*) by MHV3 of erythrocytes from different species, titre of haemagglutination. I':ryl hl'licytes
|", {]
21i,, l:
27" ("
tluman O Fowl Mouse I)ay-ohl chick Ferret Hamster
I/I 1/ I tl 1/16 1/32 I/8
I/IV; I/32
!/32
1/61 I/32
l/Hi
1/8
1/S
I/8
1/16
1132
I*) Using It.2 % ( v / v ) e r y t h r o ( ' y es at p l l 6.2.
was observed at 4, 20 and 37 ° C except with mouse e r y t h r o c y t e s , which did not agglutinate at 20 and 37 ° C. E r y t h r o c y t e s did not elute from agglutinated preparations left for several h at 37 ° C. The l)reliminary studies reported here indicate t h a t MHV.~ m a y be included a m o n g those coronaviruses causing haemagglutination. H a e m a g glutination inhibition studies and infectivity assays are now being undertaken to confirm the above results and establish a ral)id method of measuring a n t i b o d y levels to MHV.
SU313IAI~Y Partially purilie(I preparations of mouse het)atitis virus type 3 (MHV.0 agglutinated fowl, day-old chick, ferret, h a m s t e r and htmmn 0 e r y t h r o c y t e s at 4, 20 and 37 ° C and mouse e r y t h r o c y t e s at 4 ° C. I{EY-WORI)S: Mouse hepatitis, MHV:,; H a e m a g g l u t i n a t i o n , Coronavirus.
lil,;FI,'AiI£NCI:.S
[1] I~IN,HA.~I, R. W., MADiIE, M. 11. & "I'vm~VH., I). A., |laemagglutination bv avian infeetioas bronchitis virus, a eoronavirus. ,I. 9en. Virol., 1973, 28, 381-390. [2] CLAmmY:,D. H. & CASAI.S, .I., Techniques for haemagglutination inhibition with arthrop,~cl-borne viruses. ,liner..I. /rop. Med., 1958, 7, 561-573. [3] l)t'Lt~v:t:(:(), R. & '/o(vr, M., Plaque formation and isolation of pure lines with poliomyelitis virus. J. exp. 5led., 1964, 99, 167-182. [41 GLEDHH.L, A. W., DmK, G. W. A. & NIVEN, H. S. F., Studies on mouse hepatiffs virus (MHVa). Prof. 6lh Cony. int. Microbiol., Rome, 1953, 3, 219-225. [5] KAvE, H. S. & I)OWDEL, W. R., Some characteristics of haemagglutination of certain strains o f , IBV-like ,, virus. J. inject. Dis., 1969, 120, 576-581. Ann. IZirol, (Inst. Past.), 131 E, no 4, 1980.
36
521)
!). P. V~'AI,KEI-I AND G. M. CLI,'ATOI/
[6] Mcln'rosu, K., Co,'onavirus: a comparative review. Curt'. Top. 31it'robiol. hmmmol., 1974, 63, 85-12{}. [7} PtAZZA, M., Fxperimentai viral hepatitis. Ch. C. Thomas Publ., Springfield (I11., USA), 1969. {81 Sll.xlWt.:E, l{. L., MEmTS, C. A. ,~ BAss, E. P., Characterization of a calf diarrhoeal coronavirus. Amer. J. vel. Res., 1!;76, 37, 1031-1041. [9] TYI~I~ELI.,1). A..]., ALMI':IDA,,]. D., BERRY, I). M. cg: .~I¢:IN'I'OSII,[<.., Coronaviruses. Nahn'r (I.ond.), 1968, 220, 650.
PRELIMINARY NOTE
HAEMAGGLUTINATION BY
MOUSE
HEPATITIS
VIRUS
TYPE
3
by D. P. Walker and G. M. Cleator
Department o/Bacteriology and Virolofly, Universil!l o/Jianchesler, Manchester ( Em.lland)
BESU.~IE L ' H I ~ M A G G L U T I N A T I O N PAR LE V I R U S T Y P E D E L ~ H E P A T 1 T E I)E LA S O U R I S
3
Une suspension partiellement purifi6e tie virus (le I'h6patite de la souris (MHV3) agglutine les 6rythrocytes tie poule, 1)oussin d'uu jour, furet, hamster, et les 6rythrocytes humains du groupe 0, aux temp6ratures de 4, 20 et 37 ° C, tandis que les 6rythrocytes de souris ne sont agglutinOs qu'fi 4 ° C. .'~'IoTs-CL~S : H6patite (le la souris, MHV.~ ; H6magglutination, Coronavirus.
INTF~ODUCTION Heretofore, mouse hepatitis virus type 3 (MHV3), a coronavirus causing a fatal infection of mice, has not been observed to agghltinate erythrocytes [4, 7, 9]. However, other coronaviruses do cause agglutination of red cells from several species, including rat, chicken, hamster and mouse, but some, such as avian infectious bronchitis virus (IBV) must tirst be purified in sucrose gradients [6, 8, 1, 5]. Previous t)relinfinary work in this laboratory had shown that untreated extracts from mouse livers containing MHV3 gave inconsistant llacmagglutination an(I thus, in the present study, partially purified preparations of the virus were tested for haemagglutinating properties.
Manuserit re(:u le 28 juin 1980, acceptO le 26 n o v e m b r e 1980.
518
!). P. WAI,KEI~ AND G. M. CLEATOB.
MATEI~IAI~S AND METHODS
Culture and purification o/ oirtts. MIIVa (obtained from Dr .I.S. Porterlleld, Sir Williams Dunn School of Pathology, Oxford, l:ngland) was propagated by th- intraperitoneal inoculation of 21-~lay old Manchester strain mice. Livers were removed 72 h after infection. macerated in I ml of l)ulb:~eco lal phosphate buffered saline ,, A ,, (PBS-A) per liver, and centrifugated at 1,500 .q for 10 rain. Supernatant fluids (3 ml) were overlaid onto 20 to 60 % (v/v) m~crose gradients (55 ml) prepared in distilled water and centrifuged at 70,000 .q for 48 h. The gradients were fractionated in 3-ml aliqnots which were diluted four fold in PBS-A and centrifuged for 2 h at 30,000 g. Sedimented material from each fraction was resuspended in PBS-A ((1.5 nil}, and screened for infectivity in mice. Control preparations of uninfected mouse livers were processed as above.
Erylhrocyles. Ervthrocvtes were collected into Alsever's solution, washed in PBS-A and resusp'ended'to 8 "i, (v/v) by haematocrit.
ltaemayffiulinalion lesls. Haemagglutination tests were carried out as described except that each well (WHO plates) contained 0.2 and 0.2 ml of a 0.2 % (v/v) erythrocytc suspension. Fractions virus were pooled and tested at 4, 20 and 37° C, using pH 6.4, 6.8 and 7.2. s a l s 12],
by Clarke and Casml of virus dilution containing infectious values 5.75, 6.0, 6.2,
R E S U L T S AViD DISCUSSION Coronavirus-like particles and ,;mall amounts of amorphous material were seen by electron microscopy in pooled materials recovered from virus-containing fractions detectc(! towards the middle of the sucrose gradients. Haemagglutination was detected only in those fractions which contained infectious virus (titre of pooled material 10s.s LDs0/ml in mice). Optimal haemagglutination titres using mouse and ferret erythrocytes were obtained at pH values between 5.75 and 6.4, and erythrocytes from all species were thence tested at pH 6.2 (table I). Haemagglutination was not observed with erythrocytes from the following species: sheep, rabbit, rhesus monkey, rat, gerbil, pigeon, cat and guinea-pig. Haemagglutination was not detected in control material from fractions of sucrose gradients used to ,, partially purify )) uninfected liver material. In common with other coronaviruses, MHV3 agglutinates erythrocytes from several species and, as with IBV, it is necessary to partially purify the preparations of virus to obtain consistent results. Haemagglutination IBV = infectious bronchitis virus. M H V a = m o u s e h e p a t i t i s v i r u s t y p e 3. P B S - A = p h o s p h a t e - b u f f e r e d s a l i n e ,, A ,.
I lAI';MAGGIAVI'INATION BY MHV3
519
]'ABIA~ 1. --- Haemagglutination (*) by MHV3 of erythrocytes from different species, titre of haemagglutination. I':ryl hl'licytes
|", {]
21i,, l:
27" ("
tluman O Fowl Mouse I)ay-ohl chick Ferret Hamster
I/I 1/ I tl 1/16 1/32 I/8
I/IV; I/32
!/32
1/61 I/32
l/Hi
1/8
1/S
I/8
1/16
1132
I*) Using It.2 % ( v / v ) e r y t h r o ( ' y es at p l l 6.2.
was observed at 4, 20 and 37 ° C except with mouse e r y t h r o c y t e s , which did not agglutinate at 20 and 37 ° C. E r y t h r o c y t e s did not elute from agglutinated preparations left for several h at 37 ° C. The l)reliminary studies reported here indicate t h a t MHV.~ m a y be included a m o n g those coronaviruses causing haemagglutination. H a e m a g glutination inhibition studies and infectivity assays are now being undertaken to confirm the above results and establish a ral)id method of measuring a n t i b o d y levels to MHV.
SU313IAI~Y Partially purilie(I preparations of mouse het)atitis virus type 3 (MHV.0 agglutinated fowl, day-old chick, ferret, h a m s t e r and htmmn 0 e r y t h r o c y t e s at 4, 20 and 37 ° C and mouse e r y t h r o c y t e s at 4 ° C. I{EY-WORI)S: Mouse hepatitis, MHV:,; H a e m a g g l u t i n a t i o n , Coronavirus.
lil,;FI,'AiI£NCI:.S
[1] I~IN,HA.~I, R. W., MADiIE, M. 11. & "I'vm~VH., I). A., |laemagglutination bv avian infeetioas bronchitis virus, a eoronavirus. ,I. 9en. Virol., 1973, 28, 381-390. [2] CLAmmY:,D. H. & CASAI.S, .I., Techniques for haemagglutination inhibition with arthrop,~cl-borne viruses. ,liner..I. /rop. Med., 1958, 7, 561-573. [3] l)t'Lt~v:t:(:(), R. & '/o(vr, M., Plaque formation and isolation of pure lines with poliomyelitis virus. J. exp. 5led., 1964, 99, 167-182. [41 GLEDHH.L, A. W., DmK, G. W. A. & NIVEN, H. S. F., Studies on mouse hepatiffs virus (MHVa). Prof. 6lh Cony. int. Microbiol., Rome, 1953, 3, 219-225. [5] KAvE, H. S. & I)OWDEL, W. R., Some characteristics of haemagglutination of certain strains o f , IBV-like ,, virus. J. inject. Dis., 1969, 120, 576-581. Ann. IZirol, (Inst. Past.), 131 E, no 4, 1980.
36
521)
!). P. V~'AI,KEI-I AND G. M. CLI,'ATOI/
[6] Mcln'rosu, K., Co,'onavirus: a comparative review. Curt'. Top. 31it'robiol. hmmmol., 1974, 63, 85-12{}. [7} PtAZZA, M., Fxperimentai viral hepatitis. Ch. C. Thomas Publ., Springfield (I11., USA), 1969. {81 Sll.xlWt.:E, l{. L., MEmTS, C. A. ,~ BAss, E. P., Characterization of a calf diarrhoeal coronavirus. Amer. J. vel. Res., 1!;76, 37, 1031-1041. [9] TYI~I~ELI.,1). A..]., ALMI':IDA,,]. D., BERRY, I). M. cg: .~I¢:IN'I'OSII,[<.., Coronaviruses. Nahn'r (I.ond.), 1968, 220, 650.