- Email: [email protected]
Isolation of a virus, MVBr1, from Mycoplasma bovirhinis
I (,3
FI:.~IS Micr.bi~d~,~y t.eller,, ? (198fi) 163--163 O C*~pyJighl I:ederaliol] t)l I(ii,ropt'an Mic~,~bi~]t~.t:it'al S,Wiclics Iqlbli'~hcd l)3' I I,ievier/Nollh-Iloll:ll)d Ilit~metlit.d Prc~,i
ISOLATION OF A VIRUS, 51VBrl, FROM MYCOPLASAIA B O V I R l l l N I S C.J. HOWARD, R.N.GOURLAY and $.C,,. WVLD ,.I,eri¢~tltural ]~¢'~¢fll'ch C~u t~cil, h~srirll!,' I'or Re~'~m'h on A p~hnal l)i~eflw~. Corot,ton. ,Vr. A'c'whurr. It~'r~ shire, R(; I A ¢).V.V. I,'.A'. Received t3 Ntwember 1979 A¢¢,apled 29 Noven'tber 1979
1. Introduction The fir,~l isolatit~n of a virus from art organism tff the class M~llicutes was hi ] 970 from Acholeplasma laidlawff [ 1I. Subsequent studies led to the isolatiotl from the same Acholeplasma species of two other morphologically distiller viruses [2,3]. A virus has also been isolated From another genus within the Mollicutes, Spiroplasma [4.I , and examination by electron microscopy has indicated Ihat several dislinct viruses can apparently infect this genus [5I. There have been reports of the isolatitm of viruses fron~ organisms of the genus M),copla~ma I6-8] but all of these isolations have been made on lawns of A. laidlawfi and the virus isolates have been found to produce plaques only on A. laidlawii and not oa the original Mycoplasma species Froru whicll they were apparently derived. Since spontaneous plaques ar~ known to occur on these A. laidlawff lawns such claims for the isolation of viruses from the genus Mycoplasma must be open to doubt [2,6]. Viruslike particles have aIso been observed by electron microscopy in certain Alycoplasma species [7,8] but these have not been isolated. We rep~rt here the isolation of a virus from M. bo~'irhinis which can be propagated on that species. This represents the first indisputable isolation of a virus from this/iO,coplasura species and indeed from any organism of the genus/tfycoplasma or family Mycoplasm~taceae. 2. ~,laterials and Methods Mycopiasmas were grown in glucose-serum broth or on the solid medium prepared as described previ-
ously [91 excep! that in the solid mediutn 10% v/v horse serum (Flow I.~lboratories, Scotland) was sometimes substiluled ft)r foet:d calf serum. ,,1. laMlawii, strains M1302/68, 511305/68, BNI. M221/69, TGI-(C) and $2 aud 3/. bovirhiltis strains PG43,CI55 and OIOC have been described [10--12 I. M. hurirhinis slraios L866 and Ca302 were istdated from the respiratory tract of calves, cloned by filtra. lion and picking sia~,le colonies 3 times and identified by the indirecl inmlunoflut~reseeut arltibody tcclmiquc [I 31. The methods used to produce lawns of royceplasma for the propngatiou of virus were as already described I3,14,15]. An tisera to M. boHrhinis strain PG43 and A. faidlawii strains PG8 ;and PG9 were obtained from the National Institute of Allergy and Infectious Diseases, Bethcsda, MD, USA. Antiserum to/If, bovirhhtis straia PG43 was also prepared in a rabbit [16]. Prt~duclion of antisera to the viruses MVL1, MVL2. MV.Lg.pS2. L172, MVL3 and BNI virus has been described [3,
ISl. Electron microscopy on stock virus and the plague inhibition and serum neutralisation tests were performed as previously reported [3,15].
3. Results and Discu~ion
3.1. Orightal isolation of virus A nasopharyngeal swab taken from a calf anlongst a group with respiratory disease was titrated in glucose serum broth to isolate mycoplasrnas. After 5 days incubation at 37°C drops from the brt~th cultures were put on to solid medium which was incubated
I t,4
:Jemhit':dly at 37~C. 3 days I:lter tylfical my c,,plasm;t c~h~fies were visible ¢~n the s~litt medium bnl the u~yc~plasma growll~ ~ppeared t~ be peppered with s[~Olllallo.lns plaques. These typical myc~q~l;lsma ctlloilies were identified ;asM. bta'irhinis by lhe indirecl fluorcscenl amibody test. Two clones of virus were isolated by picking single plaques lille O. 15 M phosphate.b~! ffcred saline, pli 7.2. The suspet~sions were filtered thrt~ugh a 220 nm Millipore membrane and the filtrate iilmied on lawns ofM. hovirhinis P(;43. Single plaques were picked 3 times and a stock of virus was grown ot~ 3L bovirhb~is PG43 which was slorcd at -.-70"C is1 sm:~lt volumes until the putative new virus was examined.
1
3.2. Properties of the virus Fig. 2. i:lectrun m[crographs of MVBrl virus, ne~rlrivdy
The virus produced cle:lr plaques 1-2 mm in diameter on lawns of.M. bol,irhinis strain PG43 (Fig, 1). Examination of negatively stained virus by electron microscopy (Fig. 2) indicated Ihat it had a polyhedral head approx. 72 mm across (measured between apexes) and a tail approx. 71 nm long. The tail Itad a narrow portion near the head and a wide portitm, approx. 19 nm wide, ending ill a base plate. Tail fibres appeared to be present. I~ is therefore mor-
stained with pho.~ph~tungsticacid. phologically distinct froth A. Ioidlawii viruses MVLI arid MVL2, and resembles MVL3 and BN1 virus, but has a considerably longer tail. The virus was titrated on lawns of six A. Iaidtawii strains which support multiplication of tile previously isolated virtlses. No plaques were produced on any of these strains. The satne stock of virus (iitre 1 - I07 pfutml on PG43) was titrated on A/. bovirhb~is strain~ PG43, Ca302, 1,866, Ol 0C and C155, Approxhn~tely the same number of plaques were produced on strains PG43 and C155. No plaques were visible on the other 3 strains. Antisera to A. laidlawii viruses MVL1, MVL2, MVLg.pS2-L172, MVL3 and BNi were tesicd by the plaque inhibition test, on M. bovirhinis strain PG43, for their ability to inhibit growth of the new virus. None Ilad any inhibitory activity. Amisera to MVL3 and BNI were also tested by the serum ncutralisation test for activity against the new virus isolate. No neutralisation of virus was observed, Finally an examination was macle of the effect of exposure to UV (254 rim) irradiation. This reduced the number of plaque,forming units by 99.99%. 4, Conclusions
Fig. 1. Plaques produced by MVBrl virus ohM. borirhiuis strain PG43, stained with 0.1% neutral red.
This report records the first isolation of a virus from M, bovirhhtis and also the first recovery from an
t~rganism ~f the family Mycot~l:tsmalace:~c which is capable of pr~ducing plaques ~n lawas ~f the species o f origill. The observation shows that M.~'t'oplttstna spp., and hence organisms with a genome size as small as 5 , 10 s daltons, can be parasitised by viruses. The virus, provisionally named MVBr I (Mycoplasma virusbovirhinis 1). is morphologically a tailed bacleriophage.
Acknowledgements We thank Mrs. A.P. Bland for the electron micrograph and Mr. I. J e b b e t t for producing the photograph.
References [1 ] Gourla),, R.N. (1970) Nalure (London) 225, 1165. 121 Gourlay, R.N. (1974) CRC Crit. Rev. Microbiol. 3, 315-33|. [31 Gourlay, R.N., Wyld, S,G. and Bland, A,P. (1979) J. Gen. Virol. 42,315-322,
t 41 ('~1~', ILM,. Milch~:ll, W.O. :lml (~ar~l~l, C.t . (1977) 151 Ct~lc. R.M. ~1979) in The Myc~l'~l;l~masIltatile, M.F. and Razin. SL.,eds.), Vnl. I. Cell Bi~logy, pp. 385 410. A¢;idt.lllJc Prexs, Lontloll. [6] Clyde, W.A. 11973) Anla. N.Y, Acad. Sci. 225, 159. lfi0. [7 ] Maniloff, J., Dan, I. alld Chrislen~;en, J.R. 11977) Adv. Viru~ Re.,;.21,343-380. 181 I~,ahman, A.A. and Selhi, K.K. (1979) I.[xpericnlia 35, 1029--1030. 191 Gotlrla}', R.N. and Lt'ach. R.11. (1970) J. Med. ,~licr~bi~l. 3,111-123. [I O] Guurlay, R.N, and Wyld, S.G. (1972) J. Gen. Vir,31. 14, 15=-23. { I 1 ] Liska, 13. (1972) Studia Ilitlphys. 34, 151 - 155. [12] tloward, C.J., IItownlie, J., C,outlay, R.N. and Ct~lli~ls. J. (1975) J. Ilyg. 74,261 --270. [13] Ros~udal, S. and Black, F,T. (1972) Acta Pathol. Microbiol. Seand. B 80,615-622. I 141 Gourlay, R.N. and l'yld, S.G. (I 973) J. Gen. Virol. 19, 279-283. [ 151 GourlaY, R.N., Wyld, S.G., Garwes. D,J'. and pocock, D.It. (1979) Arch. Virol, 6I, 289-296. 1161 ltoward, C3., Gourlay, R,N. and Collins, J. (1974) J. Ilyg. 73,457-466.
FI:.~IS Micr.bi~d~,~y t.eller,, ? (198fi) 163--163 O C*~pyJighl I:ederaliol] t)l I(ii,ropt'an Mic~,~bi~]t~.t:it'al S,Wiclics Iqlbli'~hcd l)3' I I,ievier/Nollh-Iloll:ll)d Ilit~metlit.d Prc~,i
ISOLATION OF A VIRUS, 51VBrl, FROM MYCOPLASAIA B O V I R l l l N I S C.J. HOWARD, R.N.GOURLAY and $.C,,. WVLD ,.I,eri¢~tltural ]~¢'~¢fll'ch C~u t~cil, h~srirll!,' I'or Re~'~m'h on A p~hnal l)i~eflw~. Corot,ton. ,Vr. A'c'whurr. It~'r~ shire, R(; I A ¢).V.V. I,'.A'. Received t3 Ntwember 1979 A¢¢,apled 29 Noven'tber 1979
1. Introduction The fir,~l isolatit~n of a virus from art organism tff the class M~llicutes was hi ] 970 from Acholeplasma laidlawff [ 1I. Subsequent studies led to the isolatiotl from the same Acholeplasma species of two other morphologically distiller viruses [2,3]. A virus has also been isolated From another genus within the Mollicutes, Spiroplasma [4.I , and examination by electron microscopy has indicated Ihat several dislinct viruses can apparently infect this genus [5I. There have been reports of the isolatitm of viruses fron~ organisms of the genus M),copla~ma I6-8] but all of these isolations have been made on lawns of A. laidlawfi and the virus isolates have been found to produce plaques only on A. laidlawii and not oa the original Mycoplasma species Froru whicll they were apparently derived. Since spontaneous plaques ar~ known to occur on these A. laidlawff lawns such claims for the isolation of viruses from the genus Mycoplasma must be open to doubt [2,6]. Viruslike particles have aIso been observed by electron microscopy in certain Alycoplasma species [7,8] but these have not been isolated. We rep~rt here the isolation of a virus from M. bo~'irhinis which can be propagated on that species. This represents the first indisputable isolation of a virus from this/iO,coplasura species and indeed from any organism of the genus/tfycoplasma or family Mycoplasm~taceae. 2. ~,laterials and Methods Mycopiasmas were grown in glucose-serum broth or on the solid medium prepared as described previ-
ously [91 excep! that in the solid mediutn 10% v/v horse serum (Flow I.~lboratories, Scotland) was sometimes substiluled ft)r foet:d calf serum. ,,1. laMlawii, strains M1302/68, 511305/68, BNI. M221/69, TGI-(C) and $2 aud 3/. bovirhiltis strains PG43,CI55 and OIOC have been described [10--12 I. M. hurirhinis slraios L866 and Ca302 were istdated from the respiratory tract of calves, cloned by filtra. lion and picking sia~,le colonies 3 times and identified by the indirecl inmlunoflut~reseeut arltibody tcclmiquc [I 31. The methods used to produce lawns of royceplasma for the propngatiou of virus were as already described I3,14,15]. An tisera to M. boHrhinis strain PG43 and A. faidlawii strains PG8 ;and PG9 were obtained from the National Institute of Allergy and Infectious Diseases, Bethcsda, MD, USA. Antiserum to/If, bovirhhtis straia PG43 was also prepared in a rabbit [16]. Prt~duclion of antisera to the viruses MVL1, MVL2. MV.Lg.pS2. L172, MVL3 and BNI virus has been described [3,
ISl. Electron microscopy on stock virus and the plague inhibition and serum neutralisation tests were performed as previously reported [3,15].
3. Results and Discu~ion
3.1. Orightal isolation of virus A nasopharyngeal swab taken from a calf anlongst a group with respiratory disease was titrated in glucose serum broth to isolate mycoplasrnas. After 5 days incubation at 37°C drops from the brt~th cultures were put on to solid medium which was incubated
I t,4
:Jemhit':dly at 37~C. 3 days I:lter tylfical my c,,plasm;t c~h~fies were visible ¢~n the s~litt medium bnl the u~yc~plasma growll~ ~ppeared t~ be peppered with s[~Olllallo.lns plaques. These typical myc~q~l;lsma ctlloilies were identified ;asM. bta'irhinis by lhe indirecl fluorcscenl amibody test. Two clones of virus were isolated by picking single plaques lille O. 15 M phosphate.b~! ffcred saline, pli 7.2. The suspet~sions were filtered thrt~ugh a 220 nm Millipore membrane and the filtrate iilmied on lawns ofM. hovirhinis P(;43. Single plaques were picked 3 times and a stock of virus was grown ot~ 3L bovirhb~is PG43 which was slorcd at -.-70"C is1 sm:~lt volumes until the putative new virus was examined.
1
3.2. Properties of the virus Fig. 2. i:lectrun m[crographs of MVBrl virus, ne~rlrivdy
The virus produced cle:lr plaques 1-2 mm in diameter on lawns of.M. bol,irhinis strain PG43 (Fig, 1). Examination of negatively stained virus by electron microscopy (Fig. 2) indicated Ihat it had a polyhedral head approx. 72 mm across (measured between apexes) and a tail approx. 71 nm long. The tail Itad a narrow portion near the head and a wide portitm, approx. 19 nm wide, ending ill a base plate. Tail fibres appeared to be present. I~ is therefore mor-
stained with pho.~ph~tungsticacid. phologically distinct froth A. Ioidlawii viruses MVLI arid MVL2, and resembles MVL3 and BN1 virus, but has a considerably longer tail. The virus was titrated on lawns of six A. Iaidtawii strains which support multiplication of tile previously isolated virtlses. No plaques were produced on any of these strains. The satne stock of virus (iitre 1 - I07 pfutml on PG43) was titrated on A/. bovirhb~is strain~ PG43, Ca302, 1,866, Ol 0C and C155, Approxhn~tely the same number of plaques were produced on strains PG43 and C155. No plaques were visible on the other 3 strains. Antisera to A. laidlawii viruses MVL1, MVL2, MVLg.pS2-L172, MVL3 and BNi were tesicd by the plaque inhibition test, on M. bovirhinis strain PG43, for their ability to inhibit growth of the new virus. None Ilad any inhibitory activity. Amisera to MVL3 and BNI were also tested by the serum ncutralisation test for activity against the new virus isolate. No neutralisation of virus was observed, Finally an examination was macle of the effect of exposure to UV (254 rim) irradiation. This reduced the number of plaque,forming units by 99.99%. 4, Conclusions
Fig. 1. Plaques produced by MVBrl virus ohM. borirhiuis strain PG43, stained with 0.1% neutral red.
This report records the first isolation of a virus from M, bovirhhtis and also the first recovery from an
t~rganism ~f the family Mycot~l:tsmalace:~c which is capable of pr~ducing plaques ~n lawas ~f the species o f origill. The observation shows that M.~'t'oplttstna spp., and hence organisms with a genome size as small as 5 , 10 s daltons, can be parasitised by viruses. The virus, provisionally named MVBr I (Mycoplasma virusbovirhinis 1). is morphologically a tailed bacleriophage.
Acknowledgements We thank Mrs. A.P. Bland for the electron micrograph and Mr. I. J e b b e t t for producing the photograph.
References [1 ] Gourla),, R.N. (1970) Nalure (London) 225, 1165. 121 Gourlay, R.N. (1974) CRC Crit. Rev. Microbiol. 3, 315-33|. [31 Gourlay, R.N., Wyld, S,G. and Bland, A,P. (1979) J. Gen. Virol. 42,315-322,
t 41 ('~1~', ILM,. Milch~:ll, W.O. :lml (~ar~l~l, C.t . (1977) 151 Ct~lc. R.M. ~1979) in The Myc~l'~l;l~masIltatile, M.F. and Razin. SL.,eds.), Vnl. I. Cell Bi~logy, pp. 385 410. A¢;idt.lllJc Prexs, Lontloll. [6] Clyde, W.A. 11973) Anla. N.Y, Acad. Sci. 225, 159. lfi0. [7 ] Maniloff, J., Dan, I. alld Chrislen~;en, J.R. 11977) Adv. Viru~ Re.,;.21,343-380. 181 I~,ahman, A.A. and Selhi, K.K. (1979) I.[xpericnlia 35, 1029--1030. 191 Gotlrla}', R.N. and Lt'ach. R.11. (1970) J. Med. ,~licr~bi~l. 3,111-123. [I O] Guurlay, R.N, and Wyld, S.G. (1972) J. Gen. Vir,31. 14, 15=-23. { I 1 ] Liska, 13. (1972) Studia Ilitlphys. 34, 151 - 155. [12] tloward, C.J., IItownlie, J., C,outlay, R.N. and Ct~lli~ls. J. (1975) J. Ilyg. 74,261 --270. [13] Ros~udal, S. and Black, F,T. (1972) Acta Pathol. Microbiol. Seand. B 80,615-622. I 141 Gourlay, R.N. and l'yld, S.G. (I 973) J. Gen. Virol. 19, 279-283. [ 151 GourlaY, R.N., Wyld, S.G., Garwes. D,J'. and pocock, D.It. (1979) Arch. Virol, 6I, 289-296. 1161 ltoward, C3., Gourlay, R,N. and Collins, J. (1974) J. Ilyg. 73,457-466.