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A THESIS PRESENTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF PHILOSOPHY IN
VIROLOGY AND IMMUNOLOGY AT MASSEY UNIVERSITY
TOMASA CRUZ BALASSU
D,V,M,
(
UNIV, OF THE PHILIPPINES)
M.P.H. - VET.
(
UNIV, OF THE PHILIPPINES)
MARCHJ
1981
STATEf1EIH
The gradi ents depi cted in Figure 1 and the ge l depi cted in F igure 3 were done in coll aborat i on w i th Dr . A . J . Rob i nson . The rest of the work was my own .
! f301/D.A/J'
A---T . C . B a l assu
ii.
A pr e l imi nary s tudy of various o r f v i rus i s o l at e s i n New Z ea l and is pre s e nted . A me thod o f i s o l ating and pur i fy i ng the v i r u s from s c ab mater ial i� de s c r i b ed . Thi r te e n o r f v i ru s i s o late s were compared by DNA
r e s tr i c t ion endonuc lea s e analysi s . There was ext e n s ive heterogene i ty in t h e EcoR1 r e s t r i c t i on patterns , however , two o f the i so l ate s were s imi lar.
Propagat i on o f the virus .{. V!. v-<.tJL o w a s a l so at temp t ed . F i ve of the i s olat e s w e r e s uc ce s s fu l ly adapted to growth in l amb te s t i s c e l l s and two o f the s e w e re p l aque -pur i f i ed tw i c e . The c haracter i s t i c cytopath i c e f f e c t o f c e l l - adapted virus i s e a r ly rounding and c l umpi ng toge ther o f i n f e c ted c e ll s . Th i s c e l l round ing cou ld be s e e n a s e a r ly a s two hou r s po s t- i n f e c tion a nd could be inhibi ted by cyclohexamide . Ther e were two typ e s o f p laque� exh i b i ted by the i s o l at e�
i n l amb t e s t i s ce l l mono layer s , the " open " and " c lo s e d "
type s . I nc l u s i o n bod i e s w e r e a l s o s e e n i n s ta i n ed i n f e c te d mono laye r s .
3 2P- labe l l ing o f a plaque-pur i fied i s o late was s u c ce s s f u l ly pe r formed . Low pas s ag e ( 2 3 p a s s ag es) in c e l l cu l tu r e o f p laqu e-pur i f i e d i so late No . 2 d i d not a l ter the E coR1 , Barn H- I or H i nd I I I patterns o f t h e DNA when compared w i th the o r i g in a l v i r a l DNA from s c ab mater i al .
1V.
ACKNOWLEDGf'.lENTS
F i r s t o f a l l, I wou l d l ike to exp res s my s 1ncere gra t i tude t o the Min i s try o f F o re i gn A f f a i r s , New Zea l and f o r g1v1ng me th i s opportun i ty t o pursue h i gher s tud ies in th i s c ountry . I w i sh t o acknowledge the suppor t �nd adv i ce o f Ms S arah C ra i g , Mr Don Car s on and Mr Chr i s Wr i gh t . The f inan c i a l supp o r t extended b y the Dep t . o f Ve ter inary Pa th o l o gy and Pub l i c Heal th made th i s research po s s ib le and o f wh i ch I am very gratefu l .
My mo s t s 1ncere thank s are due t o my superv1 s o r , Dr. A . J . Rob in s on , for h i s enc oura gemen t and gu i dance through o u t
the c ou r se o f th i s wo rk . I am a l s o gra te fu l to the techn i c a l a s s i s t ance o f Ms 1. Fray and Ms G. E l l i s .
A s pec i a l thank i s due to Mr R . D i xon for h i s mod i f ied
techni que o f May-Grunwal d-Giem s a s ta in wh i ch wa s very u s e fu l . The va luab le a s s i s t ance o f Mr Peter W i l db o re and the
pho tograph i c wo rk o f Mr Tom Law are s in cerel y appre c i a ted and a cknowled ged . I am a l s o thank ful t o Mr s Jan S ch rama and Mr Rex Fau l d ing who se serv i c e s were o f mu ch hel p in the c omp l e t ion o f th i s work .
A grate fu l acknmvledgmen t i s a l s o made to the MAF Near
D iv i s i o n veter inar i an s for prov i d ing the s amp les needed for th i s s tudy . Fur ther a cknowledgmen t i s due to the Department o f S c ien t i f i c and Indu s t r i a l Resear ch for the u se o f the e lec t r on mi c r o s c ope wi th s pec i a l thanks t o Mr Doug Hop c r o f t .
F inal l y , I w i sh t o a cknowledge the fr iend s h i p and
h o s p i ta l i ty o f the member s o f s t a f f and fel l ow gradua te s tu den t s o f the Dep t . o f Veter inary P a tho l o gy and Pub l i c Hea l th . The f ine and c ompeten t typ ing o f Mr s V . Lobb i s a l s o acknowledged .
PAGE
Statement i i
Ab s trac t i i i
Acknm..r l e dgmen t s iv
Tab l e o f Con ten t s V
L i s t o f F igur e s v i i
In t rodu c t ion ix
Ch ap ter One - Rev i ew o f L i tera ture 1
Chap ter T<;vo
Con tagious ec thyma ln s h e ep, goa t s , and man
Or f in o ther hos t / exp er imen t a l an ima l s
S cabby mou th i n New Zea l and D ia gno s i s
Tre a tmen t and Con tr o l
Phy� i co-cheu i c a l prope r t i e s o f the V l ru s
Mu l t i p l i c i ty o f s t r a ins
Growth o f o rf virus in c e l l cul ture
- M a t e r i a l s and me th o d s
I s o l a t i on and pur i f i c a t i on o f o r f
1
6 9 1 0 1 1
1 5 1 7 1 9
2 7
V l ru s 2 7
Sp r ay drop l e t c ount ing o f o r f
v i ru s par t i c l e s 29
Measuremen t o f d en s i ty o f
frac t ions from aryl g r a d i e n t s . 3 0
DNA extrac t ion 3 0
Re s t r i c t i on endonu c l eas e d i ge s t ion
and ge l e l e c trophore s i s o f DNA 3 1 Prop aga t ion o f o r f virus i n c e l l
c u l tur e 3 1
P l aque-pur i f i c a t ion o f v i ru s
i s o l a t e s 3 3
3 2P - l ab e l l ini o f o r f virus DNA 3 4 E f f ec t o f cyc l ohexam ide o n o r f CPE 3 6
Cover s l ip prepara t i on s 3 6
TABLE OF CONTENTS
PAGE
Chapter Thr e e - Re s u l t s 38
V i ru s purificat ion Par t icle coun t i ng
Res t r i ction e ndonuc l e a s e a n a lys i s
Growth i n c e l l cu lture Cytopa thic effe c t ( CPE ) i n
c e l l c u l ture
P laque -purifi cat ion and
38 38
38 4 0 4 2
t i tration of the virus 4 7 Exam i nation of May-Grunwald-
G i emsa s tained prepar a�ions 4 7 Effe c t of cyclohexamide o n orf
CPE 48
Chap ter Four - D i s cu s s i on 4 8
Chapter F ive - Summ ary 5 6
Appendix I - Li s t of Mate r i a l s 58
Appendix I I - L i s t of Equ ipment and Apparatus 61 Appendix I I I - Compo s i t ion of Buffe r s and So lutions 63
B ib l i og r a phy 68
v i
FIGURE
l S o d ium me t r i z oa t e ( l e f t ) and Na d i a t r i z o a t e ( r igh t ) grad i e n t s c on ta in ing virus b and s . A m inor b and \vh i ch i s mo re d i s t ine t in Na
AFTER PAGE
me t r i z o a te i s s een b e low the ma in b and . 38
2 A spray drop l e t con taining or£ v1rus
p a r t i c l e s and l a t e x b eads . 3 8
3 Agar o s e gel e l e c t rophore s i s o f t h i r t e e n o r f v i rus DNAs d i ges t e d w i th EcoR1 r e s tr i c t i on
endonu c l e a s e . E l e c tro phore s i s wa s in a 3 8
0 . 7 % a ga ro s e s l ab gel for 4 hour s a t 8 0 V . 4 Agaro s e ge l e l e ctrophores i s of i s o l a t e
N o . 2. E l e c troph o r e s i s wa s in a 0 . 7 % a garo s e s l ab g e l for 4 hours a t 8 0V .
(A) Lane s con t a in v i ru s DNA s d e r ived f rom
o r i g ina l sca b l e s ions d iges t e d wi th E c oR 1(1 ), 40
B arn H-1(2) and H in d III(3).
(B) Lanes show an au t o radiograph o f th e s ame g e l contain ing c o rre s ponding 3 2 p-l abel led DNA d i ges t s der ived f rom a p l aque-
p ur i f i e d v i ru s p a s s aged 23 t im e s in LT c e l l s .
5 Unino cu l a ted c on t r o l l amb t e s t i s mono l ayer . 46
X 134 .
6 L amb t e s t i s mono l aye r , 2_lz hou r s a f ter
i n fe c t ion wi th orf no . 1 0 P 1 0 showing e a r l y 4 6 c e l l round ing . X 134 .
7 A mor e genera l i s ed c e l l round ing s een 5
hou r s a f ter in fe c t i on , l amb t e s t i s c e l l s . 46
X 134 .
8 a LT , 48 hours p o s t - in fe c t ion . Mo s t c e l l s
have d e tached f r om the f l a sk. X 134 . 46 8b LT , 48 hours p . i . showing c l ump o f
rounded up c e l l s . N o t e tha t s ome c e l l s 4 6 have be come en l arged and ep i th e l i o i d .
xsoo.
9 LT monolayer i n fe c t e d wi th s c ab-derived
v i ru s i s o l a t e No . 1 9 P4 . No t e tha t s ome 46 ce l l s have numerous t iny c e l l u l ar pro c e s s e s .
xsoo.
FIGURE
1 0 Un ino cu l a te d con t r o l f o e t a l bov ine lun g mono l aye r . (FBL ) . X l 3 4 .
l l FBL, 5 hours a f t e r i n fec t ion wi th s c ab
d e r iv e d i s o l a t e No. l2 P5 , showing rounded c e l l s w i th t iny proc e s s e s . X 5 00 .
l 2 a FBL , 2 4 h ours a f t e r i nfe c t i on . Note tha t th ere i s l e s s c l umping o f rounded c e l l s and c e l l s tha t rema ined a t tached to the f l a sk b e c ome s p ind l e-sha p e d . X 1 34 . l2b FBL , 2 4 hours p o s t-in f e c t ion showing
c lump ing o f rounded up c e l l s . X 500 . 1 3a Open type o f p l aqu e in LT mono l ayer s even
days p o s t- inocu l a t ion wi th i s o l a t e No . 1 0 ( s ec ond p l a que-pur i f i c a t ion) . X 1 34 . 1 3b C l o s e d type o f p l aque in LT mon o l ayer
s even day s a f t e r inocu l a t ion wi th i s o l a t e No . 1 2 P l l ( f i r s t p l aque-pu r i f i ca t ion) X 1 34 . 1 4 Un ino c u l a t e d con t rol LT mon o l ayer . May
Grunwa l d-Giems a , X 1 2 5 .
l 5 a LT , one hour pos t- ino cu l a t i on w i th No . l OP l O . May-Grunwa l d- G i ems a , X 1 2 5 .
1 5b LT , one hour po s t- inocu l a t ion a t h i gh e r magn i f i c a t i on . May-Grunwa l d-G i ems a , X 3 1 0 . 1 6 LT , three h ours a f te r ino cu l a t i on , showing
the appe aranc e of a f ew rounded up c e l l s . May-Grunwa l d-G i ems a , X 3 1 0 .
l 7 a LT , 6 hours p o s t- ino cu l a t i on . Note tha t mo s t o f the ce l l s hav e rounded up and
s ta ined mor e in t en s e l y than the contro l . May-Grunwa l d-G i ems a , X 1 25 .
viii
AFTER PAGE
46 46
46
46
47
47
55
55 55 55
55
l 7b H i gh e r magn i f i c a t ion o f the above . X 3 1 0 . 55 l 8 a LT , 24 h ours a f te r i n fe c t ion , showing
c lump ing o f rounde d u p c e l l s . May-Grunwa l d - 55 G i em s a , X 1 2 5 .
l 8b H ighe r magn i f i c a t i on o f the above . X 3 1 0 . 55 1 9
2 0
LT, 2 4 h our s a f t e r i n fe c t i on . I t can b e s een that s ome c e l l s contain p aranuc l e a r inc l u s i on b o d i e s s u rround e d b y a "ha l o"
May-Grunwal d-Giem s a, X 3 1 0 .
LT , 2 4 hours p . i . showing an enl arged c e l l (X) c on t a in ing s ev e r a l nuc l e i ( a sync y t ium?) May-Grunwa l d-Giems a , X 3 1 0.
55
55
Orf i s common among s he ep i n New Z e a l and and the i nc i dence in m a n appears to be incre a s ing . Th ere i s a v ac c i ne
avai la b l e for sheep . A l though the v a c c i ne prote c t s l amb s from the d i s e a s e i t perpe tu ate s the v iru s i n the·
e nv i ronment . There i s a n e ed for a l ternative vacci n a t ion m e t hod s .
I t i s the a im of thi s project to deve lop me thods o f i so l a t i ng , propagating a n d pur i f y i n g orf v i r u s i n c e l l
cul t ure an d ident i f y i n g i so l a t e s by re s tri c t ion endonuc l e a s e a na l y s i s i n preparation for v a c c i ne s tudi e s .
