UNIVERSITI PUTRA MALAYSIA
DETECTION AND DIFFERENTIATION OF MALAYSIAN NEWCASTLE DISEASE VIRUS ISOLATES BY
RNA-POLYMERASE CHAIN REACTION AND CYCLE SEQUENCING
NG BAN KIM
FSAS 1995 3
DETECTION AND D I FFERENTIAT I ON OF MALAY S IAN
NEWCAS TLE DISEASE VIRUS I SOLATES BY RNA-POLYMERASE CHAIN REACTION
AND CYCLE SEQUENCING
By
NG BAN KIM
Thesis Submitted in Fu l f i l ment of the Requirements for the Degree of Master of Science in the Faculty of
S cience and Environmental S tudies, Universiti Pertanian Malaysia
June 1995
ACKNOWLEDGEMENTS
I s incere l y t hank my s up e rv i s o ry c o mm i t te e m e mber s ,
As s o c . Pro f. Dr . Kh a t ijah Mohd . Yu s o f f ( ch a irman),
A s s oc . P r o f . Dr . A i n i I de r i s and As s oc . Pro f . Dr . N o r a n i
Abd. S am a d for t h e i r guidance and h e lp f u l d i s c u s s i o n a n d
advi c e throughout the c o u r s e o f my s tudy .
I am very much gra t e f u l t o Lau Chin Hoon f o r
t ea c h ing m e t h e cyc l e s equen c ing t echn ique, f o r h i s
e n c o uragement and thought f u l c omme n t s dur ing t h e
p r oc e s s o f r e s e ar c h .
I appr e c i a t e t h e a s s i s t a n c e of Adam Ahm ad i n
prep a r ing t h e c h i cken t i s s u e s and Abd Ghan i H a s h im f o r
exce llent photography .
Th i s study w a s s upporte d by I RPA Grant
No . 1-07-05-027 and TWAS Grant No . 93-074 RG/ B IO /A S .
i i i
TABLE OF CONTENTS
ACKNOWLEDGEMENTS
L I ST OF
L I ST OF
LIST OF
L I S T OF
ABSTRACT
AB STRAK
CHAPTER
I
II
TABLES
FIGURES
PLATES
ABB REVIAT IONS
INTRODUCTION
LI TERATURE REVIEW
Newc a s t l e Dis eas e Virus (NDV)
M orpho l ogy
Viru l e n c e Variat i o n
Molec u lar Marker o f Viru l en c e o f NDV
D i agnosis of Newcastle Disease
Polyme rase Chain Reac tion
D eveloping a Diagn o s t i c PCR A s s ay
Obtaining Nuc l e otid e S equen c e I n f o rmation
Primer D e s ign
Op timi s ation of pe R
iv
PAGE
iii
vii
viii
ix
x
xi
xiii
1
5
5
5
7
8
9
13
14
14
15
16
I I I
I V
Sampl e P repara t i on Method
peR Product D e t e c t i o n Methods
Qua l it y C o n t r o l of P C R
Cyc l e Sequenc i n g
MATERI ALS AND METHODS
V i r u s I s o l a t e s /St r a i n s and G r owth
C h i c ken T i s s u e Spe c imen
Labor a tory D e s i gn f o r PCR
Samp l e P r e p a r a t i on for RNA- peR from A l l ant o i c F l u i d
Samp l e P r e p a ra t i on f o r RNA- peR f rom T i s s ue
P r im e r s
RNA - p e R f o r A l l an t o i c F l u i d S am p l e s
RNA- PCR f o r T i s s u e Samp l e s
Sequenc ing o f RNA-PCR P roduct s
RESULTS
Amp l i f ic a t i on o f Haemagg l u t i n i n -
1 6
1 6
1 7
17
19
19
21
22
22
23
24
24
2 6
27
29
Neuram i n i da s e ( HN ) Gene Fragm e n t 2 9
Seque n c ing o f H N G e n e Amp l if i e d Fragm e n t s 30
RNA - pe R f o r T i s s ue Samp l e s 35
Opt i m i s a t i on of PCR 35
D e t ec t i on o f NDV i n V a r i o u s T i s s u e s 37
Amp l i f i c a t i on o f Fus i o n ( F ) Gene Fra gment from D i f f er e n t NDV Str a i n s o r I s o l a t e s 42
v
Sequencing of F Gene Amplified Fragments 42
v DISCUSSION
VI CONCLUSION
BIBLIOGRAPHY
APPENDICES
VII'2\.
LIST OF PUBLICATIONS/PAPERS PRESENTED
vi
48
54
55
59
63
64
LIST OF TABLES
Tabl e Page
1 Func t i on of NDV Coded P r o t e i n s 6
2 M a l ays i an Newc a s t l e D i s e a s e Vir u s I s o l a t e s U s ed In Th i s S tudy 20
3 P r imers U s e d f o r RNA - P C R a n d S e quenc ing 25
4 RNA-peR f o r D i f ferent Typ e s o f T i s s ue s 40
5 Nuc l e o t ide and Am i n o A c i d S eque n c e a t t h e F G e n e C l e avage S i t e o f Var io u s NDV str a i n s o f D i f f e r en t P at h otypes 47
v i i
LIST OF FIGURES
Figure
1 Partial HN Gene Sequence (mRNA sense) of Malaysian NDV Isolates and Vaccine Strain V4-UPM
2 Partial F Gene Nucleotide Sequence (mRNA sense) of NDV Strain AF2240
viii
Page
33
46
LI ST OF PLATES
P l a t e P a g e
1 RNA- P C R of H N G e n e o f D i f f erent NDV S t r a in s / I s o l a t e s 3 1
2 Auto r a d iograph o f a P o lyacry l a m ide/Urea G e l s howing S equence s o f HN Gene PCR-Amp l i f i e d DNAs f rom I s o l a t e s 4 8 4 ( a ) a n d 5 7 5 ( b ) 3 2
3 RNA - PCR o f F Gene o f NDV S t r a in AF2 2 4 0 I n f ect e d S p l e e n a t D i f f erent Cycl e Number 36
4 RNA- PCR of F Gene of NDV S t r a in AF224 0 I n f ect e d S p l e e n a t D i f f erent MgC12 C oncen t ra t i on s 3 8
5 RNA- PCR o f F Gene o f NDV S t r a in AF22 4 0 I n f ect ed S p l e e n a t D i f f e rent Ann e a l ing Temp e r a t u r e s 3 9
6 RNA- PCR o f F Gene o f I n f ect e d and Un i n f ect e d Sp l e en s 4 1
7 RNA- PCR o f F Gene o f D i f f e r ent NDV S t r a in s / I s o l a t e s 4 3
8 Aut oradi ograph o f a P o lyacryl am i d e /Urea G e l s howing S e quence o f F Gene PCR-Amp l i f i ed DNA f rom NDV S t r a in AF22 4 0 f r o m I n f ect ed S p l e en 4 4
ix
EID 5 0
ELISA
F
HA
HI
HN
ICPI
IVPI
L
Le
M
MDT
Me
NAP
ND
NDV
NP
peR
RNA- PCR
RNAs in
UPM
Ve
LIST O F ABBREVIATIONS
Egg i n f ect ive do s e 5 0%
Enzyme l inked immuno s orbent a s s ay
Fu s i on
Haemagg l u t i n a t i o n
Haemagg l u t i n a t i o n - inh ib i t i on
Haemagg l u t i n i n - n e ur am i n i d a s e
Int racerebr a l pathogenicity index
Int ravenous p a t h ogenicity index
Large
Len t ogenic
Membr ane
Mean death t im e
M e s o g e n ic
Nucleocap s id- a s s oc i a t e d pro t e in
Newca s t l e d i s e a s e
N e wca s t l e d i s e a s e v i r u s
Nuc l e ocap s id p r o t e in
P olym er a s e cha i n r e act i o n
RNA - p o l ymera s e cha i n r e act i on
RNas e inh i b i t o r
Un ive r s it i P e r t an i an M a l ay s i a
V e l ogenic
x
Abs tract of thes is submi t ted to the Senate 0 f Universiti Pertanian Malaysia in fulfilment of the requirements for the degree of Master of Science.
DETECTION AND DIFFERENTIATION OF MALAYSIAN NEWCASTLE
DISEASE VIRUS ISOLATES BY RNA-POLYMERASE CHAIN REACTION
AND CYCLE SEQUENCING
By
NG BAN KIM
June 1995
Chairman: Dr. Khatijah Mohd. Yusoff
Faculty: Science and Environmental Studies
This study was undertaken to develop diagnostic
tests for Newcastle disease virus (NDV) based on RNA-
polymerase chain reaction (RNA-PCR) and cycle sequencing
techniques as a supplement to the presently av ailable
te st s .
Two RNA-peR cycle sequencing systems each targeted
at the haemagglutinin-neuraminidase (HN) and fusion (F)
genes were developed. RNA-PCR amplification of a 398
base pairs (bp) HN gene fragment was performed on
total RNAs extracted from infected allantoic fluid of 9
Malaysian NDV field isolates, vaccine strain V4-UPM and
velogenic strain AF2240. Sequence analysis over 113
bases in the amplified fragment showed variations among
these isolates/strains. However, identical sequences
were obtained from some of the field isolates within a
particular pathotype and these were thought to be of the
same strain of NDV.
xi
RNA-peR amplification of a 242 bp F gene fragment
was performed on total RNAs which were isolated from
several types of tissues (spleen, brain and lung) from
six non-vaccinated chickens which were challenged with
the velogenic strain AF2240. The spleen was found to
have the highest number of samples positive by PCR.
Spleens from uninfected chickens as well as those from
vaccinated and challenged chickens which were
slaughtered were all PCR negatives. The challenged virus
was thought to have been neutralised by the antibodies
produced by the chickens. The identity of the PCR
products amplified from the spleens w'ere confirmed by
cycle sequencing. Similarly, this test was also
performed on RNAs from infected allantoic fluid of 11
different NDV strains/isolates. These samples were also
PCR positives.
The 18 nucleotide at the cleavage site of the F
gene amplified fragment was sequenced for 4 NDV strains.
The deduced amino acid sequences for strains AF2240
(velogenic) and S (mesogenic) were 112Arg-Arg-Gln-
Arg/Lys-Arg-Phel17 and 112GlY-Arg/Lys-Gln-G1Y-Arg-Leul17
for strains F (lentogenic) and V4-UPM (avirulent).
Therefore, the velogenic and mesogenic strains could be
distinguished from the lentogenic and avirulent strains
by sequencing the cleavage site.
xii
Abstrak tesis yang dikemukakan kepada Senat Universiti Pertanian Malaysia untuk rnernenuhi keperluan Ijazah Master Sains.
PENGESANAN DAN PEMBEZAAN ISOLAT VIRUS NEWCASTLE DISEASE
MALAYSIA MELALUI TINDAKBALAS RANTAI POLIMERASE-RNA DAN
PENJUJUKAN KITARAN
Oleh
NG BAN KIM
Jun 1995
Pengerusi: Dr. Khatijah Mohd. Yusoff
Fakulti: Sains dan Pengajian Alarn Sekitar
Kajian ini telah dijalankan untuk rnenghasilkan
ujian diagnosis bagi virus Newcastle disease (NDV)
berdasarkan teknik tindakbalas rantai polirnerase-RNA
(RNA-peR) dan penjujukan kitaran sebagai tambahan kepada
ujian yang sedia ada.
Dua sistern RNA-peR penjujukan kitaran setiap satu
ditujukan pada gen hemaglutinin-neuraminidase (HN) dan
pertaupan (F) telah dihasilkan. Amplifikasi rnelalui
RNA-peR serpihan gen HN yang bersaiz 398 pasangan bes
(bp) dijalankan ke atas RNA total yang diekstrak
daripada cecair alantoik yang ada jangkitan dari 9
isolat NDV �lalaysia, strain vaksin V4-UPM dan strain
velogenik AF2240. Analisis jujukan terhadap 113 bes
dalam serpihan yang diamplifikasi menunjukkan variasi di
antara isolat/strain. Bagaimanapun, jujukan yang
identikal didapati pada sesetengah isolat dari patotaip
tertentu dan difikirkan adAlah strain NDV yang sarna.
xiii
Amplifikasi melalui RNA-peR serpihan gen F yang
bersaiz 242 bp dijalankan ke at as RNA total yang
diasingkan daripada beberapa jenis tisu (limpa, otak dan
paru-paru) dari enam ayam tanpa diberi vaksin yang
dicabar dengan strain velogenik AF2240 . Limpa didapati
rnernpunyai bilangan sampel yang p e R positif yang
tertinggi.
limpa dari
Limpa dari ayam yang tanpa j angki tan serta
ayam yang disembelih setelah diberi vaksin
dan dicabar didapati
Virus pencabar difikirkan
peR negatif kesemuanya.
telah dineutralkan oleh
antibodi yang
produk peR
dihasilkan oleh ayam tersebut. Identiti
yang diamplifikasi dari limpa disahkan
ini juga telah melalui penjujukan kitaran. Ujian
dilakukan ke atas RNA daripada cecair alantoik yang ada
jangkitan dari 11 strain/isolat NDV yang berlainan.
Kesemua sampel tersebut juga didapati peR positif.
18 nukleotida pada tapak pemotongan gen F telah
dijujuk untuk 4 strain NOV. Jujukan asid amino yang
ditentukan untuk strain AF2240 (velogenik) dan S
(mesogenik) adalah 112Arg-Arg-Gln-Arg/Lys-Arg-Phe117 dan
untuk strain F (lentogenik) dan V4-UPM (avirulen) adalah
112Gly-Arg/Lys-Gln-Gly-Arg-Leul17. Oleh itu, strain
velogenik
lentogenik
dan mesogenik
dan avirulen
pemotongan tersebut.
dapat dibezakan dari
dengan menjujuk
xiv
strain
tapak
CHAPTER I
I NTRODUCTION
Newc a s t l e d i s e a s e v i r u s ( NDV ) , a m ember o f t h e
Paramyxov_iridae f am i l y , i s an e c o n om i c a l ly i mp o r t a n t
p athogen o f pou l t ry . The v i r a l g e n o m e c on s i s t s o f a
n e g a t ive s e n s e s in g l e - s t r anded RNA m o l e c u l e o f
approx imat e ly 15 k i l ob a s e ( kb ) i n l ength . The s ix gene
produ c t s encoded by the genome a r e t h e nuc l e opro t e i n
( NP ) , pho sphopr o t e i n (P ) , m a t r i x ( M ) p r o t e i n , fu s i on ( F )
pro t e in , haemagg l u t i n in -n euram i n i da s e ( HN ) pro t e in and
t h e l arge ( L ) p r o t e i n ( S ams on, 1 9 8 8 ) .
NDV s t r a i n s a r e known t o d i s p l ay var i a t ions i n many
b i o l og i c a l propert i e s i n c lud ing p a thogen i c i t y t owards
t h e i r ho s t s . The s e s t r a ins a r e be ing c la s s i f ie d into
three ma j or groups depending on the s ever i ty of the
d i s e a s e in s u s cept i ble c h i c kens . S t r a i n s c a u s ing h i gh
mort a l i ty a r e t e rmed ve l ogen i c . The m e s ogen i c s t r a i n s
a r e thos e t h a t c a u s e u p t o 50% m o rt a l i t y i n young and
s u s c ep t i b l e c h i c k e n s and s er i ou s l y d e c r e a s ing egg
produc t i on . S t r a in s that produce m i l d d i s e a s e in
i n f e c t e d ho s t s a r e known as l e n t ogen i c . I n addi t io n
1
t o t hese t h ree groups, some s t r ains c ause no disease
a t all and a re cl assi fied as avirulen t. The s t r ains
vari ations
di fferences
in
in
virulence a re
susceptibilities
prim arily
o f t he Fo
due to
p recursor
protein to c leavage by host ce ll proteases into its
a c tive form. Recent studies h a ve revealed a re l a tionship
bet ween s t r ain
composition at
p athogenic i t y and
t he c leavage site o f
the
t he
amino acid
F 0 protein.
The amino a cid sequence at t he cle av a ge si te is
A r g-Ar g-Gln-Ar g(Lys)-Arg-Phe for velogeni c and mesogeni c
str ains and Gly-Ar g(Lys)-Gln-G ly-Arg-Leu for lentogeni c
and avirulent s t r ains (A lexander, 1990b). Thus , t he
velogenic and mesogenic str ains could be distinguished
from t he len togenic and avirulent s t r ains by sequencing
t he cleav a ge s i te.
Newcast le disease (ND) c an c a use gre a t economic
losses to t he pou l try indus try. It is t hus impor t an t to
h ave met hods whi c h c an p rovide r apid and spe c i fic
diagnosis in order to prevent and control the disease.
Clinical signs typical of ND produ ced by a b ird is not a
definitive proo f t h a t t he a gent responsible for t he
disease is NDV. There is a possibilit y t h a t t he
in fec ting NDV is o f an aviru lent or vac cine strain w hi c h
i s asymptom a ti c and the observed c lini c al symp toms
simi l ar to ND are in f a c t c a used by o t her p a thogens
(A lexander, 1988). There fore , birds showing c linic a l
3
s igns o f ND s h o u l d b e d i agno s ed t o demo n s tra t e the
p r e s e n c e of R v i ru l en t NDV s tr a in .
The d e t e c t i on and p at h o typing o f NDV i s o l at e s i s
r out i n e l y c a r r i e d o u t t hrough a t im e c on s um ing p r o c es s
t h a t i nvo lve s i s o l a t i on , ident i f i c a t i on and v i r u l e n c e
charac t e r i s a t i on by o n e o r mor e o f the f o l l ow ing
b i o l og i c a l t e s t s: m e a n d e a t h t im e (MDT ) , i n t r a c e r ebr a l
p athoge n i c ity index (IC PI) and intravenous p at hogen i c i ty
index (IVPI) (Al exander, 1988) . In r e c ent y e a r s ,
a l t ernat ive m e t h o d s b a s e d on ant i -pept ide a n t ibo d i e s
(Hodder e t a l . , 1993) or
a l . , 1992;
o l i gonu c l e o t ide
J a re c k i-B l a c k and
prob e s
K ing, ( J a r ec k i - B l a c k e t
1993) have
i n f e c t ions ,
b e en deve l oped.
further t e s t s
Apa r t f r o m c o n f i rm in g NDV
for d i s t ingu i s h ing v i r u s
s t r a i n s are a l s o imp o r t ant i n t r a c ing t h e o r i g i n o f a
part i c u l a r str a i n f r o m an i nf e c t ed f lo c k . For t h i s
purpo s e,
ant ibo d i e s
d i a gn o s t i c proc edur e s
(Sr i n i v a s appa e t a l . ,
u s ing monoc l ona l
1986; Erde i e t a l . ,
1987) and o l igonu c l e o t ide f ingerpr i n t ing (McMillan and
Hans on , 1 9 8 2 ) have b e e n deve l op e d .
The po lym e r a s e c h a i n r e a c t i o n (peR) t ec hn ique w h i c h
i s spec i f i c , r a p i d a n d s en s i t ive h a s b e e n u s ed f o r t h e
d e te c t ion o f m a ny vet e r i nary imp o r t ant v i ru s e s
inc l uding NDV . Je s t in and Je s t in (1991) have deve l oped a
P C R t e s t wh i c h c o u l d s p ec i f i c a l ly d e t e c t NDV i s o l at e s
f rom t h e i n f e c t e d a l l an t o i c f lu id o f embryona t e d c h i c k e n
eggs . The t arget s equence for amp l i fication was l ocat ed
wi t h in the F gene.
I n a prev i ou s s tu dy , t he aut h o r had devel oped an
RNA - po lymeras e chain reac t i on ( RNA- peR ) as say bas ed on
t he HN gene f o r detec t ing NDV in i n fec ted a l l ant o i c
f lu i d ( Ng , 1993). Th i s RNA- peR as say was u sed t o amp l i fy
a s egment o f the HN gene f r om s everal reference NDV
s t ra in s . I f po s i t ive res u l t s were obtained for al l t he
reference NDV s t rains , t he as s ay s h o u l d a l s o be
appl i cable t o the l ocal NDV i s o lates . A l s o , d i agn o s i s o f
NDV direc tly f rom c h i c ken t i s s ue w o u l d be fas ter t han
t hat from a l l ant o i c f lu i d
required. Th i s projec t was
f o l lowing objec t ives :
as v ir u s growing i s not
thus carr ied out w i th the
1. to s t udy t he feas i b i l i ty o f us i ng the HN gene RNA- peR
assay t o detec t Malay s ian f ie l d i s o l ates o f NDV ;
2. to d i fferen t iate between var i o u s l ocal NDV i s o lates
by sequencing the HN gene peR amplified-DNAs; and
3. to develop an RNA-peR cycle s equencing system based
o n the F gene f o r t he rap i d detec t i on and patho t yp i ng
o f NDV d i rec t ly from i n fect ed ch i c ken t i s s ues .
CHAPTER I I
L ITERATURE REVI EW
Newcastl e Disease Virus ( NDV )
NDV i s t h e agent c aus ing ND in av i an s pec ies .
The v i rus a l s o c a l led a v i an pa r amyxovirus type I,
is a member o f t h e ParamyxDvirus genus from t he
ParamyxDviridae f am i ly . To date , m any NDV i s o l a tes w i th
w i d e var i a t i ons in b i o l ogi c a l proper t i e s have b e en
i dent i f i e d wo rldw i de .
Morpho l ogy
NDV i s a l arge pl e omorph i c envel oped v i r u s wh i c h
varies i n s i ze from 1 50 t o 4 00 nm . It s genome c ons i s t s
o f a s ing l e
approx imat e l y 1 5
s tr anded
k i l oba s e
nega t i ve
( kb ) in
po l a r ity
length . The
RNA o f
genome
encodes s ix s truct ur a l pro te ins namely : nuc leoc apsid
( NP ) pro tein , pho s phopr o t e in ( P ) , l a rge (L) protein,
mat r i x (M) pro t e in, fus i on ( F ) protein and
h aemagglut inin-neu r am inida s e ( HN ) protein . The func t i on
o f each o f the pro tein i s summ a r i s ed in Table 1.
5
Table 1
Function of NDV Coded Proteins
Protein
nucleocapsid
phosphoprotein
large
matrix
fusion
haemagglutininneuraminidase
Abbrev.
(NP)
(P)
(L)
(Fl
(HN)
(Source: Samson, 1988).
Function
major structural component of nucleocapsid: complexed with RNA genome
associated with nucleocapsid, role in transcription / replication.
RNA directed RNA polymerase.
virus assembly organiser, moderates transcription.
fusion of virus and host membrane.
attachment to cellular receptor and receptor destroying activity.
The F and HN proteins are synthesised as precursor
proteins and are cleaved by host cell proteases to form
functional proteins (Samson, 1988).
Virulence variation
NDV strains can cause distinct clinical signs and
different degrees of disease severity in susceptible
chickens. Based on this biological characteristic NDV
strains have been grouped into three pathotypes:
1. the viscerotropic-velogenic strains which cause
either haemorrhagic lesions in the intestinal tract
or neurological and respiratory signs with high
mortality;
2. the mesogenic strains which cause respiratory and
sometimes nervous infection with low mortality; and
3. the lentogenic strains which cause mild or inapparent
respiratory infection.
In addition to these three pathotypes, some strains
cause inapparent enteric infection and are classified as
avirulent. This classification, however, is merely a
guide because there is always some degree of overlap in
the clinical signs produced (Alexander, 1990a).
Molecular Marker of Virulence of NDV
The variation in virulence among NDV strains
has been correlated in part with the variations in
susceptibilities of the F 0 precursor protein to
proteolytic cleavage to form an active protein (Nagai
et al., 1976; 1979). The Fo protein of velogenic and
mesogenic strains is readily cleaved by proteases of a
wide range of host cells while the Fo protein cleavage
does not occur in most cell types infected with
lentogenic and avirulent strains. Another protein which
also plays a role in determinating the virulence of NDV
strains is the HN protein. There are three different
sizes of primary translation HN polypeptides: 616, 577,
or 5 7 1 amino acids depending on the position of the stop
codons (Sakaguchi et al. t 1 9 8 9 ). Both the smaller HN
polypeptides are synthesised by either velogenic,
mesogenic or lentogenic NDV strains and are already in
their active forms. In contrast, the 616 amino acid HN
polypeptide is found only in avir ulent strains and the
extended amino acids 11 ave to be removed for it to become
biologically active. Since the F 0 protein of velogenic
and mesogenic strains can be activated in a wide range
of host cells and their HN protein is already
synthesised in their active form, these strains are able
to spn�ad through the host and cause a more severe
disease than the lentogenic and avirulent strains.
Rec e nt nucle otide seq ue ncing studie s co ver ing
NDV s t rains o f all pat hotypes have re ve a l e d a
re l ati ons hi p bet w e e n viru l ence var i at i ons and t he
am i no ac i d c om po s i t i o n at the c l e avage s i te o f the
pr o t e i n. The am i no aci d s e quenc e at the
c l e avag e s ite o f ve l o g e ni c and m e s ogeni c s tr ains i s
Arg - Arg - G l n- Arg ( Ly s ) - Ar g - P he w hi l e f o r l e nto geni c and
avi rul e nt s trains the s e que nc e at that site i s
G ly - Arg ( Ly s ) - G l n-Gl y - Arg - L e u ( A l exander, 1990 b ) . Thus,
the var i ati on i n am i no aci d com po s i t i o n at the c l e avage
s ite m ay be use d as a m arke r to di s t i ngui s h t he
ve l o genic and m e s o g e nic s trains f rom the l e nto genic and
avirul e nt s tr ai ns . The avi rul ent s tr a i ns can be f urthe r
dis ti ngui s he d f rom the l e nto gen i c strai ns by dete rm i n i ng
the po s i t i o n o f s t o p c o do n i n the HN g e ne .
Diagnosis of Newcastle Disease
The une quivo c al m e t ho d o f di agnosing ND i nvo l ve s a
rather complex procedure which consists of three s tage s .
Ini tial l y, the virus i s i s o l at e d from sus pe c t ed c lini c a l
s pe c i m e n i n e ither c e 11 cultures o r m o r e comm o nly i n
embr yonat e d chi c ke n e gg s . Then. al l anto i c f l u i d o bt aine d
f r o m the s e e g g s betw e e n 5 to 7 days po s t i no cul ati o n i s
te s t e d f or haem ag l utinat i o n ( HA) ac ti v i t y w i th chi c ke n
red b l oo d c e l l s . H A pos i t i ve s am pl e s i ndic ate the
10
p r e s en c e o f NDV o r one o f t h e o t h e r avi a n
p a r amyxovi rus e s o r i n f luenza vi ru s . HA negat i ve s amp l e s
a r e p a s s a ged f o r a t l e a s t one more t im e . Is o l a t i on o f
NDV i s s ub s equent ly c o n f i rmed b y a h a emagglut in a t i on
inhib i t i o n ( HI ) t e s t us ing known p o s i t i ve a n t i s e r um .
However , o t h e r avi an p a ramyxovirus s er o t yp e s
p a r t i c u l a r ly v i r us e s o f PMV - 3 s e r o type may show s ome
i nh i b i t i o n w i t h NDV ant i s er um . Mono c lo n a l ant i b od i e s
d i r e c t e d aga in s t h ighly c on s erved ep i to p e s o f NDV
ant igens have b e e n produced by s ever a l r e s e a r c h groups
( Rus s e l l and A l exander , 1 983 ; Is h i da e t a l . , 1 98 5 ;
M e u l emans e t a I . , 1 987 ; Lana e t a I . , 1 988 ) t o r ep l a c e
the ant i s erum for c o n f irming NDV i s o l a t i on . F i n a l l y,
t h e v i r u l e n c e o f t h e i s o l at e d NDV s t r a in i s d e termined
by one o r m o r e of the f o l l ow ing b i o l og i c a l t e s t s :
mean death t ime ( MDT) , i n t r a c erebr a l p a thogen i c i ty index
( IePI) and i n t r aveno u s p athogen i c i ty index ( IV PI ) . The s e
are b i o l og i c a l t e s t s wh i ch me a s u r e t h e s e r i o u s ne s s o f
the d i s ea s e c a u s e d by the i s o l a t ed NDV s tr a i n t o
embryonated c h i c k en e ggs o r c h i c kens . NDV s t r a i n s a re
c l a s s i f i ed i n t o t h r e e major pathotype s : ve l ogeni c ,
me s ogen i c and l en t o ge n i c b a s e d on c a l c u l a t e d index va l u e
( Al exander , 1 988 ) .
S e r o l o g i c a l t e s t s f o r NDV wh i c h may b e u s e d t o
de t e c t the virus o r t o mon i t o r immune r e s p on s e i n
v a c c i n a t i o n a r e a l s o ava i l ab l e and t h e s e i n c l ud e virus
neutra 1 i z a t i on , s ing l e r a d i a l immunodi f f u s i on and agar
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