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

DEDICATED TO MY PARENT,

BROTHERS AND SISTERS

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