Diagnostic Virology Protocols

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iagnos tic V iro log y P rotocols


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M E T H O D S IN M O L E C U L R M E D I C I N E T

J o h n M . W alker S~RIES

E ITOR

29. DNA Vaccines: Methods and Protocols edited by Douglas B. Lowrie and Robert Whalen 1999

28. Cytotoxic Drug Resistance Mechanisms edited by Robert Brown and Uta BOger-Brown 1999

27. Clinical Applicat ions of Capillary Elec trophoresi s edited by Stephe n M. Palfrey 1999

26. Quanti tative PCR Protocols edited by Bernd Kochanowski and and Udo Reischl 1999

25. Drug Target ing edited by G. E. Francis and Cristina Delgado 1999

24. Antiviral Chemotherapy Protocols edited by Derek Kinchington and Raymond F. SchinazL 1999

23. Peptidomimet ics Protocols edited by Wieslaw M. Kazmierski 1999

22. Neurodegeneration Methods and Protocols edited by Jean Harry and Hugh A. Tilson 1999

21. Adenovirus Methods and Protocols edited by William S. M. Wold 1998

20. Sexually ransmitted DiseasesProtocols edited by Rosanna Peeling and P. Frederick Sparling 1999

19. Hepatiti s C Protocols edited by Johnson Y. N. Lau 1998

18. Tissue Engineering Methods and Protocols edited by Jeffrey R. Morgan

and Martin Yarmush 1999

17. HIV Protocols edited by Nelson Michael and Jerome H. Kim 1999

16. Clinical Applica tions of PCR edited by E M. Dennis Lo 1998

15. Molecular Bacteriology: Protocols and Clinical Applications edited by Neil WoodJord

and Atan P. Johnson 1998

14. Tumor Marker Protocols edited by Margaret Hanausek and Zbigniew Walaszek J998

13. Molecular Diagnosis of Infectious Diseases edited by Udo Reischl 1998

12. Diagnostic Virology Protocols edited by John R. Stephenson and Alan Warnes 1998

11. Therapeut ic Applications of Rihozymes edited by Kevin J Scanlon t 998

10. Herpes Simplex Virus Protocols edited by S. Moira Brown andAlasdair R. MacLean 1998

9. Lectin Methods and Protocols edited by Jonathan M. Rhodes and Jeremy D. Milton 1998

8. Helicobacter pylori Protocols edited by Christopher L. Clayton and Harry L. 72 Mobley 1997

7. Gene Therapy Protocols edited by Paul D. Robbins 1997

6. Molecular Diagnosis of Cancer edited by Finbarr E. Cotter 1996

5. Molecular Diagnosis of Genetic Diseases edited by Rob Elles 1996

4. Vaccine Protocols edited by Andrew Robinson Graham H. Farrar and Christopher N. Wiblin 1996

3. Priori Diseases edited by Harry F. Baker and Rosalind M. Ridley t 996

2 Human Cell Culture Protocols edited by Gareth E. Jones 1996

1. Antisense Therapeuties edited by Sudhir Agrawal 1996


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iag n o stic V iro lo g y

Protocols

dited by

J o h n R S t e p h e n s o n

Lond on S choo l o f Hyg iene and Tropical Medicine

University o f London UK

nd

A l a n W a r n e s

M icron Bioproducts Cam berley Surrey UK

Humana Press ~ Totowa New Jersey


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r e f a c e

T h e a c c u r a t e a n d r e l ia b l e d i a g n o s i s o f t r a n s m i s s i b l e d i s e a s e s is t h e m o s t

p o w e r f u l w e a p o n a v a i l a b l e t o e n s u r e t h e i r c o n t r o l , a n d i n s o m e c a s e s

e r a d ic a t io n . T h e d e t e c t io n o f p a r a si te s in c l in i c al c a s e s, c o m p a n i o n a n d f a r m

a n i m a l s, a n d i n t h e e n v i r o n m e n t is r e la t iv e l y e a s y s i n c e m a n y o f t h e m a re

v i s i b l e t o t h e n a k e d e y e , a n d t h o s e t h a t a r e n o t a r e r e a d i l y d e t e c t e d b y l i g h t

m i c r o s c o p y . F u n g a l i n f e c t i o n s c a n s i m i l a r ly b e d e t e r m i n e d . B a c t e r i a a r e

s o m e w h a t h a r d e r to d e te c t. A l t h o u g h t h e i r p r e s e n c e c a n f r e q u e n t l y b e d e t e c t e d

b y l ig h t m i c r o s c o p y , d i ff e r e n ti a l d ia g n o s i s , b e y o n d t h e i r g r o s s m o r p h o l o g y , is

a l m o s t a l w a y s i m p o s s i b l e . H o w e v e r , m o s t b a c te r ia l p a t h o g e n s c a n b e c u l t u r e d

i n t h e l a b o r a t o r y a n d c a n b e a c c u r a t e l y i d e n t i f i e d b y c o m b i n a t i o n s o f a se r i es

o f s i m p l e t e st s s u c h a s m o r p h o l o g y , s ta i n in g , a n t i b io t ic s e n s i ti v it y , b i o c h e m i c a l

a n a l y s e s , n u t r i e n t d e p e n d e n c e , a n d p h a g e s e n s it iv i ty .

V i r u s e s , h o w e v e r , h a v e p r o v e d m u c h m o r e d i f f ic u l t ; t h e i r s iz e a n d

a b s o l u t e d e p e n d e n c e o f th e h o s t c e ll f o r p r o p a g a t i o n h a v e r e n d e r e d u s e l e s s th e

m e t h o d s t r a d i t i o n a l l y u s e d f o r o t h e r m i c r o o r g a n i s m s . U n t i l t h e d e v e l o p m e n t

o f ti s s u e c u l t u r e in th e m i d d l e o f th i s c e n t u r y , d ia g n o s i s w a s e n t i r e l y d e p e n d e n t

o n t h e s k i ll a n d e x p e r i e n c e o f t h e c l in i c i a n . B u t t h is w a s a n u n r e l i a b l e p r o c e s s

s i n c e m a n y o f th e c o m m o n v i r u s in f e c t io n s e x h ib i t s i m i l a r c l in i c a l s y m p t o m s ,

s u c h a s c o ry z a , e x a n t h e m a , v o m i t i n g , d i a r rh e a , n e u r a l g i a , a n d l e t h a r g y . I n d e e d

m a n y v i r a l i n f e c t i o n s d i s p l a y c l i n i c a l s i g n s t h a t a r e i n d i s t i n g u i s h a b l e f r o m

b a c t e r i a l o r p a r a s i t i c i n f e c t i o n s . I n a f e w c a s e s a n e x p e r i e n c e d p a t h o l o g i s t

c o u l d d e t e c t v i ra l i n f e c ti o n s w h e r e m a c r o s c o p i c i n c l u s i o n b o d i e s w e r e p r e s e n t

o r w h e r e i n f e c t e d ce ll s d i s p l a y e d a d is t in c t iv e m o r p h o l o g y e .g . , m e a s l e s g i a n t

c e il p n e u m o n i a ) .

W i t h t h e i n t r o d u c t i o n o f e l e c t r o n m i c r o s c o p y i n t h e 1 9 6 0 s, t h e d i r e c t

v i s u a l i z a ti o n o f v i ru s p a r ti c le s i n c l in i c a l s p e c i m e n s b e c a m e p o s s i b l e . H o w e v e r ,

e v e n w i th t h e i n c r e a s i n g s o p h i s t i c a t io n o f e le c t r o n m i c r o s c o p y , o n l y t h o s e

v i r u s e s w i t h d e f i n e d a n d r o b u s t s tr u c tu r e s e .g ., a d e n o v i r u s e s , p o l i o v i r u s e s ,

h e r p e s v i r u s , a n d r o t a v i r u s e s ) c o u l d b e r e l ia b l y o b s e r v e d . D i a g n o s i s b y e l e c t r o n

m i c r o s c o p y o f d is e a s e s c a u s e d b y o t h e r v i r us e s , e s p e c i a ll y e n v e l o p e d v i r u s e s

s u c h a s i n f l u e n z a v i r u s , m e a s l e s v i r u s , a n d y e l l o w f e v e r v i r u s h a s n o t b e e n

p o s s i b l e . T h e o n l y n o ta b l e e x c e p t i o n s o c c u r i n c a s e s w h e r e i n f e c t e d c e ll s

c o n t a i n c l e a r l y i d e n t i f i a b l e i n c l u s i o n b o d i e s o r v i r u s - r e l a t e d s t r u c t u r e s , s u c h


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vi reface

as t he d i s t i nc t ive nuc l eocap s ids s ee n in mea s l e s - in fe c t ed ce l ls . I n sp i te o f i ts

l im i t a t io n s , e l e c t r o n m i c r o s c o p y h a s b e e n t h e o n l y r e l i ab l e d i a g n o s t i c t o o l f o r

s u c h n o n c u l t u r a b l e v i ru s e s a s a s tr o v i r u s e s a n d N o r w a l k - l i k e a g e n t s .

B e c a u s e d i r e c t v ir u s c u l t u r e f r o m s u c h e a s i l y o b t a i n a b l e c l i n i c a l m a t e r i a l

a s s e rum, s a l i va , o r u r ine i s nea r ly a lways t oo un re l i ab l e fo r rou t ine use , t he

m a j o r i t y o f v ir a l d i a g n o s e s h a v e r e s te d o n t h e d e t e c t i o n o f a s p e c i f i c i m m u n e

response . A l though se ro logy i s i n mos t ca ses s a t i s f ac to ry , i t i s nea r ly a lways

u s e f u l o n l y f o r r e t r o s p e c t i v e a n a l y s e s a n d f o r m o n i t o r i n g v i r u s s p r e a d i n

p o p u l a t io n s . S p e c i f i c i m m u n e r e s p o n s e s c a n o n l y b e d e t e c t e d s o m e t i m e a f te r

the i n i t ia l v i rus i n f ec t ion and thus s e rod iag nos i s do es no t no rm a l ly bene f i t t he

p a t ie n t . S u c h a s s a y s h a v e t r a d i ti o n a l l y d e p e n d e d o n d e t e c t i n g a n t i b o d i e s t h a t

i n h i b i t b i o l o g i c a l f u n c t i o n i n g i n t h e v i ru s a n d i n c l u d e p l a q u e r e d u c t i o n

n e u t r a l i z a t i o n t es ts P R N T ) , h e m a g g l u t i n i n i n h i b i t io n a s s a y s H I ) , a n d

c o m p l e m e n t fi x a ti o n C F ) a s sa y s . T h e s e a s s a y s c a n b e s p e c i f ic a n d re l ia b l e in

the hand s o f an expe r i enced l abo ra to ry worke r , bu t t hey a r e exp ens ive , l abo r ious

a n d , i n t h e c a s e o f s u c h h u m a n p a t h o g e n s a s y e l l o w f e v e r v i r u s a n d r a b i e s

v i rus , po t en t i a l l y dange rous , r equ i r ing soph i s t i ca t ed con ta inmen t f ac i l i t i e s .

S e v e r a l t e c h n o l o g i c a l i n n o v a t i o n s , d e v e l o p e d o v e r t h e l a s t d e c a d e o r s o ,

a r e no w com ing toge the r t o r evo lu t ion i ze d i agnos ti c v i ro logy . So l id -phase a s says

s u c h a s R I A r a d i o im m u n e a s sa y ), E L I S A e n z y m e - l in k e d i m m u n o s o r b a n t

a s s a y ), a n d l a t e x - a g g l u t i n a ti o n te c h n o l o g y , h a v e b e c o m e i n c r e a s i n g l y p o p u l a r

s ince they use l e s s ma te r ia l and can be r ead i ly and cheap ly adap ted to au tom a ted

l a b o r a to r y p r o t o c o ls . T h e e x p l o s i v e d e v e l o p m e n t in c o m p u t e r t e c h n o l o g y a n d

p o w e r f u l s o f t w a r e , c o m b i n e d w i t h t h e r a p i d f a l l i n c o s t , b u t i n c r e a s i n g

s o p h i s t ic a t i o n a n d a c c e p t a b i l it y o f r o b o t i c a n a l y z e r s , i s r a d i c a l ly c h a n g i n g t h e

n a t u r e o f t h e h o s p it a l p a t h o l o g y l a b o r a to r y . I n a d d i ti o n t o d e v e l o p m e n t s i n

a n a l y t i c a l t e c h n o l o g y , th e n a t u r e o f t h e b i o l o g i c a l r e a g e n t s a v a i l a b l e t o t h e

c l i n ic a l p a t h o lo g i s t h a s i m p r o v e d d r a m a t i c a ll y . S y n t h e t i c p e p t id e s c a n b e m a d e

tha t a r e pu re , i nexpens ive , and h igh ly spec i f i c an t igens , r ead i ly adap tab l e t o

au to m a ted l abo ra to ry sys t em s . Fo r v i r a l an t igens t ha t a r e unsu i t ed t o syn the t i c

p e p t id e c h e m i s t r y , a w i d e v a r i et y o f r e c o m b i n a n t D N A t e c h n o l o g i e s a r e

a v a i l a b l e t h a t c a n m a k e a n y v i r a l a n t ig e n , o r f r a g m e n t t h e r e o f , in h i g h y i e l d s

and to an accep tab l e s t anda rd o r pu r i t y . These two t ech no log ie s , t aken toge the r ,

can r em ov e one o f t he ma jo r i nh ib i t i ons on v i r a l d i agnos is : t ha t i s, t he d i f f i cu l ty

o f p r o v i d i n g r e l i a b l e b a tc h e s o f h ig h - q u a l i t y d i a g n o s t i c r e a g e n t s . M o r e o v e r ,

t h e r e is n o n e e d n o w t o g r o w l a r g e a m o u n t s o f d a n g e r o u s p a t h o g e n s f o r t h e

p r o d u c t i o n o f a n a l y t ic a l re a g e n t s . N o t o n l y h a s t h e d e s i g n a n d p r o d u c t i o n o f

v i r a l a n ti g e n s b e e n d r a m a t ic , b u t t h e a d v e n t o f m o n o c l o n a l a n t i b o d i e s h a s a ls o

m a r k e d l y i m p r o v e d t h e s e n s it iv i ty a n d r e li a b il it y o f d e t e c t io n s y s t e m s e m p l o y e d

b y E L I S A a s s a y s a n d s i m i l a r t e c h n o l o g i e s . T h u s i t is n o w p o s s i b l e t o a n a l y z e


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r e f a c e v fi

the immune response in an individual to give details of each viral protein the

immune response is raised against and also provide information on the about

the nature of the antibodies involved. This analysis can be done in hours and

not days and can be automated and performed in a laboratory requiring only

low levels o f biological containment.

In the past virus diagnosis has been less valuable than similar tests for

bacterial and parasitic infections since the technology has been too slow to

directly affect the treatment of the patient from whom the sample was taken.

The invention and rapid evolution of PCR technology has for the first time

enabled pathologists to consider viral diagnosis to affect treatment. PCR is so

sensitive that with the appropriate carefully designed controls it can detect

and ident ify viral genomes in the early stages of infection. Furthermore the

product of the PCR reaction can have its entire nucteotide sequence determined

at later date for the unequivocal confirmation of the diagnosis. Viral antigens

can also be detected using specific monoclonal antibodies bound to a var iety

of conjugates although this technology is significantly less sensitive than PCR.

Such accurate information early in a viral infection can determine whether

one of the increasing number of antiviral drugs should be used and the period

of treatment minimized to avoid unnecesary side effects. In addition the

unnecessary use of antibiotics can be avoided i fa viral infection can be reliably

identified. The inappropriate use of antibiotics is a matter of increasing concern

since overprescription has been implicated in the rise of antibiotic-resistant

bacteria.

Viral diagnosis is therefore entering a new and exciting era in which

many new technologies are combining to enable both the researcher and the

clinician to use rapid accurate sensitive and robust analyses. In

Diagnostic

Virology Protocols

we have brought together well-tried diagnostic protocols

that use at least one of the modern technologies now available. Chapters

covering all the major groups of human viral pathogens have been included

as well as those introducing and assessing the utility a number of modern

technologies. Viruses causing diseases of veterinary importance have not

been included but in many cases protocols for similar viruses causing human

diseases should be easily adaptable. We hope the information provided in

Diagnostic Virology Protocols

will be of equal value to researchers and

clinician alike and be usable by both experienced workers and those just

entering the field.

John R Stephenson

l a n W a r n e s


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onten ts

P re fa ce . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . v

Contr ibutors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

x i

1 R a p i d D e te c t io n o f A d e n o v i r u s fr o m F e c a l S p e c i m e n s

T a n v i r T a b is h , A la n W a rn e s, a n d S tu a r t C la r k

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 A I p h a v i r u s e s

Jo hn 7 . Roehr ig, Teresa M. Brown, A l ison J. John son, N ick

Karabatsos, Denise A. Martin, Ca rl J. Mitchell , a nd Ro ge r S. N asc i . . 7

3 D e t e c t io n o f H u m a n C a l i c i v i ru s e s a n d A s t r o v i r u s e s in S t o o l s

b y R T - P C R

X i J i a n g a n d D a v i d O . M a t s o n . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . 19

4 E n t e r o v i r u s e s a n d R h i n o v i r u s e s

P e t e r M u i r . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. 29

5 R a p i d D e t e c t io n a n d I d e n t if ic a t io n o f D e n g u e V i r u s e s b y R e v e r s e

T r a n s c r i p t a s e / P o l y m e r a s e C h a i n R e a c t i o n

De n n is W . T ren t, Gw o n g -J e n C h a n g , A . Va n ce Vo rn d a m ,

a n d R o b e r t S . L a n c i o t t i

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51

6 H e p a t i t is V i r u s e s

Gi r i s h J . Ko twa l . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . 63

7 H e r p e s v i r u s e s

Mark J . Espy , P . Shawn Mi tche l l , Dav id H. Pers ing ,

a n d T h o m a s F . S m i th

. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .

89

8 D i a g n o s i s a n d D i re c t A u t o m a t e d S e q u e n c i n g o f H IV -1

F r o m D r ie d B lo o d S p o t s D B S ) C o l l e c t e d o n F i lt e r P a p e r

Sh a ro n Ca s s o l, S ta n le y Re ad , B ru c e G . W e n ig er , R i c h a rd P i lo n ,

Ba rb a ra L e u n g , a n d T h e re s a Mo

. . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . .

103

9 P C R fo r t h e D e t e c t i o n o f I n f lu e n z a V i r u s e s in C l i n ic a l M a t e r ia l

J o a n n a S . E l l is a n d D a v id W . G . B ro w n . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . 119

1 0 L y s s a v i r u s e s :

Specia l Em phasis on Rabies Virus

H e r ve B o u r h y

. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . .

129

11 M e t h o d s fo r D e t e c t in g A n t i - M e a s l e s , M u m p s , a n d R u b e l la V i r u s

A n t i b o d i e s

I l k k a J u l k u n e n , I r j a Da v id k in , a n d Ch r i s t i a n Ok e r -B lo m ............. 143

ix


10/369

x Contents

1 2 P a p i l l o m a v i r u s e s

Sandra H. Kirk and David

T Y

Liu ................................................. 159

1 3 D i a g n o s i s o f P a r v o v i r u s B 1 9 D N A b y D o t B l o t H y b r i d iz a t i o n

Karen E. Hicks Stuart Beard Bernard J. Cohen

and Jonathan P. Clewley ............................................................. 173

1 4 P o l i o v i r u se s : Concurrent Serotyping and Intratypic Differentiation

of Polioviruses

David J. Wood ................................................................................... 189

1 5 P o x v i r u s e s

Hermann Meyer Susan L. Ropp and Joseph J. Esposito .......... 199

1 6 R e s p i r a to r y S y n c y t i a l V i r u s a n d O t h e r P e d ia t r ic R e s p i r a t o r y V i r u s

In fe c t i o n s

G. L. Toms ......................................................................................... 213

1 7 D e t e c t i o n a n d M o l e c u l a r E p i d e m i o l o g y o f R o t a v i r u s b y R N A G e l

E l e c t r o p h o r e s i s

Lennart Svensson ............................................................................. 223

1 8 D e t e c t i o n o f P C R A m p l i f ie d S a n d f l y F e v e r S i c i li a n V i r u s R N A

b y C o l o r i m e t r i c E n z y m a t i c I m m u n o a s s a y

M. Soft Ibrahim and Connie S. Schmaljohn

..................................

231

1 9 T i m e R e s o l v e d F l u o r e s c e n c e

Pekka Halonen and Timo L~vgren ................................................. 245

2 0 E n z y m e L i n k e d I m m u n o s o r b e n t A s s a y s

David M. Kemeny

..............................................................................

257

21 D y n a m i c A n a l y s i s o f V i ra l P o p u l a t i o n s b y D i r e c t D N A S e q u e n c i n g

a n d S o l i d P h a s e T e c h n o l o g y

Joakim Lundeberg Jacob Odeberg and Mathias Uhlen ............ 277

2 2 R o b o t i c A n a l y z e r s

Dale R. Pfost and Richard G. Keightley ......................................... 287

2 3 T h e D e t e c t io n o f E n t e r o v i r u s e s in W a t e r a n d A s s o c i a t e d M a t e r i a ls

U s i n g th e P o l y m e r a s e C h a i n R e a c t i o n

Peter Wyn-Jones and Jane Sellwood ............................................ 301

2 4 R e c o m b i n a n t A n t i g e n s in V i ra l D i a g n o s i s

John R. Stephenson and Alan Wames .......................................... 315

2 5 N A S B A : A Method for Nucleic Acid Diagnostics

Dianne van Strijp and Pierre van Aarle

.........................................

331

2 6 O i i g o n u c l e o t i d e s

Stina Syrj~nen ................................................................................... 341

In de x . . . . . . . .. . . . . . . . .. . . . . . . . .. . . . . . . . .. . . . . . . . .. . . . . . . . .. . . . . . . . .. . . . . . . . .. . . . . . . . .. . . . . . . . .. . . . . . . . .. . . . . . . . .. 367


11/369

on t r i bu to r s

P I E R RE V A N A A R L E ° Organon Technika, Boxtel, The Ne therla nd s

S T U A R T B E A R D ° Virus Reference Laboratory, Cen tral Pub lic Hea lth

Laboratory, London, UK

H E R V E B O U R H Y • Pa steu r Insti tute, Paris, Fra nc e

D A V I D W . G . B R O W N ° Virus Reference Laboratory, Cen tral Pub lic Health


T E R E S A M . B R O W N °

D epa rtme nt o f Health a nd Human Services, Centers

fo r D isease C ontrol and Prevention, Fo rt Collins, CO

S H A R O N C A S S O L ° Division o f Infect ious D iseases, D epa rtm ent o f Medicine,

Ottawa G enera l Hospital, Ottawa, Ontario, C anada

G W O N G JE N C H A N G ° Division o f Vector-Borne Infect ious Diseases, Cen ters


S T U A R T C L A R K • M icron Bioproducts, Cam berly, Surrey, U K

J O N A T H A N P C L E W L E Y ° irus Reference Laboratory, Cen tral Pub lic He alth


B E R N A R D J C O H E N °

irus

Reference Laboratory, Cen tral Pu blic Hea lth


I R J A D A V I D K I N ° Na tiona l Pu blic H ealth Insti tute, Helsinki, Fin lan d

J O A N N A S E L L I S °

irus

Referenc e Laboratory, Cen tral Pu blic Hea lth


J O S E P H J . E S P O S I T O ° CD C Poxvirus Sec tion , Centers fo r Disease Control

an d Prevention, A tlanta, GA

M A R K J . E S P Y •

D epar tmen t o f Cl inical Microbiology, M ayo Clinic ,

Rochest er , M N

P E K K A H A L O N E N ° Department of Virology, Universi ty of Turku, Finland

K A R E N E . H I C K S ° Virus Referen ce Laboratory, Cen tral Pu blic Health


M . S o F t IB RA H 1M ° Virology Division, USAM RID, Fo rt Detrick, Frederick, M D

X I J I A N G ° Ce nter fo r P ediatric Research, Eas tern Virginia Me dica l School ,

Children s H osp ital o f the K ing 's Daughters, Norfolk, VA

A H S O N J . JO H N S ON °

Dep artment o f Heal th and Huma n Services , Centers


xi


12/369

x i i o n t r i b u t o r s

I L K K A J U L K U N E N •

Na tiona l Pu blic H ealth Insti tute, Helsinki, F inla nd

N I C K K A R A B A T SO S

Dep artment o f Heal th and Hum an Serv ices , Centers

fo r D isease Control and Prevention, Fo rt Collins, CO

R I C H A R D G . K E I G H TE E Y °

Orchid Biocomputer, Prin ceton ,NJ

D A V I D M . K E M E N V °

D epartm ent of Immunology, K ing s C ollege Scho ol

o f Medicine, London, UK

S A N D R A H . K I k K • D epar tme nt o f Li fe Sciences, N ott ingham Trent

University, Nottingham, UK

G I R I S H J . K O T W A L °

Dep artment o f M icrobiology and Immunology,

University o f Lo uisville Sch ool of Medicine, Louisville, K Y

R O B E R T S . L ANC 1 OTT I °

Division of Vector-Borne Infectious Diseases,

Cen ters fo r D isease C ontrol and Prevent ion, Fo rt Collins, CO

B A R B A R A L E U N ~ °

Brit ish Columbia Centre fo r Exce l lence in HIV/AIDS,

St. P au l s Hospital, Vancouver, British C olumbia, Canada

D A V I D T . Y . L I U °

D epar tmen t o f Ob stetrics and G ynaecology, City H osp i tal

o f Nottingham, U K

T I M O L 6 V G R E N °

D epa rtm ent o f Biotechnology, University o f Turku, Fin land

J O A K I M L U N D E B E R G °

D epartm ent of Biochemistry , Ro yal Inst i tute of

Technology, Stockholm, Sweden

D E N I S E A . M A R T I N °

Dep artment o f Heal th and Hum an Serv ices , Centers

fo r D isease Co ntrol and Prevention, Fo rt Collins, CO

D A V I D O . M A T S O N °

Cen ter fo r Pediatric Research, Eastern Virginia

M edica l School, Ch ildren s H osp i tal of the K ing s D aughters,

Norfolk, VA

HE R M ANN M E Y E R °

Federal Arm ed Forces Medical Academy, M unchen, G ermany

C A R L J . M I T C H E L L °

Departm ent o f Heal th and H uman Serv ices , Centers

fo r Disea se Con trol and Prevention, Fo rt Collins, CO

P . S H A W N M I T C H E L L °

D epar tmen t of Cl inical Microbiology, M ayo Clinic ,

Roches te r , M N

T H E RE S A M O °

Brit ish Columbia Centre fo r E xcel lence in HIV /AIDS,

St. P au l s Hospital, Vancouver, British C olumbia, Canada

PETER MUIR ° D ivision o f Microbiology, D ep artm ent o f Virology,

St. Thom as s Hospital, London, U K

R O G E R S . N A S C I •

Dep artment o f Heal th a nd Human Serv ices , Centers

.[or D isease C on trol an d Prevention, Fo rt Collins, CO

J A C O B O D E B E R G °

D epar tmen t of Biochemistry , Ro yal Inst itute

o f Technology, Stockholm, S we den

C ~tR IS TIA N O ~ R B L o M °

VTT Biotechnology and Fo od Research, Espoo, Finland


13/369

o n t r i b u t o r s x i f i

DAVID H. PERS1NG ° D epartm ent o f Cl inical Microbiology M ayo C linic

Rochester MN

DALE R. PFOST °

Orch id Biocomputer Prin ceton NJ

RICHARD PILON °

Division o f Infect ious Diseases D epa rtme nt of Pediatrics

H osp i tal fo r S ick Children a nd the University o f Toronto Ontario

Canada

STANLEY READ ° Division o f Infectious D iseases Depa rtment o f Pediatrics

Hospital fo r Sick Children and the University o f Toronto

Ontario Canada

JOHN Y. ROEHRIG

°

Dep artment o f Heal th and Hum an Services Centers

fo r D isease Control and Prevention Fo rt Collins CO

SUSAN L. RoPP ° CD C Poxvirus Section Centers fo r Disease Co ntrol

an d Prevention Atlanta GA

CONNIE S. SCHMALJOHN Virology Division USAM RID Fo rt De trick

Frederick MD

JANE SELLWOOD

Read ing PHLS Ro yal Berks hire Hospital Reading U K

THOMAS F. SMITH ° D epartm ent o f Cl inical Microbiology M ayo C linic

Rochester MN

JOHN R. STEPHENSON

°

D epartm ent o f Infect ious a nd Tropical Diseases

Londo n Schoo l o f Hyg iene and Tropical Medicine The Universi ty

o f London UK

DIANNE VAN STRIJP

°

Organon Technika Boxtel The Ne therla nd s

LENNART SVENSSON ° Depa rtment o f Virology Swedish Inst itu te fo r

Infectiou s D iseas e Control K aro linska Insti tute Stockholm Sw ede n

STINA SYRJ ~NEN

°

Inst i tute o f Den tistry Universi ty o f Turku Fin land

TANVIR TABISH

°

M icron Bioproducts Cam berly Surrey U K

G. L. TOMS •

School of Patho logy Science University o f Ne w cast le M edica l

School Newcastle-Upon-Tyne U K

DENNIS W. TRENT

° M cCul ley Cuppan LLC Sal t Lake Ci ty UT

MATHIAS UHLI~N

Dep artment o f Biochemistry Royal Ins t itu te o f

Technology Stockholm Sweden

A. VANCE VORNDAM

°

Division o f Vector-Borne Infect ious Diseases Cen ters

fo r D isease Con trol and Prevention Fo rt Collins CO

ALAN WARNES

°

Micron Bioproducts Cam berley Surrey U K

BRUCE G. WENIGER °

Centers fo r Disease Control and Prevention Atlanta GA

DAVID J. WOOD ° Division o f Virology NIBSC South Mimms Po tters Bar

Hertfordshire UK

PETER WYN-JONES

°

School o f Health Sciences University o f Sunderland UK


14/369


15/369

2 Tabish, Warnes, and Clark

eukaryottc systems, and this technology 1s described m further detail m

Chapter 24.

Recent work has indicated that monoclonal antibodies (MAbs) specific for

types 40 and 4 1 can detect those adenovtrus types causmg gastroenterms, but

not those causing respiratory or ocular mfecttons (4). Subsequently, enzyme

tmmunoassays (EIAs) based on the use of these MAbs have been developed to

detect the types 40 and 41 directly from fecal samples. However, there are

reports that a number of other adenovnuses are also responsible for gastroen-

teritis, and consequently an assay that detects adenovnus group antigen may

be better suited for screening purposes (5-7).

Although there are a number of EIAs available for the detection of

adenovuuses, most require sophisticated laboratory equipment, which 1snot

always avatlable to all laboratortes We, therefore, descrtbe a latex agglutma-

tion assay for the raped detection of adenovn-uses m stool specimens that can

be as sensitive as electron microscopy (8), which is still used as the confirma-

tory test for positive fecal samples. Latex agglutination can be performed

rapidly taking only 2 mm, with 3 min for sample preparation; the result is easy

to visualize, with no requu-ement for support equipment. Upon mixmg the

extracted sample, with polyclonal antibody-coated latex particles, cross-link-

ing occurs between the anttbodies coated to the latex and the virus partrcles,

resulting in the productton of visible aggregates.

Owmg to the widespread use of adenovtral vectors as deltvery systems m

gene therapy, there is now the opportunity to utilize the rapid latex assay as an

on-line monitoring system to estimate viral load in cell culture. This has the

benefits of being able to maximize viral yield for the production of stocks,

using an assay that takes only 2 mm to perform.

2. Materials

1. Beckman52-21 centrifuge (Fullerton, CA).

2. Dialysis tubing

BDH Stze 5 Dram 24/32-19.0 mm (BDH, Poole Dorset, UK)

3. Nalgene glass iber pre-filters: Sybron International (Rochester,NY).

4 Amicon 76mm Ultracentrifugation cell (Amicon Ltd, Stonehouse, Gloucester-

shire, UK)

5. Ultrafiltration membrane (PM30 43mm) (Amicon).

6. Roller mix.

7. Sonicator MSE Soniprep 150 (MSE Scientific Instruments, Crawley, UK)

8. Sephacryl S-400-HR (Sigma S400-HR) (Sigma Chemical Company Ltd, Poole,

Dorset, UK).

9 Glycme-buffered salme (GBS). 100 mA4 glycine, 171 mM sodium chloride,

15 mA4 sodium azide, pH to 8.2 with 10 A4 sodmm hydroxide solution (BDH).

10. Latex; Prolabo K080: 0.8 pm (Estapor, Manchester, UK).

11 Glacial acetic acid (BDH).


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Detection of Adenovirus

3

12. n-Octanoic acid (BDH)

13. Phosphate-buffered saline (PBS): 150 rnA4sodium chlortde, 4 rnM potassium

dihydrogen phosphate, 11 mM disodium hydrogen phosphate dehydrate (BDH).

14. Ethylenedtamine tetra-acetic acid (EDTA): 10 mM stock solution

15 Acetate buffer, pH 4.0. 100 mM sodium acetate, 5 75 mM glacial acetic acid

16 Acetate buffer, pH 4.8: 100 mA4 sodium acetate, 5 75 n&Y glacial acetic acid

17. 0.1 Bovine serum albumin (BSA) solution (Bayer Diagnostics, Nankakee, IL)

m GBS

18 Normal rabbit serum (NRS).

19. Cell growth media (EMEM): 10 fetal bovine serum, 1X nonessential ammo

acids, 10 nM HEPES,2 rnM L-glutamme.

20. Beta-propiolactone.

2 1. Extraction buffer: available from Microgen Bioproducts Ltd (Camberley, Surrey).

22 Mixing cards (Microgen Bioproducts).

23. Filtration units (Microgen Bioproducts).

3. Methods

The following list of protocols describes the procedures involved in purify-

ing hexon proteins required as immunogen to raise polyclonal anti-adenovirus

hexon antisera.

3.1. Production of Adenovirus

1 Grow a Hep2 cell monolayer to 80 confluency in 175 cm2 tissue-culture flasks

m cell-growth media sparged with 95 sir/5 CO2 at 37°C.

2. Infect cells with a multtpltcity of infection of 0.01 of adenovuus type 5 and mcu-

bate for 1 h at 37°C.

3. Remove the vnus and wash the cells with prewarmed PBS.

4. Add 60 mL of fresh prewarmed cell-growth media, gas wtth 95 sir/5 CO2

and incubate at 37°C

5. Examme the cell monolayer daily and harvest, when the cell monolayer shows

100 cytopathic effect.

6. Freeze thaw the culture four times and then inactivate the vnus with 0.4 beta-

propiolactone.

7. Store crude vnus stockpreparationsat-20°C.

3.2. Purification of Adenovirus Hexon Protein

and Production of Polyvalent Sera

A

key stage is the purification of hexon protein and the subsequent prodwc-

tion of polyvalent sera; although the precise method cannot be mentioned here,

purification processes are now well-advanced and detailed elsewhere (9). The

use of genetic engineering should also not be forgotten because these methods

now offer the productton and purification of protems as diagnostic antigens

with relative ease (see Chapter 24). The purified adenovirus hexon protem can


17/369

4 Tabwh, Warnes, and Clark

be evaluated for purity using polyacrylamlde gel electrophoresis (PAGE) or

immunoblotting if polyclonal antibodies are available (see

Note 1).

Polyclonal anti-hexon sera can then be obtained by immumzation of am-

mals, and the serum stored at -4OOC.

3.3. Fractionation of IgG

from Rabbit Antiserum Using n-Octanoic Acid

1 Pour the rabbit antiserum mto appropriate plastic containers; add twice the vol-

ume of acetatebuffer, pH 4.0.

2. Stir each pot vigorously at room temperature with the aid of a magnetic stirrer

3. For every 10 mL of rabbit serum, add 0 75 mL n-octanolc acid in a dropwlse manner.

4 Leave containers to stir at room temperature for 30 mm

5. Centrifuge m Beckman J2-21, for 20 mm, at 15,300g and 10°C

6. Decant and retam the supernatant.

7 Resuspend the precipitate m 10 mL of acetate buffer pH 4 8 per 10 mL of original

serum volume.

8. Centrifuge at 15,300g for 20 min at 10°C

9. Pool the supernatant with that retained in Subheading 3.3.6.

10. Boil the dialysis tubing m 10 mM EDTA for 2 mm m a glass beaker, allow to

cool, and wash thoroughly with dlstllled water

11 Filter treated serum through a Nalgene glass-fiber pre-filter to remove coarse

precipitate and plpet mto the dialysis tubing

12. Dialyze against GBS, mltlally for 4 h at 4”C, followed by a further 16 h at the

same temperature.

13 Check pH of the dialyzed material, if pH is 8 O-8 2, then proceed, if not, then

dialyze for a further period until the desired pH has been achieved

14. Store the purified anti-adenovirus IgG at -7O’C

3.4. Manufacture of Test Antibody-Coated Latex

The method for the production of antibody-coated latex particles has

already been published in detail (IO), as described here; a moditkatlon of

this method is currently used at Microgen Bioproducts Ltd. This provides

hexon-specific polyvalent antibody-coated latex particles produced in

Sub-

heading 3.2.

1. Wash the latex particles three times with PBS by centrlfugatlon at SOOOg for 20

min at 4°C

2. Resuspend at a concentration of 0.4 m PBS.

3. Add an equal volume of anti-adenovlrus antibody at 16 mg/mL

4. Shake at room temperature for 2 h

5. Wash coated latex particles three times with PBS by centrifigation at 8000g for 20

mm at 4°C

6. Resuspend to a final concentration of 0.7 in PBS and store at 4°C.


18/369

Detection of Adenovirus

5

Table 1

Comparison of Adenovirus Latex Against EM

Adeno latex

+

-

EM results

+

53

3a

-

lb 79

OThis sample was retested wrth other commercial assays

and was shown to be positive

“On retesting wrth other commercial assays 2 of the 3

samples were also shown to be negatrve

3.5. Manufacture of Control Antibody-Coated Latex

As part of the assay, it is essential that a control latex is always used so that

the test can be correctly evaluated and nonspecific reactions detected. The con-

trol rabbit IgG is derived from normal rabbit serum (NRS) as described m

Sub-

heading 3.3.

and processed as described m

Subheading 3.4.

3.6. Test Procedure

1 Prepare an approximate 10 suspension of the fecal sample by transferring 0.1 g

(0 1 mL) of sample into 1 mL of extraction buffer in a stoppered tube (supplied as

a component of the filter pack). Mix the contents well

2. Allow the reagents to reach room temperature

for l-2 min before

processing.

3. Remove the stopper and fit integral filter/dropper unit

4. Holding the whole assembly vertically, dispense 1 drop of clear filtrate onto each

of 2 wells on the test slide.

5. Add 1 drop of well-mtxed test latex reagent to one well and 1 drop of control

latex reagent to the other

6. Mix the contents of each well using a separate mixing sttck for each sample,

covering the entire area of the well

7. Gently rock the slide and observe for agglutination for up to 2 min

8.

A

posrttve result is indtcated by agglutinatron of the test latex reagent wrth no

agglutinatton of the control latex reagent.

9. The result 1s negattve if no agglutination of either the test latex reagent or the

control latex reagent IS observed within the 2-min test period

10.

Agglutmatton both in the test and control latex Indicates a nonspecific result and

the sample should be retested.

3.7. Expected Results

To contrast the efficacy of the assay the following results were obtained

when a panel of fecal samples was independently assessed by electron mtcros-

copy (EM) (see

Table 1).


19/369

6 Tabish, Warnes, and Clark

These results produced a sensitivity of 95 and a specificity of 99 com-

pared to the “Gold standard” EM. Other commercial kits are available from

other companies (see Note 2).

4. Notes

1 If polyclonal or MAbs are available, the purified antigen should be characterized

usmg both PAGE and immunoblotting. If fusion protems are produced via genetic

engineering techniques, then most protein end products can be visualized used

supplied anttbodies against the tagging protein

2 Commercial assays based on latex agglutination are available from Microgen

Bioproducts (Adenoscreen); Onon, Finland (Adenolex); Bioktt, Spain (Adenogen)

References

1, Hrerholzer, J C (1991) Antigenic relationships among the 47 human adenovnuses

determined m reference horse sera

Arch Vzrol 121, 179-l 97

2 Albert, M J (1986) Enteric adenovnuses

Arch Vzrol 88,

1-17

3 Cepko, C. L., Whetstone, C. A , and Sharp, P. A. (1983) Adenovnus hexon mono-

clonal antibody that is group specific and potentially useful as a diagnostic reagent.

J Clan. Mcroblol 17(2), 360-364.

4 Grydsuk, J. D., Fortsas, E , Petric, M , and Brown, M. (1996) Common eprtope on pro-

tem VI of enteric adenovuuses from subgenera A and F

J Gen Vzrol 77(8),

18 l-l 89

5. Bhan, M. K., Raj, P., Bhandart, N., Svensson, L., Stmtzmg, G., Prasad, A. K.,

Jayashree, S., and Snvastava, R. (1988) Role of enterrc adenovnuses and rotavuuses

m mild and severe acute enterms. Pedzatr Infect Du. J 7(5), 320-323

6 Petrtc, M (1995) Cahcivrruses, astrovnuses and other drarrherc viruses, m

Manual of

Clznzcal Mzcroblology,

6th ed. (Murray, P. R , Baron, E. J., Pfaller, M. A , Tenover, F.

C , and Yolken, R. H., eds.), ASM, Washmgton, DC, pp. 1017-1024

7. Noel, J , Mansoor, A., Thaker, U , Herrmann, J., Perron-Henry, D , and Cubitt,

W D. (1994) Identification of adenovnuses m faeces from patients with drar-

rhoea at the Hospitals for Sick Children, London, 1989-l 992

J, Med Vu-01 43(l),

- 84-90

8.

Grandlen, M., Pettersson, C. A., Svensson, L., and Uhnoo, I (1987) Latex agglu-

tmatron test for adenovnus dragnosrs m diarrhea1 drsease J.

Med Vzrol 23(4),

311-316.

9. Doonan,

S.

(ed.) (1996)

Methods zn Molecular Biology Protein Purlficatron Pro-

tocols.

Humana, Totowa, NJ.

10. Sanekata, T., Yoshida, Y , and Okada, H. (1981) Detection of rotavnus m faeces

by latex agglutination

J Immunol Methods 41,377-385


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2

Alphaviruses

John T. Roehrig, Teresa M. Brown,

Alison J. Johnson, Nick Karabatsos,

Denise A. Martin, Carl J. Mitchell, and Roger S. Nasci

1. Introduction

Alphaviruses are enveloped, positive-stranded RNA viruses that are the etio-

logic agents of severe encephalitis and polyarthritis. These viruses can be

divided into six or seven serocomplexes (1). Four of these serocomplexes-

represented by eastern equine encephalitis (EEE), western equine encepha-

litis (WEE), Venezuelan equine encephalrtts (VEE), and Semliki Forest

viruses+omprise the most medically important alphaviruses. The VEE

serocomplex can be further divided into at least six subtypes (1 to 6), with

subtype 1 having at least five different varieties (1 AB, lC, lD, lE, and

1F). The importance of VEE virus subtyping 1s that varieties 1AB and 1C

vu-uses cause epidemic/epizootic VEE infection, whereas disease caused

by other VEE viruses is endemic/enzootic. Ross River, Chikungunya,

Mayaro, and Getah viruses are members of the Semliki Forest serocomplex.

Sindbis and Ockelbo viruses are members of the WEE virus serocomplex. A

newly emerging alphavirus, Barmah Forest, may represent a new serocomplex

of alphaviruses.

Laboratory diagnosis of human alphavirus infections has changed greatly

over the last few years. In the past, identification of alphavirus antibody relied

on four tests:hemagglutmation-mhibition, complement fixation, plaque reduc-

tion neutralizatton test, and the indirect fluorescent antibody (IFA) test. Posi-

tive identification using these immunoglobulin M- (IgM-) and IgG-based

assays required a fourfold increase in titer between acute and convalescent

serum samples. A number of very good procedural reviews contam the specif-

ics of these older assays 2).

From Methods m Molecular Medmne Vol 12 Diagn osbc Wology Protoc ols

EdRed by J R Stephenson and A Warnes 0 Humana Press Inc Totowa NJ

7


21/369

8

Roehrig et al.

With the advent of solid-phase antibody-binding assays, such as enzyme-

linked immunosorbent assay (ELISA), the diagnostic algorithm for identifica-

tion of viral activity has changed. Rapid serologic assayssuch as IgM-capture

ELISA (MAC-ELISA)

are now employed early in infection (3). In many cases,

a positive MAC-ELISA with an acute serum sample precludes the need for

testing of a convalescent serum sample. Early m infection, IgM antibody is

more serocomplex specific, while later in infectton, IgG antibody is more

serocomplex crossreactive. Inclusion of monoclonal antibodies (MAbs) with

defined virus specificities in these solid-phase assayshas allowed for a level of

standardization that was not previously possible. All tests described in this

chapter are equally applicable to all alphaviruses.

Virus isolation and identification have also been useful in defining viral

agents in serum, cerebrospinal fluid (CSF), or mosquito vectors. Although vnus

isolation still depends upon growth of an unknown virus in cell culture or neo-

natal mice, virus identification has also been greatly facilitated by the avatl-

ability of virus-specific MAbs for use m IFA assays. Similarly, MAbs with

avidities sufficiently high to allow for specific binding to virus antigens in a

complex protein mixture (e.g., mosquito pool suspensions) have enhanced our

ability to rapidly identify virus agents

in situ.

Although polymerase chain reac-

tion has been developed to identify a number of viral agents, such tests have

not yet been developed for routine rapid identification of alphaviruses in the

clinical setting.

2. Materials

2.1. General ELISA Materials List

The following materials list is employed m all subsequentELISA procedures:

1. 96-Well Immulon 2 microtiter plates (Dynatech Industries, Inc., Chantilly, VA).

2 Carbonate-bicarbonate (PH 9.6) coating buffer: 1.59 g of Na$Os, 2.93 g NaHCOs

in 1 L of distilled water (4).

3. Phosphate-buffered saline (PBS): BBL FTA buffered saline (9.23 g/L, Becton

Dickinson, Cockeysville, MD)

4. Blocking buffer: 5% skim milk, 0.5% Tween-20 m PBS.

5. Rinse buffer: 0.05% Tween-20 in PBS.

6. ELISA plate-reader.

7. Refrigerator.

8. Humid incubator, 37°C.

2.2. Antigen Detection ELlSA in Virus-infected Mosquitoes

1. Grinding apparatus Ten Broeck homogenizers r mortars and pestles).

2. Microcentrifuge with accompanying 1.5-mL microcentrifuge tubes

3 Probe sonicator.


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Alphaviruses

9

4 BA- 1 dduent. 1X cell culture medium M 199,0.05 MTris-HCl, 1% bovme serum

albumm, 0.35 g/L NaHCO,, final pH 7.6. Filter sterilize

5. Lysls buffer: 5% Tween-20 in PBS

6. Substrate: 3-3’,5-5’-tetra-methyl benztdme (TMB) Commercial source: TMB-

ELISA reagent (Gibco-BRL, Gaithersburg, MD)

7. Stopping reagent: 1 N H,SO,.

8 Positive control antigen (sucklmg mouse bram [SMB] antigen of either EEE

virus strain NJ-60, or WEE virus strain Fleming) Procedures for preparation of

SMB antigens have been previously described (2)

9. Capture antibody* Murine MAb, lA4B-6, for EEE vnus or 2A3D-5, for WEE

virus ($6).

10. Detector antibody: Murine MAb, lB5C-3, conjugated to horseradish peroxtdase

(HRP) for EEE virus, or 2BlC-6, conjugated to HRP for WEE virus (5,6).

11 Polyclonal control antibodies: Procedures for producing murine polyclonal

anttvirus antibodies for use in the inhibition assay have been previously

described (2).

2.3. IgM-Capture ELBA (MAC-ELBA)

1 Previously tttered goat antihuman IgM capture antibody (Cappel Labs, Organon

Tekmka, Durham, NC)

2 Previously tttered vn-us and control SMB antigens (2).

3. Prevtously tttered HRP-conjugated MAb detector, 2A2C-3 (5)

4 Known-posmve human serum or CSF samples reactive with test viruses to serve

as positive controls.

5 Known-negative human serum or CSF samples to serve as negative controls

2.4. IgG ELlSA

1 Capture antibody* Murme MAb, EEE lA4B-6 (5)

2. Previously titered virus and control SMB antigens (2).

3. Detecting antibody: Goat antthuman IgG (Fc-spectfic)-alkaline phosphatase (AP)

coqugate (Jackson Immunochemtcals, West Grove, PA).

4. Known-positive human serum samples reactive wtth test vu-uses to serve as posi-

tive controls.

5. Known-negative serum samples to serve as negative controls

6. Substrate: 3 mg/mL Sigma 104 m 1 MTris-HCl, pH 8.0 (Sigma, St. LOUIS, MO).

7. 3 MNaOH (120 g in 1 L water) to stop reaction.

2.5. IFA Assay

1. Unconjugated MAbs of various specificity (Table 1).

2. Fluoresceinated anttmouse antibody (Jackson Imrnunochemtcals).

3 Sodium azide (as preservative).

4. Penictllin-streptomycin,

5. PBS.

6. Counterstain: Trypan blue diluted 1:4000 in PBS.


23/369


24/369

Alphaviruses 11

enizers. Method two employs homogenization in mortars and pestles (14). The

method of trrturatron seems to be less important than the pool size. Prelrmi-

nary data indicate that as pool srze increases from 25 to 50 or 100, the ELISA

signal 1s diminished. The diminution of ELISA signal with larger pools is

probably associated with the larger concentrations of irrelevant material m the

larger pools

2. Following trtturation, centrifuge the suspension in a micromge at 15,000g for 2 min.

At this point, a small amount of the sterile supernatant can be removed for subse-

quent virus isolation in plaque assay in Vero cells. Split the remaming volume

mto two aliquots. Reserve one ahquot for confirmation of testing. The second

aliquot serves as antigen for the antigen-capture ELISA.

3. Also prepare at least SIX independent pools of normal, nonmfected mosquitoes to

serve as ELISA-negative antigens.

4. Resuspend one 0.25-mL ahquot (including the pellet) Somcate each sample m

a biosafety cabinet using a microprobe at 100 W for 10 s. Centrifuge in a

microfuge at 15,000g for 2 min. Transfer supernatant to new veal. Immedt-

ately before ELISA testing, add 10 uL lysis buffer per 100 mL mosquito

pool sample. Addition of the lysis buffer frees virus antigens from larger

parttcles. Incubate 15 mm at room temperature (RT). After incubation, centrr-

fuge in a microfuge at 15,OOOg for 2 min The supernatant from this centrifuga-

tion will be the mosquito pool antigen used in the antigen-capture ELISA (see

Subheading 3.1.2.)

3.7.2. Antigen-Capture ELBA

1. Drlute capture antibody (1:20,000 of MAb lA4B-6 for EEE, vnus or 1:5000 of

MAb 2A3D-5 for WEE virus ($61) m coating buffer. Coat wells of a 96-well

Immulon 2 microtrter plate with 100 mL capture antibody per well. Incubate

coated plates overnight at 4°C.

2. Rinse plates 5 times with ELISA rinse buffer

3. Block plates with 300 yL per well blocking buffer for 1 h at 37°C. Repeat the

rinse step.

4. Add 100 mL per well detergent-treated mosquito pool antigen. Test mosquito

pools m triplicate. Incubate plates overnight at 37% Include space for SIX nor-

mal uninfected mosquito pool homogenates. These normal homogenates will be

used to calculate test background. Also include space for positive control antigen

diluted 1:lOOO and treated with lysis buffer (step 4, Subheading 3.1.1.) Use

posrtrve control antigen at 100 mL per well. Repeat the rinse step.

5. Add 100 pL per well detector antibody lBSC-3-HRP-conjugate, diluted 1.1000

for EEE virus detection or 2BlC-6-HRP conjugate diluted 1:5,000 m ELISA rinse

buffer for WEE virus. Incubate 1 h at 37°C

6. Rinse 10 times with ELISA rinse buffer.

7. Add 100 mL per well substrate (TMB-ELISA) Incubate 30 mm at RT and stop

the reaction with 50 mL per well 1 N H2S04. Measure the absorbance at 450 nm

(A4s0 nm) in a microplate reader.


25/369

12 Roehrig et al.

3.1.3. Inhibition Assay

All mosquito pools presumed to be positive for viral antigen should be tested

m the inhibition assay.

1. Dilute EEE, or WEE, and St. Louis encephalitis virus polyclonal antibodtes 1:20

in PBS

2. Mix 100 yL mosquito supernatant with 20 uL of either EEE vuus or WEE vu-us

polyclonal antibody. Also mix 100 uL mosqutto supematant with 20 uL St Louis

encephalitis vuus polyclonal antibody, incubate at 37°C for 1 h If there is enough

mosquito supematant, do the procedure m duplicate.

3. Add mixture to ELISA plate (see step 4 in Subheading 3.1.2.) Incubate over-

night at 4°C. Perform ELISA as in Subheading 3.1.2.

4. If the mean absorbance value of the pool is reduced by 50% or more when it is

premcubated wtth the polyclonal anttalphavuus anttbody, sample is constdered

specific for alphavirus antibodies.

3.1.4. Data Analysis

1. Derive the mean Ad5s ,,,

for each duplicate or triplicate and also the mean of the

six normal mosquito pool samples The negative cutoff will be twice the Adso ,,, of

the mean of the six normal mosquito pools. Any experimental pool with a mean

Adso ,,, greater than twice the mean of the Ads,, “,,, of the SIX negative control pools

should be considered presumptive for the presence of EEE or WEE virus antigen

These pools should be tested in the inhibition assay (see Notes l-4).

2 The experimental sensitivity of this assay is 3.5-4 0 loglo PFU per 0.1 mL. Pools

with titers lower than this cutoff will give negative or variable results.

3.2. IgM-Capture ELISA (MAC-ELBA)

Assays that detect virus-specific IgM are advantageous because they detect

antibodies produced within days of infection, obviating the need for conva-

lescent-phase specimens in many cases. The MAC-ELISA is the optimum

approach to detect IgM because capturing the antiviral IgM antibody negates

the competitive effects seen with antiviral IgG in the more standard mdtrect

ELISA format. The MAC-ELISA is simple, sensitive, and applicable to serum and

CSF samples.False-positive reactions owing to rheumatoid factor are minimized.

3.2.1. ELISA

1, Coat 96-well Immulon 2 plates wtth 75 yL per well of goat antihuman IgM in

coating buffer, pH 9.6. Coat enough wells to test each sample against both posl-

tive and negative antigens m triplicate. Do not use the outer wells on the

plate. Incubate overnight at 4°C. Wash plates in microplate washer five times

with rinse buffer.

2. Block plates with 300 uL per well blocking buffer. Incubate covered plates at RT

for 30 min. Repeat wash step.


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Alphaviruses

13

3. Add 50 yL per well of the patient’s serum drluted 1:400 m rmse buffer or

patient’s CSF undiluted to six wells. Incubate for 1 h at 37’C Also test appropri-

ately diluted positive control human serum and a normal human serum. Repeat

wash step

4. Dilute virus-infected SMB antigen in rinse buffer accordmg to previous titration.

Add 50 pL per well to three wells of each test sample. To the other three wells

add 50 pL per well of normal SMB antigen diluted in the same manner. Incu-

bate overnight at 4°C. Repeat wash step.

5. Add 50 PL per well of HRP-coqugated MAb, 2A2C-3, diluted as per the previ-

ous titration in the blocking buffer. Incubate 1 h at 37°C

6 Repeat wash step twice

7. Add 75 pL per well of TMB substrate. Incubate at RT for 10 min.

8. Add 50 mL per well of 1 A4 H,SO, to stop the reaction Allow to sit at RT for 1

mm Read plates in microtiter plate-reader using 450 nm.


Calculate the positive/noise (PIN) values as follows:

[Average 450 nm reading of patient’s serum plus antigen (P)]

[Average 450 run reading of normal human serum plus antigen (N)]

The

PIN

ratio must be at least 2.0. The positive human serum control

P/N

ratio should be at least 2.0 and the normal human serum control P/N ratio

should be less than 2.0. If any OD readings or control serum P/N values fall

outside these threshold values, the test must be repeated. All patient

PINvalues

greater than or equal to 2.0 should be reported as positive with the understand-

ing that P/Nvalues

between 2.0 and 2.5 could represent false positive reactions

(see Notes 5-8).

3.3. IgG ELBA

Rapid testing for IgG antibody in a solid-phase assayprecludes the necessity

for other IgG measuring tests such as hemagglutination-inhibition, complement

fixation, and plaque reduction neutralization tests. Serologic crossreactivity in

the IgG response to the alphaviruses makes the IgG ELISA less specific than

the MAC-ELISA. The ELISA assay design of this IgG test allows for concur-

rent application with the MAC-ELISA. The use of a MAb capturing antibody

allows for easy standardization of antigen quantities between laboratones.

3.3.1. ELISA

1. Dilute MAb lA4B-6 l.lO,OOO m coating buffer and coat wells of a 96-well

microtiter plates with 75 yL overnight at 4°C. Coat enough wells to test each

sample againstboth positive and negative antigens n triplicate. Wash plates n

microplate washer 5 times with rinse buffer.


27/369

Roehrig et ai.

.

2 Block plates with 300 pL blocking buffer per well for 30 mm at RT. Repeat

wash step.

3. Add 50 mL per well of appropnate SMB vuus or control antigen (see Subhead-

ing 3.2.1., step 4) diluted in rinse buffer and incubate overmght at 4°C. Rmse

plate five times with rinse buffer.

4 Add 50 pL per well unknown sera diluted 1:400 m rinse buffer and Incubate 1 h

at 37°C. Rinse plates 5 times with rinse buffer.

5. Add 50 pL goat antihuman IgG (Fc-specific)-AP conjugate diluted 1 *lOOO in

rinse buffer per well and incubate 1 h at 37°C.

6 Rinse plates 10 times with rinse buffer.

7. Add 75 mL per well of substrate (Sigma 104) and incubate 30 mm at RT. Stop

color development, if necessary, by adding 25 pL 3M NaOH per well, and read

absorbance at 405 nm.


P/N ratios are determined as m Subheading 3.2.2. Ratios greater than or

equal to 2.0 are considered positive with the understandmg that P/N values

between 2.0 and 2.5 could represent false positive reactions (see Notes P-11).

3.4. IFA Assay

Immunofluorescence tests provide a useful means of identifying viral anti-

gen directly m clmlcal specimens and of providing specific immunologic lden-

tification of isolates m the laboratory (15-17). If the antigen 1s known, the

presence of specific antibodies m a test serum may also be documented. After

mcubatlon of antiserum and antigen, the presence of a reaction is detected by

observation of fluorescence in a microscope that is equipped with a source of

ultraviolet light. A sequence of filters 1sused to generate excitation light of

optimal wavelength and to block light of harmful wavelengths before vlewmg.

3.4.1. infecting Ceils and Preparing Spot Slides

1. Select a cell culture type appropriate for the virus to be used. Inoculate a mono-

layer culture less than 1 wk old with the vu-us seed stock

2. Incubate at 37°C and observe dally for virus cytopathic effects When It mvolves

at least 25% of the cell sheet, harvest the cells, saving the media for virus seed

(if necessary).

3. Dilute the harvested cells so that sufficient cells are added to each spot on the

shde Add about 10 pL of diluted cells to each well on the slide.

4. Allow shdes to air dry at least 2 h FIX slides m cold acetone for 15 mm, dry, and

store at -70°C

3.4.2. iFA Assay

1. Remove antigen slides from -70°C and allow to air dry or optionally, refix in

cold acetone for 10-15 mm.


28/369

Alphavmses

15

2 Dilute all antibodies m PBS with 0.1% sodium azide and 2% penicillin/strepto-

mycin Dilute MAb ascites to appropriate concentrations Add l&12 mL of

diluted antibody to one well on the spot slide Run all necessary control slides

(see Subheading 3.4.3.)

3 Incubate m a moist chamber for 1 h at 37“C

4 Wash slides for 15 mm in PBS Allow to air dry.

5 Add 10-12 mL ofpretiterated antlmouse antibody (see Subheading 3.4.3.1.) conju-

gated to fluorescem lsothlocyanate made up m 1:4000 trypan blue with 0.1% sodmm

azlde to each well. Incubate for 1 h at 37°C in a moist chamber. Repeat wash step

6. Add mounting solution and coverslips. Examine slides by fluorescence microscopy

no later than 24 h aRer completing procedure. Store slides at 4°C. A positive reaction

appears as apple-green fluorescence against a background of red counterstamed cells

3.4.3. Test Controls

3.4.3.1.

TITRATING

MAss

AND CONJUGATES

1 Each new lot of MAb and commercial conjugate must be titrated before use This

is best done in a box titration Choose several antiviral antibodies for which you

have homologous antigen slides.

2 Serially dilute MAb m PBS with 0 1% sodmm azlde and 2% pemclllm-strepto-

mycm, starting at 1: 100 in twofold dilutions to 1.10,240

3. Refix antigen slides as for indirect assay protocol.

4 Add 1615 mL of each detecting antibody in the dilution series to one spot on the

antigen slide Incubate at 37°C for 1 h. Wash slides as in Subheading 3.4.2.

5. Prepare a dilution series for the fluorescein-isothiocyanate-conjugate in 1:4000

trypan blue with 0.1% sodium azlde.

6. Add 10-15 pL of each dilution m the series to one set of the dllutlons of detectmg

antibody. Incubate at 37°C for 1 h Wash slides as m step 4

7. Affix coverslips as for the indirect assay protocol

8. Read slides with a fluorescence microscope. There must be at least 100 cells per

well to assessaccurately the extent of the antibody-antigen reaction Optimal con-

Jugate dilution is that dilution yielding 4+ fluorescence at the highest antibody dllu-

tion. This dilution 1sused in all subsequent tests performed with this conjugate lot.

3.4.3.2. NORMAL TISSUE CULTURE CELL SLIDE

A slide of uninfected cells prepared in the same manner as for infected cells

must be run in the same manner as the unknowns in all tests. This slide indi-

cates any nonspecific reaction between the antibody and normal tissue culture

cells of the type used.

3.4.3.3.

SERUM AND MABS

1. Use normal sera from the species in which the antibody was produced to show

the level of nonspecific fluorescence between the species and the tissue culture

cell type used.


29/369

16

Roehrig et al.

2. Use a homologous antigen slide and an unrelated antigen slide for each MAb

used in the test. Use the homologous MAb and another unrelated antibody on

each slide. This will demonstrate that the MAb is specific for the antigen and

shows no crossreactivtty. Use a normal MAb that is an asctte produced from

mice inoculated with parental Sp2/0-Ag14 myeloma cells. Alternatively, any

MAb specific for an antigen other than alphaviruses (e.g., flavivuuses) can be

used as a negative control. This preparation will show if the procedure for pro-

ducmg the MAb causes any nonspecific reactions between the antibody and the

tissue culture cells used.


When the slides are read, each well is ranked upon the following scale: 4+

(positive cells fluoresce intensely); 3+ (positive cells fluoresce brightly); 2+

(positive cells fluoresce to some degree, less than brtghtly); I+ (cells fluoresce

dully); f (varying degrees of fluorescence that may or may not be specific);

-(no fluorescence of the cells; cells appear red from the counterstain). Positive

wells must be ranked 2+ or higher. Results are reported as a simple positive or

negative by IFA (see Notes 12 and 13).

4. Notes

4.1. Antigen Capture ELISA

1. If you are unsure about the results because absorbance values are close to the

negative cutoff, retest the pool, or use a backup test such as plaque assay in

Vero cells.

2. The serologic reactivities of the MAbs used m these assays are shown m Table 1.

3 Occasionally, high backgrounds with uninfected control mosquitoes may be

observed. In this case the test should be repeated.

4 For this and all other ELISA assays, MAb reagents are in the form of mouse-

ascitic luids. MAb-enzymeconjugatesare commercialpreparationsusing ascetic

fluids supplied by our laboratory (Jackson Immunochemicals). Reagent potency

may vary depending upon preparations and should be independently determined

before use.

4.2. IgM-Capture ELISA

5. Store all diagnostic specimens at -20°C prior to and after testing. Avoid repeated

freeze-thaw cycles, which tend to inactivate IgM.

6 This test is used if serum or CSF samples have been drawn within 45 d of onset

7 In the event that a very early CSF or serum IS negative by this test, a convalescent

serum specimen must be requested and tested before that patient is reported as

negative for serological evidence of recent viral mfection. Without testing of a

convalescent specimen, a negative result may reflect testing of an acute-phase

specimen obtained before antibody response.


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Alphaviruses 17

8. Occasionally the test serum will be highly positive when tested with the normal

SMB antigen. The reason for this is unknown. If this happens, the test should be

repeated. If high backgrounds persist, another test must be used.

4.3. IgG ELISA

9. We have tried a number of detector antibodies m this test The IgG (Fc-spe-

cific)-AP conmgate gives us the best results with the lowest backgrounds.

10 Using the lA4B-6 MAb as capture antibody for all alphaviruses allows for easy

antigen standardization.

11. Remember that antialphavirus IgG is in general more crossreactive than IgM; there-

fore, the specificity of this test is less than that of MAC-ELISA

4.4. IFA Assay

12. If any of the controls do not perform within the expected reaction range, the test

must be repeated.

13 Unlike normal polyclonal antiviral antibodies, MAb reagents are of extremely

high potency. Be sure to dilute them out appropriately. Using MAb

reagents at low dilutions results in false-positive staining This high activity

is why tt is imperative to quantitate MAb dilution by endpoint box titration

prior to use.

References

1. Roehrig, J. T. (1986) The use of monoclonal antibodies m studies of the structural

proteins of alphavnuses and flaviviruses, in The

G-uses The Toguvrrzdae and

Flaviviridae (Schlesinger, S. and Schlesinger, M. J., eds.), Plenum, New York, pp.

25 l-278.

2. Tsai, T. H (1992) Arboviruses, in

Manual of Clinical Laboratory Immunology,

4th ed (Rose, N R , Marcario, E C., Fahey, J L., Friedman, H , and Penn, G M.,

eds ), American Society for Microbiology, Washington, DC, pp. 6066 18.

3. Monath, T. P., Nystrom, R. R., Bailey, R. E., Calisher, C. H., and Muth, D. J. (1984)

Immunoglobulm M antibody capture enzyme-linked nnmunosorbent assay for

diagnosis of St Louis encephalitis. J Clm Muzroblol 20,784-790.

4. Voller, A., Bidwell, D., and Bartlett, A. (1976) Microplate immunoassay for the

immunodtagnosis of vu-us mfections, in

Handbook of Clznzcal Immunology

(Rose,

N R and Friedman, H H., eds.), American Society for Microbiology, Washmg-

ton, DC, pp. 506-5 12.

5. Roehrig, J. T., Hunt, A. R., Chang, G.-J , Sheik, B., Bolm, R A., Tsar, T F , and

Trent, D. W. (1990) Identification of monoclonal anttbodies capable of differenti-

ating antigemc varieties of eastern equine encephahtis viruses.

Am J Trap. Med

Hyg 42,394-398.

6. Hunt, A. R and Roehrig, J. T. (1985) Biochemical and biological characteris-

tics of epitopes on the El glycoprotein of western equine encephalitis virus

Vzrology 142,334-344.


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7 Roehrtg, J T , Bolin, R A , Hunt, A R., and Woodward, T M (1991) Use of a

new synthetic peptide derived monoclonal antibody to differentiate vaccine from

wild-type Venezuelan equine encephalomyelms vnuses. J Clan Mlcroblol 29,

63@631.

8. Roehrtg, J. T., Day, J. W , and Kinney, R. M. (1982) Anttgemc analysts of the

surface glycoprotems of a Venezuelan equme encephalomyelms virus (TC-83)

using monoclonal antibodies. Vzrologv 118,269278.

9. Roehrig, J T and Mathews, J H (1985) The neutralization site on the E2 glyco-

protem of Venezuelan equine encephalomyelms (TC-83) vuus IS composed of

multiple conformationally stable epitopes. firology 142,347-356.

10 Rico-Hesse, R., Roehrtg, J. T., and Dickerman, R. W. (1988) Monoclonal anti-

bodtes define antigemc variation within the ID variety of Venezuelan equine

encephalitis virus Am J Trop Med Hyg 38, 187-194

11. Karabatsos, N., Lewis, A. L., Cahsher, C. H., Hunt, A. R., and Roehrig, J. T

(1988) Identtficatton of Highlands J vuus from a Florida horse. Am J Trop Med

Hyg 39,603-606

12. Schmaljohn, A L., Johnson, E. D., Dahymple, J. M., and Cole, G. A. (1983) Non-

neutralizing monoclonal antibodies can prevent lethal alphavuus encephahtts

Nature 297, 70-72

13. Boere, W. A. M., Harnsen, M , VmJe J , Benaissa-Trouw, B J , KraaiJeveld, C

A , and Snippe, H. (1984) Identificatton of distmct determmants on Semliki For-

est virus by using monoclonal antibodies with different antiviral activities

J Vwol. 52,575-582.

14 Tsai, T. F., Bolin, R. A., Montoya, M., Bailey, R E , Francy, D. B., Jozan, M , and

Roehrig, J. T. (1987) Detection of St. Louis encephalitis virus antigen m mosqut-

toes by capture enzyme mmmnoassay. J Clan. Mtcroblol. 25,370-376.

15. Wulff H. and Lange J. V. (1975) Indirect tmmunofluorescence for the diagnosis

of Lassa fever infection. Bull WHO 52,429-436

16 Wulff, H., Lange, J. V., and Webb, P. A. (1978) Interrelationships among

arenavuuses measured by indirect immunofluorescence. Intervlrology 9,344-350

17. Riggs J. L. (1979) Immunofluorescent staining, in Diagnostic Proceduresfor Viral,

Rickettsia, and Chlamydlal Infectzons, 5th ed. (Lennette, E. H. and Schmidt, N. J.,

eds ), American Public Health Association, Washington, DC


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2 Jiang and Matson

7 ,7 kb

RNA

2C 3C 30

C V O R Fs : ~ : : : c a ~ ~ 5 _ 5 _ _ _ :

~ t O , R ~

3O

C? 3C

,;

---7ZZI::2

c~std

Fig I GeHomic organiza t ion of Ht~CV and as t ro vim s The top l ine represents the

o f ca l i c iv ims (CV) and ast rOv irus (As t )a re shown b e low the 2C, 3 C and 3D m o t i f

a r e c o n s e rv e d i n m o s t s i n g le o st ra n d ed R N A v i ru s e s i a c u d i n g p ic o rn a v iru S e s ,

cal{civTruses~ and aS~rov iruses A flame=shift m echan ism is re spon sible :fro-the exp res-

s ion off ice second open readi~g f lame (ORF) of ast rovi ra l genom e:

5 ' m t h e c a p s l d g e n e o n t h e g e n o m e . I n a s t r o vi r u s e s, a f r a m e s h i f l m e c h a n i s m i -

r e q u i r e d f o r e x p r e s s i o n o f t h e e nt i re n o n s t r u c t u m l g e n e s ( 5. D~ A m i n o a c i d

s e q u e n c e m o l i t~ ~ 2C . 3 C . a n d 3 D~ c o m m o n l y f i mn d i n t h e o t h e r s i n g l e - s t r a n d e d

R N A v i ru s t h rn i |i e s, s u c h a s p i c o rn a \ u 'u s es , a ls o a r e fo u n d in t h e g e n o m e s o f

a s m w i ru s e s a n d H u C V s . b u t th e 2(_ r e g i o n i n t h e a st ro v i r a l g e n o m e i s n o t c l e a r

5- .ZIO.1I) . T he nuc leo t ide se que nce s enc od ing these motil~s a re h ighl5 co t , -

se rved wi f l f in a ~i~n~ily b m y a w s i g n i f i c a n l l y a m o n g t h e t h m f l i e s .

T h i s c h a p t e r d e s c ri b e s a m e t h o d f or d e t e c ti n g a s m w i m s e s a n d l t u C V s m

s t o c f l s p e c H T ~ e n s u s i n g t h e r e v e r s e t r a n s c r~ p t i o n -p o l y me ra s e

ch in

r e a c t i o n

R T~P(7 R k R h n i l a r m e t h o d s l i k e l y c a n b e a p p l i e d t o o t h e r R N A v i ru s e s in s t o o l

spec imens_ il" t he appropr ia~ .e nuc leo f ide p r im ers a re used . T he m e th od s a re

d i v i d e d i m o t w o pa r~ s: e x ~ r a c tm n o f v i ra l R N A t m m s t o ol s p e c i m e n s a n d

R T - P C R a m p l i f i c a t i o n a n d d e te c t i o n o f v ir al R N A . W e e m p h a s i z e t h e e •

n o n , d v~ ra l R N A f ro m s to o l s p e c i me n s , b e c a u s e t h e q ua [it> o f t h e v i r a l R N A

s fl~e m o s t i m p o r t a m e l e m e n t f o r s u c c e s s o f t h e m e t h o d . H u m a n s t o ol s p e c >

m e n s c o n t a i n a n u m b e r o f u n c h a m c t e d z e d i n h i b i t o r s o f r e v e r s e t ra n sc r ip ~ a se

a n d )'7~q p o l y m e ra s e R e m o v a l o f t h e s e i n h i b i mrs w i t h o u t t os s o f t h e vi r a l R N A

~s e s s e n t i a l I n a d d i t i o n v ir a l R N A i s e a s i l y d e g ra d e d i f s t o o l s p e c i me n s c o n -

r ain R N a s e . T h e c e t y l t r i m e t h y l a m m o n i u m b r o m i d e { U T A B } m e t h o d d e s c r i b e d

here e f t ]c ien i{y re m ov es inh ib i to rs o f t he enz3 rues fY{ma s too l an d genera t e , .

M g h q u a l i t y v i r a l R N A fo r R T -P C R o T h i s m e t h o d w a s d e v e l o p e d o r ig i n a l I 3 fb r

the de t e c t ion ~ fN or w a l k v i rus ( /2~ . i n l he l as t seve ra l yea rs . {1 has been ad ap te d

h> ma n } / a b o rm o r i e s fo r d e t e c t i o n a n d g e n e t i c a n a ly s i s o f o t h e r c a / i c iv i ru s e s .

a s ~ r o v i r u s e s _ a n d p i c o m a w r u s e s 1,3 ,13-I6) . T h i s c h a p t e r d e s c r i b e s m c e m

mo d J f i c a t i o n s o f {h e me t h o d b a s e d o n t h e s e s t u d i e s .


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Human Caliciviruses and Astroviruses

21

2. Materials

2.1. Reagents and Equipment Used for Extraction

of Viral RNA from Stools

1 Freon (1,1,2-trichloro- 1,2,2-trifluoroethane); also called Genetron (DuPont,

Wilmington, DE). store at room temperature.

2. 2X PEG solution. prepare 16% polyethylene glycol-6000 or 8000,O 8 MNaCl m

sterile distllled water. Aliquot and store at room temperature.

3. 2X Proteinase K digestion buffer: 0.2 M Tris-HCl, pH 7 5, 25 mM ethylenedl-

amme tetraacetic acid (EDTA), 0 3 A4 NaCl, 2% (w/v) sodmm dodecyl sulfate

(SDS). Aliquot and store at room temperature.

4 Proteinase K stock solution make 10 mg/mL of protemase K m 1X protemase K

digestion buffer. Ahquot and store at -20°C.

5 10% CTAB solution. prepare 10% CTAB (also called hexadecyl, tnmethyl-

ammonium bromide, Sigma H5882, Sigma, St. Louis, MO) m distilled water.

Store at room temperature This solution may crystallize at room temperature

Warm at 55°C to dissolve the crystals prior to use.

6. 4 A4 NaCl solution* prepare 4 M NaCl solution with dlstilled water and store at

room temperature

7. Water-saturated phenol: any commercially available phenol, phenol/chloroform,

or phenol/chloroform/lsoamyl alcohol that 1ssaturated with distilled water, blotech

research grade, peroxide-free. Store at 4°C.

8 Chloroform: molecular biology grade, peroxlde-free Store at room temperature.

9. 4 A4 Sodium acetate. prepare 4 M sodium acetate solution in distilled water and

store at room temperature

10 Ethanol solutions* prepare 100 and 70% solutions m dIstIlled water and store

at -20°C

11 Mlcrocentrlfuge and Eppendorf 1 5-mL reaction tubes

2.2. Reagents and Equipment Used in RT-PCR

1. 10X PCR buffer. 100 mM Tris-HCl, pH 8.3, 15 mM MgCl,, 500 mA4 KC1 Ali-

quot and store at -20°C.

2. RNasin: 40,000 U/mL (Promega, Madison, WI). Store at -20°C

3. Deoxynucleoslde trlphosphate mixture (dNTPs): combme 10 mMdATP, dGTP,

dCTP, and dTTP (Promega). Ddute to 0.5 mM each and store at -20°C.

4. AMV-RT. avlan myeloblastosls virus reverse transcriptase, 20 U/pL (Life Scl-

ences, Inc. 007-5, St Petersburg, FL). Store at -20°C.

5. Amp11

Taq:

recombmant

Taq

DNA polymerase, 5 U/pL (Perkm-Elmer/Cetus

N801-0060, Foster City, CA) Store at -20°C.

6. Primers for type-specific and type-common detection of CahcIvIruses.

7. Primers for type-specific and type-common detection of astrovu-uses

8 Mineral oil.

9. Programmable thermocycler.

10 Eppendorf 0.5-mL reaction tubes


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22

Jiang and Matson

11 6X Sample buffer: 0.25% bromophenol blue, 0.25% xylene cyanol, 40% (w/v)

sucrose in distilled water. Store at 4°C

12. 5X TBE buffer: add 54 g Tris base, 27.5 g boric acid and 20 mL of 0.5M EDTA

(pH 8 0) into distilled water to make 1 L final vol. Store at room temperature.

13 Ethidium bromide: prepare a stock solution of 10 mg/mL m water Store at room

temperature and keep away from light

14 SeaKem agarose: make 0 5%1% SeaKem agarose powder (FMC BloProducts,

Rockland, ME) in 1X TBE buffer Boil for 3-4 mm and pour the gel

15 Equipment for agarose gel electrophorests. submarine gel electrophorests appa-

ratus and power supply.

16. UV light illummator

3. Methods

3.1. Storage of Stool Specimens

Stool specimens can be kept at 4°C for weeks after collection. For long-term

storage, -2OOC or -70°C is recommended. Avoid multrple freezing and thaw-

ing of stool samples and exposure to hrgh pH because viral parttcies tend to

degrade under these conditions,

3.2. Extraction of Viral RNA from Stools

Extractron of viral RNA from stool specimens IS the crmcal step of the

method. The viral RNA 1s particularly susceptrble to RNase after samples

are

treated with protemase K, which removes the viral capsld and exposes

the viral RNA. Process the samples as quickly as possible according to the

followmg protocol. To prevent cross-contaminatron and contammation by

carryover PCR products, the location for extracting viral RNA should be

separated from that for amplificatron, detection, and clonmg of PCR products

(see Note 6).

1. Place 300 mL of stool suspension (lO-50% m water) in an Eppendorf tube

Extract once with an equal volume of freon by vortexmg for 30 s followed by

centrlfugatron for 5 mm m a microcentrtfuge Remove the supernatant (do not

disturb the Interface) and transfer to a new Eppendorf tube (see Note 1).

2 Add 300 mL 2X PEG buffer to the supernatant at a final concentratton of 8%

PEG and 0.4 M NaCl. Incubate the sample for 30 mm at 4°C and then centrifuge

in a microcentrtfuge for 15 min at 4°C.

3. Remove the supematant by aspiration

Documents

Diagnostic Virology Protocols