Molecular biological methods in the diagnosis of viral disease

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Reviews

Experientia 43 (1987), Birkh/iuser Verlag, CH-4010 Basel/Switzerland 1189 40 Richman, D. D., and Wahl, G. M., Nucleic acid probes to detect viral

diseases, in: Concepts in Viral Pathogenesis, Ii, pp. 301-309. Eds A.L. Notkins and M. B. A. Oldstone. Springer, New York--Berlin- Heidelberg 1986.

41 Rindler, M.J., Ivanov, I. E., Plesken, H., Rodriguez-Boulan, E., and Sabatini, D. D., Viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same Golgi apparatus dur- ing their intracellular transport in doubly infected Madin-Darby canine kidney cells. I. Cell Biol. 98 (1984) 1304--1319.

42 Rodriguez-Boulan, E.J., and Sabatini, D.D., Asymmetric budding of viruses in epithelial monolayers: a model for the study of epithelial polarity. Proc. natl Acad. Sci. USA 75 (1978) 5071- 5075.

43 Rubin, D.tt., and Fields, B.N., The molecular basis of reovirus virulence: the role of the M2 gene. J. exp. Med. 152 (1980) 853-868.

44 Scheid, A., and Choppin, P. W., Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity by proteolytic cleavage of an inactive precursor protein of Sendal virus. Virology 57 (1974) 475-49(I.

45 Scholtissek, C., Influenza A viruses with noncleaved hemagglutinin are not internalized after adsorption. ArchsVirol. 90(1986) 159 163.

46 Shafritz, D. A., Shouval, D., Sherman, H. I., Hadziyannis, S.J., and Kew, M.C., Integration of hepatitis B virus DNA in the genome of liver cells in chronic liver disease and hepatocellular carcinoma. New Engl. J. Med. 305 (1981) 1067 1073.

47 Sharpe, A.H., Chen, L.B., and Fields, B.N., The interaction of mammalian reoviruses with the cytoskeleton of monkey kidney CV-

1

cells. Virology 120 (1982) 399-411.

48 Singer, R.H., Lawrence, J.B., and Villnave, C., Optimization of in situ hybridization using isotopic and non-isotopic detection methods. Bio Feature 4 (1986) 230-249.

49 Sixbey, J.W., Vesterinen, E.H., Nedrud, J.G., Raab-Traub, N., Walton, L. A., and Pagano, J. S., Replication of Epstein-Barr virus in human epithelial cells infected in vitro. Nature 306 (1983) 480-483.

50 Skehel, J., Bayley, P,, Brown, E., Martin, S., Waterfield, M., White,

J., Wilson, I., and Wiley, D., Changes in the conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion. Proc. natl Acad. Sci. USA 79 (1982) 968-972.

51 Steeg, H. van, Kasperaitis, M., Voorma, H. A., and Benne, R., Infec- tion ofneuroblastoma cells by Semliki Forest virus. The interference of viral capsid protein with the binding of host messenger RNA's into initiation complexes is the cause of the shut-off of host protein synthesis. Eur. J. Biochem. 138 (1984) 473M78.

52 Tashiro, M., and Homma, M., Pneumotropism of Sendal virus in relation to protease-mediated activation in mouse lungs. Infect. Im- mun. 39 (1983) 879-888.

52a Tashiro, M., Ciborowski, P., Klenk, H.-D., Pulverer, G., and Rott, R., Role of Staphylococcus protease in the development of influenza pneumonia. Nature 325 (1987) 536-537.

53 Trachsel, H., Sonenberg, N., Shatkin, A.J., Rose, J. K., Keong, K., Bergman, J.E., Gordon, J., and Baltimore, D., Purification of a factor that restores translation of vesicular stomatitis virus mRNA in extracts from poliovirus infected HeLa cells. Proc. natl Acad. Sci.

USA 77 (1980) 770-774;

54 Tycko, B., and Maxfield, F.R., Rapid acidification of endocytic vesicles containing cq-macroglobulin. Cell 28 (1982) 643-651.

55 Week, P.K., and Wagner, R.R., Inhibition of RNA synthesis in mouse myeloma cells infected with vesicular stomatitis virus. J. Virol.

25 (1978) 770780.

56 Wolf, J. L., and Bye, W.A., The membranous epithelial (M)cell and the mucosal immune system. A. Rev. Med. 35 (1984) 95 112.

57 Yamada, A., Tsurudome, M., Hishiyama, M., and Ito, Y., Inhibition of host cellular ribonucleic acid synthesis by glycoprotein of Mumps virus. Virology 135 (1984) 299 307.

0014-4754/87/11/121185-0551.50 + 0.20/0

~') Birkh/i, user Verlag Basel, 1987

Molecular biological methods in the diagnosis of viral disease

b y H. W o l f

M a x yon Pettenkofer Institute, University o f Munich, Molecular and Tumorvirology, Pettenkoferstrasse 9a, D-8000 Munich 2 (Federal Republic o f Germany')

Summary, M o l e c u l a r b i o l o g y allowed c o n s i d e r a b l e i m p r o v e m e n t s in d i a g n o s t i c p r o c e d u r e s by p r o d u c t i o n o f new a n d m o r e specific s o n d s for the d e t e c t i o n o f traces o f viruses, b o t h o n the nucleic acid a n d p r o t e i n levels, a n d by d e t e r m i n i n g the i m m u n e r e s p o n s e o f the h o s t to specific antigens. I m p r o v e m e n t s in sensitivity a n d i m p r o v e d c o r r e l a t i o n to the stage o f viral disease are already evident f r o m several a p p l i c a t i o n s a n d strongly suggest a b r o a d a p p l i c a t i o n o f these a p p r o a c h e s .

Key words'. Nucleic acid h y b r i d i z a t i o n ; s y n t h e t i c peptides; c o m p u t e r analysis; a n t i g e n selection; r e c o m b i n a n t a n t i g e n s ; r a p i d diagnosis,

Introduction

D e v e l o p m e n t o f virus-specific t h e r a p y a n d the need for fast differential diagnosis for intensive medical care challenge q u i c k diagnosis w h i c h s h o u l d also allow c o n c l u s i o n s for the stage o f disease.

Classic m e t h o d s o f viral disease diagnosis are b a s e d o n the c u l t i v a t i o n o f agents, direct d e m o n s t r a t i o n o f viral antigens, e.g., b y i m m u n o f l u o r e s c e n c e , a n d the d e t e c t i o n o f specific a n t i b o d i e s . M a n y o f the p r o c e d u r e s c u r r e n t l y in use h a v e the d i s a d v a n t a g e t h a t the r e a d i n g o f results requires well-trained p e r s o n n e l a n d disease-related advice is o f t e n n o t given o r c o m e s t o o late. This is p a r t i c u l a r l y true for c h r o n i c neoplas- tic or r e a c t i v a t e d acute virus-related diseases. A d d i t i o n a l p r o b l e m s arise, e.g. f o r m H e p a t i t i s B, t h r o u g h l o n g transi- tions f r o m a n t i g e n o r a n t i b o d y excesses in c o n n e c t i o n with a c o m p l e t e exclusion o f r e m a i n i n g infectiosity.

Detection o f genetic material o f viruses by nucleic acid hybridi- zation

L A B E L S :

1) Isotopes: H 3, $35, p32, 1125 2) Reactive groups: A l l y l a m i n e - U T P

3) Haptens: Sulfonylation, Digoxin, Biotin

I N T R O D U C T I O N O F L A B E L S : - nick transl~tJon: f o r labels l , 2, 3 - random prime: for labels 1, 2, 3 - eDNA: for labels 1, 2, 3

- c R N A using SP6 or T7 Polyrnerases with appropriate promotor-containlng vectors:

for labels 1, 2 " ~ , ~ - -

- chemical modification, e.g. lodination, Photobiotin:

for labels 1, 3 - sandwich hybridization:

for labels 1, 2, 3

C o n s i d e r a b l y influenced b y the findings o f t u m o r virology, Figure 1. Introduction of label in nucleic acids for use as hybridization i.e., t h a t viruses persist in cells a n d c a n c h a n g e these w i t h o u t probe 1, 7, 8,13,14,16,19, 23, 24

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t ~90 Experientia 43 (1987), Birkh~user Verlag, CH 4010 Basel/Switzerland Reviews Serologic parameter of EBV-related diseases based on specific proteins produced by recombinant DNA technology.

Diagnosis Antibody class Antigen (ag-complex) Antibody class Antigen (ag-complex)

Acute infection Past infection Chronic infection Nasopharyngeal carcinoma

IgM BMRF- 1 (EA) IgG BMRF- 1 (EA)

IgM BcLF- 1 (VCA)

lgG BcLF- 1 (VCA)

IgG BKRF-I(EBNA-1)

IgG BcLF- 1 (VCA)

(in absence oflgG to BKRF-I (EBNA-1) and/or BLLF-1 MA as well as lgM to BMRF-1)

IgA BALF-2(EA) IgA BLLF-1 (MA)

t o t a l H B V D N A p o s .

N N g

HBE AG pos. 1051 805 77 (20-100)

Anti HBE pos. 360 141 16 (=-64)

HBE AG neg.,

Anti HBE neg. 175 36 21 (0-47)

Figure 2. Detection of HBV-DNA in serum with various serological parameters (Seelig et al. , including some of our own unpublished data). 20

the presence of structural proteins, the detection of viral nucleic acids was seen as a useful test for acute and chronic active diseases.

F o r this method to be widely used, the availability of appropriate probes leaving a detectable signal is an absolute requirement. Several methods have been applied to introduce a label into virus-specific D N A segments, derived almost ex- clusively from segments of the viral genomes cloned into the appropriate plasmid vectors. As a detection system, [32p]-labeled nucleic acids together with autoradiography are frequently used because of their low price a n d high-signal den- sity. Non-radioactive detection systems are being increasingly applied as the sensitivity is gradually improved and the possibilities of long-term storage a n d reduced environmental pol- lution are solved (fig. 1). The requirement to have a multitude of specific probes can cause logistic problems. Introduction of specific sequences in bacteriophages with single-stranded D N A in c o m b i n a t i o n with labeled universal probes to the phage portion allows the economic establishment of a large collection of unlabeled yet detectable probes 24.

In the case of Hepatitis B, viral D N A in serum can be de- tected with simple procedures. Serum samples can be applied to simple filtration units or directly to m e m b r a n e filters a n d tested for nucleic acids. Evaluation of test series from several laboratories demonstrates that nucleic acid hybridization is the best p r o o f of potential infectiosity of patients 2~ (fig. 2).

The difficulties in the diagnosis of acute diseases becomes evident with the d e m o n s t r a t i o n of cytomegalovirus in i m m u - nosuppressed individuals such as organ transplant recipients or those infected by h u m a n immunodeficiency virus (HIV).

Several methods have been compared. Virus isolation is too slow in any case. Short-term culture a n d detection o f viral antigens or nucleic acids in these cultures have a considerably accelerated diagnosis which is i m p o r t a n t in the face of virus- specific therapy. Because testing of several samples is usually little more work compared to tests with single samples, it seems worth the consideration to routinely screen over lon- ger periods persons at high risk for cytomegalovirus infection for virus release.

Using the example of bone marrow transplant patients, considerable variations of virus load could be demonstrated TM ~3.

A coordination of immunosuppressive therapy a n d / o r anti- viral therapy is possibly more effective t h a n waiting for the viral disease to reach its final stages. I m p o r t a n t for cytomegalovirus-related diagnosis is the use of specific segments o f viral D N A as a probe for several regions of viral D N A

cross-reacted with h u m a n D N A . In addition, care must be taken to avoid false-positive results by incorrect preparation of test samplesl?.

D e m o n s t r a t i o n of viral nucleic acids is o f interest and importance for virus-associated neoplasias of man, papillomavirus-associated carcinomas (e.g. of the cervix uteri) a n d Epstein-Barr virus (EBV)-associatd nasopharyngeal carci- n o m a (NPC) in particular. Detection of viral genomes can be done as described in figure 3. Cellular material can be ob- tained from biopsies, swabs m or collected with the help of simple aspiration devices is.

Frequently the distinction, whether tissue or lymph nodes represent m a l i g n a n t or benign growth or reactive processes, is difficult. In the case of virus-associated condition, the status of viral nucleic acids m a y allow i m p o r t a n t conclusions.

D N A of papillomavirus, for example, is integrated into the host genome (fig.4C) in a specifc m a n n e r while it is still present as a circular molecule (fig. 4B) in warts and condy- lomas 4. D N A of EBV in l y m p h o m a cells is present in a

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Figure 3. Nucleic acid hybridization for the detection of cytomegalovirus with urine samples of bone marrow transplant recipients. A low speed and a high speed pellet were prepared in consecutive order from 5 ml of urine collected at the weeks post transplantation given at each lane. For each sample low speed and high speed pellets are tested using a cloned fragment of C M V - D N A 13. A sample was considered positive when the high speed pellet yielded a stronger signal than the low speed pellet. K1 K3 represent samples taken from three different patients.

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Experientia 43 (1987), Birkh/iuser Verlag, CH 4010 Basel/Switzerland 1191 RE

I circular form (fig. 4B) while cells replicating virus contain

linear viral genomes (fig. 4A). Differentiation of these forms of viral D N A can help to distinguish m a l i g n a n t proliferative from reactive forms.

The tissues to be tested can be analyzed with the help of restriction enzymes for the presence of linear, circular or integrated viral D N A (fig. 4).

F o r diseases involving latency, sensitivity of detection can be a serious problem. Besides considerable progress in introduction a n d detection of signals in the hybridization probe (fig. 1) 2 , specific amplification of the relevant (viral) sequences in the test material has been developed as a powerful e n h a n c e m e n t of sensitivity (fig.4) m. The principle of the test 12 involves the addition of (synthetic) oligonucleotides complementary to two areas of both strands leaving a space of a few h u n d r e d nucleotides in between. The 'gap' can be filled in the presence of the Klenow fragment of D N A polymerase a n d nucleosidetriphosphates. The use of heat-stable D N A polymerase

(T. aquaticus)

allows several consecutive cycles o f amplification by a sequence of heating and anneal- ing/sequencing steps (fig. 5).

In the case of N P C it was necessary to correlate the presence of viral nucleic acid with the epithelial t u m o r cells. This was possible using frozen t u m o r sections a n d hybridizing viral D N A to the viral sequences in situ followed by autoradiography 22. This powerful technique was used for a multitude of questions and, with appropriate improvements 5, has been very useful for elucidation of pathogenetic mechanisms as shown for sudden deafness of m a n 6.

Immunological procedures

Classic serological procedures are n o t at all reduced in the overall importance by the possibilities of nucleic acid hybridization; rather, it becomes evident that inclusion of antibody class-specific tests and the use of well-characterized antigen systems allows considerably improved diagnostic judgement. F o r example, it was possible to develop test systems for the EBV-related NPC, which allows early diagnosis and which leads to considerable improvements of prognosis.

The increasing force for rationalization in the laboratories a n d the desire for exact d o c u m e n t a t i o n can only be fulfilled when pure antigens permitting machine-readable tests are available.

G e n e technological methods and chemical synthesis of segments of viral gene products offer the possibility of pure reagents, even for those agents which are difficult to grow in tissue culture.

RE

d e n o t u r e . ~ / a d d p r i m e r

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Figure 5. Amplification of selective segments of DNA in test material.

RE: restriction enzyme site.

The value of these techniques will be explained on the example of diagnosis of EBV a n d HIV-related diseases. I m m u n o - fluorescence only rarely allows detection of antibodies for specific proteins because these tests are neither machine- readable n o r available for documentation. Series of immu- noprecipitations with radioactive-labeled EBV-infected cells using sera from N P C patients, patients with infectious mononucleosis and sera from healthy EBV antibody-positive individuals were made in order to identify those viral proteins which seemed useful for identification of the particular health status due to the regular presence of the respective antibodies.

Parallel to the selection of antigens, proteins were mapped on the viral genome using hybridselected translation2< The identified genes were then cloned into bacterial plasmids such that transfected bacteria would produce these products.

Because m a n y viral proteins are n o t stable in heterologous expression systems, strategies were developed which allowed the identification of those protein segments responsible for their recognition by the i m m u n e system and to selectively express these segments (fig. 6) ~s. Repeated application of this

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Figure 4. Detection of the stage of viral DNA in infected tissue. Extracted total DNA is digested with the appropriate restriction enzymes, electro- phoretically separated by molecular weight on agarose gels, transferred to membranes and hybridized with a specific fragment of the viral genome.

The hatched band will be the only one visible in the excess of cellular DNA through the hybridization detection and by its molecular weight directed location allow the distinction of the stages of viral DNA 17.

F R I I I L

250

F R I V

F R V

F R V l l

~ C O O H

Figure 6. Computer analysis of an EBV gene (p138; BALF-2 EA). Pre- diction of secondary structure (c~ helix ~ , pleated sheet ~aM/, random coil /~A and fl-turn ~ ) with various algorithms (e.g. Refs. 3, 9) are superimposed with further parameters, here hydropathy (9 hydrophilic, hydrophobie). Areas with fl-turns in hydrophilic environment were found to be preferred areas for eliciting humoral immune response 2s. FR IVII: subcloned fragments of the p 138 gene.

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1192 Experientia 43 (1987), Birkhfiuser Verlag, CH4010 Basel/Switzerland Reviews t e c h n o l o g y allowed the d e v e l o p m e n t o f a n t i g e n kits w h i c h

p e r m i t diagnosis o f v a r i o u s diseases c a u s e d b y E B V (table).

T h e described a p p r o a c h is o f special i m p o r t a n c e also for diagnosis o f H I V infections. Because W e s t e r n blots derived f r o m viruses are often difficult to i n t e r p r e t due to the presence o f diffuse b a n d s o f g l y c o p r o t e i n s a n d v a r i a t i o n s in qual- ity. W i t h respect to the security c o n c e r n s for m a s s cultures o f H I V , tests h a v e been d e v e l o p e d o n the basis o f r e c o m b i n a n t p r o d u c t s . A c o m b i n a t i o n o f such p r o d u c t s allows definite diagnosis even in q u e s t i o n a b l e cases ( S o u t s c h e k - B a u e r , M o t z a n d Wolf, in prep,). A t the same time r e c o m b i n a n t a n t i g e n s , w h i c h m a y c o n t a i n the m a j o r a n t i g e n i c deter- m i n a n t o f viral p r o t e i n s , c a n be the source o f inexpensive a n d simple first screening tests ( M o t z , S o u t s c h e k - B a u e r a n d Wolf, in prep.).

O n e a l t e r n a t i v e to a n t i g e n s p r o d u c e d b y gene t e c h n o l o g y is the chemical synthesis o f peptides. A d i s a d v a n t a g e o f this m e t h o d is t h a t only s h o r t segments o f a p r o t e i n c a n be repre- sented. A p p l i c a t i o n o f a p p r o p r i a t e c o m p u t e r p r o g r a m s for e p i t o p e selection a n d the use o f several a n t i g e n s in one test c a n very effectively limit these d i s a d v a n t a g e s 25. A n a d v a n - tage o f this m e t h o d is t h a t n o p r o b l e m s w i t h foreign antigens exist a n d t h a t a n t i g e n s b e c o m e a v a i l a b l e shortly after a se- q u e n c e o f a gene is finished. This t h e n b e c o m e s p a r t i c u l a r l y e v i d e n t w h e n a new t e c h n i q u e for synthesis is used. W i t h this m e t h o d peptides are linked via ester b o n d a g e so stably to the c a r r i e r t h a t they r e m a i n b o u n d even t h r o u g h r e m o v a l o f the p r o t e c t i v e g r o u p s ( M o d r o w a n d Wolf, in prep.). It is a fea- t u r e o f this m e t h o d t h a t peptides need n o t be r e m o v e d f r o m the c a r r i e r followed b y t i m e - c o n s u m i n g p u r i f i c a t i o n a n d c o n s e q u e n t b i n d i n g to surfaces w h i c h always causes the re- d u c t i o n o f antigenicity. T h u s fast a n d inexpensive test systems can be d e v e l o p e d w h e n simple pieces o f e q u i p m e n t such as the millititer system f r o m M i l l i p o r e is used (fig. 7).

membrane- sealed 9 6 - w e l l plate

standard 96-well plate

vacuum

Figure 7. Solid phase ELISA test with peptide antigen coupled to resin beads (Modrow and Wolf, in prep.). Using modifications of Fmoc and tBoc chemistry of peptide synthesis resins containing deprotected oligo- peptides can be produced. These particles can be used in suspension to react with antibodies from serum samples and thus allow the development of diagnostic tests. The use of microtiter plates with membrane-mounted bottom including a fitted suction device for both incubation and washing greatly facilitates this technique of antibody detection.

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2 Burns, J., Chan, V.T.W., Jonasson, J. A., Fleming, K. A., Taylor, S., and McGee, O.D., Sensitive system for visualising biotinylated DNA probes hybridised in situ: rapid sex determination of intact cells. J. clin. Path. 38 (1985) 1085--1092.

3 Chou, P.Y., and Fasman, G. D., Prediction of the secondary structure of proteins from their amino acid sequence. Adv. Enzym. 47 (1978) 45 148.

4 Dfirst, M., Schwarz, E., and Gissmann, L., Integration and persis- tence of human papillomavirus DNA in genital tumors, in: Viral Etiology of Cervical Cancer, pp. 273 280. Eds R. Peto and H. zur Hausen. Banbury Report 21, Cold Spring Harbor Laboratory 1986.

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with nucleic acid hybridization in situ. J. Virol. 57 (1986) 335 339.

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7 Feinberg, A.P., and Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.

Analyt. Biochem. 132 (1983) 6.--13.

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