Vol.

167,

March

No.

2, 1990

BIOCHEMICAL

BIOPHYSICAL

AND

RESEARCH

COMMUNICATIONS

Pages

16, 1990

425-430

RAPID DETECTION OF INFLUENZA VIRUS El BY THE POLYMERASE CRAIN REACTION AlbCric

Bressoud,

Jeannette

Otto

Ealler*

Whitcomb,

Charareh

and Peter

Cerutti

Department of Carcinogenesis, Swiss Institute Cancer Research, 1066 Epalinges/Lausanne, *Department Albert

of Virology,

Ludwig

Clinical

Institute

Freiburg,

for Experimental Switzerland

of Medical

University,

Pourzand,

Microbiology

Eermann Berderstr.

IGN,

11, 6866

West Germany

Received January 8, 1990 We applied a combination of reverse transcription (RT) with the polymerase chain reaction (PCR) for a rapid detection of influenza virus HI subtype. We amplified a 441 bp segment of relatively high genetic stability of the hemagglutinin gene. Experimental conditions were established using plasmid DNA and infected cell cultures. The test was applied to 28 nasopharyngeal lavages from patients, two of which were positive for influenza virus Hl. When the amplified DNA of a positive sample was sequenced we found 97% homology with the recent strain A/USSR/70. 0 1990Academrc Press,Inc. The aim of the

detection

virus often

remains escapes

variation dase our fic

of

(5,6,7).

work

was to

influenza

develop

A virus

its

high

for

the

its two surface We have applied

specificity

other

in

oriented

The

in man. continued

of

heat

primers

for A

virus

antigenic

flanking

denaturation,

a speci-

hybridization PCR allows the DNA. Because of

PCR has been used successfully

viruses

including

HPV (Whitcomb variation of

A virus. in the

HIV,

(2,3)

rhinovirus

et al, unpublished). influenza A virus can repre-

for its detection. "Antigenic the surface glycoproteins

each strain of type in major modifications

method Influenza

antigens hemagglutinin and neuraminithe polymerase chain reaction (PCR) in

and sensitivity of

and in our laboratory The continued antigenic

within sists

and rapid material.

of the primers with Taq polymerase, of unique sequences in total genomic

detection

sent a difficulty minor changes

a simple

in clinical

an important cause of morbidity immuno-surveillance because of the

With two oppositely test (I). DNA segment, repeated cycles

and extension amplification

(4)

our of

drift" occurring

consists in gradually

Secondly "antigenic shift" glycoproteins causing the

conappea-

0006-291X/90

425

$1.50

Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.

Vol.

167,

No.

BIOCHEMICAL

a "new virus". segments of

rance of of eight fourth

2, 1990

segment

importance

The

of

this

genome

single

codes

for

AND

glycoprotein

in

of

the

known

seven

we have

MATERIALS

relatively primers was

chosen

influenza

high

segment

sequences

of

from

96 to

base

to

The the

of influenza amplify by

PCR a region

genetic

of

stability.

A a

this The

sites of the oligoBy comparison of

hemagglutinin base

composed of

Because

the classification we have chosen and

is polarity.

hemagglutinin.

(RT)

COMMUNICATIONS

A virus negative

stable sequences for the of particular importance.

nucleotide the

RESEARCH

RNA of

viral

viruses and their pathogenicity, combination of reverse transcription which possesses segment relatively selection nucleotide

of

stranded the

BIOPHYSICAL

HI

(8,9,10,11)

536.

AND METHODS

Oligonucleotide primers for RT and PCR of a portion of the hemagglutinin gene of influenza virus Hl were synthesized on a DNA synthesizer (Bio Applied Systems) and purified on Sephadex NAP-24 columns. The relevant portion of the hemagglutinin gene and the positions of the primers are shown in Figure IA. E. coli strain HP11 was transformed with plasmid pSP64HAM (recei(14) containing a cDNA insert of the hemagglutived from P. Palese) nin gene from influenza virus A/PR/8/34. Plasmid DNA was purified on a CsCl gradient, ethanol precipitated and resuspended in IOmM Tris pH 7.4, 0.1 mM EDTA buffer (TE). Isolation

of

viral

RNA

Influenza virus Hl (A/WSN/33) (8) suspensions in phosphate buffered saline containing 0.2% albumin were extracted three times with acidic guanidium thiocyanate-phenol-chloroform (12). Viral RNA was precipitated in the presence of t-RNA carrier (lug/d) by the addition of 2.5 volumes 90% ethanol at -2OO. The pellet was washed with 80% ethanol, dried and resuspended in sterile water. Reverse

transcription

and amplification

of

viral

RNA

Extracted viral RNA or viral RNA in sonicates of infected cells (sonication under paraffin oil for 15 seconds at level 2 on a Branson Sonifier B15) was reverse transcribed at 42O for 30 min in a total volume of 5ul containing 50mM Tris HCl pH 8.3, 50mM KCl, 7mM BSA, ImM each dATP, dTTP, dCTP, dGTP, 0.1% TritonNC12 r 17Oug/ml x-100, 1mM DTT, IOU Rnasin (Promega), 10mM 2-mercaptoethanol, 0.2ug of each primer and 7U AMV Reverse Transcriptase (Pharmacia). cDNA obtained by reverse transcription or plasmid DNA were amplified in a total volume of 25~1 containing 50mM Tris HCl pH 8.3, 50mM 170ug/ml BSA, ImM each dATP, MgC12, 7mM KCL, 10mM 2-mercaptoethanol, dTTP, dCTP, dGTP, 0.4ug of each primers, 3% DMSO and IU Taq Polymerase (Biofinex, Praroman, Fribourg, Switzerland). The reaction mixtures were subjected to 30-40 cycles of amplification at two temperatures (Ampligene Apparatus, Moretronic, Switzerland). Each cycle consisted of 97 set incubation at 590 and 85 set at 910. Amplified samples were electrophoresed in a 2% agarose gel containing 8.7ug/ml ethidium bromide and 1 x TBE buffer. 426

Vol.

167,

RESULTS

No.

2, 1990

developed

virus

HI

cription

(RT)

tion (PCR). 96 to base

RNA in of

pensions,

For 536)

amplification DMSO. For

re

which

clinical

RESEARCH

allows

material

the

by

COMMUNICATIONS

conditions

were

viral chosen

it

cDNA,

sonicates

the

necessary

to

a suspension

of

was

diluted

experiments from canine band after

the

of

the

441

bp

band

with

from

10B2

acidic

gels

detected virus

in sonicates Hl(A/WSN/33).

3% virus guani-

in fold

low6

relationship the virus

on the

a

virus susand simian

shown to

1

at 441 bp on a 28 cycles of

RNA with

experiment

re extraction of the RNA and RT/PCR. A linear observed between the relative concentrations of intensities

reac-

bp segment (base virus (see Figure

in

In the

virus

chain

in a buffer containing and amplification of

extract

thiocyanate-phenol-chloroform.

trans-

441

a visible obtained

pg SP64HAM plasmid reverse transcription

Influ-

reverse

viral RNA extracted of cultured bovine,

amplimer pair bromide gel was

of

polymerase a the

established

1

was

detection

combining

this purpose we have chosen of the hemagglutinin gene of

from efficient

suspension IB

BIOPHYSICAL

RNA by AMV RT with

and RNA in

cells. With the 2% agarose-ethidium

in

a method

viral

Optimal test A). plasmid containing

dium

AND

AND DISCUSSION

We have enza

BIOCHEMICAL

determined

Figubefo-

was and the

by

densito-

of

cultured bp frag-

metry. Viral RNA bovine (MHDK) ment

of

the

could cells cellular

be directly infected with H-globin

gene

(base

positions

A

201

717 to

926,

see

A 44 89 134 179 224 269 314 359 404 449 494 539

CCUACUGGUCCUGW AAUAUGUAUAGGCUA CACAGUACUCGAGAA GCUCGAAGACAGCCA AGCCCCACUACAAW GGGAAACCCAGAAUG CUACAWGUAGAAAC AGGAGAULIUCAUCGA AGUGUCAUCAUUCGA AUGGCCCAACCACAA UGAGGGGAAAAGCAG GAAGGAGGGCUCAUA

FIGURE ~-

AUGUGCACWGCAGC CCAUGCGAACAAWC GAAUGUGACAGUGAC CAACGGAAAACUAUG GGGGAAAUGUAACAU CGACCCACUGCWCC ACCAAACUCUGAGAA CUAUGAGGAGCUGAG AAGAWCGAAAUAW CACAAACGGAGUAAC UUUWACAGAAAUW CCCAAAGCUGAAAAA

P I i

UGCAGAUGCAGACAC AACCGACACUGWGA ACACUCUGWAACCU UAGAWAAAAGGAAU CGCCGGAUGGCUCW AGUGAGAUCAUGGUC UGGAAUAUGWAUCC GGAGCAAWGAGCUC UCCCAAAGAAAGCUC GGCAGCAUGCUCCCA GCUAUGGCUGACGGA UUCUUAUGUGAACAA

\

20-

+

2 $

\

lo-

+ \

4 kJ

0

I 1 o-2

10-4

10-S

VIRUS DILUTIONS

1

A: the hemagglutinin Sequence of (PR/8/34) chosen for amplification the amplimers are underlined. -B:

portion of (positive

Relationship between virus concentration band on an agarose gel by the RT/PCR 421

and assay.

influenza strand).

intensity

virus Positions

of

the

AH1 of

441

bp

Vol.

167,

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ref 13) was amplified simultaneously and served as internal conand 6 of the gel shown in Figure 2A contain the trol. Lanes 4,5, results with 2000, 200 and 20 infected MBDK cells. The diagnostic 441 bp band originating conditions the

from

and attests

@-globin

to

few years Department

cDNA is

sensitivity

(MK) cells

visible

of the

which

under assay.

all three As expected

intensity with decreasing to sonicates of cultured had been infected

over

the

with fresh isolates of influenza Hl (obtained of Microbiology, University of Lausanne). Viral

could

be detected

(data

not

occurred

viral

bands at 201 bp diminish in The same protocol was applied

cell number. canine (MDCK) and simian last the

the

shown).

from RNA

in all 10 cell sonicates which had been analyzed These results indicated that no major changes had

recently

in

Type

HI

virus

in

the

primer

sequences

which

were used for the RT and PCR of the hemagglutinin gene. The test was applied to the analysis of 28 fresh nasopharyngeal lavages

from

patients

who were suspected

to

be infected

with

Influ-

to contain free virus enza virus A. Since such samples were expected as well as infected cells they were extracted with acidic guanidium thiocyanate-phenol-chloroform. As shown in Figure 2A, lanes 7 and 8, the

presence

of viral

RNA HI was detected

in two of

the

28 samples.

A 260 bp fragment clinical Bluescript

of the amplified viral sequence of a positive by the dideoxy method (15) using sample was determined plasmid as vector. We found 85% homology between this

sequence

and

1933

corresponding

Homology

(8)).

isolated amplified

the

of

sequence of A/WSN/33 (isolated in 97% was found with A/USSR/70 (which had been

in 1970 (10)) demonstrating the close sequence of our most recent isolate.

PCR was also performed order to further increase sible to obtain a visible amplificiation DNA a visible appeared

only

relationship

with

the

in the presence of [alpha 32PldATP in the sensitivity of the test. It was pasband on autoradiograms after 18 cycles of

from 1Opg sp64HAM plasmid. With the same amount of fluorescent band on an ethidium bromide agarose gel after

ments with infected was observed in the

25 cycles

of

amplification.

However,

cells, no significant increase test system using radioactivity.

in experi-

in sensitivity Figure 2B shows

autoradiograms of the amplified viral DNA fragments obtained Hl-infected canine and simian cell strains. Our results demonstrate that the combination of RT with allows the rapid and sensitive detection of influenza clinical samples without the need to grow the virus on eggs or in cell cultures. The choice of primer sites in more stable portions of the hemagglutinin gene reduces 428

from PCR

virus Hl in embroynated genetically the risk of

Vol.

167,

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

1234567

COMMUNICATIONS

8

bp 998 631 506 396 298 221 154

bp 441

123456

B bp 998 -

$3: x 506 -

bp 441

396 344 298 --

FIGURE ~A: -

2

Amplification polynucleotide

of Influenza chain reaction.

Hl

reverse

transcription

1:

Molecular EcoRI.

Lane

2:

RNA extracted mers).

Lane

3:

1 pg SP64HAM

Lane

4-6:

Influenza Hl (WSN33) infected bovine specific amplimers and amplimers sequence). Lane 4, 2000 cells; lane 5, 200 cells;

MBDK cells for bovine

Influenza Hl positive cific amplimers).

lavage

7,0:

standard,

by

Lane

Lane

weight

Virus

from

pBR322

virus

plasmid

digested

suspension

with

(virus

and Hinf

specific

I and ampli-

DNA.

nasopharyngeal

lane

6,

(virusS -globin 20 cells.

(virus-spe-

-B. Amplification of using ]alpha-32P]

influenza dATP.

virus

Lane

1:

Molecular EcoRI.

weight

Lane

2-6:

Inlfuenza cells.

Hl

Hl

standard,

(recent

stains)

by reverse pBR322 infected

transcription digested canine

and

with and

Hinf simian

PCR

I and

Vol.

167,

false

No.

negatives.

internal Sequencing tion

of

BIOCHEMICAL

2, 1990

base of new

It

should

pair in the the amplified mutations

which

AND

be noted primer viral may

BIOPHYSICAL

that

sequence sequence have

RESEARCH

COMMUNICATIONS

a mutational change of an can be tolerated by PCR. readily

occurred

allows relative

the to

detec-

previous

isolates. ACKNOWLEDGMENT This

work

was

supported

by the

Swiss

National

Science

Foundation.

REFERENCES Saiki, R.K., Gelfand, D.H., Stoffels, S., Scharf, S-J., Higuchi, (1988) Primer-directed R ., Horn, G.T., Mullis K.B., Erlich, H.A. enzymatic amplification of DNA with thermostable DNA polymerase. Science 239, 487-491. Murakawa, G.J., Zaia, J.A., Spallone, P.A., Stephens, D.A., Kap(1988) Direct detection lan, B.E., Wallace, R.B., Rossi, J.J. of HIV-l RNA from AIDS and ARC patient samples DNA 7, 287-295. B.J. (1988) Detection Byrne B.C., Li, J.J., Sninsky, J., Poiesz, of HIV-l RNA sequence by in vitro DNA amplification. Nucleic Acids Res. 16, 4165. Gama, R.E., Hughes, P.J., Bruce, C.B., Stanway, G. (1988) Polymerase chain reaction amplification of rhinovirus nucleic acids from clinical material. Nucleic Acids Res. 19, 9346. Klenk, H.D., Rott, R. (1988) The molecular biology of influenza virus pathogenicity. Advances in Virus Res. 34, 247-281. Air, G.M., Laver, W.G. (1986) The molecular basis of antigenic variation in influenza virus. Advances in Virus Res. 31, 53-101. Lamb, R.A., Choppin, P.W. (1983) The gene structure and replication of influenza virus. Ann. Rev. Biochem. 53, 467-506. Hiti, A.L., Davis, A.R., Nayak, DP. (1981) Complete sequence analysis shows that the hemagglutinin of the HO and H2 subtypes of human influenza virus are closely related. Virology

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2.

3.

4.

5. 6. 7. 8.

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Nature 10

113-114.

Winter, G., Fields, the haemagglutinin

9.

‘.

11 , ,.

12.

13. 14.

15.

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S., Brownlee, gene of a human

G.G. Nucleotide influenza virus

sequence ~1 subtype.

of

7375.

Flucy, R., Caton, A.J., Cox, N.J., Kendal, A.P, Brownlee, G.G. (1983) Antigenicity and evolution amongst recent influenza viruses of HlNl subtype. Nucleic Acids Res. 11, 7191-7203. Caton, A.J., Brownlee, G.G, Jonathan, Y.W., Gerhard W. (1982) The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (Hl subtype). Cell 31, 417-427. Chomczynski, P., Sacchi, N. (1987) Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Bioch. 162, 156-159. Schimenti, J.C., Duncan, C.H. (1984) Ruminant globin gene structures suggest an evolutionary role for Alu-type repeats. Nucleic Acids Res. 12, 1641-1653 Young, J.F., Desselberger, U., Graves, P., Palese, P., Shatzman, A . I Rosenberg, M. (1983) Cloning and expression of influenza virus genes. In: Laver W.G. Ed. The origin of pandemic influenza viruses. Elsevier, 129-138. Sanger, F., Nicklens, S., Coulson, A.R. (1987) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. 74,

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