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et al, unpublished).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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