CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
SEROTYPE-SPECIFIC IDENTIFICATION OF
ENTEROVIRUS 71 BY RT-PCR
FIELD OF THE INVENTION
The present invention relates to reagents and methods for the
detection and analysis of enterovirus 71. In particular, the present invention
relates to nucleic acids, as well as kits and methods comprising these nucleic
acids, for detecting, amplifying, and sequencing nucleic acids of enterovirus
71.
BACKGROUND
io The two leading agents responsible for large outbreaks of hand-foot-
and-mouth disease (HFMD), a common, usually benign, rash illness in
children, are the enteroviruses coxsackievirus A16 (CA16) and enterovirus 71
(EV71 ). CA 16 and EV71 are closely related genetically, but EV71 is also
associated with severe neurologic disease such as encephalitis, meningitis,
~5 cranial nerve palsies, Guillan-Barre syndrome, and poliomyelitis-like
syndrome much more frequently than is CA16 (reviewed in Alexander, J.P., et
al., ~'Enterovirus 71 infections and neurologic disease - United States, 1977-
1991." J. Infect. Dis. 169;: 905-908 (1994); Chumakov, M., et al.,
"Enterovirus
71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria,"
2o Arch. Virol., 60:329-340 (1979); Gilbert et al., "Outbreak of enterovirus
71
infection in Victoria, Australia, with a high incidence of neurologic
involvement," Pediatr. Infect. Dis. J., 7:484-488 (1988); Landry et al.,
"Fatal
enterovirus type 71 infection: rapid detection and diagnostic pitfalls,"
Pediatr.
Infect. Dis. J., 14:1095-2000 (1995)). Two recent EV71 outbreaks in Asia
25 (Malaysia, 1997, and Taiwan, 1998) have involved thousands of HFMD cases
associated with severe neurologic disease and rapid death (CDC, "Deaths
among children during an outbreak of hand, foot and mouth disease --
Taiwan, Republic of China, April-July 1988," Morbidity and Mortality Weekly
Report,47:629-632 (1998); Chang et al., "Fulminant neurogenic pulmonary
30 oedema with hand, foot, and mouth disease," Lancet, 352:352-367 (1998);
Lum et al., "Fatal enterovirus 71 encephalomyelitis," J. Pediartrics 133:795-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
798 (1998); World Health Organization, "Outbreak of hand, foot and mouth
disease in Sarawak. Cluster of deaths among infants and young children,"
Wkly. Epidemiol. Rec., 72:211-212 (1997).
Enterovirus 71 exhibits a wide variation in clinical presentation. EV71
has been associated with severe central nervous system disease with a case-
fatality rate of 0% to 6% (Landry et al., 1995). During a large EV71 outbreak
in Bulgaria in 1975 (705 reported cases), there were 149 cases of paralytic
disease and 44 fatalities. Forty-five cases of EV71 infection were reported in
the United States in 1987, including eight cases of paralysis and one
fatality.
io (Alexander J.P., et al., 1994), and virus circulation was widespread, with
isolates reported in at least seventeen states.
Because of this wide variation in clinical presentation as well as
increased public health concerns associated with EV71 and its potential for
greater neurovirulence, it is important to be able to rapidly identify the
specific
~5 strain of EV71 during an HFMD outbreak and to be able to rapidly
discriminate between EV71 and CA16. Since early symptoms are similar for
HFMD associated with infections of different strains of EV71 or CA16, the
etiologic diagnosis depends on virus isolation and serotyping. Standard
serotyping involves neutralization tests with monospecific antiserum, which
2o are quite limited in their availability. Furthermore, antigenic typing is
often
hampered by non-neutralizable virus due to aggregation (Schmidt et al., "An
apparently new enterovirus isolated from patients with disease of the central
nervous system," J. Infect. Dis., 129:304-309 (1974); Blomberg et al., "New
enterovirus type associated with epidemic of aseptic meningitis and/or hand,
25 foot, and mouth disease," Lancet, 12:112-113 (1974); Nagy et al.,
"Virological
diagnosis of Enterovirus type 71 infections: experiences gained during an
epidemic of acute CNS diseases in Hungary in 1978," Arch. Virology, 71:217-
227 (1982). A second typing method using immunofluroescence techniques
with commercially available monoclonal antibodies has also been employed.
3o End-labeled nucleic acid probes in the VP2 region have also been used in
diagnostic tests for EV71 and CA16 (Kitamura et al., "Serotype determination
of enteroviruses that cause hand-foot-and-mouth disease; Identification of
_2_
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
enterovirus 71 and coxsackievirus A16 from clinical specimens by using
specific probe," Kansenshogaku Zasshi, 71:715-723 (1997)).
Since enterovirus serotypes are defined by neutralization using
immune sera directed against the capsid proteins, nucleotide and amino acid
sequences of the capsid region correlate with serotype (Oberste et al.,
"Molecular evolution of the human enteroviruses: Correlation of serotype with
VP1 sequence and application to Picornavirus classification," J. Virology,
73:1941-1948 (1999a)). In particular, the VP1-coding region has been shown
to specifically correlate with serotype (Oberste et al., 1999a), and this
region
io has been successfully targeted for the development of molecular typing
reagents (Kilpatrick et al., "Group-specific identification of polioviruses by
PCR using primers containing mixed-base or deoxyinosine residues at
positions of codon degeneracy," J. Clin. Microbiol., 34:2990-2996 (1996);
Kilpatrick et al.,"Serotype-specific identification of polioviruses by PCR
using
primers containing mixed-base or deoxyinosine residues at positions of codon
degeneracy, J. Clin. Microbiol., 36:352-357 (1998); Oberste et al., "Typing of
human enteroviruses of partial sequencing of VP1," J. Clin. Microbiol.,
37:1288-1293 (1999b)).
Despite the existence of typing methods, there remains a need for
2o reagents which can be used in a diagnostic assay to rapidly and accurately
distinguish EV71 and CA16, and which can be used to identify the strain of
EV71. A need also exists for a method for rapidly and accurately identifying
the strain of EV71 and differentiating between EV71 and CA16. Such
reagents and methods will allow the clinician to improve the speed and
accuracy of processing large numbers of clinical samples. Such reagents
and methods will also aid the clinician in patient management, eliminate
unnecessary tests, improve the speed and accuracy of diagnosis and
prognosis, help control enterovirus 71 infection, and reduce the use of
unnecessary antibiotics.
3o Accordingly, the present invention provides nucleic acids which can be
used as primers in amplification and sequencing reactions to rapidly
(generally within approximately 6 hours) amplify and sequence EV71 nucleic
acids. A preferred group of nucleic acids of the present invention when used
-3-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
as primer pairs in amplification reactions, detect EV71 with a high degree of
specificity and sensitivity. With these preferred primer pairs, the
specificity of
amplification methods of the present invention is such that target EV71
nucleic acids are amplified to a detectable level, whereas no detectable
product is obtained when CA16 nucleic acids are used. The nucleic acid
primers of the present invention contain mixed bases or deoxyinosine
residues at positions of codon degeneracy.
DESCRIPTION OF THE RELATED ART
Polymerise chain reaction primer sets containing mixed-base and, in
some cases, deoxyinosine residues is set forth in D. Kilpatrick, "Poliovirus
specific primers and methods of detection utilizing the same," U.S. Pat. No.
5,691,134. The disclosed polymerise chain reaction primer sets distinguish
between the three different serotypes of poliovirus and differentiate
polioviruses from nonpolio enteroviruses. Amplification reactions utilizing
the
~5 disclosed primers do not amplify EV71 nucleic acid. Further, the primer
sets
have nucleotide sequences which are different from those of the nucleic acids
of the present invention.
A polymerise chain reaction primer set containing mixed-base and
deoxyinosine residues is set forth in D. Kilpatrick et al. (1996). This
2o polymerise chain reaction primer set appears to differentiate polioviruses
from nonpolio enteroviruses. Amplification reactions utilizing the disclosed
primers do not amplify EV71 nucleic acid. Further, this primer set has
nucleotide sequences which are different from those of the nucleic acids of
the present invention.
25 None of the above-described publications teaches or describes the
primers, or methods and kits using these primers.
SUMMARY OF THE INVENTION
The present invention provides nucleic acids which can be used as
primers in amplification and sequencing reactions to rapidly amplify and
3o sequence target EV71 nucleic acids. Examples of these nucleic acid primers
are set forth in the Sequence Listing as SEQ ID NOS:1-12. A preferred group
-4-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
of nucleic acids of the present invention (i.e., SEQ ID NOS: 1-4), when used
as primer pairs in amplification reactions, detect EV71 with a high degree of
specificity and sensitivity. With these preferred primer pairs, the
specificity of
amplification methods of the present invention is such that target EV71
nucleic acids are amplified to a detectable level, whereas no detectable
product is obtained when CA16 nucleic acids are used.
The present invention also provides purified nucleic acids which are
complementary to nucleic acids having a nucleotide sequence selected from
SEQ ID NOS 1-12.
io The present invention also provides purified nucleic acids which are
substantially the same as the above-described nucleic acids. These nucleic
acids may vary from the above-described nucleic acids by one or more
nucleotide substitutions, additions and/or deletions, or by the addition of an
advantageous feature therein, such as, for example, a radiolabel or other
~5 label for nucleic acid detection or immobilization, so long as they retain
the
ability of the above-described nucleic acids.
The present invention also provides a method for detecting the
presence or absence of EV71 in a sample containing nucleic acids, including
clinical samples. The method comprises amplifying the nucleic acids present
2o in the sample with a primer pair comprising nucleic acids within the
present
invention, and determining the presence or absence of an amplification
product having a size which is characteristic for EV71, thereby determining
the presence or absence of EV71 in the sample.
The present invention still further provides a kit for determining the
25 presence or absence of EV71 in a biological sample. The kit contains the
nucleic acid primers disclosed in the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a dendrogram generated by neighbor-joining method with
the DNADIST distance measure (Phylip 3.5). The phylogram was calculated
3o based on the nucleotide divergence of the VP1 gene (position 2442 to 3332).
The last four or five characters of strain name indicate the state or country
and two digit year of isolation. Branch lengths are proportional to the number
-5-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
of nucleotide differences and the frequencies with which the branches for
genotype A, B, and C appeared in 1000 bootstrap replications (i.e. 898, 543
and 999, respectively). Clades with bootstrap numbers are expressed in
percentile. The marker bar denotes a measurement of the relative
phylogenetic distance. (The branch length for the outgroup CA16 was
reduced by 0.75 for space consideration.)
Figure 2 is an alignment of genotype-consensus VP1 amino acid
sequences. The EV71 consensus sequence shows amino acid residues that
are identical in at least 85% of all strains (upper case letters) and those
that
are identical in at least 50%, but less than 85% of all strains (lower case
letters). Sites that are identical in all strains of all genotypes are double-
underlined; those that are identical in all strains of genotypes B and C, but
different in BrCr-CA-70, are single-underlined. The genotype consensus
sequences indicate sites of at least 85% consensus among all strains of a
given genotype (hyphens) and sites that are characteristic of one or more
genotypes (upper case, 85% consensus within genotype; lower case 50% to
85% consensus within genotype).
Figure 3 illustrates the position of PCR primers relative to the amino
acid sequences of VP3 and VP1 (Brown et al., "Complete nucleotide
2o sequence of Enterovirus 71 is distinct from poliovirus," Virus Res:, 39:195-
205
(1995); Poyry et al., "Molecular analysis of coxsackievirus A16 reveals a new
genetic group of enteroviruses," Virology, 292:982-987 (1994)). Primer
position and sense are indicated by arrows. Amino acids at the annealing
sites for primers 159S and 162A are boxed. Amino acids at the annealing
sites for primers 92S and 93A are underlined. Dots indicate sequences not
shown. Numbers above sequence indicate relative amino acid positions in
VP3 and VP1.
Figure 4 shows ethidium bromide-stained gel sections containing
amplification products produced by RT-PCR of EV71 RNA in the Examples
3o described hereinbelow using the primer pair 159S/162A or the primer pair
92S/93A. Sources of templates for the products in each lane were as follows:
(A) (Primer pair 159S/ 162A and EV71 genotype A and genotype B strains),
1, CA70-BrCr; 2, NY72-2228; 3, AUS74-2610; 4, MN78-10181; 5, CA79-
-6-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
2258; 6, TN80-2114; 7, OH82-2381; 8, OK87-6910; 9, AL88-8149; 10, MS87-
7423; (B) (Primer pair 159S/ 162A and EV71 genotype C strains), 1, AK87-
7238; 2, MA87-0915; 3, TX89-9978; 4, TX91-0443; 5, NC94-1997; 6, VA95-
2132: 7, AUS95-2640; 8, MA97-2381; 9, OK97-2354; 10, M098-2814; (C)
Primer pair 92S/93A and genotype A and genotype B strains as listed in
panel A. (D) Primer pair 92S/93A and genotype C strains as listed in panel B.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention may be understood more readily by reference to
the following detailed description of the preferred embodiments of the
io invention, and to the Example, Tables, and Sequence Listings included
therein.
Abbreviations for "nucleotides" follow the nomenclature described by
the Nomenclature Committee for the International Union of Biochemistry,
"Nomenclature for Incompletely Specified Bases in Nucleic Acid Sequences,"
i5 Eur. J. Biochem. 150:1-5 (1985), in which "A" represents adenine residues,
"C" represents cytosine residues, "T" represents thymine residues, "G"
represents guanine residues, "I" represents deoxyinosine residues, "M"
represents adenine or cytosine residues, "R" represents adenine or guanine
residues and "Y" represents cytosine or thymine residues.
2o Definitions
The terms "nucleic acid" and "oligonucleotide" refer to primers, probes,
and/or oligomer fragments to be detected, and are generic to
polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to
polyribonucleotides (containing D-ribose), and to any other type of
25 polynucleotide which is an N glycoside of a purine or pyrimidine base, or
modified purine or pyrimidine base. The terms "nucleic acid" and
"oligonucleotide" are used interchangeably herein. These terms refer only to
the primary structure of the molecule. Thus, these terms include double- and
single-stranded DNA, as well as double- and single-stranded RNA. Nucleic
3o acids and oligonucleotides can be prepared by any of several well-known
methods. For example, they may be prepared by cloning and restriction of
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
desired sequences, or by direct chemical synthesis by the phosphotriester
methods described by Narang et al., " Meth. Enzymol. 68:90-99 (1979) and
Brown et al., Meth. Enzymol. 68:109-151 (1979); by the
diethylphosphoramidite method described by Beaucage et al., Tetrahedron
Lett. 22:1859-1862 (1981); or by the solid support method described in U.S.
Patent No. 4,458,066 and Matteucci, M. D., and Caruthers, M. H., J. Am.
Chem. Soc. 103, 3185 (1981). A review of nucleic acid syntheses methods is
provided in Goodchild, Bioconjugate Chemistry 1(3):165-187 (1990).
The term "primer" refers to an oligonucleotide, whether natural or
io synthetic, which is capable of hybridizing with a template nucleic acid
(i.e., the
nucleic acid being amplified), and which is capable of initiating the
synthesis
of a DNA extension product having a nucleotide sequence which is
complementary to the template nucleic acid strand in the presence of four
different nucleoside triphosphates and an agent for polymerization (i.e., DNA
~ 5 polymerise or reverse transcriptase) present in an appropriate buffer and
at a
suitable temperature. The length of primers typically ranges between about
and about 100 nucleotides. Short primer molecules generally require
cooler temperatures to form sufficiently stable hybrid complexes with the
template. Primers need not reflect the exact sequence of the template
2o nucleic acid, but must be sufficiently complementary (at least 85%,
preferably
at least 90%, and more preferably at least 95% complimentary) to hybridize
with the template.
Extensions which are not capable of hybridizing with the target nucleic
acid may generally be added to primers to allow the performance of a variety
25 of postamplification operations on the amplification product without
significant
perturbation of the amplification itself. For example, a primer can
incorporate
an additional feature, such as a radio- or non-radioactive label (biotin,
etc.)
which will allow for the detection or immobilization of the amplification
product, but which will not alter the basic property of the primer (that of
acting
3o as a point of initiation of DNA synthesis). For another example, a primer
may
contain an additional nucleic acid sequence at the 5' end which will not
hybridize to the target nucleic acid, but which will facilitate the cloning of
the
amplified nucleic acid product.
_g_
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
The phrase "selectively hybridize" for the present invention indicates
that the stringency of hybridization conditions can be set such that a nucleic
acid hybridizes with target nucleic acid sequences present in EV71, but not
with nucleotide sequences present in CA16. For example, the primers 93A
and 162A of the present invention selectively hybridize with target EV71
nucleic acids. This is demonstrated by the results of Example 2 which
indicate that either of these primers, in combination with another primer that
does not selectively hybridize, can be used to amplify EV71 target nucleic
acids under conditions wherein CA16 nucleic acids are not amplified.
The term "hybridization" refers to the formation of a duplex structure by
two single-stranded nucleic acids due to fully (100%) or less than fully (less
than 100%) complementary base pairing. Hybridization can occur between
fully complementary nucleic acid strands or between less than fully
complementary nucleic acid strands which contain regions of mismatch due
~ 5 to one or more nucleotide substitutions, deletions or additions.
Hybridization
can also occur when a nucleic acid is comprised of one or more modified
nucleotides, such as inosine. Depending on the length of a primer of the
present invention, the primer can generally range from between about 85%-
90%, and more preferably 95% complementary bases and full
2o complementarity with a target region of a nucleic acid and still hybridize
therewith.
The term "purified" means that the nucleic acids are of sufficient purity
so that they may be employed, and will function properly, in the methods of
the present invention, as well as in a clinical, diagnostic, experimental or
other
25 procedure, such as reverse transcription/polymerase chain reaction,
Southern
or dot blot hybridization, or gel electrophoresis. Many procedures are known
by those of ordinary skill in the art for purifying nucleic acids prior to
their use
in other procedures.
The term "substantially the same as" refers to a nucleic acid having a
3o nucleotide sequence which is similar to the nucleotide sequence of one of
the
nucleic acids set forth in the Sequence Listing as SEQ ID NOS:1-77, and
which retains the functions of such nucleic acid, but which differs from such
nucleic acid by the substitution, deletion and/or addition of one or more
_g_
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
hybridizing or non-hybridizing nucleotides, andlor by the incorporation of
some other feature into the nucleic acid, such as a radiolabel or other label
(biotin, etc.) for nucleic acid detection or immobilization. These nucleic
acids
will have the ability of the primers whose nucleotide sequences are set forth
in
s the Sequence Listing to detect in a biological sample during amplification
reactions. such as reverse transcription/polymerase chain reaction, nucleic
acids present in EV71. Modifications at the 5'- end of a nucleic acid can
include. for example, the addition of an isotope, such as 32P, or a chemical,
such as digoxigenin, for detection when using a commercial kit, such as the
Boehringer-Mannheim Dig/Genius detection system. In addition, restriction
enzyme sites and/or cloning sites can be added to the 5'- end of a nucleic
acid (from about 6 to more than about 12 nucleotides) for the direct cloning
of
the amplified product.
The phrase "primer pair" refers to two primers that each hybridize to
different target sequences on different strands of a DNA molecule to prime
amplification of a target nucleic acid. The primers are oriented upon
hybridization with the target nucleic acid with their 3' ends pointing towards
each other and prime enzymatic extension along the nucleic acid target in the
presence of the four deoxyribonucleotide triphosphates. The primers of a
2o primer pair when used in an amplification reaction are capable of
amplifying a
target nucleic acid located between the primers.
The phrases "target region" and "target nucleic acid" refer to a region
of nucleic acid that is to be amplified. detected, sequenced, or otherwise
analyzed.
25 The phrase "target sequence" refers to the sequence to which a primer
hybridizes.
Nucleic acid sequences and the relative genomic position of the
"VP1"and "VP3" genes are disclosed in GenBank entries referenced in Brown
and Pallansch, 1995 and Poyry et al., 1994.
3o The term "amplification reaction mixture" refers to a mixture comprising
four different nucleoside triphosphates and an agent for polymerization,
preferably DNA polymerase or reverse transcriptase, present in an
appropriate buffer.
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
Oliqonucleotide Primers Used for RT-PCR and Sequencing of Target
EV71 Nucleic Acids
In one aspect, the present invention provides purified nucleic acids
which hybridize with nucleic acids present in the EV71 VP1 gene and which
function as primers for amplification and sequencing of target nucleic acids
of
the EV71 VP1 gene. To account for the amino acid sequence variation within
EV71 strains and for codon degeneracy, primers preferably contain sites of
mixed-base composition and deoxyinosine at sites of fourfold codon
degeneracy. When these primers are used in an amplification reaction, EV71
1o target nucleic acids are amplified for further analysis, such as for
nucleic acid
sequence analysis. These nucleic acids allow a clinician to rapidly and
accurately detect and characterize, using amplification reactions and, in some
cases, nucleic acid sequencing, the EV71 strain that may be present in a
biological sample.
Examples of the nucleic acids of the present invention include DNA
primers having the nucleotide sequences set forth below, and/or in the
Sequence Listing. (SEQ ID NOS:1, 2, and 5-53).
Table 1. EV71 Amplification and Sequencing Primers
Primer Sequence ID No. I PositionUse
159S ACYATGAAAYTGTGCAAGG 1 2385-2403 P
197S CTCTCGATAGTTTCTTCAGCAG 5 2674-2695 S
172S TTCAGTAGGGCAGGCTTGGTAGG 6 2691-2714 P
204A CTGCTGAAGAAACTATCGAGAG 7 2698-2679 S
161 S CTGGGACATAGAYATAACWGG 8 2766-2785 P
162A CCRGTAGGKGTRCACGCRAC 2 2869-2850 P
198A CCGTCATAGAACCATTGATAAG 9 3048-3037 S
163S GAGCAYAARCAGGAGAAAGAYC 10 3078-3100 S
169S ATAYATGAGAATGAAGCAYGT 11 3194-3215 S
174A GCTGACCAAACTTTCCAAGGG 12 3348-3328 P
I
NP1A GCICCICAYTGITGICCRAA 13 3355-3336 P
-11-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
In Table 1, the column labeled "Primer," "A" indicates an antisense or
antigenome polarity primer, and "S" indicates a sense or genome polarity
primer. In the column labeled "sequence," Y = C or T; R = A or G; I = inosine,
K=G or T, W=A or T. In the column labeled "Position," the numbered position
is relative to 7423-MS-87 (Brown and Pallansch et al., 1995). Primer NP1A is
discussed in Oberste, et al.,1999a. In the column labeled "Use," "P" (PCR)
and "S" (sequencing) indicates the preferred use for a given primer: of
course,
any of the primers could be used for either PCR or sequencing.
For amplification reactions such as PCR, sense-oriented primers( such
as 159S, 161 S, and the like) can be paired with antisense primers ( such as
174A, 198A, and the like) in any arrangement as long as they span a genomic
region which produces an amplicon reasonably large enough to be analyzed,
preferably on an agarose gel (approximately 100 by in length). The following
are effective primer pairs for amplification and sequencing: 159S/162A;
~5 159S/204A; 161S/NP1A; 169S/NP1A; 163S/ 174A; 172S/174A; and
197S/198A.
The consensus nucleotide sequence (SEQ ID N0:1 ) is the degenerate
primer containing mixed-base nucleotide positions for primer 159S and
denotes the four possible combinations (species) of nucleotides that are
2o found in SEQ ID NOS:S-8, as set forth below:
SEQ ID N0:14 5'-ACC ATG AAA CTG TGC AAG
G
SEO ID N0:15 5'-ACC ATG AAA TTG TGC AAG
G
SEQ ID N0:16 5'-ACT ATG AAA CTG TGC AAG
G
SEQ ID N0:17 5'-ACT ATG AAA TTG TGC AAG
G
25 The consensus nucleotide sequence set forth in SEQ ID N0:2 for
primer 162A denotes the sixteen possible combinations (species) of
nucleotides that are found in SEQ ID NOS:18-33, as set forth below:
SEQ ID NO: 18 5'-CCA GTA GGG GTA CAC GCA AC
SEQ ID NO: 19 5'-CCA GTA GGG GTA CAC GCG AC
3o SEQ ID NO: 20 5'-CCA GTA GGG GTG CAC GCA AC
SEQ ID NO: 21 5'-CCA GTA GGG GTG CAC GCG AC
-12-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
SEQ ID NO: 22 5'-CCA GTA GGT GTA CAC GCA
AC
SEQ ID NO: 23 5'-CCA GTA GGT GTA CAC GCG
AC
SEQ ID NO: 24 5'-CCA GTA GGT GTG CAC GCA
AC
SEQ ID NO: 25 5'-CCA GTA GGT GTG CAC GCG
AC
SEQ ID NO: 26 5'-CCG GTA GGG GTA CAC GCA
AC
SEQ ID NO: 27 5'-CCG GTA GGG GTA CAC GCG
AC
SEQ ID NO: 28 5'-CCG GTA GGG GTG CAC GCA
AC
SEQ ID NO: 29 5'-CCG GTA GGG GTG CAC GCG
AC
SEQ ID NO: 30 5'-CCG GTA GGT GTA CAC GCA
AC
~o SEQ ID NO: 31 5'-CCG GTA GGT GTA CAC GCG
AC
SEQ ID NO: 32 5'-CCG GTA GGT GTG CAC GCA
AC
SEQ ID NO: 33 5'-CCG GTA GGT GTG CAC GCG
AC
The consensus nucleotide sequence set forth in SEQ ID N0:8 for
primer 161 denotes the four possible combinations (species) of nucleotides
~5 that are found in SEQ ID NOS:34-37, as set forth below:
SEQ ID NO: 5'-CTG GGA CAT AGA CAT AAC AGC
34
SEQ ID NO: 5'-CTG GGA CAT AGA CAT AAC TGC
35
SEQ ID NO: 5'-CTG GGA CAT AGA TAT AAC AGC
36
SEQ ID NO: 5'-CTG GGA CAT AGA TAT AAC TGC
37
-13-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
The consensus nucleotide sequence set forth in SEQ ID N0:10 for
primer 163 denotes 8 possible combinations (species) of nucleotides that are
found in SEQ ID NOS:38-45, as set forth below:
SEQ ID NO: 38 5'-GAG CAC AAA CAG GAG AAA GAC
SEQ ID N0:39 5'-GAG CAC AAA CAG GAG AAA GAT
SEQ ID NO: 40 5'-GAG CAC AAG CAG GAG AAA GAC
SEQ ID NO: 41 5'-GAG CAC AAG CAG GAG AAA GAT
SEQ ID NO: 42 5'- GAG CAT AAA CAG GAG AAA GAC
SEQ ID NO: 43 5'-GAG CAT AAA CAG GAG AAA GAT
1o SEQ ID NO: 44 5'-GAG CAT AAG CAG GAG AAA GAC
SEQ ID NO: 45 5'-GAG CAT AAG CAG GAG AAA GAT
The consensus nucleotide sequence set forth in SEQ ID N0:11 for
primer 169 denotes the four possible combinations (species) of nucleotides
that are found in SEQ ID NOS:46-49, as set forth below:
SEQ ID N0:46 5'-ATA CAT GAG AAT GAA GCA CGT
SEQ ID N0:47 5'-ATA CAT GAG AAT GAA GCA TGT
SEQ ID N0:48 5'-ATA TAT GAG AAT GAA GCA TGT
SEQ ID N0:49 5'-ATA TAT GAG AAT GAA GCA CGT
The consensus nucleotide sequence set forth in SEQ ID N0:13 for
2o primer NP1A denotes the four possible combinations (species) of nucleotides
that are found in SEQ ID NOS:50-53, as set forth below:
SEQ ID N0:50 5'-GCI CCI CAC TGI TGI CCA
AA
SEQ ID N0:51 5'-GCI CCI CAT TGI TGI CCA
AA
SEQ ID N0:52 5'-GCI CCI CAC TGI TGI CCG
AA
SEQ ID N0:53 5'-GCI CCI CAT TGI TGI CCG
AA
EV71 Serotype-Specific Primer Pairs
In another aspect of the invention, preferred nucleic acids are
disclosed which form primer pairs wherein one of the primers of the primer
-14-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
pairs selectively hybridizes to EV71 nucleic acids. When these primer pairs
are used in an amplification reaction, EV71 target nucleic acids are
amplified,
but not CA16 nucleic acids. Determination of the desired nucleotide
sequence for these EV71 serotype-specific primers is facilitated, in part, by
data obtained using the oligonucleotide primers used for RT-PCR and
sequencing of EV71 nucleic acid described above.
Analysis of amino acid sequences in and around the VP1 region of
EV71 and CA16 strains reveal the presence of serotype-specific sequence
motifs (Figs. 2-3). Pairs of EV71-specific PCR primers can be designed using
1o conserved motifs which amplify many strains of EV71. In a preferred
embodiment, these pairs of primers are designed so that a target nucleic acid
of EV71 is amplified, but no amplification product is detectable for CA16
nucleic acid. Preferably, at least one primer of the primer pair selectively
hybridizes to a region of VP1. Most preferably, one primer of the primer pair
hybridizes to sequences at the carboxyl-terminus of the VP3 gene and the
other primer of the primer pair selectively hybridizes near the center of VP1
gene (Fig. 3). To account for the amino acid sequence variation within EV71
strains and for codon degeneracy, primers preferably contain sites of mixed-
base composition and deoxyinosine at sites of fourfold codon degeneracy.
2o These preferred nucleic acids, especially when used in the disclosed primer
pairs, allow a clinician to rapidly and accurately determine by amplification
reactions, such as reverse transcription/polymerase chain reaction, whether
or not EV71 is present in a biological sample.
Examples of the nucleic acids of the present invention include DNA
primers having the nucleotide sequences set forth below, and/or in the
Sequence Listing (SEQ ID NOS:1-4, and 54-77):
Table 2. Preferred Group of EV71-Specific Oligonucleotide
Primers Used in this Study
PrimerSequence Position Seq Amino acids
ID
No. tar eted
159S ACYATGAAAYTGTGCAAGG 2385-24031 TMKLCKD
162A CCRGTAGGKGTRCACGCRAC 2869-28502 VACTPTG
-15-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
92S GTIGARYTITTYACITAYATG 2808-2828 3 VELFTYM
~
93A ~ ACIYCICCIGTRGGIGGIGTRCA2859-2879 4 CTPTG(Q/R/E)V
In Table 2, column labeled "Primer," "A" indicates an antisense or
antigenome polarity primer, and "S" indicates a sense or genome polarity
primer. In the column labeled "sequence," Y = C or T; R = A or G; I =
insosine; K = G or T. In the column labeled "Position," the numbered position
is relative to 7423-MS-87 (Brown and Pallansch et al., 1995).
Generally, these primers are intended to be used in primer sets for the
identification of EV71. For example, primers 159S and 162A are preferably
i o used together as a primer set in an amplification reaction to specifically
identify EV71. Likewise, primers 92S and 93A are preferably used together
as a primer set in an amplification reaction to specifically identify EV71.
These pairs of primers are degenerate primer sets that can be used to
distinguish between EV71 and CA16.
~5 The consensus nucleotide sequence set forth in SEQ ID N0:3 for
primer 92S denote the sixteen possible combinations (species) of nucleotides
that are found in SEQ ID NOS:54-69, as set forth below:
SEO ID N0:54 5'-GTI GAA CTI TTC ACI TAC ATG
SEO ID N0:55 5'-GTI GAA CTI TTC ACI TAT ATG
2o SEQ ID N0:56 5'-GTI GAA CTI TTT ACI TAT ATG
SEQ ID N0:57 5'-GTI GAA CTI TTT ACI TAC ATG
SEQ ID N0:58 5'-GTI GAA TTI TTT ACI TAT ATG
SEQ ID N0:59 5'-GTI GAA TTI TTC ACI TAT ATG
SEQ ID N0:60 5'-GTI GAA TTI TTC ACI TAC ATG
25 SEQ ID N0:61 5'-GTI GAA TTI TTT ACI TAC ATG
SEQ ID N0:62 5'-GTI GAG CTI TTC ACI TAC ATG
SEQ LD N0:63 5'-GTI GAG CTI TTC ACI TAT ATG
SEQ ID N0:64 5'-GTI GAG CTI TTT ACI TAT ATG
SEQ ID N0:65 5'-GTI GAG CTI TTT ACI TAC ATG
3o SEQ ID N0:66 5'-GTI GAG TTI TTT ACI TAT ATG
SEQ ID N0:67 5'-GTI GAG TTI TTC ACI TAT ATG
SEQ ID N0:68 5'-GTI GAG TTI TTC ACI TAC ATG
SEQ ID N0:69 5'-GTI GAG TTI TTT ACI TAC ATG
-16-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
The consensus nucleotide sequence set forth in SEQ ID N0:4 for
primer 93A denotes the eight possible combinations (species) of nucleotides
that are found in SEQ ID NOS:70-77, as set forth below:
SEQ ID N0:70 5'-ACI CCI CCI GTA GGI GGI GTA
CA
SEQ ID N0:71 5'-ACI CCI CCI GTA GGI GGI GTG
CA
SEQ ID N0:72 5'-ACI CCI CCI GTG GGI GGI GTG
CA
SEQ ID N0:73 5'-ACI CCI CCI GTG GGI GGI GTA
CA
SEQ ID N0:74 5'-ACI TCI CCI GTA GGI GGI GTA
CA
SEQ ID N0:75 5'-ACI TCI CCI GTA GGI GGI GTG
CA
~o SEQ ID N0:76 5'-ACI TCI CCI GTG GGI GGI GTG
CA
SEQ ID N0:77 5'-ACI TCI CCI GTG GGI GGI GTA
CA
In order to compensate for the high level of degeneracy of EV71
genomic nucleotide sequences which encode the VP1 and VP3 proteins,
degenerate codon positions on the EV71 genome template were matched by
~5 mixed bases (i.e., more than one base used at a particular nucleotide
position) or by deoxyinosine residues on the polymerise chain reaction
primers. This was done even though some investigators have reported
unsatisfactory losses in polymerise chain reaction sensitivity and diagnostic
specificity when using degenerate primers. Because deoxyinosine residues
2o can pair with all four of the nucleotide bases, deoxyinosine residues were
used in those positions where 3 or 4 different nucleotides were possible. The
use of deoxyinosine residues in primers is discussed in F. Martin et al.,
"Base
Pairing involving Deoxyinosine: Implications from Probe Design," Nucleic
Acids Res., 13, 8927-8938 (1985), in M. Batzer et al., "Enhanced Evolutionary
25 PCR using Oligonucleotides with Inosine at the 3'-Terminus," Nucleic Acids.
Res., 19, 5081 (1991 ); in S. Case-Green, "Studies on the Base Pairing
Properties of Deoxyinosine by Solid Phase Hybridisation to Oligonucleotides,"
Nucleic Acids Res., 22(2), 131-136 (1994); and in E. Ohtsuka et al., "An
Alternative Approach to Deoxyoligonucleotides as Hybridization Probes by
3o Insertion of Deoxyinosine at Ambiguous Codon Positions," J. Biol. Chem.,
260, 2605-2608 (1985).
Deoxyinosine residues were incorporated into the polymerise chain
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
reaction primers to match all template positions having a possible fourfold
degeneracy. Other template positions with twofold degeneracy were
complemented by twofold mixed residues. The positions of the primer
nucleotide sequences in which either mixed bases or deoxyinosine residues
were used are shown in the nucleotide sequences set forth hereinabove, and
in the Sequence Listing, for primers 159S (SEQ ID N0:1), 162A (SEQ ID
N0:2), 92S (SEQ ID N0:3), and 93A (SEQ ID N0:4). Table 2 indicates the
amino acid residues which are encoded by the nucleotide sequence
recognized by each of these primers.
io As is described in more detail in the Examples, the oligonucleotide
primers used for RT-PCR and sequencing of EV71 nucleic acid described
above were used to determine the nucleotide sequence of the VP1 region of
113 EV71 strains. Using the EV71 serotype-specific primer pairs, all strains
of EV71 tested were detected, whereas all CA16 stains tested were not
~5 detected.
One of ordinary skill in the art can use the teachings of this invention to
devise other primers that can be used to amplify and sequence target EV71
nucleic acids. The current invention teaches that primers constructed to
hybridize in the VP1 gene region of EV71 can be successfully used to amplify
2o and sequence this region in a given EV71 isolate. Furthermore, the current
invention facilitates the construction of other primers for amplifying EV71
target nucleic acids by providing fourteen examples of nucleotide sequences
that are effective for this amplification. Finally, the current invention
facilitates
the construction of other primers for amplifying target nucleic acids in the
25 EV71 genome by providing the sequence of the VP1 gene for 113 strains of
EV71 (available in GenBank sequence database, accession numbers
AF009522 to AF009559 and AF 135867 to AF 135949, AF 135911, AF 135935,
and AF135941 to AF135950). Therefore, primers can be constructed from
highly conserved nucleotides across many strains of EV71 that will amplify
3o EV71 target nucleic acids from many strains of EV71.
Not only do the teachings of the current invention facilitate the
construction of primers for amplifying EV71 target nucleic acids, these
teachings facilitate the construction of primers which selectively amplify
EV71
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
target nucleic acids and not those of other enteroviruses, such as CA16. For
example, by comparing the nucleotide sequences of the VP1 gene across the
113 strains of EV71 to other nucleotide sequences of other enteroviruses,
such as CA16, primers can be constructed which selectively hybridize to
EV71 target nucleic acids. Alternatively, amino acid sequence comparisons
can be used to construct primers that selectively amplify EV71 target nucleic
acids based on nucleotide coding sequences for these amino acid
sequences. In fact, Example 2 of this invention exemplifies the use of amino
acid information to construct EV71-specific primers. Computer programs that
facilitate such nucleotide sequence comparisons across strains and species
are readily available and well known to those of ordinary skill in the art.
Specific nucleic acids within the scope of the invention include, but are
not limited to, the nucleic acids described herein. Contemplated equivalents
of the nucleic acids described herein include nucleic acids which otherwise
~5 correspond thereto, and which have the same general properties thereof,
wherein one or more simple variations are made which do not adversely
affect the function of the nucleic acids as described herein.
Modified Nucleic Acids
Other examples of the nucleic acids of the present invention include
2o DNA molecules which are substantially the same as the nucleic acids having
the nucleotide sequences set forth below, and in the Sequence Listing as
SEQ ID NOS:1-77.
Modifications to the nucleic acids set forth as SEQ ID NOS:1-77 (e.g.,
one or more nucleotide substitutions, additions, and/or deletions, or the
25 addition of some beneficial component to the nucleic acids, such as a
radiolabel or non-radiolabel for nucleic acid detection or immobilization) can
be made so long as they do not prevent these nucleic acids from annealing to
cDNAs prepared from the conserved target EV71 sequences from Which they
were derived. For the oligonucleotide primers used for RT-PCR and
30 sequencing of EV71 nucleic acid described above, such modified nucleic
acids are within the scope of the present invention if they have the ability
to
function as primers in amplification or sequencing reactions for EV71 nucleic
_19_
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
acids. For the EV71 serotype-specific primer pairs described above, such
modified nucleic acids are within the scope of the present invention if they
have the ability, when used with a second EV71 serotype-specific primer, to
detect EV71, but not CA16.
Computer programs are readily available to the skilled artisan which
can be used to compare modified nucleotide sequences to nucleotide
sequences of many strains of EV71 and CA16 to select the most appropriate
sequences for priming amplification. The specificity of these sequences for
EV71 and sensitivity of the sequences for various strains of EV71 can be
determined by conducting a computerized comparison with known
sequences. Preferably these sequences are catalogued in GENBANK, a
computerized database, and the search is performed using the FASTA tool of
the Genetics Computer Group (Madison, WI), which facilitates searching the
catalogued nucleotide sequences for similarities to the nucleic acid in
~5 question.
The modified nucleic acids of the invention have at least 85%
homology (and preferably at least 90%, 95%, 97%, 98%, or 99% homology)
with the nucleotide sequences set forth in the Sequence Listing as SEQ ID
NOS:1-77, or at least 85% complementarity (and preferably at least 90%,
20 95%, 97%, 98%, or 99% complementarity) with the nucleic acid sequences to
which nucleic acids having the nucleotide sequence set forth in the Sequence
Listing as SEQ ID NOS:1-77 hybridize.
The nucleic acids of the present invention can be used as primers in
amplification reactions to detect EV71 or as primers in reverse transcription
of
25 viral RNA from EV71 isolates. These oligonucleotides are typically between
about 10 and about 100 nucleotides in length, preferably between about 12
and about 30 nucleotides in length, and most preferably between about 15
and about 25 nucleotides in length. An optimal length for a particular
application is readily determined in the manner described in H. Erlich, PCR
3o Technology, Principles and Replication for DNA Amplification, (1989).
Several computer software programs are available to facilitate primer design,
for example, T. Lowe, "Computer Program for Selection of Oligonucleotide
Primers for Polymerase Chain Reactions," Nucl. Acids. Res., 18:1757-1761
-20-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
(1991 ) and RT-PCR, Methods and Applications Book 1, (Clontech
Laboratories, Inc. (1991 )).
If used as primers in an amplification reaction, at least two nucleic
acids of the invention which hybridize with different regions of nucleic acid
present in EV71 should be employed, so as to amplify a desired region.
These two nucleic acids should hybridize to opposite strands of the EV71
DNA in reverse orientation such that they direct extension toward each other.
Nucleic acids present in EV71 can, thus, be detected with the nucleic acids of
the present invention utilizing a nucleic acid amplification technique, such
as
1o reverse transcription/polymerase chain reaction as taught in the Examples
described hereinbelow.
Where the detection method employed uses a nucleic acid
amplification technique, EV71 polymerase chain reaction primers which
hybridize to EV71 nucleic acids are utilized. The presence of an amplification
product having a size which is characteristic for EV71 after the performance
of the amplification technique, such as a reverse transcription/polymerase
chain reaction, indicates the presence in the sample of EV71. The lack of a
detectable amplification product after the performance of the amplification
technique indicates that EV71 is not present in the sample. Amplification
2o products may be separated, for example, by electrophoresis on
polyacrylamide or agarose gels, and visualized with ethidium bromide
staining.
As is described in the Examples, the degenerateEV71 polymerase
chain reaction primers of the present invention can be utilized in polymerase
chain reactions in the combinations of nucleotide sequences set forth in the
Sequence Listing as SEO ID NOS:14-17 (for Primer 159S); SEQ ID NOS:18-
33 (for Primer 162A); SEQ ID NOS:34-37 (for Primer 161); SEQ ID NOS:38-
45 (for Primer 163); SEQ ID NOS:46-49 (for Primer 169);SEQ ID NOS:50-53
(for Primer NP1A); SEQ ID NOS:54-69 (for Primer 92S); and SEQ ID
3o NOS:70-77 (for Primer 93A).
It is contemplated that the nucleic acids of the present invention can be
utilized in any of a number of nucleic acid detection techniques including,
but
not limited to, reverse transcription/polymerase chain reaction, isothermal
-21-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
DNA amplification, liquid hybridization, etc. It is also contemplated that the
nucleic acids of the present invention can be labeled or tagged for use in
radioactive, chemiluminescence, fluorescent, or other detection systems. In
general, the nucleic acids of the present invention may be prepared and
tested for the ability to hybridize with an EV71 target nucleic acid in the
manner described herein, or by modifications thereof, using readily-available
starting materials, reagents, and equipment. However, a preferred method
for preparing and testing these nucleic acids is described hereinbelow in the
Examples.
Amplification and Hybridization
In another aspect, the present invention provides a method for
detecting the presence or absence of EV71 in a sample suspected of
containing nucleic acids of enterovirus 71, said method comprising:
(a) providing, in combination, (1) a primer pair comprising a first primer
that hybridizes to nucleic acids of enterovirus 71 and a second primer that
hybridizes to nucleic acids of enterovirus 71, and (2j an amplification method
reaction mixture;
(b) amplifying nucleic acids from the sample suspected of containing
nucleic acids of enterovirus 71 using the primer pair and amplification method
2o reaction mixture; and
(c) determining the presence or absence of an amplification product
having a size which is characteristic of enterovirus 71, thereby determining
the presence or absence of enterovirus 71 in the sample.
In a preferred embodiment, the second primer selectively hybridizes to
nucleic acids of enterovirus 71. In a further preferred embodiment, the first
primer and the second primer recognize target sequences in an enterovirus
71 region selected from the group consisting of VP1 and VP3, preferably the
second primer recognizes a target sequence in the VP1 region of enterovirus
71. One of ordinary skill in the art, preferably with the aid of a computer
3o program, could use the teachings of this disclosure to devise primers that
hybridize to the VP1 or the VP3 genes of all known strains of EV71.
Furthermore, one of ordinary skill could use this disclosure to construct
_22_
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
primers that hybridize to EV71 and not to CA16. Preferably, these primers
that hybridize to EV71 but not CA16, hybridize to a portion of the VP1 gene of
EV71.
In a specific embodiment of this preferred embodiment, the first primer
is a purified nucleic acid comprising the nucleotide sequence set forth in the
Sequence Listing as SEQ ID N0:1, or a nucleic acid that is substantially the
same as the purified nucleic acid comprising the nucleotide sequence set
forth in the Sequence Listing as SEQ ID N0:1; and the second primer is a
purified nucleic acid comprising the nucleotide sequence set forth in the
Sequence Listing as SEQ ID N0:2, or a nucleic acid that is substantially the
same as the purified nucleic acid comprising the nucleotide sequence set
forth in the Sequence Listing as SEQ ID N0:2. In another specific
embodiment of this preferred embodiment the first primer is a purified nucleic
acid comprising the nucleotide sequence set forth in the Sequence Listing as
i5 SEQ ID N0:3, or a nucleic acid that is substantially the same as the
purified
nucleic acid comprising the nucleotide sequence set forth in the Sequence
Listing as SEQ ID N0:3; and the second primer is a purified nucleic acid
comprising the nucleotide sequence set forth in the Sequence Listing as SEQ
ID N0:4, or a nucleic acid that is substantially the same as the purified
nucleic
2o acid comprising the nucleotide sequence set forth in the Sequence Listing
as
SEQ ID N0:4. .
A number of amplification methods are known in the art that could
successfully employ the nucleic acid primers of the current invention to
amplify EV71 nucleic acids. Preferably, the amplification method is a reverse
25 transcription/polymerase chain reaction. The polymerase chain reaction (for
amplifying DNA) and the reverse transcription polymerase chain reaction (for
amplifying cDNA generated from RNA, as would be used in amplification
reactions performed with an RNA virus) are rapid methods for increasing the
copy number of, and sensitively detecting, specific nucleic acid sequences.
3o These methods may be used for the rapid detection of viruses from clinical
samples, such as feces, nasal wash, rectal swab, cerebrospinal fluid, throat
swab, lung biopsy tissue, and like materials. These specimens may be
collected by methods known in the art, such as by the methods described in
-23-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
T. Chonmaitree et al., "Comparison of Cell Cultures for Rapid Isolation of
Enteroviruses," J. Clin. Microbiol., 26:2576-2580 (1988), and in C. Hall,
"Clinically Useful Method for the Isolation of Respiratory Syncytial Virus,"
J.
Infect. Dis. , 131:1-5 ( 1975).
The nucleic acids present in a sample which are being amplified may
be single- or double-stranded DNA or RNA. If the starting material is RNA,
such as in EV71, reverse transcriptase is used to prepare a first strand cDNA
prior to conventional polymerase chain reaction.
General information concerning polymerase chain reaction, and the
amplification of specific sequences of nucleic acids, is present in U.S.
Patent
No. 4,683,195; U.S. Patent No. 4,683,202; U.S. Patent No. 4,965,188; U.S.
Patent No. 5,578,467; U.S. Patent No. 5,545,522; U.S. Patent No. 5,624,833;
F. M. Ausubel et al., "Current Protocols in Molecular Biology," Greene
Publishing Associates and Wiley-Interscience, (John Wiley and Sons, New
York (1987; updated quarterly)); H. Rotbart, "Enzymatic RNA Amplification of
the Enteroviruses," J. Clin. Microbiol., 28:438-442 (1990); E. Kawasaki,
"Amplification of RNA," 21-27, in M. Innis et al., PCR Protocols (Academic
Press, New York (1990)); and Rossolini et al., "Use of Deoxyinosine-
Containing Primers vs. Degenerate Primers for Polymerase Chain Reaction
2o Based on Ambiguous Sequence Information," Mol. Cell Probes, 8:91-98
(1994). The amplification of cDNA generated from RNA using a reverse
transcription/polymerase chain reaction is described in U.S. Patent No.
5,310,652 and U.S. Patent No. 5,322,770.
Commercial vendors, such as Perkin Elmer (Norwalk, Connecticut),
market polymerase chain reaction reagents and publish polymerase chain
reaction protocols.
In each cycle of an amplification reaction, a double-stranded target
nucleic acid sequence present in a sample is denatured and, due to the
presence of a large molar excess of the primers, primers are annealed to
3o each strand of the denatured target sequence. The primers, oriented with
their 3' ends pointing towards each other, hybridize to opposite strands of
the
target sequence and, due to the action of DNA polymerase, prime enzymatic
extension along the nucleic acid template in the presence of the four
-24-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
deoxyribonucleotide triphosphates. The two primers anneal to opposite ends
of the target nucleic acid sequence, and in orientations such that the
extension product of each primer is a complementary copy of the target
nucleic acid sequence and, when separated from its complement, can
hybridize to the other primer. The end product is then denatured again for
another cycle. After this three-step cycle has been repeated between about
25 and about 40 times, preferably about 30 times, amplification of a nucleic
acid segment by more than one million-fold can be achieved. Each cycle, if
100% efficient, would result in a doubling of the number of target sequences
present, thereby leading to exponential increases in the concentration of
desired nucleic acid sequences.
The primers of the present invention are complementary to a cDNA
generated by reverse transcription of an EV71 nucleotide sequence.
Denaturation of nucleic acid strands usually takes place at about 94'C.
~5 The normal annealing (about 55 to about 60=C) and extension (about 65 to
about 72~C) temperatures generally used for in vitro amplification by reverse
transcription/polymerase chain reaction are generally unsuitable for use with
the degenerate primers of the present invention because the presence of
deoxyinosine residues results in low annealing temperatures with many
2o poliovirus cDNA templates. The optimal annealing temperature was
determined to be approximately 42'C, near the temperature optimum for
avian myeloblastosis virus reverse transcriptase. Annealing temperatures
above about 46'C or below about 38'C generally reduce the yield of the
specific amplicon, and increase the generation of nonspecific amplification
25 products. Thus, the annealing temperature which may be employed with the
primers and methods of the present invention ranges from about 38'C to
about 46-C, and is preferably about 42°C. The extension temperature was
decreased to 60°C, which is still within the range for high Taq
polymerase
activity, in order to minimize dissociation of the primers from the templates.
3o The extension temperature which may be employed with the primers and
methods of the present invention ranges from about 56°C to about 64'C,
and
is preferably about 60°C. When these preferred annealing and extension
conditions were used in the Examples described hereinbelow, sequences of
-25-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
all EV71 cDNA templates tested were efficiently amplified. Examples of
suitable reaction times are from about 15 seconds to about 1 minute
denaturing, preferably about 45 seconds; from about 30 seconds to about 1.5
minutes of annealing, preferably about 45 seconds; and from about 30
seconds to about 1.5 minutes of extension, preferably about 1 minute. Of
course, as those skilled in the art realize, modification of both temperature
and duration of each portion of the annealing and extension cycles, as well as
the minutes of total cycles, can be varied.
Suitable assay formats for detecting amplification products or hybrids
formed between probes and target nucleic acid sequences in a sample are
generally well known and are described, for example, in F. M. Ausubel et al.,
1987: Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y. (1985). Examples of these
assay formats include dot-blot and reverse dot-blot assay formats. In a dot-
~5 blot format, amplified target DNA is immobilized on a solid support, such
as a
nylon membrane. The membrane-target complex is incubated with labeled
probe under suitable hybridization conditions, unhybridized probe is removed
by washing under suitable conditions, and the membrane is monitored for the
presence of bound probe. An alternate format is a "reverse" dot-blot format,
2o in which the amplified target DNA is labeled and the probes are immobilized
on a solid support, such as a nylon membrane. The target DNA is typically
labeled during amplification by the incorporation of labeled primers therein.
One or both of the primers can be labeled. The membrane-probe complex is
incubated with the labeled amplified target DNA under suitable hybridization
25 conditions, unhybridized target DNA is removed by washing under suitably
stringent conditions, and the filter is then monitored for the presence of
bound
target DNA.
Conventional techniques of molecular biology and nucleic acid
chemistry which may be employed in the preparative and testing processes of
3o the present invention are fully explained in the literature. See, for
example, F.
M. Ausubel et al., 1987; Sambrook et al., Molecular Cloning-A Laboratorx
Manual, supra.; J. Watson et al., Molecular Biology of the Gene (Fourth
Edition, The Benjamin/Cummings Publishing Company, Inc. 1987);
-26-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
Oligonucleotide Synthesis (M. J. Gait, ed., 1984); and Nucleic Acid
Hybridization (B. D. Names and S. J. Higgins. eds., 1984).
Kits
The present invention also provides a kit for detecting enterovirus 71
by nucleic acid amplification comprising one the primer pairs described
hereinabove. Preferably, the kit will contain one (or more) of the primer
pairs
specifically described in the Example 2, and instructions describing the use
of
these primer pairs in the detection of enterovirus 71. If desired. the kit may
also include, for example, suitable buffers, PCR enzymes, standards, and the
i o like.
The following Examples are intended to describe and illustrate the
methods for the preparation of primers within the present invention, the
methods for using these primers in reverse transcription/polymerase chain
reactions to amplify EV71 nucleic acids and to rapidly and accurately detect
i5 EV71 in a sample, while not detecting CA16. The Examples are intended to
be merely illustrative of the present invention. and not limiting thereof in
either
scope of spirit. Those of skill in the art will readily understand that
variations
of the conditions and processes of the procedures described in the Examples
can be used to prepare and test these primers.
2o Unless otherwise stated, all percentages are by weight. All references
cited in the present specification are hereby incorporated by reference.
EXAMPLE 1
MOLECULAR EPIDEMIOLOGY AND EVOLUTION OF ENTEROVIRUS 71
STRAINS ISOLATED FROM 1970-1998
25 Viruses Analyzed
The 113 EV71 strains examined in this study are listed in Table 3, with
year and state or country of isolation, and, if known, associated clinical
symptoms ("NA" indicates unknown or not reported). The strains were
isolated between 1970 and 1998 at the Centers for Disease Control and
3o Prevention (CDC), Atlanta, Georgia, from 25 different laboratories of state
health departments in the United States, and from 5 national enterovirus
-27-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
laboratories in other countries. Viruses were isolated from original clinical
specimens by using a variety of cell lines and further propagated in
rhabdomyosarcoma cells prior to sequencing. Most isolates were typed by
neutralization assay with monospecific rabbit anti-EV71 antiserum.
Table 3.
Code in Fi j Year Disease Accession
ure 1 ~ Location No.
BrCr-CA-70 1970 California Encephalitis022521
2228-NY-72 1972 New York NA AF135867
2604-AUS-74 1974 Australia Meningitis AF135883
2605-AUS-74 1974 Australia Meningitis AF135884
2608-AUS-74 1974 Australia Meningitis AF135885
2609-AIDS-741974 Australia I MeningitisAF135886
2610-AUS-74 1974 Australia I NA AF135887
2229-NY-76 1976 New York NA AF135868
2230-NY-76 1976 New York NA AF135869
2231-NY-77 1977 New York NA AF135870
2232-NY-77 1977 New York NA AF135871
2234-NY-77 1977 New York NA AF135872
2235-NY-77 1977 New York NA AF135873
2236-NY-77 1977 New York NA AF135874
2237-NY-77 1977 New York NA AF135875
2238-NY-77 1977 New York NA AF135876
~ 2239-NY-77 1977 New York NA AF135877
10181-NM-78 1978 New Mexico NA AF138675
1011-ND-79 1979 North Dakota NA AF135864
2241-NY-79 1979 New York NA AF135878
2243-NY-79 1979 New York I NA AF135879
2258-CA-79 1979 California Tremor AF135880
2114-TN-80 1980 Tennessee NA AF135866
2952-SD-81 1981 South Dakota NA AF135888
I
3663-MA-82 1982 MassachusettsNA AF135889
3885-UT-82 1982 Utah NA AF135891
3874-ND-82 1982 North Dakota NA AF135890
3982-OH-82 1982 Ohio Rash AF135892
3984-OH-82 1982 Ohio NA AF009538
2259-CA-82 1982 California Diarrhea AF135881
-28-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
Code in Fi Year I Location Disease Accession
ure 1 No.
4224-MA-82 1982 MassachusettsEncephalitis AF135893
4323-UT-83 1983 Utah NA AF135894
4599-OR-83 ~ 1983Oregon CNS disorder AF135895
4644-AR-83 1983 Arkansas NA AF135896
4826-CT-83 1983 Connecticut ~ NA AF135897
4827-CT-83 1983 Connecticut NA AF009529
5115-TX-83 1983 Texas NA AF135898
0667-CHN-85 1986 China HFM AF135934
2260-CA-86 1986 California Fever AF135882
2623-AUS-86 1986 Australia HFM AF135945
6762-OK-86 1986 Oklahoma NA AF135900
1410-CA-86 1986 California Paralysis AF009525
0915-MA-87 1987 California Meningitis AF009549
0916-MA-87 1987 MassachusettsNA AF009550
1061-TN-87 1987 Tennessee NA AF009528
1413-CA-87 1987 California Paralysis AF009527
2219-IA-87 1987 Iowa Meningitis AF009539
2246-NY-87 1987 NewYork Paralysis AF009542
6910-OK-87 1987 Oklahoma Rash AF135901
7234-AK-87 1987 Alaska Paralysis AF009522
7235-AK-87 1987 Alaska Respiratory AF135902
7237-AK-87 1987 Alaska Diarrhea AF135951
7238-AK-87 1987 Alaska Rash ' AF135952
7289-NC-87 1987 North CarolinaNA AF135903
I
7298-AK-87 1987 Alaska Fatal AF135904
7423-MS-87 1987 Mississippi Paralysis U22522
7628-PA-87 1987 Pennsylvania Paralysis AF009530
7629-PA-87 1987 Pennsylvania GastroenteritisAF009531
7630-PA-87 1987 Pennsylvania GastroenteritisAF009532
7631-PA-87 1987 Pennsylvania GastroenteritisAF009533
7632-PA-87 1987 Pennsylvania GastroenteritisAF135905
7633-PA-87 1987 Pennsylvania GastroenteritisAF009534
7635-WA-87 1987 Washington Meningitis AF135906
7673-CT-87 1987 Connecticut NA AF009535
7962-PA-87 1987 Pennsylvania Paralysis AF009523
7968-PA-87 1987 Pennsylvania NA AF009524
8102-WA-87 1987 Washington Meningitis AF009526
-29-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
Code in Fi Year Disease Accession
ure 1 I Location No.
8209-MD-87 1987 Maryland NA AF009536
8279-PA-87 1987 Pennsylvania NA AF009537
2222-IA-88 1988 Iowa Fever AF009540
8149-AL-88 1988 Alabama NA AF135907
8495-VA-88 1988 Virginia NA AF135953
9166-TX-89 1989 Texas NA AF135954
9243-OK-89 1989 Oklahoma NA AF135955
9323-TX-89 1989 Texas NA AF135956
9541-TX-89 1989 Texas NA AF009557
9718-TX-89 1989 Texas NA AF135957
9837-WA-89 1989 Washington NA AF135958
9873-NM-89 1989 New Mexico NA AF135959
9978-TX-89 1989 Texas Rash AF009558
0359-TX-90 1990 Texas NA AF135931
0390-TX-90 1990 Texas Otitis media AF135932
1411-CA-90 1990 California NA AF009551
0443-TX-90 1990 Texas NA AF135933
0925-OR-91 1991 Oregon Tremors AF009547
0926-OR-91 1991 Oregon NA AF009548
2261-CA-91 1991 California Meningitis AF135938
2583-CAN-91 1991 Quebec. CanadaNA AF135944
2262-CA-92 1992 California Meningitis AF135939
~ 2251-NY-93 1993 New York NA AF009543
1873-CT-94 1994 Connecticut Fatal AF009559
1919-NM-94 1994 New Mexico Rash AF009552
1924-AZ-94 1994 Arizona NA AF009553
1997-NC-94 1994 North CarolinaNA AF135936
2006-CT-94 1994 Connecticut Rash AF009554
i
2007-CT-94 1994 Connecticut NA AF009555
2253-NY-94 1994 New York NA AF009544
2254-NY-94 1994 New York NA AF009545
2263-CA-94 1994 California Paralysis AF135940
2264-CA-94 1994 California Meningitis AF009546
6658-COL-94 1994 Colombia Paralysis AF135899
2037-MD-95 1995 Maryland NA AF009556
2132-VA-95 1995 Virginia NA AF135937
2640-AUS-95 1995 Australia NA AF135946
30-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
Code in Fi ~ Year Disease I Accession
ure 1 I Location No.
2641-AUS-95 1995 I Australia ~ HFM AF135947
2642-AUS-95 1995 Australia EncephalitisAF135948
2644-AUS-95 1995 Australia NA AF135949
0731-MAA-97 1997 Malaysia Fatal AF135911
0756-MAA-97 1997 Peninsular NA I AF135935
2286-TX-97 1997 Texas NA AF135941
2355-OK-97 1997 Oklahoma NA AF135942
2381-MA-97 1997 MassachusettsFatal AF135943
2814-MO-98 1998 Missouri Meningitis AF135950
1o Olig~onucleotide Synthesis
Synthetic oligonucleotide degenerate primers used to amplify nucleic
acid present in enterovirus 71 by reverse polymerase/transcription chain
reactions, and having the nucleotide sequences set forth hereinabove in
Table 2, and in the corresponding sequences in the Sequence Listing, were
prepared by the ~-cyanoethyl phosphoramidite method using an automated
synthesizer (Model 380A, Applied Biosystems, Foster City, CA), as described
by Sinha et al., "Polymer Support Oligonucleotide Synthesis XVIII; Use of ~3-
cyanoethyl-N,N-dialkylamino-N-morpholino phosphoramidite of
Deoxynucleosides for the Synthesis of DNA fragments Simplifying
2o Deprotection and Isolation of the Final Product,"Nucleic Acids Research,
12,
4539-4557 (1984).
Reverse TranscriptionlPolymerase Chain Reaction
Viral RNA was extracted from 200 NI of cell culture supernatant using
UItraSpec III (Biotecx, Houston, Tx.) and resuspended in 20 p1 of water or
with the Qiamp Viral RNA kit (Qiagen Inc.,Valencia, Calif.). The primers used
for reverse transcription-polymerase chain reaction (RT-PCR) and
sequencing are listed in Table 2. The following were effective primer pairs
for
amplification and sequencing: 159S/162A; 159S/204A; 161S/NP1A;
169S/NP1A; 163S/ 174A; 172S/174A; and 197S/198A. The VP1 gene was
3o amplified as a series of overlapping fragments in a one-tube RT-PCR
reaction
containing 2 NI of RNA, 20 pmol of each primer, 100 mM each dNTP, 2 mM
-31-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
MgClz, 67 mM Tris-HCI (pH 8.8), 17 mM (NHQ)2S04, 1 mM ~-mercaptoethanol,
0.2 mg/ml gelatin, 10 U placental RNase inhibitor (Boehringer Mannheim
Biochemicals, Indianapolis, Ind.), 12 U AMV reverse transcriptase
(Boehringer Mannheim), and 5 U Taq polymerise (Boehringer Mannheim), in
a total volume of 50 p1. The amounts of primer used in each reaction
depends on the number of species of that primer. Generally, 5 pmoles per
species were used in each reaction. VP1-specific cDNA was synthesized by
incubation of the reaction mixture for 30 min at 42°C and 3 min at
94°C, and
amplified by 30 cycles of 94°C for 45 sec, 42°C for 45 sec, and
68°C for 1
io min. DNA fragments used for sequencing were gel-purified by using the
QIAquick gel extraction kit (Qiagen). Cycle sequencing was performed with
the Prism Ready Reaction Dyedeoxy Terminator Cycle Sequencing kit
(Perkin-Elmer Corporation-Applied Biosystems, Foster City, Calif.). All
sequences were determined on both strands.
Sequence analysis
The assembled complete VP1 sequences were compared with one
another using the GAP and PILEUP programs. Genetics Computer Group,
Program Manual for the GCG Package version 9.0" (1996) (Genetics
Computer Group, Madison, Wis). Phylogenetic trees were constructed by the
2o neighbor-joining method using PHYLIP 3.57. Felsenstein, J., "PHYLIP -
phylogeny inference package," (version 3.5), Cladistics, 5:164-166 (1989).
Branch lengths were determined by the maximum likelihood method
implemented in Puzzle (Strimmer, K., and A. V. Haeseler, "Quartet puzzling:
a quartet maximum likelihood method for reconstructing tree topologies," Mol.
Biol. Evol., 13:964-969 (1996)). The reliability of the neighbor-joining tree
was
estimated by bootstrap analysis with 1000 pseudo-replicate data sets.
Previously sequenced EV71 strains BrCr-CA-70 and 7423-MS-87 were also
included in the analyses. The VP1 sequence of the CA16 prototype strain, G-
10 (Poyry, T. et al., 1994), was included in the phylogenetic analysis as an
outgroup.
The nucleotide sequence data generated in this analysis have been
deposited in the GenBank sequence data base, accession numbers
-32-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
AF009522 to AF009559 and AF135867 to AF135949, AF135911, AF135935,
and AF135941 to AF135950.
Estimation of genetic distance and evolutionary rate
Because of the lack of a true "founder" strain and the apparent
presence of multiple lineages, sequences were selected based on their
relationships, as depicted in Figure 1, in order to estimate evolutionary
rate.
Genetic distances were calculated by pairwise comparison using the Kimura
2-parameter method of the Distances program (Genetics Computer Group,
"Program Manual for the GCG Package version 9.0" (1996)), using the oldest
strain in each set as a reference. Two separate analyses were performed,
one using all three positions (representative of both synonymous and
nonsynonymous substitutions) and a second analysis using only synonymous
substitutions. The evolutionary rate was calculated by linear regression of
genetic distance from the earliest isolate versus year of isolation. The
i5 synonymous substitution rate (Ks) was calculated from the number of
nucleotide substitutions per synonymous site by using the computer program
Diverge (Genetics Computer Group, "Program Manual for the GCG Package
version 9.0" (1996)) based on a method of Li, W. H. et al. "A new method for
estimating synonymous and nonsynonymous rates of nucleotide substitution
2o considering the relative likelihood of nucleotide and codon changes," Mol.
Biol. Evol.. 2:150-174 (1985). (The nonsynonymous rates [Ka), the number of
nonsynonymous substitutions per nonsynonymous sites, were less than 3 x
10-' and were not included in the data).
Results
25 The complete VP1 gene sequences (891 nucleotides) were
determined for 113 EV71 strains isolated in the United States, Australia,
Colombia, China, Canada, and Malaysia from 1970 to 1998. A phylogenetic
tree constructed by the neighbor-joining method indicated that EV71 strains
were monophyletic with respect to other enterovirus serotypes (Figure 1 ).
3o The strains clustered in three distinct lineages (genotypes), designated A,
B,
and C. Genotype A contained a single member, BrCr-CA-70, the EV71
-33-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
prototype, and differed from all other isolates by 16.5% to 19.7 %. Genotype
B was represented by 65 strains isolated from 1972 to 1997 in the United
States, Australia, Colombia, and Malaysia (Sarawak, island of Borneo).
Genotype C, represented by 47 strains isolated from 1986 to 1998, included
s viruses from the United States, Australia, China, Canada, and Malaysia
(mainland).
Genotypes B and C were further subdivided into clusters within each
genotype, two for genotype B (Figure 1 B) and two for genotype C (Figure 1 C).
Cluster B1 contained isolates from the United States and Australia during the
1970s, as well as a few U.S. isolates from the 1980s (2114-TN-80, 5115-TX-
83, 6762-OK-86, and 6910-OK-87). Strains in cluster B1 were more diverse
than the B1 strains, differing by up to 8.3% within the cluster and by 6.9% to
11.1 % from other genotype B strains. Cluster B2 contained strains isolated in
the U.S. from 1981 to 1987, including most isolates from the 1987 nationwide
~5 EV71 outbreak. Strain 6658-COL-94 was genetically distinct from all other
genotype B strains (5.8% to 11.1 % difference), but differed from strains of
genotype C by 15.5% to 17.2%. Strain 0731-MAA-97, a typical representative
of many Sarawak, Malaysia strains, was also distinct from other genotype B
strains by 6.5% to 10.5% and differed from genotype C strains by 17.1% to
20 18.3%. The earliest genotype C strains in our collection were isolated in
China in 1985 and Australia in 1986 (Figure 1 C). Genotype C isolates
differed from those of genotype B by 15.5% to 18.7%. Cluster C1 was
composed of isolates from the United States and Australia from 1986 to 1995,
as well as a 1997 isolate from peninsular Malaysia. Cluster C2 was
25 composed of U.S. and Australian isolates from 1995 to 1998. A 1985 isolate
from China appeared to be intermediate between clusters C1 and C2.
Viruses in cluster C1 differed from one another by 1.0% to 6.3% and from
those in cluster C2 by 6.1 % to 10.1 %, while isolates in cluster C2 differed
from one another by 0.7% to 1.1 %.
3o Among all the EV71 isolates, 82% of the predicted VP1 amino acid
residues were invariant (Figure 2). In comparison, the VP1 amino acids of
Echovirus 30 isolates are at least 88% identical. The EV71 prototype strain,
BrCr-CA-70 (genotype A) was 94.2% to 96.0% identical in VP1 amino acid
-34-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
sequence to all other EV71 isolates. Genotype B isolates were at least
97.9% identical to one another, whereas the genotype C isolates were 98.9%
identical to one another. Residues 58, 184, 240 and 289 varied among
different genotypes, but were invariant within a genotype. At four other sites
(residues 43, 124, 249, and 292), the predominant amino acid at the site
differed between genotypes B and C (Figure 2).
For the calculation of evolution rates, monophyletic clusters were
identified that spanned a period of at least 10 years ( Figure 1 and Table 4).
Within each cluster, one from genotype B and one from genotype C, the rate
io was calculated by plotting the number of nucleotide changes between each
strain and the oldest strain in the lineage versus the year of isolation (data
not
shown). Synonymous and nonsynonymous changes were plotted separately
for each of the two data sets. The slope of the linear regression line fitted
to
the data points is the calculated rate of evolution in substitutions per
~5 nucleotide per year. The overall evolutionary rates for all codon positions
were 4.2 x 10-3 and 3.4 and 10-3 substitutions per nucleotide per year for B
and C genotypes, respectively. Approximately 93% of all substitutions in VP1
occurred in the third position, 98% of all substitutions in the third position
were
synonymous, consistent with the very small number of amino acid changes
20 observed among EV71 isolates. The synonymous substitution rates at the
third codon position were 1.6 x 10-2 and 1.2 x 10'2 substitutions per
nucleotide
per year, for the B and C genotypes, respectively (Table 4).
TABLE 4. Estimation of the Nucleotide Substitution Rate in the
Vp1 Region of Ev71
Substitution
Rate (substitutions/year/nucleotide)a
25 Data Set Synonymous 2 z
sites R All sites I R
B 1 b 1. 5 x 10'2 0. 74 4.2 x 10-' 0.68
C 1 ' 1.2 x 10'z 0.85 3.4 x 10-' 0. 73
Average 1.35 x 10-z 3.71 x 10-'
(B1 + C1)
30 a RZ denotes linear regression coeffcient
-35-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
° 25 isolates, 1972 - 1987
39 isolates. 1986-1987
' weighted average for sets B1 and C1
Example 2.
SEROTYPE-SPECIFIC IDENTIFICATION OF ENTEROVIRUS 71 BY PCR
Viruses Ana~zed
The 124 EV71 strains examined in this study include the 113 strains
listed in Table 3 and the following additional isolates, with year and state
or
country of isolation: OK97-2354, CA87-3105, CA88-3104, CA90- 3103,
1o MD87-9256, and TA198-2731, TA198-2732, TA198-2733, TA198-2734, TA198-
2735, and TA198-2785. The 125 EV71 strains were isolated between 1970
and 1998 at the Centers for Disease Control and Prevention, Atlanta,
Georgia, from 25 different laboratories of state health departments in the
United States, and from 5 national enterovirus laboratories in other
countries.
Thirty-two coxsackievirus A16 (CA16) strains tested included CA16-G10-51
(prototype), TX88-8799, PA88-8888, IL89-0255, PA89-9544, PA89-9263,
PA89-9280, PA89-9281, PA89-9282, PA89-9283, TN89-9328, TN89-9330,
KY89-9352, OR89-9354, OR89-9359, TX89-9538, SD89-9704, MN89-9712,
WA89-9840, WA89-9838, WA89-9389, NC89-9853, PA90-9876, NM92-1450,
2o TX92-1576, TX92-1577, TN92-1603, PA94-5753, CT94-2004, CT94-
2005,TX95-2147, and AZ97-2446. A panel of enterovirus prototype strains
wuas tested and included echoviruses 1 to 9, 11 to 21, 24 to 27, and 29 to 33;
coxsackievirus A1 to A6, A8 to A22, A24, and B1 to B6; Polio Sabin1, Polio
Sabin2, and Polio Sabin3; and enteroviruses 68, 69,70, and 71. Viruses were
isolated from original clinical specimens by using various cell culture lines
and
further propagated in human rhabdomyosarcoma cells. Most isolates were
typed by neutralization assay; all EV71 strains and six (20%) of the CA16
strains were sequenced in the VP1 gene region by dideoxy sequencing.
PCR amplification and analysis of EV71 strains
3o Viral RNA was extracted from 200 NI of cell culture supernatant using
by UItraSpec III (Biotecx) and resuspended in 20 NI of water or extracted with
-36-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
the Qiamp Viral RNA kit (Qiagen). The primers used for RT-PCR are listed in
Table 2. RT-PCR amplifications were performed in a one-tube RT-PCR
assay containing 2 NI of RNA, 20 pmol of each primer, 100 mM each dNTP, 2
mM MgClz, 67 mM Tris-HCI (pH 8.8), 17 mM (NH4)zS04, 1 mM b-
mercaptoethanol, 0.2 mg/ml gelatin, 10 U placental RNase inhibitor
(Boehringer Mannheim), 12 U AMV reverse transcriptase (Boehringer
Mannheim), and 5 U Taq polymerise (Boehringer Mannheim), in a total
volume of 50 NI. In the reactions using inosine-containing primers, 80 pmol of
each primer was used. VP1-specific cDNA was synthesized by incubation of
the reaction mixture for 30 min at 42°C and 3 min at 94°C, and
amplified by
30 cycles of 94°C for 45 sec, 42°C for 45 sec, and 60°C
for 1 min. Reaction
products (10 NI each) were visualized by ethidium bromide staining and UV
transillumination following electrophoretic separation in 12% polyacrylamide
gels for 71-by products or in 1% agarose gels for 484-by products.
~ 5 Results
Analysis of amino acid sequences in and around the VP1 region of
EV71 and CA16 strains revealed the presence of serotype-specific sequence
motifs (Figure 3). Two pairs of EV71-specific PCR primers were designed
using conserved motifs at the carboxyl-terminus of VP3 and near the center
2o of VP1 (Figure 3). To account for the amino acid sequence variation within
EV71 strains and for codon degeneracy, all four primers contain sites of
mixed-base composition, and primers 92S and :;A contain deoxyinosine at
sites of fourfold codon degeneracy.
Primers 92S and 93A were directed to sequences encoding the motifs
25 VELFTYM (VP1-123 to VP1-129) and CTPTG(E/Q/R)V (VP1-140 to VP1-
146), respectively (Figure 3 and Table 2). These primers produced a 71-by
amplification product from EV71 RNA templates derived from 20
geographically and temporally distinct strains (Figure 4 and Table 2). Primers
92S and 93A did not amplify RNA templates from any of 19 CA16 strains, nor
3o did they amplify templates from the prototype strains of 60 other serotypes
(Table 5).
-37-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
Table 5.
Primer EV71 strains CA16 strains testedPrototypes tested
Pair tested
positive/total Positive/totat Positive/total
159S/162A125/125 0/31 3/61'
92S/93A 20/20 0/19 1/61'
a Prototypes listed in Methods; EV71 prototype provided a strong
signal; two prototypes (CA3 and Echovirus 32) gave very weak positive
signals with the primer pair 159S/162A.
A second EV71-specific primer pair, 159S and 162A, was designed for
use in sequencing and molecular epidemiology studies to provide for the
to rapid genotyping of EV71 isolates during outbreaks. Primers 159S and 162A
were directed to sequences encoding the motifs TMKLCKD (VP3-225 to VP3-
232) and VACTPTG (VP1-138 to VP1-144), respectively (Figure 3 and Table
2). These primers produced a 484 by amplification product from EV71 RNA
templates derived from 125 geographically and temporally distinct strains
(Figure 4, Table 3, and Table 5. Primers 159S and 162A did not amplify RNA
templates from any of 31 CA16 strains (Table 5).
-38-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
SEUUE:?CE ~3S:i~JG
.."..~C=..'7CE LiJ. _:?o
<liC> THE GOVERNMENT OE THE UNITED S=':RTES 0=.:iERIC=, AS
REcRESENTED BY THE SEC RET_a.R'f Or THE ~EPAR'='NI.~T O~"
HEALTH AND HUMAN SER:'T___S, C=.'?TERS :OR CISE S
CONTROL AND PREV~VTI0~1
3rocan, _Bet r y
Kilpatrick, David
Oberste, M. Steven
Pallansch, Mark
<120> Seretype-specific Ide:-aificatio~. o~-_'
enterovirus 71 by PCR
<13G> 00'50
<=cG> 93
<_,',,> ~~~~.~.,.'_Q for 5~rii-..w... ...=svc.. _."
< L _ .. > 1
<2ii> i9
<2~2> ~~?t-i
<2~.~> n__~__..'_3i S2:a2.~...,
<22G>
<22~> ~Ri:~ER
Y = C or T; R = A or ~~ ; K = G cr -~ ; vd = ~ ~= T
<4GG> 1
acyatgaaay tgtgcaagg
<21G> 2
<211> 20
<212> DNA
<213> Artificial Seauence
<220>
<223> PRIMER
Y = C or T; R = A c. G ; K = G or T ; W = A or T
1
WO 01/34848 CA 02391362 2002-05-10 pCT/US00/29021
....r~=3~~'C~ '_rC3C~Cr3C 2~
<2_..,> 3
<211> 21
<212> ~Na
<213> _'-. _rtific~_31 jequence
<220>
<22.1> ~odified ease
<222> (1)...(21)
<223> I=inosine
<223> PRIMER
Y=~. or T; R=? or C , .C=.-~_ _, '.v=.-.-';orT
<_~~;> 3
'=_~3=yi= =~%3Ci=3_.'3t ~3 21
L.:
<L_-> .......
<i~~> =.~ ____..i3~ .. . ....._...
<2L-J>
<---> .-..c.-~.? f_ease
<222> ,'!.. (23)
<223> '_ - _::os l ne
<223> : RI:~!ER
y =_ C or T; R = A or G ; K = .. cr ., .v = n. or
<:~'30> l
..cI'vcIcclg tr~gI99I9t rca 23
<210> 5
<211> 22
<21 2> CNA
<213> Artificial Sequence
<220>
<223> PRIMER
Y = C or T; R = A or G ; K = G or T; W = A or T
2
WO 01/34848 CA 02391362 2002-05-10
PCT/US00/29021
G~ ~,:> J
~7aud LttCttC3gC ag 2'
. _ ._ _ g
<2=J> 6
<211> 23
<2_2> WA
<2'~3> Artificial Sequence
<220>
<223> PRI.~7~R
Y = C or T; R = A or G ; K = G or : , n1 = A or T
<1n0> E
__..~?taggg caggcttggt agg
<Li.r..> 7
<2=:> L2
<2~_2> will
<="-=> Fr=~--~=al Sequence
<2~'u>
<=«> =j.T_:~:=R
_ -CCr '.. R=..OrG ; =<_.--.- ., ., -.
- _ . ~a aa~_~_:.gag ag
<2':C> 8
<21:> 21
<212> D~iA
<213> Artificial Sequence
<220>
<223> PRT_MSR
Y = C or T; R = A or G ; K = G or T; :4 = .. or T
<400> 8
,; ,
L 1
ctgggacata gayataacwg g
<210> 9
<211> 22
<212> DNA
<213> Artificial Sequence
3
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
<220>
<223> ?RIi~IER
Y = C or T; R = A or G ; K = G or T; G': = A or T
<400> 9
_cgtcataga accattgata ag 22
<210> 10
<211> 22
<212> CNA
<213> Artificial Sequence
<220>
<223> PRIMER
Y = C or T; R = A or G ; K = G or T, %~ = A or T
<400> 10
;ag.cay3arc aggagaaaga yc 22
<210> 11
<211> 21
<212> DNA
<2'_3> Artificial Sequezc°
<220>
<223> PRIMER
': = C c. T, K = ~ or G ; ., = G or ~'; -4 = ~ or T
<z0~> 11
?=ay~~gaga atgaagcayg t 21
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> PRIMER
Y = C or T; R = A or G ; K = G or T; W = A or T
<400> 12
gctgaccaaa ctttccaagg g 21
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
<<=:~> '_3
<211> 20
<212> DNA
<213> ,==ificial Sequence
<220>
<221> T.odified base
<222> (1)...;20)
<223> I = inosi.~.e
<223> PRIMER
_ = C or T; R = A cr ~ ; K = ~ or ~, :~i = A or T
<400> i3
3clcclcaft gl~g=ccraa
<210> '_.
<211> 19
15 <212> DNA
<2_3> =..~_ificiai Sequence
<==>
?> ?P,I_~.ER
_ - .. .~ T; R = a cr G ; .C = G o_ ., :1 = .. ._ T
20 ,__>> ,_.
..___ , _..~ _gtgcaagg 19
<~=J> 1:.
<21 1>
<=_2> DNA
<213> ~rt~-ficial Sequen::e
<220>
<223> P RI:~:ER
'i = C or T; R = A or G ; K = G or T; :~1 = A or T
<400> 15
a~ca~gaaat tgtgcaagg 19
<210> 16
<211> 19
<212> DNA
5
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
<213> Ar~ifioia= S=quenca
<220>
<223> PR='-'ER
Y = C or ?'- R = A or G ; K = G or T; W = .. or T
<400> 16
ac=atgaaac tgtg~aagg 19
<210> 1~
<211> 19
<2i2> v~;r'1
<213> Ar=ificial S2uence
<220>
<223> ?RI~~'.=R
'! = C or _. R = A o_ ~ . K = .. _L . n4 = '>,
<400>
3-~=a.gaaat =~'_~~-.'33Q~ .7
<21u>
<2~y;> L ,
<212> ~'I=..
<="-3> Ar=-~i~ia= S=cv.:e~:ce
2O <LcJ>
<223> =3I_~:~R
'! = C or . R. = A or G ; K = ~ or . , 'l = .. or T
<~~;,0> 13
_~37r37:~~ =a'-a'-'3~'_33~ 2~~
<210> 19
<211> 2C
<212> ~i'iA
<213> Artificial Sequence
<220>
<223> PRIMER
Y = C or T; R = A or G ; K = G or T; W = A or T
<400> 19
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
CCagtaggg3 -3CaCgCgdC 2~
<210> 20
<2i1> 20
<2i2> DNA
<213> Artificial Sequence
<220>
<223> ?R.IMER
Y = C or T; R = A or « ; K = ~ or : ; 's4 = A or T
<400> 20
o:.agtagggg tgcscgcaac 20
<210> 2i
<211> 20
<212> ANA
<213> artificial Sequence
<220>
<223> =RIMER
'.' = C or T; R = .. .,_ _ , IC = 3 0_ , ,~ - .. .,_ _
~,> ~1
<~~,v G
..cag~agg9:~ tgcacgcgac 20
<2i0> 22
<2i~> 20
<212> DNA
<213> ArtlflClal SeUUenCe
<220>
<223> ?RIMER
Y = C or T; R = A or G ; K = G or T; b~ _ :, or T
<900> 22
ccagtaggtg tacacgcaac 20
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence
WO 01/34848 CA 02391362 2002-05-10 PCT/US00/29021
<220>
<223> °RIMER
Y = C or T; R = A or G : :C = .- ..~ _ , W = =; cr '='
<400> 23
5 ~Cagtaggtg r_3C3CgCgaC
<210> 24
<211> 20
<212> ~':A
<2i3> Artificial Sequence
p <220>
<223> °RT_MER
Y = C or T; R = A or G ; ~ _ ,, or : , ~~ _ _. or T
<40G> 24
CCagtaggtg -gC3CgCa3C 2~
~5 <G:~J> G~
<L1=> 2~
<21 2> ~~i'l.
<21 3> A_ ____C131 je~~l:2.~.Ce
< G G J >
2p <223> __._._=R.
'!' = C Cr .. R = -1 ~r ,a : v = ~. .._ , .. - ._ ..~
<-100> 2~
ccagtaggcg tgcacgcgac 2~,
<2~0> 26
<211> 20
<212> DMA
<213> Artificial Sequence
<220>
<223> ?RIMER
Y = C or T; R = A or G ; :C = G or T; W = A or T
<900> 26
ccggtagggg tacacgcaac 20
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
< 2 ='; > 2 7
<2i1> 20
<212> Di'IA
<213> Artificial Sequence
<220>
<223> PRIMER
Y = C or T; R = A or G ; K = G or T; W = A ~r T
<400> 27
ccggtagggg t3C3C3cgac 20
<210> 28
<211> 20
G212> ANA
<213> Artificial Sequence
G220>
G223> PRIMER
'r = C or T; R = A or G ; :C = G or T; rT = .. or T
2~3
- :7t°~:'9~ ~'-d',:gCadC 2v
<<':~_~> 2 7
G2_~_> 20
< 2 : 2 > D~1A
<2'_3> A=tificial Sequence
<220>
G223> PRIMER
Y = C or T; R = A or G ; K = ~ or T; W = A or T.
G900> 29
ccggtagggg tgcacgcgac 20
G2i0> 30
<211> 20
<212> DNA
<213> Artificial Sequence
G220>
<223> PRIMER
9
WO 01/34848 CA 02391362 2002-05-10 PCT/US00/29021
Y = C or T.: R = A or G ; K = G cr :'; :~I = A cr T
<400> 30
ccggtaggtg tacacgcaac
<210> 31
5 <211> 20
<212> D~IA
<213> Arti~icial Seauence
<220>
<223> PRIMER
10 _ = C or T; R = A or G ; K = G or ., ~1 = A or T
<400> 31
ccggtaggtg tacacgcgdc
<210> 32
<211> 2C
15 <212> DcIA
<213> A__i_°ic_a1 Segeence
<G2~>
<223> PRI'dER
_ C or _, R = A Or G T =~ = G or :, %v = A c_ T
20 <C0> 32
2 '~
ag3:g tu~~.~3:.~1.~3a~
<21~> 33
<211> 20
<212> ~_.._
<213> yeti=icial Sexuence
<220>
<223> PRIMER
Y = C or T; R = A or G : K = G or T; W = A or T
<400> 33
30 ccggtaggtg tgcacgcgac
<210> 34
<211> 21
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
<212> DNA
<213> Artificial Sequence
<220>
<223> PRIMER
Y' = C or T; R = A or , ; K = G or T; W = A or T
<400> 34
~_g~~aCata gacataacag c 21
<210> 35
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> PRIMER
C or T; R = A or ~G ; ~C = ~ or T, Sv = A or T
<~:JO> 35
=gggacata gacataactg c 21
<<l~> 36
<211> 2i
<2i2> DNA
<=13> Ar~ificial Sequence
<L20>
<223> PRIMER
Y = C or T; R = A or G ; :C = G or ~, W = A or T
<4C0> 36
.. ggacata gatataacag c
<210> 37
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> PRIMER
Y = C or T; R = A or G ; K = G or T; W = A or T
WO 01/34848 CA 02391362 2002-05-10
PCT/US00/29021
<y00> 37
21
c~gggacata gatataactg c
<210> 38
<211> 21
<212> DD?A
<213> Artificial Sequence
<220>
<223> PRIMER
Y = C or T; R = A or G ; K = G or ~'; ~rI = A or T
<400> 38
21
gagcacaaac aggagaaaga c
<210> 39
<211> 21
< 2 i 2 > D.dA
<213> Artificial Seque:~ce
<220>
<223> ?RI~IER
Y = .. or T; R = A or G ; K = _ __ _, .. - __ .._
<;0C> ~9
ga~~acaaac aggagaaaga t 2
<210> 40
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> PRIMER
Y = C or T; R = A or G ; K = G or T; '~I = A or T
<400> 40
21
gagcacaagc aggagaaaga c
<210> 41
<211> 21
<212> DNA
<213> Artificial Sequence
BIZ
WO 01/34848 CA 02391362 2002-05-10 PCT/US00/29021
=2J>
<223> FRIi~IER
Y = ;. or T: R = A or G ; K = G or :, W = ?. ;.r T
<40C> 41
21
gag~~acaagc aggagaaaga t
<210> 42
<211> 21
<212> D«A
<213> Artificial Sequence
<220>
<223> rRIMER
Y = C or T, R = A or G ; K = G or T ; W = A
<4~0> 42
21
~3~~~3taa3s. 3gya~33ag3 C
<210> 43
<211> 21
<2'_2> ANA
<2=3> Artificial Sequence
<220>
<223> ?~I_-.~R
Y = C cr T; R = A or G ; K = G or _ , %~ = r o_ T
<400> 43
21
caaca_aaac aggagaaaga t
<210> 44
<211> 21,
<212> DNA
<213> Artificial Sequence
<220>
<223> ?RIMER
Y = C or T; R = A or G ; K = G or T; W = A or T
<400> 44
21
gagcataagc aggagaaaga c
~3
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
«~u> 45
<211> 21
<212> DNA
<213> Arcifi~cial Sequer.~e
<220>
<223> PRIMER
Y = C or T; R = A or G ; K = G or T; W = A or T
<~O~C> ~3
gagcataagc aggagaaaga t 21
<210> 46
<211> 21
<212> DNA
<213> Artificial Seque.~.ce
<220>
<223> ?RIMER
Y = C or T; R = .. or C , .C = G c_ T; ;'7 = A or T
<~00>
J,=a~~.3t~3gd at'.3aay.~_aCg t 21
<21C>
<<11> 21
<2i.2> DivA
<21j> ArtlflClal J'2q;ie :.'.e
<2L~>
<223> PRIvER
'f = C o- T; R = A or G ; K = G cr T; %'1 = A or T
<40C> y7
atacatgaga atgaagcatg t 21
<210> 48
<211> 21
<2_12> DNA
<213> Artificial Sequence
<220>
<223> PRT_MER
1~-
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
~~ = C or T; R = A cr C ; ~ = C c~ . , .. - A or T
<~00> 48
3t3=a=g3g3 atgaagcatg t 21
<210> 49
<211> 21
<2i2> DMA
<2i3> 'r~rtiLiu:131 S'eCj2lenCe
<220>
<223> PRIMER
Y = C or T; R = A or C ; :C = .: or T; f4 = ~ or T
<~:00> 4a
g3gd 3tgd3gC3Cg t 2i
<~il:> 70
<G:_> LV
<21%> 2~aA
<21 3> :-._ _i_°ici31 Sequa.~.ce
~~2~>
<=21> ~;cd-f_e case
i
<=-%> , ,...,20;
«23> . - ~..~s=_ne
<-23> =RI:~"ER
_ = C or T; R = A or . , ~ _ C or ., ;v = y cr
<400> 50
G
_~3Ct gItgTCC333
g~I~::T~ 0
<210> 51
<211> 20
<212> '~. NA
<213> Artificial Seauence
<220>
c221> modified base
<222> (1)...(20)
<223> I = inosine
~S
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
<223> °RI~IER
Y = C or T; R = A or G ; :C = G or T; ri = , :,r T
<y~0> 51
3cIccicatt gItgIccaaa 20
<210> 52
<211> 20
<212> ~~lA
<213> =~rtificiai Sequence
<220>
<221> modified base
<222> (1)...(20)
<223> I - inosine
<223> ?RIhIER
Y = C or T; R = A or G ; ~C = G or T, ;i = ., cr
<~~0> 52
a~~~~_~a-~t gItglccgaa 20
<2=J> 53
<2_1> 20
<212> OVA
<213> :rt_ficiai Sequence
<220>
<221> modified base
<222> (1)...(20.'
<223> I = inosine
<223> PRI:QER
Y = C or T; R = A or G ; K = G or T , n = A or T
<400> 53
gcI~:,cIcatt gItgIccgaa 20
<210> 54
<211> 21
<212> DNA
<213> Artificial Sequence
WO 01/34848 CA 02391362 2002-05-10 PCT/US00/29021
<22fJ>
<221> moth°ied 03se
<222> !1)...r,21)
<223> I = _nosi::~
<223> ?g.I;>1FR
Y = C or T; R = A or G ; K = G or ., L4 = A or
<900> 54
2i
gtIgaac~it ~caci=acac g
<210> ~5
<211> 21
<212> DNA
<213> Artificial Sequence
..-,C>
<LG
<221> ~,oa1==ed base
<222> ~:'_)....'.21)
_ ~osi.~...
<<_>> . _
<_'23> . ~.__':_~.
,~' _ ~ - r
_ - .. ~_ .. ~ = A or s~ ; ~ _ ~~ .._ _. __ _
<1~v> ~~
~tlgaac=I_ _____=a=at g __
<L_~> 70
<.~..il> 2t
<212> DNA
<213> Ar=-='~=.=al Sequence
<220>
<221> modi=fied base
<222> 61)...;21)
<223> I = inosine
<223> °RIMER
Y = C or T; R = A or G ; K = G or T; '~ ° A or T
<400> 56
21
gtIgaactIt ttacItatat g
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
<210> 57
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<221> modified base
<222> (1)...(21)
<223> I = inosine
<223> PRIMER
Y = C or T; R = A or G ; iC = G or T; W = A or
<400> 57
gtIgaactIt ttacItacat g 21
<2iC> 58
<211> 21
<212> DNA
<213> Artificial Seque:~~
<22C>
<22=> :no.dified base
<222> (1)...(21)
2~ <223> i - l:iCSin2
<223> S RIB?ER
= C Cr T; R = :, Cr ~ . K = G or T. :'i = A Cr T
«00> ~8
gtIgaattIt ttacItatat g 21
<210> >9
<211> 21
<212> DNA
<213> _Artificial Sequence
<220>
<221> modified base
<222> (1)...(21)
<223> I = inosine
<223> PRIMER
WO 01/34848 CA 02391362 2002-05-10 pCT/US00/29021
': = C or : ; R = A o~ G ; :C = ~ or T; %u = A or ':
<4c0> sa
7=_~~d3Ct-_ t.~_aCltatdt ~ 21
<210> 60
<211> 21
<212> DNA
<2i3> Artificial Sequence
<220>
<22i> modified base
<222> (i)...;21i
<223> I = inosine
<223> PRIMER
':' = C or T; R = A or .. , ~ = C c_ , fi _ .. or T
<9 ~C> 60
- " t 2~
:xaarmt tca..i acat y
<G..J>
<2=i> 2i
< % 12 > DN?
<<'~3> Arrificial Sequence
2p <22~>
<221> modified base
<222> ;1)...(21)
<223> I = inosine
<223> PRIMER
-~ = C or T; R = A or G ; R = ~ ~~ . , ;v = A or T
<900> 61
gtIcaattIt ttacItacat g 21
<210> 62
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
-79
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
<221> modified base
<222> (1)...(20)
<223> I - inosine
<223> PRi:~IER
Y = C or T; R = A or G ; K = G or T; l = ~ or
<40C> 62
gtl.3ag:ytit t~3~Ita~at g 21
<210> 63
<21i> 21
<212> OC.'_
<213> Artificial Sequence
<220>
<22:> ;codified base
<222> (1)...(21)
<223> I - _.ncsine
<223> °:cILIER
_ = C ~_ T; R = ~ cr ~ . =C = .. .._ . .. -
03
_ , ___ _~aclt3t3t , __
<=IO> 64
<211> 21
<G! %> UiVA
<213> Artificial Sequence
<220>
<221> modified base
<222> (1)...(21)
<223> I = inosine
<223> PRIMER
Y = C or T; R = A or G ; K = G cr T; Ud = A or T
<400> 64
gt_TgagctIt ttacItatat g 21
<210> 65
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
<211> 21
<212> DNa
<213> ~.rti=ici31 Sequen~=a
<220>
<221> ~:odified base
<222> (i)...(21)
<223> I = inosine
<223> PRIMER
Y = C or T; R = A or G ; K = G or T; W = A or T
<400> 63
~;~tI~3~~~Ctlt tt3CIt3C3t ~ 21
<2i0> 66
<211> 2
<212> DNA
<2i3> A='_i__..i31 Seque~_ce
<220>
<22".> :~.,.di ~_ed cas.--.-.
<222> ;- ; . . . ;2-~;
<_2?> I - ~~osi~:e
<=2 ;> _'-P,i:~:=~.
'. _ .. cr T; R = A or G ; K = G or T; ri = A or T
<;00> 50
2
~'Iy3~~=It rt3Cit3~3t ~ 1
<210> 07
<211> 21
<212> DNA
<213> Arti~icial Sequence
<220>
<221> modified base
<222> (1)...(21)
<223> I = inosine
<223> PRIMER
Y = C or T; R = A or G ; K = G or T; W = A or T
WO 01/34848 CA 02391362 2002-05-10 PCT/US00/29021
<4C0> 6~
21
gtIgagttIt tcacItatat g
<210> 63
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<221> :codified base
<222> (1)...(21)
<223> I = _.~.osine
<223> PRIMER
Y = C or :; R = A or G ; K = G c_ T; .'d = A cr
<400> 68
21
g=IgagttIt tcacltacat g
<210> 69
<211> 21
<212> :~~I~=
<213> artificial Segue.~..°
<220>
2~ <221> :.~.Odifi2d bd52
<222> !1)...(2i)
<223> I = i:~osine
<223> FRIh?ER
Y = C c_ T; R = A Or G ; .C = G o=' =. :~1 = =~ c_ T
<400> 09
21
gtIgagttIt ttacltacat g
<210> ~0
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<221> modified base
<222> (1)...(23)
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
<GG~S> 1 = lnOSlrie
<223> PRT_i~!ER
Y = C or T; R = A or G ; K = G or T, W = A or T
<400> 70
aci~clccIg taggIggIgt aca 23
<2i~> 71
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<221> modified base
<222> (1)...l23)
<223> I = izosine
<223> ?RIMER
Y = C or T; R = A or G ; K = ~~ c_ T; %4 = ~ or T
<0> 71
ail=~Ic~Ig taggIgglgt gca 23
<210> 72
<<'__> 23
2O <L1L> DNA
<213> Artificial Seauence
<220>
<221> :codified base
<222> (1)...(23)
<223> I = inosine
<223> PRIMER
Y = C or T; R = A or G ; K = G cr T, ~r1 = A or T
<400> 72
acIccIccIg tgggIggIgt gca 23
<210> 73
<211> 23
<212> DNA
23
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
<%~3> ~__-__~'_a1 Sequence
<220>
<221> mca'__'ied base
<222> (1)...(231
<223> i = inosine
<223> PRIN:ER
Y = ~ or T'; R = A or G ; K = G or T; .1 = .. or
<4C0> 73
acT_ccIcc.ig =gyq=39I9t aca Z
<2iC>
<211> 23
<2'_2> CNA
<213> ArLi~icial Sequence
<220>
G22'_> :;cdi°ied base
GLL2> ~~ ~ ) . . . (23)
G=e3> . ~i:i73i::2
- T or T; 3 = ~ or G ; K = ~ or _, :i =- ..
<=,:.:>
3C..:I~~-7 L3;3~~3'a1C)L 3~3 __
77
<'ri> 23
<2i2> DDIA
<~13> Ar=i=icial Sequence
G220>
<221> :~c,~'.if'_ed base
G222> (1i...(23)
<223> I = inosine
G223> PRIMER
Y = C or T; R = A or G ; K = G or T; W = A or T
<aCp> 75
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
acI=.-._.._lg =aggl~,~=~= ;Cs 23
<210> 76
<2~1> 23
<212> DNA
<213> Arti~iCia1 Seque..~_
<220>
<221> modiried base
<222> (i)...(23)
<223> I = ir:osi~e
1O <L23> ?RI'~GfZ
Y = C or T; R = A or .. . :~ _ C cr =, .'1 = A or '_'
<.~00> 76
____~_.._I3 t", ~y=0~ '_'~a 23
<210>
<2'_1> 23
<212> D'iA
<213> ~______..___ .. , _..C2
G220>
<221> :~,CCII~eC CeS2
<222> ., ,...,-3?
<223> I - _:~cs=~_
<223> . RT__v.'~R
Y = C cr ~ ; ~ = A or G ; K = G o_ , sJ = A cr T
<.~00> 77
aCitCICCI~ t.3a9=~~=yt 3Ca 23
<210> 78
<211> 7
<212> PRT
<213> enterovirus 71
<400> 78
Thr Met Lys Leu Cys Lys Asp
1 5
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
<210> 79
<211> 7
<212> PRT
<213> enterovirus 71
<400> 79
-,'ai Ala Cys Thr Pro Thr Gly
5
<210> 8C
<211> 7
<2i2> PRT
<2i3> enterovirus 71
<;00> 80
;a_ Clu Leu Phe Thr Tyr ~?et
<2i0> 91
<211> 9
<212> PRT
<2_3> e::L2ro:~irus 7i
' 0~;,'> 81
-; s -.._ _ ro .'.__ Cly G1 n Arg ~Clu '. __
5
<2:~~> 32
<2'_1> 7
<2i2> PRT
<213> enterovirus 71
<~00> 82
Cys Tzr Pro Thr Gly Arg val
5
<210> 83
<211> 7
<212> PRT
<213> enterovirus 71
CA 02391362 2002-05-10
WO 01/34848 PCT/US00/29021
<;0;:>> ~3
r_y5 T!-:r Pro Tnr =iy Glu val
z ~-
