virus Research, 20 (1991) 159-179 Elsevier ADONIS 016817029100100X

VIRUS

159

00681

Detection and identification of vaccine-related polioviruses by the polymerase chain reaction Chen-Fu Yang, Lina De, Brian P. Holloway, Mark A. Pallansch and Olen M. Kew Division of viral and Rickettsial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia, U.S.A. (Accepted

16 April 1991)

Summary

We have used the polymerase chain reaction (PCR) to obtain sensitive detection and identification of poliovirus RNA genomes. Primer pairs were designed to permit identification of each Sabin poliovaccine strain by the electrophoretic mobilities of the amplified DNA products (Sabin 1: 97 bp; Sabin 2: 71 bp; Sabin 3: 44 bp). The compositions of samples containing mixtures of vaccine strains could be readily determined by PCR. When the amplified products were visualized by ethidium bromide fluorescence, as few as 250 genomic copies in the original sample could be detected. When PCR was used in combination with strain-specific 32P-labeled oligonucleotide probes, the limit of detection was I 2.5 poliovirus genomes, exceeding the sensitivity of poliovirus isolation in cell culture by at least lOO-fold. PCR amplifications may be performed on virion RNAs extracted directly from clinical specimens, potentially eliminating the requirement for virus isolation in routine identifications while yielding reliable results within 8 h.

Vaccine-related polioviruses; Genotype-specific detection

Polymerase

chain reaction;

In vitro amplification;

Correspondence to: C.F. Yang, Respiratory and Enterovirus Branch, G17 Rickettsial Diseases, Centers for Disease Control, Atlanta, GA 30333, U.S.A.

Division

of Viral

and

160

Introduction

Poliovirus isolates are currently identified by either of two independent approaches. The first distinguishes vaccine-derived and wild polioviruses by their antigenic properties (Nakano et al., 1963; van Wezel and Hazendonk, 1979). Serodifferentiation methods have become increasingly refined with the development of comprehensive panels of specific neutralizing monoclonal antibodies (Ferguson et al., 1982; Osterhaus et al., 1983; Crainic et al., 1983). Nevertheless, equivocal results are still obtained with many clinical isolates. The ambiguities arise when virus populations of clinical isolates contain substantial proportions of vaccine-derived variants having antigenic properties indistinguishable from those of wild polioviruses (Nakano et al., 1963; Nakano et al., 1978; Minor et al., 1986a; Crainic et al., 1983). Antigenic reversion during replication in the human intestine is an intrinsic property of the Sabin oral poliovaccine strains, occurring at high frequencies with the type 1 strain (Nakano et al., 1963; Crainic et al., 1983) and at lower frequencies with the strains of serotypes 2 and 3 (Nakano et al., 1978; Minor et al., 1986a). The second fundamental approach encompasses several different methods for comparing sequence relationships among poliovirus RNA genomes (approximately 7.5 kb in length). Molecular methods in current use include ribonuclease T, oligonucleotide fingerprinting (Nottay et al., 1981; Kew et al., 19841, partial genomic sequencing (Rico-Hesse et al., 1987; Kew et al., 19901, and hybridization with synthetic oligodeoxynucleotide probes (da Silva et al., 1991; Kew et al., 1990). Although all of these molecular techniques are highly reliable, only probe hybridization is well-suited for routine diagnostic applications. Both the above antigenic and molecular methods depend upon the prior isolation of polioviruses in cell culture, a process that typically requires 1 to 3 weeks. Because many clinical specimens contain heterotypic poliovirus mixtures, further separation procedures are needed before intratypic identifications can be performed. Most difficult to characterize are homotypic mixtures obtained from patients concurrently infected with vaccine-derived and wild polioviruses. Specimens containing homotypic mixtures have been encountered in countries where oral poliovaccine was used in epidemic or highly endemic areas (unpublished results). The comparatively slow procedures of in vivo amplification of polioviruses in cell culture may be replaced by rapid in vitro amplification of viral RNA sequences by the polymerase chain reaction (PCR). Since its introduction (Saiki et al., 1985, 19881, PCR has quickly become an important tool of both basic research and clinical diagnostics (Kwok and Sninsky, 1989). The unsurpassed sensitivities and technical simplicity of PCR-based detection methods have favored their application to the identification of numerous medically important viruses (Kwok and Sninsky, 19891, including human rhinoviruses (Gama et al., 1989), enteroviruses (Hyypia et al., 1989; Chapman et al., 1990; Olive et al., 1990; Rotbart, 1990) and hepatitis A virus (Jansen et al., 1990). In this report, we describe the development of PCR methods for the rapid,

161

efficient detection and identification of poliovirus genomes. Primer pairs were designed for the specific amplification of sequences characteristic of each Sabin poliovaccine strain. The components of mixtures of vaccine-derived viruses could be readily identified by the electrophoretic mobilities of the amplified doublestranded DNA products. When used in combination with 32P-labeled oligonucleotide probes, the limit of detection by PCR was I 2.5 poliovirus RNA molecules. Amplifications may be performed on virion RNAs extracted directly from clinical specimens, thereby bypassing virus isolation and giving accurate identifications within 8 h. We have routinely used these new reagents to identify polioviruses in both clinical isolates and environmental samples.

Materials and Methods Viruses

Poliovirus isolates (Tables l-3) had been previously characterized by neutralization with hyperimmune equine sera and partial genomic sequencing (Rico-Hesse et al., 1987; Kew et al., 1990). All reference strains and many of the clinical isolates were further analyzed by RNase T, oligonucleotide fingerprinting (Nottay et al., 1981; Kew et al., 1984) and by hybridization with genotype-specific oligodeoxynucleotide probes (da Silva et al., 1991; Kew et al., 1990). Three sets of poliovirus reference strains were examined: (1) the Sabin oral poliovaccine strains LSc 2ab (Sabin 11, P712 Ch 2ab (Sabin 2) and Leon 12 a,b (Sabin 3) (obtained at passage levels of SO + 2; Sabin and Boulger, 19731, (2) strains Mahoney, MEF-1, and Saukett of the inactivated poliovaccine (Salk et al., 19541, and (3) the original serotype reference strains Brunhilde, Lansing and Leon (Bodian et al., 1949). Viruses were propagated in HeLa or RD monolayers to produce high-titer inoculation stocks. Stool suspensions and rectal swabs (Table 4) were clinical samples that had been previously shown to contain vaccine-related polioviruses by molecular characterization of cell culture isolates. Preparation of RNA

Freeze-thaw lysates of RD cell cultures infected with clinical isolates, stool suspensions (10% w/v; clarified at 8000 x g, 30 min), or rectal swabs (80-400 ~1 per sample) were thoroughly mixed (4: 1) with 5 X lysis buffer [250 mM Tris-HCl (pH 8.3), 350 mM KCI, 25 mM MgCl,, 2.5% NP-40, 25 mM vanadyl ribonucleoside complex (Bethesda Research Laboratories, Gaithersburg, MD)], and incubated on ice for 10 min. The mixtures were extracted three times with equal volumes of phenol-chloroform-isoamyl alcohol (25 : 24 : l), and the aqueous phases transferred to a new microcentrifuge tube. Aqueous-phase extracts from infected cell cultures were used directly in the PCR reactions. RNAs extracted from stool or rectal swabs were precipitated by addition of 2.5 volumes of ethanol and resuspended in l/10 the original sample volume in 10 mM Tris-HCI (pH 7.8), 0.1 mM EDTA.

162 TABLE 1 Type 1 polioviruses characterized

by PCR

Strain a*b

Amplification products

Donor’

Sabin 1 (97 bp)

Sabin 2 (71 bp)

Sabin 3 (44 bp)

Reference viruses PVl/Sabin 1 PVl/Mahoney/USMZ PVl/Brunhilde/LJSA39

+ + +

-

-

RM JHN NIH

Vaccine-related isolates PV1/0246/GUT90 PV1/9825/USA89 PV1/9703/ELS89 PV1/9360/VEN89 PV1/9240/HON89 PV1/0074/PER88 PV1/8316/MEX88 PV1/8315/MEX88 PV1/8284/HON88 PV1/8221/GUT87

+ + + + + + + + + +

_ _ _ -

_ _ _ -

PC MAP JRC RS PC LB JRG JRG CMM PC

Wild isolates PV1/0116/ECU90 PV1/9884/THA89 PV1/9475/ZAI89 PV1/9025/BRA88 PV1/8771/OMA88 PV1/7180/SOA86 PV1/7166/MEX86 PV1/7064/IND86 PV1/6535/NEP86 PV1/0109/CHN86

_ -

_ _

_ _ -

JB BI AV EdS PM BS MLZ KD MAP SG

a*c See footnotes to Table 4 for country and donor name abbreviations. b Independently characterized by partial genomic sequencing.

Generally, 1 to 2 ~1 samples were used for PCR amplification. Poliovirus RNAs used for titrations were extracted from purified virions, and the RNA concentrations determined spectrophotometrically from the relationship A,, (1 mg/ml) = 22.0 (Rueckert, 1976). Oligonucleotide synthesis

Synthetic DNA molecules were prepared by the P-cyanoethyl phosphoramidite method (Sinha et al., 1984) using an automated synthesizer (Model 380A, Applied Biosystems, Foster City, CA). Detritylated oligonucleotide products were purified by high performance liquid chromatography (Becker et al., 1985>, and characterized by electrophoresis on 20% polyacrylamide sequencing gels. Three sets of

163 TABLE 2 Type 2 polioviruses characterized Strain a*b

by PCR Amplification products Sabin 1 (97 bp)

Reference viruses PV2/Sabin 2 PV2/MEF-l/EGY42 PV2/Lansing/USA37 Vaccine-related isolates PV2/0042/ELS90 PV2/0078/PER89 PV2/9897/GUT90 PV2/9819/BRA89 PV2/9818/PER89 PV2/9579/USA89 PV2/9364/GUT89 PV2/8238/GUT88 PV2/8018/GUT87 PV2/6886/GUT83

-

Wild isolates PV2/0176/PER89 PV2/7079/IND86 PV2/6876/COL86 PV2/1534/IND82 PV2/6683/YUG78 PV2/0710/KEN70 PV2/0637/IND68 PV2/058O/COL64 PV2/0390/IND60 PV2/0177/ZAI60

_ -

Donor ’

Sabin 2 (71 bp)

Sabin 3 (44 bpl

+

-

RM JHN NIH

+ + + + + + + + + +

-

JRC LB PC EdS LB MAP PC PC PC JRC

-

-

_ -

-

EdS KD JB TJJ DM MHH MHH JHN JHN JHN

a,C See footnotes to Table 4 for country and donor name abbreviations. b Independently characterized by partial genomic sequencing.

Sabin strain-specific PCR primer pairs (Sab/PCR-1, tion probes (Sab/PCR-PI were prepared:

Sab/PCR-2)

Sabl/PCR-1

(2584 - 2601)

5’-TCCACTGGCITCAGTGTT-3’

Sabl/PCR-2

(2505 - 2523)

5’-AGGTCAGATGCTTGAAAGC-3’

and hybridiza-

Sabl/PCR-P

(2535 - 2565)

5’-CGTTGCCGCCCCCACCGTCGGACTGTG-3

Sab2/PCR-1

(2580 - 2595)

5’-CGGCTTGTGTCCAGGC-3’

Sab2/PCR-2

(2525 - 2544)

5’~CCGTTGAAGGGATTACTAAA-3’

Sab2/PCR-P

(2547 - 2577)

5’-GCTATTGGTGGAAGTCGGGGGAACCAATGCA-3

Sab3/PCR-1

(2562 - 2580)

5’-TAAGCTATCCTGTTGCC-3’

Sab3/PCR-2

(2537 - 2553)

5’-AGGGCGCCCTAACIYTG-3’

Sab3/PCR-P

(2544 - 2574)

5’-ATCCTGTTGCITCGGGACGACAAAG?TAGG-3’

’

’

164 TABLE

3

Type 3 polioviruses

characterized

Strain a,b

by PCR Amplification

products

Donor

Sabin 1

Sabin 2

Sabin 3

(97 bp)

(71 bp)

(44 bp)

Reference viruses PV3/Sabin 3 PV3/Saukett/USA52 PV3/Leon/USA37

-

_ -

+ + +

RM JHN JHN

Vaccine-related isolates PV3/0040/ELS90 PV3/0131/MEX89 PV3/0044/GUT89 PV3/9896/GUT89 PV3/9847/MEX89 PV3/9442/NIC89 PV3/9441/GUT89 PV3/8774/TRT88 PV3/8239/GUT88 PV3/8024/GUT87

_ _ _ _ -

+ + + + + + + + + +

JRC JRG PC PC JRG JRC PC BH PC PC

Wild isolates PV3/0174/MEX90 PV3/9033/BRA88 PV3/8854/COL88 PV3/7840/PER86 PV3/7095/IND86 PV3/6699/ECU86 PV3/6184/FIN84 PV3/V3/SPA83 PV3/Vl/EGY82 PV3/1525/IND82

_ _ _ _ _

_ _ _ _ _

JRG MJO JB RML KD AA TH JK JK TJJ

a,c See footnotes b Independently

to Table 4 for country and donor characterized by partial genomic

-

-

-

-

’

name abbreviations. sequencing.

The numbers in parentheses indicate the intervals within the VP1 region matching the primers and probes (Fig. 0, following the consensus numbering system of Toyoda et al. (1984).

Isotopic labeling of probes

‘Oligodeoxynucleotide probes were 5’-end labeled in 10 ~1 reactions (37°C 30 min) containing 100 ng probe (- 20 pmol), 100 &i (15 to 25 pmol) [Y-~~PIATP (New England Nuclear, Boston, MA), 20 mM Tris-HCI (pH 7.61, 10 mM MgCl,, 5 mM dithiothreitol and 5 U T4 polynucleotide kinase (Pharmacia P-L Biochemicals, Piscataway, NJ). Radiolabeled probes were purified by electrophoresis on 20%

165 TABLE 4 Clinical specimens characterized

by PCR

Clinical specimen a

Original clinical material

Identity of virus isolate b

Donor ’

PV1/0084/PER89 PVlj9342jHON89 PV1/9225/GUT89 PV1/8453/USA89 PV2/8763/HON88 PV2/8452/USA88 PV2/6902/GUT86 PV2/6899/GUT86 PV3/0123/COL90 PV3/9237/GUT89 PV3/8940/MEX89 PV3/8761/HON88

Stool Stool Stool Stool Stool Stool Rectal swab Rectal swab Stool Stool Stool Rectal swab

Sabl-rel Sabl-rel Sabl-rel Sabl-rel Sab2-rel Sab2-rel SabZ-rel Sab2-rel Sab3-rel Sab3-rel Sab3-rel Sab3-rel

LB CMM PC MAP CMM MAP JRC JRC JB PC JRG CMM

Country abbreviations: BRA, Brazil; CHN, China; COL, Colombia: ECU, Ecuador; EGY, Egypt; ELS, El Salvador; FIN, Finland; GUT, Guatemala; HGN, Honduras; IND, India; KEN, Kenya; MEX, Mexico; NEP, Nepal; NIC, Nicaragua; OMA, Oman; PER, Peru; SOA, South Africa; SPA, Spain; THA, Thailand; TRT, Trinidad and Tobago; USA, United States; VEN, Venezuela; YUG, Yugoslavia; ZAI, Zaire. Isolates were identified by dot-blot hybridization of virion RNAs using Sabin strain-specific oligonucleotide probes (da Silva et al., 1991). Sabl-rel, Sabin l-related; Sab2-rel, Sabin 2-related; Sab3-rel, Sabin 3-related. Donors: A. Alava, L. Belalnde, J. Boshell, P. Caceres, J.R. Cruz, K. Dave, S. Gu, M.H. Hatch, J.R. Hernandez, T. Hovi, B. Hull, B. Innis, T. Jacob John, J. Kapsenberg, D. Magrath, R. Mauler, R. MCndez Lopez, CM. Mejia, P. Minor, J.H. Nakano, M.J. Oliveira, M.A. Pallansch, J. Ruiz Gomez, R. Salas, B. Schoub, E. da Silva, A. Vernon, M.L. Z&rate. NIH: NIAID/NIH/USPHS.

polyacrylamide gels and recovered from the gels by standard extraction procedures (Kew et al., 1984). In vitro amplification

Amplification reactions were carried out in two steps. cDNA transcripts were prepared by incubation (42”C, 20 min) of RNA templates in 20 ~1 reaction mixtures containing 50 mM Tris-HCl (pH 8.3), 70 mM KCl, 5 mM MgCl,, 10 mM dithiothreitol, one or more PCR-1 primers (50 pmol each), 200 PM each of dATP, dCTP, dGTP, dTTP (Pharmacia), 10 U placental ribonuclease inhibitor (Boehringer Mannheim Biochemicals, Indianapolis, IN), and 2.5 U avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer Mannheiml. cDNA products were diluted to 100 ~1 containing (final concentration) one or more PCR-2 primers (50 pmol each) and 2.5 U of the thermostable DNA polymerase from Thermus aquaticus (Taq DNA polymerase; Perkin Elmer-Cetus, Norwalk, CT> and overlaid with mineral oil. Programmed amplification cycles (denaturation: 94°C 30 s; annealing: 62°C 45 s; extension: 72°C 1 min) were performed by a DNA thermal

166 Sabl/PCR-2 >

Sabin

1

AUG AWGACAACACAGUCCGUGAAACG

AUGCWGAAAGC

GGG WAGGUCAG

SabZ/PCR-2 > Sabin

2

--A

A-U

---

--U

A-U

-AA

G-C

---

A--

---

G-G GCC G--

--A

GGG

U--

A--

***

UC’U GAA G--

***

GCA CAG _O

-W

ACU AM

A-B

SakwPCR-2 Sabin

3

J

2500

Sabin

1

G-U

I

I

I

I

2510

2520

2530

2540

GCC CU-

-4,

I

2550

Sabl/PCR-1 < GUG GGG GCG GCA ACG UCU AGA GAC GCU CUC CCA AAC ACU GAA GCC AGU GGA

SabZ/PCR-1 i Sabin

2

u-- -W

C-C

C-G

--U

--C

-CC

A-U

AGC --G

--U

GGA CAC A-G

--U

G-U

C-G

--C

--U

---

__-

__C

Sabf/PCR-1 Sabin

3

->


, 71 bp (Sabin 21, and 44 bp (Sabin 3) (Fig. 2; Tables l-3). Specific product bands were obtained only when the corresponding template RNAs were present in the amplification reactions. When RNA was omitted from the amplification reactions, no product bands were detected. Because the amplification products from the three Sabin strain templates are clearly resolved by electrophoresis, the compositions of vaccine strain mixtures may be rapidly determined by PCR. When all three primer pair sets were included in

168

W

M

Sl

s2

s3

Sl s2

Sl S3

s2 S3

Sl s2 S3

N

M

97 _$ 71 +

44 +

Fig. 2. Specificity of amplification of Sabin strain RNA templates (Sl, Sabin 1; S2, Sabin 2; S3, Sabin 3) in reactions containing indicated templates (10 ng each) and all three sets of PCR primer pairs. Products were resolved by electrophoresis on 12.5% polyacrylamide gels, stained with ethidium bromide, and visualized by ultraviolet fluorescence. Symbols: M, molecular weight markers (Hue111 digest of pBR322, base pair lengths: 587/540/504/458/434/267/234/213/192/184/124/123/104/ 89/64/57/51/21/18/11/8; Boehringer Mannheim); Sl, S2, S3, Sabin templates added to reaction mixtures; N, negative control, no template added to reaction mixtures.

the amplification reactions, all combinations identified (Fig. 2).

of Sabin strain templates were easily

Vaccine-related clinical isolates

Vaccine-related clinical isolates may differ markedly from their parental vaccine reference strains. The oral poliovaccines frequently undergo extensive genetic and antigenic evolution during replication in the human intestine (Nakano et al., 1963; Crainic et al., 1983; Kew and Nottay, 1984; Minor et al., 1986a). The extent of the changes may impede intratypic differentiation based on antigenic traits. To evaluate the ability of the Sabin strain PCR primers to accurately identify vaccine-related isolates, the RNAs of 30 clinical isolates (10 of each serotype) previously characterized by partial genomic sequencing (Rico-Hesse et al., 19871, were analyzed by PCR. Sequences of all vaccine-related isolates were efficiently ampli-

169

fied by the corresponding primer pairs (Fig. 3; Tables 1-3). In addition, the oligonucleotide probes formed specific hybrids with the amplified DNA products (Fig. 3). In separate PCR experiments, well-characterized vaccine-related isolates known to be intertypic recombinants or antigenic variants were correctly identified by serotype and Sabin strain derivation (not shown). Thus, identifications by PCR and probe hybridization are apparently unimpaired by the genetic changes that often accompany natural replication of the oral poliovaccines.

MSlSZS3

MSlS2S31

1

2

2

3

3

4

5

6

7

8

9

10

4

5

6

7

8

910N

N

M

Sl

S2

53

1

2

3

4

5

6

7

8

9

10

N

4

5

6

7

8

9

10

N

Fig. 3. Specific amplification of the genomes of Sabin-related clinical isolates (Tables l-3) by PCR. Viral RNAs were extracted directly from clarified freeze-thaw lysates of infected cells and amplified in reaction mixtures containing all three primer pair sets. After 30 amplification cycles, DNA products were separated by electrophoresis on 12.5% polyacrylamide gels and visualized by ethidium bromide fluorescence (left panels) or hybridized in solution to the corresponding 32P-labeled Sabin strain probes (Materials and Methods) before electrophoresis and the hybrids detected by autoradiography (right panels). Each lane contains the products obtained from approximately lo3 infected cells (left panels) or 5 infected cells (right panels). Sources of templates in reaction mixtures are given after each lane number: Sabin l-related isolates. 1: 0246 2 : 9825 3 : 9703 4 : 9360 5 : 9240 6 : 0074 7: 8316 8 : 8315 9: 8284 10:8221. Sabin 2-related isolates. 1:0042 2:0078 3:9897 4:9819 5:9818 6:9.579 7:9364 8:8238 9:8018 10 : 6886. Sabin 3-related isolates. 1: 0040 2: 0131 3 : 0044 4: 9896 5 : 9847 6 : 9442 7 : 9441 8 : 8774 9: 8239 10:8024. M: molecular weight markers; Sl, S2, S3: reaction mixtures containing purified Sabin strain RNAs; N: reaction mixtures lacking RNA.

170

Standard wild poliovirus strains

Our primer pairs were further tested for their capacities to yield specific products with the genomes of laboratory strains widely used as reference wild polioviruses (Tables 1-3). RNA templates of the inactivated poliovaccine strains (Mahoney, MEF-1, and Saukett) and the serotype reference strains (Brunhilde, Lansing and Leon) were added to amplification reaction mixtures containing the three primer sets. Templates of the type 1 (Mahoney and Brunhilde) and the type 3 (Saukett and Leon) reference strains yielded PCR products that had identical mobilities to the corresponding Sabin strain products (Tables 1 and 3). In contrast, no amplification products were obtained with the genomes of the type 2 strains MEF-1 and Lansing (Table 2). These observations are readily interpretable from the genetic relationships among the reference poliovirus strains. Mahoney and Sabin 1, which share > 99% nucleotide sequence homology, have completely identical primer binding sequences (Nomoto et al., 1982). Brunhilde, also related to Sabin 1 (98% nucleotide homology within capsid region; unpublished results), has sequences that mismatch with the primers at only one position (mismatching Sabl/PCR-1). Similarly, the genomes of Leon and Sabin 3 (99.9% homologous) have perfectly matched Sab3/PCR primer binding sequences (Stanway et al., 1984). Sequences of Saukett, distantly related to Sabin 3 Wnnunen and Hovi, 1989; Kew et al., 19901, are amplified at lower efficiencies (indicated by reduced product yields) by the Sab3/PCR primers. DNA synthesis is primed from Saukett templates despite 2 mismatches in Sab3/PCR-1 and 3 mismatches in Sab3/PCR-2 (unpublished results). The genomic templates of MEF-1 and Lansing, both quite divergent from Sabin 2 (La Monica et al., 1986; Kew et al., 1990), are not amplified by the Sab2/PCR primer pairs. Multiple mismatches to the Sab2/PCR primers occur within the corresponding sequences of both type 2 templates (Sab2/PCR-1: 9 for MEF-1, 5 for Lansing; Sab2/PCR-2: 8 for MEF-1, 6 for Lansing). Wild poliovirus isolates

Numerous distinct genotypes of wild polioviruses currently exist in nature (Rico-Hesse et al., 1987; Kew et al., 1990). The genomic sequences of the independent genotypes are very heterogeneous, differing by as much as 23% within their capsid regions (Toyoda et al., 1984; Hughes et al., 1986; La Monica et al., 1986; Rico-Hesse et al., 1987; Kew et al., 1990). This extensive sequence diversity among wild poliovirus genomes raises the concern that some wild polioviruses might share sufficient local sequence homology with a vaccine strain within the regions corresponding to the PCR primer binding sites to permit cross-amplification of their genomes by our Sab/PCR primers. Such results could present serious diagnostic difficulties since wild polioviruses, having the potential for epidemic spread, would be incorrectly identified as vaccine-related strains, which are not associated with outbreaks. To further assess the specificities of our Sab/PCR primer sets, the RNAs of 30 independent wild isolates (10 of each serotype) were templates in reaction mixtures containing all three Sabin strain-specific PCR primer pairs. The 30 isolates

171

tested represent many different genotypes, having widely separate geographic origins (Rico-Hesse et al., 1987; Kew et al., 1990). Representatives of the major poliovirus genotypes currently endemic to North and South America, Asia and Africa were included. Despite the wide range of sequence variability among these wild isolates, our Sabin strain-specific PCR primers did not support amplification of their sequences as detected by ethidium bromide staining or hybridization with 32P-labeled probes (Fig. 4; Tables l-3). Thus, our Sab/PCR primers do not cross-amplify the sequences of the wild poliovirus genotypes most frequently obtained as clinical isolates. The only “wild” polioviruses whose sequences were efficiently amplified were historical reference strains known to be related to the Sabin vaccine strains. Background products

Minor products were occasionally generated in our amplification reactions (see Figs. 2 and 3). Conditions favoring the production of secondary bands include (1) high input template concentrations, (2) increased numbers (> 30) of thermal cycles, (3) primer annealing temperatures below 60°C or (4) use of templates extracted directly from clinical specimens such as stool. Some of the bands cross-hybridized with the corresponding Sabin-specific PCR probes, suggesting that they were produced in side reactions involving the same sequences normally amplified. The presence of background bands did not interfere with precise identification of viral genomes, and the sequence structures of the bands were not investigated further. Sensitivities

When tested with lysates of infected cells, amplification with our PCR primers permits detection of less than 1 infectious unit of vaccine-related poliovirus.

Fig. 4. Nonamplification of wild poliovirus (Tables l-3) genomic sequences by the Sabin strain-specific PCR primer pairs. Viral RNAs were added to reaction mixtures containing all three primer pair sets. After 30 thermal cycles, the mixtures were analyzed for amplified products as described in Fig. 3. Products were visualized after polyacrylamide gel electrophoresis by ethidium bromide fluorescence (left panel) or autoradiography (right panel). Each lane contains the products obtained from approximately lo3 infected cells (left panel) or 5 infected cells (right panel). Sources of templates in reaction mixtures are given after each lane number: Type 1 isolates. 1:0116 2: 9884 3: 9475 4: 8771. Type 2 isolates. 5:0176 6:7079 7:6876 8:1534. Type 3 isolates. 9:0174 lo:9033 11:7839 12:7094. M: molecular weight markers; Sl, S2, S3: reaction mixtures containing purified Sabin strain templates; N: reaction mixtures lacking RNA.

172

However, infectivity assays detect only a small fraction of the virions present in poliovirus preparations, as specific infectivities can vary from 50 to > 1000 particles per infectious unit. Therefore, we used purified poliovirus RNAs to determine the sensitivity limits of virus detection with our PCR primer pairs. Serial lo-fold dilutions (1O-3 to 10e9 ng; corresponding to 250,000 to 0.25 RNA molecules) of each Sabin strain RNA were used as templates for in vitro amplifica-

SABIN 1 log [ng RNA]

log [ng RNA]

log [ng RNA]

log [ng RNA]

SABIN

2

SABIN

3

log [ng RNA]

log [ng RNA]

Fig. 5. Sensitivity of detection of Sabin poliovaccine strain RNAs by PCR. Serial lo-fold dilutions of purified virion RNAs were added to reaction mixtures containing the corresponding primer pair sets. In vitro amplification and analyses of products were as described in Fig. 3. The average quantities of virion RNA present in the reaction mixtures at each dilution are indicated above the sample lanes. At high template dilutions, the proportion of PCR reaction mixtures containing RNA molecules is calculated from the Poisson distribution. At an average input RNA mass of 10-s ng, 92% of reactions contain 2 1 genome. At lOmy ng, 78% of samples contain no genomes. The mass of one poliovirus genome (MW = 2.5~ 10’) is approximately 4.2~ lo-’ ng.

173

tion with the corresponding primer pair. A portion of the amplification products were hybridized in solution to 32P-labeled oligonucleotide probes before polyacrylamide gel electrophoresis. Products were visualized by ethidium bromide fluorescence and autoradiography (Fig. 5). Specific product bands were obtained at very low levels of input template RNA. When visualized by ethidium bromide fluorescence, the lower limit for detection of Sabin 1 and Sabin 2 genomes was between 10m5 ng (2.500 molecules) and lo-’ ng (250 molecules). The smaller amplification product from the Sabin 3 template was

Sl s2 MS31

2

3

4

5

6

7

8

9101112N

Fig. 6. Utrect detection by PCK ot Sabm-related pottovuuses m clmtcal specimens. Nucleic acid fractions, extracted from samples of stool suspensions or rectal swabs shown to contain Sabin-related virus by cell culture isolation (Table 4j were amplified by 30 PCR cycles. DNA products were resolved on 12.5% polyacrylamide gels and visualized by ethidium bromide fluorescence. Sources of templates in reaction mixtures are given after each lane number: Samples containing Sabin l-related viruses: 1: 0084 2:9342 3:9225 4:8453. Samples containing Sabin 2-related viruses: 5:8763 6:8452 7:6902 8:6899. Samples containing Sabin 3-related viruses: 9:0123 lo:9237 11:8940 12:8761. M: molecular weight markers; Sl, S2, S3: reaction mixtures containing purified Sabin strain RNAs; N: reaction mixtures lacking RNA.

174

detected with less sensitivity, requiring between low4 ng (25,000 molecules) and lop5 ng (2500 molecules) in the PCR reactions to yield prominent ethidium-stained bands. Sensitivities approached the theoretical limit when PCR was combined with hybridization and autoradiography. Radiolabeled hybrids were detected when the amplification reactions contained as little as lo-’ ng (2.5 molecules) of each Sabin strain template. No product bands were obtained at dilutions yielding average template inputs of 10V9 ng or less (average compositions = I 0.25 genome/ sample). Direct detection of polioviruses in clinical specimens

The high sensitivities of PCR for detecting purified Sabin strain RNA templates suggested that in vitro amplification could be used to detect polioviruses directly in clinical material. To evaluate the feasibility of this approach, we analyzed RNAs extracted from 24 stool suspensions and rectal swabs which had been previously shown to contain Sabin-related viruses by isolation. Of the 24 specimens, 19 (79%) were positive for Sabin strain sequences by PCR; the amplification products of 12 of these specimens (4 of each serotype; Table 4) are shown in Fig. 6. In every positive reaction, the major product bands indicated the presence of the genome of the vaccine virus that had been isolated in cell culture. Indications of the presence of other vaccine strain genomes were obtained with some amplification reactions (for example, see minor 71 bp bands in lanes 9-12, Fig. 6). Possibly these other genomes were contained in inviable particles or were parts of minority virus populations that were overgrown by the predominant types during virus cultivation. We do not know why 21% of our clinical samples were negative by PCR. Some stool samples contain components that interfere with PCR reactions (Wilde et al., 1990). Olive et al. (1990) have emphasized the importance of prompt processing of stool samples after collection in order to obtain maximal rates of PCR positivity for enterovirus genomes. Our specimens, which had been sent to us months or years earlier from domestic and foreign laboratories, were not specially treated or stored. Prolonged storage at -20°C of original clinical material is generally accompanied by a decline in virus titer and may also be associated with a loss of PCR signal. Nevertheless, these preliminary experiments underscore the potential value of PCR for the direct detection and identification of polioviruses in clinical samples.

Discussion

PCR offers several key advantages over other methods for the detection and identification of polioviruses in clinical specimens. Chief among these is the capacity for predictable and very high specificities. Each of our PCR identifications is based upon three independent selective steps: efficient base-pairing by two separate primers binding different variable genomic intervals combined with the

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requirement that the predominant DNA products have the predicted chain lengths. An additional selective step is imposed when detection is by hybridization to specific probes. Stringencies of selection may be further controlled with the choice of genomic targets, the design of probes and primers (including the use of nested primer sets), the number and characteristics of the amplification cycles, and the conditions for probe hybridization. This exceptional degree of analytical flexibility permits the systematic application of conditions that take into account the relative variabilities of the genomic intervals targeted for amplification. We were aided in the design of our reagents by knowledge of the X-ray crystal structure of the poliovirion (Hogle et al., 1985). The structural domain selected for sequence amplification, while potentially able to induce neutralizing antibodies (Emini et al., 1983; Chow et al., 19851, appears to be under very limited immune pressure during natural infections, as it is internalized within native virions and only transiently externalized during the attachment phase of the replication cycle (Fricks and Hogle, 1990). Although the extent of variability within this domain is large, the rate of evolutionary divergence appears to be moderate. Thus, our Sab/PCR primers were able to prime amplification of the sequences of distant Sabin strain relatives, such as Brunhilde and Saukett, but not the sequences of contemporary wild polioviruses. It is important that the sequences targeted for amplification reside within the capsid region, so that the products obtained by PCR accurately reflect the serotypes of the viruses present in the specimens. A large proportion of vaccine-related clinical isolates are intertypic recombinants (Kew and Nottay, 1984; Minor et al., 1986a). However, intertypic recombinants with crossover sites within the capsid region have not been observed (King, 1988). Because the PCR target sequences are flanked by genetic intervals encoding type-specific antigenic sites, loss of linkage between PCR identity and serotype would require a double crossover within the capsid region, which is presumably a very rare event. Identification of serotype, either by direct or indirect means, is a key diagnostic function because of the implications of virus type to human immunity. A second major advantage of PCR is its extreme sensitivity. When our amplification products were detected by hybridization with 32P-labeled deoxyoligonucleotide probes, fewer than 2.5 genomic copies could be amplified to detectable levels. This level of sensitivity, approaching the theoretical detection limit of one template copy, is potentially 20- to lOO-fold higher than that of virus isolation in cell culture. Moreover, the poliovirus particle yield from a single infected cultured cell (approximately 105; Schaffer and Schwerdt, 1959) is more than four orders of magnitude higher than our threshold of detection by PCR. Attainment of optimal sensitivities with stool and other clinical specimens requires further refinements in sample preparation. The selection of short genomic intervals for amplification contributes to the analytic sensitivity of our system. Since AMV reverse transcriptase has comparatively low processivity (Berger et al., 19831, short nucleotide intervals are most efficiently transcribed in the initial step of the chain reaction. Moreover, because the Sabin strain-specific primer pairs are separated along the genome by less than

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100 nucleotides, even partially degraded RNA templates may be amplified. Consequently, detectability does not require any remaining virus infectivity. The proportion of each vaccine strain genome covered by the primer and probe sets is much smaller (approximately 0.9%) than the fraction resolved as characteristic RNase T,-resistant spots (lo-15%) in oligonucleotide fingerprinting (Kew et al., 1984). However, the percentage of the genome sampled by PCR is similar to that encoding surface residues (about 0.5%) that form neutralizing antigenic sites (Minor et al., 1986b; Page et al., 1988). Thus, the standard methods of serodifferentiation are based upon variation within an even smaller proportion of the genome (Nakano et al., 1963; van Wezel and Hazendonk, 1979; Ferguson et al., 1982; Osterhaus et al., 1983; Crainic et al., 1983). A third key advantage of PCR is that analyses are rapid and easy to perform. The high sensitivity of PCR-based detection can permit the direct amplification and identification of polioviruses from clinical specimens. While poliovirus isolation in cell culture will continue to be an important diagnostic procedure, especially for obtaining isolates for additional characterization, its routine use is comparatively slow, laborious, and expensive. Direct virus detection by PCR yields definitive results within a few hours. PCR also offers substantial economies over conventional methods in the analysis of mixtures of vaccine-derived viruses. Many clinical samples contain poliovirus mixtures because the oral poliovaccines are usually given in trivalent formulations. When the primer pairs are used in combination, the compositions of clinical specimens containing mixtures of vaccine-related viruses can be quickly determined from the mobilities of the product bands. We have used the Sabin strain-specific PCR primers and probes described here to identify poliovirus isolates from the United States and neighboring countries in North and South America. Vaccine-related isolates are positively identified with our Sab/PCR primer and probe sets. Wild poliovirus genomes can be identified by their lack of amplification in reaction mixtures containing these primers. However, confirmation of the identities of wild polioviruses required virus isolation and typing, usually followed by partial genomic sequencing (Rico-Hesse et al., 1987). A more direct approach is the selective amplification of wild poliovirus sequences. To this end, we have prepared primer and probe sets specific to each of several wild poliovirus genotypes (C.F. Yang et al., in preparation). These reagents have been used for the differential detection of wild polioviruses in clinical specimens and environmental samples that also contained Sabin-related viruses. Two kinds of primer and probe sets are useful for poliomyelitis-endemic countries: one for detecting Sabin-related strains and another specific for the indigenous wild poliovirus genotypes. Use of both sets increases the chances for detecting wild polioviruses, especially in samples also containing vaccine-related polioviruses. The principal challenge to the widespread implementation of PCR in routine diagnostics is the high potential for obtaining false-positive results if reagents and specimens are not handled proficiently, as even one contaminating template may be greatly amplified. Strict precautions must therefore be taken to avoid carryover of amplified DNA into subsequent reactions, and negative controls must be included each time diagnostic PCR tests are performed (Kwok and Sninsky, 1989).

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New developments in reagents and techniques (Sarkar and Sommer, 1990) should surmount these impediments and broaden the applicability of PCR to diagnostic virology.

Acknowledgements We thank Mary Flemister, Beverlie Hamby and George Marchetti for the preparation of poliovirus isolates, and Edwin George for assistance in preparing synthetic oligodeoxynucleotides. We are grateful for the assistance of Su-Ju Yang, who performed many of the PCR amplifications. We thank Chin-Yih Ou for constructive comments and suggestions. Lina De was supported by the Poliomyelitis Eradication Initiative of the Expanded Program on Immunization, Pan American Health Organization, Washington. This report is dedicated to the memory of our colleague, friend and teacher, James Nakano.

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