Complete nucleotide sequence of enterovirus 71 is distinct from poliovirus

ELSEVIER

Virus Research 39 (1995) 195-205

Virus Research

Complete nucleotide sequence of enterovirus 71 is distinct from poliovirus 1 B e t t y A. B r o w n *, M a r k A. P a l l a n s c h Respiratory and Enteric Viruses Branch, G17, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centersfor Disease Control and Prevention, Atlanta, GA 30333, USA

Received 29 June 1995;revised 16 August 1995;accepted 17 August 1995

Abstract Enterovirus 71 (EV71) is capable of causing paralytic disease indistinguishable from poliomyelitis due to poliovirus. To determine the relationship of EV71 to poliovirus and other enteroviruses, two strains of EV71 have been cloned and sequenced. The EV71 strains had only 46% amino acid identity with the polioviral P1 capsid region and 55% with the entire polyprotein. There were no regions of high similarity that might account for their respective ability to cause paralytic disease. The two strains, a neurovirulent isolate (EV71/7423/MS/87) and the prototype strain (EV71/BrCr), share 81% nucleotide identity and 95% amino acid identity. Sequence comparisons in the coding region between the two EV71 strains and other picornaviruses indicate that EV71, coxsackievirus A16 and coxsackievirus A2 comprise a distinct genetic group within the enteroviruses. Keywords: Enterovirus 71; Viral diversity; Poliovirus; Coxsackievirus A16;

1. Introduction Enterovirus 71 (EV71) is a human enterovirus within the family Picornaviradae that was first isolated and characterized from cases of neurological disease occurring in California from 1969 to 1973 (Schmidt et al., 1974). The neurovirulence of EV71 was again manifested in the 1975 outbreak in Bulgaria when 44 people died

* Corresponding author. Tel.: +1 (404) 639-2749; Fax: +1 (404) 639-1307; E-mail: [email protected]. 1Accession Numbers: EV71/BrCr: U22521; EV71/7423/MS/87: U22522. 0168-1702/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0168-1702(95)00087-9

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of paralytic disease (Chumakov et al., 1979; Shindarov et al., 1979). Subsequent epidemics of central nervous system (CNS) disease have been associated with EV71 in New York, Australia, Europe and Asia (Kennett, 1974; Blomberg et al., 1974; Chonmaitree et al., 1981; Nagy et al., 1982; Samuda et al., 1987; Gilbert et al., 1988). In Japan, two epidemics of hand, foot and mouth disease (HFMD) associated with EV71 occurred in 1973 and 1978, but the incidence of CNS disease was low in those epidemics (Hagiwara et al., 1978a; Tagaya et al., 1981). The spectrum of disease associated with EV71 has now been shown to include HFMD, aseptic meningitis, encephalitis, paralysis, and frequently inapparent infections. Viruses of the same serotype may be associated with outbreaks with different clinical manifestations which suggests strain differences could contribute to the pathogenic potential of the virus (Ishimaru et al., 1980). Since EV71 can produce a disease both clinically and pathologically resembling poliomyelitis, it has been hypothesized that EV71 might be genetically related to poliovirus. The precise nature and regions of relatedness might provide clues to the neurovirulence of both viruses. To date, no relationship between EV71 and poliovirus has been identified; however, cross-immunofluorescence studies suggest that EV71 and coxsackievirus A16 (CA16) share a common capsid epitope (Hagiwara et al., 1978b). Poyry et al. (1994) reported sequence similarity between these two viruses in the VP1 and 2A region. In this report we have determined the complete nucleotide sequence of the prototype strain EV71/BrCr and another neurovirulent strain of EV71 (7423/MS/87) and demonstrate that EV71/7423/ MS/87, EV71/BrCr and CA16 constitute a distinct genetic group of enteroviruses.

2. Materials and methods

2.1. Propagation of virus EV71 (strain 7423/MS/87) was isolated from an eighteen month-old boy in Mississippi who displayed paralytic illness in 1987 (CDC:MMWR 1988). EV71 (strain BrCr) was isolated from a 2-month-old infant suffering from aseptic meningitis (Schmidt et al., 1974). Both EV71 isolates were purified by limiting dilutions in HLF (human lung fibroblast) cells. Virus was propagated in HLF cells and harvested by freezing and thawing when total cytopathic effect was seen (approximately 72 h post-infection). Virus in the clarified supernatant was pelleted by centrifugation in a SW27 rotor at 100,000 × g for 4 h through a 30% sucrose cushion containing 1 M NaC1, 0.025 M Tris (pH 7.5) (Rueckert and Pallansch, 1981) In order to prepare virion RNA for synthesis of hybridization probes, the virus was further purified in CsC1 density gradients (Jamison et al., 1966). 2.2. Extraction of RNA The 100,000 x g viral pellet was resuspended in TNE (.01 M Tris (pH 7.8) 0.15 M NaCI, and 0.002 M EDTA) and heated for 1 min at 60°C in the presence of 4%

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197

SDS and 140 mM/3-mercaptoethanol (Hamby et al., 1987). The RNA was purified by phenol extraction and ethanol precipitated. To prepare virion RNA for synthesis of hybridization probes, CsCl-banded virions were dialyzed in TNE and the RNA purified as described previously. 2.3. cDNA synthesis and cloning strategy

In an initial experiment to obtain EV71-specific sequence, the 3' end of viral RNA was sequenced by extension of an oligo-(dT) primer using avian myeloblastosis virus (AMV) reverse transcriptase in the presence of dideoxy chain termination inhibitors as described previously (Zimmern and Kaesberg, 1978; Biggin et al., 1983; Rico-Hesse et al., 1987). From this sequence analysis an EV71-specific oligonucleotide primer was designed (5'-CCTCAAGTTCTCGAAGTTCGGAA-3') and used to direct cDNA synthesis. Synthesis and cloning of the cDNA was performed according to the procedure described by Gubler and Hoffman (1983) with a slight modification. The first strand was synthesized in a 40 /xl reaction containing 50 mM Tris-HCl (pH 8.3), 75 mM KC1, 3 mM MgC12, 10 mM dithiothreitol (DqT), 1 mM each of 4 dNTPs, 50 U cloned MMLV Reverse Transcriptase and 2/.~g RNA. The reaction mixture was incubated at 37°C for 1 h and then mixed with 280 #l of the solution for the second-strand synthesis at a final concentration of 25 mM Tris-HC1 (pH 8.3), 100 mM KCI, 10 mM (NH4)2SO4, 5 mM MgC12, 250/xM each of 4 dNTPs, 250 U / m l DNA polymerase I, 8.5 U / m l RNase H, 30 U / m l DNA ligase. The reaction mixture was incubated at 16°C for 2 h. To create blunt-end double-stranded cDNA the product was treated with T4 DNA polymerase and then extracted with phenol/chloroform and ethanol precipitated. The cDNA was inserted into SmaI-digested Bluescript SK + with 50 U / m l T4 DNA ligase in a reaction mixture containing 50 mM Tris-HCl (pH 7.6) 10 mM MgCI2, 10 mM D'IT, and 1 mM ATP at 16°C for 12 h. The recombinant plasmids were introduced into the E. coli XL-1 Blue by the method of Hanahan (1983). 2.4. Screening of recombinant clones

Clones were obtained by screening recombinant cDNA libraries with two types of EV71-specific hybridization probes. One probe, corresponding to N7282 to N7305, had been 5' end-labeled with 32p and T4 polynucleotide kinase (Sambrook et al., 1989). A second probe was 5' end-labeled EV71 RNA which had been fragmented and labeled with 32p ATP using T4 polynucleotide kinase (Coffin and Billeter, 1976). 2.5. Reverse transcriptase PCR (RT-PCR amplification)

DNA fragments of various lengths were amplified by the RT-PCR assay with virus-specific primers and Taq polymerase (Perkin-Elmer). PCR primers were designed from sequences obtained from the cDNA clones of EV71/7423/ MS/1987 and EV71/BrCr. Reactions contained approximately 0.5 /zg RNA, 20

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pmol of each primer, 100/~M of each of the four deoxynucleotide triphosphates, 2 mM MgC12, 1 × reaction buffer containing 67 mM Tris-HC1 (pH 8.8), 17 mM (NH4)2504, 1 mM /3-mercaptoethanol, 6 ~M EDTA, 0.2 m g / m l gelatin, 10 U placenta ribonuclease inhibitor, 5 U Taq polymerase and 12 U AMV Reverse Transcriptase (Boehringer, Mannheim) in a total volume of 100/zl. Each reaction was incubated in a Perkin Elmer Cetus DNA 9600 thermal cycler according to the following protocol: 30 min at 42°C, 5 min at 94°C, then 35 cycles of 94°C for 1 min, 42°C for 1 min and 65°C for 2 min, then finally 7 min at 65°C. The DNA fragments used as sequencing templates were purified by electrophoresis on 0.7% Nusieve (FMC Bioproducts) and excised bands were purified by Qiaquick (Qiagen), and then used in dideoxynucleotide sequencing reactions. Each fragment was sequenced in both directions. PCR amplicons were sequenced from four regions of E V 7 1 / 7 4 2 3 / M S / 8 7 spanning N1797 to N3108; N2275 to N3498; N3232 to N5210; N7196 to N7445. PCR amplicons were sequenced from regions of E V 7 1 / B R C R spanning N1 to N818; N720 to Nl120; N984 to N1348; and N7206 to N7445.

2.6. DNA sequencing Sequences were determined from PCR DNA or cDNA clones by the chain termination method (Sanger et al., 1982) using ABI fluorescent sequencing techniques. The primer extension method was used and the sequence was determined from both strands.

2. 7. Sequence determination of the 5' and 3' ends of genomic RNA The 3' end of viral RNA was sequenced following the method of Lambden et al. (1992). In this procedure a synthetic primer 1 was ligated to the 3' end of virion RNA. Subsequently a complementary primer 2 was annealed to primer 1 to promote cDNA synthesis. A DNA fragment spanning approximately 260 nucleotides of the 3' end was produced by PCR amplification using a virus-specific primer 3 and primer 2. Virus-specific primer 3 used in the determination of the 3' end of EV71/Brcr was 5'-CATAATGGAAAACAGGAGTATGAG-3', which corresponds to N7206N7229. Virus-specific primer 3 used in the determination of the 3' end of E V 7 1 / 7 4 2 3 / M S / 8 7 was 5'-CCTGCTGGCTTGGCACAATGGC-3', which corresponds to N7196-N7218. Genomic 5' end RNA sequences were determined by extension of sequencing primers with AMV reverse transcriptase in the presence of dideoxy chain termination inhibitors (Zimmern and Kaesberg, 1978; Biggin et al., 1983; Rico-Hesse et al., 1987). The primers used were 5'-GGTATAAAACAGGCGTACAAIGGTACCG3', the complement of N64 to N88 in the genome of EV71/BrCr, and 5'GGATATAAAACAGGCGCACAAGG-3', the complement of N69 to N91 in the genome of EV71/7423/MS/87.

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2.8. Computer analysis Nucleotide comparisons were made by using the GAP program of the Genetics Computer Group sequence analysis package (GCG). Multiple sequence alignments were generated by using the PILEUP program of GCG. Palmenberg's computer analysis with adjusting alignments according to secondary structure was also employed to generate alignments (Palmenberg, 1989). Phylogenetic trees were constructed using the DNAML program in the PHYLIP 3.5 package (Felsenstein, 1989).

3. Results

3.1. Sequence of EV71 The sequencing strategy with the individual clones and PCR amplicons is shown in Fig. 1. The base composition of the EV71/7423/MS/87 genome without the poly(A) tract showed a composition of 27.7% A, 24.1%G, 23.6% C, and 25.1% U and thus shows the slight A excess always seen in enteroviruses. An analysis of dinucleotide frequencies revealed the rarity of CG (3.2%) which is consistent with

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4

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Fig. 1. Nucleotide sequencing strategy. The position of cDNA clones and PCR amplicons, relative to EV71 RNA, is shown. (A) Inserts in recombinants M31 and M76 plasmids contained the coding sequence spanning N12 to N223 and N5126 to N7304. PCR amplicons were sequenced from regions of E V 7 1 / 7 4 2 3 / M S / 8 7 spanning N1797 to N3108; N2275 to N3498; N3232 to N5210; and N7196 to N7445. (B) Inserts in recombinants B40, B5 and B30 plasmids contained the coding sequence spanning N1292 to N3605, and N3333 to N5607, and N5126 to N7285. PCR amplicons were sequenced from regions of EV71/BrCr spanning N984 to 1348; N720 to 1120; N1 to N818; and N7206 to N7445. Each insert and amplicon was sequenced from both strands.

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Table 1 Comparison of nucleotide identities of EV71/MS/7423 with other enterovirus members (in %)

EV71/BrCr Coxsackie A16 Coxsackie A21 Coxsackie B1 Polio 1M Rhino 1B

G a

5'NTR

P1

P2

P3

3'NTR

81 77 58 60 58 53

85 86 70 83 71 62

82 68 53 53 53 52

77 82 57 61 58 51

80 79 61 61 61 54

92 79 50 46 41 43

The comparisons were done with the GAP program of the UWGCG package with a gap weight of 5.0 and gap lengths of 0.3. Reference strains are listed in the legend of Fig. 2A. a G represents genome.

a typical picornavirus. A frequency of 5.3% would be expected on a random basis considering base composition (Toyoda et al., 1984; Iizuka et al., 1987). If the 5'NTR is examined as a whole, EV71/7423/MS/87, EV71/BrCr, and CA16 show about 85% identity while other enteroviruses are less similar (see Table 1). When E V 7 1 / 7 4 2 3 / M S / 8 7 is compared to EV71/BrCr, CA16, Coxsackieviruses B1 (CB1), B3 (CB3), B4 (CB4), and B5 (CB5) in the conserved region spanning N412 to N652, there is a 90 to 93% identity. This identity drops markedly to 60% to 74% identity in the hypervariable region (N653 to N747). EV71/7423/ MS/87 shows 70% and 74% identity to EV71/BrCr and CA16 in this hypervariable region. The methionine codon which initiates translation is predicted to occur at position N747 and gives rise to a polypeptide of 2194 amino acids. The prototype strain has a similar coding region. A comparison of the entire coding region of E V 7 1 / 7 4 2 3 / M S / 8 7 and other enteroviruses is given in Table 1. The identity at the nucleotide level ranges from 81% to 77% identity with EV71/BrCr and CA16 to 58% with poliovirus. In turn, this translates to a 95% and 89% identity at the amino acid level with EV71/BrCr and CA16 and a 55% identity with poliovirus for the entire polyprotein (Table 2).

Table 2 Comparison of amino acid identities of EV71/MS/7423 with other enterovirus members (in %)

EV71/BrCr Coxsackie A16 Coxsackie A21 Coxsackie B1 Polio 1M Rhino 1B

G a

P1

P2

P3

VP1

VP2

VP3

VP4

95 89 55 57 55 46

97 79 47 46 46 44

94 95 55 64 59 44

94 95 62 63 62 49

93 71 38 39 36 37

99 84 56 53 55 52

99 84 47 46 45 44

100 78 61 49 58 44

The comparisons were done with the GAP program of the UWGCG package with a gap weight of 3.0 and a g a p length 0.10. Reference strains are listed in the legend of Fig. 2A. a G represents genome.

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The four structural proteins of the virion are derived from the P1 region. These capsid proteins confer on the virus distinct antigenic properties and permit unique receptor recognition. VP1 is the capsid protein which displays the most marked divergence with an amino acid identity that ranges from 93% for EV71/7423/

0

PV2 PV3

CA24 CA21

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CB4

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EV70

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CA16 EV711BrCr EV71174231MS/87

0.10

Fig. 2. (A) Phylogenetic tree demonstrating the genetic relatedness of enterovirus strains in the 5'NTR region (DNAML; Felsenstein, 1989). Sequences used for comparative analysis were PVIMahoney (Gen Bank accession no. J02281; Kitamura et al., 1981); PV2Lansing (M12197; La Monica et al., 1986); PV3Leon (K01392; Stanway et al., 1984); CA21 (D00538;Hughes et al.,1989);CA24 (D90457; Supanaranond et al., 1992); CA9 (D00627; Chang et al., 1989); F~hol2 (X77708; Kraus, W. unpublished data); CB3 (M33854; Klump et al., 1990); CB5 (X67706; Zhang et al., 1993); SVDV(X54521; Seechurn et al., 1990); CB4 (X05690; Jenkins et al., 1987); CB1 (M16560; Iizuka et al., 1987); EV70 D00820; Ryan et al., 1990); CA2 (L28146; Poyry et al., 1994); CA16 (U05876; Poyry et al., 1994); EV71/BrCr (U22521) EV71/7423/MS/87 (U22522); and Rhino 1B (D00239; Hughes et al., 1988). (B) Phylogenetie tree, constructed by the DNAML method (Felscnstein, 1989), based on 2585 bp from the P1 region of enterovirus strains.

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MS/87 compared to EV71/BrCr, 71% when compared to CA16, and 36% compared to poliovirus (Table 2). Nucleotide sequences of the P2 and P3 coding region of various enteroviruses were compared. The identity of EV71/7423/MS/87 in the P2 region compared to EV71/BrCr and CA16 is 77% and 82%, respectively. This identity increases to about 95% for all three at the amino acid level (Table 2). Similar levels of identity are in the nucleotide and amino acid sequences in the P3 region. The 3' non-coding region is relatively short (less than 100 nucleotides) and EV71/7423/MS/87 has 92%, 79%, and 41% identity, respectively, when compared to EV71/BrCr, CA16, and poliovirus.

3.2. Genetic diversity To compare the sequence relationship of EV71 to the other enteroviruses, phylogenetic trees were generated for each region of the enterovirus genome (both nucleotide alignments and amino acid alignments). There are basically two groups of viruses in the dendogram of the 5' NTR region (Fig. 2A). EV71/7423/MS/87 falls in the group with EV71/BrCr, CA16, CB1, CB3, CB4, CB5, CA9, Echovirus 12 (El2), and swine vesicular disease virus (SVDV). These share nucleotide identities ranging from 78% to 86% compared to EV71/7423/MS/87. The second group, which includes the polioviruses, CA24, CA21, and EV70, share 68% to 73% compared to EV71/7423/MS/87. When the P1, P2, and P3 coding regions are aligned and analyzed at either the nucleotide or amino acid level, the EV71 cluster (comprised of EV71/7423/ MS/87, EV71/BrCr, and CA16) falls into a unique group. This relationship of the P1 region is displayed as a dendogram in Fig. 2B. This pattern recurs in trees based on the P2 and P3 regions with minimum variation (not shown). In contrast to the genetic relationship in the 5'NTR region of two groups, there are four genetic groups in the P1 region. These four genetic groups include a poliovirus-like group, a coxsackievirus B-like group, EV70, and a distinct group with EV71, and CA16 and CA2. (The CA2 sequence is only available in the P1 and 2A regions.)

4. Discussion

The complete nucleotide and polyprotein sequences of EV71/7423/MS/87 and EV71/BrCr have been obtained and reflect typical enterovirus organization and composition. Overall these two EV71 strains share 81% nucleotide identity and 95% amino acid identity. Typically, the P1 region encoding the capsid proteins is the most variable coding region of the genome between different groups of enteroviruses. For instance, the three poliovirus serotypes (PoliolMahoney, Polio2Lansing, and Polio3Leon) share 72-74% nucleotide identity in the P1 region. CA16, the most closely related virus to EV71, shares 68% nucleotide identity with EV71 in the P1 region.

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EV71, CA16, and CA2 comprise a distinct genetic group when analyzed in the coding region. However, this close relationship is not apparent in sequence comparisons in the 5'NTR since there are only two groups. Sequence conservation among all the enteroviruses in the 5' NTR indicates that this region of the genome is less able to tolerate changes during the evolutionary process. In the 5'NTR, EV71 and CA16 are closely related to the coxsackie B-like group of viruses which includes CA9, CB1, CB3, CB4, CB5, SVDV, and El2, while they are distinct from this group in the remainder of the genome. The genetic similarity of EV71 and CA16 is consistent with the fact that both cause hand, foot, and mouth disease. EV71, however, has been more frequently associated with meningitis and paralytic disease than has CA16. On the other hand, genetic relatedness did not correlate with ability to cause paralytic poliomyelitis. EV71 and poliovirus are not closely related genetically. Sequence analysis indicates that the neurovirulent E V 7 1 / 7 4 2 3 / M S / 8 7 strain shares only 58% nucleotide identity with poliovirus and identifies no areas of high similarity even in areas corresponding to postulated poliovirus receptor sites (Colston and Racaniello, 1994). It is possible that the mechanisms of neurovirulence are different from poliovirus or that the genetic basis for neurovirulence resides on a small portion of the genome which our present comparison studies would not identify. Studies of the genetics of tissue tropism, cellular receptors, and regulatory proteins may allow us to determine genetic links between disease manifestations of different enteroviruses.

Acknowledgements We thank Eddie George and Melissa Olsen-Rasmussen for supplying oligonucleotides. We thank Tuija Poyry for CA16 sequence information. We thank Steve Monroe for assistance with sequence analysis. We also thank our collaborators Chen-Fu Yang and Edson da Silva for helpful discussion on virus growth and cloning strategies.

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