Haolin Ni, 1 N. Jack Burns, 2 Gwong-Jen J. Chang, 2 Ming-Jie Zhang, 2 Mark R. Wills, 3 Dennis W. Trent, ~ Peter G. Sanders 4 and Alan D. T.

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1 Journal of General Virology (1994), 75, Printed in Great Britain 1505 Comparison of nucleotide and deduced amino acid sequence of the 5' non-coding region and structural protein genes of the wild-type Japanese encephalitis virus strain SA14 and its attenuated vaccine derivatives Haolin Ni, 1 N. Jack Burns, 2 Gwong-Jen J. Chang, 2 Ming-Jie Zhang, 2 Mark R. Wills, 3 Dennis W. Trent, ~ Peter G. Sanders 4 and Alan D. T. BarretP* 1 Department of Pathology F-05, University of Texas Medical Branch, Galveston, Texas , 2 Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, Fort Collins, Colorado , U.S,A. 3Department of Medicine, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ and ~ Molecular Microbiology Research Group, School of Biological Sciences, University of Surrey, Guildford GU2 5XH, Surrey, U.K. Nucleotide sequences of the 5' non-coding region and the structural protein genes of the live, attenuated Japanese encephalitis vaccine virus strains SA and SA and the wild-type parental strain SA14/ USA were determined. SA differed from SA14/ USA by 13 nucleotides and eight amino acids whereas SA differed from SA14/USA by 15 nucleotides and eight amino acids. A comparison of the 5' noncoding region and amino acid sequences of the structural proteins of these two attenuated vaccine strains and of vaccine strains SA /PHK and SA /PDK with three sequences of their wild-type parent SA14 virus was performed. This revealed only two common amino acid substitutions at positions 138 and 176 in the envelope (E) protein. The substitution at E138 was predicted to cause a change in the secondary structure of the E protein. These two amino acid substitutions in the E protein may contribute to attenuation of the Japanese encephalitis vaccine viruses. Effective inactivated vaccines to control Japanese encephalitis (JE) have been developed but they are expensive and require repeated booster vaccinations. Several attempts have been made to develop a live attenuated JE virus vaccine (Rohitayodhin & Hammon, 1962; Kodama et al., 1968; Takehara et al., 1969; Yu et al., 1973). The most promising attenuated JE virus vaccine is the Chinese SA virus derived from the wild-type strain SA14 (Yu et al., 1973). The safety and efficacy of this vaccine have been confirmed in human vaccinees (Yu et al., 1981, 1988; Ao et al., 1983). The JE virus SA14 strain was isolated from mosquitoes collected in Sian, China, in 1954 (Li, 1986). The first attenuated variant, , was obtained after passage of the virulent parent virus SA14 11 times by intracerebral inoculation of newborn mice, followed by 100 passages in primary hamster kidney (PHK) cells (Yu et al., 1962; Li, 1986). Passage histories of the SA14 attenuated viruses are summarized in Fig. 1. The SA virus was The nucleotide sequence data presented here have been submitted to the GenBank database and assigned the accession numbers U02367 (SA14-2-8), U04521 (SA14-5-3), and U04522 (SA14/USA). attenuated by irradiation of virus with ultraviolet light followed by plaque purification. SA virus was derived from virus by additional plaque purification passages in PHK cells. SA /PHK virus was obtained by a further passage of the SA virus in suckling mice and by plaque purification in PHK cells (Yu et al., 1981 ; Li, 1986). Eckels et al. (1988) passaged SA /PHK virus nine times in primary dog kidney (PDK) cells to prepare the SAI4-14-2/PDK vaccine. All of these candidate vaccine strains are attenuated for humans and are not encephalitogenic in adult mice inoculated intracerebrally with 106 p.f.u, of virus (Chen & Wang, 1974; Yu et al., 1981 ; Wills et al., 1992). JE virus is a member of the Flavivirus genus. It has a positive-sense ssrna genome, approximately 11 kb in length, which encodes three structural proteins, capsid (C), membrane (M) and envelope (E) and seven nonstructural proteins (Brinton, 1986). The flavivirus C protein forms the structural component of the nucleocapsid. It elicits no neutralizing antibody and has groupreactive antigenic determinants (Chambers et al., 1990). The M protein is a component of the virus envelope and is believed to interact with the cell surface and elicit neutralizing activity (Brinton, 1986; Heinz, 1986). The E SGM

2 1506 Short communication SA14 Virulent parent strain I Isolated from mosquitoes in China I 1 passages in newborn mice 100 passages in PHK cell culture 3 plaque purifications Irradiation by u.v. and plaque purification in PHK cells Clone First attenuated variant I 6 animal passages 6 plaque purifications SA passages in suckling mice 2 plaque purifications SA /PHK 9 passages and plaque purification in PDK cell culture SA /PDK SA Fig. 1. The derivation of live attenuated vaccine clones of JE virus by passage of the wild-type strain SA14 in cell culture. protein contains the viral haemagglutinin. It induces protective immunity and mediates receptor-specific virus attachment to the cell surface (Brinton, 1986). This protein plays a major role in the pathogenicity of the virus by determining the cellular tropism and/or affecting virus penetration into susceptible cells (Gollins & Porterfield, 1984; Heinz, 1986). The molecular basis of JE virus attenuation has not been elucidated, although genomes of both the virulent parental strain SA14 and the attenuated vaccine viruses SA /PHK (Aihara et al., 1991) and SA /PDK (Nitayaphan et al., 1990) have been sequenced and compared. The nucleotide sequences of SA14 published by the two groups are not identical and nucleotide differences between parent and attenuated viruses SA /PHK or SA /PDK were identified throughout the genome. Aihara et al. (1991) found 57 nucleotide changes, coding for 24 amino acid substitutions, between SA14 (referred to here as SA14/ JAP) and SA /PHK. Nitayaphan et al. (1990) reported 45 nucleotide changes, coding for 15 amino acid substitutions, between SA14 (referred to here as SA14/ CDC) and SA /PDK viruses. Both groups reported that most of the amino acid substitutions were present in the virus structural proteins. Unfortunately the initial attenuated clone of SA14 virus, , no longer exists. Therefore to elucidate the molecular basis of the attenuation of JE virus, we cloned and sequenced the 5' non-coding region and structural protein genes of attenuated vaccine viruses SA and SA Since the published nucleotide sequence of SA14/CDC differed from SA14/JAP the structural protein genes of the parent SA14 virus (referred to here as SA14/USA) were again cloned and sequenced. The nucleotide and deduced amino acid sequences of SA and SA viruses were compared to the published sequences of the two SA attenuated vaccine strains and that of wild-type parental SA14. The biological, antigenic and immunogenic properties of vaccine strains SA14-2-8, SA and SA have been described previously. It has been shown that all of these viruses are attenuated for mice (Sil et al., 1992; Wills et al., 1992, 1993) and these viruses were used in the studies reported here. JE viruses were grown in mosquito C6/36 cells and viral RNA was extracted as previously described (Chomczynski & Sacchi, 1987; Jennings et al., 1993). Four sense oligonucleotide primers (covering nucleotides 1 to 34, 8 to 28, 895 to 916 and 1276 to 1303) and four antisense primers (nucleotides 1298 to 1317, 1180 to 1201, 2463 to 2483 and 3453 to 3477) were designed on the basis of the published SA14 genomic sequence (Nitayaphan et al., 1990). Viral RNA was reverse-transcribed, converted into cdna and sequenced using published methods (Nitayaphan et al., 1990; Lewis et al., 1992; Jennings et al., 1993). Computer analyses of nucleic acid sequence information and deduced amino acid sequences were accomplished using the MICROGENIE (Queen & Korn, 1984) and CLUSTAL (Higgins & Sharp, 1988) programs. Comparison of the nucleotide sequence of SA14/USA with that of the SA14/CDC sequence reported by Nitayaphan et al. (1990) revealed differences at positions 1052 (G to A), 1708 (G to A), 1921 (C to U), 2293 (A to G) and 2441 (G to A). These resulted in three amino acid differences at positions E244 (glutamic acid to glycine), E315 (alanine to valine) and E439 (lysine to arginine) of the E protein (Table 1, Fig. 2). Sequences of the SA14/USA and SA14/JAP (Aihara et al., 1991) viruses differed by four nucleotides at positions 1052, 1217, 1708 and 1977, which introduced two amino acid differences at positions 244 (glutamic acid to glycine) and 334 (proline to serine) of the E protein (Table 1). These sequence differences are presumably due to the passage history of the SA14 virus used in the three studies. SA14/USA was a mouse brain preparation of SA14 virus whereas SA14/CDC was a plaque-purified virus, derived from the mouse brain preparation containing SA14/USA virus following three passages in PDK cell culture (Eckels et al., 1988; Nitayaphan et al., 1990). SA14/JAP virus was derived by plaque purification of SA14 virus in baby hamster kidney (BHK)-21 cells (Aihara et al., 1991). Since these three preparations of

3 m Short communication 1507 Table 1. Comparison of nucleotide and amino acid differences among JE virus strains SA14, SAI4-14-2/PHK, SA /PDK, SA and SA Position Nucleotide Amino acid NT* AAt SA14 SA14 SA14 SA14- SA14- SAI4 SA14 SAI4 SAI4 SA14 /USA /CDC /JAP~ /USA /CDC /JAP /PHK{ /PDK 20 5'NCR C C C C C G NDII 'NCR U U U A A A U C65 U U U C C C U Leu Leu Leu 576 prm U U U U U C U Arg Arg Arg 578 prm C C C C U C C Cys Cys Cys 989 E4 G G G U U G G Leu Leu Leu 1052 E25 G A A A A A A Leu Leu Leu 1061 E28 U U U C C C C Asp Asp Asp 1217 E80 C C U U C C C Ala Ala Ala 1296 El07 C C C U U U C Leu Leu Leu 1354 E126 U U U U U U C lle Ile Ile 1360 E128 G G G G G G A Arg Arg Arg 1389 E 138 G G G A A A A Glu Glu Glu 1503 E176 A A A G G G G Ile Ile Ile 1506 E177 A A A G A A A Thr Thr Thr 1512 E179 A A A A A A G Lys Lys Lys 1661 E224 U U U U U U C Pro Pro Pro 1704 E243 G G G G A G G Glu Glu Glu 1708 E244 G A A A A A A GLU Gly Gly 1769 E264 G G G A G G G Gln Gln Gln 1813 E279 A A A U U U A Lys Lys Lys 192l E315 C U C U U U U Ala Val Ala 1977 E334 C C U C C C C Pro Pro SER 2051 E358 C C C C C C U Asn Asn Asn 2293 E439 A G A G G G G Lys Arg Lys 2441 E488 G A G A A A A Gly Gly Gly SA14- SA14- SA14 SA /PHK /PDK SER SER SER Leu Arg Arg Arg Arg Cys Cys Cys Cys Leu Leu Len Len Leu Leu Leu Leu Asp Asp Asp Asp Ala Ala Ala Ala PHE PHE PHE Leu Ile Ile Ile THR Arg Arg Arg LYS Lys Lys Lys Lys Val Val Val Val ALA Thr Thr Thr Lys Lys Lys GLU Pro Pro Pro Pro Glu LYS Glu Glu Gly Gly Gly Gly HIS Gln Gln Gln MET MET MET Lys Val Val Val Val Pro Pro Pro Pro Asn Asn Asn Asn Arg Arg Arg Arg Gly Gly Gly Gly * Nucleotide position. t Amino acid position; 5'NCR, 5' non-coding region; prm, M precursor protein. :~ Sequenced by Aihara et al (1991). Sequenced by Nitayaphan et al. (1990). It ND, Not determined due to the sense primer for reverse transcriptas~pcr being in this region. Bold type indicates the changes common to all four vaccine virus strains. SA14 virus are virulent for mice it would appear that the amino acid changes at E244, E315, E334 and E439 are not involved in the attenuation of SA14 virus. Comparison of the E protein amino acid sequences of SA14/USA, SA14/CDC and SA14/JAP viruses with those of five published wild-type JE virus strains, Nakayama (McAda et al., 1987), JaAOrS892 (Sumiyoshi et al., 1987), Beijing-1 (Hashimoto et al., 1988), Sarawak (Cecilia & Gould, 1991) and Kamiyama (Hasegawa et al., 1992), revealed that the amino acid at position E244 is variable; some strains have glutamic acid and others have glycine (data not shown). However, amino acids at positions E315 and E439 of SA14/CDC virus were shared with the four vaccine strains and not SA14/USA or SA14/JAP viruses. The amino acid at position E334 was proline in all the wild-type strains except SA14/JAP virus (data not shown). These results suggest that the SA14/USA virus sequence represents the dominant RNA genome of wild-type parental SA14 viruses. The nucleotide sequences of the 5' non-coding region and the structural protein genes of SA and SA viruses were determined and compared with those of the virulent parent virus SA14 and its attenuated vaccine clones SA /PHK and SA /PDK (Aihara et al., 1991; Nitayaphan et al., 1990; Table 1 and Fig. 2). The nucleotide change at position 39 in the 5' non-coding region of SA /PHK and SA /PDK viruses was present in the SA vaccine clone but not in the SA virus. This suggests that the change in the 5" non-coding region does not play a role in the attenuation of the virulence of JE virus. The 5' non-coding region of SA virus was found to have a unique nucleotide change at position 20. The sequence of SA virus differed from the published sequence of SA /PDK virus at five positions: nucleotides 20, 576, 578, 989 and Only nucleotide 1704 caused an amino acid difference, at position E243. SA differed from the published sequence of SA /PHK virus at six positions: nucleotides 20, 576, 989, 1217, 1506 and Only nucleotides 1506 (E177) and 1769 (E264) coded for amino acid differences (Table 1). Thus vaccine virus

4 1508 Short communication BHK-2I SA14/JAP~ ceils SA14 E244GIy E334 Ser E439 Lys... brain tissue E244 Glu SA14/USA E315 Ala E334 Pro..."x / "k ~ E L y ~ "~ SA14/CDC ~C65 keu -~ ~r ~23~ alu : ~Y SA ~I0378 ~]u 2 Ph: "~ E334 Pro E176 lie ---) Val SA E439 Lys ---) Arg ~ E279 Lys -~ Met E126 Ile* ~ Thr EI28 Arg* ~ Lys E138 Glu ---) Lys E176 lie ---) Val C65 Ser E179 Lys* ---) Glu El07 Phe SA E138 Lys (PHK) m 76 Val E177 Thr* ---) Ala E264 Gln* ---) His E279 Lys -~ Met C65 Ser El07 Phe E138 Lys SA E176 Val (PDK) z177 Ala -~ Thr E243 Glu* --~ Lys E264 His -~ Gin E279 Met Fig. 2. Flow diagram showing the amino acid differences between wildtype SA14 and its vaccine virus derivatives. The amino acid to the left of the arrow refers to that found in the progenitor of each virus and the other amino acids shown indicate the residue of the virus at that position. Amino acid substitutions marked (*) are unique for the virus. All four vaccine viruses had the same amino acid residues as those of SA14/CDC at E315 and E439. Amino acids in bold type refer to common substitutions shared by the four vaccine strains. SA differed from SA14/USA virus by 15 nucleotides and eight amino acids over the 5' non-coding region and structural protein genes. SA virus differed from its parent virus (SA14/USA) by 13 nucleotides and eight amino acids. These amino acid substitutions occurred at positions E126, E128, E138, E176, E179, E244, E315 and E439. Of the nucleotide differences present in the SA virus, substitutions at positions 1052 (E25), 1061 (E28), 1389 (E138), 1503 (E176), 1708 (E244), 1921 (E315) and 2293 (E439) were present at the same positions in the other attenuated JE virus vaccine strains (Table 1). Nucleotide changes at positions 1052 and 1061 did not result in amino acid substitutions. Nucleotide 1708 in SA virus was the same as that present in the SA14/JAP virus at this position. SA /PHK, SA /PDK and SA vaccine viruses have seven identical amino acid substitutions compared with the sequences of the SA14/USA and SA14/JAP viruses. These included one in the C protein (C65) and l six in the E protein (E107, E138, E176, E279, E315 and E439; Table 1 and Fig. 2). When the amino acid sequence of the SA virus was included in the comparison, four common amino acid substitutions were found (Table 1 and Fig. 2). These were at positions E138 (glutamic acid to lysine), E176 (isoleucine to valine), E315 (alanine to valine) and E439 (lysine to arginine). These attenuated JE vaccine viruses shared two common amino acids with the virulent SA14/CDC virus at positions E315 and E439. This suggests that these two amino acids are not involved in the attenuated phenotype. Comparison of 5' non-coding region and structural protein genes of four vaccine strains and wild-type JE virus strains revealed only two common amino acid substitutions, E138 and E176, in the vaccine strains. Nucleotide variations at positions 1354, 1360, 1512, 1661 and 2051 of SA virus resulted in three unique amino acid substitutions in the SA virus at positions E126, E128 and E179. The 13 nucleotide and nine amino acid differences between SA and SA /PHK viruses presumably resulted from the ultraviolet light irradiation treatment of SA virus and the subsequent 13 animal passages and six plaque purifications. The unique nucleotide difference at position 576 of SA virus did not result in an amino acid substitution. The deduced amino acid sequences of the parent SA14 virus and the attenuated derivative viruses were analysed by the Novotny method (Novotny & Auffray, 1984), which calculates hydrophobicity and can be used to predict protein conformation and any changes in secondary structure. This program predicted that the amino acid substitution at position E138 (glutamic acid to lysine) would cause a change in the charged residues profile and alpha-helix propensity of the E proteins of the attenuated viruses compared with parental SA14. Amino acid substitution at C65 of SA /PHK, SA /PDK and SA vaccine viruses would change the hydrophobicity profile and beta-sheet propensity of the C protein secondary structure. A unique difference was noted in the SA /PDK virus at amino acid 243 in the E protein, where lysine was present instead of glutamic acid in all other JE virus strains (data not shown). Haemagglutination (HA) assays using different strains of JE virus showed that the optimum ph for agglutination of gander red blood cells was 6-0 (Wills et al., 1992), whereas SA /PDK virus had a much broader optimum ph range (6.0 to 6.2; data not shown). It is possible that the lysine residue at E243 is involved in determining the optimum ph for the HA activity of the SA /PDK virus. Monoclonal antibody (MAb) reactivities of the wildtype parental SAI4 and attenuated vaccine derivatives

5 Short communication 1509 have been compared. E protein antigenic changes among the attenuated viruses have been found to be identical (Sil et al., 1992). By indirect immunofluorescence and HA inhibition tests, two E-protein reactive MAbs (T60 and V23), prepared against SA virus, recognized only vaccine strains derived from SA14 (SA /PHK, SA /PDK, SA and SA14-5-3). Five E-protein reactive MAbs (K10, K13, K24, K43 and J44), prepared against wild-type JE viruses, did not react with any vaccine virus strains (Sil et al., 1992). This indicates that common E protein epitope(s) are present on all vaccine strains derived from SA14, possibly due to common amino acid change(s). This hypothesis has been investigated by comparing the sequences of the parental SA14 virus and four attenuated clones. All of the attenuated vaccine strains have two identical amino acid substitutions at positions E138 and E176 (Table 1). Therefore, one or both amino acid substitutions in these clones could be responsible for the three-dimensional structure changes in the E protein that alter wild-typespecific epitopes and generate new vaccine-specific epitopes. The predicted change in secondary structure of the E protein caused by the glutamic acid to lysine substitution at position E138 supports this proposal. The two common amino acid substitutions in the E protein at positions E138 and E176 may also contribute to attenuation. The attenuated viruses were found not to bind dopamine D2 and/or 5HT~ subtype receptors recognized by the antagonist ligand spiperone, whereas the wild-type virulent parent strain SA14 did (Barrett et al., 1991). Infectious cdna clones of JE virus must be used to determine whether or not these amino acid substitutions are directly involved in the attenuation of the vaccine virus. This work was funded in part by grant MIM/V22/181/27, from the WHO Vaccine Development Programme, to A. D. T. 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