**3. Genotypic characteristics**

#### **3.1. Gene sequence of SA14-14-2 compared to its parent SA14 and other attenuated derivatives.**

When the full-length gene sequence of SA14-14-2 was compared to parental SA14, 57-66 nu‐ cleotide substitutions were found to be scattered all over the genome except prM. These coded for 24-31 amino acid substitutions, of which 8 were in E protein[1, 61] and were stud‐ ied to be the critical amino acid mutations involved in virus attenuation [3, 13] (Table 9). Among the 8 substituted amino acids observed in SA14-14-2 virus, only 3 substitutions ap‐ peared in the unstable virus SA14-12-1-7; while those highly and stable strains, SA14-9-7, SA14-5-3 and SA14-14-2 PDK viruses had 6 changes of the 8 amino acids [Ni H et al.,1994, 8]. Two of the 8 amino acid substitutions at position E-177 (T,Threonine→A,Alanine) and E-264 (Q, Glutamine→H, Histudine) were unique to SA14-14-2 virus [8] (Table 10). The contribu‐ tion of these two amino acid changes to the biological properties of SA14-14-2 virus requires further study.

Development of Japanese Encephalitis Attenuated Live Vaccine Virus SA14-14-2 and its Charcteristics http://dx.doi.org/10.5772/ 52980 191


**Table 9.** Comparison of amino acid differences between JE attenuated vaccine SA14-14-2 and its parent SA14 strain reported by Aihare and Zeng

Sequences were reported by [1] and [61]

and used to infect weanling mice by i.c. inoculation. In order to enhance the virus titer of the mosquito suspension (M-1), it was passed once in BHK cells (M-1 C-1) and then infected mice by i.c. and s.c. inoculation. The full E protein gene of the M-1 C-1 virus was sequenced and compared to that of its parent SA14-14-2 virus. As shown in Table 8, none of the mice died after bitten or i.c. inoculation with virus titers of 4.2 and 7.2 log10pfu/ml, and only one nucleotide in the virus E protein gene changed resulting in one amino acid substitution (E447 A→G) which was not reverse mutation. And the eight critical amino acids remained unchanged. The similarity of the virus full E gene sequence compared to that of the parent SA14 was 99.9% [34]. This result demonstrated that the SA14-14-2 virus is phenotypic and

**Suckling mice 2.5 weeks mice**

**Table 8.** Virulence and E gene sequence of the SA14-14-2 virus after *Culex tritaeniorhynchus* mosquitoes IT passage. a One intrathoracical passage (SA14-14-2 M-1) and one BHK-21 cell passage (M-1 C-1).b By the infected mosquitoes c

M-1C-1 107.2 0/10 0/10 0/8d E-447

**3.1. Gene sequence of SA14-14-2 compared to its parent SA14 and other attenuated**

When the full-length gene sequence of SA14-14-2 was compared to parental SA14, 57-66 nu‐ cleotide substitutions were found to be scattered all over the genome except prM. These coded for 24-31 amino acid substitutions, of which 8 were in E protein[1, 61] and were stud‐ ied to be the critical amino acid mutations involved in virus attenuation [3, 13] (Table 9). Among the 8 substituted amino acids observed in SA14-14-2 virus, only 3 substitutions ap‐ peared in the unstable virus SA14-12-1-7; while those highly and stable strains, SA14-9-7, SA14-5-3 and SA14-14-2 PDK viruses had 6 changes of the 8 amino acids [Ni H et al.,1994, 8]. Two of the 8 amino acid substitutions at position E-177 (T,Threonine→A,Alanine) and E-264 (Q, Glutamine→H, Histudine) were unique to SA14-14-2 virus [8] (Table 10). The contribu‐ tion of these two amino acid changes to the biological properties of SA14-14-2 virus requires

Pfu/ml bittenb ic ic sc mutation similarity

**E gene sequence**

(A→G)

99.9%

genetic stable and could not be transmitted after mosquito passage.

**Virusa Virus titer Virulence tested in**

No. dead /no. testedd No. reversion/no. attenuating amino acidSC Subcutaneously

SA14-14-2 M-1 104.2 0/16c 0/16

**3. Genotypic characteristics**

**derivatives.**

190 Encephalitis

further study.

#### **3.2. Stability of gene sequence of SA14-14-2 virus strain**

SA14-14-2 virus at PHK cells passage 8 (PHK8) was serially passed to PHK17 or given one i.c. passage in suckling mouse (HKC8SM1). The E protein gene of the viruses was sequenced and compared to that of SA14-14-2 PHK8 and parental SA14. At passage 17, all the 8 attenuating amino acid residues in SA14-14-2 PHK8 were retained no change, while two new nucleotide mutations were found at NT-1970 (TG), and NT-2169 (AG), which resulted in two ami‐ no acid changes at positions E-331 (SR) and E-398 (KE)( Table 11). The two substituted amino acids, Arg(R) and Glu(E), were not the residues of parental SA14, Ser(S) and Lys(K), this suggests that the two changes were not reverse mutation [9]. After one passage of the PHK8 virus in suckling mouse brain (SA14-14-2 HK8SM1), 7 of the 8 amino acids remained unchanged, one at E107 (F→L) was reverse mutation, while 3 other amino acid mutations appeared at E-83, E-318 and E-327, which were not reverse mutations (Table 11).

Li et al. [27] and Gao et al.[11] performed similar studies by additional passage of the SA14-14-2 vaccine seed virus (early passage) in PHK cell culture to passage 20 and passage 18, respectively. The E protein gene sequences of the various passaged viruses were se‐ quenced and compared to the original seed virus.The results demonstrated no reverse muta‐

Development of Japanese Encephalitis Attenuated Live Vaccine Virus SA14-14-2 and its Charcteristics

http://dx.doi.org/10.5772/ 52980

193

Xu et al [52] studied full-length sequence stability of the SA14-14-2 virus by passing the early passages seed virus(PHK8) on PHK cells to passage 22 (PHKC22) and its full-length genome was sequenced. By comparing the full sequences of the PHKC22 with the original primary seed virus of SA14-14-2 in Genbank (D90195), the result showed that there were only 8 nucleo‐ tide differences (one in E, 5 in non-structure-region and 2 in 3'-NTR) leading to 4 amino acids changed, which were not reverse mutations. The homology of the nucleotides and amino acids between the viruses of passage 22 viruses and the primary seed virus in Genbank was 99.93% and 99.88%, respectively (Table 12). These results demonstrated that the genotype of SA14-14-2

**Position Nucleotide change Amino acid change**

 E-389 a→c D→N NS2a-69 t→c none NS3-343 t→a W→R NS4a-57 c→t T→I NS4b-130 g→t none NS5-639 g→t Q→H

**Table 12.** Nucleotide and amino acid changes in the PHK cells passage 22 compared to the sequence of SA14-14-2 in

Wills et al. [48] investigated the ability of SA14-14-2 (PHK) and other 4 attenuated vaccine clones SA14-2-8, SA14-5-3, SA14-14-2 (PDK) to induce a humoral immune response in Balb/c

of the live viruses. Mice were bled 14 and 28 days postinoculation. Anti-JE serum antibody levels were measured using hemagglutination inhibition (HAI) and neutralization (N) tests. The results demonstrated that the live SA14-14-2 (PHK) elicited good HAI and N responses

N response by 14 days postinoculation and neutralizing antibody was not detectable with a

pfu at 14 days postinoculation, with the 28-days sera showing no re‐

pfu of the SA14-14-2 (PDK) virus evoked only a poor

and 106

pfu

mice. The mice, 6-8 weeks old, were inoculated by intraperitoneal route with 103

vaccine virus was very stable during multiple cell culture passages.

10701(3'-NCR) -→g 10784(3'-NCR) t→c

Nt Aa

tion of the 8 attenuating amino acids.

Genbank (D90195)

at dose of 103

**4. Immunogenicity**

**4.1. Humoral immune response**

and 106

duction in N titer. In comparison, 106


**Table 10.** Comparison of the amino acid differences in the E protein gene of JE SA14-14-2 PHK vaccine virus with the other attenuated derivatives and the parental SA14


**Table 11.** Substitutions of amino acid of SA14-14-2 strain after passage in PHK cell or suckling mice. HKC8V2, HKC 8 passages, vero cells 2 passages ;HKC17V2, HKC 17 passages, vero cells 2 passages ;HKC8SM1, HKC 8 passages, suckling mice one i.c. passage

Li et al. [27] and Gao et al.[11] performed similar studies by additional passage of the SA14-14-2 vaccine seed virus (early passage) in PHK cell culture to passage 20 and passage 18, respectively. The E protein gene sequences of the various passaged viruses were se‐ quenced and compared to the original seed virus.The results demonstrated no reverse muta‐ tion of the 8 attenuating amino acids.

Xu et al [52] studied full-length sequence stability of the SA14-14-2 virus by passing the early passages seed virus(PHK8) on PHK cells to passage 22 (PHKC22) and its full-length genome was sequenced. By comparing the full sequences of the PHKC22 with the original primary seed virus of SA14-14-2 in Genbank (D90195), the result showed that there were only 8 nucleo‐ tide differences (one in E, 5 in non-structure-region and 2 in 3'-NTR) leading to 4 amino acids changed, which were not reverse mutations. The homology of the nucleotides and amino acids between the viruses of passage 22 viruses and the primary seed virus in Genbank was 99.93% and 99.88%, respectively (Table 12). These results demonstrated that the genotype of SA14-14-2 vaccine virus was very stable during multiple cell culture passages.


**Table 12.** Nucleotide and amino acid changes in the PHK cells passage 22 compared to the sequence of SA14-14-2 in Genbank (D90195)
