**2. Phenotypic characteristic**

#### **2.1.** *In vitro* **phenotypic characteristics**

SA14-14-2 attenuated virus replicates well in primary hamster kidney (PHK) cell, C6/36 mosquito cells, continuous African green monkey kidney (Vero) cells, Rhesus monkey kid‐ ney (LLC-MK2) cells and baby hamster kidney (BHK21) cell lines. SA14-14-2 virus showed homogeneous small plaques (≤1mm) when grown in above - mentioned cells, while SA14 wild strain showed heterogeneous and larger plagues (2-3mm) [57][1].

Many attenuated viruses are temperature-sensitive, often showing restricted growth in vi‐ tro at 39-40℃, some strains were even sensitive to 37°C. SA14-14-2 strain was not temperaturesensitive, showing no reduction in infectivity at 37°C or 40°C. SA14-14-2 strain was also thermostable as the parental SA14 virus. In liquid status reduction of virus titer was 3.5log10TCID50 after heated at 50℃ for 50 minutes and virus could be detected 4 hours after further incubation at 50°C, a result similar to that observed with SA14 parent strain. SA14-14-2 was more thermostabe than the SA14-5-3 [57].

### **2.2. Ability of** *in vivo* **virus replication**

cine from one manufacturer [35] and of 60000 children given vaccine from another manufacturer [15] reported no cases of temporally associated encephalitis. The most common adverse effect associated with vaccination was fever, which was reported in less than 0.2% of vaccinated

Daily examination of 867 vaccinated children for fever (>38℃) disclosed low rates with on‐ set distributed evenly over the 21-day observation period, without clustering as might have been expected if onset were associated with a specific incubation period. Temperature eleva‐

A block-randomized coherent study of 13,266 vaccinated and 12,951 nonvaccinated children followed prospectively for 30 days has shown that no cases of encephalitis or meningitis were detected in either groups, and rates of fever, allergic, respiratory symptoms were similar in the two groups [33]. Moreover no case of encephalitis associated with the live vaccine has been reported so far from the large scale vaccination in other areas of China [66] and in countries

The immunogenicity and protection efficacy in humans have been studied several times in China and outside China. Neutralizing antibody were produced in 85%~100% of non-im‐ mune subjects studied in China [2, 58, 20, 64] and 98%, 92% and 95% studied in Korea [38], Philiphines [12] and Thailand [6], respectively after a single dose of vaccination. Several efficacy trials of SA14-14-2 vaccine in China from 1988 to 1999 in 1 to 10 year-old children have consistently yielded high protection rates, above 95%,[66, 5, 43]. One study in Guizhou prov‐ ince [67] and another in Anhui province [66] have shown the protection efficacy persisted for at least 11 and 5 years respectively following an immunization schedule of one primary dose at one age and one booster dose at two ages. Case-control study for evaluation the efficacy of SA14-14-2 vaccine has been studied. A case control study conducted in 1993 in Sichuan province, China in children <15 years measured vaccine effectiveness of routinely delivered SA14-14-2 vaccine at 80% for a single-dose and 97.5% for a two-dose given at a one year interval [14]. In 1999, the SA14-14-2 vaccine was given as a single dose to over 220000 residents of the Terai region of Nepal in an effort to reduce the impact of an emerging epidemic of JE. A casecontrol study demonstrated 99.12% efficacy [4] followed by a 5-year efficacy of 96% [41]. In 2000, a case-control study in Chongqing city, China found a 93% efficacy after one dose vaccination [42]. Besides, Kumar [23] reported a case control study in India, where 9.3 mil‐ lion children were immunized with SA14-14-2 vaccine in 2007, demonstrated a 94.5% vac‐

SA14-14-2 attenuated virus replicates well in primary hamster kidney (PHK) cell, C6/36 mosquito cells, continuous African green monkey kidney (Vero) cells, Rhesus monkey kid‐ ney (LLC-MK2) cells and baby hamster kidney (BHK21) cell lines. SA14-14-2 virus showed homogeneous small plaques (≤1mm) when grown in above - mentioned cells, while SA14

wild strain showed heterogeneous and larger plagues (2-3mm) [57][1].

outside China [38, 4].The vaccine is well tolerated in subjects as young as 8 months.

children, with lower rates for rash and other systemic symptoms.

tions were limited to a single day in most cases.

184 Encephalitis

cine efficacy after a single dose.

**2. Phenotypic characteristic**

**2.1.** *In vitro* **phenotypic characteristics**

Yu et al. [55] studied the replication ability of SA14-14-2 virus in young mice (2.5 weeks) by subcutaneous(s.c.) inoculation followed by recovering virus from spleens and subcu‐ taneous tissues of the infected mice for 2 weeks post inoculation. In those mice infected with the parent SA14 strain, viruses were recovered from the both tissues from day 4 to day 10 post infections, while those mice inoculated with SA14-14-2 strain, viruses were isolated in both tissues as well but limited within a short period from day 2 to day 4 or day 6 post infections (Table 4). Wu et al. [49] performed a similar study for recovering virus from brains and sera of the infected mice. Mice that infected with as less as 2.7log10 pfu/mL of the parent SA14 virus, high titers (≥6.0log10 pfu/mL) of virus was detected in the brains and low tit‐ ers (2.7 – 3.4log10 pfu/mL) in the sera, whereas mice inoculated with as high as 6.2log10 pfu/ mL of the SA14-14-2 ,, virus was neither detected in the brains nor in the sera of the mice. Lee et al. [24] examined virus growth of SA14-14-2 live virus and the parent SA14 virus strains in mice after i.p. inoculation. The results showed that SA14 virus was detected in sera and in spleens with peak titers of 3.24log PFU/ml and 4.3 logPFU/g, respectively as well as brains with peak titer of 6.42logPFU/g. On the other hand, SA14-14-2 virus was detected only in the spleens with extremely low titer (1.7logPFU/ml) but not detected in the sera and brains of all ten inoculated mice.


**Table 4.** *In vivo* replication of SA14-14-2 strain in mice. a Subcutaneous tissue

Further investigation for viremia induction of SA14-14-2 virus using guinea pigs animal model has been studied by Liu et al [31]. Guinea pigs intraperitoneally (i.p.) injected with 4.0log10 pfu/mL of parent virus SA14 and other 4 JEV virulent strains induced viremia to virus titers of 1.0-3.0log10 pfu/mL and lasted for 3 days post infection. However, the animals inoculated with SA14-14-2 strain containing virus titer equal to that of the virulent strains, no viremia was detected from day 1 to day 10 post inoculation (Table 5)

Besides, a viremia clinical study in India has demonstrated the absence of any viremia activity in adult population up to 15 days after administration of a single dose of the live JE SA14-14-2 vaccine.

of the virus inoculated intracerebrally results in death. Mice are more susceptible than mon‐ key to JE virus following inoculation by the peripheral route. Rhesus monkeys show four

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

**2.** "survival but showing neurological signs", i.e. the inoculation of SA14 95th PHK cell

**3.** "survival, without showing neurological symptoms, but with fever" i.e. the inoculation

**4.** "healthy", no fever, no symptom, no death, i.e. when the vaccine virus SA14-14-2 was

Neurovirulence of JE SA14-14-2 strain was tested using these animal models with virus tit‐ ers of 7.0~8.0log10 pfu/ml. Weanling mice inoculated with the virus by i.c. or s.c. inoculation did not cause death. SA14-14-2 was tested by standard intrathalamic and intraspinal combi‐ nation inoculation method in monkeys. Monkeys showed no mortality or morbidity and on‐ ly a minimal degree of CNS inflammation around the injection sites [30] (Table 6). Further, neuropathogenicity was tested in immune- deficient or immune-suppressed animals, athy‐ mic nude mice or mice treated with cyclophosphamide. No deaths or histopathologic abnor‐ malities were observed after intraperitoneal or subcutaneous inoculation of a viral dose greater than 107.0 TCID50/ml. Although cyclophosphamide increases susceptibility of mice to virulent JE strains, immunosuppression with cyclophosphamide did not lead to encepha‐ litis in mice inoculated pheripherally with SA14-14-2 virus [56, 18]. The strain also did not

**Mice Rhesus Monkeys**

**Died/tested**

**Histopathological score (neuronal lesions)ab**

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187

**Histopathological score (neuronal lesions)a**

10-4 8/8 2-4 0/1 2-3 10-5 ND ND 2/2 2-4 10-6 8/8 2-3 2/2 2-4 10-7 8/8 2-4 2/2 2-4 10-8 8/8 2-4 ND ND

IC 10-1 ND ND 2/2 2-4

SC 10-1 30/30 2-4(day 5) ND ND

IC 1:5 0/30 0-2 0/4 0-1 SC 1:5 0/30 0(1)c ND ND

**Table 6.** Comparative neurovirulence of attenuated SA14-14-2 and parent SA14 Japanese encephalitis viruses in 3 week-old mice and adult rhesus monkeysIC, intracerebral; SC, subcutaneous. ND, Not determineda) 0, No lesion; 1, ≤5%; 2, 6-20%; 3, 21-50%; 4, >50% of neurons died.b) Inoculation in thalami bilaterally (each 0.5ml) and lumbar

grades of response to the different attenuated JE viruses by i.c. inoculation [68]:

**1.** "death", the inoculation with parental SA14 virus;

passage virus (SA14 HKC-95) ;

kill weanling hamsters by i.c. inoculations [55].

spinal cord (0.2mL).c) One mouse showed a few dead nerve cells.

**Dilution Died/tested**

**Inoculation route**

with SA14-12-1-7 virus ;

inoculated.

**Virus strain (Virus titer, pfu/ml)**

SA14 parent (6.15×108)

SA14-14-2( 8×106)


**Table 5.** Viremia in guinea pigs after i.p. inoculation with different JEV virus strains. ND, Not determined

These results indicated that growth of the attenuated live virus in vivo was significantly re‐ duced in contrast to the growth of parent SA14 virus, and SA14-14-2 strain showed a lack of viremia and neuroinvasion.

#### **2.3. Neuroattenuation phenotype**

Mice and rhesus monkeys are highly susceptible to the wild virulent Japanese encephalitis virus (JEV) inoculated by intracerebral route. Approximately 1~10 plaque forming unit (pfu) of the virus inoculated intracerebrally results in death. Mice are more susceptible than mon‐ key to JE virus following inoculation by the peripheral route. Rhesus monkeys show four grades of response to the different attenuated JE viruses by i.c. inoculation [68]:

**1.** "death", the inoculation with parental SA14 virus;

Besides, a viremia clinical study in India has demonstrated the absence of any viremia activity in adult population up to 15 days after administration of a single dose of the live

> No.1 2.04 2.18 0 ND ND No.2 2.08 2.59 0 ND ND No.3 2.23 2.56 0 ND ND No.4 1.70 2.21 0 ND ND

> No.1 1.00 1.78 0 0 0 No.2 1.78 1.70 0 0 0 No.3 1.78 1.60 0 0 0 No.4 1.00 1.90 0 0 0 No.5 1.30 1.95 0 ND ND

> No.1 1.90 1.30 0 0 0 No.2 2.00 1.00 0 0 0 No.3 1.95 1.48 0 0 0 No.4 1.95 1.48 0 0 0 No.5 2.48 ND ND ND ND

> No.1 2.92 ND ND ND ND No.2 2.41 2.85 0 0 0 No.3 2.94 3.40 0 0 0 No.4 2.53 3.00 0 0 0 No.5 3.02 ND ND ND ND

No.1 0 0 0 0 0 No.2 0 0 0 0 0 No.3 0 0 0 0 0 No.4 0 0 0 0 0 No.5 0 0 0 0 0

**Table 5.** Viremia in guinea pigs after i.p. inoculation with different JEV virus strains. ND, Not determined

These results indicated that growth of the attenuated live virus in vivo was significantly re‐ duced in contrast to the growth of parent SA14 virus, and SA14-14-2 strain showed a lack of

Mice and rhesus monkeys are highly susceptible to the wild virulent Japanese encephalitis virus (JEV) inoculated by intracerebral route. Approximately 1~10 plaque forming unit (pfu)

**Viremia titer by day after inoculation (log10 pfu/mL)** 1 3 5 7 10

JE SA14-14-2 vaccine.

186 Encephalitis

P3

02-41

HLJ02-144

SA14

SA14-14-2

viremia and neuroinvasion.

**2.3. Neuroattenuation phenotype**

**Virus strain Animal No.**


Neurovirulence of JE SA14-14-2 strain was tested using these animal models with virus tit‐ ers of 7.0~8.0log10 pfu/ml. Weanling mice inoculated with the virus by i.c. or s.c. inoculation did not cause death. SA14-14-2 was tested by standard intrathalamic and intraspinal combi‐ nation inoculation method in monkeys. Monkeys showed no mortality or morbidity and on‐ ly a minimal degree of CNS inflammation around the injection sites [30] (Table 6). Further, neuropathogenicity was tested in immune- deficient or immune-suppressed animals, athy‐ mic nude mice or mice treated with cyclophosphamide. No deaths or histopathologic abnor‐ malities were observed after intraperitoneal or subcutaneous inoculation of a viral dose greater than 107.0 TCID50/ml. Although cyclophosphamide increases susceptibility of mice to virulent JE strains, immunosuppression with cyclophosphamide did not lead to encepha‐ litis in mice inoculated pheripherally with SA14-14-2 virus [56, 18]. The strain also did not kill weanling hamsters by i.c. inoculations [55].


**Table 6.** Comparative neurovirulence of attenuated SA14-14-2 and parent SA14 Japanese encephalitis viruses in 3 week-old mice and adult rhesus monkeysIC, intracerebral; SC, subcutaneous. ND, Not determineda) 0, No lesion; 1, ≤5%; 2, 6-20%; 3, 21-50%; 4, >50% of neurons died.b) Inoculation in thalami bilaterally (each 0.5ml) and lumbar spinal cord (0.2mL).c) One mouse showed a few dead nerve cells.

### **2.4. phenotypic stabilities**

#### *2.4.1. Stability of plaque morphology*

Small homogeneous plaque (≤1mm) morphology was retained through 8 -17 PHK cell pas‐ sages studied by Jia et al. [17]. As reported by Aihara, plaque-size phenotypes did not change during plaque purification in BHK21 cells and propagation in C6/36 cell [1]. Eckels et al. [7] showed that SA14-14-2 strain had a homogeneous small plaque morphology, with no large plaques seen when passed 7 times in LLC-MK2 cells.

Neuroattenuation after long-term cold storage has been studied. Fourteen lots of lyophilized live JE vaccines manufactured year by year since 1987 were stored at low temperature (-20℃). After 15 years, neurovirulence of the vaccine viruses were studied in year 2002. The results showed that viruses in the all 14 lots were avirulent for i.c. inoculated mice. [22]

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

Mosquito infection and transmission with SA14-14-2 virus have been done using *Culex tri‐ taeniorhynchus* mosquitoes, the most important JEV vector species, by oral feeding with meals containing the virus or intrathoracial(IT) inoculation with the virus. The mosquitoes did not become infected by oral feeding with meals containing 6.06log10pfu/ml virus and on‐ ly one of the 34 groups (3.13%) of the infected mosquitoes became infected after feeding with meals containing 6.18 log10pfu/ml virus, reaching a low virus titer of 1.24log10pfu/ml. However, most of the mosquitoes (10 of 14 groups, 71.43%) became infected after feeding meals containing 7.85log10pfu/ml of virulent JE virus strain Nakayama(Nak), reaching high‐ er titers of 3.33-4.79 log10pfu/ml (Table 7) [62]. The result indicated that SA14-14-2 virus is restricted in its ability to infect and replicate in the *Culex tritaeniorhychus* mosquito vector.

> **No. groups tested**

6.06 15(4-31)1 345(15-36)2 0(0%)

Nakayama 7.85 14(3-11) 215(11-26) 10(71.43%)(3.33-4.79)

**Table 7.** Growth of SA14-14-2 virus and wild virulent JEV Nak strain in *Culex tritaeniorhynchus* mosquitoes by oral

**3.** Virus titers in the mosquito suspensions of the positive groups (log10pfu/ml)

Mosquitoes were exposed to virus-containing meals for oral ingestion. Fully engorged mos‐

However, virus could replicate at low level by intrathoracical (I.T.) inoculation of the *Cx.tri‐ taeniorhynchus* mosquitoes [62], reaching titers of 2.0~3.72 log10pfu/ml over 2~20 days after inoculation. In contrast, mosquitoes IT infected with its parent SA14 virus exhibited higher ability of replication, reaching titers of 3.0~4.85 log10pfu/ml over the same periods [10].

The ability of transmission by the IT infected mosquitoes was studied later. Two groups of the mosquitoes were infected IT with SA14-14-2 virus, 8 days after infection one group of infected mosquitoes was used to infect suckling mice by direct bite, another group of infect‐ ed mosquitoes was made in a suspension (M-1), in which the virus content was measured,

6.18 34(2-11) 573(10-39) 1(3.13%)(1.24)3

**Total number of mosquitoes tested**

**No. group Positive (%)**

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189

**2.5. Growth characteristics and ability of transmission in mosquitoes**

**Virus strain Meals containing**

SA14-14-2

infections.

**virus(log10pfu/ml)**

quitoes were then collected after a period of extrinsic incubation.

**1.** Times(days) of extrinsic incubation periods;

**2.** Numbers of mosquito per group;

#### *2.4.2. Stability of neuroattenuation*

Wang et al. [44] studied neuroattenuation stability by serial passages the SA14-14-2 strain from passage 8 to passage 23 in PHK cells, virulence was tested every 2-3 passages by intracerebral or subcutaneous inoculation in mice (12 ~ 14g). None of the PHK-passaged viruses containing virus doses of 8.0~9.0 log10TCID50 caused death in these mice. Jia et al. [16] passed the SA14-14-2 virus in PHK cells for 17 passages, neurovirulence and neuroin‐ vasion were determined at the 8th, 15th, 17th passage. No animal showed illness at any the passage level.

Eckels et al. [7] performed the neuroattenuation stability by serial passage the SA14-14-2 vi‐ rus in Beagle canine kidney cells for a total of 15 passages, the passage 15 virus was attenu‐ ated for young mice causing no symptoms or death by i.c. inoculation. Wang et al. [45] passaged the SA14-14-2 HKC5 virus in primary dog fetal kidney cell cultures for 11 passag‐ es. Each of the passaged virus was tested for its pathogenicity in weanling mice, all the mice survived by either i.c. or s.c. inoculation with a virus dose containing 7.5-8.0 log10TCID50/0.2ml. These results showed that over many passages of the SA14-14-2 virus in PHK cells or primary dog kidney cells, neuroviruence reversed to the virulence of parental SA14 was not observed.

Wu et al [49] studied on the stability of SA14-14-2 vaccine seed virus by i.c. passage in suck‐ ling mice, viruses recovered from the mice brain of passage 1 only caused a few weanling mice death following i.c. inoculation with a high dose of 8.7 log10pfu/ml virus (LD50≥7.7 log10pfu), showing no reversion to the virulence of SA14 parent virus (i.c.LD50≥0.28 log10pfu).

Athymic nude mice (nu/nu) inoculated intra-peritoneally with 8.2log10TCID50/ml of SA14-14-2 virus did not fall ill. Attempts to recover virus from brain, liver, spleen, kidney, heart and lymph nodes were made over a period of 3 weeks. In three independent experi‐ ments viruses were recovered only in one experiment from brain at the 10th, 14th and 21st day and kidney tissue at 6th day. Virus isolated from the brains on day 21st after intraperitoneal inoculation, was enhanced once in PHK cell to a virus titers of 6.7-7.2 log10TCID50/ml and then tested for neurovirulence and neuroinvasiveness in normal mice. Tests were repeated three times and showed that the recovered viruses were avirulent to 10-12g mice by i.c. or intraperitoneal inoculation, maintaining the attenuated phenotype as SA14-14-2 [56].

Neuroattenuation after long-term cold storage has been studied. Fourteen lots of lyophilized live JE vaccines manufactured year by year since 1987 were stored at low temperature (-20℃). After 15 years, neurovirulence of the vaccine viruses were studied in year 2002. The results showed that viruses in the all 14 lots were avirulent for i.c. inoculated mice. [22]

#### **2.5. Growth characteristics and ability of transmission in mosquitoes**

**2.4. phenotypic stabilities**

188 Encephalitis

*2.4.1. Stability of plaque morphology*

*2.4.2. Stability of neuroattenuation*

passage level.

SA14 was not observed.

log10pfu).

large plaques seen when passed 7 times in LLC-MK2 cells.

Small homogeneous plaque (≤1mm) morphology was retained through 8 -17 PHK cell pas‐ sages studied by Jia et al. [17]. As reported by Aihara, plaque-size phenotypes did not change during plaque purification in BHK21 cells and propagation in C6/36 cell [1]. Eckels et al. [7] showed that SA14-14-2 strain had a homogeneous small plaque morphology, with no

Wang et al. [44] studied neuroattenuation stability by serial passages the SA14-14-2 strain from passage 8 to passage 23 in PHK cells, virulence was tested every 2-3 passages by intracerebral or subcutaneous inoculation in mice (12 ~ 14g). None of the PHK-passaged viruses containing virus doses of 8.0~9.0 log10TCID50 caused death in these mice. Jia et al. [16] passed the SA14-14-2 virus in PHK cells for 17 passages, neurovirulence and neuroin‐ vasion were determined at the 8th, 15th, 17th passage. No animal showed illness at any the

Eckels et al. [7] performed the neuroattenuation stability by serial passage the SA14-14-2 vi‐ rus in Beagle canine kidney cells for a total of 15 passages, the passage 15 virus was attenu‐ ated for young mice causing no symptoms or death by i.c. inoculation. Wang et al. [45] passaged the SA14-14-2 HKC5 virus in primary dog fetal kidney cell cultures for 11 passag‐ es. Each of the passaged virus was tested for its pathogenicity in weanling mice, all the mice survived by either i.c. or s.c. inoculation with a virus dose containing 7.5-8.0 log10TCID50/0.2ml. These results showed that over many passages of the SA14-14-2 virus in PHK cells or primary dog kidney cells, neuroviruence reversed to the virulence of parental

Wu et al [49] studied on the stability of SA14-14-2 vaccine seed virus by i.c. passage in suck‐ ling mice, viruses recovered from the mice brain of passage 1 only caused a few weanling mice death following i.c. inoculation with a high dose of 8.7 log10pfu/ml virus (LD50≥7.7 log10pfu), showing no reversion to the virulence of SA14 parent virus (i.c.LD50≥0.28

Athymic nude mice (nu/nu) inoculated intra-peritoneally with 8.2log10TCID50/ml of SA14-14-2 virus did not fall ill. Attempts to recover virus from brain, liver, spleen, kidney, heart and lymph nodes were made over a period of 3 weeks. In three independent experi‐ ments viruses were recovered only in one experiment from brain at the 10th, 14th and 21st day and kidney tissue at 6th day. Virus isolated from the brains on day 21st after intraperitoneal inoculation, was enhanced once in PHK cell to a virus titers of 6.7-7.2 log10TCID50/ml and then tested for neurovirulence and neuroinvasiveness in normal mice. Tests were repeated three times and showed that the recovered viruses were avirulent to 10-12g mice by i.c. or

intraperitoneal inoculation, maintaining the attenuated phenotype as SA14-14-2 [56].

Mosquito infection and transmission with SA14-14-2 virus have been done using *Culex tri‐ taeniorhynchus* mosquitoes, the most important JEV vector species, by oral feeding with meals containing the virus or intrathoracial(IT) inoculation with the virus. The mosquitoes did not become infected by oral feeding with meals containing 6.06log10pfu/ml virus and on‐ ly one of the 34 groups (3.13%) of the infected mosquitoes became infected after feeding with meals containing 6.18 log10pfu/ml virus, reaching a low virus titer of 1.24log10pfu/ml. However, most of the mosquitoes (10 of 14 groups, 71.43%) became infected after feeding meals containing 7.85log10pfu/ml of virulent JE virus strain Nakayama(Nak), reaching high‐ er titers of 3.33-4.79 log10pfu/ml (Table 7) [62]. The result indicated that SA14-14-2 virus is restricted in its ability to infect and replicate in the *Culex tritaeniorhychus* mosquito vector.


**Table 7.** Growth of SA14-14-2 virus and wild virulent JEV Nak strain in *Culex tritaeniorhynchus* mosquitoes by oral infections.

Mosquitoes were exposed to virus-containing meals for oral ingestion. Fully engorged mos‐ quitoes were then collected after a period of extrinsic incubation.


However, virus could replicate at low level by intrathoracical (I.T.) inoculation of the *Cx.tri‐ taeniorhynchus* mosquitoes [62], reaching titers of 2.0~3.72 log10pfu/ml over 2~20 days after inoculation. In contrast, mosquitoes IT infected with its parent SA14 virus exhibited higher ability of replication, reaching titers of 3.0~4.85 log10pfu/ml over the same periods [10].

The ability of transmission by the IT infected mosquitoes was studied later. Two groups of the mosquitoes were infected IT with SA14-14-2 virus, 8 days after infection one group of infected mosquitoes was used to infect suckling mice by direct bite, another group of infect‐ ed mosquitoes was made in a suspension (M-1), in which the virus content was measured, 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 genetic stable and could not be transmitted after mosquito passage.

**Position SA14-14-2**

SA14 aa nt Zeng Aihara C-65 292 S S L E-107 1296 F F L E-138 1389 K K E E-176 1503 V V I E-177 1506 A A T E-264 1769 H H Q E-279 1813 M M K E-315 1921 V V A E-334 1977 P S S E-439 2293 R R K NS1-292 3351 S S G NS1-339 3493 M M R NS1-351 3528 H H D NS1-354 3539 K K N NS1-392 3652 V V A NS2B-63 4403 D D E NS2B-65 4408 G G D NS2B-87 4475 F L L NS3-59 4782 V V M NS3-73 4825 K K R NS3-105 4921 G G A NS3-343 5634 R W R NS4A-27 6634 I T I NS4B-106 7227 V V I NS5-31 7768 G A A NS5-45 7809 S R R NS5-195 8261 I M M NS5-386 8832 Y Y H NS5-636 9593 H Q Q NS5-671 9688 A A V NS5-731 9898 G D D NS5-759 9954 P A A NS5-767 9978 V L L

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

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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]


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