JGV Papers in Press. Published April 23, 2015 as doi:10.1099/vir.0.000160
Journal of General Virology Genotype-specific neutralization determinant in envelope protein: implications for the improvement of Japanese Encephalitis vaccine --Manuscript Draft-Manuscript Number:
VIR-D-15-00192R1
Full Title:
Genotype-specific neutralization determinant in envelope protein: implications for the improvement of Japanese Encephalitis vaccine
Short Title:
JEV genotype-specific neutralization determinants
Article Type:
Standard
Section/Category:
Animal - Positive-strand RNA Viruses
Corresponding Author:
Cheng-Feng Qin Beijing Institute of Microbiology and Epidemiology Beijing, CHINA
First Author:
Qing Ye
Order of Authors:
Qing Ye Yan-Peng Xu Yu Zhang Xiao-Feng Li Hong-Jiang Wang Zhong-Yu Liu Shi-Hua Li Long Liu Hui Zhao Qing-Gong Nian Yong-Qiang Deng E-De Qin Cheng-Feng Qin
Abstract:
Japanese encephalitis (JE) remains the leading cause of viral encephalitis in children in Asia and is expanding its geographic range to larger areas in Asia and Australasia. Five genotypes of Japanese encephalitis virus (JEV) co-circulate in its geographically affected areas. Especially, the emergence of genotype I (GI) JEV has displaced genotype III (GIII) as dominant circulating strains in many Asian regions. However, all approved vaccine products are derived from GIII strains. In the present study, bioinformatic analysis revealed GI and GIII JEV strains shared two distinct amino acid residues within envelope (E) protein (E222 and E327). By using reverse genetic approaches, A222S and S327T mutations were demonstrated to decrease the liveattenuated vaccine (LAV) SA14-14-2 induced neutralizing antibody in human, without altering viral replication. Then, A222S or S327T mutations were rationally engineered into the infectious clone of SA14-14-2, respectively, and the resulting mutant strains remained the same genetic stability and attenuation characteristics as their parent strain. More importantly, immunization of mice with LAV-A222S or LAV-S327T elicited increased neutralizing antibodies against GI strains. Together, these results demonstrated that E222 and E327 are potential genotype-related neutralization determinants, and are critical in determining the protective efficacy of live JE vaccine SA14-14-2 against circulating GI strains. Our findings will aid in the rational design of next generation of live-attenuated JE vaccine capable of providing broad protection against all JEV strains belonging to different genotypes.
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Title Page
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Genotype-specific neutralization determinant in envelope protein: implications
3
for the improvement of Japanese Encephalitis vaccine
4 5
Qing Yea†, Yan-Peng Xua†, Yu Zhang a, Xiao-Feng Lia,b, Hong-Jiang Wanga,
6
Zhong-Yu Liua, Shi-Hua Lia, Long Liua,c, Hui Zhaoa,b, Qing-Gong Niana, Yong-Qiang
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Denga, E-De Qin a, Cheng-Feng Qin a,b,c*
8
a
9
Beijing 100071, China
Department of Virology, Beijing Institute of Microbiology and Epidemiology,
10
b
State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, China
11
c
Graduate School, Anhui Medical University, Hefei 230032, Chin
12
†These authors contribute equally to this work.
13 14
*Corresponding author. Mailing address: Department of Virology, Beijing Institute of
15
Microbiology and Epidemiology, Beijing 100071, China, Email:
[email protected] 16
Content category: Animal RNA viruses
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Running title: JEV genotype-specific neutralization determinants
18 19
Number of words in the summary: 229
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Total number of words in the main text: 3455
21
Number of Figures: 5
1
1 2
ABSTRACT
3
Japanese encephalitis (JE) remains the leading cause of viral encephalitis in
4
children in Asia and is expanding its geographic range to larger areas in Asia and
5
Australasia. Five genotypes of Japanese encephalitis virus (JEV) co-circulate in its
6
geographically affected areas. Especially, the emergence of genotype I (GI) JEV has
7
displaced genotype III (GIII) as dominant circulating strains in many Asian regions.
8
However, all approved vaccine products are derived from GIII strains. In the present
9
study, bioinformatic analysis revealed GI and GIII JEV strains shared two distinct
10
amino acid residues within envelope (E) protein (E222 and E327). By using reverse
11
genetic approaches, A222S and S327T mutations were demonstrated to decrease the
12
live-attenuated vaccine (LAV) SA14-14-2 induced neutralizing antibody in human,
13
without altering viral replication. Then, A222S or S327T mutations were rationally
14
engineered into the infectious clone of SA14-14-2, respectively, and the resulting
15
mutant strains remained the same genetic stability and attenuation characteristics as
16
their parent strain. More importantly, immunization of mice with LAV-A222S or
17
LAV-S327T elicited increased neutralizing antibodies against GI strains. Together,
18
these results demonstrated that E222 and E327 are potential genotype-related
19
neutralization determinants, and are critical in determining the protective efficacy of
20
live JE vaccine SA14-14-2 against circulating GI strains. Our findings will aid in the
21
rational design of next generation of live-attenuated JE vaccine capable of providing
22
broad protection against all JEV strains belonging to different genotypes.
23 24
Key words: Japanese encephalitis virus; Genotype; Live attenuated vaccine; Neutralization
25
2
1 2
INTRODUCTION
3
Japanese encephalitis (JE), a typical mosquito-borne viral disease, is one of the most
4
important causes of viral encephalitis resulting in about 68,000 cases and
5
10,000-15,000 deaths worldwide annually (Solomon et al., 2003; van den Hurk et al.,
6
2009). Japanese encephalitis virus (JEV) is transmitted by Culex mosquitos between
7
vertebrate hosts, particularly pigs and birds, and humans are occasional dead-end
8
hosts. Clinical manifestation of JEV infection ranges from undifferentiated febrile
9
illness and aseptic meningitis to acute encephalitis, and death. About half of survivors
10
have severe neurological sequelae (Solomon et al., 2000). The geographical range of
11
JE is expanding to larger areas in Asia and Australia during the last decade (Hanna et
12
al., 1996; Mackenzie et al., 2004). To date, JE affects more than 25 countries, where
13
~60% of the global population lives at risk of JEV infection.
14
JEV is a member of the family Flaviviridae, genus Flavivirus, together with other
15
important human pathogens including dengue virus (DENV), West Nile virus (WNV),
16
yellow fever virus (YFV) and tick-borne encephalitis virus (TBEV). JEV has an
17
approximately 11 kb positive-sense, single-stranded RNA genome, which contains a
18
single open reading frame that encodes three structural proteins (capsid, premembrane,
19
and envelope) and seven nonstructural proteins (NS1 to NS5), flanked by the 5’ and 3’
20
untranslated regions (UTRs). Based on the envelope (E) gene sequence or the
21
complete genome, JEV can be further divided into five genotypes (GI to GV) (Uchil
22
& Satchidanandam, 2001), and each genotype has a specific geographic distribution
23
pattern. Currently, GI and GIII strains represent the most prevalent genotypes in many
24
epidemic countries. In the first half of 20th century, the majority of JEV isolates from
25
human belong to GIII; however, since the last two decades, GI virus has gradually
26
replaced GIII as the dominant circulating strains in many areas of Asia including
27
China, Vietnam, Japan, India and Thailand (Ma et al., 2003; Nga, 2004; Nitatpattana
28
et al., 2008; Pan et al., 2011; Parida et al., 2006; Sarkar et al., 2012). The mechanism
29
underlying the genotype displacement as well as the impact of the genotype shift on
30
transmission/outbreak control strategies remains largely unknown (Han et al., 2014; 3
1
Schuh et al., 2013; Schuh et al., 2014).
2
Vaccination has been proven the most effective method to prevent and control JE,
3
and the incidence has decreased significantly in many Asian countries. Currently,
4
several inactivated vaccines and live attenuated vaccines have been licensed for
5
clinical use (Beasley et al., 2008; Halstead & Thomas, 2010). The inactivated
6
vaccines are cultivated in mouse brain, primary hamster kidney cells or Vero cells.
7
The live attenuated vaccine SA14-14-2 was first licensed in 1989 in China, and now
8
has been widely used in JEV-endemic countries in Asia including China, South Korea,
9
Thailand, India, Nepal and Sri Lanka (Gatchalian et al., 2008; Yu, 2010). More than
10
300 million children have been immunized with LAV SA14-14-2, and the safety and
11
efficacy profile has been well established (Bista et al., 2001; Hennessy et al., 1996;
12
Kumar et al., 2009; Liu et al., 1997; Tsai et al., 1998). Very recently, a recombinant
13
chimeric LAV based on the yellow fever vaccine strain (17D) backbone carrying the
14
prM and E gene of JEV SA14-14-2 (JE-CV) has been licensed in multiple countries
15
including Australia, Thailand, Malaysia and Philippines (Bonaparte et al., 2014;
16
Feroldi et al., 2014; Monath et al., 2015). However, all the licensed JE vaccines,
17
including SA14-14-2, are derived from GIII viruses. Previous clinical trials with GIII
18
vaccines in areas where heterologous genotypes are circulating have showed
19
effectiveness. It has been also expected that immunity induced by GIII vaccines
20
would provide cross protection against the infection of other JEV genotypes. However,
21
various immunological assays with antisera or monoclonal antibodies have
22
demonstrated the existence of antigenic differences among genotypes of JEV strains
23
(Hasegawa et al., 1994; Hasegawa et al., 1995; Kimura-Kuroda & Yasui, 1983;
24
Kobayashi et al., 1985; Wills et al., 1993). Data from inactivated GIII vaccines
25
demonstrated reduced neutralization capability against heterologous GI viruses
26
(Beasley et al., 2004; Erra et al., 2013; Fan et al., 2012; Ferguson et al., 2008; Kurane
27
& Takasaki, 2000). Mice immunized with SA14-14-2 showed decreased
28
neutralization antibody titers against GI viruses (Liu et al., 2011). Recently, we also
29
demonstrated that the LAV vaccination induced reduced levels of neutralizing
30
antibody against GI viruses in human (Ye et al., 2014). Especially, several groups 4
1
reported JE cases in individuals who have been vaccinated with LAV SA14-14-2 (Hu
2
et al., 2013; Sarkar et al., 2013; Zhang et al., 2011). Therefore, the rapid spread and
3
replacement of GI viruses might depress the protective efficacy induced by current JE
4
vaccines, and an improved JE vaccine that would confer broad protection against all
5
circulating genotypes, especially GI viruses, is needed.
6
Flavivirus E glycoprotein is the principal antigen in eliciting neutralizing
7
antibodies and plays a critical role in inducing protective immunity against virus
8
infection. The E protein contains three discernible domains (DI, DII and DIII) (Luca
9
et al., 2012). Numerous neutralizing epitopes have been identified at the tip of DII
10
(Oliphant et al., 2006), in the hinge region between DI and DII (de Alwis et al., 2012;
11
Teoh et al., 2012), and on the DIII (Austin et al., 2012; Gromowski & Barrett, 2007;
12
Oliphant et al., 2005). Previous investigation on DENV has revealed genotype- and
13
strain-dependent epitopes that impacts neutralization and protection (Shrestha et al.,
14
2010; Sukupolvi-Petty et al., 2013).
15
In the present study, by combining bioinformatic analysis and reverse genetic
16
technology, we identified two JEV genotype-specific neutralization determinants
17
within E protein (E222 and E327). And the corresponding mutations (A222S and
18
S327T) were rationally engineered into the LAV strain SA14-14-2 to generate an
19
improved version of JE LAV with enhanced neutralization capability against GI
20
circulating strains.
21 22 23
RESULTS
24
E222 and E327 were distinct in JEV GI and GIII strains. A total of 40
25
envelope gene sequences of JEV strains were retrieved from GenBank, the detailed
26
information about the origin, collection date and host of the JEV strains used in this
27
study was shown in Table S1. Phylogenetic analysis and multiple sequence alignment
28
of E protein identified two genotype-specific amino acid residues (Fig. 1a and b). The
29
amino acid 222 and 327 of E protein (E222 and E327) are alanine (A) and serine (S)
30
in all GIII strains including LAV SA14-14-2 respectively, while all GI strains contain 5
1
serine (S) and threonine (T) at the same positions, respectively. Besides, E222 is
2
serine (S) in GII viruses, or alanine (A) in GIV and GV viruses; E327 is threonine (T),
3
leucine (L) or glutamine (Q) in GII, GIV and GV viruses, respectively. Then, all
4
available sequences of complete E gene of JEV were retrieved from GenBank, and
5
E222 and E327 were confirmed as GI- and GIII- specific in all 570 E protein
6
sequences (Fig. S1).
7
To figure out the three dimensional location of E222 and E327, the two residues
8
were mapped to the crystal structure of JEV E protein (Fig. 1c) and the reconstructed
9
virion (Fig. 1d). It was found that E222 and E327 locate in DII and DIII, respectively.
10
E327 was clustered with other epitopes recognized by known neutralizing antibodies
11
(Fig. 1c). The reconstructed virion model revealed that E327 is largely exposed on the
12
surface of mature virion, while E222 was buried inside (Fig. 1d), suggesting that these
13
two amino acids may function in different mechanisms.
14
A222S and S327T mutations decreased LAV-induced neutralization. To
15
clarify the potential role of E222 and E327 in neutralization, single and double
16
mutations of these two residues were introduced into a full-length infectious clone of
17
GIII JEV (pAJE70). After standard in vitro transcription and transfection procedure,
18
all three mutant viruses (A222S, S327T, and A222S/S327T) were recovered in
19
BHK-21 cells. RT-PCR and sequence analysis confirmed that the corresponding
20
mutations were successfully engineered into JEV genome. Immunofluoscence
21
staining using anti-JEV mouse sera showed that A222S, S327T or A222S/S327T
22
generated similar levels of viral protein expression with the WT virus (Fig. 2a). There
23
is no significant difference in plaque morphology among the four viruses (Fig. 2b).
24
Growth curves in BHK-21 cells showed that WT and the mutant viruses A222S,
25
S327T, and A222S/S327T replicated efficiently, and peaked at 48 h post-infection
26
with titers of 106.94 PFU/ml, 106.40 PFU/ml, 106.79 PFU/ml and 106.69 PFU/ml,
27
respectively (Fig. 2c). These results demonstrated that A222S and S327T mutations in
28
E protein did not significantly impact viral replication.
29
Then, to clarify whether these mutations affect JEV neutralization, a panel of
30
human sera from volunteers immunized with LAV SA14-14-2 was used to assess the 6
1
neutralizing antibody titers against WT and each mutant virus. As shown in Fig. 3, all
2
human sera could efficiently neutralize the three mutant viruses together with the WT
3
virus (Fig. 3). However, the PRNT50 titers against each mutant virus significantly
4
decreased in comparison with that against the WT virus for selected sera (#1, #2, and
5
#5). These results showed that single mutation in either E222 or E327 would affect
6
the neutralization capability against GI and GIII JEV.
7
LAV-A222S and LAV-S327T induced higher neutralizing antibody titers
8
against GI strains. Further, to clarify whether these genotype-specific mutations
9
would enhance the neutralization capability of LAV SA14-14-2 against GI viruses,
10
A222S and S327T mutation was individually introduced into the infectious clone of
11
LAV SA14-14-2 by standard reverse genetic approach. As expected, the resulting two
12
mutant viruses, named LAV-A222S and LAV-S327T, respectively, were successfully
13
rescued with similar viral protein expression efficiency (Fig. 4a) and plaque
14
morphology (Fig. 4b) in BHK-21 cells in comparison with LAV. Growth kinetics in
15
BHK-21 and C6/36 cells demonstrated that LAV-A222S and LAV-S327T replicated
16
similarly with LAV SA14-14-2 in mammalian cells but exhibited a slightly decreased
17
multiplication capacity in mosquito C6/36 cells (Fig. 4c).
18
The immunogenicity of LAV-A222S and LAV-S327T were further tested in mice
19
in comparison with LAV SA14-14-2. Groups of 3-week-old mice were immunized
20
with equal dose of LAV, LAV-A222S or LAV-S327T, respectively, and neutralizing
21
antibody response against representative GI circulating strains SX06 and SH53 were
22
determined, respectively. The results showed that both LAV-A222S and LAV-S327T
23
could elicit higher titers of neutralization antibodies against GI strains SX06 and
24
SH53 than that by SA14-14-2 (Fig. 5a and b). These results further demonstrated the
25
introduction of A222S or S327T mutation into the LAV SA14-14-2 enhanced the
26
neutralization capacity against GI strains.
27 28
DISCUSSION
29
In our study, phylogenetic analysis and sequence alignment of all available JEV E
30
protein sequences revealed E222 and E327 are conserved within, meanwhile distinct 7
1
between GI and GIII viruses. The introduction of mutations into the two residues
2
significantly changed the neutralization capability of human sera, demonstrating the
3
critical role of E222 and E327 in JEV genotype-specific neutralization. To our
4
knowledge, E222 and E327 are first reported as the only known genotype-specific
5
neutralization determinants of JEV. Structural modeling revealed that E327 was in the
6
lateral ridge in DIII and largely exposed on the mature virion (Fig. 1c and d),
7
clustered with the residues (G302, E306, S331, D332, G333, I337, F360 and R387)
8
recognized by JEV-specific neutralizing MAbs (Cecilia & Gould, 1991; Goncalvez et
9
al., 2008; Lin & Wu, 2003; Wu et al., 2003; Wu et al., 1997) , indicating that E327
10
probably directly influences neutralization by interacting with other identified
11
neutralizing epitopes nearby. Structure modeling indicated that E222 is not exposed
12
on the surface of mature JEV virion (Fig. 1d), whether any conformational interaction
13
with the identified neutralizing epitopes remains unknown. In addition, structural
14
analysis revealed the diversification of flavivirus surface structure in virus life-cycle,
15
and the hidden epitopes may expose on the surface through the conformational
16
changes in viral glycoprotein arrangement (Lok et al., 2008; Zhang et al., 2004).a
17
These genotype-dependent epitopes are JEV specific, and completely different
18
from that of DENV (Bernardo et al., 2009; Brien et al., 2010; Kochel et al., 2002;
19
Shrestha et al., 2010; Wong et al., 2007). Previous studies using monoclonal
20
antibodies indicated that the potent neutralizing capacity was impacted by the
21
genotype-dependent conformational change/exposure of the CC’ loop epitope in DIII
22
(Austin et al., 2012). The natural occurring genotype- or strain- dependent amino acid
23
variations in DIII have been demonstrated to affect the binding and neutralization of
24
type-specific MAbs of DENV (Pitcher et al., 2012; Sukupolvi-Petty et al., 2013;
25
Wahala et al., 2010).
26
The LAV SA14-14-2 possesses advantages of satisfactory immunogenicity, less
27
dosage of vaccination and low cost of production compared to inactivated vaccine. It
28
now takes the largest JE vaccine market worldwide, and further improvement in
29
vaccine efficacy would be meaningful. Based on the infectious cDNA clone of LAV
30
SA14-14-2, the newly identified genotype-specific neutralization determinants were 8
1
rationally engineered. The mutant viruses LAV-A222S and LAV-S327T showed
2
similar plaque morphology and replicated efficiently in Vero cells with peak titers of
3
105.9 PFU/ml and 106.5 PFU/ml (data not shown), indicating the potential capability of
4
large-scale production and industrialization at low cost. Both mutants exhibited
5
potential genetic stability after serial passages in Vero cells (data not shown). For
6
immunogenicity test, immunization with LAV-A222S and LAV-S327T were capable
7
of eliciting protective neutralizing antibody against the circulating GI viruses SX06
8
and SH53, with increased antibody titers compared to those induced by native LAV
9
SA14-14-2. These properties support LAV-A222S and LAV-S327T as an improved
10
version of JEV LAV.
11
Until now, the mechanisms of circulating JEV genotype replacement are not fully
12
understood. E protein is the phylogenetic basis for JEV genotype division and the
13
major component on virion surface as well as the critical factor of virulence and
14
immunity, therefore it is considered as one of the most important subject investigated.
15
Phyletic and adaptive evolutionary analyses predicted the positively selected sites
16
within GI and GIII alignment as potential genetic determinants affecting the genotype
17
distribution, besides, it has been suggested that increased viral multiplication in avian
18
and mosquito as well as the more efficient replication and transmission cycle may be
19
responsible for the genotype replacement (Han et al., 2014; Schuh et al., 2014). In
20
this study, the recovered JE viruses LAV-A222S and LAV-S327T with GI mutations
21
showed a slightly decrease in replication efficiency in C6/36 cells, and the possible
22
role of host adaptions remains to be determined. Moreover, naturally occurring
23
sequence variation within or between genotypes may influence the antibody binding
24
and neutralization capacity (Austin et al., 2012; Sukupolvi-Petty et al., 2013; Wahala
25
et al., 2010). More data of antigen variation surveillance and evolution analysis are
26
required for the explanation of the genotype shift.
27
Our results have important implications for the evaluation and design of next
28
generation JE vaccine candidate. Currently, the JE chimeric live attenuated vaccine
29
JE-CV has been used in Asian countries as well as SA14-14-2 (Bonaparte et al., 2014;
30
Feroldi et al., 2014), and several flavivirus chimeric live attenuated vaccine carrying 9
1
JEV E protein is under development (Gromowski et al., 2014; Sjatha et al., 2014).
2
These two amino acid mutations A222S and S327T should be taken into consideration
3
for the rational design and improvement of a new JEV vaccine with potential
4
protection capability against all circulating JEV strains.
5 6
MATERIALS AND METHODS.
7
Cells and viruses. BHK-21 and Vero cells were cultured in Dulbecco’s modified
8
Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum
9
(FBS) in growth medium in 5% CO2 environment at 37℃. C6/36 cells were grown in
10
RPMI 1640 (Invitrogen) medium supplemented with 10% FBS at 37℃ in 5% CO2.
11
LAV strain SA14-14-2 (GIII) was from Chengdu Institute of Biological Products, and
12
its parental strain SA14 was from National Institute for Food and Drug Safety. GI JEV
13
strains SX06 and SH53 were isolated in Shanxi, in 2006, and Shanghai, in 2001,
14
respectively (Ye et al., 2014). All JEV strains were propagated in BHK-21 cells
15
cultured in DMEM with 2% FBS. Virus stocks were stored in aliquots at −80℃ until
16
use. Virus titers were determined by standard plaque forming assay in BHK-21 cells
17
as described before (Li et al., 2013a).
18
Phylogenetic analysis and sequence alignment. A total of 40 E protein gene
19
sequences of JEV strains (Table S1), which were isolated from a variety of geographic
20
regions in Asia, were collected from GenBank.
21
Multiple sequence alignment (MSA) and phylogenetic analysis were performed
22
by CLUSTALW (http://www.ebi.ac.uk/Tools/clustalw2/index.html) and MEGA
23
version 5.0 software (http://www.megasofteware.net). The phylogenetic tree was
24
constructed by the neighbor-joining method and the maximum composite likelihood
25
model.
26
Structure modeling. The crystal structure of JEV SA14-14-2 E protein (PDB
27
accession code, 3P54) was used to exhibit the modeled structure of the E protein.
28
Considering the similarity among mosquito borne flaviviruses, the E protein monomer
29
was superimposed onto the DENV-2 virion model (PDB accession code, 1THD)
30
based on cryo-EM reconstruction to generate a rational model for JEV virion. All the 10
1
identified JEV neutralizing epitopes were mapped onto JEV E protein and the
2
modeled virion (Luca et al., 2012). The structure demonstration and graphic rendering
3
were performed by using PyMOL.
4
Construction and characterization of recombinant JEV. All plasmids were
5
constructed using standard molecular clone protocols and confirmed by DNA
6
sequencing. Single mutation (A222S or S327T) or both mutations (A222S and S327T)
7
in E protein were introduced into the infectious full-length JEV (GIII) cDNA clone
8
pAJE70 previously described (Ye et al., 2012) by site-directed mutagenesis based on
9
overlapping PCR. The constructed plasmids were named pAJE70-A222S,
10
pAJE70-S327T and pAJE70-A222S/S327T, respectively. The mutations mentioned
11
above were introduced into the infectious full-length cDNA clone of LAV SA14-14-2
12
(named LAV) in the same fashion.
13
Xho I-linearized plasmids were purified and used as DNA templates for in vitro
14
transcription as previously described (Li et al., 2013a) in the presence of m7GpppA
15
cap analog with RiboMaxTM Large Scale RNA Production system (Promega). RNA
16
transcripts were transfected into BHK-21 cells with Lipofectamine 2000 (Invitrogen).
17
Four days after transfection, culture supernatants were harvested and seeded into
18
BHK-21 monolayer to prepare virus stocks. Recovered viruses derived from pAJE70,
19
pAJE70-A222S, pAJE70-S327T and pAJE70-A222S/S327T were named WT, A222S,
20
S327T and A222S/S327T. Mutant viruses based on LAV SA14-14-2 were named
21
LAV-A222S and LAV-S327T, respectively.
22
Indirect immunofluorescence assay were performed in BHK-21 cells using
23
anti-JEV mouse sera as previously described (Li et al., 2013b). Growth curves of all
24
recombinant viruses were generated by infecting confluent BHK-21 and C6/36 cells
25
in a 24-well plate. Cell supernatants were collected per 24 h post-infection and virus
26
titers were determined by standard plaque assay in BHK-21 cells.
27
Neutralization assay. A panel of human serum samples were collected from
28
volunteers vaccinated with LAV and subjected to 50% plaque reduction neutralization
29
test (PRNT50) (Ye et al., 2014). Briefly, 2-fold serial dilutions of serum sample were
30
inactivated at 56℃ for 30 min, and then mixed with equivalent JEV suspension and 11
1
incubated at 37℃ for 90 minutes. The PRNT50 titers were calculated by the method of
2
Karber (Traggiai et al., 2004). PRNT50 >1:10 was considered protective (Hombach et
3
al., 2005).
4
Animal immunization. To evaluate the immunogenicity of mutant vaccines,
5
groups of 3-week-old female BALB/c mice were injected by s.c. route with equal
6
dose (105 PFU/mouse) of LAV-A222S or LAV-S327T, and LAV group was set as
7
control. A booster dose was given 30 days after primary immunization, and mice sera
8
were collected 30 days post the second immunization. Sera samples were inactivated
9
and stored at -20℃ and subjected to neutralization assay by PRNT50.
10 11
Statistic analysis. Significant difference of neutralization titers was analyzed by one-way ANOVA using Graphpad Prism 5.0.
12 13 14 15
ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No.31300151 and No.31470265).
16
12
1 2
REFERENCES
3 4 5
Austin, S. K., Dowd, K. A., Shrestha, B., Nelson, C. A., Edeling, M. A., Johnson, S., Pierson, T. C.,
6 7 8 9
Beasley, D. W., Li, L., Suderman, M. T., Guirakhoo, F., Trent, D. W., Monath, T. P., Shope, R. E.
10 11
Beasley, D. W. C., Lewthwaite, P. & Solomon, T. (2008). Current use and development of vaccines
12 13 14
Bernardo, L., Fleitas, O., Pavon, A., Hermida, L., Guillen, G. & Guzman, M. G. (2009).
15 16 17
Bista, M. B., Banerjee, M. K., Shin, S. H., Tandan, J. B., Kim, M. H., Sohn, Y. M., Ohrr, H. C.,
18 19 20
Bonaparte, M., Dweik, B., Feroldi, E., Meric, C., Bouckenooghe, A., Hildreth, S., Hu, B., Yoksan,
21 22 23
Brien, J. D., Austin, S. K., Sukupolvi-Petty, S., O'Brien, K. M., Johnson, S., Fremont, D. H. &
24 25
Cecilia, D. & Gould, E. A. (1991). Nucleotide changes responsible for loss of neuroinvasiveness in
26 27 28
de Alwis, R., Smith, S. A., Olivarez, N. P., Messer, W. B., Huynh, J. P., Wahala, W. M., White, L.
29 30 31
Erra, E. O., Askling, H. H., Yoksan, S., Rombo, L., Riutta, J., Vene, S., Lindquist, L., Vapalahti, O.
32 33 34 35
Fan, Y. C., Chen, J. M., Chiu, H. C., Chen, Y. Y., Lin, J. W., Shih, C. C., Chen, C. M., Chang, C.
Diamond, M. S. & Fremont, D. H. (2012). Structural basis of differential neutralization of DENV-1 genotypes by an antibody that recognizes a cryptic epitope. PLoS Pathog 8, e1002930.
& Barrett, A. D. (2004). Protection against Japanese encephalitis virus strains representing four genotypes by passive transfer of sera raised against ChimeriVax-JE experimental vaccine. Vaccine 22, 3722-3726.
for Japanese encephalitis. Expert Opinion on Biological Therapy 8, 95-106.
Antibodies induced by dengue virus type 1 and 2 envelope domain III recombinant proteins in monkeys neutralize strains with different genotypes. Clin Vaccine Immunol 16, 1829-1831.
Tang, J. L. & Halstead, S. B. (2001). Efficacy of single-dose SA 14–14–2 vaccine against Japanese encephalitis: a case control study. The Lancet 358, 791-795.
S. & Boaz, M. (2014). Immune response to live-attenuated Japanese encephalitis vaccine (JE-CV) neutralizes Japanese encephalitis virus isolates from south-east Asia and India. BMC Infect Dis 14, 156.
Diamond, M. S. (2010). Genotype-specific neutralization and protection by antibodies against dengue virus type 3. J Virol 84, 10630-10643.
Japanese encephalitis virus neutralization-resistant mutants. Virology 181, 70-77.
J., Diamond, M. S., Baric, R. S. & other authors (2012). Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions. Proc Natl Acad Sci U S A 109, 7439-7444.
& Kantele, A. (2013). Cross-protective capacity of Japanese encephalitis (JE) vaccines against circulating heterologous JE virus genotypes. Clin Infect Dis 56, 267-270.
C., Chang, G. J. & other authors (2012). Partially neutralizing potency against emerging genotype I virus among children received formalin-inactivated Japanese encephalitis virus vaccine. PLoS Negl Trop Dis 6, e1834.
13
1 2 3 4
Ferguson, M., Johnes, S., Li, L., Heath, A. & Barrett, A. (2008). Effect of genomic variation in the
5 6 7 8
Feroldi, E., Pancharoen, C., Kosalaraksa, P., Chokephaibulkit, K., Boaz, M., Meric, C.,
9 10 11 12
Gatchalian, S., Yao, Y., Zhou, B., Zhang, L., Yoksan, S., Kelly, K., Neuzil, K. M., Yaich, M. &
13 14 15
Goncalvez, A. P., Chien, C. H., Tubthong, K., Gorshkova, I., Roll, C., Donau, O., Schuck, P.,
16 17 18
Gromowski, G. D. & Barrett, A. D. (2007). Characterization of an antigenic site that contains a
19 20
Gromowski, G. D., Firestone, C. Y., Hanson, C. T. & Whitehead, S. S. (2014). Japanese encephalitis
21 22
Halstead, S. B. & Thomas, S. J. (2010). Japanese encephalitis: new options for active immunization.
23 24 25
Han, N., Adams, J., Chen, P., Guo, Z. Y., Zhong, X. F., Fang, W., Li, N., Wen, L., Tao, X. Y. &
26 27 28
Hanna, J. N., Ritchie, S. A., Phillips, D. A., Shield, J., Bailey, M. C., Mackenzie, J. S., Poidinger,
29 30
Hasegawa, H., Yoshida, M., Fujita, S. & Kobayashi, Y. (1994). Comparison of structural proteins
31 32
Hasegawa, H., Yoshida, M., Kobayashi, Y. & Fujita, S. (1995). Antigenic analysis of Japanese
33 34 35
Hennessy, S., Liu, Z., Tsai, T. F., Strom, B. L., Wan, C. M., Liu, H. L., Wu, T. X., Yu, H. J., Liu, Q.
challenge virus on the neutralization titres of recipients of inactivated JE vaccines--report of a collaborative study on PRNT50 assays for Japanese encephalitis virus (JE) antibodies. Biologicals 36, 111-116.
Hutagalung, Y. & Bouckenooghe, A. (2014). Primary immunization of infants and toddlers in Thailand with Japanese encephalitis chimeric virus vaccine in comparison with SA14-14-2: a randomized study of immunogenicity and safety. Pediatr Infect Dis J 33, 643-649.
Jacobson, J. (2008). Comparison of the immunogenicity and safety of measles vaccine administered alone or with live, attenuated Japanese encephalitis SA 14-14-2 vaccine in Philippine infants. Vaccine 26, 2234-2241.
Yoksan, S., Wang, S. D. & other authors (2008). Humanized monoclonal antibodies derived from chimpanzee Fabs protect against Japanese encephalitis virus in vitro and in vivo. J Virol 82, 7009-7021.
dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus. Virology 366, 349-360.
virus vaccine candidates generated by chimerization with dengue virus type 4. Vaccine 32, 3010-3018.
Clin Infect Dis 50, 1155-1164.
other authors (2014). Comparison of Genotypes I and III in Japanese encephalitis virus reveal distinct differences in their genetic and host diversity. J Virol.
M., McCall, B. J. & Mills, P. J. (1996). An outbreak of Japanese encephalitis in the Torres Strait, Australia, 1995. Med J Aust 165, 256-260.
among antigenically different Japanese encephalitis virus strains. Vaccine 12, 841-844.
encephalitis viruses in Asia by using monoclonal antibodies. Vaccine 13, 1713-1721.
M. & other authors (1996). Effectiveness of live-attenuated Japanese encephalitis vaccine (SA14-14-2): a case-control study. Lancet 347, 1583-1586.
14
1 2 3
Hombach, J., Solomon, T., Kurane, I., Jacobson, J. & Wood, D. (2005). Report on a WHO
4 5
Hu, Q., Chen, B., Zhu, Z., Tian, J., Zhou, Y., Zhang, X. & Zheng, X. (2013). Recurrence of
6 7 8
Kimura-Kuroda, J. & Yasui, K. (1983). Topographical analysis of antigenic determinants on
9 10
Kobayashi, Y., Hasegawa, H. & Yamauchi, T. (1985). Studies on the antigenic structure of Japanese
11 12 13
Kochel, T. J., Watts, D. M., Halstead, S. B., Hayes, C. G., Espinoza, A., Felices, V., Caceda, R.,
14 15
Kumar, R., Tripathi, P. & Rizvi, A. (2009). Effectiveness of one dose of SA 14-14-2 vaccine against
16 17 18
Kurane, I. & Takasaki, T. (2000). Immunogenicity and protective efficacy of the current inactivated
19 20 21
Li, S. H., Dong, H., Li, X. F., Xie, X., Zhao, H., Deng, Y. Q., Wang, X. Y., Ye, Q., Zhu, S. Y. &
22 23 24 25
Li, X. F., Deng, Y. Q., Yang, H. Q., Zhao, H., Jiang, T., Yu, X. D., Li, S. H., Ye, Q., Zhu, S. Y. &
26 27 28
Lin, C. W. & Wu, S. C. (2003). A functional epitope determinant on domain III of the Japanese
29 30 31
Liu, X., Yu, Y., Li, M., Liang, G., Wang, H., Jia, L. & Dong, G. (2011). Study on the protective
32 33 34
Liu, Z. L., Hennessy, S., Strom, B. L., Tsai, T. F., Wan, C. M., Tang, S. C., Xiang, C. F., Bilker, W.
35
Lok, S. M., Kostyuchenko, V., Nybakken, G. E., Holdaway, H. A., Battisti, A. J., Sukupolvi-Petty,
consultation on immunological endpoints for evaluation of new Japanese encephalitis vaccines, WHO, Geneva, 2-3 September, 2004. Vaccine 23, 5205-5211.
Japanese encephalitis epidemic in Wuhan, China, 2009-2010. PLoS One 8, e52687.
envelope glycoprotein V3 (E) of Japanese encephalitis virus, using monoclonal antibodies. J Virol 45, 124-132.
encephalitis virus using monoclonal antibodies. Microbiol Immunol 29, 1069-1082.
Bautista, C. T., Montoya, Y. & other authors (2002). Effect of dengue-1 antibodies on American dengue-2 viral infection and dengue haemorrhagic fever. Lancet 360, 310-312.
Japanese encephalitis. N Engl J Med 360, 1465-1466.
Japanese encephalitis vaccine against different Japanese encephalitis virus strains. Vaccine 18 Suppl 2, 33-35.
other authors (2013a). Rational design of a flavivirus vaccine by abolishing viral RNA 2'-O methylation. J Virol 87, 5812-5819.
other authors (2013b). A Chimeric Dengue Virus Vaccine using Japanese Encephalitis Virus Vaccine Strain SA14-14-2 as Backbone Is Immunogenic and Protective against Either Parental Virus in Mice and Nonhuman Primates. J Virol 87, 13694-13705.
encephalitis virus envelope protein interacted with neutralizing-antibody combining sites. J Virol 77, 2600-2606.
efficacy of SA14-14-2 attenuated Japanese encephalitis against different JE virus isolates circulating in China. Vaccine 29, 2127-2130.
B., Pan, X. P. & other authors (1997). Short-term safety of live attenuated Japanese encephalitis vaccine (SA14-14-2): results of a randomized trial with 26,239 subjects. J Infect Dis 176, 1366-1369.
15
1 2 3
S., Sedlak, D., Fremont, D. H., Chipman, P. R. & other authors (2008). Binding of a neutralizing
4 5
Luca, V. C., AbiMansour, J., Nelson, C. A. & Fremont, D. H. (2012). Crystal structure of the
6 7 8
Ma, S. P., Yoshida, Y., Makino, Y., Tadano, M., Ono, T. & Ogawa, M. (2003). Short report: a major
9 10
Mackenzie, J. S., Gubler, D. J. & Petersen, L. R. (2004). Emerging flaviviruses: the spread and
11 12 13 14
Monath, T. P., Seligman, S. J., Robertson, J. S., Guy, B., Hayes, E. B., Condit, R. C., Excler, J. L.,
15 16 17
Nga, P. T. (2004). Shift in Japanese encephalitis virus (JEV) genotype circulating in northern Vietnam:
18 19 20
Nitatpattana, N., Dubot-Peres, A., Gouilh, M. A., Souris, M., Barbazan, P., Yoksan, S., de
21 22 23
Oliphant, T., Nybakken, G. E., Engle, M., Xu, Q., Nelson, C. A., Sukupolvi-Petty, S., Marri, A.,
24 25 26
Oliphant, T., Engle, M., Nybakken, G. E., Doane, C., Johnson, S., Huang, L., Gorlatov, S.,
27 28 29
Pan, X. L., Liu, H., Wang, H. Y., Fu, S. H., Liu, H. Z., Zhang, H. L., Li, M. H., Gao, X. Y., Wang,
30 31
Parida, M., Dash, P. K. & Tripathi, N. K. (2006). Japanese encephalitis outbreak, India, 2005. Emerg
32 33 34
Pitcher, T. J., Gromowski, G. D., Beasley, D. W. & Barrett, A. D. (2012). Conservation of the
35
Sarkar, A., Taraphdar, D., Mukhopadhyay, S. K., Chakrabarti, S. & Chatterjee, S. (2012).
antibody to dengue virus alters the arrangement of surface glycoproteins. Nat Struct Mol Biol 15, 312-317.
Japanese encephalitis virus envelope protein. J Virol 86, 2337-2346.
genotype of Japanese encephalitis virus currently circulating in Japan. Am J Trop Med Hyg 69, 151-154.
resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat Med 10, S98-109.
Mac, L. M., Carbery, B. & other authors (2015). Live virus vaccines based on a yellow fever vaccine backbone: standardized template with key considerations for a risk/benefit assessment. Vaccine 33, 62-72.
implications for frequent introductions of JEV from Southeast Asia to East Asia. J Gen Virol 85, 1625-1631.
Lamballerie, X. & Gonzalez, J. P. (2008). Change in Japanese encephalitis virus distribution, Thailand. Emerg Infect Dis 14, 1762-1765.
Lachmi, B. E., Olshevsky, U. & other authors (2006). Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein. J Virol 80, 12149-12159.
Mehlhop, E., Marri, A. & other authors (2005). Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med 11, 522-530.
J. L. & other authors (2011). Emergence of genotype I of Japanese encephalitis virus as the dominant genotype in Asia. J Virol 85, 9847-9853.
Infect Dis 12, 1427.
DENV-2 type-specific and DEN complex-reactive antigenic sites among DENV-2 genotypes. Virology 422, 386-392.
16
1 2
Molecular evidence for the occurrence of Japanese encephalitis virus genotype I and III infection
3 4 5 6
Sarkar, A., Banik, A., Pathak, B. K., Mukhopadhyay, S. K. & Chatterjee, S. (2013). Envelope
7 8
Schuh, A. J., Ward, M. J., Brown, A. J. & Barrett, A. D. (2013). Phylogeography of Japanese
9 10 11
Schuh, A. J., Ward, M. J., Leigh Brown, A. J. & Barrett, A. D. (2014). Dynamics of the emergence
12 13 14 15
Shrestha, B., Brien, J. D., Sukupolvi-Petty, S., Austin, S. K., Edeling, M. A., Kim, T., O'Brien, K.
16 17 18 19
Sjatha, F., Kuwahara, M., Sudiro, T. M., Kameoka, M. & Konishi, E. (2014). Evaluation of
20 21
Solomon, T., Dung, N. M., Kneen, R., Gainsborough, M., Vaughn, D. W. & Khanh, V. T. (2000).
22 23
Solomon, T., Ni, H., Beasley, D. W. C., Ekkelenkamp, M., Cardosa, M. J. & Barrett, A. D. T.
24 25 26 27
Sukupolvi-Petty, S., Brien, J. D., Austin, S. K., Shrestha, B., Swayne, S., Kahle, K., Doranz, B. J.,
28 29 30
Teoh, E. P., Kukkaro, P., Teo, E. W., Lim, A. P., Tan, T. T., Yip, A., Schul, W., Aung, M.,
31 32 33
Traggiai, E., Becker, S., Subbarao, K., Kolesnikova, L., Uematsu, Y., Gismondo, M. R., Murphy,
34 35 36
Tsai, T. F., Yu, Y. X., Jia, L. L., Putvatana, R., Zhang, R., Wang, S. & Halstead, S. B. (1998).
associated with acute encephalitis in patients of West Bengal, India, 2010. Virol J 9, 271.
protein gene based molecular characterization of Japanese encephalitis virus clinical isolates from West Bengal, India: a comparative approach with respect to SA14-14-2 live attenuated vaccine strain. BMC Infect Dis 13, 368.
encephalitis virus: genotype is associated with climate. PLoS Negl Trop Dis 7, e2411.
and establishment of a newly dominant genotype of Japanese encephalitis virus throughout Asia. J Virol 88, 4522-4532.
M., Nelson, C. A., Johnson, S. & other authors (2010). The development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1. PLoS Pathog 6, e1000823.
chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection-enhancing antibody response. Microbiol Immunol 58, 126-134.
Japanese encephalitis. J Neurol Neurosurg Psychiatry 68, 405-415.
(2003). Origin and Evolution of Japanese Encephalitis Virus in Southeast Asia. J Virol 77, 3091-3098.
Johnson, S., Pierson, T. C. & other authors (2013). Functional analysis of antibodies against dengue virus type 4 reveals strain-dependent epitope exposure that impacts neutralization and protection. J Virol 87, 8826-8842.
Kostyuchenko, V. A. & other authors (2012). The structural basis for serotype-specific neutralization of dengue virus by a human antibody. Sci Transl Med 4, 139ra183.
B. R., Rappuoli, R. & Lanzavecchia, A. (2004). An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med 10, 871-875.
Immunogenicity of live attenuated SA14-14-2 Japanese encephalitis vaccine--a comparison of 1- and 3-month immunization schedules. J Infect Dis 177, 221-223. 17
1 2 3
Uchil, P. D. & Satchidanandam, V. (2001). Phylogenetic analysis of Japanese encephalitis virus:
4 5
van den Hurk, A. F., Ritchie, S. A. & Mackenzie, J. S. (2009). Ecology and geographical expansion
6 7 8
Wahala, W. M., Donaldson, E. F., de Alwis, R., Accavitti-Loper, M. A., Baric, R. S. & de Silva, A.
9 10 11
Wills, M. R., Singh, B. K., Debnath, N. C. & Barrett, A. D. (1993). Immunogenicity of wild-type
12 13
Wong, S. S., Abd-Jamil, J. & Abubakar, S. (2007). Antibody neutralization and viral virulence in
14 15 16
Wu, K. P., Wu, C. W., Tsao, Y. P., Kuo, T. W., Lou, Y. C., Lin, C. W., Wu, S. C. & Cheng, J. W.
17 18 19
Wu, S. C., Lian, W. C., Hsu, L. C. & Liau, M. Y. (1997). Japanese encephalitis virus antigenic
20 21 22
Ye, Q., Li, X. F., Zhao, H., Deng, Y. Q., Xu, Y. P., Wang, H. Y., Liang, G. D. & Qin, C. F. (2014).
23 24 25
Ye, Q., Li, X. F., Zhao, H., Li, S. H., Deng, Y. Q., Cao, R. Y., Song, K. Y., Wang, H. J., Hua, R. H.
26 27
Yu, Y. (2010). Phenotypic and genotypic characteristics of Japanese encephalitis attenuated live
28 29 30
Zhang, J. S., Zhao, Q. M., Guo, X. F., Zuo, S. Q., Cheng, J. X., Jia, N., Wu, C., Dai, P. F. & Zhao,
31 32 33
Zhang, Y., Zhang, W., Ogata, S., Clements, D., Strauss, J. H., Baker, T. S., Kuhn, R. J. &
envelope gene based analysis reveals a fifth genotype, geographic clustering, and multiple introductions of the virus into the Indian subcontinent. Am J Trop Med Hyg 65, 242-251.
of Japanese encephalitis virus. Annu Rev Entomol 54, 17-35.
M. (2010). Natural strain variation and antibody neutralization of dengue serotype 3 viruses. PLoS Pathog 6, e1000821.
and vaccine strains of Japanese encephalitis virus and the effect of haplotype restriction on murine immune responses. Vaccine 11, 761-766.
recurring dengue virus type 2 outbreaks. Viral Immunol 20, 359-368.
(2003). Structural basis of a flavivirus recognized by its neutralizing antibody: solution structure of the domain III of the Japanese encephalitis virus envelope protein. J Biol Chem 278, 46007-46013.
variants with characteristic differences in neutralization resistance and mouse virulence. Virus Res 51, 173-181.
Reduction of neutralization antibody against heterologous circulating strains in adults immunized with Japanese encephalitis live vaccine. Hum Vaccin Immunother 10.
& other authors (2012). A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1' formation and contributes to attenuation. J Gen Virol 93, 1959-1964.
vaccine virus SA14-14-2 and their stabilities. Vaccine 28, 3635-3641.
J. Y. (2011). Isolation and genetic characteristics of human genotype 1 Japanese encephalitis virus, China, 2009. PLoS One 6, e16418.
Rossmann, M. G. (2004). Conformational changes of the flavivirus E glycoprotein. Structure 12, 1607-1618.
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Figure legends
3
Fig. 1. E222 and E327 were distinct in JEV GI and GIII strains. (a)
4
Neighbor-Joining Tree was constructed by Tamura-Nei model with gamma-
5
distribution of among-site with 40 envelope gene sequences of JEV isolates. (b)
6
Sequence alignment of representative GI and GIII strains. The genotype specific
7
residues 222 and 327 were highlighted in multicolor. (c) Structural model of JEV
8
monomer E protein. Domain I, II and III were colored in palecyan, yellow and blue,
9
respectively. All the identified neutralizing epitopes were colored in green, and the
10
genotype specific neutralization epitope E222 and E327 in envelope protein were
11
colored in red and hotpink, respectively. (d) The pseudo-virion model of JEV based
12
on the reconstruction of DENV-2 cryo-EM density map.
13
Fig. 2. Recovery and characterization of A222S and S327T mutants. (a) Indirect
14
immunofluorescence assay of WT, A222S, S327T and A222S/S327T viruses infected
15
BHK-21 cells. The expression of E protein was detected using anti-JEV mouse sera at
16
24 h and 48 h post-infection. (b) Plaque morphology of WT, A222S, S327T and
17
A222S/S327T in BHK-21 cells. (c) Growth curves of WT, A222S, S327T and
18
A222S/S327T in BHK-21 cells. BHK-21 cells were infected with viruses at an MOI
19
of 0.01. Culture supernatants were harvested at 24, 48 and 72 h post-infection. Virus
20
titers were determined using standard plaque assays in BHK-21 cells and shown as
21
mean±SD from two independent experiments.
22
Fig. 3. Neutralizing antibody titers with LAV SA14-14-2 immunized human sera.
23
PRNT50 titers of human sera against WT, A222S, S327T and A222S/S327T were
24
shown in histogram as mean±SD from two independent experiments. The statistical
25
significance was analyzed by one-way ANOVA and indicated by asterisks (* P