RESEARCH ARTICLE
Nerome et al., Journal of General Virology 2018;99:897–907
DOI 10.1099/jgv.0.001081

Development of a Japanese encephalitis virus genotype V
virus-like particle vaccine in silkworms
Kuniaki Nerome,1,* Ryoji Yamaguchi,2 Naoyuki Fuke,2 Uda Zahli Izzati,2 Kenichi Maegawa,1 Shigeo Sugita,3Kazunori Kawasaki,4 Kazumichi Kuroda5 and Reiko Nerome1

Abstract
To counter the spread of multiple Japanese encephalitis virus (JEV) variants harboured in alternative host species and highly
neurotoxic variants with new antigenicity, such as genotype V (Muar), methods for developing more effective and low-cost
vaccines against a variety of epidemic JEV strains are required. Here, we successfully synthesized large amounts of a Muar
virus-like particle (MVLP) vaccine for JEV in silkworm pupae by using a Bombyx mori nuclear polyhedrosis virus recombinantconsisting of JEV codon-optimized envelope (E) DNA. In particular, histopathological examination suggested that MVLP was
efficiently synthesized in body fat tissues as well as epithelial cells. Quantitative analysis indicated that one silkworm pupa
produced 724.8 µg of E protein in the MVLP vaccine. Electron microscopic examination of purified MVLP vaccine defined atypical MVLP morphological structure. Detailed MVLP antigen assessment by immune-electron microscopy revealed that the
majority of MVLPs were covered with approximately 10 nm projections. Boosted immunization with MVLP antigens in miceand rabbits tended to show improved plaque inhibition potency against homologous Muar and heterologous Nakayama, butless potency to Beijing-1 strains. Notably, mixed immune rabbit antisera against Nakayama and Muar VLP antigens led to anincrease in the low antibody reaction to Beijing-1. Additionally, a stopgap divalent JEV vaccine consisting of MVLP and
Nakayama VLP and its immune mouse serum significantly increased plaque inhibition titre against Muar, Nakayama andBeijing-1 strains. These findings suggested that low-cost MVLP vaccines prepared in silkworm pupae are suitable forproviding simultaneous protection of individuals in developing countries against various JEV strains.

INTRODUCTION
Many severe neurological diseases are caused by transmission of Japanese encephalitis viruses (JEVs) from mosquito
vectors, although most have shown a tendency of decreasing
prevalence in humans upon immunization with suitablevaccines and improvement of ecological circumstance.
However, some different JEV variants are still circulating inswine populations, mosquitos and birds, thus maintaining
the mosquito–swine or mosquito–bird ecosystem. For
example, the active circulation of numerous JEV variants inthe swine population was defined following a large-scalevirological surveillance in Japan, conducted with the cooperation of local governments [1]. In particular, several antigenic variants exhibiting slightly different evolutionary
locations within genotype I evolutional branch clusters were
found to co-circulate in the swine population. Furthermore,
two new isolates with distinct neurovirulence andneuroinvasiveness were isolated from swine at the same
farm on the same day of 2004 [1], meaning there is thepotential for JEV to re-emerge in humans despite
vaccination.
JEVs have been actively circulating in humans and swine inother South Asian countries as well [2]. In particular,
among the numerous isolates analysed, most were reported
to belong to genotype III until the 1990s, including isolates
from China, Taiwan, India, Japan, Australia, Korea andIndonesia [3]. However, the major genotype of JEV isolated
in Japan changed from genotype III to genotype I in theearly 1990s and recently a similar genotype shift has also
been confirmed in South Korea, northern Vietnam, China,
Taiwan and Thailand [4]. Moreover, the isolation of genotype IV virus in Indonesia has attracted further attention
owing to the potential for future epidemics [3]. Consistent
with these findings, Japanese encephalitis disease has also

Received 5 March 2018; Accepted 5 May 2018
Author affiliations: 1The Institute of Biological Resources, Okinawa, Japan; 2Laboratory of Veterinary Pathology, Department of Veterinary, Faculty ofAgriculture, University of Miyazaki, Miyazaki, Japan; 3Equine Research Institute, Japan Racing Association, Tochigi, Japan; 4National Institute ofAdvanced Science and Technology (AIST), Osaka, Japan; 5Division of Microbiology, Nihon University School of Medicine, Tokyo, Japan.
*Correspondence: Kuniaki Nerome, rnerome_ibr@train.ocn.ne.jp
Keywords: Japanese encephalitis genotype V virus; vaccine; silkworm; virus-like particle.
Abbreviations: CPE, cytopathic effect; E, envelope; HE, haematoxylin-eosin; JEV, Japanese encephalitis virus; MDNA, Muar DNA; MNPV, multinucleocapsid nuclear polyhedrosis virus; MVLP, Muar virus-like particle; NVLP, Nakayama virus-like particles.
001081 ã 2018 The Authors

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been reported in several Asian countries, with 50 000–
175 000 confirmed new cases per year consisting of different
age groups [5–7] and incidence rates ranging from 0.003 to
3.7 % across various populations in Indonesia, Nepal, India,
Bangladesh, Korea and China [8].
Historically, JEV vaccines have played an important role inelimination of JEV epidemics since the first use of a mousebrain-derived genotype III JEV vaccine in Japan in 1945 [9].
Subsequently, vaccine quality has markedly improved, withmouse brain-derived vaccines having been replaced by cell
culture-derived vaccines [10–16]. In particular, the safety
and efficacy of a Vero cell-derived genotype III JEV vaccine
has been confirmed [14]. Conversely, the increased incidence of infection may be attributable in part to decreased
protective immunity in humans and constant circulation of
the virus in swine and bird vectors. Many antigenic and virulent JEV variants are co-circulating in mosquitos, swine
and humans in Southeast Asian countries [7, 8], highlighting the need for wide distribution of more effective and lowcost vaccines against a variety of epidemic JEV strains
throughout many parts of Asian countries. In particular, thespread of a genotype V virus possessing new antigenicity
and high neurovirulence characteristics necessitates suitableand effective countermeasures against this variant. For
example, the high pathogenicity of the genotype V Muar
virus, which was first isolated from a patient with encephalitis in Malaysia in 1952 [17], has been investigated in detailin a mouse model [18, 19]. In addition, this virus has been
circulating constantly in the mosquito population in different parts of Asia, producing new threats mainly in Asian
countries [20, 21]. However, the present JEV vaccine may
be ineffective against JEV genotype V Muar strain owing toantigenic differences [18, 22].
Accordingly, in this study, we aimed to develop a genotype
V vaccine of JEV using our baculovirus (multinucleocapsid
nuclear polyhedrosis virus; MNPV) genetic engineering system [23, 24]. As a result, we achieved successful productionof a JEV-Muar virus-like particle (MVLP) vaccine in silkworm Bombyx mori pupae. In addition, the biological,
morphological and immunological characteristics of the
JEV-MVLP vaccine were subsequently characterized.

RESULTS
Design and characterization of chimeric JEV DNA(genotypes III and V)
For production of MVLP vaccines against JEV genotype V(Muar strain), codon-optimized DNA consisting of Muar
envelope (E) and frequently used silkworm-specific codons
was designed based on computer gene analyses as described
previously [23, 24].
Expression of codon-optimized MNPV recombinant
DNA gene
Expression of recombinant JEV-MNPV was assessed in
B. mori ovarian (Bm-N) cells (Fig. 1a, b). The appearance ofcytopathic effects (Fig. 1a RV-CPE), which differedmarkedly from that of normal control cells (Fig. 1a NC),
indicated infection of recombinant virus in Bm-N cells.
Apparent synthesis of E protein in the recombinant-infected
cells was also confirmed in Bm-N cells, showing clear fluorescence in the recombinant virus-infected cells (Fig. 1b
Ab-MVLP, Ab-JEV), which was apparently comparable tothat of normal cells (Fig. 1b NC).

Histopathological examination in infected pupae
Pathological specimens of recombinant JEV-MNPVinfected pupae were separated into two main tissues. As
shown in Fig. 1(c) with haematoxylin-eosin (HE) staining,
the epithelium under the skin showed expanded nuclei from4 days after infection, with epithelial cell nuclei (indicatedby an arrow) showing marginalized chromatin with single
prominent nucleoli (Fig. 1c E). A similar nuclear change(indicated by an arrow) was detected in body fat cells(Fig. 1c F). In the course of the 4 day infection, the numberof recombinant JEV-MNPV-immunoreactive epithelial cells
increased (Fig. 2a E, indicated by arrows). Similar positive
cells were also confirmed in body fat tissues (Fig. 2a F, indicated by arrows). As shown in Fig. 2(a) F, the number of
positive epithelial cells reactivated with anti-Tag monoclonal antiserum (indicated by arrows) increased over time in
the body fat tissue. The above positive cells were quite different from those of negative (noninfected) control cells.
Extraction of MVLPs and their morphology
For extraction and preparation of MVLPs, JEV-MNPV was
infected into 30 silkworm pupae, and MVLPs wereextracted via three cycles of sonication [23, 24]. As shownin Table 1 60 ml of crude pupae homogenates exhibited ahaemagglutination (HA) titre of 512 in an ordinary PBS
sample containing Mg++ and Ca++ ions, with a total protein
concentration of 906 mg ml 1 (Table 1). Conversely,
authentic Nakayama and Muar strains of JEV did not show
HA activity in PBS containing Mg++ and Ca++ ions, whereas
Muar-infected homogenates exhibited marked HA activity.
The migration profile in slab gel electrophoresis is shown in
Fig. 2(b); the 53 kDa E protein was estimated to be present
at a concentration of 21 744 µg ml 1, with an estimated production level of 724 µgpupa 1, indicating that abundantamounts of E protein were produced (Table 1). Unfortunately, we could not directly identify the above 53 kDa protein in the present study, however, as shown in Fig. 2(c), theposition of MVLP E protein was indirectly confirmed with
antiserum to the Beijing-1 control virus in Western blot
analysis (Fig. 2c). This partially purified sample was further
purified by 10–50 % sucrose gradient centrifugation [23, 24]
and MVLPs were determined as a marked white band in
Fig. 2(d) (indicated by an arrow). As a result, the homogenate produced two protein peaks in the sucrose gradient(Fig. 2e). The two peaks of protein concentration and HA
activity coexisted in the same 12 fractions, suggesting that
the Muar E protein apparently exhibited HA activity.

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Fig. 1. Examination of expression of MVLP antigens in Bm-N cells and their synthesis in recombinant-infected pupae. (a) Expressionof recombinant MVLP in Bm-N cells based on cytopathic effect (CPE) and haemadsorption (HAD) tests in normal control cells (NC),recombinant-infected CPE (RV-CPE) and recombinant-infected HAD (RV-HAD) cells. (b) Expression test based on fluorescent (FA) antibody test in normal (NC), recombinant-infected cells treated with antibody to MVLP (Ab-MVLP) and recombinant-infected cells treatedwith an antibody to the JEV vaccine (Ab-JEV). (c) MVLP production analysis in MVLP-recombinant-infected pupae based on histopathological examination during a 4 day period. E, epithelium. F, body fat. (c) E, marginalized chromatin and single dense nucleoli observedin epithelial cells from 4 days after infection and noninfected (normal) control cell. (c) F, lack of eosinophilic cytoplasmic granules, marginalized chromatin and single dense nucleoli observed from 4 days after infection in body fat tissue and also noninfected (normal)
control cell. (c) E, arrows indicate epithelial nucleus and (c) F, body fat nucleus.

Morphological examination of JEV-MVLP
As marked, HA titres were present in the crude silkworm
homogenate (Fig. 2c) and fractions 9–13 (Fig. 2c) werepooled and examined by electron microscopy. As shown in
Fig. 3(a), MVLPs exhibited a variety of shapes under high-magnification electron microscopy. Notably, we observed
many clear, long projections (spikes) on the surfaces of
MVLPs that measured approximately 80–100 nm in diameter. For estimation of the lengths of spikes on the surface of
VLPs, 10 spikes were selected and their length was

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Fig. 2. Immunopathology of epithelial cells and body fat tissues in silkworm pupae infected with recombinant MVLP. (a) IHC. E, intense
nuclear membrane and cytoplasmic immunoreactivity with an anti-DYKDDDK Tag monoclonal antibody in infected epithelial cells andnoninfected (normal) control cell. F, nuclear membrane and cytoplasmic immunoreactivity with an anti-DYKDDDK Tag monoclonal antibody in infected body fat tissue and also noninfected (normal) control cell. Arrows in E and F, epithelial cells stained with Tag monoclonal antibody. (b) Separation of MVLP antigen and identification of E protein. (c) Identification of E protein of JEV control (JEV C) by
Western blot analysis with antiserum to Beijing-1 strain. (d) Separation of MVLP antigen by sucrose density gradient centrifugation.
(e) Fractionation of MVLP antigen and examination of biological activities.

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Table 1. Recovery of MVLP-E protein from recombinant virus-infected pupae
No. of pupae30

Vol (ml)

60

HA titre

512

measured (Fig. 3a, b). The lengths were distributed between
7.89 and 16.62 nm, with a mean length estimated as approximately 11 nm (Fig. 3b).
Next, we used an immuno-gold electron microscope to confirm the presence of E protein on these spikes. Immunogold-treated MVLPs showed clear distributions across alarge number of gold spots around MVLPs (Fig. 3c, indicated by white arrows). Electron micrographic imaging
showed that the immune-gold bound to a large number of
MVLPs. The clear immune-gold binding to the surface of
MVLP suggested the presence of specific E proteins on the
MVLP particles. For further detailed observation, two
MVLP particles were selected and their spikes measured(Fig. 3d, e). For short distances of approximately 9–14 nm,
these spikes were considered to have bound directly to
MVLP structures.

Plaque neutralization using immune antisera
prepared in mice and rabbits immunized with
MVLP and Nakayama (N)VLP antigens
Plaques produced by Muar, Nakayama and Beijing-1 strainsof JEV in the presence of rabbit antiserum clearly distinguished at a 40–10, 240-fold antiserum dilution as exhibiting plaque inhibition titres against homologous Muar andheterologous Nakayama and Beijing-1 strains (Fig. 4a). For
example, plaques of Muar and Nakayama strains at 40
serum dilution had completely disappeared, indicating
100 % plaque inhibition. In contrast, the plaques of the Beijing-1 strain at 40 serum dilution were similar to those of
the virus control. This result indicated potential antigenic
differences between Muar or Nakayama strains and Beijing1 strains (Fig. 4a). Similarly, the plaque inhibition curve of
the Muar strain markedly differed from those of the
Nakayama and Beijing-1 strains (Fig. 4b–d). The plaqueinhibition titres are summarized in Fig. 4(e). The plaqueinhibition titres against the Muar, Nakayama and Beijing-1
strains were 10, 240, 256 and 40, respectively.
The immune responses of anti-Muar VLP antisera obtained
from mice immunized with three doses of MVLP vaccines
are shown in Fig. 5(a). Preimmune control mouse serum
did not show apparent inhibition (Fig. 5a). Plaque inhibition titres against the three strains increased graduallyaccording to dose increase, although the antibody titre
against Beijing-1 was low. This tendency was also observedin rabbit antisera against MVLP antigens (Fig. 4e). We also
prepared rabbit antiserum against the MVLP vaccine using
Freund’s complete adjuvant-MVLP vaccine, which demonstrated a considerably higher 50 % plaque inhibition titre
against the homologous Muar strain, showing more thanProtein
Total (mg ml 1)

Total E (mg ml 1)

E (mg head 1)

906

21 744724

10 000-fold plaque reduction (Fig. 4e); the same antiserum
demonstrated a nearly 2560-fold plaque inhibition titre
against the Nakayama strain (Fig. 4e). However, this mouseantiserum still showed a low titre against the Beijing-1 virus.
To address whether the low immune response against the
Beijing-1 strain might be improved, mouse antiserum
obtained from three-dose mice and rabbit antiserum weremixed and their antisera were again tested. As shown in
Fig. 5(b), the antibody titre against the Beijing-1 strain
increased nearly twofold. Additionally, preimmune rabbit
serum did not show apparent inhibition.

Experimental preparation of the divalent JEVvaccineTo confirm this finding, Nakayama VLP and Muar VLP
antigens were mixed and their antigen contents were
adjusted to contain 20 µg E protein/ml, and the mixture wasused to immunize mice three times at 1 week intervals. As
shown in Fig. 5(c), the immune serum obtained after
4 weeks showed almost similar antibody responses to thethree strains at levels similar to those of the mixed antiserum (Fig. 5c).

DISCUSSION
Recently available JEV vaccines have played an importantrole in decreasing JEV epidemics caused by JEV variants
belonging to genotypes I–IV. Nevertheless, the developmentof additional, low-cost vaccines is required to provide protection for populations in developing countries. In this context, silkworm-derived vaccines may constitute suitableprophylactic agents. However, genotype V viruses were firstisolated from a human in Malaysia (1952) and from mosquitoes in China (2009), with the genotype V JEV genome
identified in mosquitoes in China (2009) and in South
Korea as well, whereas immune efficacy against this virus isvery low. Specifically, Tajima et al. [18] found that mouseantisera to the inactivated Nakayama genotype III JEV vaccine showed efficient plaque neutralization titres to thehomologous Nakayama strain, whereas the antibody titresto genotype V Beijing-1 and Muar strains were much lower[18]. Consistent with the above report, in JEV-vaccinatedmice, the protection levels against genotype III and V JEV
were high and low, respectively [24]. Furthermore, seroconversion levels in different age groups of healthy individuals
immunized with the currently available JEV vaccine were
extremely low against genotype V JEV [22].
To address this issue, in the present study we demonstrated
that potent antibodies against both the Muar genotype V
virus and the Nakayama strain could be produced throughDownloaded from www.microbiologyresearch.org by
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Fig. 3. Electron microscopic examination of purified MVLP antigens produced in silkworm pupae. (a–c) Electron micrograph of a sample treated with immune-gold. White arrows indicate typical MVLP antigens covered with black immune-gold (indicated by whitearrows). (d), (e) Estimation of the length of E projection through transmission of MVLP vaccine detected with electron microscopy. Identification of E protein spikes with a gold-conjugated JEV antibody; black points indicate the edges of the E protein projection reactedwith the JEV-specific antibody.

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Nerome et al., Journal of General Virology 2018;99:897–907

Fig. 4. Plaque inhibition profiles of antisera prepared in rabbits immunized with the partially purified MVLP vaccine against Muar,
Nakayama and Beijing-1 strains of JEV. (a) Challenge viruses and plaque inhibition patterns. VC, virus control. (b–d) Plaque inhibition
curves of Muar, Nakayama and Beijing-1 strains. (e) Summary plaque inhibition titres of Muar, Nakayama and Beijing-1 strains.

immunization with MVLP vaccines prepared in silkworm
pupae. First, a series of experiments demonstrated that thecodon-optimized and synthetic Muar DNA (MDNA) geneof the JEV Muar strain allowed increased production of an
E protein-associated MVLP vaccine. We also found that asingle pupa could produce 724 µg of E protein. It was further evident that nuclear changes could be detected in theepithelium and body fat tissues, suggesting increased production of MVLP. These changes were consistent with the
immunoreactivity observed in both tissues, indicating

active production of MVLP antigens in the epithelium andbody fat.
Additionally, in this study we evaluated the tentative
length of the E protein on the surface of MVLPs. Consistent with previous evidence, most E protein spikes were
found to stretch upward from the surface of MVLPs andcould be characteristically identified using immune-electron microscopy. Although the VLP structures of JEV andother flaviviruses have been reported in other vaccine studies, the detailed structures of VLP E proteins have not

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Fig. 5. Immune responses in mice and rabbits immunized with MVLP and NVLP vaccines produced in silkworm pupae. (a) 50 % plaqueinhibition antibodies produced in mice immunized with three doses of vaccine. (b) 50 % plaque inhibition antibody titres of mixed antisera against MVLP rabbit and NVLP mouse antisera. (c) Serial antibody production in mice immunized with three doses of divalent
MVLP and NVLP vaccine.

been extensively studied [25–27]. In the present report, we
identified a variety of E protein spikes ranging from 7.8 to
16.6 nm in length (mean length: 11 nm). However, virion
surface structures of flaviviruses have been reported tochange during growth from an immature to mature stage[28, 29], with the arrangement of the spikes also influencing virus particle maturation [30]. For example, the surface
structure of immature flavivirus shows prominent andirregular spikes, which markedly differ from those of
mature particles [28]. The spikes observed in the MVLPs

in the present study were somewhat similar to those of thesurface of immature flaviviruses, showing clear and long E
spikes.
Anti-mouse serum to MVLPs tended to exhibit a low
immune response against both homologous Muar and heterologous Nakayama strains after one dose of immunization, with considerably less reaction to the Beijing-1 strain;
however, this lower activity tended to increase after threeimmunization doses, reaching high reactivity with the former strains. Notably, such cross-potency, in contrast to the

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standard JEV vaccines, may afford the potential for developing potent, divalent vaccines consisting of Muar and
Beijing-1 strains or Muar and Nakayama strains. Indeed,
test divalent JEV vaccines prepared in the present study
could substantively mutually raise antibody titres against allof the above three strains of JEV. Together, our findings
thus suggest low-cost MVLP vaccines prepared in silkworm
pupae as suitable for providing simultaneous, potent protection of individuals in developing countries against various
JEV strains.
Because the antibody to the Muar strain highly reacted withhomologous Muar and heterologous Nakayama strains, but
reacted to a lesser extent with the Beijing-1 strain, a divalentvaccine consisting of Muar and Beijing-1 strains or Muar
and Nakayama strains would be ideal. In fact, the test divalent JEV vaccine prepared in the present study apparently
could produce a mutual antibody titre against the above
three strains of JEV.

METHODS
Construction of chimeric JEV-MDNA and generationof JEV-recombinant MNPV
Silkworm codon-optimized chimeric MDNA was designed
using the E gene of genotype V JEV-Muar strain by introduction of frequently used silkworm codons. In total, 433
silkworm codons and prM plus 1500 Muar virus E codons
were used for preparation of target chimeric MDNA for
vaccine production. The resulting codon-optimized synthetic MDNA containing 2001 codons was used for preparation of recombinant baculovirus (JEV-MNPV), andsubsequent procedures were performed as described previously [23, 24, 31].
Cells and viruses
The cells and viruses used in the present study were basically similar to those described in our previous reports[23, 24, 31]. Vero cells (cat. no. JCRB 9013) were purchasedfrom the Japanese Collection of Research Bioresources(JCRB) cell bank and were used for propagation of the
Nakayama, Beijing-1 and Muar JEV strains, which wereobtained from the National Institute of Infectious Diseases(Tokyo, Japan).
Description of fluorescent-labelled antibodies andWestern blot
Bm-N cells infected with the recombinant virus (JEVMNPV) were maintained in MEM containing 10 % calf
serum at 25  C in a 5 % CO2 incubator for 1–3 days. Briefly,
experimental procedures were as described previously[24, 31]. The wased Bm-N cells on the cover slip glass weretreated with mouse or rabbit antisera against JEV, followed
by treatment with fluorescent isothiocyanate (FITC) conjugated anti-mouse or rabbit antibodies (West Grove, PA,
USA). For Western blot analysis, goat polyclonal anti-JEV
rabbit serum was obtained from NIID (Tokyo, Japan) and
FITC-conjugated rabbit anti-goat IgG-AP antibodies (Santa
Cruz Biotechnology) were used. The final signal was

detected using ECL plus reagents (GE, Healthcare, Port
Washington, NY, USA).

Histopathological examination
Infected silkworm pupae from 1 to 4 days after infection
were collected and fixed in 10 % neutral buffered formalin
or Methyl Carnoy’s fixative solution, processed by conventional methods, and embedded in paraffin. For histopathological examination, 4 µm sections were cut and stainedwith haematoxylin-eosin (HE). For detection of recombinant JEV-MVLP, continuous sections of each sample were
subjected to immunohistochemistry analysis. Antigen
retrieval was omitted in Methyl Carnoy’s fixed samples. Following blocking steps, sections were incubated overnight at
4  C with an anti-DYKDDDDK Tag monoclonal antibody
as the primary antibody, and for 30 min at 37  C with the
EnVision+Dual Link System-HRP (Dako) as a secondary
antibody, with PBS rinses prior to each step. Colour wasdeveloped by reaction with 3,3¢-diaminobenzidine substrates and counterstaining with haematoxylin.
Extraction and purification of MVLP antigen fromrecombinant-infected silkworm pupae
homogenates
The recombinant baculovirus (JEV-MNPV) was inoculated
into silkworm pupae. After 3–4 days, 0.01 % formalin andphenylthiourea were added and the inactivated pupae were
homogenized with three cycles of sonication, as described
previously [24]. The resulting homogenates were treated as
described previously [23, 24, 31].
Haemagglutination test
In a recent study, we found that silkworm-derived MVLP
and NVLP could apparently agglutinate chicken RBC in
PBS free of Mg++ and Ca++ ions [PBS ( )] and we performed the haemagglutination (HA) test as described previously [23, 24].
Morphological characterization of MVLPs using
electron microscopy
Purified MVLP fractions showing high HA activity werecollected, and the sucrose component was removed throughcolumn filtration (PD-10 column; GE Healthcare). The
resulting HA-MVLP samples were negatively stained with
2 % phosphotungstic acid. Samples were observed under an
H-7600 electron microscope for identification of spikes on
the surface of MVLPs, and another sample was treated with
5 % skim milk in PBS blocking solution. 10 µl of primarypolyclonal JEV rabbit sera antibody (100dilution)
obtained from NIID (Tokyo, Japan) was applied on the gridfor 10 min at 37  C, and the grids were then rinsed twicewith blocking solution. Secondary antibodies [10 nm goldconjugate goat polyclonal anti-rabbit IgG (HTL; BBI Solutions); 20dilution] in blocking solution were applied on
the grid for 30 min at room temperature. After rinsing with
distilled water, the grids were stained with 2 % phosphotungstic acid solution for 90 s. The grids were then observed
under a transmission electron microscope (Hitachi-HT

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Nerome et al., Journal of General Virology 2018;99:897–9077700) at 80 kV. Spike length was observed and measured
based on the electron micrographs through reciprocationfrom electron micrographs, and 10 spikes were selected for
calculation.

Immunization with JEV-MVLP antigens in rabbits
and mice
Preparation of antisera against JEV-MVLP and JEVNakayama VLP antigens was performed as described previously [31]. Briefly, partially purified MVLP antigen was
adjusted to contain 40 µg ml 1 dose 1 and this MVLP antigen was immunized into mice. The immunizations wererepeated three times at weekly intervals and all blood samples were obtained 1 week after the last immunization. In
the case of divalent JEV vaccines, 40 µg each of Nakayama
and MVLP antigens were mixed together and immunized
into mice as described for the Muar VLP vaccine.
50 % plaque reduction neutralization tests (PRNT50)
Growth inhibition of immune sera against MVLP and
NVLP prepared in mice and rabbits was undertaken by
PRNT50 in Vero cells as described previously [31]. Briefly,
each challenge virus (Nakayama, Beijing-1, Muar) was
mixed with an equal volume of fourfold diluted antisera(1 : 40–1 : 10 240) and then incubated at 35  C for 90 min.
Subsequently, overlay medium containing 1 % methylcellulose was added and the cells were incubated at 35  C for 4–
6 days. The cells were fixed using a 3.7 % formalin/PBS
solution and stained using methylene blue. The PRNT50
titre was estimated as described previously [31].
Funding information
This work was supported by a grant from the Okinawa Communicable
Disease Research Hub Formation Promotion Project by Okinawa
Prefectural Government Commissioned Project for fiscal year 2016
and 2017.
Acknowledgements
Authors are grateful to Ms Sayaka Matsuda (Institute of Biological
Resources) for her skilful technical support.
Conflicts of interest
We are applying for a patent based on the results described in thepresent report.
Ethical statement
Mouse and rabbit experiments were approved and performed inaccordance with the Fundamental Rules for Animal Experiments andthe Guidelines for Animal Experiments Performed at The Institute of
Biological Resources published by the Animal Welfare and Animal
Care Committee including the Animal Ethics Committee of the Instituteof Biological Resources, Okinawa, Japan.
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