R

CH

a

ARRAA

KCGRB

1

aeA(s4ACispatttv[dftatta

0d

Vaccine 27 (2009) 2845–2850

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

ecombinant glycoprotein based vaccine for Chandipura virus infection

.H. Venkateswarlu, V.A. Arankalle ∗

epatitis Division, National Institute of Virology, Microbiological Containment Complex, Sus Road, Pashan, Pune 411021, India

r t i c l e i n f o

rticle history:eceived 22 November 2008eceived in revised form 13 February 2009ccepted 24 February 2009vailable online 11 March 2009

a b s t r a c t

Chandipura virus (CHPV) has emerged as an important pediatric encephalitis-causing pathogen with veryhigh mortality in India. No specific vaccine or treatment is available till date. We attempted to preparea candidate vaccine employing recombinant CHPV Glycoprotein (rGp). The Glycoprotein gene (G-gene)of CHPV was expressed using Baculovirus expression system. The rGp was purified by HPLC and used for

eywords:handipura virusgene

ecombinant vaccine

mice immunization, 3 doses, and 4 weeks apart. One microgram rGp was found to be optimum. Sero-conversion was observed as early as 2nd week by detecting anti-CHPV IgG antibodies. Antibody titreswere immunogen-concentration dependent. Intracerebral challenge of the immunized mice with 100LD50 of the homologous strain demonstrated 90% protection. In in vitro neutralization, antibodies from theimmunized mice were able to neutralize heterologous viruses. There was 60% T cell proliferation observed

d mice can

aculovirus expression system against rGp in immunizerepresents an ideal vaccin

. Introduction

Encephalitis caused by Chandipura virus (CHPV) has emerged asn important pediatric health problem in India as evidenced by thepidemics of the disease with high mortality in 2003 in the state ofndhra Pradesh (183/329, 55%) [1], in 2004 in the state of Gujarat

20/26, 78.4%) [2] and in 2007 in Maharashtra and Andhra Pradeshtates (our unpublished observations). These fatalities occur within8 h of appearance of clinical symptoms, majority being within 24 h.s a result, documentation of the presence of IgM antibodies toHPV (IgM-anti-CHPV-IgM) remains infrequent and the diagnosis

s mainly dependent on the detection of the viral RNA in the serumamples. Though the virus was first isolated from the sera of twoatients during an outbreak of febrile illness in 1965 in India [3]nd subsequently from a child with acute encephalitis in 1980 [4],he epidemic potential of CHPV causing encephalitis and high mor-ality in children was recognized only in 2003. CHPV is transmittedo humans by sandflies [5]. Retrospective studies revealed that theirus was highly prevalent at least since 1955 in India [3], Sri Lanka6] and Africa (Nigeria, Senegal) [7,8]. During the epidemics of theisease [1,2] and one year study of sporadic encephalitis in childrenrom endemic areas [9], it was clearly shown that CHPV infec-ion is highly endemic in these areas. Detection of IgM-anti-CHPV

mong febrile cases indicate clinical spectrum of Chandipura infec-ion. However, of the infected cases what proportion progresseso encephalitis is not clear. For obvious reasons, studies related tontiviral therapy or vaccine development were not vigorously pur-

∗ Corresponding author. Tel.: +91 20 26006323; fax: +91 20 25871895.E-mail address: varankalle@yahoo.com (V.A. Arankalle).

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.02.089

e. The study shows that rGp induces both arms of immune response anddidate for further evaluations.

© 2009 Elsevier Ltd. All rights reserved.

sued earlier. However, as CHPV has recently emerged as a seriouslocalized problem in India, immunization of the children from theaffected areas was considered to be helpful in preventing encephali-tis associated rapid and high mortality.

CHPV is a member of the family Rhabdoviridae and genusVesiculovirus. It is an enveloped, bullet shaped, single stranded RNAvirus with genome size of approximately 11 kb [10]. Viral genomecodes for five polypeptides, namely, Nucleocapsid protein N, Phos-phoprotein P, Matrix protein M, Glycoprotein G and Large proteinL. N protein encapsidates genomic RNA into a nuclease resistantform to protect it from cellular RNAses. L with the help of P pro-tein acts as a viral RNA dependent RNA polymerase (RdRp), Matrixprotein glues the encapsidated genome RNA with the outer mem-brane envelope. G-protein spikes out of the membrane and actsas a major antigenic determinant [11–13]. Studies on Vesicularstomatitis virus (VSV) and Rabies virus (both belong to family Rhab-doviridae) revealed that the G-protein could be an excellent vaccinecandidate [14–18]. Therefore, CHPV G protein was the immuno-gen of choice for vaccine development. We report here the utilityof recombinant glycoprotein (rGp) expressed using Baculovirusexpression system in protecting mice from the intracerebral chal-lenge of the virus and propose this protein to be an ideal vaccinecandidate for Chandipura encephalitis.

2. Materials and methods

2.1. Construction of recombinant plasmids

Complete G gene (1593 bp, position 2-1566, 524 aa) from super-natant of Vero E6 cell line infected with CHPV (isolate CIN034627,GenBank accession No: AY382603) representing a strain from

2 alle /

2tTATwsabDA(swc

2

ipyc1s

2

alobauIwmT

2

clHHceetp

2

(c53afaFwl

846 C.H. Venkateswarlu, V.A. Arank

003 epidemic in Andhra Pradesh was amplified by reverseranscription-PCR according to the protocol described earlier [1].he primers used were: Forward primer 5′ ATG ACT TCT TCA GTGCA ATT AGT GTG ATCC 3′ and Reverse primer 5′ TCA TAC TCT GGCCT CAT GTT GAA GGG CTT 3′. The resultant PCR product (∼1.6 kbp)as TA-cloned in pGEM T Easy vector (Promega, Madison, USA) and

ub-cloned in to pFAST Bac1 expression vector (Invitrogen, CA, USA)ccording to manufacturer’s instructions. The insert was confirmedy sequencing of both the strands using multiple primers and Bigye Terminator cycle sequencing Ready Reaction Kit (version 3.1,pplied Biosystems, Foster city, CA) and an automated sequencer

ABI 3130 xl, Applied Biosystems, Foster city, CA). In order to con-truct recombinant bacmid (rBac), recombinant pFAST Bac1 vectoras transformed into maximum efficiency DH-10 Bac competent

ells.

.2. Expression of CHPV G-gene in Baculovirus expression system

rBac was transfected in Spodoptera Frugiperda (SF9) cells accord-ng to the standard protocol and after 72 h of post-transfection,ellet and supernatant were harvested. To determine the maximumield and immunoreactivity of rGp at different time-points, the timeourse, fractions of both pellets and supernatants were collected at2 h interval post-infection (PI) up to 96 h. All the fractions wereubjected to ELISA for the detection of rGp as described later.

.3. SDS-PAGE and Western blot

Supernatant and cell pellet of infected Sf9 cells were harvestedt 48 h post-infection. Cell pellets as well as supernatants were ana-yzed on 10% SDS-PAGE. Gels were either stained directly or blottednto nitrocellulose membrane. Blots were blocked with phosphate-uffered saline (PBS), pH 7.4 containing 5% nonfat milk powder O/Nt 4 ◦C. Anti-CHPV-IgG-positive human serum at 1:20 dilution wassed as a primary antibody. Secondary antibody was anti-human-

gG antibody conjugated to horseradish peroxidase (HRP). Blotsere developed using diaminobenzidine and H2O2. 2.5% nonfatilk powder in PBS was used as diluent and PBS containing 0.05%

ween 20 was used for washing.

.4. Purification of rGp

Fifteen milliliters of the rGp expressing Sf9 cell supernatant wasoncentrated to 5 ml with low protein binding Amicon filters (Mil-ipore Corporation, MA 01821, USA). 2.5 ml of rGp was loaded oniPrep 16/60 sepharose Gel-filtration column (AKTA BASIC 100PLC system, Amersham Pharmacia, USA). Fractions of 0.5 ml wereollected and coated at 1:10 dilution and screened for the pres-nce of rGp by using anti-CHP-IgG positive and negative sera forach fraction. The ELISA was carried out as described below. Frac-ions showing rGp activity were pooled and concentration of theurified rGp was measured by Lowry’s method.

.5. Mice immunization

The purified rGp was used for immunization of mice10 mice/group, Swiss albino, Female, 6–8 weeks old). Initially, fouroncentrations of rGp were used for immunization, i.e., 100 ng,00 ng, 1 �g and 2 �g. One micrograms of rGp was adsorbed on.25 �g of AlPO4 (Sigma chemicals, St. Louis, MO, USA) by vortexingt a low speed for 1 h at RT followed by centrifugation at 5000 rpm

or 5 min at RT. The pellet was re-suspended in 0.01 M PBS, pH 7.2nd diluted to the required rGp dose at the time of immunization.or further experiments, a total of 3 doses of rGp (1 �g/mice), 4eeks apart, were used for immunization. Serum samples were col-

ected periodically by retro-orbital plexus bleeding and subjected

Vaccine 27 (2009) 2845–2850

to ELISA for IgG-anti-CHP detection/quantitation as well as tissueculture based in vitro virus neutralization test (NT).

2.6. ELISA for the detection/quantitation of IgG-anti-rGpantibodies

Mice sera were screened for the presence of IgG-anti-rGp anti-bodies in ELISA employing rGp as a coating antigen. Infected Sf9supernatant containing rGp was diluted 1:10 in 50 mM carbonatedbuffer (pH 9.5) and coated as 100 �l/well. Following coating at 37 ◦Cfor 2 h, blocking solution (10% donor calf serum, 0.5% Tween 20, 0.5%gelatin) was added to each well and incubated at 37 ◦C for 30 min.After washing with wash solution (0.01 M PBS, 0.5% Tween 20), micesera diluted 1:25 in blocking solution were added (100 �l/well) todifferent wells according to the protocol. A 1:25 dilution of pre-immune sera served as negative controls. Incubation was continuedat the same temperature for 30 min. All the wells were washed threetimes and each well was added with horseradish peroxidase con-jugated goat anti-mouse IgG (Sigma chemicals, St. Louis, MO, USA)at 1:10,000 dilution and incubated at 37 ◦C for 30 min. Unboundconjugate was removed by washing 4 times. The enzyme substrate(O-phenylenediamine and urea peroxide) was added (200 �l/well)and allowed to incubate at RT in dark for 8–10 min. The reactionwas stopped by the addition of 4N H2SO4 and the absorbance wasmeasured at 492 nm (Labsystems Multiskan MS, MTX Lab SystemsInc., Virginia, USA). A serum sample was considered to be reactivewhen the optical density (OD) value was ≥cut-off value. The cut-offvalue for the anti-rGp IgG antibodies was calculated as mean ODvalues for the three negative controls multiplied by 3. The CHPVspecific IgG antibody titers were determined for individual mousefrom each group. The reciprocal of the highest dilution that had anabsorbance ≥than the cut-off value was taken as the IgG-anti-CHPVantibody titer. All the analyses were carried out on the geomet-ric mean titers (GMTs) and log-transformed antibody titers withstandard error.

2.7. Tissue culture based in vitro virus neutralization test (NT)

Initially, tissue culture infectious dose (TCID50) of CHPV stock(CIN034627 strain), propagated in Vero E6 cell line was determined.Equal volumes of mice serum and 100 TCID50 CHPV stock weremixed and incubated for 1 h at 37 ◦C. 100 �l of the above mixturewas added on monolayer of Vero E6 cells incubated at 37 ◦C andobserved periodically for 48 h for cytopathic effects (CPE). Anti-body negative and positive controls and virus alone control wereincluded in every test. When control wells demonstrated evidenceof extensive replication of the virus, at 35–48 h, the wells werestained with amido-black. A sample was considered positive forneutralizing antibodies when no CPE observed in the correspond-ing well. The reciprocal of the highest dilution that showed no CPEin at least two wells out of four used was taken as the NT CHPVantibody titer.

2.8. Lymphocyte proliferation assay (LPA)

To assess the cell-mediated immunity, mice were sacrificed 2–3weeks after the last dose by cervical dislocation and spleen cellswere harvested. 1 × 105 cells/well were cultured in quadruplicatesin 96-well flat bottom plate (Nunc, Denmark) in RPMI medium(Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovineserum (Invitrogen, Carlsbad, CA, USA) at 37 ◦C with 5% CO2. The

positive and negative controls included cells stimulated with T cellmitogen phytohaemagglutinin (PHA) (Sigma chemicals, St. Louis,MO, USA) at the concentration of 10 �g/ml or with the mediumalone respectively. Following dose–response experiment, 20 �g/mlrGp (optimum concentration) was added to the respective wells

C.H. Venkateswarlu, V.A. Arankalle / Vaccine 27 (2009) 2845–2850 2847

F m theu d at 48a t show

ft(bwec

2

9a

2

limlo

3

3

svor

ig. 1. A–C: (A) Depicts ELISA reactivity of the supernatant/pellet-derived rGp froninfected and recombinant baculovirus infected SF9 cells and supernatants collectentibody positive human serum (C). IgG-anti-CHPV antibody negative serum did no

or specific proliferation. Cells were pulsed at 96 h with 1 �Ci ofritiated thymidine for 24 h. Cells were harvested onto GF/C filterWhatman, UK) and the incorporated radioactivity was measuredy �-counter (LKB Pharmacia, Sweden). The stimulation index (SI)as determined as ratio of counts per minutes (CPM) in the pres-

nce of rGp/CPM in absence of rGp. Mice with SI value ≥ 3 wereonsidered to be the responders [19].

.9. Statistical analysis

ELISA and NT titers were compared using t-test employing SPSS.0 software. Non-responders in each group were included in thenalyses.

.10. Intracerebral challenge of immunized mice with CHPV

The homologous strain of CHPV (CIN034627) was used as a chal-enge virus. The lethal dose 50 (LD50) of the stock was determinedn 16–18 weeks old Swiss albino female mice by Reed and Munch

ethod [20]. The immunized mice were bled periodically and chal-enged intracerebrally at 2 weeks after the 3rd dose with 100 LD50f the virus. All the challenged mice were observed for 21 days.

. Results

.1. PCR amplification and cloning of G gene

The complete G-gene of CHPV was amplified by reverse tran-cription PCR and the product was TA cloned into pGEM T Easyector and subsequently in the pFAST Bac1 expression vector. Therientation of the insert in both the vectors was checked withestriction enzymes and both the clones were sequenced com-

SF9 cells harvested at different time intervals. (B and C) Document SDS-PAGE ofh post-infection (B) and Immunoblot analysis of the same employing IgG-anti-CHPVany reactivity (photograph not shown).

pletely. 100% identity was noted with the sequence of the sameisolate deposited in the GenBank (accession No: AY382603).

3.2. rGp expression and characterization

Sf9 cells were infected with CHPV-G-recombinant baculovirusand temporal expression of G protein was studied. ELISA was usedto screen the presence of rGp in both cells and supernatants har-vested at different time intervals of post-infection (12–96 h). Themaximum expression of rGp was observed at 48 h PI and contin-ued at significant levels thereafter (Fig. 1A). The majority of the rGpwas detected in the supernatant. Both SDS-PAGE and Western blotshowed the presence of a single band of expected size of approxi-mately 60-kDa in the supernatant of the infected cells (Fig. 1B andC).

3.3. Purification of rGp

Fig. 2 depicts elution profile of the serum-free concentrate ofthe rGp positive SF9 cell culture supernatant loaded on gel filtra-tion HPLC column. All the fractions were subjected to ELISA forrGp detection. Among these, only fractions corresponding to a sin-gle peak (Peak: 13.61) were scored as reactive. Similar to culturesupernatant (Fig. 1), this rGp peak was confirmed by SDS-PAGEand Immunoblotting to be a single protein of approximately 60-kDa (expected size of rGp, data not shown). This protein was usedfor mice immunization.

3.4. Humoral immunity

Both pre- and post-immunization mice sera were subjected toELISA and NT for the detection of anti-CHPV antibodies. Both assays

2848 C.H. Venkateswarlu, V.A. Arankalle / Vaccine 27 (2009) 2845–2850

Fig. 2. Elution profile of the serum-free concentrate of rGp positive SF9 culture supernatant loaded on gel filtration HPLC column. Peak: 13.61 corresponded to rGp.

Table 1Percent seroconversion in mice immunized with three doses of different concentrations of rGp at 4 weeks interval (0, 4 and 8 weeks). Antibody testing was done by bothELISA and NT prior to every immunogen dose and 2 weeks after the last dose.

rGp concentration Percent seroconversion

Pre-1st dose Pre-2nd dose Pre-3rd dose 2 weeks after the last dose

ELISA NT ELISA NT ELISA NT ELISA NT

1512

dPAtwwrtp

wb

Fw

00 ng 0 0 20 1000 ng 0 0 30 20�g 0 0 40 40�g 0 0 40 40

etected antibodies as early as 2 weeks after 1st dose (Table 1).re-immunization sera were scored negative by both the tests.s compared to NT, ELISA detected seroconversion earlier when

he dose of the immunogen was low (100 ng). Antibody responseas immunogen concentration dependent, increasing graduallyith dose from 100 ng to 1 �g. Both 1 and 2 �g gave comparable

esponse. Percentage sero-conversion increased after each dose and

he maximum (90%) sero-conversion was observed at 10th week ofost-immunization, i.e., 2 weeks after the last dose, either 1 or 2 �g.

Fig. 3 compares anti-CHP-antibody titers by ELISA and NT at 2eeks after the last dose. Similar to seroconversion rates, the anti-ody titers were function of the concentration of the immunogen

ig. 3. The Geometric mean of reciprocal anti-CHPV antibody titers (log 10) at 10eeks, i.e., 2 weeks after the third dose of rGp. Bar represents standard error.

50 40 40 4050 50 60 5070 70 90 9080 80 90 90

used, 1 and 2-�g rGp producing comparable titers. The maximumtiters in NT and ELISA were 1:320 and 1:1200 respectively. Neutral-izing antibody titers were consistently lower than the ELISA titers.As evident from Fig. 4, irrespective of the dose of rGp used for immu-nization, the anti-CHP-antibody titers remained almost constantduring the observation period of 6 months.

To assess the efficacy of the antibodies generated by immuniz-ing with the 2003 isolate-derived rGp against the viruses isolated in1965, 2004 and 2007 neutralization tests were performed employ-ing different virus isolates (Table 2). Serum samples from fiveimmunized mice were screened in NT with the homologus as wellas heterologus CHPV isolates. An excellent cross protection wasrecorded; NT titers employing different isolates did not differ sig-nificantly (p = 0.423–0.510).

3.5. Cell mediated immunity

To assess the T cell-response, spleens from 10 mice from eachgroup harvested at 2–3 week after the last immunization were used.The optimum concentration of rGp for the stimulation of spleen cellwas found to be 20 �g/ml. Fig. 5 records stimulation indices for the

Fig. 4. The Geometric mean of reciprocal IgG-anti-CHPV antibody titers (log 10)(ELISA) and anti-CHPV neutralizing antibody titers (log 10) (NT) in mice immunizedwith different concentrations of rGp at 1, 3, 5 and 6 months after the last dose. Barrepresents standard error.

C.H. Venkateswarlu, V.A. Arankalle / Vaccine 27 (2009) 2845–2850 2849

Table 2NT titers of serum samples from immunized mice employing homologous and heterologus CHPV isolates.

Mice no. NT titer against isolate 034627 NT titer against isolate 653514 NT titer against isolate 2004 NT titer against isolate 076324

1 80 40 40 202 320 1603 20 404 160 805 320 160

Fdv

miw2

3

yt5t1wplaanw

4

dwd5

TC

r

1512A

ig. 5. Stimulation indices measured in spleenocytes from mice immunized withifferent concentrations of three doses of rGp and stimulated with rGp antigen. SIalues ≥3 indicate responders.

ice immunized with different doses of rGp. Similar to humoralmmune response, the lymphocyte proliferative response increased

ith the dose of the immunogen, 60% mice immunized with 1 or�g rGp responding with high SI values.

.6. Intracerebral virus challenge experiments

An intracerebral challenge of 100LD50 of the homologus strainielded satisfactory results. Survival was directly proportional tohe immunogen dose, 20 and 40% mice surviving with 100 and00 ng doses respectively. Both 1 and 2 �g doses gave 90% pro-ection. Mortality in control, unimmunized group infected with00LD50 virus was 100% (2–7 days). The surviving mice immunizedith different doses of the rGp were healthy during the observationeriod of 21 days. The immunized mice succumbing to the chal-

enge died at times similar to the controls. As evident from Table 3,n ELISA titer of 1:40 and NT titer of 1:20 was predicting protectiongainst the intracerebral challenge. Overall, 1 �g purified recombi-ant G protein expressed employing Baculovirus Expression Systemas shown to be a promising candidate vaccine.

. Discussion

This study reports utility of rGp of CHPV as the vaccine can-idate for Chandipura encephalitis, a recently emerged diseaseith high mortality in children from three Indian states. As theisease progression is very rapid, 47% mortality within 24 h and5% at 48 h of the appearance of clinical symptoms [1], develop-

able 3omparison of survival following intracerebral challenge of CHPV with antibody titers as

Gp concentration No. of mice showing ELISA titers ≥1:40 (n = 10) No. o

00 ng 2 200 ng 4 4�g 9 9�g 9 9lPO4 alone 0 0

a The same mice showed ELISA titres ≥1:40 and NT titers ≥1:20 and survived the intrac

160 32020 4080 160

160 80

ment of a prophylactic vaccine was thought to be the priority overthe evaluation of antivirals. Considering the documented immuno-genicity of glycoproteins of two other rhabdoviruses, rabies [21]and VSV [22,23], glycoprotein of CHPV was the protein of choice.Our data demonstrates the efficacy of the recombinant glyco-protein produced employing the baculovirus expression system.It was pleasing to note that the protein was predominantlypresent in the supernatant of the SF9 cells. In the serum-freemedium almost entire protein was the protein of our interest.The activity of rGp was present in a single peak of expected sizewhen HPLC-based gel filtration was carried out. Thus, purifica-tion of protein was relatively easy and the yield was also high(Figs. 1A and 2).

Following confirmation of immunoreactivity of the rGp in ELISAand Western blot, mice were immunized with three doses of dif-ferent concentrations of the protein (100 ng to 2 �g) and bothhumoral as well as cell-mediated responses were monitored. Aslow as 100 ng rGp was able to induce IgG-anti-CHPV antibodiesand neutralizing antibodies in 50% each of the immunized mice, asevidenced by ELISA and NT respectively. Anti-CHP antibody titersrose with increase in the immunogen dose. However, both 1 and2 �g doses of rGp yielded comparable titers and 1 �g was consid-ered the optimum concentration of the immunogen. We were ableto demonstrate T cell responses in immunized mice (rGp concen-tration dependent) with a maximum of 60% response with 1 or2 �g doses. Thus, the recombinant protein generated both arms ofimmunity. However, we need in-depth studies employing bettertools to clarify the cell-mediated immune responses during vacci-nation and disease.

Based on the results of intracerebral challenge with 100LD50 ofthe homologus virus strain, utility of rGp as vaccine candidate forCHPV infection was obvious. With both 1 and 2 �g dose schedule,90% mice survived the challenge of the virus. The two mice notprotected by the vaccine died on 4 (1 �g) and 7 (2 �g) days post-infection; with 500 ng rGp five mice died during an interval of 2–7days post-infection, similar to unimmunized controls. It is pertinentto note here that rabies glycoprotein sub-unit vaccine at as lowdose as 360 ng could offer protection to 87.5% immunized mice afterintracerebral challenge [24].

When anti-CHPV antibody titers as determined by NT or ELISAwere compared with the protection offered against the intracere-

bral challenge of CHPV in mice, it was estimated that an ELISA titerof 1:40 or NT titer of 1:20 could be considered protective. ThoughCHP viruses isolated so far are closely related to each other [25,2and our unpublished observations], we used viruses isolated dur-ing 1965, 2004 and 2007 epidemics, i.e., all the viruses isolated so

estimated by ELISA and NT.

f mice showing NT titers ≥1:20 (n = 10) Survival statusa (100 LD50) (n = 10)

24990

erebral challenge.

2 alle /

fi(uta2(tsa

uchap

A

osCe

R

[

[

[

[

[

850 C.H. Venkateswarlu, V.A. Arank

ar, for in vitro NT for estimating neutralizing antibody titers in micemmunized with rGp based on 2003 strain. The homologus strain2003) was used as the standard virus. With all the different virusessed, similar neutralization titers were obtained, suggesting protec-ion against all the known strains of CHP virus. Percent nucleotidend (amino acids) homologies with the 2003 strain were 99.0 (99.2,004 strain, 98.1 (99.2, 2007 strain) and 96.3 (98.0, 1965 strain)our unpublished data). Thus the G gene was conserved amonghe viruses isolated during 1965–2007. Experiments using differenttrains of Chandipura viruses, though closely relate to each otherre under progress.

These encouraging results make us believe that rGp expressedsing baculovirus expression system will provide an excellentandidate vaccine for controlling Chandipura encephalitis withigh mortality in children from the endemic areas from Indiand should be immediately subjected to the required evaluationrotocols.

cknowledgements

The authors thanks Dr. A.C. Mishra, Director, National Institutef Virology for all the encouragement and Mr. Jadi R.S. for constantupport. Financial support for this work was provided by the Indianouncil of Medical Research (ICMR). Venkateswarlu CH acknowl-dges ICMR for providing Senior Research Fellowship.

eferences

[1] Rao BL, Basu A, Wairagkar NS, Gore MM, Arankalle VA, Thakare JP, et al.A large outbreak of acute encephalitis with high fatality rate in children inAndhra Pradesh, India, in 2003, associated with Chandipura virus. Lancet2004;364:869–74.

[2] Chadha MS, Arankalle VA, Jadi RS, Joshi MV, Thakare JP, Mahadev PV, et al. Anoutbreak of Chandipura virus encephalitis in the eastern districts of Gujaratstate, India. Am J Trop Med Hyg 2005;73:566–70.

[3] Bhatt PN, Rodrigues FM. Chandipura: a new Arbovirus isolated in India frompatients with febrile illness. Indian J Med Res 1967;55:1295–305.

[4] Rodrigues JR, Singh PB, Dave DS, Prasan R, Ayachit V, Shaikh BH, et al. Isolationof Chandipura virus from the blood in acute encephalopathy syndrome. Indian

J Med Res 1983;77:303–7.

[5] Dhanda V, Rodrigues FM, Ghosh SN. Isolation of Chandipura virus from sandfliesin Aurangabad. Indian J Med Res 1970;58:179–80.

[6] Peiris JS, Dittus WP, Ratnayake CB. Seroepidemiology of dengue and otherarboviruses in a natural population of toque macaques (Macaca sinica) at Polon-naruwa Sri Lanka. J Med Primatol 1993;22:240–5.

[

[

Vaccine 27 (2009) 2845–2850

[7] Fontenille D, Traore-Lamizana M, Trouillet V, Leclerc A, Mondo M, Ba Y, et al.First isolations of arboviruses from Phlebotomine sandflies in West Africa. AmJ Trop Med Hyg 1994;50:570–4.

[8] Traore-Lamizana M, Fontenille D, Diallo M, Bâ Y, Zeller HG, Mondo M, etal. Arbovirus surveillance from 1990 to 1995 in the Barkedji area (Ferlo) ofSenegal, a possible natural focus of Rift Valley fever virus. J Med Entomol2001;38:480–92.

[9] Tandale BV, Tikute SS, Arankalle VA, Sathe PS, Joshi MV, Ranadive SN, et al.Chandipura Virus: a major cause of acute encephalitis in children in NorthTelangana, Andhra Pradesh, India. J Med Virol 2008;80:118–24.

[10] Marriott AC. Complete genome sequences of Chandipura and Isfahanvesiculoviruses. Arch Virol 2005;150:671–80.

[11] Banerjee AK. Transcription and replication of rhabdoviruses. Microbiol Rev1987;51:66–87.

12] Banerjee AK. The transcription complex of vesicular stomatitis virus. Cell1987;48:363–4.

[13] Barr JN, Whelan SP, Wertz GW. Transcriptional control of the RNA-dependent RNA polymerase of vesicular stomatitis virus. Biochim Biophys Acta2002;1577:337–53.

[14] Cantlon JD, Gordy PW, Bowen RA. Immune responses in mice, cattle and horsesto a DNA vaccine for vesicular stomatitis. Vaccine 2000;18:2368–74.

[15] Mackett M, Yilma T, Rose JK, Moss B. Vaccinia virus recombinants: expres-sion of VSV genes and protective immunization of mice and cattle. Science1985;227:433–5.

[16] Yilma T, Breeze RG, Ristow S, Gorham JR, Leib SR. Immune responses of cattleand mice to the G glycoprotein of vesicular stomatitis virus. Adv Exp Med Biol1985;185:101–15.

[17] Lambot M, Blasco E, Barrat J, Cliquet F, Brochier B, Renders C, et al. Humoral andcell-mediated immune responses of foxes (Vulpes vulpes) after experimentalprimary and secondary oral vaccination using SAG2 and V-RG vaccines. Vaccine2001;19:1827–35.

[18] Yuan Z, Zhang S, Liu Y, Zhang F, Fooks AR, Li Q, et al. A recombinant pseudoRabies virus expressing Rabies virus Glycoprotein: safety and immunogenicityin dogs. Vaccine 2008;26:1314–21.

[19] Deshmukh TM, Lole KS, Tripathy AS, Arankalle VA. Immunogenicity of candi-date hepatitis E virus DNA vaccine expressing complete and truncated ORF2 inmice. Vaccine 2007;30:4350–60.

20] Reed LJ, Muench HA. Simple method of estimating fifty per cent endpoints. AmJ Hyg 1938;27:493–7.

21] Rupprecht CE, Hamir AN, Johnston DH, Koprowski H. Efficacy of a vaccinia-Rabies Glycoprotein recombinant virus vaccine in raccoons (Procyon lotor). RevInfect Dis 1988;10:S803–9.

22] Rosenthal KL, Zinkernagel RM. Cross-reactive cytotoxic T cells to serologicallydistinct vesicular stomatitis virus. J Immunol 1980;124:2301–8.

23] Kelley JM, Emerson SU, Wagner RR. The Glycoprotein of vesicular stomatitisvirus is the antigen that gives rise to and reacts with neutralizing antibody. JVirol 1972;10:1231–5.

24] Fekadu M, Shaddock JH, Ekström J, Osterhaus A, Sanderlin DW, Sundquist B, etal. An immune stimulating complex (ISCOM) subunit Rabies vaccine protectsdogs and mice against street Rabies challenge. Vaccine 1992;10:192–7.

25] Arankalle VA, Prabhakar SS, Madhukar WA, Hanumaih, Dattatraya PS, ChandraMA. G, N, and P gene-based analysis of Chandipura viruses, India. Emerg InfectDis 2005;11:123–6.