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IJBSTR REVIEW PAPER (RVP-3) VOL 1 [ISSUE 2] FEBRUARY 2013

Rabies Vaccine Production from Different Viral Infected Cell Line

and Vaccine Production
Praveen Gaurav Singh and S. K. Singh
ABSTRACT- Bullet-shaped rabies virus is classified as Lyssavirus genus in Rhabdoviridae family which transferred by animals like
Dogs, Raccoons, Skunks, Foxes, Coyotes, Bat to Human. Virus has a single-stranded, negative-sense RNA. Among of 7 member of
Lyssavirus genus, only serotype 1 infects humans. Human rabies have two form; furious and Paralytic rabies. The first rabies vaccine
is developed by two French scientists, Louis pasture and Emile Roux in 1885. Various types of rabies virus strain i.e. CVS, HEP, PV,
ERA, PM, CPZ, BOLIVAR etc. are grown and isolated from cultured cell i.e. Pig kidney cells, Chick embryo fibroblasts, Dog
kidney cells, BHK-21 cells, Human diploid cells etc. The preparation of rabies vaccines involve production of seed virus followed by
preparation of final vaccine that include live attenuated vaccine and inactivated vaccine (Vaccines containing animal brain tissues,
Avian vaccines, and Cell-cultured vaccines).These strains are inactivated by acetylethylenimine, ethylenimine, -propiolactone or
ultraviolet irradiation. The potency of inactivated vaccine is determined by single radial immunodiffusion and NIH potency test. In
dog inactivated PV strain by ethylenimine have higher antigenic value and potency. BHK-1 is frequently used for the production of
vaccine using suspension culture in bioreactor ranging from 20 to 1000 lt. and potency rate is 6 IU for 20 lt. In Europe and USA,
human fibroblast is used in Human diploid cell culture vaccine (HDCV).
Key words: Rabies virus; Rabies vaccine; Rabies strain; Rabies infection; Cell lines; NIH potency test; Vaccine production.
Introduction

Single-stranded
negative-sense
RNA
of
approximately 12 kb rabies virus has 12 viral
species (RABV; geno-type I) belongs to the genus
Lyssavirus in the family Rhabdoviridae. These are
responsible for greater than 55,000 human deaths
annually throughout the world. Humans are infected
by RABV through the saliva of infected carnivores
and bat in America. In Latin America, common
vampire bat (Desmodusrotundus) and several other
Neotropical bat species are major cause of rabies
infection [5], [48], [49]. In Ontario, Canada, more
than 54,800 animals were infected with rabies, from
1954 to 2006 (no including clinical cases). Red foxes
(Vulpesvulpes; 46%) and striped skunks (Mephitis
mephitis; 20%) are infected with an Arctic variant of
rabies virus from the majority (66%) cases [36].
Author Praveen Gaurav Singh is currently
pursuing master of technology program in
biotechnology
engineering
in
Lovely
Professional University, Chiheru, Kapurthala,
Punjab,
India,
PH-9041382361.
E-mail:
singhpraveengaurav@gmail.com
Co-Author S. K. Singh is currently Assistance
Professor in School of Biosciences and
Biotechnology
in
Lovely
Professional
University, Chiheru, Kapurthala, Punjab, India,
PH-01123456789.
E-mail:
author_name@mail.com

In China surpass 3,000 human rabies cases per year

reported in the recent year [51]. In United States and
Puerto Rico, 6,292 cases of wildlife rabies were
reported in 2004. Coyotes (Canislatrans), gray fox
(Urocyoncinereoargenteus), red fox (Vulpesvulpes),
and skunks (Mephitis spp. and Spirogolaputorius)
raccoons (Procylon lo-tor), bats (Lasionycterus spp.,
Myotis spp., and Pipstrellus spp.) are involved in
these rabies infection [35]. Mostly deaths due to
rabies infection were reported for near about 40%
death of children aged below 15 years or 15 years in
Asia [52]. In eastern Ontario, 135 cases are found
with
raccoon
rabies
infection
after
the
implementation of oral virus-rabies glycoprotein (VRG) vaccine between 1999 and 2005 [40]. Rabies
infected dogs are responsible for more than 90% of
human death [47]. The World Health Organization
(WHO) estimated that 40,000-70,000 death every
year in all over world due to the rabies virus [31].
2 Rabies Virus Structure and Infection
2.1 Structure
In Rhabdoviridae, such as RV and vesicular
stomatitis virus (VSV) the nonsegmented negativestranded RNA is enclosed within bullet-shaped
envelope structure [10], [14]. The 12-kb genome
encodes only five different type protein closely

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associated with nucleoprotein or nucleoocapsid proteinn

(N), matriix protein (M)), phosphoprootein (P), virall
polymerasee (V) and the single
s
transmem
mbrane proteinn
0], [12], [47].T
There are ninee
or glycoprrotein (G) [10
moter, bind w
with the sugarr
nucleotide per N prom
NA by virall
phosphate backbone of the RN
nucleocapssidprotein (N).. This N-RNA template havee
important role for repliication and trranscription off
R
RNA and are catalyzed by
b the viral RNA-dependent
t
L). Polymerrase cofactorr
RNA poolymerase (L
(phosphoprotein (P)) hellped in the binnding of RNA-ymerase (L) to N-RNA
A
dependent RNA poly
wn as internnal core orr
template.thhis is know
ribonucleooprotien (RNP)) complex andd formed thee
e
compponent, virall
RV virionn with the external
lipoproteinn envelope whiich is composeed of M and G
protein[4], [12], [47]. Th
he RV M and G protein aree
budding and assemblyy and M aree
essential for
f
n
(NC). The G
surroundinng a helical nucleocapsid
protein plaayed important role in the inteeraction with a
d pH-dependeent membranee
cellular reeceptor(s) and
hen the amounnt of virions iss
fusion. If G is deleted th
aree
G
reduced about 30-fold. Spike G-proteins
ve at least one subunit of thee
amphiphiliic proteins hav
oligomericc protein. This is inserted iinto the lipidd
bilayer off the viral env
velope anchorring the spikee
The spike G-proteein involved inn
to the membrane.
m
the stimulaation of virus--neutralizing aantibody as ann
Rabies viruss
antigen [10], [12], [14], [55].R
he ERA strainiis a 505 aminoo
glycoproteein (RGP) of th
acid type I membrane gllycoprotein thaat oligomerizess
mers and that contains three A
Asn-X-Ser/Thrr
into trimm
potential N-glycosylatio
on sites (seqquons) in thee
extracellular domain [25]]

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2.2 Structure
S
of N-RNA
N
Coplex

Fig.2. Ribbon Diiagram of N-R

RNA of Rabies Virus
[50].
In thhis ribbon diagrram of N-RNA
A, RNA is shoown as
blackk coil with different color oof N-protomerr. The
A ring structuure as
11-nuucleotide oliggomer N-RNA
wed from the boottom in Ribbon diagram [50]]
view

Fig.33. The helical ssecondary struccture of N-prottomer

D are representted by
(Ribbbon Diagram). NTD and CTD
dark and light blue color respectivvely [50].

Fig.1. Show
w the rabies virus
v
envelope with Matrix
(M) proteinn, Glycoproteiin (G), Nucleeocap
protein and lipid [54].

NTD
D (N-terminall core domainn) have 32 too 233
residdues of aminoo acid. Foldeed helical strructure
consiist 6 helices annd connected bby a large loopss.CTD
(C-teerminal core doomain) have 2336 to 356 aminno acid
residdues. CTD is ccomposed of 111 helices joinned by
loop tighter than NT
TD [50].

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2.3 Infection
Rabies viruses can cause the fatal diseasedue to the
replication in central nervous system, so known as
neurotropic virus. RV invades the central nervous
system (CNS) from peripheral sites, due to this RV
known as neuroinvasivenes. This process expressed
thepathogenicity of rabies virus [47]. Rabies or
Rabies related virus caused hindlimb paralysis in
immunocompetent in mice and hamsters [15].

ISSN 2320-6020

reeze-dried fixed-virus strain Flury LEP grown in

cultures of chicken fibroblasts. Both vaccines
induced the Induces neutralizing antibodies and
cellular immunity. Imovax are stored at 35 to 46F.
Do not freeze and RabAvert are stored at 36 to
46F. Protect from light [55].

Many types of commercial product of rabies vaccine

are available.

2.4 Virus Survival

References

Rabies virus are survived at 5 degrees C for 114

hours, when spread in a thin layer onto surface like
metal, leaves or metal. Rabies virus is infected the
mammalian for 24 hours at 240C when spread o glass
and leaves and 48 hours on metal. But due to
exposure of sunlight virus are inactivated within 1.5
hours at 300C [54]

[1] R. Fischman et al. Recovery from Experimental

Rabies by Adoptive Transfer of Immune Cell.
J. gen. Virol. (1981), 56, 25-3l.

3 Rabies Vaccines
3.1 OspA lipoprotein
OspA lipoprotein is found on outer surface protein of
Borrelia burgdorferi sensu stricto ZS7 and expressed
by Escherichia coli. OspA lipoprotein contains
lipoprotein OspA and use as vaccine against Lyme
diease. Single polypeptide chain of OspA consist of
257 amino acids with lipds covalently bonded to N
terminus and is conjugated with aluminum hydroxide
as an adjuvant. The protein chemical formula of this
vaccine is C1198H2012N322 - -O422S2 with average
molecular weight of 27743.1000. In vitro condition,
this vaccine has 1.2 hour half-time (Tested in
mammalian reticulocytes). The hydrophobicity and
isoelectric point of this protein is -0.65 and 6.72
respectively [53].
3.2 Imovax (Human Diploid Cell) & RabAvert
(Chicken Fibroblasts)
Imovax- The imovax (trade name) are present in the
form of powder for injection. PM-1503-3M strain of
rabies virus (freeze-dried suspension) are cultured in
human diploid cell culture for vaccine production and
available at least 2.5 international unit rabies
antigen/ml.
RabAvert- The RabAvert are present in the form of
powder for injection. RabAvert are produced by the

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[54]http://www.wormsandgermsblog.com/2012/07/ar
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