Download IJBT 4(2) 284-286

Indian Journal of Biotechnology
Vol. 4, April 2005, pp. 284-286
Short Communications
Differentiation of rabies fixed and street
viruses using RT-PCR coupled with
restriction endonuclease analysis
Praveen K Gupta*, V K Chaturvedi, P C Verma and
K D Pandey
Division of Biological Products, Indian Veterinary Research
Institute, Izatnagar 243 122, India
Received 6 November 2003; revised 24 May 2004;
accepted 5 June 2004
A method based on RT-PCR and restriction endonuclease
digestion of amplified PCR product was used to differentiate
rabies laboratory “fixed” and “street” viruses. The primer sets
from glycoprotein and nucleoprotein gene of rabies virus were
used to amplify 406 and 533 bp amplicons, respectively. After
amplification in RT-PCR, the PCR product was digested with
HaeIII restriction endonuclease and analysed for the presence or
absence of restriction site on amplicons. The presence of HaeIII
restriction site on amplicons indicated rabies fixed virus while
absence indicated street virus.
Keywords: rabies, virus, differentiation, street, fixed, RT-PCR,
restriction endonuclease analysis
IPC Code: Int. Cl.7 C12N15/10, 15/47; C12R1:93
Rabies is a disease of central nervous system that
constantly produces victims in human beings as well
as in domestic and wild animals. Rabies virus belongs
to Lyssavirus genus in the Rhabdoviridae family. The
genus Lyssavirus consists of 6 genotypes, of which
genotype 1 includes the classical rabies viruses,
including majority of both laboratory “fixed” and
“street” viruses1. The fixed virus is a laboratory
passaged attenuated virus with predictable properties,
in terms of incubation period, pathological and
clinical effects. In contrast, the virus isolated from
naturally infected animals is termed as street virus.
The street virus may have incubation periods varying
from 2 weeks to more than 1 year and variable
clinical effects2. The vaccines available today against
rabies are killed vaccines prepared from laboratory
fixed virus.
__________________
*Author for correspondence:
Tel: 91-581-2301613, extn 2117; Fax: 91-581-2301584
E-mail: [email protected]
The diagnostic tests for detection of rabies in
clinical samples are fluorescent antibody test (FAT),
mouse inoculation test (MIT) and reversetranscription polymerase chain reaction (RT-PCR) 3-5.
These methods can only detect presence of virus in
clinical samples but cannot differentiate between
street and laboratory fixed virus. In this study, the
authors report differentiation of street and fixed rabies
viruses using RT-PCR coupled with restriction
endonuclease analysis of the amplified product.
The laboratory fixed rabies viruses used in this
study were the mouse brain adapted challenge virus
standard (CVS) strain and BHK-21 adapted PV-11
strain. The street rabies viruses were brain tissue
samples received from Pasteur Institute of India,
Coonoor, India. All the tissue samples used in this
study were found positive in MIT and RT-PCR5.
Total RNA was isolated from all the laboratory
fixed and street rabies viruses using Trizol LS reagent
(Life Technologies, New York, USA) following
manufacturer’s instructions. The isolated total RNA
samples were dissolved in DEPC-treated RNase free
distilled water. The primer sets used in this study
were from two different genes (glycoprotein, G and
nucleoprotein, N) of rabies virus. The primer set from
G gene (forward, 5’- TAA TCC CAG AGA TGC
AAT CA -3’; reverse, 5’- CCT CAG AGT CTG GTC
TCA CC -3’) was used to amplify 406 bp amplicon.
Similarly, the primer set from N gene (forward, 5’ACT GAT GTA GAA GGG AAT TG -3’; reverse,
5’- GAA CGG AAG TGG ATG AAA TA -3’) was
used to amplify 533 bp amplicon5.
For RT-PCR reaction, 8-10 µg of total RNA from
each sample was used. The single tube RT-PCR
reaction was performed using G or N gene primer sets
following the method described earlier5. Briefly, RNA
samples mixed with G or N gene primer set and other
ingredients were incubated at 42°C for 30 min for RT
reaction and then PCR was done in the same tube
following 35 amplification cycles with denaturation at
94°C for 1 min, annealing for 1 min and extension at
72°C for 5 min. The annealing temperatures were 55
SHORT COMMUNICATIONS
285
1
234
5
and 50°C for primer set for G and N gene,
respectively.
An aliquot of 5 III from each sample was analysed
for amplification on 1% agarose gel. For restriction
digestion, 5 III of PCR product was digested with 10
units of Haem restriction endonuclease (Promega,
USA) in 25 III volume at 37°C for 2 hrs. The digested
PCR fragments were separated on 1% agarose gel
along with 100 bp DNA ladder (Pro mega, USA) and
analysed.
The 533 bp amplicon from N gene. and 406 bp
amplicon from G gene from reported nucleotide
sequences were analysed for Haem restriction site using
MapDraw software (DNAStar, USA). For HaeIII
restriction site analysis of 406 bp amplicon from G gene,
GenBank accession numbers used were: AF042824
(CVS-B2c), AF042823 (CVS-N2c), AJ506997 (CVS),
M31046 (SAD B-19), M13215 (PV) , M38452 (ERA),
M32751 (Flury), M81059 (Street 1), M81060 (Street 2),
M81058 (Street 3) and U52947 (Street 4). For RaeIII
restriction site analysis of 533 bp amplicon from N gene,
GenBank accession numbers used were: D42112 (CVS),
M13215 (PV), M31046 (SAD B19) and ABOI0491
(Nishigahara).
The diagnostic tests available for detection of rabies
in field samples, viz. FAT, MIT, virus isolation,
histopathological
examination
of Negri bodies,
immunoperoxidase staining and PCR based diagnosis,
though sensitive and reliable, are unable to differentiate
between rabies fixed and street viruses".
Therefore, for differentiation of rabies fixed and street
viruses, the amplicons from G and N gene were
amplified and analysed for the presence of HaeIII
restriction site: Both the groups (fixed and street viruses)
yielded 406 bp amplified amplicon. When the PCR
products from rabies fixed viruses i.e. CVS and PV
strains were subjected to restriction digestion with
HaeIII restriction endonuclease, there was presence of
restriction site in the amplicon due to which the PCR
product of 406 bp was digested into fragments (Figs 1 &
2). When the amplified products from street viruses
(field samples) were subjected to Haem digestion, the
absence of restriction site was found in all the samples
used in this study (Figs 1 & 2). This indicated that
HaeIII restriction site on 406 bp amplicon was present
only in fixed viruses and absent in street viruses. The
street viruses (field samples) used in this study were
406 bP7
~533bp
Fig. I-Restriction
endonuclease analysis of PCR amplified 406
bp amplicon from G gene and 533 bp amplicon from N gene from
rabies "street" and "fixed" viruses; Lane 1: 406 bp PCR product
digested with HaeIII, Lane 2: 406 bp PCR product digested with
HaellI, Lane 3: 100 bp DNA ladder marker, Lane 4: 533 bp PCR
product digested with HaeI!, Lane 5: 533 bp PCR product
digested with HaeIII.
already confirmed as rabies isolates using MIT and RTPCR5. When the amplicon from nucleotide sequences
submitted to GenBank for fixed viruses (CVS, PV,
ERA, HEP-Flury) and few of the reported street viruses
(Street 1-4) were analysed using DNA Star software, the
restriction site for HaeIII restriction endonuclease was
found at nucleotide position 225 in fixed viruses while
absent in street viruses (Fig. 2). One additional
restriction site at position 235 was also found in CVS.
But the resultant 10 bp fragment could not be located in
1% agarose gel.
.
Similarly, the amplified amplicon (533 bp) from N
gene demonstrated the presence of restriction site for
Haem at nucleotide position 280 in fixed viruses
(CVS and PV) used in this study. However, no site
was found in PCR amplified products from street
viruses (Figs 1 & 2). The same ampl icon from
reported fixed viruses (from GenBank sequences)
when analysed using DNA Star software, the presence
of HaeIII restriction site for fixed viruses was well
corroborated (Fig. 2).
The results from this study indicated that the
presence of HaeIII restriction sites on amplicon from
G or N gene at nucleotide position 225 and 280,
respectively, for rabies fixed virus while absence for
INDIAN J BIOTECHNOL, APRIL 2005
286
Fig. 2⎯HaeIII restriction patterns of 406 bp and 533 bp amplicons from glycoprotein and nucleoprotein genes of rabies virus,
respectively.
street virus. This finding will be of help in grouping a
rabies field isolate (sample) either in fixed or street
rabies viruses.
Acknowledgement
Authors are thankful to the Director, Indian
Veterinary Research Institute, Izatnagar for providing
necessary facilities to carry out the work. We are also
thankful to Dr Y U B Rao, Pasteur Institute of India,
Coonoor, India for providing MIT positive rabies
brain samples.
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