Biosci. Biotech. Res. Comm. 10(1): 121-124 (2016)
Polymerase chain reaction based detection of Grasserie
virus, BmNPV in silkworm,
Bombyx mori
Rashmi P. Joshi and I. A. Raja
Research Laboratory of Zoology. Shri Shivaji College. Akola. 444001
Silkworm, Bombyx mori is a purely domesticated insect since long, which make it a quite delicate venture, easily
susceptible to viral and other diseases. The viral diseases are dif cult to manage due to a very short life cycle of silk-
worm. One of the most effective solutions is a timely detection of such infection so that to stop spread of the disease.
In the present study a polymerase chain reaction (PCR) with a set of speci c primers to the Grasserie virus gene region
was used to diagnose B. mori nucleopolyhedro virus (BmNPV) infection which were made available from Euro ns
Genomics India Pvt Ltd Bangalore. DNA was extracted from the mid gut tissue of experimental 5
instar larvae of
silkworms and ampli ed. After ampli cation the samples were loaded on 1% Agarose gel and electrophoresis was
run at 65 volts. The gel was stained using ethidium bromide and visualized under UV illuminator. Results of PCR
ampli cation helped us to detect Grasserie BmBPV infection.
Since 4,500 years, silkworm, Bombyx mori has become a
purely domesticated insect. Like other domesticated ani-
mals, it is a quite delicate venture easily susceptible to a
number of seasonal diseases, (Govindan et al., 1998 and
Prasad, 1999). Occurrence of seasonal disorders and dis-
eases is a periodic surge in disease incidence, correspond-
ing to seasons or other calendar periods (Rane, 1911).
In tropical countries Grasserie also known as the
hanging disease is one of the most destructive diseases
of silkworms. The causative agent is Borrelina bomby-
cis virus, of the family Baculoviridae. The Baculoviri-
dae comprises only 2 genera nucleorpolyhedorsis virus
(NPVs) and granulovirus (GVs). In this infection the
virus multiplies and forms polyhedra in the nucleus
of infected cells. Infection mainly takes place through
wounds and feeding of polyhedral contaminated mul-
berry leaves. The high temperature, humidity and their
sudden  uctuation, bad ventilation, ineffective disinfec-
tion of rearing house and rearing appliances, starvation
and inadequate larval spaces as well excessive moisture
*Corresponding Author:
Received 10
Dec, 2016
Accepted after revision 20
March, 2017
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Rashmi Joshi and Raja
in the rearing bed affect spreading of the disease. The
majority of baculovirus host are within the order Lepi-
doptera. They have also been isolated from orders Dip-
tera, Hymenoptera, Coleoptera and some crustaceans,
(Hong et al., 2000).
According to Mallika (2006) the Grasserie infected
silkworm show disease symptom during the  nal stage
of larval development and die without cocoon produc-
tion resulting in the waste of expense, time and labour
work therefore accountable for considerable economic
losses in the Indian silk industry. The incidence of Gras-
serie is reported in the silkworm rearing areas of the
entire district of Akola from Vidarbha region of Maha-
rashtra, throughout the year. This infection is dif cult
to cure due to a very short life cycle of silkworm. The
greatest way to manage Grasserie disease is to prevent
disease infection. However, the presumable most effec-
tive solution for the control of Grasserie disease is to
detect viral infection as early as possible in order to
stop spread of the disease in rearing units. Lack of rapid
and accurate disease detection technique causes severe
spread of Grasserie disease seasonally (Mallika, 2006).
Earlier, techniques have been developed to detect this
viral disease such as the enzyme-link immunosorbent
assay (ELISA) (Vanapruk et al., 1992), DNA hybridiza-
tion (Attathom et al., 1994), colloidal textile dye-based
dipstick immunoassay (Nataraju et al., 1994), and west-
ern blot analysis, (Chaeychomsri et al., 1995).
PCR is an extremely sensitive technique which ampli-
es target DNA sequences and PCR ampli cation of
conserved fragment enabled the detection of low level
of viral DNA (Mallika, 2006). It has been employed for
the detection of viral DNA such as human virus (Umlauft
et al., 1996), aminal virus (Peng et al., 1998) and plant
virus (Levesque, 2001). No such detection study so far
has been carried out for Grasserie virus in silkworms
from, Maharasthra. So in the present study we used PCR
technique and polyhedrin gene (polh) to detect early
infection of Grasserie virus (BmNPV) in silkworm Bom-
byx mori. This study will help to prevent the spread of
the Grasserie, and to eradicate this viral disease during
silkworm rearing.
The experimental silkworms were collected from the
local farmers in Akola district and were dissected for
the midgut tissue. The identi cation of diseased worms
infected with Grasserie in the  elds initially was made
on the basis of gross pathology. Initially the skin shows
oily and shining appearance with progress of infection,
skin becomes thin and fragile and the midgut appeared
milky white with inter-segmental swelling (Photo plate-
I).The larvae infected with Grasserie in the rearing cent-
ers were found to be slightly sluggish.
For reliable and distinct PCR product in rapid detec-
tion, a set of speci c primers procured from Euro-
ns Genomics India Bangalore, which is the
cloned nucleotide sequence within BmNPV polyhedrin
Primers – (bp -424 bp)
Forward primer: 5’ AATTCGCAGTGAAACCCG 3’
Reverse primer: 5’ AGAGTCTGTGCCGATGT
3’(Mallika, 2006)
The oligonucleotide sequences of forward primer began
from position 221-240 of polh ORF and reverse primer
began from 616-644 of polh ORF. These primers ampli-
ed a 424bp PCR product.
Using these primers PCR was performed on the basis
of studies by Mallika (2006) and using the prescribed
protocol for DNA extraction (Insect DNA extraction kit
Nucleopore, Genetix ltd.). DNA extracted from the mid-
gut tissue of the non infected healthy and infected 5
instar larvae of silkwormsare ampli ed with primers by
speci c polh BmNPV isolates
PCR Protocol: 1μl DNA sample (~50μl)
• Sterile water : 31μl
• Buffer : 5μl
• MgCl
: 2μl
• Template DNA : 1μl
• Forward primer : 1μl
• Reverse primer : 1μl
• Taq DNA : 1μl
After ampli cation the samples were loaded on 1% Aga-
rose gel and electrophoresis was run at 65 volts. The gel
was then stained with ethidium bromide and visualized
under UV illuminator (Gel Doc Machine).
The speci c pathogens that are dif cult to culture in
vitro or require a long cultivation period present in
the infected silkworms, was diagnosed by PCR. Simi-
lar method was earlier used for detection of Lyman-
tria dispar NPV (LdNPV) on the surface of an egg in
Gypsy moth, by Burand et al., (1992).It was preceded,
with extraction of DNA from experimental silkworms,
PCR ampli cation, followed by detection of amplicons
by visualization. Mid gut tissues of infected silkworm
moths were used to illustrate the Grasserie disease detec-
tion by PCR.
On Visualization the Gel, (Photo plate-II) it is reported
that DNA extracted from Grasserie BmNPV infected
silkworm yielded the ampli cation product of ~424bps
Rashmi Joshi and Raja
PHOTO PLATE-I: Grasserie infected larvae
Rashmi Joshi and Raja
(Lane 1, 2, 3, 4, 5, i.e. BmNPV polh gene con rmed pres-
ence of Grasserie BmNPV infection but not in lane 6
and 7 indicating infection other than grasserie. The lane
8 loaded with DNA extracted from healthy non infected
control larvae no PCR ampli cation product was found.
The PCR product obtained was ~424bps for Grasserie
as expected and were in accordance to that obtained
from the DNA extracted from BmNPV(polh gene), in
Lane M.
As PCR products were speci c to the virus used as the
DNA template therefore no nonspeci c sequences were
observed. Strong intensity of PCR product bands were
clearly visualized on the gel. These studies provide proof
that PCR is a competent tool for detecting virus of Gras-
serie disease in silkworm.
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PHOTO PLATE-II: Gel plate showing PCR ampli ca-
tion of DNA from 5
instar larvae of silkworm infected
with Grasserie causing BmNPV. Lane M DNA Marker
Lane 1 BmNPV detected, Lane 2 BmNPV detected Lane
3 BmNPV detected, Lane 4 BmNPV detected, Lane 5
BmNPV detected, Lane 6 BmNPV not detected,Lane 7
BmNPV not detected, Lane 8 Control healthy - BmNPV