Biotechnological

Communication

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

ABSTRACT

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.

KEY WORDS:

BOMBYX MORI, GRASSERIE

, NUCLEOPOLYHEDRO VIRUS (BMNPV),

PCR, POLYHEDRIN GENE (POLH).

121

INTRODUCTION

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

ARTICLE INFORMATION:

*Corresponding Author: rashmisawalkar75@gmail.com

Received 10

Dec, 2016

Accepted after revision 20

March, 2017

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122 POLYMERASE CHAIN REACTION BASED DETECTION OF GRASSERIE VIRUS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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.

MATERIAL AND METHODS

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 pvt.ltd Bangalore, which is the

cloned nucleotide sequence within BmNPV polyhedrin

gene.

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).

RESULTS AND DISCUSSION

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

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS POLYMERASE CHAIN REACTION BASED DETECTION OF GRASSERIE VIRUS 123

Rashmi Joshi and Raja

PHOTO PLATE-I: Grasserie infected larvae

124 POLYMERASE CHAIN REACTION BASED DETECTION OF GRASSERIE VIRUS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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

absent