Abol-Ela, S.

Fédière, Gilles

Giannotti, J.

Monsarrat, Annie

Taha, A.A.

Zeddam, Jean-Louis

Name not available

A small RNA virus isolated from the maize stem borer Sesamia cretica Led. (Lepidoptera : Noctuidae) in Egypt

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Egypt. J. Biol. P, Cont. 1(2),1991,115 - 119 i 
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A SMALL RNA VIRUS ISOLATED FROM THE MAIZE STEM BORER 
SESAMIA CRETICA LED. (LEPIDOPTERA : NOCTUIDAE) IN EGYPT, 
G. FEDERE, A.A. TAHA, X,LERY, J. GIANNOTTI, J,L. ZEDDAM, A. MONSARRAT 
AND S. ABOL-ELA. 
, Entomovirology laboratory - ORSTOM - Faculty of Agriculture, Cairo University, Giza, 
Egypt. 
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i '  ABSTRACT 
A small RNA virus was isolated from larvae of the maize swm borer Sesamia 
cretica Lederer (Lepidoptcra : Noctuidae) , Some properties of his virus ( Maize 
Stem Borer Virus : MSBV ) have been studied. Elecuon microscopic observations 
of the purified suspention showed the presence of non-enveloppzd isometric viral 
particles, 30 nm in diameter, The viral genome was composed OF R N A ,  the virus 
capsid contained three major proteins ( VPI, VP3, VP4) with molecular weights 
of 60 000,45 O00 and 2S O00 as well as one minor (VP2) with molecular weight 
oc 58'000 daltons , Immunodiffusion tests showed that MSBV s'as serologically 
unrelated to certain inseci Picoma-like viruses. This new virus could be 
provisionally arranged among the unclassified small RNA v ù ~ s e s  of invertebrales 
and represents a great interst as a biological control agent I 
KEY WORDS : Sesumio creticn , Noctuidae RNA virus , Bioconuol agent 
INTRODUCTION 
Com borers are considered among the major pests in Egypt. This group of pests are not 
easily accessible by the methods of chcmical control, hence the real insect suppression requires 
further control measures which have the ability to act in their targst host , Among the major 
com borer in Egypt, Sesamia crerica Lederer, 1857 (Lepidoptera : Noctuidae) is the most 
frequently observed, but two other lepidopterous larvae are serious pests : Ostrìnia nubilalis 
Hubn. and Chilo agamemnon Bles. (Awadallah, 1974) . The maize stem borer S. cretica is a 
polyphagous insect on graminaceous, especially Zea mays , Saccharum oficiarum and 
Sorghum vulagare ( Mostafa, 1981) , 
Control of this pest was limited in the use of chemical insecticides which represent at term 
damage for the environment. Biological control by insect viruses had to be considered . Within 
the genus Sesamia only three viruses have been described for the species S, calamisris 
Hampson (jacquemard et al., 1985), Le. Nodamuravhs, Cytoplasmic Polyhedrosis Virus and 
Nuclear Polyhedrosis Virus, but no viruses were recorded in S. cren'ca. 
The present investigation, aims to characterize a small isomehc virus isolated from dead 
infected larvae of S. crerìca collocted from maize fields . Therefore, physicochemical 
properties, serology and electron microscopy of this virus designated Maize Stem Borer Virus 
(MSBV) were studied in order to provide its classification . 
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ORSTOM Fonds Documentaire 
4 ,  LARVAE STOCKS . .  . 
S. cretica larvae used in this investigation were originated from EI Badrashin in the Nile 
valley and Kafr EI Sheikh in May and June 1990, They were reared in the laboratory on 
semisynthetic media ( Bordat, 1980 ). 
VIRUS STRAIN 
virus was purifid and propagated in laboratory reared larvae infected pzr os, 
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Dead infected larvae were collected from maize fields at El Badrashin in May 1990, The' 
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PURIFICATION OF THE VIRUS 
Extract of insects, either for infectioily assays or for virus characterization, were 
perapred by homogenizing the infected larvae in 0.05 h4 Tris-Buffer ( TB ), pH 7,8, containing 
0.5 & Sodium Dodecyl Sulfate. The extract was squeezcd Lhmugh cheesecloth and the 
emulsion was centrifuged at 8000 8 for 10 minutes, The supcrnatant fluid was kept and the 
pellet was re-extract twice after rc-suspending and sonificating in  TB, The resulting 
supernatants were mixed and the \ h s  was pelleted by cenmifuging at 145000 g for 1 h 30 at 
4OC. The pellet was allowed to resuspcnd overnight in  small \'alum of TB. 
The partially purified virus suspension was deposit4 on a 15 to 35 % ( W/IV ) sucrose 
gradient in TB and cenvifuged for 2 hours at 200000 g. The band containing virus was 
collected and the particales wcrc rc-purified by an additional cycle of cenbifugation an sucrose 
gradicnt. The purified virus particles were then concentrated as above and stored at-30°C, 
ELECTRON MICROSCOPY 
7.4, 
DETERMINATION OF THE CKEhlICAL COhPOSITION OF THX VIRION 
Virus samples were tested for nucleic acid by the colorimetic method of Mejbaum ( 1939 ) 
using orcinol and the diphenylanine reaction ( Giles and Meyers, 1965 ) 
Purificd virus suspention was negatively, stained with 2 % ( WfiV ) uranyl acetate, pH 
SPECTROPHOTOMETRIC ME. AS UREmhTS 
U.V. absorption of purified virus was examined through wave lenghts between 320 and 220 
nm , The average ratio of optical densities at 260 and 280 nm ( maximum absorption of nucleic 
acids and proteins respectively ) was mesured . 
ELECTROPHORESIS OF VIRUS POLYPEPTIDES IN SDS POLYACRYANMIDE 
GELS 
Molecular weight and number of proteins were assessed by comparing their 
electrorphoretic mobilities in  9% polyacrylamide gels (Weber and Osbom ,1969) with those of 
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standard marker proteinse : phosphorylase b (MW : 94000),bovone serum albumine ( MW : 
67000 ) , ovalbumine { h4w : 43000 ) , carbonic anhydsrase (" : 30000),trypsin inhibitor 
(Mw:20 loo). 
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ANTISERA AND SEROLOGICAL TESTS 
Antisera were prepared in rabbits by intraveinal injection of 1 ml antigen ( 500 mg / ml ) 
and intramuscular injection twice at weekly intervals with virus preparation emulsified in 
Freund's complete adjuvant. Gel immunodiffiusian tests were carried out in 1 % agrose using 
0.9 % NaCl ( Outcherlony, 1648 ). The antiserum títer was determined and the highst was 
selected. Reciprocal comparison of MSBV with the Picorna- like virus of Latoiu viridissima 
(fediere et al. , 1990 ) and Trunuca ryfisquamara ( Fedieral et al., 1991 ) and thier antisera 
were realised. 
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RESULTS AND DISCUSSION 
Examination of purified viral suspension by electron microscope revealed large number of 
isomemc particles, 30 nm in diameter ( Fig , 1 ). Uv extinction spectra of the virus showed a 
r: 
N 
-". 5 )  
maximum absorption at 260 nm and a minimum absorption at 240 nm,  which characterize the 
viral nucleoproteins. The average ratio of extinction at 260 nm to that at 250 nm was 1.4 , 
Purified virus particales reacted negatively with diphenylalanine and positively with orcinol, 
which indicated the RNA nature of the viral nuclic acid. Electrophoresis of the viral proteins in 
9 % polyacrylamide gels revealed three major bands with molecular weights of 60 000 ( vP1 ) 
,54 O00 ( VB3 ) and 28 o00 dalrons ( VB4 ) as well as one minor band with molecular weight 
of 58 ax> dlatons ( VB2 ) ( Fig. 2 ). 
Immunodiffusion tests showed that MSBV virions reacted strongly with the homologous 
antiserum titrated by 1/128. Serological companion between MSBV and two Picoma- like 
viruses of Latoia viridissima and Turnaqua r&squamata rcvcaled Lhat these viruses were 
serologically different. Although a large number of small RNA viruses was already isolated 
from insects, only a few number of these viruses were classified among three families : 
Picomaviridae, Nodaviridae and Teraviridae whereas the athcrs bclong ta the group of 
unclassified small RNA viruses of invertebrates ( Mattews, 1982 ), 
Date recorded in the present investigation show that MSBV is a small, nonenvelapped, 
isomemc virus, 30 nm in diameter, Its genome is composed of RNA and its capsid i s  formcd 
of three majors and one minor structural polypeptides, From the physical and chemical 
properties of this RNA virus of. S, cren'ca, it is suggested to arrange i t  among the unclassified 
viruses waiting for a complete characterization. Observations throught experimentally 
infections realised for the production of large amcunt of virus f necessary for its 
characterization ) and the natural epizootics observed in plantation, show that MSBV is highly 
pathogenic and give it a great interest for its use as biologicai control agent against S.cretìca, 
this aspect will be developed in a next investigation. 
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REFERENCES 
AWADALLAH, W.H. 1974. Biological studies on com 
Faculty of Agriculture - Cairo University. 
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borers in Egypt. Ph.D, T h e h  , 
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BORDAT, D. 1980, Sesamiu culamìsris HMPS : une technique pratique d elevage de masse 
sur milieu artificiel. Agr. Trop., 35 ; 35-40. 
FEDIERE G., R. PHILIPPE, J.C, VEYRUNES and P. MONSARRAT. 1990. Biological 
control of oil palm pest Latoia viridissima Holland( Lepidoptera, Limacodidae 
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FEDIERE, G., X. LERY, K.N. KOUASSI, S. HERDER, J,C, VEYRLWES and D, 
MARIAU. 1991 , Etude dun nouveau virus RNA a chez Tui&co 
Rufisquamura ( Lepidoptera Notodontiedae ) defoliateur du palmier a huile en 
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GILES K.W. and A. MEYERS. 1965. An improved diphenylalanine method .for the 
estimation of deoxyribonucleic. acid Nature, 206; 93, 
JACQUEhlARD P., G, CROIZIER, A. AMARGIER, J,C. VEYRUNES, L, CROIZIED, 
D. BORDAT and B. VERCAMBRE, 1985, Presence de vois virus chez 
Sesam'u calamistis Hampson ( Lepidoptere : Agrotidae ) a Iilz de la Reunion. 
MATTHEWS R.E.F. 1982. Classification and nomenclaturc of viruses. Intervuology, 17; 
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MEJBAUM, M,Z. 1939, Ubcr die bestimmung kleiner pcntoscmcngen. insbesonder in 
derivaten der adenylsaure. Z. Physiol. 258 : 1 17-120. 
MOSTAFA F.F. 1981, Biological and ecological studics on the pink borer Sesamia crerica 
Led. (Lepidoptera : Noctuidae ). Ph.D, thesis , Faculty of .4grkulture Cairo 
Universicy Egypt, 
OUCHTEREONY O, 1984. Antigen antibody raection in gels , Ark , Keminer. Geol. B:, 26 ; 
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WEBER K. and M. OSBORN. 1969 . The reliability of molecular weight determination by 
dodceyl sulfate polyacrylamide gel clccuophoreisis. J. B i d  Ckm.,  244 ; 4406- 
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Agr. Trop., 40; 66-7 1. 
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Fig. 2 : Electrophmtic analysis of polypeptides in 9% polyacrylamide-SDS gelq 
Lane A : MSBV 
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A small RNA virus isolated from the maize stem borer Sesamia cretica Led. (Lepidoptera : Noctuidae) in Egypt

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