MOLECULAR AND SEROLOGICAL STUDIES ON TOMATO SPOTTED WILT VIRUS

Abstract

Tomato spotted wilt vifl:IS (TSWV) is one of the most serious pathogens affecting the cultivation of many crops causing serious econonVc losses. A new disease on Physalis peruviana caused by TSWV has been reported in Egypt. TSWV contains a tripartite RNA I

genome. The small RNA segment, S, encodes the nucleoprotein (N) and a nonstructural protein (NSs) in an overlapping reading frame. The nu¢leoprotein gene (N) was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) from total nucleic acid extracts I I ' of TSWV-infected Physalis peruviana, Datura mete/, Nicotiana rustica and Pluchea

' I .discoridis plants. The two primers TSWVl (5'ATGTCTAAGGTTAAGCTC3') and

TSWV2 (5' TTAAGCAAGTTCTGTGAG 3') amplified a PCR product of about 777 bp. . I • The PCR product obtained was subsequently cloned into pGEM-T Easy vector for I sequencing. The nucleotide sequence of N gene of the Egyptian isolate was compared at

both the nucleotide and amino-acid levels with other available TSWV isolates available in I

the gene bank to determine its homology tree. The Egyptian Physalis peruviana isolate of

I TSWV shared a high degree (97-100%) of amino acid sequence identity in the N gene with

other isolates from the gene bank.

The nucleoprotein gene located on the ambisense S RNA segment of tomato spotted wilt virus (TSWV), was cloned and expressed linto pBAD-C terminal 6xHis Tag

TOPO expression vector in Escherichia coli. The 1 I

ambisence S RNA encoded a

nucleoprotein (N) of 250 amino acids of approximately 28 kDa.