1.11.42
MOLECULAR CHARACTERISATION OF MUSHROOM BACILLIFORM VIRUS (MBV)

MJ KIRBY1, S MUTHUMEENAKSHI2, PR MILLS2 and GD FOSTER1

1School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 lUG, UK; 2Microbial Biotechnology, HRI, Wellesbourne, Warwick CV35 9EF, UK

Background and objectives
A disease of the commercial mushroom Agaricus bisporus attributed to a virus (La France disease) was first described in 1950. A number of virus-like particles were detected in infected cells by electron microscopy, one of them being MBV (a bacilliform virus 19x50 nm [1]). MBV contains a ssRNA genome (4009 nucleotides long) of positive polarity which encodes four major open reading frames (ORFs) [2]. ORF 4 encodes the coat protein of the virus and it is thought that ORF 3 encodes a protein which is, or constitutes a part of, the RNA polymerase [2]. Until recently MBV had only been detected in diseased mushrooms as a co-infection with spherical viruses (LIV); however, MBV has also been found as a single infection in apparently healthy mushrooms [3]. The aim of this research is to determine whether MBV plays a role in La France disease, possibly by attenuating the symptoms associated with LIV. It is also unknown whether infection solely by MBV alters the phenotype of the fungal host making it susceptible to LIV infection.

Results and conclusions
Two UK sources (HRI and Leicester) of Agaricus bisporus were used for virus extraction. A PCR and cloning approach was adopted due to a poor virus titre. Total mushroom RNA was extracted and viral cDNA made and amplified using polymerase chain reaction (PCR) with primers designed to the published sequence of MBV [2]. Both the HRI and Leicester MBV amplification products share about 98% sequence homology with the published MBV (Australian) sequence [2]. PCR primers had been designed to produce a full length cDNA clone from which transcripts can be synthesized under the control of a T7 promoter. RNA produced from these constructs has been electroporated into Agaricus protoplasts (from mushrooms lacking MBV) to investigate by RT-PCR, and by using MBV specific antisera, whether these MBV clones are replication competent. Northern blot results shows the presence of several subgenomic RNAs suggesting that not only the coat protein (ORF4) is encoded in this way. Apart from the full-length MBV clone (ORF1, 2, 3 and 4) we have generated clones containing either ORF1 and 2 or ORF 1, 2 and 3 or ORF4 which will be used in in vitro translation studies to enable us to determine whether the suspected ORFs produce proteins in vitro and in vivo.

References
1. Tavantzis SM, Romaine CP, Smith SH, 1980. Virology 105, 94-102.
2. Revill PA, Davidson AD, Wright PJ, 1994. Virology 202, 904-911.
3. Romaine CP, Schlagnhaufer B, 1995. Applied and Environmental Microbiology 2322-2325.