Molecular and Biological Characterization of Mycovirus Identified from Environmental Fungal Isolates

Abstract

Mycoviruses ubiquitously infect a wide range of fungal hosts in the world. The current study reports the development of a database for the existing mycoviruses and the characterization of a novel double stranded RNA virus, termed Alternaria alternata botybirnavirus 1 (AaBbV1), infecting a Pakistani strain, 4a, of a phytopathogenic fungus Alternaria alternata that was identified by screening of environmental fungal isolates. A total of 120 fungal isolates were screened for mycoviral infection with the infection percentage of 8.3 %. A combined approach of next generation and conventional terminal end sequencing of the viral genome revealed that the virus is a distinct member of the genus Botybirnavirus. This virus comprised of two segments (dsRNA1 and dsRNA2) of sizes 6127 bp and 5860 bp respectively. The dsRNA1-encoded protein carrying the RNA- dependent RNA polymerase domain showed 61% identity to the counterpart of Botrytis porri botybirnavirus 1 and lower levels of amino acid similarity with those of other putative botybirnaviruses and the fungal dsRNA viruses belonging to other families. The dsRNA2- encoded protein also showed resemblance with corresponding proteins of botybirnaviruses. Electron microscopy showed AaBbV1 to form spherical particles of 40 nm in diameter. Biochemical analyses showed that two structural proteins encoded by dsRNA1 and dsRNA2 underwent processing to some extent during particle purification, resulting in the appearance of multiple smaller products. Phylogenetic analyses of structural proteins suggested that their coding region might have been duplicated once and maintained without recombination. Protoplast fusion technique allowed the introduction of AaBbV1 into a virus free Japanese strain of A. alternata and demonstrated symptomless infection by the virus.