Molecular and biological properties of two putative new cytorhabdoviruses infecting Trifolium pratense

Three pairs of generic primers designed for specific reverse transcription polymerase chain reaction amplification of part of the cytorhabdoviruses genomes were successfully tested with a number of red clover samples. Two infected plants showing irregular vein clearing, tissue necrosis and stunted growth symptoms were selected for further studies. Subsequently, two novel plant rhabdoviruses, tentatively named Trifolium pratense virus A (TpVA) and Trifolium pratense virus B (TpVB), were detected in red clover plants. Sequence analyses of nucleotide sequences of their genomic RNAs obtained after high-throughput sequencing confirmed their affinity to members of the genus Cytorhabdovirus. While TpVA was transmitted by mechanical inoculation to Nicotiana occidentalis 37B and subtransferred to Physalis floridana plants, crude sap transmission of TpVB to a range of herbaceous host plants failed. The bacilliform shape of virions, which has a cytoplasm-limited distribution, their sizes, and the phylogenetic relationships, support assignment of TpVA and TpVB to two distinct species of the genus Cytorhabdovirus.