Molecular Variation And Characterization Of Pennisetum Mosaic Virus

Two Pennisetum mosaic virus isolates (PenMV-B and PenMV-C) from perennial whitegrass (Pennisetum centrasiaticum Tzvel) in North China were characterized at the molecular level. The complete nucleotide (nt) sequences of the two isolates have been determined. The viral genome of both isolates comprised 9611 nts excluding the 3'-terminal poly (A) sequence, potentially encoding a single polyprotein of 3065 amino acids with a calculated Mr of 349,575 Da, flanked by 5'- and 3'-NTRs with 172 and 244 nucleotides respectively. Nine putative cleavage sites were identified by a sequence comparison of this viral polyprotein with those of other potyvimses. The result showed that the putative cleavage site between P3 and 6K.1 for this virus was E/H, which is different from those of any other potyvimses. Several specific motifs were also present in the polyprotein of PenMV. Sequence comparisons of the complete nucleotides and amino acids suggested that the vims was closely related to some monocot-infecting potyvimses such as Sorghum mosaic virus (SrMV), Sugarcane mosaic virus (SCMV) and Maize dwarf mosaic virus (MDMV). Sequence comparison and phylogenetic analysis confirmed our previous report that PenMV is a distinct potyvirus within the SCMV subgroup.The complete sequences of PenMV-B and PenMV-C share identities of 88.5% and 96.8% at the nucleotide and amino acid levels, respectively; However, the nucleotide sequences in the NIb-coding region was only 85.0% identical, which was the lowest level of identity observed in comparison to other regions of two PenMV isolates. The nucleotide sequence alignment showed that PenMV-B, PenMV-C and PenMV-A shared identities of 92.1% to 93.0% in the CP gene and 96.3% to 99.2% in the 3' NTR, respectively; but only shared identities of 85.0% to 88.4% in the NIb region, which is distinct from other potyvimses where the NIb was among the most conserved regions.GenBank/EMBL search using BLAST and phylogenetic analyses were performed separately for the nucleotide sequences of each functionally distinct part of the genome. These sequences of two PenMV isolates and other vimses and isolates grouped inconsistently for each part of the genome in these analyses. The alignment results indicated that the isolates were most closely related to MDMV-BG for the 5'-NTR and ZeMV for the 3'-NTR; however, they were most closely related to SrMV-YH in the P1 gene SCMV-A in the NIa gene, SCMV-SD in the NIb gene and SrMV-YH in the CP gene, though they were more closely related to SrMV-H in the whole genome than to any other vimses and isolates. One could speculate that this was the result of one or more possible recombination events between the ancestors of SCMV, MDMV and SrMV or ZeMV in the distant past. Moreover, a high mutation rate during the evolution of this viral genome could have occurred following various selective pressures.The expression vector pET-PenMV CP containing coat protein (CP) gene of Pennisetum mosaic virus was constructed by cloning PenMV-B CP gene into the plasmid pET22b(+) and being transferred into E. coli BL21(DE3). The results of SDS-PAGE and Western blot showed that the expression of specific fusion protein of about 36 kDa was enhanced by IPTG induction and the protein was highly immunogenic. The specific fusion protein was purified by Ni2+-affinity chromatography and wasinjected into rabbit to raise antiserum. The antiserum titer was 1/8192 by antigen coating plate-ELISA (ACP-ELISA) and has been proved useful for identification of PenMV in plant materials.