A multigene phylogentic analysis of Potato virus Y and its application in strain identification.

The objective of this study is to develop a rapid and accurate multigene phylogenetic analysis to identify Potato virus Y (PVY) strains. The phylogenetic relationships of strains within the PVY species were evaluated with isolate-strain association using five datasets of concatenated sequences from the P1, HC-pro, VPg and CP genes to determine the best dataset for PVY strain identification. Results from phylogenetic analyses and Bayesian tip-association significance (BaTS) tests indicated that the major PVY strains could be distinguished using the P1, VPg and CP concatenated sequences datasets but not the remaining concatenated sequence datasets. Phylogenetic trees reconstructed from the concatenated sequences of P1, VPg and CP genes revealed that the ML and NJ trees had broadly similar topologies and that both were better than the maximum clade credibility tree (MCC). Additionally, the full genome of HLJ26, one isolate randomly selected for the multigene phylogenetic analysis, was clustered with high confidence among members of the PVYNTN-NW (SYR-Ⅱ) strain, which includes isolates of SYR-Ⅱ-2-8, SYR-Ⅱ-Be1 and SYR-Ⅱ-DrH. This suggests that it was a PVYNTN-NW (SYR-Ⅱ) isolate. Recombination analysis of this isolate identified four putative recombination joints in the P1, HC-pro/P3, VPg and the 5'-terminus of CP. This pattern is similar to that observed in the genomic structure of PVYNTN-NW (SYR-I), supporting the classification of this isolate as the PVYNTN-NW strain (SYR-Ⅱ). Simultaneously, two expected fragments of approximately 1 000 and 400 bp in size were also amplified from the isolate by a multiplex RT-PCR, consistent with the expected band pattern of the PVYNTN-NW (SYR-Ⅱ) strain. This further supports the utility of the multigene phylogenetic method in identifying PVY strains. We propose that the major PVY strains could be distinguished accurately using multigene phylogenetic analysis based on the concatenated sequences from the P1, VPg and CP genes.

[Comparative analysis of population genetic structure of Potato virus Y from different hosts].

Nucleotide sequences of P3 and pipo genes of Potato virus Y (PVY) from potato and tobacco were compared to investigate the effect of hosts on the population genetic structure. Meanwhile, mutation, natural selection and gene flow were evaluated to determine evolutionary forces responsible for the population genetic dynamics. The fixation indices of population differentiation (FST) of PVY from tobacco and potato were 0.116 and 0.120, respectively with significant difference, suggesting a moderate genetic differentiation between the two populations. Genetic variation analysis showed that nucleotide identities in P3 and pipo genes among the viral isolates from tobacco were respectively in the range of 85.2%-100% and 76.5%-100% while that from potato were respectively in the range of 95.7%-100% and 93.0%-100%, indicating higher genetic variation in PVY from tobacco than that from potato. Moreover, purifying selection was detected on the majority of polymorphic sites within P3 gene, suggesting that most of mutations in the gene were harmful and consequently being eliminated by natural selection. Conversely, positive selection was detected on two polymorphic sites, suggesting that these two mutations were beneficial to PVY. Neither purifying nor positive selection was detected in pipo gene, indicating neutral evolution of the gene. The values of gene flow (Nm) between PVY populations from tobacco and potato in P3 and pipo genes were 1.91 and 1.83, respectively, suggesting strong gene flow also contributes significantly to the population genetic dynamics of PVY population. In summary, this study indicates there was a significant genetic variation in PVY hosted by tobacco and potato, and mutation, natural selection and gene flow all contribute to the genetic diversity and population dynamic of the virus.

Anti-TMV activity of malformin A1, a cyclic penta-peptide produced by an endophytic fungus Aspergillus tubingensis FJBJ11.

Plant-associated microorganisms are known to produce a variety of metabolites with novel structures and interesting biological activities. An endophytic fungus FJBJ11, isolated from the plant tissue of Brucea javanica (L.) Merr. (Simaroubaceae), was proven to be significantly effective in producing metabolites with anti-Tobacco mosaic virus (TMV) activities. The isolate was identified as Aspergillus tubingensis FJBJ11 based on morphological characteristics and ITS sequence. Bioassay-guided isolation led to the identification of a cycli penta-peptide, malformin A1, along with two cyclic dipeptides, cyclo (Gly-L-Pro) and cyclo (Ala-Leu). Malformin A1 showed potent inhibitory effect against the infection and replication of TMV with IC50 values of 19.7 and 45.4 µg·mL⁻¹, as tested using local lesion assay and leaf-disc method, respectively. The results indicated the potential use of malformin A1 as a leading compound or a promising candidate of new viricide.

[Sequence variation and protein structure of pipo gene in Potato virus Y].

The objectives of this study were to understand the sequence variation and the putative protein structure of pipo gene in the Potato virus Y (PVY) collected from Solanum tuberosum. The pipo gene in PVY was cloned using a pair of degenerate primers designed from its conserved region and its sequences were used to re-construct phylogenetic tree in Potyvirus genera by a Bayesian inference method. An expected fragment of 235 bp was amplified in all 20 samples by RT-PCR and the pipo genes in the 20 samples assayed shared more than 92% nucleotide sequence similarity with the published sequences of PVY strains. Among the 20 pipo gene sequences, 13 polymorphic sites were detected, including 4 parsimony informative sites and 9 singleton variable sites. These results indicate that PVY pipo gene is highly conserved but some sequence variations exist. Further analyses suggest that the pipo gene encodes a hydrophilic protein without signal peptide and transmembrane region. The protein has theoretical isoelectric points (pI) ranging from 11.26 to 11.62 and contains three highly conserved regions, especially between aa 10 and 59. The protein is likely located in the mitochondria and has a-helix secondary structure. Bayesian inference of phylogenetic trees reveals that PVY isolates are clustered in the same branch with high posterior probability, while Sunflower chlorotic mottle virus (SoCMoV) and Pepper severe mosaic virus (PepSMV) are closely related, consisting with the classification of Potyvirus genera using other approaches. Our analyses suggest that the pipo gene can be a new marker for phylogenetic analysis of the genera. The results reported in this paper provide useful insights in the genetic variation and the evolution of PVY and can stimulate further research on structure and function of the PIPO protein.

Complete genome sequence of narcissus late season yellows virus infecting Chinese narcissus in China.

The complete genome sequence of a Chinese narcissus isolate of narcissus late season yellows virus from Zhangzhou, China (NLSYV-ZZ), was determined to be 9,651 nucleotides in length, excluding the 3'-terminal poly (A) tail, by amplification and sequencing of virus RNA. The viral genome contains a single long open reading frame of 9,315 nucleotides encoding a polyprotein of 3,105 amino acids. The polyprotein was predicted to be cleaved into ten mature proteins by three viral proteases. Complete genome sequence comparison and phylogenetic analysis indicated that NLSYV-ZZ was most closely related to narcissus yellow stripe virus (NYSV), which was also isolated from narcissus. These viruses shared 69.9 % identity in their complete nucleotide sequences and 77.0 % identity in their polyprotein amino acid sequences.

Expression of rice gall dwarf virus outer coat protein gene (S8) in insect cells.

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBac™1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.

Genetic diversity and population structure of rice stripe virus in China.

Rice stripe virus (RSV) is one of the most economically important pathogens of rice and is repeatedly epidemic in China, Japan and Korea. The most recent outbreak of RSV in eastern China in 2000 caused significant losses and raised serious concerns. In this paper, we provide a genotyping profile of RSV field isolates and describe the population structure of RSV in China, based on the nucleotide sequences of isolates collected from different geographical regions during 1997-2004. RSV isolates could be divided into two or three subtypes, depending on which gene was analysed. The genetic distances between subtypes range from 0.050 to 0.067. The population from eastern China is composed only of subtype I/IB isolates. In contrast, the population from Yunnan province (southwest China) is composed mainly of subtype II isolates, but also contains a small proportion of subtype I/IB isolates and subtype IA isolates. However, subpopulations collected from different districts in eastern China or Yunnan province are not genetically differentiated and show frequent gene flow. RSV genes were found to be under strong negative selection. Our data suggest that the most recent outbreak of RSV in eastern China was not due to the invasion of new RSV subtype(s). The evolutionary processes contributing to the observed genetic diversity and population structure are discussed.