[Construction and characterization of an infectious clone of Soybean mosaic virus isolate from Pinellia ternata].

Soybean mosaic virus (SMV), one of the major viral diseases of Pinellia ternata (Thunb.) Breit., has had a serious impact on its yield and quality. The construction of viral infectious clones is a powerful tool for reverse genetics research on viral gene function and interaction between virus and host. To clarify the molecular mechanism of SMV infection in Pinellia ternata, it is particularly important to construct the SMV full-length cDNA infectious clone. Therefore, the infectious clone of Soybean mosaic virus Shanxi Pinellia ternata isolate (SMV-SXBX) was constructed in this study by Gibson in vitro recombination system, and the healthy Pinellia ternata leaves were inoculated by Agrobacterium infiltration, further through mechanical passage and RT-PCR, confirming that the 3' end of the SMV-SXBX infectious clone had a stable infectivity when it contained 56-nt of poly(A) tail. This method is not only convenient and efficient, but also avoids the instability of SMV infectious clones in Escherichia coli. The construction of SMV full-length infectious cDNA clones laid the foundation for further study on the molecular mechanism of SMV replication and pathogenesis.

Complete genomic sequence of crow-dipper mosaic-associated virus, a novel macluravirus infecting Pinellia ternata.

A new macluravirus infecting Pinellia ternata in China was identified by high-throughput sequencing (HTS) and tentatively named "crow-dipper mosaic-associated virus" (CrdMV). The complete genome sequence of CrdMV was determined by reverse transcription (RT) PCR and rapid amplification of cDNA ends (RACE) PCR. The genomic RNA of CrdMV consists of 8,454 nucleotides (nt), excluding the poly(A) tail at the 3' end. CrdMV has a genomic structure typical of macluraviruses, with large open reading frame encoding a polyprotein of 2,696 amino acids (aa). CrdMV shares 54.40%-59.37% nt sequence identity at the genome sequence level, 48.00%-58.58% aa sequence identity, at the polyprotein sequence level and 37.27%-49.22% aa sequence identity at the CP sequence level with other members of the genus Macluravirus. These values are well below the species demarcation threshold for the family Potyviridae. Phylogenetic analysis based on the amino acid sequences of polyproteins confirmed that CrdMV clusters closely with broad-leafed dock virus A (BDVA, GenBank accession no. KU053507). These results suggest that CrdMV should be considered a distinct member of the genus Macluravirus.

Prokaryotic expression of CP gene of Fritillary virus Y infecting Thunberg fritillary and antiserum preparation.

OBJECTIVE: To prepare antiserum against Fritillary virus Y (FVY) CP for detecting FVY and study serological relationships with other viruses. METHODS: Specific primer was designed according to Genbank (accession: AM039800) to amplify CP gene of FVY infecting Thunberg fritillary. Sequence relationship with other potyviruses was made by Blast. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys E strain. The object protein was purified by 12% SDS-PAGE firstly and subsequently 5% - 20% gradient SDS-PAGE. The antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis. The reactivity of the antiserum produced to FVY CP was tested by Western blot against the over-expressed coat proteins of 17 potyviruses. The ability to combine with nature FVY particles was confirmed by ELISA analysis. RESULTS: It shared 81.2% nucleotide acids identities with TrVY (Tricyrtis virus Y, AY 864850) CP gene, 68.1% with SMV-P (Soybean mosaic virus Pinellia strain, AJ507388. 2) CP gene and 67.2% with ZYMV (Zucchini yellow mosaic virus Luan isolate) CP gene. The prepared antiserum was special to FVY CP, also reacted moderately to the expressed CP of SMV-P (Soybean mosaic virus Pinellia strain) and weakly to that of ZYMV (Zucchini yellow mosaic virus Luan isolate). CONCLUSION: The antibody could combine to nature FVY particles and the antiserum is suitable for FVY detection by ELISA in large scale.

Molecular characterization and evolutionary analysis of soybean mosaic virus infecting Pinellia ternata in China.

Twenty-nine Pinellia ternata specimens were collected from representative areas in China, including the major production provinces of Zhejiang, Henan, Shanxi, Hunan, Shandong and Hubei. Seven isolates related to soybean mosaic virus (SMV), which could be pathogenic on P. ternata and some soybean [Glycine max (L.) Merr.] cultivars, were detected using double antibody sandwich immunosorbent assay (DAS-ELISA) and RT-PCR amplification performed with degenerate primer of potyviruses. It is revealed that the common potyvirus infecting P. ternata is, indeed, only SMVs rather than Dasheen mosaic virus (DsMV) as previously reported. Further molecular phylogenetic analysis of the coat protein (CP) genes of these SMV isolates from P. ternata and G. max, along with some other potyvirus members, such as DsMV and Watermelon mosaic virus (WMV) reconstructed the evolutionary route on both nucleotide and amino acid levels. Similarity and homology of nucleotide sequences for SMV CP genes demonstrated high host correlation and low partial habitat correlation, while those of amino acid sequences also showed that the host correlation was more notable than the habitat correlation. The amino acid sequence of conserved region within CP determines the main function, which shows high homology between species. This study outspreaded from the viruses themselves and their relationship to the infected hosts and revealed the evolutionary strategies, especially the rapid variation or recombination of SMV of P. ternata, in order to adapt itself naturally to the special host.

[Detection of two viruses infecting Pinellia ternata in China].

OBJECTIVE: To study viruses infecting Pinellia ternata in China. METHOD: Symptom observation, DAS-ELISA and RT-PCR detection were applied. RESULT AND CONCLUSION: During a survey in early spring, SMV and CMV were both commonly distributed as main viruses infecting P. ternata collected from different areas in China. But DsMV was the virus which infected P. ternate in natural condition. The infection ratio of cultivated P. ternate by SMV and CMV were 71.4% and 14.3% respectively for 21 samples collected from Ningbo, Zhejiang province; 100% and 44.4% for 18 samples from Xiaoshan, Zhejiang province; 61.9% and 33.3% for 21 samples from Hebei province; 50.0% and 41.7% for 12 samples from Anhui province; 16.7% and 16.7% for 12 samples from Sichuan province; 31.3% and none for 16 samples from Beijing. And the infection ratio of 25 wild samples from different areas of China infected by SMV and CMV were both 20.0%.

The '6K1' protein of a strain of Soybean mosaic virus localizes to the cell periphery.

The '6K1' protein of the Pinellia isolate of Soybean mosaic virus was cloned into a prokaryotic expression vector and a polyclonal antiserum raised to the expressed fusion protein. In immunogold labeling of thin sections of infected leaves of Pinellia ternata, specific labeling occurred at the cell periphery. This might suggest that the potyvirus '6K1' protein plays some role in viral cell-to-cell movement but the lack of transmembrane domains suggests that it does not conform to currently-recognized patterns of viral movement proteins.

A virus related to soybean mosaic virus from Pinellia ternata in China and its comparison with local soybean SMV isolates.

A potyvirus isolated from Pinellia ternata in China was characterised and shown to be related to Soybean mosaic virus (SMV). The virus was pathogenic on P. ternata and some soybean cultivars, whereas the local soybean SMV isolate HH5 did not infect P. ternata. Western blot experiments demonstrated a serological relationship between the virus from Pinellia, SMV and Watermelon mosaic virus (WMV). The complete nucleotide sequences of the Pinellia virus (isolate P-1, 9735 nt) and of the Chinese soybean SMV isolates HH5 (9585 nt) and HZ (9588 nt) were determined. A 1733 nt sequence at the 3'-terminus of a second isolate from Pinellia (isolate P-2) was also determined. The predicted polyprotein of isolate P-1 has 83% amino acid (aa) identity with those of published SMV sequences. In many parts of the genome, aa identity was about 90% but it was much lower in the P1 protein region (24-29%), where it more closely resembled Dasheen mosaic virus (62%). The partial sequence of isolate P-2 had 91% nt identity to P-1 and both isolates resembled a recent sequence in the public databases (AF469171) wrongly named Zantedeschia mosaic virus. The two complete SMV soybean sequences had 93-95% nt identity with those of the previously sequenced isolates and >97% amino acid identity. Phylogenetic analysis and comparisons of coat proteins suggest that the Pinellia, WMV and SMV potyviruses should probably be treated as strains of the same species.