Z. F. Fan

Complete sequence of the genomic RNA of the prevalent strain of a potyvirus infecting maize in China

Summary. The complete nucleotide sequence of the prevalent strain of a potyvirus isolated from maize in Beijing, China was determined and compared with other closely related potyviruses. The viral genome comprises 9595 nucleotides, excluding the poly (A) tail, and encodes a putative polyprotein of 3063 amino acid residues. Sequence comparison of the coat proteins showed that this isolate was most closely related to most other potyviral isolates infecting maize across China with identities of about 99% and thus represented the prevalent strain. It was also closely related to most isolates of Sugarcane mosaic virus (SCMV) infecting maize in Europe with maximum identity of about 95% at the amino acid level. The polyprotein sequence of the Beijing isolate shares identities of 98% with those of two other Chinese maize isolates and shares identity of 69% with Maize dwarf mosaic virus-Bulgarian isolate, respectively. Phylogenetic analyses of the sequences indicated that the Beijing isolate can be tentatively referred to as a prevalent strain of SCMV.

Real-time TaqMan RT-PCR for detection of maize chlorotic mottle virus in maize seeds.

Maize chlorotic mottle virus (MCMV) causes corn lethal necrosis disease, and can be transmitted through infected maize seeds. It remains a challenge to detect this virus in the seeds to prevent its introduction and infection. For this purpose, a real-time TaqMan RT-PCR procedure for efficient detection of MCMV was developed. The sensitivity of the method was 4 fg of total RNA or 25 copies of RNA transcripts, which was approximately ten-fold higher than conventional RT-PCR gel electrophoresis method. The successful detection of MCMV in maize seeds suggested the feasibility of this procedure for routine testing.

Molecular characterization of a distinct potyvirus from whitegrass in China.

Summary. A potyvirus isolated from perennial whitegrass (Pennisetum centrasiaticum Tzvel.) in North China was characterized at the molecular level. The 3′ terminal nucleotide (nt) sequence of 1669 nt of the viral RNA genome has been determined, which covered the coding region of the C-terminal part of the large nuclear inclusion protein (NIb, RNA polymerase), capsid protein (CP) gene and the 3′ nontranslated region (NTR). The CP gene consisted of 909 nt (including the stop codon) encoding 302 amino acid residues, and the 3′ NTR was 241 nt in length excluding the polyadenylated tract. Sequence comparison of the amino acids of CPs showed that this virus was most closely related to Sorghum mosaic virus and Maize dwarf mosaic virus with percent identities of 77% to 78% while that of the 3′ NTRs suggested that it was most closely related to Zea mosaic virus with identity of 72%. This virus isolate was to some extent closely related to other members of the Sugarcane mosaic virus subgroup of potyviruses for the CP amino acid sequences. Phylogenetic analyses of the sequences indicated that this virus isolate represented a distinct potyvirus, and the name Pennisetum mosaic virus (PenMV) is proposed.

Rapid and sensitive detection of Banana bunchy top virus by loop-mediated isothermal amplification

A sensitive loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of Banana bunchy top virus (BBTV) infection. The reaction was performed in a single tube at 63°C for 90 min, with an improved closed-tube detection system by adding the SYBR Green I dye to the inside of the tube lid prior to amplification. The detection limit of the LAMP assay was approximately 1 pg/μl plasmid DNA when mixed with extracted DNA from healthy banana plant, and no cross-reaction with other banana-infected pathogens was observed. Real-time turbidimetry was used to monitor the amplification result in the tubes, and it was shown that this LAMP assay was about 100-fold more sensitive than PCR. The results demonstrated that this LAMP method should be useful for both banana disease monitoring and mass propagation of virus-free banana plantlets.

The genomic sequence of Wisteria vein mosaic virus and its similarities with other potyviruses

Summary.The complete nucleotide sequence of a Beijing isolate of Wisteria vein mosaic virus was determined to be 9695 nucleotides in length excluding the poly(A) tail. Sequence analysis predicted a single large open reading frame of 9279 nucleotides potentially encodes a polyprotein of 3092 amino acids. Phylogenetic analysis based on the genomic and deduced amino acid sequences support the current status of Wisteria vein mosaic virus (WVMV) as a distinct virus of the genus Potyvirus and a member of the Bean common mosaic virus (BCMV) subgroup. Sequence comparisons of WVMV and other members of the BCMV subgroup showed that WVMV is most closely related to both soybean mosaic virus and watermelon mosaic virus.

Molecular variation and recombination in RNA segment 10 of rice black-streaked dwarf virus isolated from China during 2007-2010

Rice black-streaked dwarf virus (RBSDV) is the causal agent of rice black-streaked dwarf and maize rough dwarf diseases in China. The only open reading frame encoding the viral outer capsid protein on S10 RNA of 21 RBSDV isolates was sequenced for phylogenetic and recombination analysis. Four natural recombinants were detected, and the recombinant breakpoints were identified. In addition, the distribution of synonymous and non-synonymous nucleotide changes revealed that the virus had been evolving under purifying selection.

The genomic sequence and biological properties of Pennisetum mosaic virus, a novel monocot-infecting potyvirus

The complete nucleotide sequences of two isolates of Pennisetum mosaic virus (PenMV) were determined. The viral genome comprised 9,611 nucleotides (nt) excluding the 3′-terminal poly(A) sequence, with the capacity of encoding a single polyprotein of 3,065 amino acids. The large open reading frame is flanked by a 172-nt 5′-untranslated region (UTR) and a 244-nt 3′-UTR. Sequence comparisons and phylogenetic analyses of the complete genome and polyproteins suggest that PenMV is closely related to other monocot potyviruses such as Maize dwarf mosaic virus, Sorghum mosaic virus and Sugarcane mosaic virus (SCMV), and thus represents a distinct potyvirus within the SCMV subgroup. The host range of PenMV is limited to Gramineae, and the virus naturally infects maize, sorghum and some wild grasses, causing mosaic symptoms on the leaves. This virus could be transmitted by both mechanical inoculation and by at least four species of aphids.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF APPLE DIMPLE FRUIT VIROID IN CHINA

In a survey of apple virus diseases in China, typical symptoms of Apple dimple fruit viroid (ADFVd) infections were observed. The presence of ADFVd in samples of symptomatic fruits and symptomless leaves from different apple trees was confirmed by RT-PCR, sequencing and dot blot hybridization. Sequence alignment and RNA structure analysis showed that, compared with isolates and variants reported in Italy, there were two nucleotide variations and an alteration of the secondary structure in the ADFVd variants we isolated. Phylogenetic analysis revealed the four variants from China clustered in a group separate from previously reported isolates and variants from Italy and Japan. This is the first report of the identification and molecular characterization of ADFVd in China.

OCCURRENCE OF BEET WESTERN YELLOWS VIRUS IN JAPANESE HOP (HUMULUS SCANDENS) IN CHINA

Beet western yellows virus (BWYV) naturally infects Beta vulgaris, Raphanus sativus, Capsella bursa-pastoris and a number of common weed species. In autumn 2011, Japanese hop [Humulus scandens (Lour.) Merr] plants showing mosaic symptoms on young leaves were observed in the West Campus of the China Agricultural University of Beijing (China). Total RNA was extracted from symptomatic leaves and screened by RT-PCR with degenerate primer pairs to detect poty-, potex-, tobamo-, cucumo-, polero- and tospoviruses. A specific RT-PCR product (1,384 bp) was obtained only with polerovirus primers designed to amplify a fragment including the partial RNA-dependent RNA polymerase (RdRp) gene, an intergenic non coding region and the coat protein (CP) gene (Zhou et al., 2011). Sequence analysis (GenBank accession No. KC210049) confirmed the presence of BWYV in the analysed samples. The CP gene of the BYWV isolate under study had the highest identity with a BWYV isolate from Inner Mongolia (92.1%) (EU636991) and another BWYV isolate from Beijing (90.6%) (ADR74372.1). whereas the partial RdRp sequence had the highest identity with a BWYV isolate from Beijing (98.3%) (EF051249.1). No RT-PCR products were obtained from healthy plants. To our knowledge, this is the first report on the natural occurrence of BWYV in H. scandens. As H. scandens is one of the most common weeds in China, management of BWYV in or near sugar beet fields or other crop hosts should include the removal of this weed.