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Complete nucleotide sequence of strawberry vein banding virus Chinese isolate and infectivity of its full-length DNA clone.

ABSTRACT: Strawberry vein banding virus (SVBV) is a double-stranded DNA plant virus, which has been found in North America, Australia, Brazil, Japan, Europe and several provinces of China. Infected strawberry plants exhibit mild vein-banding symptoms and chlorosis along the veins. It is one of the most economically important diseases in Asiatic, European and North American strawberry-growing areas.The complete genome of an SVBV Chinese isolate (SVBV-CN) was isolated and cloned from a naturally infected strawberry (Fragaria?×?ananassa cv. Sachinoka) sample found in Shenyang city of Liaoning province. Sequence analysis revealed a complete genome of 7864 nucleotides (nts) that indicated SVBV-CN was most closely related to SVBV from the United States (SVBV-US) with a sequence similarity of 85.8 %. Two major clades were identified based on phylogenetic analysis of the complete genome sequences of caulimoviruses. SVBV-CN clustered together with SVBV-US, whereas other caulimoviruses formed a separate branch. Agrobacterium-mediated inoculation of Fragaria vesca with an infectious clone of SVBV-CN results in systemic infection with distinct symptoms of yellowing bands along the main leaf veins. This suggests that the SVBV-CN infectious clone can recapitulate the symptoms observed in naturally infected strawberries, and therefore is likely the causal agent of the original disease observed in strawberries. Furthermore, strawberry plants inoculated with the infectious clone using vacuum infiltration developed symptoms with a very high infection rate of 86-100 % in 4-5 weeks post-inoculation. This compares to an infection rate of 20-40 % in 8-9 weeks post-inoculation using syringe-inoculation.The complete nucleotide sequence of SVBV from a naturally infected strawberry was determined. Agroinfiltration of strawberry plants using an infectious clone of SVBV-CN resulted in symptoms typically found in infected strawberries from Shenyang city of Liaoning province in China. This is the first report describing an infectious clone of SVBV-CN, and that vacuum infiltration can be potentially used as a new and highly efficient means for inoculation of strawberry plants.

SUBMITTER: Feng M

PROVIDER: S-EPMC5052798 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Complete nucleotide sequence of strawberry vein banding virus Chinese isolate and infectivity of its full-length DNA clone.

Feng Mingfeng M Zhang Hanping H Pan Yuan Y Hu Yahui Y Chen Jing J Zuo Dengpan D Jiang Tong T

Virology journal 20161006 1

<h4>Background</h4>Strawberry vein banding virus (SVBV) is a double-stranded DNA plant virus, which has been found in North America, Australia, Brazil, Japan, Europe and several provinces of China. Infected strawberry plants exhibit mild vein-banding symptoms and chlorosis along the veins. It is one of the most economically important diseases in Asiatic, European and North American strawberry-growing areas.<h4>Findings</h4>The complete genome of an SVBV Chinese isolate (SVBV-CN) was isolated and ...[more]

PMID: 27716385

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Project description:Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.

Dataset Information

Complete nucleotide sequence of strawberry vein banding virus Chinese isolate and infectivity of its full-length DNA clone.

Publications

Complete nucleotide sequence of strawberry vein banding virus Chinese isolate and infectivity of its full-length DNA clone.

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