Identification And Molecular Biological Characterization Of Viruses Infecting Water Chestnut

Water chestnut (Eleocharis dulcis) is a nutritious vaule of aquatic vegetables cultivated all over the world, whose yield and quality will be decreased when it infected with viruses. In recent years, high throughput sequencing of small RNA (sRNA) in combination with bioinformatics is an effective method to identify and characterize novel viruses. From year 2013 to 2015, water chestnut samples from Tuanfeng county of Hubei province and Guangxi Academy of Agricultural Sciences, respectively, were investigated to infect with viral diseases. The cladode tissue samples were collected for high-throughput sequencing of sRNAs, bioinformatics analysis, colning and sequencing analysis. The results revealed that one endogenous DNA virus integrated into host water chestnut genome and 2 positive-sense single strand RNA viruses were identified. Further, their molecular characterization were determined based on PCR amplification and sequence analysis. The obtaining results are shown as the followings:1. This is the first report of an endogenous DNA virus (designated as Water Chestnut Soymovirus-1, WCSV-1) belonging to a new species of Soymovirus infecting water chestnut host.The full-length genomic sequence of water chestnut DNA virus was 7,535 nucleotides (nt), and shared 42-52% nucleotide sequence identity with that of members in the Caulimoviridae family. The DNA virus contained nine open reading frames (ORFs) encoding MP, CP, RT, transactivator and another 5 hypothetical proteins, respectively, with conserved domains characteristic of caulimoviruses. Phylogenetic analyses at the full-length genome and polyprotein ami no acid levels indicated that it belonged to the genus Soymovirus. The DNA virus was tentatively designated Water Chestnut Soymovirus-1(WCSV-1). Phylogenetic analyses of the putative proteins encoded by ORF I, IV, V, VI and VII revealed that WCSV-1 was a new member, distinct from other well established species in the Soymovirus genus. The sRNA bioinformatics analyses showed that the majority of the vsRNAs were 22 nt in length with a preference for U at in the 5’-terminal nucleotide position. The vsRNAs were unevenly distributed over both the positive and negative strands of the entire WCSV-1 genome. In addition, different tissue samples of water chestnut collected from different regions of China were all PCR positive for WCSV-1 sequences, Southern blot analysis of genomic DNA extracts isolated from cladode, bulb and root tissue samples of different E.dulcis cultivars from different origins using probes from ORF I and ORF IV, respectively, demonstrated that the WCSV-1 sequence integrated into the host genome.2. Two types of+ssRNA viruses infecting water chestnut (designated as WCV-1 and WCV-2) are identified, whose molecular characterization and taxonomy are further determined.Two positive single-strand RNA viruses from water chestnut, tentatively named as water chestnut virus-1 (WCV-1) and water chestnut virus-2 (WCV-2), respectively, were determined by sRNA sequencing and bioinformatics. The viral genomic sequence was amplified based on the primers designed from virus-derived small RNA (vsRNA) assembled contigs. The WCV-1 partial genome was 12,663 nt in length, with 8 ORFs encoding proteins including Met, Hel, RdRp, HSP70h, HSP90h. Phylogenetic analyses of RdRp and HSP70h at the amino acid (aa) level indicated that WCV-1 shared 30%-39% and 27%-37% sequence identity with those of members belonging to the Closteroviridae family, respectively. The sequence analysis results demonstrated that WCV-1 was a new member and distinct from other well established species in family Closteroviridae.In addition, the WCV-2 partial genomic sequence was 9,269 nt encoding one large ORF with the conserved CPs, NTP-binding protein, Pro and RdRp domains, respectively. Based on pair-wise comparison of aa sequence, WCV-1 shared 55%-57% identify with the members in genus Waikavirus at aa level. The phylogenetic analysis based on Pro-pol aa sequences indicated that WCV-1 was clustered into an independent branch of genus Waikavirus, revealing that it was a new member in genus Waikavirus of Secoviridae family.Furthermore, the viral detection of water chestnut samples from Tuanfeng county of Hubei province and Guangxi Academy of Agricultural Sciences were performed by PCR with primers designed based on sequences of RdRp region of WCV-1 and HSP70h of WCV-2, respectively. The results showed that 1 of 21 samples from Hubei was positive for WCV-1, meanwhile 2 of 10 samples from Guangxi was positive for WCV-1, which of one sample had a mixed infection with WCV-1 and WCV-2. The WCV-1 nucleotide sequence similarity is 98%-99% between infected samples from Hubei and Guangxi province, respectively.Finally, it is the first reports of the obtaining results of the investigation and identification of viruses infection with water chestnut in China, which can provide significant molecular information with the detection and prevention control of water chestnut viral disease.