Rice tungro bacilliform virus (RTBV) belongs to genus Tungrovirus within the family Caulimoviridae harbors circular double-stranded DNA (dsDNA). Rice tungro disease (RTD) caused by RTBV, responsible for severe rice yield losses in South and Southeast Asia. Here, we performed a systematic evolutionary and codon usage bias (CUB) analysis of RTBV genome sequences. We analysed different bioinformatics techniques to calculate the nucleotide compositions, the relative synonymous codon usage (RSCU), and other indices. The results indicated slightly or low codon usage bias in RTBV isolates. Mutation and natural selection pressures have equally contributed to this low codon usage bias. Additionally, multiple factors such as host, geographical distribution also affect codon usage patterns in RTBV genomes. RSCU analysis revealed that RTBV shows mutation bias and prefers A and U ended codons to code amino acids. Codon usage patterns of RTBV were also found to be influenced by its host. This indicates that RTBV have evolved codon usage patterns that are specific to its host. The findings from this study are expected to increase our understanding of factors leading to viral evolution and fitness with respect to hosts and the environment.
The DNA genomes of isolates of rice tungro bacilliform virus from Bangladesh, India, Indonesia, Malaysia and Thailand were cloned and compared with that of the type isolate from the Philippines. Restriction endonuclease maps revealed differences between the isolates and cross-hybridization showed that they fell into two groups, those from the Indian subcontinent and those from south-east Asian countries. The genomes of isolates from the Indian subcontinent contained a deletion of 64 bp when compared with those from south-east Asia. The implications of this variation are discussed.
Multivariate analyses were performed using 13 morphological traits and 13 molecular markers (10 SSRs and three ISSRs) to assess the phylogenetic relationship among tungro resistant genotypes. For morphological traits, the genotypes were grouped into six clusters, according to D(2) statistic and Canonical vector analysis. Plant height, days to flowering, days to maturity, panicle length, number of spikelet per panicle, number of unfilled grain per panicle and yield were important contributors to genetic divergence in 14 rice genotypes. Based on Nei's genetic distance for molecular studies, seven clusters were formed among the tungro resistant and susceptible genotypes. Mantel's test revealed a significant correlation (r = 0.834*) between the morphological and molecular data. To develop high yielding tungro resistant varieties based on both morphological and molecular analyses, crosses could be made with susceptible (BR10 and BR11) genotypes with low yielding but highly resistant genotypes, Sonahidemota, Kumragoir, Nakuchimota, Khaiyamota, Khairymota and Kachamota. The chi-square analysis for seven alleles (RM11, RM17, RM20, RM23, RM80, RM108 and RM531) of SSR and five loci (RY1, MR1, MR2, MR4 and GF5) of three ISSR markers in F2 population of cross, BR11×Sonahidemota, showed a good fit to the expected segregation ratio (1:2:1) for a single gene model.
Coat protein genes CP1, CP2 and CP3 of an isolate (MaP1) of rice tungro spherical virus (RTSV) from Malaysia were isolated, cloned and sequenced. Comparative analysis indicated that MaP1 isolate is closely related to the Philippine isolate.
Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and a member of the Caulimoviridae family and closely related to viruses in the Badnavirus genus. The coat protein of RTBV is part of the large polyprotein encoded by open reading frame 3 (ORF3). ORF3 of an RTBV isolate from Malaysia was sequenced (accession no. AF076470) and compared with published sequences for the region that encodes the coat protein or proteins. Molecular mass of virion proteins was determined by mass spectrometry (matrix-assisted laser desorption/ionization-TOF) performed on purified virus particles from three RTBV isolates from Malaysia. The N- and C-terminal amino acid sequences of the coat protein were deduced from the mass spectral analysis, leading to the conclusion that purified virions contain a single coat protein of 37 kDa. The location of the coat protein domain in ORF3 was reinforced as a result of immunodetection reactions using antibodies raised against six different segments of ORF3 using Western immunoblots after SDS-PAGE and isoelectrofocusing of proteins purified from RTBV particles. These studies demonstrate that RTBV coat protein is released from the polyprotein as a single coat protein of 37 kDa.
The DNA of three biological variants, G1, Ic and G2, which originated from the same greenhouse isolate of rice tungro bacilliform virus (RTBV) at the International Rice Research Institute (IRRI), was cloned and sequenced. Comparison of the sequences revealed small differences in genome sizes. The variants were between 95 and 99% identical at the nucleotide and amino acid levels. Alignment of the three genome sequences with those of three published RTBV sequences (Phi-1, Phi-2 and Phi-3) revealed numerous nucleotide substitutions and some insertions and deletions. The published RTBV sequences originated from the same greenhouse isolate at IRRI 20, 11 and 9 years ago. All open reading frames (ORFs) and known functional domains were conserved across the six variants. The cysteine-rich region of ORF3 showed the greatest variation. When the six DNA sequences from IRRI were compared with that of an isolate from Malaysia (Serdang), similar changes were observed in the cysteine-rich region in addition to other nucleotide substitutions and deletions across the genome. The aligned nucleotide sequences of the IRRI variants and Serdang were used to analyse phylogenetic relationships by the bootstrapped parsimony, distance and maximum-likelihood methods. The isolates clustered in three groups: Serdang alone; Ic and G1; and Phi-1, Phi-2, Phi-3 and G2. The distribution of phylogenetically informative residues in the IRRI sequences shared with the Serdang sequence and the differing tree topologies for segments of the genome suggested that recombination, as well as substitutions and insertions or deletions, has played a role in the evolution of RTBV variants. The significance and implications of these evolutionary forces are discussed in comparison with badnaviruses and caulimoviruses.
Rice tungro spherical virus (RTSV) has an RNA genome of more than 12 kb with various features which classify it as a plant picornavirus. The capsid comprises three coat protein (CP) species, CP1, CP2 and CP3, with predicted molecular masses of 22.5, 22.0 and 33 kDa, respectively, which are cleaved from a polyprotein. In order to obtain information on the properties of these proteins, each was expressed in E. coli, purified as a fusion to the maltose-binding protein and used for raising a polyclonal antiserum. CP1, CP2 and CP3 with the expected molecular masses were detected specifically in virus preparations. CP3 is probably the major antigenic determinant on the surface of RTSV particles, as was shown by ELISA, Western blotting and immunogold electron microscopy using antisera obtained against whole virus particles and to each CP separately. In some cases, especially in crude extracts, CP3 antiserum detected several other proteins (40-42 kDa), which could be products of CP3 post-translational modification. No serological differences were detected between the three CPs from isolates from the Philippines, Thailand, Malaysia and India. The CP3-related 40-42 kDa proteins of the Indian RTSV isolate have a slightly higher electrophoretic mobility (42-44 kDa) and a different response to cellulolytic enzyme preparations, which allows them to be differentiated from south-east Asian isolates.
Rice tungro disease (RTD) is one of the most destructive diseases of rice in South and Southeast Asia. RTD is routinely detected based on visual observation of the plant. However, it is not always easy to identify the disease in the field as it is often confused with other diseases or physiological disorders. Here we report the development of two serological based assays for ease of detection of RTD. In this study we had developed and optimized an indirect ELISA and dot-blot assay for detection of RTD. The efficiency of both assays was evaluated by comparing the specificity and sensitivity of the assays to PCR assay using established primer sets. The indirect ELISA showed 97.5% and 96.6%, while the dot-blot assay showed 97.5% and 86.4% sensitivity and specificity, respectively, when compared to established PCR method. The high sensitivity and specificity of the two assays merit the use of both assays as alternative methods to diagnose RTD. Furthermore, the dot-blot assay is a simple, robust, and rapid diagnostic assay that is suitable for field test for it does not require any specialized equipment. This is a great advantage for diagnosing RTD in paddy fields, especially in the rural areas.
Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes.