Genome organization and phylogenetic relationship of <Emphasis Type="Italic">Pineapple mealybug wilt associated virus-3</Emphasis> with family Closteroviridae members

The nucleotide sequence of Pineapple mealybug wilt associated virus-3 (PMWaV-3) (Closteroviridae: Ampelovirus), spanning seven open reading frames (ORFs) and the untranslatable region of the 3′ end was determined. Based on the amino acid identities with orthologous ORFs of PMWaV-1 (54%–73%) and PMWaV-2 (13%–35%), we propose PMWaV-3 is a new species in the PMWaV complex. PMWaV-3 lacks an intergenic region between ORF1b and ORF2, encodes a relatively small, 28.8 kDa, coat protein, and lacks a coat protein duplicate. Phylogenetic analyses were used to analyze seven different domains and ORFs from members of the family Closteroviridae. Two distinct clades within the recognized genus Ampelovirus were observed; one that includes PMWaV-3 and PMWaV-1 and several GLRaVs and another that includes PMWaV-2 and GLRaV-3, the type member of the genus Ampelovirus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular characterization of polish blueberry red ringspot virus isolate

In this study, we determined the complete sequence of the genomic DNA of a Polish isolate of Blueberry red ringspot virus (BRRSV24) and compared it with a Czech (Darrow 5), and the US isolates of the virus and those of other Caulimoviridae family. The genomic DNA of BRRSV24 consists of 8,265 nucleotides and encodes eight open reading frames (ORFs). The sequence homologies of the eight ORFs of BRRSV24 were from 95 to 98% in respect of Darrow 5 and from 91 to 98% in respect of the US isolates at the amino acid level. This high level of amino acid sequence identity within the coding regions among the Czech, the US and Polish BRRSV isolates is suggestive of their common origin.     

Complete genomic sequences of <Emphasis Type="Italic">Tomato yellow leaf curl Mali virus</Emphasis> isolates infecting tomato and pepper from the North Province of Cameroon

Complete genomic sequences of Tomato yellow leaf curl Mali virus isolates infecting tomato and pepper from the North Province of Cameroon. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation,characterization and genome sequence of a birnavirus strain from flounder <Emphasis Type="Italic">Paralichthys </Emphasis><Emphasis Type="Italic">olivaceus</Emphasis> in China

A birnavirus strain, Paralichthys olivaceus birnavirus (POBV), was isolated and characterized from cultured flounder in China, and its complete genomic sequence was subsequently determined. The virus could induce cytopathic effects (CPE) in four of seven fish cell lines and was resistant to chloroform, 5-iodo-2′-deoxyuridine, acid and alkaline pH, and heat treatment. Purified virus particles had a typical icosahedral shape, with a diameter of approximately 55–60 nm. The genomic segments A and B of POBV were 3,091 and 2,780 bp in length and shared many of the features of the members of the family Birnaviridae. Segment A contained two partially overlapping ORFs encoding a polyprotein, pVP2-VP4-VP3, and a nonstructural protein, VP5, while segment B had only one ORF encoding for the VP1, a viral RNA-dependent RNA polymerase (RdRp). This is the first report about a birnavirus strain from a new non-salmonid host in China and its complete genome sequence. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The complete genome sequences of POBV reported here have been deposited in the GenBank database under accession no EU161285 and EU161286, respectively.     

Genome characterization and genetic diversity of beet curly top Iran virus: a geminivirus with a novel nonanucleotide

Beet curly top Iran virus (BCTIV) was previously reported as a distinct curtovirus in Iran. Complete nucleotide sequences of three BCTIV isolates, one each from central, southern, and south eastern Iran were determined to be 2844, 2844, and 2845 nt long, respectively. BCTIV shared highest nucleotide sequence identity (52.3%) with Spinach curly top virus (SpCTV) and lowest identity (46.6%) with Horseradish curly top virus (HrCTV). The BCTIV genome comprises three virion-sense (V1, V2, and V3) and two complementary-sense (C1 and C2) ORFs. ORFs C3 and C4 were not found in BCTIV genome. Based on a comparison of nucleotide sequence identity of individual genes, the three virion-sense ORFs were 72.7–79.9% related to the corresponding ORFs of curtoviruses, whereas no significant relationship was found between the C1 and C2 ORFs of BCTIV and curtoviruses. These two ORFs, however, were only distantly related with those of mastreviruses. Similar to the latter viruses, the BCTIV genome comprises two intergenic regions. The BCTIV large intergenic region included a sequence capable of forming a stem loop structure and a novel nonanucleotide (TAAGATT/CC) with a unique nick site. Phylogenetic analysis using deduced amino acid sequence of individual ORFs revealed that the V2 and V3 ORFs are monophyletic and the V1 ORF is classified with the related ORF of curtoviruses. Whereas the two complementary-sense ORFs are grouped with those of mastreviruses. Computer-based prediction suggested that BCTIV has a chimeric genome which may have arisen by a recombination event involving curto- and mastrevirus ancestors. Percent nucleotide sequence identities of the coat protein gene of ten isolates of BCTIV, collected from a wide range of geographical regions in Iran, varied from 87.1 to 99.9, with the isolates being distributed between two subgroups. Based on biological and molecular properties, BCTIV is proposed as a new member of the genus Curtovirus.     

The complete genome sequences for a novel enterovirus type,enterovirus 96, reflect multiple recombinations

Enterovirus 96 (EV-96) is a recently described genotype in the species Human enterovirus C. So far, only partial genome sequences of this enterovirus type have been available. In this study, we report complete genome sequences for two EV-96 strains isolated from healthy children during enterovirus surveillance in Finland. Sequence analysis revealed substantial nucleotide divergence between EV-96 strains and suggested several recombination events between EV-96 and other HEV-C types. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers FJ751914 and FJ751915.     

Complete genomic RNA sequence of the Polish <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> isolate belonging to the US2 strain

We have determined the complete nucleotide and amino acid sequences of the Polish Pepino mosaic virus (PepMV) isolate marked as PepMV-PK. The PepMV-PK genome consists of a single positive-sense RNA strand of 6412-nucleotide-long that contains five open reading frames (ORFs). ORF1 encodes the putative viral polymerase (RdRp), ORFs 2–4 the triple gene block (TGB 1–3), and ORF5-coat protein CP. Two short untranslated regions flank the coding ones and there is a poly (A) tail at the 3′ end of the genomic RNA. Thus, the genome organization of PepMV-PK is that of a typical member of the genus Potexvirus. Phylogenetic analysis based on full-length genomes of PepMV sequences showed that PepMV-PK was most closely related to the Ch2 isolate from Chile. Comparison of PepMV-PK and Ch2 showed the following nucleotide identities: 98% for the RdRp, 99% for the CP genes, and 98, 99, and 98% for the TGB1, TGB2, and TBG3, respectively. This high level of nucleotide sequence identity between the Chilean and Polish PepMV-PK isolates suggest their common origin.     

Genetic basis of the <Emphasis Type="Italic">ovc</Emphasis> phenotype of <Emphasis Type="Italic">Neurospora:</Emphasis> identification and analysis of a 77 kb deletion

The ovc mutant of Neurospora crassa accumulates more carotenoids than the wild type in the light, is sensitive to high osmotic pressure and exhibits an altered aerial development. The three traits are complemented by a single gene, cut-1, but only the two latter are exhibited by a mutant of this gene carrying a premature stop mutation. Targeted cut-1 deletion results in a normal carotenoid content, confirming the involvement of at least a second gene in the carotenoid-overproducing phenotype of the ovc strain. Molecular analysis of ovc genomic DNA indicates the absence of a large DNA segment affecting the gene cut-1. A PCR walking approach allowed the identification of a deletion extending along 77,078 bp on linkage group IV. The break-points are located in ApA/TpT sequences, suggesting the involvement of UV-induced thymine dimers in the origin of the deletion. The ovc mutant lacks 21 predicted ORFs, including cut-1 as the only known genetic marker, and four ORFs from a 22-member transmethylase gene family. Ten ORFs have no similarity with any predicted gene from other species. Three of them are closely related by sequence and linkage, evoking ancestral gene duplications.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Complete genomic sequence analyses of Apple stem pitting virus isolates from China

The complete genomic sequences of a Chinese ASPV isolates KL1 and KL9 were determined from ten overlapping cDNA clones. The genomes of both isolates were 9265 nucleotides excluding the poly (A) tail and contained five open reading frames (ORFs). The identities between two complete genomes were 92.5% at nt level. Multiple alignment of the amino acid sequences showed that 110 aa variations between two genomic sequences and the variable domains mainly distributed in 5′-terminal of ORF1, ORF3, and ORF5, respectively. Two complete genomic sequences shared 71.4–77.3% identities with other ASPV isolates at nt level. Phylogenetic relationship analysis of the coat protein genes revealed that ASPV isolates had high variables and formed three groups. All ASPV isolates from apples were clustered to group I, whereas pear were clustered to groups II (except NC_003462) and both KL1 and KL9 were clustered to group III. Nucleotide sequences diversity analysis showed that the between-population d _NS /d _S ratio 0.092 was similar to these for within-group (0.092–0.095); there was no geographic differentiation between ASPV isolates.     

Molecular cloning and nucleotide sequence of the coat protein gene of a Cuban isolate of potato leafroll virus and its expression inEscherichia coli

Total RNA from infectedPhysalis floridana was isolated to generate complementary DNA corresponding to the coat protein (CP) gene of a Cuban isolate of potato leaf roll virus (PLRV). This cDNA was amplified by the polymerase chain reaction (PCR) and cloned into the bacterial expression vectors pEX(1–3) for fusion protein expression inE. coli. The product was detected by antibodies specific for the PLRV CP. The coding sequence of the CP gene was determined, and the predicted length of the CP was 208 amino acids (23 kD). The nucleotide sequences and deduced amino acid sequences were compared with the other PLRV isolates and found to be 97–99.5% identical at both the nucleotide and amino acid sequence level of other isolates. Comparison of the deduced amino acid sequences of the PLRV_cub CP revealed considerable homology to other luteoviruses. We believe that the protocol described could be applicable to other plant viruses of low abundance or of cumbersome isolation, since this method is less time consuming than the traditional methods of cloning coat protein genes of plant viruses with known sequences.     

Genetic analysis of Iranian population of Potato leafroll virus based on ORF0

Potato leafroll virus (PLRV) is a destructive virus of potatoes and responsible for high yield losses wherever potatoes are grown. In this study, DNA fragments containing ORF0 from each of nine PLRV isolates was sequenced. Sequence analysis data using 36 isolates from 12 different countries including 14 Iranian isolates showed that the identities of ORF0 at both nucleotide and amino acid levels between the Iranian isolates were 96?C100?% and these isolates were more similar to the European PLRV isolates than to the other isolates. Furthermore, phylogenetic and population genetic analysis were carried out on the basis of full-length ORF0 and overlapping and non-overlapping regions of ORF0 and ORF1 (ORF0/1) which revealed that PLRV isolates were not geographically resolved. Also, we identified negative selection with different ratios for each of the mentioned genomic regions suggesting effects of F-box motif and -1 frameshift on ORF0 non-overlapping region and ORF0/1 in the selection pressure, respectively. Five recombination events were detected in the Iranian, Australian, and European isolates suggesting an important role for this phenomenon in influencing genetic diversity within this virus population.     

Siegesbeckia yellow vein virus is a distinct begomovirus associated with a satellite DNA molecule

Summary Leaf samples of Siegesbeckia glabrescens showing yellow vein, enation, and stunting symptoms were collected in Guangdong province, China. A specific 500-bp product was consistently detected in total DNA extracts, amplified with universal primers specific for members of the genus Begomovirus. Comparison of partial DNA sequences revealed that these virus isolates were identical, and therefore isolates GD13, GD24 and GD27 were selected for further sequence analysis. The complete nucleotide sequences of GD13, GD24 and GD27 were all found to be 2768 nucleotides (nts) long, with two open reading frames (ORFs) in the virion-sense strand and four ORFs in the complementary-sense strand, typical of the Old World begomoviruses. Sequence identities among the three isolates ranged from 99.7 to 99.8%. When compared with other reported sequences of begomoviruses, GD13 was most closely related to papaya leaf curl China virus (AJ876548), with a sequence identity of 76.8%. In addition, all isolates were found to be associated with DNAβ molecules. The complete DNAβ sequences of isolates GD13, GD24 and GD27 were determined. Sequence analysis showed that they had highest sequence identity with DNAβ of Eupatorium yellow vein virus (AJ438938) (44.0, 43.9 and 45.6% identity). GD13, GD24 and GD27 are considered to be isolates of a distinct begomovirus species for which the name Siegesbeckia yellow vein virus (SgYVV) is proposed.     

Nucleotide sequence analysis of the ORF0 region of Potato leafroll virus isolates from Tunisia

Potato leafroll virus (PLRV) isolates from potato plants from different regions of Tunisia were investigated for the ORF0 variable region of genomic RNA using PCR, nucleotide sequencing and sequence analysis. Based on the ORF0 variable region nucleotide sequence, individual Tunisian isolates were more homologous as a group compared to the isolates originating from other parts of the world. There was no correlation between bioclimatic origin of the isolates and their ORF0 sequence. Alignment of the deduced amino acid sequences showed that the P0 protein was not much conserved. Unexpectedly, Tunisian isolates were found to be most homologous to Peruvian ones both at nucleotide and amino acid level. A phylogenetic tree, based on the P0 amino acid sequence, showed that all the PLRV isolates were located in two major clusters regardless of their geographic origin. In the second cluster, three sub-clusters could be distinguished. These results provide valuable information for molecular characterization of the PLRV isolates occurring in Tunisia.     

Transgenic expression of coat protein gene of <Emphasis Type="Italic">Rice tungro bacilliform virus</Emphasis> in rice reduces the accumulation of viral DNA in inoculated plants

Rice tungro, a devastating disease of rice in south and southeast Asia, is caused by the joint infection of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). In order to obtain transgenic resistance against RTBV, indica rice cultivar Pusa Basmati-1 was transformed to express the coat protein (CP) gene of an Indian isolate of RTBV. Rice plants containing the transgene integrated in low copy numbers were obtained, in which the CP was shown to accumulate in the leaf tissue. The progenies representing three independent transformation events were challenged with Indian isolates of RTBV using viruliferous Green leafhoppers, and the viral titers in the inoculated plants were monitored using DNA dot-blot hybridization. As compared to non-transgenic controls, two independent transgenic lines showed significantly low levels of RTBV DNA, especially towards later stages of infection and a concomitant reduction of tungro symptoms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Complete nucleotide sequences of the genomes of two isolates of apple chlorotic leaf spot virus from peach (Prunus persica) in China

The complete nucleotide sequences of two isolates of apple chlorotic leaf spot virus (Z1 and Z3) collected from peach in Henan Province, China, were determined. The genomes of both Z1 and Z3 were found to contain three open reading frames (ORFs). Sequence analysis showed that genomic sequences of Z1 and Z3 isolates shared 67.4%-82.9% and 67.2%-82.6% identity, respectively, with the other eight isolates of ACLSV that have been reported previously. Based on the putative amino acid sequences of the products of the three ORFs, Z1 and Z3 isolates showed the greatest identity to isolate PBM1 (GenBank accession number AJ243438) from plum and the least identity with isolate Ta Tao5 (GenBank Accession Number: EU223295) from peach. Considering the low level of sequence identity between Z1/Z3 isolate and Ta Tao5 isolate, two types of ACLSV may exist in peach.     

Complete nucleotide sequences of two isolates of cherry green ring mottle virus from peach (Prunus persica) in China

Two complete nucleotide sequences of cherry green ring mottle virus (CGRMV) isolated from peach in Hebei (Hs10) and Fujian (F9) Provinces, China, were determined. Five open reading frames (ORFs) were found in the genomes of both isolates. The F9 and Hs10 isolates shared 82.2 % and 83.4-94.4 % nucleotide sequence identity, respectively, with two CGRMV isolates from cherry. Analysis of the nucleotide and amino acid sequences from the five ORFs of both isolates showed that Hs10 shares the greatest sequence identity with P1A (GenBank AJ291761) from cherry. Phylogenetic analysis indicated that CGRMV isolates from peach and cherry are closely related to members of the genus Foveavirus.     

Molecular variation of hop mosaic virus isolates

Hop mosaic virus (HpMV), a member of the genus Carlavirus, is importance to hop production worldwide. We identified variation in nucleic and amino acid sequences among 23 HpMV isolates from Australia, the USA, the Czech Republic, South Africa and Japan using a 1,455-bp fragment covering the 3′ end of the virus genome including ORFs 4, 5 and 6. Three clusters of two or more isolates were identified in phylogenies of the total nucleotide sequence and the coat protein (ORF5) amino acid sequence. Two of these clusters combined in analyses of ORF4 and ORF6 amino acid sequences. Isolates from within and outside of Australia were found in each cluster, indicating that sequence variation was not associated with geographic source. Monitoring of HpMV variants in the field and evaluation of the impact of variants on vector association, rate of spread, and hop yield and quality can now be undertaken.     

Molecular characterization of sweet potato leaf curl virus isolate from China (SPLCV-CN) and its phylogenetic relationship with other members of the Geminiviridae

A Sweet potato-infecting sweet potato leaf curl virus (SPLCV) isolated in China was detected by Polymerase Chain Reaction (PCR). PCR products amplified from DNA-A were cloned and sequenced. The isolates of SPLCV from China(SPLCV-CN)has a genome organization similar to that of monopartite begomoviruses. The DNA-A had two ORFs (AV1 and AV2) in the virion sense and four ORFs (AC1, AC2, AC3, and AC4) in the complementary sense, separated by an intergenic region (IR) containing a conserved stem-loop motif. Three incomplete direct repeat iterons were also found within the IR. The presence of AV2 ORF supports the relationship of SPLCV-CN to the Old World gemimiviruses. Sequence comparisons showed that the DNA-A sequence of SPLCV-CN were closely related to those of sweet potato leaf curl Georgia virus-[16] (SPLCGV-[16]), Ipomoea yellow vein virus (IYVV-SI), and sweet potato leaf curl virus (SPLCV) with nucleotide sequence identity ranging from 88% to 91%. Comparison of individual encoded proteins between SPLCV-CN and that of three other SPLCV isolates showed the coat protein (AV1) shared the highest amino acid sequence identity (93%–96%), suggesting the coat protein of these viruses may have identical ancestor. The relationships between SPLCV-CN and other whitefly-transmitted geminiviruses were investigated by using phylogeny of derived AV1, AC1, and AV2 amino acid sequences. In all phylogenetic trees, SPLCV-CN clustered with three other isolates of SPLCV. The analyses revealed that the four isolates of SPLCV have coat proteins which are unique from its counterparts from both the Old World and New World. The present of AV2 and phylogenic analysis of AC1 suggest that SPLCV is more close to begomoviruses from the Old World but isolates of this virus seems to form a separate subset. An erratum to this article can be found at