Diverse small circular single-stranded DNA viruses identified in a freshwater pond on the McMurdo Ice Shelf (Antarctica).

Antarctica has some of the harshest environmental conditions for existence of life on Earth. In this pilot study we recovered eight diverse circular single-stranded DNA (ssDNA) viral genome sequences (1904-3120 nts) from benthic mats dominated by filamentous cyanobacteria in a freshwater pond on the McMurdo Ice Shelf sampled in 1988. All genomes contain two to three major open reading frames (ORFs) that are uni- or bi-directionally transcribed and all have an ORF encoding a replication-associated protein (Rep). In one genome, the second ORF has similarity to a capsid protein (CP) of Nepavirus which is most closely related to geminiviruses. Additionally, all genomes have two intergenic regions that contain putative stem loop structures, six genomes have NANTATTAC as the nonanucleotide motif, while one has CCTTATTAC, and another has a non-canonical stem loop. In the large intergenic region, we identified iterative sequences flanking the putative stem-loop elements which are a hallmark of most circular ssDNA viruses encoding rolling circle replication (RCR) initiators of the HUH endonuclease superfamily. The Reps encoded by ssDNA viral genomes recovered in this study shared <38% pairwise identity to all other Reps of known ssDNA viruses. A previous study on Lake Limnopolar (Livingston Island, South Shetland Islands), using next-generation sequencing identified circular ssDNA viruses and their putative Reps share <35% pairwise identity to those from the viral genomes removed in this study. It is evident from our pilot study that the global diversity of ssDNA viruses is grossly underestimated and there is limited knowledge on ssDNA viruses in Antarctica.

Mutability dynamics of an emergent single stranded DNA virus in a naïve host.

Quasispecies variants and recombination were studied longitudinally in an emergent outbreak of beak and feather disease virus (BFDV) infection in the orange-bellied parrot (Neophema chrysogaster). Detailed health monitoring and the small population size (<300 individuals) of this critically endangered bird provided an opportunity to longitudinally track viral replication and mutation events occurring in a circular, single-stranded DNA virus over a period of four years within a novel bottleneck population. Optimized PCR was used with different combinations of primers, primer walking, direct amplicon sequencing and sequencing of cloned amplicons to analyze BFDV genome variants. Analysis of complete viral genomes (n = 16) and Rep gene sequences (n = 35) revealed that the outbreak was associated with mutations in functionally important regions of the normally conserved Rep gene and immunogenic capsid (Cap) gene with a high evolutionary rate (3.41×10(-3) subs/site/year) approaching that for RNA viruses; simultaneously we observed significant evidence of recombination hotspots between two distinct progenitor genotypes within orange-bellied parrots indicating early cross-transmission of BFDV in the population. Multiple quasispecies variants were also demonstrated with at least 13 genotypic variants identified in four different individual birds, with one containing up to seven genetic variants. Preferential PCR amplification of variants was also detected. Our findings suggest that the high degree of genetic variation within the BFDV species as a whole is reflected in evolutionary dynamics within individually infected birds as quasispecies variation, particularly when BFDV jumps from one host species to another.

The structure of LNA:DNA hybrids from molecular dynamics simulations: the effect of locked nucleotides.

Locked nucleic acids (LNAs) exhibit a modified sugar fragment that is restrained to the C3'-endo conformation. LNA-containing duplexes are rather stable and have a more rigid structure than DNA duplexes, with a propensity for A-conformation of the double helix. To gain detailed insight into the local structure of LNA-modified DNA oligomers (as a foundation for subsequent exploration of the electron-transfer capabilities of such modified duplexes), we carried out molecular dynamics simulations on a set of LNA:DNA 9-mer duplexes and analyzed the resulting structures in terms of base step parameters and the conformations of the sugar residues. The perturbation introduced by a single locked nucleotide was found to be fairly localized, extending mostly to the first neighboring base pairs; such duplexes featured a B-type helix. With increasing degree of LNA modification the structure gradually changed; the duplex with one complete LNA strand assumed a typical A-DNA structure. The relative populations of the sugar conformations agreed very well with NMR data, lending credibility to the validity of the computational protocol.

Cotton leaf curl Gezira virus-satellite DNAs represent a divergent, geographically isolated Nile Basin lineage: predictive identification of a satDNA REP-binding motif.

Cotton leaf curl Gezira virus (CLCuGV), a species of the genus Begomovirus (family Geminiviridae), was recently cloned from cotton, okra, and Sida alba plants exhibiting leaf-curling and vein-thickening symptoms in Sudan. Here, we describe a previously unknown lineage of single-stranded DNA satellite (satDNA) molecules, which are associated with CLCuGV, and are required for development of characteristic disease symptoms. Co-inoculation of cotton and Nicotiana benthamiana plants with satDNAs cloned from cotton, okra, and S. alba, together with CLCuGV as the 'helper virus' resulted in the development of characteristic leaf-curling and vein-thickening symptoms in both hosts. An anatomical study of symptomatic, virus-infected cotton leaves revealed that spongy parenchyma cells had developed instead of collenchyma cells at the sites of vein thickening. Phylogenetically, the CLCuGV-associated satDNAs from Sudan, together with their closest relatives from Egypt, form a new satDNA lineage comprising only satDNAs from the Upper and Lower Nile Basins. Analysis of satellites and their helper virus sequences identified a predicted REP-binding site consisting of the directly repeated sequence, 'CGGTACTCA', and an inverted repeated sequence, 'TGAGTACCG', which occur in the context of a 17-nucleotide motif. The conserved REP-binding motif identified herein, together with strict geographic isolation, and apparent host-restriction, may be the collective hallmark of these new satDNA-begomovirus lineages, extant in the Nile Basin.

A new class of homogeneous nucleic acid probes based on specific displacement hybridization.

We have developed a new class of probes for homogeneous nucleic acid detection based on the proposed displacement hybridization. Our probes consist of two complementary oligodeoxyribonucleotides of different length labeled with a fluorophore and a quencher in close proximity in the duplex. The probes on their own are quenched, but they become fluorescent upon displacement hybridization with the target. These probes display complete discrimination between a perfectly matched target and single nucleotide mismatch targets. A comparison of double-stranded probes with corresponding linear probes confirms that the presence of the complementary strand significantly enhances their specificity. Using four such probes labeled with different color fluorophores, each designed to recognize a different target, we have demonstrated that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide. Double-stranded probes were used in real-time nucleic acid amplifications as either probes or as primers. In addition to its extreme specificity and flexibility, the new class of probes is simple to design and synthesize, has low cost and high sensitivity and is accessible to a wide range of labels. This class of probes should find applications in a variety of areas wherever high specificity of nucleic acid hybridization is relevant.

Additional rep-encoding DNAs associated with banana bunchy top virus.

Banana bunchy top nanovirus (BBTV) has a multicomponent circular single-stranded DNA (cssDNA) genome consisting of at least six components. We have cloned, sequenced and analysed two additional cssDNA components, designated BBTV-S1 and S2, associated with a Taiwanese BBTV isolate. The sequences of BBTV-S1 and S2 comprised 1109 and 1095 nucleotides (nt), respectively, and like BBTV DNA-1, potentially encoded replication initiation proteins (Reps). However, the genome organisation of BBTV-S1 and S2 differed from that of BBTV DNA-1 in that (i) the stem sequence of the CR-SL was not conserved, (ii) the internal gene was absent and (iii) the probable TATA boxes were located 5' of the stem-loop. Further, sequence and phylogenetic analysis of the Rep genes indicated that BBTV DNA-S1 and S2 were distinct from BBTV DNA-1. When different geographical isolates of BBTV were tested for the presence of BBTV-S1/S2, these components were detected in various isolates from Vietnam, Taiwan, the Philippines, Tonga and Samoa but were not detected in isolates from Australia, Egypt, Fiji, and India. Based on these results, BBTV-S1 and S2 do not appear to be integral components of the BBTV genome and represent additional Rep-encoding DNAs associated with BBTV.

Capping DNA with DNA.

Twelve classes of deoxyribozymes that promote an ATP-dependent "self-capping" reaction were isolated by in vitro selection from a random-sequence pool of DNA. Each deoxyribozyme catalyzes the transfer of the AMP moiety of ATP to its 5'-terminal phosphate group, thereby forming a 5',5'-pyrophosphate linkage. An identical DNA adenylate structure is generated by the T4 DNA ligase during enzymatic DNA ligation. A 41-nucleotide class 1 deoxyribozyme requires Cu(2+) as a cofactor and adopts a structure that recognizes both the adenine and triphosphate moieties of ATP or dATP. The catalytic efficiency for this DNA, measured at 10(4) M(-1) x min(-1) using either ATP or dATP as substrate, is similar to other catalytic nucleic acids that use small substrates. Chemical probing and site-directed mutagenesis implicate the formation of guanine quartets as critical components of the active structure. The observation of ATP-dependent "self-charging" by DNA suggests that DNA could be made to perform the reactions typically associated with DNA cloning, but without the assistance of protein enzymes.

Two classes of replicating molecules of adenovirus type 2 DNA.

Replicating DNA of human adenovirus type 2, identified as partly single-stranded viral DNA in which [3H]thymidine is readily incorporated, was found to be separated into two fractions by chromatography on hydroxyapatite. Whereas one of the these fractions was eluted with 180 mM phosphate, the other one was eluted at the same concentration, 240 mM, as fully double-stranded DNA. The physical properties of the 180 and 240 mM fractions, in particular their buoyant densities in solutions of CsCl and Cs2SO4, were compared both before and after treatment by various enzymes such as Neurospora crassa nuclease, pancreatic ribonuclease, ribonuclease H and the Klenow fragment of DNA polymerase I of Escherichia coli, used alone or in various combinations. Unlike the 240 mM fraction, the 180 mM fraction was found to include a substantial amount of single-stranded DNA, some of it being hydrogen-bonded to RNA. Both of these features confer to the 180 mM fraction the high buoyant density in cesium salt solution which was described, for several adenoviruses, as one of the characteristic properties of replicating DNA.