Rymovirus: a cautionary tale.

A recent proposal that the genus Rymovirus be assimilated into the genus Potyvirus is examined, discussed, and rejected. It illustrates the danger of using 'sequence identity' as a proxy for phylogenetic relatedness to distinguish closely related but distinct groups of viruses.

The phylogenetics of the global population of potato virus Y and its necrogenic recombinants.

Potato virus Y (PVY) is a major pathogen of potatoes and other solanaceous crops worldwide. It is most closely related to potyviruses first or only found in the Americas, and it almost certainly originated in the Andes, where its hosts were domesticated. We have inferred the phylogeny of the published genomic sequences of 240 PVY isolates collected since 1938 worldwide, but not the Andes. All fall into five groupings, which mostly, but not exclusively, correspond with groupings already devised using biological and taxonomic data. Only 42 percent of the sequences are not recombinant, and all these fall into one or other of three phylogroups; the previously named C (common), O (ordinary), and N (necrotic) groups. There are also two other distinct groups of isolates all of which are recombinant; the R-1 isolates have N (5' terminal minor) and O (major) parents, and the R-2 isolates have R-1 (major) and N (3' terminal minor) parents. Many isolates also have additional minor intra- and inter-group recombinant genomic regions. The complex interrelationships between the genomes were resolved by progressively identifying and removing recombinants using partitioned sequences of synonymous codons. Least squared dating and BEAST analyses of two datasets of gene sequences from non-recombinant heterochronously-sampled isolates (seventy-three non-recombinant major ORFs and 166 partial ORFs) found the 95% confidence intervals of the TMRCA estimates overlap around 1,000 CE (Common Era; AD). We attempted to identify the most accurate datings by comparing the estimated phylogenetic dates with historical events in the worldwide adoption of potato and other PVY hosts as crops, but found that more evidence from gene sequences of non-potato isolates, especially from South America, was required.

Evolutionary dynamics of the N1 neuraminidases of the main lineages of influenza A viruses.

Influenza A virus infects a wide range of hosts including birds, humans, pigs, horses, and other mammals. Because hosts differ in immune system structure and demography, it is therefore expected that host populations leave different imprints on the viral genome. In this study, we investigated the evolutionary trajectory of the main lineages of N1 type neuraminidase (NA) gene sequences of influenza A viruses by estimating their evolutionary rates and the selection pressures exerted upon them. We also estimated the time of emergence of these lineages. The Eurasian (avian-like) and North American (classical) swine lineages, the human (seasonal) and avian H5N1 lineages, and a long persisting avian lineage were studied and compared. Nucleotide substitution rates ranged from 1.9x10(-3) to 4.3x10(-3) substitutions per site per year, with the H5N1 lineage estimated to have the greatest rate. The evolutionary rates of the H1N1 human lineage appeared to be slightly greater after it re-emerged in 1977 than before it disappeared in the 1950s. Comparing across the lineages, substitution rates appeared to correlate with the number of positively selected sites and with the degree of asymmetry of the phylogenetic trees. Some lineages had strongly asymmetric trees, implying repeated genotype replacement and narrow genetic diversity. Positively selected sites were identified in all lineages, with the H5N1 lineage having the largest number. A great number of isolates of the H5N1 lineage were sequenced in a short time period and the phylogeny of the lineage was more symmetric. We speculate that the rate and selection estimations made for this lineage could have been influenced by sampling and may not represent the long-term trends.

The variable codons of H5N1 avian influenza A virus haemagglutinin genes.

We investigated the selection pressures on the haemagglutinin genes of H5N1 avian influenza viruses using fixed effects likelihood models. We found evidence of positive selection in the sequences from isolates from 1997 to 2007, except viruses from 2000. The haemagglutinin sequences of viruses from southeast Asia, Hong Kong and mainland China were the most polymorphic and had similar nonsynonymous profiles. Some sites were positively selected in viruses from most regions and a few of these sites displayed different amino acid patterns. Selection appeared to produce different outcomes in viruses from Europe, Africa and Russia and from different host types. One position was found to be positively selected for human isolates only. Although the functions of some positively selected positions are unknown, our analysis provided evidence of different temporal, spatial and host adaptations for H5N1 avian influenza viruses.

Quantitative PCR measurements of the effects of introducing inosines into primers provides guidelines for improved degenerate primer design.

Polymerase chain reaction (PCR) is used to detect groups of viruses with the use of group-specific degenerate primers. Inosine residues are sometimes used in the primers to match variable positions within the complementary target sequences, but there is little data on their effects on cDNA synthesis and amplification. A quantitative reverse-transcription PCR was used to measure the rate of amplification with primers containing inosine residues substituted at different positions and in increasing numbers. Experiments were conducted using standard quantities of cloned DNA copied from Potato virus Y genomic RNA and RNA (cRNA) transcribed from the cloned DNA. Single inosine residues had no affect on the amplification rate in the forward primer, except at one position close to the 3' terminus. Conversely, single inosine residues significantly reduced the amplification rate when placed at three out of four positions in the reverse primer. Four or five inosine substitutions could be tolerated with some decline in rates, but amplification often failed from cRNA templates with primers containing larger numbers of inosines. Greater declines in the rate of amplification were observed with RNA templates, suggesting that reverse transcription suffers more than PCR amplification when inosine is included in the reverse primer.

SWeBLAST: a Sliding Window Web-based BLAST tool for recombinant analysis.

We describe a simple Perl computer tool for matching successive subsequences of a query sequence using the BLAST facilities of Genbank. SWeBLAST helps identify 'parents' of recombinant sequences, even when these are themselves unrelated, thus it is complementary to methods that compare sets of aligned homologous sequences, and avoids the significant problem of these methods in having first to decide which sequences to compare. SWeBLAST searches may also be valuable for checking the recombination history of genes proposed for use as transgenes.

The prehistory of potyviruses: their initial radiation was during the dawn of agriculture.

BACKGROUND: Potyviruses are found world wide, are spread by probing aphids and cause considerable crop damage. Potyvirus is one of the two largest plant virus genera and contains about 15% of all named plant virus species. When and why did the potyviruses become so numerous? Here we answer the first question and discuss the other. METHODS AND FINDINGS: We have inferred the phylogenies of the partial coat protein gene sequences of about 50 potyviruses, and studied in detail the phylogenies of some using various methods and evolutionary models. Their phylogenies have been calibrated using historical isolation and outbreak events: the plum pox virus epidemic which swept through Europe in the 20th century, incursions of potyviruses into Australia after agriculture was established by European colonists, the likely transport of cowpea aphid-borne mosaic virus in cowpea seed from Africa to the Americas with the 16th century slave trade and the similar transport of papaya ringspot virus from India to the Americas. CONCLUSIONS/SIGNIFICANCE: Our studies indicate that the partial coat protein genes of potyviruses have an evolutionary rate of about 1.15x10(-4) nucleotide substitutions/site/year, and the initial radiation of the potyviruses occurred only about 6,600 years ago, and hence coincided with the dawn of agriculture. We discuss the ways in which agriculture may have triggered the prehistoric emergence of potyviruses and fostered their speciation.

The VirusBanker database uses a Java program to allow flexible searching through Bunyaviridae sequences.

BACKGROUND: Viruses of the Bunyaviridae have segmented negative-stranded RNA genomes and several of them cause significant disease. Many partial sequences have been obtained from the segments so that GenBank searches give complex results. Sequence databases usually use HTML pages to mediate remote sorting, but this approach can be limiting and may discourage a user from exploring a database. RESULTS: The VirusBanker database contains Bunyaviridae sequences and alignments and is presented as two spreadsheets generated by a Java program that interacts with a MySQL database on a server. Sequences are displayed in rows and may be sorted using information that is displayed in columns and includes data relating to the segment, gene, protein, species, strain, sequence length, terminal sequence and date and country of isolation. Bunyaviridae sequences and alignments may be downloaded from the second spreadsheet with titles defined by the user from the columns, or viewed when passed directly to the sequence editor, Jalview. CONCLUSION: VirusBanker allows large datasets of aligned nucleotide and protein sequences from the Bunyaviridae to be compiled and winnowed rapidly using criteria that are formulated heuristically.

Accumulating variation at conserved sites in potyvirus genomes is driven by species discovery and affects degenerate primer design.

UNLABELLED: Unknown and foreign viruses can be detected using degenerate primers targeted at conserved sites in the known viral gene sequences. Conserved sites are found by comparing sequences and so the usefulness of a set of primers depends crucially on how well the known sequences represent the target group including unknown sequences. METHODOLOGY/PRINCIPAL FINDINGS: We developed a method for assessing the apparent stability of consensus sequences at sites over time using deposition dates from Genbank. We tested the method using 17 conserved sites in potyvirus genomes. The accumulation of knowledge of sequence variants over 20 years caused 'consensus decay' of the sites. Rates of decay were rapid at all sites but varied widely and as a result, the ranking of the most conserved sites changed. The discovery and reporting of sequences from previously unknown and distinct species, rather than from strains of known species, dominated the decay, indicating it was largely a sampling effect related to the progressive discovery of species, and recent virus mutation was probably only a minor contributing factor. CONCLUSION/SIGNIFICANCE: We showed that in the past, the sampling bias has misled the choice of the most conserved target sites for genus specific degenerate primers. The history of sequence discoveries indicates primer designs should be updated regularly and provides an additional dimension for improving the design of degenerate primers.

Universal primers that amplify RNA from all three flavivirus subgroups.

BACKGROUND: Species within the Flavivirus genus pose public health problems around the world. Increasing cases of Dengue and Japanese encephalitis virus in Asia, frequent outbreaks of Yellow fever virus in Africa and South America, and the ongoing spread of West Nile virus throughout the Americas, show the geographical burden of flavivirus diseases. Flavivirus infections are often indistinct from and confused with other febrile illnesses. Here we review the specificity of published primers, and describe a new universal primer pair that can detect a wide range of flaviviruses, including viruses from each of the recognised subgroups. RESULTS: Bioinformatic analysis of 257 published full-length Flavivirus genomes revealed conserved regions not previously targeted by primers. Two degenerate primers, Flav100F and Flav200R were designed from these regions and used to generate an 800 base pair cDNA product. The region amplified encoded part of the methyltransferase and most of the RNA-dependent-RNA-polymerase (NS5) coding sequence. One-step RT-PCR testing was successful using standard conditions with RNA from over 60 different flavivirus strains representing about 50 species. The cDNA from each virus isolate was sequenced then used in phylogenetic analyses and database searches to confirm the identity of the template RNA. CONCLUSION: Comprehensive testing has revealed the broad specificity of these primers. We briefly discuss the advantages and uses of these universal primers.

Wheat streak mosaic virus in Australia: Relationship to Isolates from the Pacific Northwest of the USA and Its Dispersion Via Seed Transmission.

Wheat streak mosaic virus (WSMV) was found for the first time in Australia in 2002. It subsequently was found widely dispersed around the continent and was shown to be seedborne in wheat. The coat protein (CP) gene sequences of nine WSMV isolates from eastern and southwestern Australia are reported, one obtained directly from infected wheat seed, three from seedlings grown from infected wheat seed, and five from infected wheat plant samples. These sequences were compared with those of 66 WSMV CP sequences, including eight previously sequenced Australian isolates. All 17 Australian sequences formed a closely knit monophyletic cluster as part of the D1 subclade of WSMV previously only reported from the Pacific Northwest of the United States. The close phylogenetic relationships of these sequences indicate that the Australian outbreak arose from a single incursion, the source of which appears to be the Pacific Northwest. Three Australian CP sequences were identical, one from the location of the post-entry quarantine facility at Tamworth, New South Wales, and two from seed that had originally been propagated at that facility. These three sequences were closest to the Pacific Northwest sequences and differed from them by as little as eight nucleotides (0.76%). The sequence of a third seedborne isolate originally from the same source differed from the other two seedborne isolates by two nucleotides, indicating that the immigrant WSMV population may have been variable. The other Australian sequences differed from the three identical ones by only one to four nucleotides. The phylogenetic pattern and small number of nucleotide differences between individual isolates from different geographic locations fit the scenario that the virus was introduced once in seed of wheat breeding material, multiplied where it was introduced, and then was dispersed over long distances around the Australian continent along standard distribution routes for wheat breeding lines, germ plasm, and crop seed. These conclusions provide a cautionary tale indicating the importance of effective monitoring of imported plant materials for exotic virus diseases during post-entry quarantine.

Molecular virology: was the 1918 pandemic caused by a bird flu?

Taubenberger et al. have sequenced the polymerase genes of the pandemic 'Spanish' influenza A virus of 1918, thereby completing the decoding of the genome of this virus. The authors conclude from these sequences that the virus jumped from birds to humans shortly before the start of the pandemic and that it was not derived from earlier viruses by gene shuffling, a process called reassortment. However, we believe that their evidence does not convincingly support these conclusions and that some of their results even indicate that, on the contrary, the virus evolved in mammals before the pandemic began and that it was a reassortant. In light of this alternative interpretation, we suggest that the current intense surveillance of influenza viruses should be broadened to include mammalian sources.

Two families of rep-like genes that probably originated by interspecies recombination are represented in viral, plasmid, bacterial, and parasitic protozoan genomes.

Two families of genes related to, and including, rolling circle replication initiator protein (Rep) genes were defined by sequence similarity and by evidence of intergene family recombination. The Rep genes of circoviruses were the best characterized members of the "RecRep1 family." Other members of the RecRep1 family were Rep-like genes found in the genomes of the Canarypox virus, Entamoeba histolytica, and Giardia duodenalis and in a plasmid, p4M, from the Gram-positive bacterium, Bifidobacterium pseudocatenulatum. The "RecRep2 family" comprised some previously identified Rep-like genes from plasmids of phytoplasmas and similar Rep-like genes from the genomes of Lactobacillus acidophilus, Lactococcus lactis, and Phytoplasma asteris. Both RecRep1 and RecRep2 proteins have a nucleotide-binding domain significantly similar to the helicases (2C proteins) of picorna-like viruses. On the N-terminal side of the nucleotide binding domain, RecRep1 proteins have a domain significantly similar to one found in nanovirus Reps, whereas RecRep2 proteins have a domain significantly similar to one in the Reps of pLS1 plasmids. We speculate that RecRep genes have been transferred from viruses or plasmids to parasitic protozoan and bacterial genomes and that Rep proteins were themselves involved in the original recombination events that generated the ancestral RecRep genes.

PATRISTIC: a program for calculating patristic distances and graphically comparing the components of genetic change.

BACKGROUND: Phylogenies are commonly used to analyse the differences between genes, genomes and species. Patristic distances calculated from tree branch lengths describe the amount of genetic change represented by a tree and are commonly compared with other measures of mutation to investigate the substitutional processes or the goodness of fit of a tree to the raw data. Up until now no universal tool has been available for calculating patristic distances and correlating them with other genetic distance measures. RESULTS: PATRISTICv1.0 is a java program that calculates patristic distances from large trees in a range of file formats and allows graphical and statistical interpretation of distance matrices calculated by other programs. CONCLUSION: The software overcomes some logistic barriers to analysing signals in sequences. In additional to calculating patristic distances, it provides plots for any combination of matrices, calculates commonly used statistics, allows data such as isolation dates to be entered and reorders matrices with matching species or gene labels. It will be used to analyse rates of mutation and substitutional saturation and the evolution of viruses. It is available at http://biojanus.anu.edu.au/programs/ and requires the Java runtime environment.

Individual sequences in large sets of gene sequences may be distinguished efficiently by combinations of shared sub-sequences.

BACKGROUND: Most current DNA diagnostic tests for identifying organisms use specific oligonucleotide probes that are complementary in sequence to, and hence only hybridize with the DNA of one target species. By contrast, in traditional taxonomy, specimens are usually identified by 'dichotomous keys' that use combinations of characters shared by different members of the target set. Using one specific character for each target is the least efficient strategy for identification. Using combinations of shared bisectionally-distributed characters is much more efficient, and this strategy is most efficient when they separate the targets in a progressively binary way. RESULTS: We have developed a practical method for finding minimal sets of sub-sequences that identify individual sequences, and could be targeted by combinations of probes, so that the efficient strategy of traditional taxonomic identification could be used in DNA diagnosis. The sizes of minimal sub-sequence sets depended mostly on sequence diversity and sub-sequence length and interactions between these parameters. We found that 201 distinct cytochrome oxidase subunit-1 (CO1) genes from moths (Lepidoptera) were distinguished using only 15 sub-sequences 20 nucleotides long, whereas only 8-10 sub-sequences 6-10 nucleotides long were required to distinguish the CO1 genes of 92 species from the 9 largest orders of insects. CONCLUSION: The presence/absence of sub-sequences in a set of gene sequences can be used like the questions in a traditional dichotomous taxonomic key; hybridisation probes complementary to such sub-sequences should provide a very efficient means for identifying individual species, subtypes or genotypes. Sequence diversity and sub-sequence length are the major factors that determine the numbers of distinguishing sub-sequences in any set of sequences.

The 'potyvirid primers' will probably provide phylogenetically informative DNA fragments from all species of Potyviridae.

A survey of gene sequences of species of Potyviridae, the most numerous family of plant viruses, has shown that the -GNNS- encoding region of the NIb gene is present in all c. 300 publicly available sequences of that region. We also report that relationships inferred from the sequence of the 1.6-2.1 kb portion of the genome between the -GNNS- encoding region and its 3' terminus reflect those of the remainder of the genome. Thus, the use for RT-PCR tests of the 'potyvirid primers' based on the -GNNS- encoding region and the 3' terminal poly-A region of the genome, will yield DNA fragments, the sequences of which are very likely to correctly identify viruses from which they were obtained.