Mark J. Gibbs

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

BackgroundPhylogenies 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.ResultsPATRISTICv1.0 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.ConclusionThe 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.

Time--the emerging dimension of plant virus studies.

Recent research has revealed that some plant viruses, like many animal viruses, have measurably evolving populations. Most of these viruses have single-stranded positive-sense RNA genomes, but a few have single-stranded DNA genomes. The studies show that extant populations of these viral species are only decades to centuries old. The genera in which they are placed have diverged since agriculture was invented and spread around the world during the Holocene period. We suggest that this is not mere coincidence but evidence that the conditions generated by agriculture during this era have favoured particular viruses. There is also evidence, albeit less certain, that some plant viruses, including a few shown to have measurably evolving populations, have much more ancient origins. We discuss the possible reasons for this clear discordance between short- and long-term evolutionary rate estimates and how it might result from a large timescale dependence of the evolutionary rates. We also discuss briefly why it is useful to know the rates of evolution of plant viruses.

Phylogenetic analysis of some large double-stranded RNA replicons from plants suggests they evolved from a defective single-stranded RNA virus.

Sequences were recently obtained from four double-stranded (ds) RNAs from different plant species. These dsRNAs are not associated with particles and as they appeared not to be horizontally transmitted, they were thought to be a kind of RNA plasmid. Here we report that the RNA-dependent RNA polymerase (RdRp) and helicase domains encoded by these dsRNAs are related to those of viruses of the alpha-like virus supergroup. Recent work on the RdRp sequences of alpha-like viruses raised doubts about their relatedness, but our analyses confirm that almost all the viruses previously assigned to the supergroup are related. Alpha-like viruses have single-stranded (ss) RNA genomes and produce particles, and they are much more diverse than the dsRNAs. This difference in diversity suggests the ssRNA alpha-like virus form is older, and we speculate that the transformation to a dsRNA form began when an ancestral ssRNA virus lost its virion protein gene. The phylogeny of the dsRNAs indicates this transformation was not recent and features of the dsRNA genome structure and translation strategy suggest it is now irreversible. Our analyses also show some dsRNAs from distantly related plants are closely related, indicating they have not strictly co-speciated with their hosts. In view of the affinities of the dsRNAs, we believe they should be classified as viruses and we suggest they be recognized as members of a new virus genus (Endornavirus) and family (Endoviridae).

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

Arising from: J. K. Taubenberger et al. 437, 889–893 (2005); see also communication from Antonovics et al.; Taubenberger et al. replyTaubenberger 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.

The potyviruses of Australia

Many potyviruses have been found in Australia. We analyzed a selected region of the coat protein genes of 37 of them to determine their relationships, and found that they fall into two groups. Half were isolated from cultivated plants and crops, and are also found in other parts of the world. Sequence comparisons show that the Australian populations of these viruses are closely related to, but less variable than, those in other parts of the world, and they represent many different potyvirus lineages. The other half of the potyviruses have only been found in Australia, and most were isolated from native plants. The sequences of these potyviruses, which are probably endemic, are on average five times more variable than those of the crop potyviruses, but surprisingly, most of the endemic potyviruses belong to one potyvirus lineage, the bean common mosaic virus lineage. We conclude that the crop potyviruses entered Australia after agriculture was established by European migrants two centuries ago, whereas the endemic plant potyviruses probably entered Australia before the Europeans. Australia, like the U.K., seems recently to have had c. one incursion of a significant crop potyvirus every decade. Our analysis suggests it is likely that potyviruses are transmitted in seed more frequently than experimental evidence indicates, and shows that understanding the sources of emerging pathogens and the frequency with which they ‘emerge’ is essential for proper national biosecurity planning.

Universal primers that amplify RNA from all three flavivirus subgroups

BackgroundSpecies 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.ResultsBioinformatic 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.ConclusionComprehensive testing has revealed the broad specificity of these primers. We briefly discuss the advantages and uses of these universal primers.

Genetic analysis of four porcine avian influenza viruses isolated from Shandong, China

SummaryA Bayesian phylogenetic analysis of eight separate gene segments indicated A/Swine/Shandong/2/2003 (H5N1), A/Swine/Shandong/na/2003 (H9N2), A/Swine/Shandong/nb/2003 (H9N2) and A/Swine/Shandong/nc/2005 (H9N2) probably represent two multiple reassortant lineages, that had not been described before, with genes coming from H5N1, H9N2 and other lineages from poultry in Asia. Amino acid motifs within the haemagglutinin sequence of A/Swine/Shandong/nb/2003 suggested it may be able to infect people, whereas the sequences of the other three isolates suggested they would not have had that capability. Our analysis emphasizes the need for a comprehensive study of the interactions between H5N1 and H9N2 viruses in Asia that includes sequencing and phylogenetic investigation.

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.

Accumulating Variation at Conserved Sites in Potyvirus Genomes Is Driven by Species Discovery and Affects Degenerate Primer Design

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.

More About Plant Virus Evolution: Past, Present, and Future

ABSTRACT Gene sequencing was invented in the 1980s, enabling the evolutionary relationships of organisms to be studied in detail. The ways in which these studies provide the intellectual framework for research into the life of viruses continues to expand. Plants, animals and other organisms present viruses with very different environments, both structurally and biochemically, and this may be the reason why so few virus groups span host kingdoms, but a few do, and studies of these reveal the shared and unique constraints and opportunities provided by different types of host, and also the diverse ways that viruses overcome the constraints. The RNA-silencing system seems to provide the primary plant defense against viruses, and although RNA-silencing mechanisms are present in all eukaryotes, they are most developed in plants where they also modulate the expression of plant genes. Plants have a rigid cellular structure with the cells connected by plasmodesmata too narrow for virions to pass through. This has required viruses to adopt specific mechanisms to aid their systemic spread within plants. The diverse measures adopted by viruses to suppress RNA silencing and to aid their spread through plants indicate that such mechanisms have evolved independently on several occasions. Likewise a great range of symbiotic, commensal, and satellite relationships are found among plant viruses, and again the diversity of the relationships, of the virus groups involved, and of the resulting phenotypes, emphasizes that viruses of plants are polyphyletic. Studies of mutations in model experimental systems, and of gene sequence variation in natural viral populations, are clarifying the mechanisms that produce “quasispecies,” even though the concept seems to be still largely misunderstood. The relative contribution of different evolutionary processes, including mutation, drift, recombination, and selection, to viral population change is becoming better understood. The taxonomies of tobamoviruses and of their principle hosts seem to be congruent, indicating that they have probably co-evolved, and hence may be of the same age, around 100 million years. However potyviruses and their hosts show no such relationships, indeed gene sequence differences in viral populations, of which the history is known, indicate that the genus Potyvirus may be only a few thousand years old. Our understanding of more distant relationships remains very speculative as it depends on comparisons of “molecular phenotypic” characters (e.g. structure and function) rather than of gene sequences. Viruses have been studied for more than a century, their molecules are well known, but our understanding of the molecular basis of plant virus biology is still in its infancy, and we have little idea of how viruses will respond to “climate change” and “transgene pollution.”