First Report of Pepper chat fruit viroid in Traded Tomato Seed, an Interception by Australian Biosecurity.

Pepper chat fruit viroid (PCFVd), a species of Pospiviroid, was first discovered in a capsicum crop in the Netherlands in 2006 (4) and was then reported only in Thailand (2) and Canada. The mechanism of international spread was not known, but movement with traded seed was suspected. PCFVd is transmissible through capsicum seed (4) and very probably through tomato seed, like other pospiviroids. The viroid causes disease in capsicum and tomato and experiments by others indicate a capacity to cause disease in potato. It poses a biosecurity threat to crops internationally. PCFVd was intercepted by the Australian Government Department of Agriculture, Fisheries, and Forestry (DAFF) in five shipments of tomato seed (Solanum lycopersicum) exported from Israel and Thailand in September and October 2012. Batches of up to 20,000 seeds were sampled from each seed lot in a shipment and total nucleic acids were extracted from sub-samples, each of about 400 seeds, following a method similar to Hoshino et al. (1). PCFVd was initially detected when reverse transcription PCR using the generic pospiviroid primers Pospi1-FW and Pospi1-RE (3) produced amplicons of 189 bp, which were then sequenced. The PCFVd specific primers AP FW1 and AP RE2 (4) were used to amplify the remainder of the viroid genome, which was directly sequenced. Overlapping sequences were aligned to produce complete sequences of 349 bases, one from seed from Thailand and two from seed from Israel (GenBank: KC762952, KC762953, KC762954). Searches of the GenBank nucleotide non-redundant database indicated close matches with sequences from PCFVd isolates from tomato in Thailand (2); alignments generated by BLAST showed the sequences differed from those from Thailand at only 2 to 18 nucleotide positions, equating to 95 to 99% identity. PCFVd sequences from seed from Thailand were almost identical (>99%) to the sequences from seed from Israel. Many sub-samples were negative, indicating that the number of contaminated seeds was very small in some shipments. The positive sub-samples as a proportion of the total number of sub-samples tested from the five shipments was 1/1, 1/5, 1/1, 12/50, and 7/50. Tomato and capsicum seed are produced in many countries and often traded through second countries. The infected tomato seed shipments intercepted by DAFF were destroyed or re-exported following Australian regulations. Other countries were informed through the International Plant Protection Convention. This pest viroid has not been intercepted by Australian authorities before and has not been detected in recent Australian survey work (data not shown). References: (1) S. Hoshino et al. Res. Bull. Plant Prot. Japan 42:75, 2006. (2) K. Reanwarakorn et al. New Dis. Rep. 24:6, 2011 (3) J. Th. J. Verhoeven et al. EJPP 110:823, 2004. (4) J. Th. J. Verhoeven et al. Virus Res. 144:209, 2009.

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.

Recombination in the hemagglutinin gene of the 1918 "Spanish flu".

When gene sequences from the influenza virus that caused the 1918 pandemic were first compared with those of related viruses, they yielded few clues about its origins and virulence. Our reanalysis indicates that the hemagglutinin gene, a key virulence determinant, originated by recombination. The "globular domain" of the 1918 hemagglutinin protein was encoded by a part of a gene derived from a swine-lineage influenza, whereas the "stalk" was encoded by parts derived from a human-lineage influenza. Phylogenetic analyses showed that this recombination, which probably changed the virulence of the virus, occurred at the start of, or immediately before, the pandemic and thus may have triggered it.

The bean common mosaic virus lineage of potyviruses: where did it arise and when?

There are more than 30 species in the bean common mosaic virus lineage of the genus Potyvirus. We have used their partial coat protein gene sequences to infer their phylogenies and have compared these with host and provenance information. Members of six species of the lineage have been isolated from crops distributed around the world, but three of these show clear links with South and East Asia. Members of the remaining species have been found in wild plants, minor crop species or ornamentals, and the majority of these have only been found in south-east and East Asia, Oceania or Australia. This phylogeographic pattern suggests that the bean common mosaic virus lineage arose in that region. Maximum-likelihood trees of the sequences were dated using the report that the initial major radiation of all potyviruses was 6,600 years ago. In this way, the bean common mosaic virus lineage was found to have first diverged 3,580 years ago, and one sub-lineage of seven species, found only in Australia, probably diverged there 2005 years ago. We discuss the ways in which the viruses could have moved from south-east Asia to Australia and note that their movement coincided with the spread of the Austronesian sea-faring/farming culture from China/Taiwan throughout the islands of the southern and eastern Pacific Ocean. Our study shows that virus isolates from wild or minimally domesticated plants, and from islands, are probably more useful indicators of the origins of viruses than those from widely grown well-travelled crop species.

The use of metallic glass scroll as an acoustic transducer element.

A study was made of the piezomagnetic and elastic properties of scrolls of Metglas 2605 SC, used as an acoustic transducer element. This study involved quasi-DC magnetic induction measurements and ultrasonic complex plane analysis of scrolls of various annealing histories. An optimum effective magnetomechanical coupling coefficient. K(eff), of 0.75, was found. The best magnetomechanical response in air obtained for metallic glass scroll during the study was for the unconsolidated case. However, preliminary experiments have shown that structural weakness and corrosion problems are significant when using the unconsolidated scrolls in water.

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.

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

A 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.

The variable codons of H3 influenza A virus haemagglutinin genes.

We have analyzed several sets of well-studied haemagglutinin (HA) gene sequences of H3 subtype influenza A viruses to identify codons that are unusually variable, using a simple pairwise sliding window method, DnDscanning. For two of the sets there were results of detailed phylogenetic modeling studies of selection already published. A third set had been the subject of an antigen mapping study, the results of which provide a completely independent benchmark of selected changes in H3 HA genes. Our analyses show that the codons with greatest DnDscan scores (i.e. the most variable) were mostly those reported in the published studies as being positively selected; indeed the DnDscan results matched the antigenic mapping results more closely than did those of the phylogenetic modeling methods. These results suggest that codons under selection can be found even when, as with some sets of virus sequences, a phylogeny is uncertain or cannot be obtained because, for example, the sequences are recombinants, or when selection is not necessarily linked with phylogeny, as in host-switching events. The program DnDscan is available at (biojanus.anu.edu.au).

A broader definition of 'the virus species'.

We propose that the formal definition of a virus species by the International Committee on Taxonomy of Viruses (ICTV) should be broadened by removing the restrictive word "polythetic" from the current definition, so that any characters can be used to define species. This change will bring the definition of virus species into line with the species definitions of cellular organisms and broaden the range of characters available for describing virus species.

Evidence that a plant virus switched hosts to infect a vertebrate and then recombined with a vertebrate-infecting virus.

There are several similarities between the small, circular, single-stranded-DNA genomes of circoviruses that infect vertebrates and the nanoviruses that infect plants. We analyzed circovirus and nanovirus replication initiator protein (Rep) sequences and confirmed that an N-terminal region in circovirus Reps is similar to an equivalent region in nanovirus Reps. However, we found that the remaining C-terminal region is related to an RNA-binding protein (protein 2C), encoded by picorna-like viruses, and we concluded that the sequence encoding this region of Rep was acquired from one of these single-stranded RNA viruses, probably a calicivirus, by recombination. This is clear evidence that a DNA virus has incorporated a gene from an RNA virus, and the fact that none of these viruses code for a reverse transcriptase suggests that another agent with this capacity was involved. Circoviruses were thought to be a sister-group of nanoviruses, but our phylogenetic analyses, which take account of the recombination, indicate that circoviruses evolved from a nanovirus. A nanovirus DNA was transferred from a plant to a vertebrate. This transferred DNA included the viral origin of replication; the sequence conservation clearly indicates that it maintained the ability to replicate. In view of these properties, we conclude that the transferred DNA was a kind of virus and the transfer was a host-switch. We speculate that this host-switch occurred when a vertebrate was exposed to sap from an infected plant. All characterized caliciviruses infect vertebrates, suggesting that the host-switch happened first and that the recombination took place in a vertebrate.

A recombinational event in the history of luteoviruses probably induced by base-pairing between the genomes of two distinct viruses.

Alignments of luteovirus readthrough protein amino acid sequences show they consist of two distinct regions, here named the N domain and the C domain. N domain sequences were classified, and comparison of this gene phylogeny to phylogenies of other luteovirus genes revealed an anomaly in the relationships between beet western yellows luteovirus, cucurbit aphidborne yellows luteovirus (CABYV), and pea enation mosaic RNA1 (PEMV1). Together with alignments of virion protein and readthrough protein amino acid sequences, these gene phylogenies indicate the anomaly to be the result of two recombinational events, probably between ancestors of CABYV and PEMV1 and leading to the transfer of RNA coding for the N domain to an ancestor of CABYV. Two likely recombination sites were identified from the alignments, one at the 5' end of the readthrough protein gene and the other at the 5' end of the sequence coding for the C domain. Alignments of the nucleotide sequences encompassing the probable recombination sites suggest that base-pairing between the genomes of the two ancestral luteoviruses, resulting from local sequence similarity at the 5' end of the readthrough protein gene, probably induced one of the interspecies recombinational events.

Spinach chloroplastic carbonic anhydrase: nucleotide sequence analysis of cDNA.

We have determined the nucleotide sequence of a cDNA encoding spinach (Spinacia oleracea) chloroplastic carbonic anhydrase (CA). The open reading frame encodes a protein consisting of a transit peptide and a mature CA protein with a predicted mass of 24, 116 daltons. This represents the first report of a nucleotide sequence of a plant CA.

The phylogeny of SARS coronavirus.

Different tree-building methods consistently place the SARS corona-virus (SARS-CoV) as a basal Group 2 coronavirus rather than as an ungrouped species as concluded by others. Detailed comparisons of the SARS-CoV genomic sequence with those of six other coronaviruses failed to find evidence of recombination or genomic rearrangement using computational methods designed for that purpose.

The haemagglutinin gene, but not the neuraminidase gene, of 'Spanish flu' was a recombinant.

Published analyses of the sequences of three genes from the 1918 Spanish influenza virus have cast doubt on the theory that it came from birds immediately before the pandemic. They showed that the virus was of the H1N1 subtype lineage but more closely related to mammal-infecting strains than any known bird-infecting strain. They provided no evidence that the virus originated by gene reassortment nor that the virus was the direct ancestor of the two lineages of H1N1 viruses currently found in mammals; one that mostly infects human beings, the other pigs. The unusual virulence of the virus and why it produced a pandemic have remained unsolved. We have reanalysed the sequences of the three 1918 genes and found conflicting patterns of relatedness in all three. Various tests showed that the patterns in its haemagglutinin (HA) gene were produced by true recombination between two different parental HA H1 subtype genes, but that the conflicting patterns in its neuraminidase and non-structural-nuclear export proteins genes resulted from selection. The recombination event that produced the 1918 HA gene probably coincided with the start of the pandemic, and may have triggered it.

Sister-scanning: a Monte Carlo procedure for assessing signals in recombinant sequences.

MOTIVATION: To devise a method that, unlike available methods, directly measures variations in phylogenetic signals in gene sequences that result from recombination, tests the significance of the signal variations and distinguishes misleading signals. RESULTS: We have developed a method, that we call 'sister-scanning', for assessing phylogenetic and compositional signals in the various patterns of identity that occur between four nucleotide sequences. A Monte Carlo randomization is done for all columns (positions) within a window and Z-scores are obtained for four real sequences or three real sequences with an outlier that is also randomized. The usefulness of the approach is demonstrated using tobamovirus and luteovirus sequences. Contradictory phylogenetic signals were distinguished in both datasets, as were regions of sequence that contained no clear signal or potentially misleading signals related to compositional similarities. In the tobamovirus dataset, contradictory phylogenetic signals were separated by coding sequences up to a kilobase long that contained no clear signal. Our re-analysis of this dataset using sister-scanning also yielded the first evidence known to us of an inter-species recombination site within a viral RNA-dependent RNA polymerase gene together with evidence of an unusual pattern of conservation in the three codon positions.

A type of nucleotide motif that distinguishes tobamovirus species more efficiently than nucleotide signatures.

The complete genomic sequences of forty-eight tobamoviruses were classified and found to form at least twelve species clusters. Individual species were not conveniently defined by 'nucleotide signatures' (i.e. strings of one or more nucleotides unique to a taxon) as these were scattered sparsely throughout the genomes and were mostly single nucleotides. By contrast all the species were concisely and uniquely distinguished by short nucleotide motifs consisting of conserved genus-specific sites intercalated with variable sites that provided species-specific combinations of nucleotides (nucleotide combination motifs; NC-motifs). We describe the procedure for finding NC-motifs in a convenient and phylogenetically conserved region of the tobamovirus RNA polymerase gene, the '4404-50 motif'. NC-motifs have been found in other sets of homologous sequences, and are convenient for use in published taxonomic descriptions.

The genome organization and affinities of an Australian isolate of carrot mottle umbravirus.

The genomic sequence of an Australian isolate of carrot mottle umbravirus (CMoV-A) was determined from cDNA generated from dsRNA. This provides the first data on the genome organization and phylogeny of an umbravirus. The 4201-nucleotide genome contains four major open reading frames (ORFs). Analysis suggests that ORF2 encodes an RNA-dependent RNA polymerase, that ORF4 encodes a movement protein, and that the virus has no coat protein gene. The functions of ORFs 1 and 3 remain unknown. ORF2 is probably translated following ribosomal frameshifting. ORFs 3 and 4 are probably translated from a subgenomic mRNA. Sequence comparisons showed CMoV-A to be closely related to pea enation mosaic RNA2 (PEMV-RNA2), but also to have affinities with the Bromoviridae. These findings shed light on the relationships between the luteoviruses, PEMV, and the umbraviruses and on the relationships between the carmo-like viruses and the Bromoviridae.

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).