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An outbreak in northwestern Turkey of prunus necrotic ringspot virus (PNRSV, genus Ilarvirus, family Bromoviridae) was sampled in 2016-2018. Gene sequences from these isolates, together with all of the gene sequence data for this virus in the GenBank database (>300 non-recombinant coat protein (CP) genes and 20 complete genomic sequences) were analysed to determine the relationship of the Turkish PNRSV isolates to those from other parts of the world. Phylogenetic and population genetic methods independently showed that the most recent common ancestor of the world PNRSV population was probably American, not Eurasian. PNRSV has spread to Turkey on several occasions, as its CP sequences are among the terminal branches of three of the most sampled CP phylogroups. The complete PNRSV genome consists of three segments (RNA1, RNA2, and RNA3), with the larger two encoding replicases and the smallest encoding the movement protein and the CP. One quarter of the RNA1 and RNA2 genes were recombinants. The phylogenies of the CP and MP genes (i.e., different regions of RNA3) were closely correlated but did not correlate with those of RNA1 and RNA2, indicating that some of the isolates were reassortants. However, the non-reassortant ancestor could not be identified, probably because none of the complete genome sequences were from isolates from the basal CP phylogroups. Our results emphasize the importance of strict quarantine, both international and local, for the world's stone fruit crops., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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.

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.

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.

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).

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.

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.

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.

Three potyviruses were identified by gene sequencing and found to be widespread in species of Apiaceae in Australia. Only celery mosaic virus was found in celery crops and in one of 180 specimens of feral carrot ( Daucus carota). Another related but distinct novel potyvirus, carrot virus Y, was the only virus found in carrot crops and all except one feral carrot. A more distantly related novel potyvirus, apium virus Y, was found in plants of sea celery ( Apium prostratum), cultivated parsley ( Petroselinum crispum) and the immigrant weed species poison hemlock ( Conium maculatum). These three potyviruses, together with celery yellow mosaic virus of South America and the closely related carrot thin leaf virus and carrot virus B of North America, form a distinct subgenus of the Potyviridae most closely related to turnip mosaic virus and two potyviruses of yam; yam mosaic virus from the Ivory Coast and Japanese yam mosaic virus. Celery mosaic and carrot virus Y are probably recent migrants to Australia, but apium virus Y may have been endemic longer. In ELISA tests using polyclonal antibodies against virions of celery mosaic virus, some isolates of carrot virus Y were indistinguishable from celery mosaic virus, whereas others gave smaller absorbancy values, and those of apium virus Y did not react. This study shows the value of virus identification based on gene sequencing for planning control measures.

The sequences of the 3'-terminal 1.8 kb of the genomes of three Australian and three Welsh isolates of ryegrass mosaic rymovirus (RGMV) were determined, as too were the virion protein genes of two New Zealand isolates of RGMV. They were compared with each other and with the published sequences of a Danish and a South African isolate by distance and maximum likelihood methods, and found to be very closely related (mean nucleotide difference 5.5%). All three Australian isolates and one from North Island of New Zealand formed one consistent cluster, and the Danish and South African isolates formed another. However the relationships between these two clusters and the other isolates were not consistent; they depended on the method of comparison used, and on the protein, gene or codon position compared. Nonetheless the European (Welsh and Danish) sequences were 2-4 times more different from one another than those from the Antipodes, suggesting that the European RGMV population may be older than the Antipodean. The Danish isolate has 39 nucleotides of the 5'-terminal region of its virion protein gene frameshifted -1 relative to the 'common' sequence. Interestingly the South African isolate has a similar frameshift, but sequence comparisons indicate that this frameshift must have occurred independently; a possible example of 'convergent frameshifting'.

A potyvirus, which we call ceratobium mosaic virus, has been detected in about one third of more than 100 plants representing c. 33 orchid genera in two collections in Australia. It was detected using RT-PCR with redundant primers that are Potyviridae-specific and have additional sequences corresponding to either the SP6 or T7 bacteriophage promoters at their 5'-termini. Thus the nucleotide sequence of the resulting PCR fragments, consisting of about 1.7 kb of the 3' portion of the viral genome, could be determined directly. Viral sequences obtained from five infected orchids indicate that they contained different isolates of a single potyvirus species most closely related to the bean common mosaic group of potyviruses, but clearly distinct from all whose virion protein genes have been reported to the international gene sequence databases.

The virion protein genes, and 3' untranslated regions, of six variants of turnip yellow mosaic tymovirus (TYMV) that produced different symptoms in their native host Cardamine robusta and in Chinese cabbage plants, have been sequenced. The sequences have been compared with each other, and with the same region of the pBL-16 clone of the Blue Lake isolate of TYMV. The sequences of the virion protein genes differed by a mean of 1.89% (range 0-2.82%), and the encoded proteins by a mean of 1.71% (range 0-3.17%). The nucleotide differences were confined to the 5'-most 60% of the gene, whereas there were amino acid differences only among residues 12 to 29 and residue 102 (numbered from the N-terminus) of the virion protein involving only hydrophobic residues at the surface of the protein. The amino acid and nucleotide differences between the seven isolates did not correlate with differences in the symptoms they caused, but confirmed earlier estimates of genetic variability in the wild populations of the virus.

A tymoyirus isolated from Malaysian crops of Calopogonium mucunoides has been shown to have virions that are serologically indistinguishable from those of clitoria yellow vein tymovirus. We have sequenced the virion protein (VP) gene of the virus and have found that although it is a member of the cluster that includes CYVV, it is the most distinct member of that cluster (< 62% sequence identity with all the others), and is clearly a separate species, which we propose should be named calopogonium yellow vein virus. Most of the serological specificity of the virions of tymoviruses seems to reside in the C-terminal hexapeptide of the virion protein.

Kennedya yellow mosaic tymovirus (KYMV) occurs along the eastern Australian seaboard in the perennial legumes Desmodium triflorum and D. scorpiurus in the north, and Kennedya rubicunda in the south. The genetic variation of more than 100 isolates of KYMV, most of them from the north, has been studied using an RNA hybrid mismatch polymorphism (RHMP) method. The method clearly separated the isolates into two groups; all the northern Desmodium isolates formed one group and all the Kennedya isolates from the south another. These sub-populations were themselves variable and the Desmodium population alone was more variable than that of the related turnip yellow mosaic tymovirus in the Kosciusko alpine area.

In the Mt. Kosciusko alpine area of Australia there are three well-separated populations of Cardamine lilacina, an endemic sward-forming perennial brassica, and these are infected with turnip yellow mosaic tymovirus. The genetic variation in these viral populations has been assessed by an RNA hybrid mismatch polymorphism method. About 100 isolates were examined; the genomic RNA of each isolate was prepared from a shoot of a single wild C. lilacina plant. RNA hybrid mismatch polymorphisms (RHMPs) were assessed in six regions of the genomes using labelled negative-strand probes transcribed from selected portions of a cloned TYMV genome. The probed region at the 3' end of the genome showed little variation and over 95% of the isolates gave the same pattern. However, other parts of the genome, including the 5' non-coding region, were much more variable. There was no significant correlation between groupings based on the RHMP patterns, and the location from which the isolates were collected, nor with the symptom type or severity shown by their host plants. The patterns of variation suggested that all three populations of the virus are a single quasi-species; at most one tenth of the isolates gave similar RHMP patterns, those of the "master copy".

Eggplant mosaic virus (EMV) and ononis yellow mosaic virus (OYMV) are two tymoviruses that have ssRNA genomes of about 6.2 kb and 6.3 kb, and which infect solanaceous and leguminous hosts, respectively. Full-length cDNA clones of these viruses were constructed with a T7 promoter adjacent to the 5' terminus of the DNA copy of the viral genome, and with unique restriction endonuclease sites at the 3' terminus. This allowed RNA to be transcribed from the DNA encoding the genome. The transcript RNA was infectious when inoculated to Nicotiana glutinosa (for EMV) and Pisum sativum (for OYMV). These clones, together with clones of turnip yellow mosaic tymovirus, which infects brassicas, have been used to construct hybrids in which the virion protein gene was exchanged between EMV or OYMV and turnip yellow mosaic virus. These and other hybrids are being used to investigate the molecular basis for host range differences in tymoviruses.

Full-length dsDNA clones that encode the genomes of two Australian turnip yellow mosaic isolates, TYMV-BL and TYMV-CL have been constructed. These clones were transcribed to give 6.3 kb capped ssRNA which infects Chinese cabbages to give symptoms indistinguishable from those produced by the parental viruses. Extensions of up to 26 nucleotides at the 3' end of the TYMV-BL clone delay infections, but virus particles isolated from these plants 4 weeks after inoculation contain RNA with the original TYMV-BL 3' terminus. A 90 nucleotide-long portion of the virion protein gene of TYMV-BL was replaced by a synthetic 90-mer primer with 16 nucleotide changes to decrease the large cytosine content (34-42%) characteristic of tymovirus genomic RNA. No reversion of any of the mutated nucleotides to cytosine occurred during 7 passages in Chinese cabbage. Hybrids between the TYMV-BL and TYMV-CL clones were also constructed, by exchanging various portions of the genome. However, it was not possible to determine definitively which part of the viral genome is responsible for the more severe symptoms caused by TYMV-BL as the hybrids gave intermediate symptoms.

The nucleotide sequences of the NS 1 genes from five Thai and three Sri Lankan dengue-2 viruses were determined by sequencing the viral RNA using synthetic oligonucleotide primers. The results were shown to be similar to four published dengue-2 NS 1 sequences and the classification of these genes was compared with the one obtained for the envelope genes of the same viruses. The classification was similar and showed that the Thai isolates could be divided into two separate groups and that the Sri Lankan isolates were distinct. We found no correlation between disease severity, serological response (1 degree or 2 degrees), or year of isolation and various aspects of NS 1 protein sequence variation; and no particular amino acid changes were correlated with virulence. The sequences were combined with those published and classified elsewhere to provide a comprehensive E/NS 1 gene taxonomy of dengue-2 virus isolates.

The nucleotide sequences of the envelope genes from five Thai and three Sri Lankan dengue-2 viruses were determined by sequencing the viral RNA using synthetic oligonucleotide primers. The results were compared with the four published dengue-2 envelope sequences to obtain a classification of these viruses, which showed that the Thai isolates could be divided into two separate groups while the Sri Lankan isolates were distinct. There was no correlation between disease severity and envelope protein sequence, or between year of isolation and sequence. No particular amino acid changes were associated with virulence or a change in hydrophilic region which could perhaps act as an epitope.