- Volume 95, Issue 3, 2014

Phylogenomic evidence suggested that recombination is an important evolutionary force for potyviruses, one of the larger families of plant RNA viruses. However, mixed-genotype potyvirus infections are marked by low levels of cellular coinfection, precluding template switching and recombination events between virus genotypes during genomic RNA replication. To reconcile these conflicting observations, we evaluated the in vivo recombination rate (r g) of Tobacco etch virus (TEV; genus Potyvirus, family Potyviridae) by coinfecting plants with pairs of genotypes marked with engineered restriction sites as neutral markers. The recombination rate was then estimated using two different approaches: (i) a classical approach that assumed recombination between marked genotypes can occur in the whole virus population, rendering an estimate of r g = 7.762×10−8 recombination events per nucleotide site per generation, and (ii) an alternative method that assumed recombination between marked genotypes can occur only in coinfected cells, rendering a much higher estimate of r g = 3.427×10−5 recombination events per nucleotide site per generation. This last estimate is similar to the TEV mutation rate, suggesting that recombination should be at least as important as point mutation in creating variability. Finally, we compared our mutation and recombination rate estimates to those reported for animal RNA viruses. Our analysis suggested that high recombination rates may be an unavoidable consequence of selection for fast replication at the cost of low fidelity.

Rosellinia necatrix megabirnavirus 1 (RnMBV1) with a bipartite dsRNA genome (dsRNA1 and dsRNA2) confers hypovirulence to its natural host, the white root rot fungus, and is thus regarded as a potential virocontrol (biocontrol) agent. Each segment has two large ORFs: ORF1 and partially overlapping ORF2 on dsRNA1 encode the major capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), whilst ORF3 and ORF4 on dsRNA2 encode polypeptides with unknown functions. Here, we report the biological and molecular characterization of this virus in the chestnut blight fungus, Cryphonectria parasitica, a filamentous fungus that has been used as a model for mycovirus research. Transfection with purified RnMBV1 particles into an RNA-silencing-defective strain (Δdcl-2) of C. parasitica and subsequent anastomosis with the WT strain (EP155) resulted in stable persistent infection in both host strains. However, accumulation levels in the two strains were different, being ~20-fold higher in Δdcl-2 than in EP155. Intriguingly, whilst RnMBV1 reduced both virulence and growth rate in Δdcl-2, it attenuated virulence without affecting significantly other traits in EP155. Western blot analysis using antiserum against recombinant proteins encoded by either ORF1 or ORF2 demonstrated the presence of a 250 kDa protein in purified virion preparations, suggesting that RdRp is expressed as a CP fusion product via a −1 frameshift. Antiserum against the ORF3-encoded protein allowed the detection of 150, 30 and 23 kDa polypeptides specifically in RnMBV1-infected mycelia. Some properties of an RnMBV1 mutant with genome rearrangements, which occurred after transfection of Δdcl-2 and EP155, were also presented. This study provides an additional example of C. parasitica serving as a versatile, heterologous fungus for exploring virus–host interactions and virus gene expression strategies.

Bacteriophages have been found to be the most abundant and also potentially most diverse biological entities on Earth. In the present study, Bacillus phages were isolated rapidly and shown to have a high degree of diversity. The genomes of a newly isolated phage, phiCM3, and a prophage, proCM3, from the Bacillus thuringiensis strain YM-03 were sequenced and characterized. Comparative genome analysis showed that the phiCM3 genome is highly similar to the genomes of eight other Bacillus phages and seven of these phages were classified as the Wβ group of phages. Analysis of the differential evolution of the genes in the Wβ-group phages indicated that the genes encoding the antirepressor and tail fibre protein were more highly conserved than those encoding the major capsid protein, DNA replication protein, and RNA polymerase σ factor, which might have diverged to acquire mechanisms suitable for survival in different microbial hosts. Genome analysis of proCM3 revealed that proCM3 might be a defective phage because of mutations in the minor structural protein, and it was not inducible by mitomycin C treatment. The proCM3 genome was similar to those of two lytic Bacillus phages in sequence, but had a different genomic structure, composed of three regions in a different order. These data suggest that the three phages might have had a common ancestor and that genome rearrangement might have occurred during evolution. The findings of this study enrich our current knowledge of Bacillus phage diversity and evolution, especially for the Wβ-group and TP21-L-like phages, and may help the development of practical applications of Bacillus phages.