Positive selection and recombination shaped the large genetic differentiation of Beet black scorch virus population.

Beet black scorch virus (BBSV) is a species in the Betanecrovirus genus, in family Tombusviridae. BBSV infection is of considerable importance, causing economic losses to sugar beet (Beta vulgaris) field crops worldwide. Phylogenetic analyses using 3'UTR sequences divided most BBSV isolates into two main groups. Group I is composed of Iranian isolates from all Iranian provinces that have been sampled. Chinese, European, one North American and some other Iranian isolates from North-Western Iran are in Group II. The division of Iranian BBSV isolates into two groups suggests numerous independent infection events have occurred in Iran, possibly from isolated sources from unknown host(s) linked through the viral vector Olpidium. The between-group diversity was higher than the within-group diversity, indicating the role of a founder effect in the diversification of BBSV isolates. The high FST among BBSV populations differentiates BBSV groups. We found no indication of frequent gene flow between populations in Mid-Eurasia, East-Asia and Europe countries. Recombination analysis indicated an intra-recombination event in the Chinese Xinjiang/m81 isolate and an inter-recombination breakpoint in the viral 3'UTR of Iranian isolates in subgroup IranA in Group I. The ω ratios (dNS/dS) were used for detecting positive selection at individual codon sites. Amino acid sequences were conserved with ω from 0.040 to 0.229 in various proteins. In addition, a small fraction of amino acids in proteins RT-ORF1 (p82), ORF4 (p7b) and ORF6 (p24) are positively selected with ω > 1. This analysis could increase the understanding of protein structure and function and Betanecrovirus epidemiology. The recombination analysis shows that genomic exchanges are associated with the emergence of new BBSV strains. Such recombinational exchange analysis may provide new information about the evolution of Betanecrovirus diversity.

A new virus, classifiable in the family Tombusviridae, found infecting Solanum tuberosum in the UK.

A novel virus was discovered in a freeze-dried collection held at SASA, UK, originating from potato (Solanum tuberosum) cv. Nadine. The complete sequence of the viral RNA was determined to be 3674 nucleotides in length encoding five predicted proteins. Based on the deduced genome organization and phylogenetic analysis, this virus represents a putative new member of the genus Alphanecrovirus, family Tombusviridae, most closely related to isolates of Olive mild mosaic virus. The virus was easily transmitted to indicator plants with symptoms that were slower to develop and less severe than those of related viruses. To distinguish this virus, the clearest symptom differences occurred with Nicotiana debneyi, Chenopodium amaranticolor and Ch. quinoa. The virus was detected with antisera to the related viruses tobacco necrosis virus A and tobacco necrosis virus D. The close association to the tobacco necrosis viruses would suggest this virus is not a new introduction to potato but in the past has been misidentified as one of these viruses. The virus isolate has been named potato necrosis virus.

Two distinct sites are essential for virulent infection and support of variant satellite RNA replication in spontaneous beet black scorch virus variants.

Spontaneous point mutations of virus genomes are important in RNA virus evolution and often result in modifications of their biological properties. Spontaneous variants of beet black scorch virus (BBSV) and its satellite (sat) RNA were generated from cDNA clones by serial propagation in Chenopodium amaranticolor and Nicotiana benthamiana. Inoculation with recombinant RNAs synthesized in vitro revealed BBSV variants with divergent infectious phenotypes that affected either symptom expression or replication of satRNA variants. Sequence alignments showed a correlation between the phenotypes and distinct BBSV genomic loci in the 3'UTR or in the domain encoding the viral replicase. Comparative analysis between a virulent variant, BBSV-m294, and the wild-type (wt) BBSV by site-directed mutagenesis indicated that a single-nucleotide substitution of a uridine to a guanine at nt 3477 in the 3'UTR was responsible for significant increases in viral pathogenicity. Gain-of-function analyses demonstrated that the ability of the BBSV variants to support replication of variant satRNAs was mainly determined by aa 516 in the P82 replicase. In this case, an arginine substitution for a glutamine residue was essential for high levels of replication, and alterations of other residues surrounding position 516 in the wtBBSV isolate led to only minor phenotypic effects. These results provide evidence that divergence of virus functions affecting pathogenicity and supporting parasitic replication can be determined by a single genetic site, either a nucleotide or an amino acid. The results suggest that complex interactions occur between virus and associated satRNAs during virus evolution.

Molecular and serological characterization of an Iranian isolate of Beet black scorch virus.

An isolate of Beet black scorch virus (BBSV) was obtained from Iranian sugar beet roots. Its genome organization closely resembles that of the previously described Chinese and North American isolates, but the nucleotide sequences of the three isolates differ considerably. Most of the nucleotide exchanges, however, are silent, and the Iranian and the Chinese isolates were serologically indistinguishable. Beets infected by the Iranian BBSV did not show black scorch symptoms, but severe root beardedness. This might have been caused by BBSV or the simultaneously present beet necrotic yellow vein virus, or both together.

Analysis of the subgenomic RNAs and the small open reading frames of Beet black scorch virus.

A full-length cDNA of the genome of Beet black scorch virus (BBSV), isolate Ningxia, was constructed and modified by site-directed mutagenesis to permit in vitro transcription of mutant viral RNAs. Two subgenomic (sg) RNAs (sgRNA1 and sgRNA2) appeared during BBSV replication. Mutagenesis revealed that sgRNA1 transcription was initiated at G2209 within the P82 polymerase subunit open reading frame (ORF) and that transcription of sgRNA2 began at G2526 within the nested p7b/p5' ORF. Initiation-codon shifting or premature termination of translation of the three ORFs (P7a, P7b and P5') encoded by sgRNA1 indicated that each of the genes was required for localized movement, accumulation of viral RNAs and formation of local lesions on the leaves of Chenopodium amaranticolor. Microscopic observations of the distribution of green fluorescent protein fused to the N-terminal portion of the capsid protein provided additional evidence that the P7a, P7b and P5' proteins are each required for cell-to-cell movement. In contrast, elimination of sgRNA2 showed that the BBSV coat protein was not required for viral RNA accumulation or the appearance of local lesions on C. amaranticolor. In addition, disruption of the small P5 ORF previously predicted by computer analysis to originate at the C terminus of the P82 ORF had no effect on disease phenotype, suggesting that this ORF may represent a cryptic, non-essential gene. These results show that BBSV has a novel cell-to-cell movement protein organization that differs in size and sequence from that of other viruses.

The complete nucleotide sequence of Beet black scorch virus (BBSV), a new member of the genus Necrovirus.

The complete nucleotide sequence of Beet black scorch virus (BBSV) was determined. The BBSV genome is composed of 3641 nucleotides and has similar organization with Tobacco necrosis virus D of 61% nucleotide identity. The 5'-proximal open reading frame (ORF) encodes a putative 23 kDa protein and a 82 kDa protein by reading-through of an amber termination codon. Three small ORFs located in the center of the genome may encode for a 4.2 kDa protein and two 7 kDa proteins. The 3'-proximal ORF encodes a 24.5 kDa protein equivalent in mass to the viral coat protein. Considering biological and molecular similarities with TNV, it is concluded that BBSV is a new member of the genus Necrovirus.