Molecular characterization of birch toti-like virus, a plant-associated member of the new family Orthototiviridae.

A novel totivirus, named "birch toti-like virus" (BTLV), was discovered in European white birch (Betula pendula) plants. The genome of BTLV is 4,967 nucleotides long and contains two overlapping open reading frames (ORFs) coding for the capsid protein (CP) and an RNA-dependent RNA-polymerase (RdRP). The encoded CP and RdRP proteins shared 46.9% and 60.2% amino acid sequence identity, respectively, with those of Panax notoginseng virus B. The presence of a putative slippery heptamer signal 82 nt upstream of the stop codon of ORF1 suggests that a -1 translational frameshifting strategy is involved in the expression of ORF2, like in other totiviruses. Phylogenetic analysis based on the CP and RdRP amino acid sequences placed this virus within a clade of plant-associated totiviruses, with taro-associated virus as its closest relative. Hence, based on its distinct host and the amino acid sequence similarity between BTLV and its relatives, we conclude that birch toti-like virus is a new member of the genus Totivirus.

Unravelling the virome in birch: RNA-Seq reveals a complex of known and novel viruses.

To unravel the virome in birch trees of German and Finnish origin exhibiting symptoms of birch leaf-roll disease (BRLD), high-throughput sequencing (HTS) was employed. In total five viruses, among which three were so far unknown, were detected by RNAseq. One to five virus variants were identified in the transcriptome of individual trees. The novel viruses were genetically-fully or partially-characterized, belonging to the genera Carlavirus, Idaeovirus and Capillovirus and are tentatively named birch carlavirus, birch idaeovirus, and birch capillovirus, respectively. The recently discovered birch leafroll-associated virus was systematically detected by HTS in symptomatic seedlings but not in symptomless ones. The new carlavirus was detected only in one of the three symptomatic seedlings. The novel putative Capillovirus was detected in all seedlings-irrespective of their BLRD status-while the Idaeovirus was identified in a plant without leaf symptoms at the time of sampling. Further efforts are needed to complete Koch's postulates and to clarify the possible association of the detected viruses with the BLR disease. Our study elucidates the viral population in single birch seedlings and provides a comprehensive overview for the diversities of the viral communities they harbor, to date.

A novel badnavirus discovered from Betula sp. affected by birch leaf-roll disease.

In declining birches (Betula sp.) from different European stands affected by the "birch leaf-roll disease" (BLRD) a novel virus is identified by means of RNA-Seq virome analysis. The virus represents a new member in the genus Badnavirus, family Caulimoviridae, tentatively named Birch leaf roll-associated virus (BLRaV) and it is the first badnavirus found to infect birch. Complete genome sequences (7,862-7,864 nucleotides) of three viral isolates of Finnish and German origin have been determined. The virus sequences show a typical badnavirus organization with three major open reading frames (ORFs) and a fourth potential ORF overlapping with the end of ORF3. ORFs 1-2-3 show low level of amino acid identity to the corresponding proteins encoded by other badnaviruses, reaching a maximum of 44% identity (ORF3). Grapevine vein-clearing virus appears as the closest badnavirus when considering the polymerase region. So far, we can exclude evidence for presence of endogenous BLRaV elements in the birch genome, while evidence for the episomal activity of BLRaV is provided. The viral population holds significant haplotype diversity, while co-infection by different BLRaV variants are observed in single hosts. BLRaV presence is associated with the BLRD in both silver (B. pendula) and downy birch (B. pubescens). These results challenge the earlier hypothesis of a causal role of Cherry leaf roll virus in BLRD. Further work is now needed to finally prove that BLRaV is the causal agent for the BLRD.

Disinfection of foot-and-mouth disease and African swine fever viruses with citric acid and sodium hypochlorite on birch wood carriers.

Transboundary animal disease viruses such as foot-and-mouth disease virus (FMDV) and African swine fever virus (ASFV) are highly contagious and cause severe morbidity and mortality in livestock. Proper disinfection during an outbreak can help prevent virus spread and will shorten the time for contaminated agriculture facilities to return to food production. Wood surfaces are prevalent at these locations, but there is no standardized method for porous surface disinfection; commercial disinfectants are only certified for use on hard, nonporous surfaces. To model porous surface disinfection in the laboratory, FMDV and ASFV stocks were dried on wood coupons and exposed to citric acid or sodium hypochlorite. We found that 2% citric acid was effective at inactivating both viruses dried on a wood surface by 30 min at 22°C. While 2000 ppm sodium hypochlorite was capable of inactivating ASFV on wood under these conditions, this chemical did not meet the 4-log disinfection threshold for FMDV. Taken together, our data supports the use of chemical disinfectants containing at least 2% citric acid for porous surface disinfection of FMDV and ASFV.

Differentiation of Cherry leaf roll virus isolates from various host plants by immunocapture-reverse transcription-polymerase chain reaction-restriction fragment length polymorphism according to phylogenetic relations.

A restriction fragment length polymorphism assay (RFLP) was developed to differentiate Cherry leaf roll virus (CLRV) isolates according to phylogenetic clades by examining restriction patterns from partial 3' non-coding region (NCR) genomic fragments (approx. 420bp). The 3' NCR fragment from 43 CLRV isolates belonging to different phylogenetic groups were compared after restriction analysis with the endonucleases Bsp143I, AluI, RsaI, EcoRI and Eco130I, and another 23 isolates were analyzed by computer assisted restriction analysis. The restriction endonucleases Bsp143I, AluI and RsaI enabled the differentiation of isolates from group B and all but two isolates belonging to group A. A major proportion of group E isolates could also be discriminated. The remainder of the group E isolates were indistinguishable from isolates belonging to phylogenetic group C or D2. Isolates belonging to group D1 could not be differentiated from two group A isolates. The method was applied successfully in an IC-RT-PCR-RFLP assay to differentiate samples from walnut, black elderberry and birch and determine their phylogenetic relationships. In future, this method will facilitate rapid phylogenetic classification of CLRV isolates detected in certain host plants by the universal immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR), and will be suitable for studying CLRV population diversity as well as genetic drift within virus populations.