First Report of Helleborus Net Necrosis Virus ( <i>Carlavirus necroretis</i> ) Associated with <i>Helleborus orientalis</i> in Australia

Hellebores (Helleborus spp.) are herbaceous perennial plants that are widely grown for their winter and early spring flowers (Eastwell et al. 2009; Liefting et al. 2010). In 2022, hellebores (Helleborus orientalis) exhibiting severe virus-like symptoms characterised by dark brown to black streaking on sepals, bracts, and stems, as well as irregularly shaped dark lesions on foliar tissues (Figure 1), were collected from a private garden located in the Blue Mountains region of New South Wales (NSW), Australia. It was noted that approximately 10% of the hellebore population in the garden exhibited symptoms. The collected symptomatic tissues were subjected to diagnostic screening at the NSW Department of Primary Industries and Regional Development Plant Health Diagnostic Service laboratory. The symptoms observed appeared consistent with those of hellebore black death, which is associated with Helleborus net necrosis virus (HeNNV, Carlavirus necroretis), a member of the Carlavirus genus (Eastwell et al. 2009; Liefting et al., 2010; Rubino 2024). A composite from multiple samples consisting of symptomatic sepal, bract, and leaf tissues was subjected to transmission electron microscopy following a negative-stain preparation. The electron microscopy analysis revealed filamentous virus-like particles measuring 650-700 nm length, with slightly flexuous and curved properties (Figure 2), which were consistent with those belonging to the Carlavirus genus. In addition, total RNA was extracted from a composite of multiple samples comprised of symptomatic sepal, bract, and leaf tissues using the Bioline ISOLATE II RNA Plant Kit (Meridian Bioscience), following the manufacturer’s instructions. The total RNA integrity was verified via High Sensitivity RNA ScreenTape Analysis (Agilent Technologies) and used to prepare an RNA-Seq library followed by sequencing as described in Maina et al. (2021), using the MiSeq cycle 2 x 251 v3 kit (Illumina). The obtained raw reads were trimmed and de novo assembled as described in Maina et al. (2021). Assembled contigs were subjected to BLASTn analysis, and a single contig of 8,534 nucleotides (nt) sharing 99.3% sequence identity with a HeNNV genome (GenBank accession NC_012038.1) was obtained. The contig of interest was represented by 2.6% of the total reads (79,136 reads out of 3,006,106 total reads), giving an average genome coverage of 1336x. The genome sequence was deposited in GenBank under accession PV165322 (isolate M22-15435). To confirm the presence of HeNNV in the original hellebore sample, specific primers targeting the HeNNV coat protein gene were designed (Sol_F-CCACTCAGCTCTCCAATTGTTA and Sol_R-CCTTCCTGTGCCTGGTTAAA) and used to conduct RT-PCR (at 57°C annealing temperature) using the original RNA sample followed by Sanger sequencing, yielding an amplicon of the expected size (604 bp) that shared 100% identity with the HTS contig. To our knowledge, this is the first detection and report of HeNNV infecting hellebores in Australia. The introduction of HeNNV to Australia may have inadvertently occurred through the importation of infected hellebore propagules. This HeNNV detection reinforces the importance of stringent biosecurity screening of imported plant propagules entering Australia. In addition, the identification of this virus contributes to the knowledge of hellebore black death aetiology. Furthermore, this report will enhance HeNNV awareness, assisting hellebore growers to adopt management strategies aimed at reducing the potential spread and impact of HeNNV disease in Australia.