In July 2019 , a sample of Rosa spp . was submitted to Fera Science Ltd. via the Royal Horticultural Society ( RHS ) gardening advice avail . The sample ( ID220 ) was sent in following the appearance of nameless symptom including mottling , chicken / blank patching , sparse texture and a pink colour in the leaves .
RNA was extract using a CTAB method acting adjust from Adams et al . ( 2009 ) , with the 4 M LiCl brooding perform overnight at 4 ° C . The sample was tested for common rose virus using RT - qPCR ( Table 1 ) . A positivist result was achieved for Rose cryptic computer virus 1 . later , the sample distribution was analyse by high throughput sequencing ( HTS ) using a TruSeq Stranded full RNA Library Prep Plant kit ( Illumina Inc. , USA ) for library preparation . A MiSeq instrument and a MiSeq Reagent Kit v3 ( 600 - cycle ) ( Illumina Inc. ) were used to start the library . The trial generated 569,452 reads for the sample , and data point was analysed as draw by Fox et al . ( 2019 ) . Three fragments of roseate natural spring midget - link up virus ( RSDaV ) were place ( 234 , 251 and 229 bp ; GenBank Accession Nos . MT993839 - MT993841 ) . A BLAST+ search found sequences with in high spirits chronological succession identicalness in both nucleotide ( 92.11 - 93.59 % identity , EU024678.1 ) and amino group dose comparisons ( 94.34 - 100 % , YP_001949737.1 ; YP_001949736.1 ; YP_001949738.1 ) . RT - PCR amplification using specific fusee ( Salem et al . , 2008 ) was performed to sustain the result , and a product of the expected size ( 418 bp ) was obtained .
To assess the scatter of RSDaV in the UK , 171 roses were analyse using the RT - PCR check . Samples were garner as part of a view of rose viruses in the UK and both symptomless and diagnostic leaf samples , ordered with computer virus infection symptom ( mottling , xanthous veining , distortion , and ringspots ) were included . Only one sample ( ID140 ) resulted convinced for RSDaV , and no symptom were identified . Previous analysis showed this sample distribution was positive for Arabis mosaic computer virus by ELISA and RT - qPCR .
The RT - PCR product ( 418 bp ) from both RSDaV - positive samples ( ID220 , 140 ) were sequenced , and nucleotide comparisons showed a 98.51 - 99.02 % identity with sequences in GenBank ( HM236366.1 ; HM2363641 ; HM236362.1 ; HM236364.1 ) . Amino acid comparison showed a 98.51 - 100 % personal identity with previously publish sequences ( ADK78852.1 ; ADK78851.1 ) .
RSDaV has previously been get in the USA ( Salem et al . , 2008 ) , Chile ( Rivera & Engel , 2010 ) , and New Zealand ( Milleza et al . , 2013 ) . This is the first write up of RSDaV in Europe . Further samples ( 4 ) were present to the RHS and Fera Science Ltd. Plant Clinic , show the antecedently described unknown symptoms . They were tested by RT - PCR and also sequence by HTS and tested negative for RSDaV. The suit of these symptoms is not believe to be of viral origin .
beginning : BSPP New Disease Reports ( I. Vazquez - Iglesias , J. Scrace , S. McGreig , H. Pufal , R. Robinson , G.R.G. Clover , I.P. Adams , N. Boonham and A. Fox )
