https://apsjournals.apsnet.org/doi/full/10.1094/PDIS-01-19-0028-PDN
Abstract.
Citrus virus A (CiVA) is a negative-sense, single-stranded RNA virus, recently identified from sweet orange (Navarro et al. 2018) and closely related to the citrus concave gun-associated virus (CCGaV), the type member of the recently described Coguvirus genus in the family Phenuiviridae, order Bunyavirales (Navarro et al. 2017, 2018). Plant-infecting coguviruses have only been discovered during the last year through high-throughput sequencing (HTS) approaches and reported in citrus (Navarro et al. 2017, 2018), watermelon (Xin et al. 2017), and apple (Wright et al. 2018). In spring 2017, leaves of various fruit tree species were collected and included in an HTS indexing assay. Double-stranded RNAs were purified from leaves as previously described (Candresse et al. 2013), and cDNA libraries were prepared and analyzed by Illumina HiSeq sequencing. Analysis of sequence data from a pear sample (Pyrus communis, cv. Packham’s Triumph, unsanitized source P1492 entered in collection in 1968) allowed the identification of 10 contigs (representing 0.03% of the sequencing reads) that shared 94 to 99% nucleotide (nt) sequence identity with the RNA1 (eight contigs) and RNA2 (two contigs) of CiVA (GenBank MG764565 and MG764566, respectively). In addition to CiVA, three known pome-fruit viruses were also detected in the sample: apple stem grooving virus, apple stem pitting virus, and apple rubbery wood virus 2 (the variety has been sanitized in 1970 in France). To confirm the presence of CiVA, total RNAs were extracted from the P1492 source and used as template for reverse transcription PCR (RT-PCR) experiments using specific primers designed from the various viral contigs and allowing to close the gaps between them. In addition, for the RNA1, the 5′ and 3′ termini were determined by RT-PCR using specific primers and terminal primers designed from the GenBank CiVA sequence, whereas only the 3′ end was similarly determined for the RNA2. The full-length RNA1 (6,690 nt) and the near complete RNA2 (2,195 nt, lacking only 545 nt at the 5′ end) sequences thus obtained have been deposited in GenBank under accession numbers MK273077 and MK273078, respectively. The pear isolate RNA1 shows 95.9% nt identity with that of the reference isolate from citrus, whereas the putative RNA-dependent RNA polymerases encoded on the viral complementary (vc) strand share 96.4% amino acid (aa) identity. The partial RNA2 of the pear isolate shares 96.8% nt identity with that of the reference citrus isolate, whereas the partial movement protein (ORF2a, viral strand) and complete nucleocapsid (ORF2b, vc strand) share respectively 95.2 and 96.8% aa identity with the corresponding proteins of the citrus CiVA isolate. In an effort to gain some information on CiVA prevalence in pear, a total of 29 unsanitized varieties were tested by RT-PCR as described above. Five of them (17%) were found to be infected, suggesting that CiVA infection was not rare in French pears 20 to 30 years ago at the time these varieties were entered in collection. To our knowledge, these results represent the first report of a natural infection of CiVA in pear, extending the host range of this virus and paralleling the recent discovery of CCGaV in apple (Wright et al. 2018). The original Packham’s Triumph pear had shown symptoms of pear vein yellows on young leaves, which is consistent with the HTS detection of apple stem pitting virus. Given the simultaneous presence of several other viruses in addition to CiVA in the P1492 pear, it is not possible to draw conclusions on potential pathogenic effects of CiVA in pear. Further efforts are now needed to clarify this point and determine the geographic distribution and prevalence of CiVA in this new host. The presence of CiVA isolates in citrus and pear also raises the question of the transmission mechanism(s) of this agent and of the potential existence of vectors.