# Novel RNA Viruses Discovered in Weeds in Rice Fields
## 3 Results
### Characterization of Positive-Sense RNA Viruses
We identified 16 novel tombusviruses in 12 weed samples: Guiyang paspalum paspaloloides tombus-like virus (GPpTV) 1 to 2, Guiyang tombus-like virus (GTV) 1 to 3, Hangzhou tombus-like virus (HTV) 1 to 2, Sanya leptochlo chinensis tombus-like virus 1 (SLCTV1) and Sanya tombus-like virus (STV) 1 to 8. These newly identified viruses have genome lengths ranging from 2591 to 4811 bp, and they share 31.6% to 80.1% aa identities with the closely related viruses (Table S2). Notably, STV1 was detected in six weed samples in Sanya (Table S2). HTV1 and HTV2 were identified in the weeds _Alternanthera philoseroides_ and _Acalypha australis_ in Hangzhou, respectively. STV8 was identified in the weed _Lecersia hexandra_ in Sanya. In addition, four novel viruses (SLCTV1, STV2, STV3 and STV7) were identified in the weed _Leptochlo chinensis_ in Sanya, and three novel viruses (STV4 to 6) were detected in the weed _Monochoria vaginalis_ in Sanya. Both GPpTV1 and GPpTV2 were discovered in the weed _Paspalum distichum_ in Guiyang, while GTV1 to 3 were discovered in the weeds _Ranunculus japonicus_, _Paspalum thunbergii_ and _Plantago depressa_ in Guiyang, respectively (Table S2). In addition, STV1, SLCTV1, GPpTV1 and GPpTV2 displayed a high abundance ranging from 0.012 to 0.139, suggesting that _Leptochlo chinensis_ was a possible host of STV1 and SLCTV1, and weed _Paspalum distichum_ was possible host of GPpTV1 and GPpTV2. Phylogenetic analysis showed that GTV1, GTV2, STV1 to -3 and SLCTV1 were assigned into the _Procedovirinae_ subfamily, GPpTV1 and HTV2 clustered within the genus _Umbravirus_, GPpTV2 clustered within the genus _Dianthovirus_ and HTV1 was located outside the genus _Umbravirus_. The other six tombusviruses (STV4 to -8 and GTV3) clustered within the previously described tombusviruses from invertebrate species by high-throughput sequencing. Importantly, SLCTV1 was closely related to out chlorotic stunt virus (OCSV) (GenBankank no. NC_003633), the only member of the genus _Avenavirus_, and SLCTV1 shared 58.7% aa identity with OCSV (Figures 4 and S5B). GPpTV2 clustered within the subfamily _Regressovirinae_ containing Carnation ringspot virus (CRSV), red clover necrotic mosaic virus (RCNMV) and sweet clover necrotic mosaic virus (SCNMV), and they shared high aa sequence similarity (61.0% to 63.1% aa identity) (Figures 4 and S5C). SLCTV1 has a similar genome structure to the closely related viruses OCSV, and GPpTV2 has a similar genome structure to the closely related viruses CRSV, RCNMV and SCNMV (Figure S5A). Previous studies have shown that CRSV, RCNMV and SCNMV caused viral disease in host plants with different symptoms, such as leaf discoloration, ring spot and stunted plant growth [38]. Thus, SLCTV1 and GPpTV2 have the potential to infect cereal crops and cause diseases. **(vi) _Nodaviruses_**. Viruses in the family _Nodaviridae_ are divided into two genera: _Alphanodavirus_ and _Betanodavirus_. The genomes of the members were composed of two molecules of single-stranded positive-sense RNA: RNA1 (3.1 kb) encoding RdRp, and RNA2 (1.4 kb) encoding capsid protein [39]. We characterized Sanya nodal-like virus (SNV) as a novel member of the family _Nodaviridae_. SNV was detected in three weeds, _Leptochoa chinensis_, _Monochoria vaginalis_ and _Cyperus difformis_ in Sanya, and it shared 54.5%  
Figure 4: ML phylogenetic analysis of the family _Tombusviridae_. Phylogenetic trees were constructed using the aligned sequences of RdRp. The names of new viruses discovered in this study are marked in red, the reference virus serial numbers and names are marked in black and the families and genera of viruses (and branches in minimized trees) obtained in this study are marked in bold. The grey triangle means that the clade is all known viruses. The viruses were found in this study are not included in this clade. This clade is large, so it’s hidden. aa identities with the closely related virus (Table S2). The genome of SNV was 3843 bp in size.