# Novel RNA Viruses Discovered in Weeds in Rice Fields
## 3 Results
### Characterization of Positive-Sense RNA Viruses
GPDV1 and XAISV1 share 72.8% and 71.1% aa identities with the closely related viruses, respectively, and displayed a high abundance ranging from 0.030 to 3.916, suggesting that _Paspulum distichum_ was the possible host of GPDV1, and _Aeschynomene indica_ is a possible host of XAISV1. Phylogenetic analysis showed that GPDV1 and XAISV1 were surrounded by members of the _Selemoviridae_ family (Figure 3). XAISV1 clustered with alfalfa enamovirus-1 (AEV-1), pea enation mosaic virus 1 (PEMV-1) and grapevine enamovirus-1 (GEV-1), with short branch lengths and sharing high similarities (57% to 86% aa identities) (Figures 3 and S3C). However, GPDV1 was assigned into the genus _Polerovirus_ and formed a small clade with ScYLV and wheat leaf yellowing-associated virus (WLaV); GPDV1 shared 62.2% and 62.9% aa identities with ScYLV and WLYaV, respectively (Figures 3 and S3B). Importantly, plants infected by AEV-1, PEMV-1 and GEV-1 exhibited obvious symptoms such as leaf chlorosis or leaf stunting [29; 30; 31]. ScYLV and WLYaV can induce yellow leaf disease in sugarcane and wheat [28; 32]. GPDV1 has a similar genome structure to the closely related viruses WLYaV and ScYLV; XAISV1 has a similar genome structure to the closely related viruses AEV-1, PEMV-1 and GEV-1. Sequence similarity analysis and phylogenetic results showed that the two novel solemoviruses may have the potential to infect weeds and cause viral diseases (Figure S3A). **(iii) _Astroviruses._** The family _Astroviridae_ comprises two genera: _Mamastrovirus_, which infects mammals, and _Avastrovirus_, which infects birds. Many members of the family _Astroviridae_ infect plants and caused various viral diseases [33; 34]. Members of the family _Astroviridae_ have genome lengths ranging from 6.8 to 7.9 kb [35]. We characterized Sanya astro-like virus 1 (SAIV1) as a novel species of the family _Astroviridae_. SAIV1 was discovered in the weed _Leresia hexandra_ in Sanya and has a genome length of 4099 bp. This virus shared 57.6% aa identity with the closely related virus (Table S2). Phylogenetic analysis showed that SAIV1 clustered with the viruses, Astroviridae sp. and Bufivirus UC1, but appeared to represent a separate evolutionary lineage (Figure S4A). Phylogenetic analysis showed that the small branch comprising these four viruses may constitute a new genus of the family _Astroviridae_. Figure 3: ML phylogenetic analysis of the family _Solemoviridae_. 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. **(iv) _Marnaviruses_**. The family _Marnaviridae_ includes seven genera, _Salisharnavirus_, _Sogarnavirus_, _Bacillaranavirus_, _Locarnavirus_, _Labymraavirus_, _Kusarnavirus_ and _Marnavirus_, with positive-sense RNA genomes of 8.6 to 9.6 kb containing one or two ORFs [36]. We identified four viruses as novel members of the family _Marnaviridae_, Sanya marna-like virus (SMaLV) 1 to 3 and Xiaogan marna-like virus 1 (XMaLV1). They have genome lengths ranging from 5717 to 9184 bp and show 46.2% to 70.2% aa sequence similarity with the closely related viruses (Table S2). SMaLV1 was identified in weeds _Leptochlo chinensis_ and _Cyperus difformis_ in Sanya, while XMaLV1 was discovered in the weeds _Bidens tripartite_ and _Digitaria singuinalis_ in Xiaogan (Table S2). The phylogenetic analysis showed that SMaLV1, SMaLV2 and XMaLV1 were assigned into the genus _Locarnavirus_, while XMaLV1 clustered within the genus _Labymravirus_ (Figure S4B). **(v) _Tombusviruses_**. The members of the family _Tombusviridae_ are divided into three subfamilies, 16 genera and have non-segmented (monopartite) linear genomes 3.7 to 4.8 kb in size [37].