Pod Pepper Vein Yellows Virus, a New Recombinant Polerovirus Infecting Pod Pepper in Yunnan Province, China

Kuangjie Zhao Ningbo University Yueyan Yin Yunnan Agricultural University Mengying Hua Ningbo University Shaoxiang Wang Wenshan Academy of Agricultural Sciences Xiaohan Mo Yunnan Academy of Tobacco Agricultural Sciences Enping Yuan Wenshan Academy of Agricultural Sciences Hongying Zheng Ningbo University Lin Lin Ningbo University Hairu Chen Yunnan Agricultural University Yuwen Lu Ningbo University Jianping Chen Ningbo University Jiejun Peng (  pengjiejun@yeah.net ) Ningbo University https://orcid.org/0000-0003-2594-9776 Fei Yan Ningbo University

We here identi ed a new recombinant of PeVYV that had identity to PeVYV-3 at 5' half of genome and had identity to TVDV at 3' part of genome.

Main Text
Pod pepper (Capsicum frutescens) is widely planted in China, especially around Wenshan city, Yunnan province, and viral diseases have now also become a major threat to pepper production in Yunnan. During July 2019, 89 pepper leaf samples were collected from three different elds in Wenshan. All had typical viral symptoms of interveinal leaf yellowing and fruit discoloration. Total RNAs were extracted from each of three pooled samples of these leaves. The total RNAs were sent for next-generation sequencing. De novo assembly generated 7 contigs, one of which was 5992 nt long and had high identities (>87.5%) to the genome of PeVYV-3 (Pepper vein yellows virus 3) (KP326573; [3] ). Primers designed from this sequence were then used to amplify a coat protein (CP) fragment of the virus (PeVYV-CP f: 5′-ATGAATACGGGAGGAGTTAGG -3′, PeVYV-CP r: 5′-CTATTTGGGGTTGTGCAGTTG -3′). Using RT-PCR, fragments of the expected size (621bp) were obtained in 58 of the 89 symptomatic samples but not from healthy plants (data not shown). The nucleotide sequences of the ampli ed fragments were all 93.4% identical to PeVYV-3. 5' and 3'RACE reactions (Invitrogen) were then performed to obtain the complete 5'and 3' terminal sequences of the new virus isolate. To avoid errors in sequence assembly, the whole viral sequence was then ampli ed using two overlapping sections with the primer pairs PeVYV-1 (PeVYV-1 f: 5′-ACAAAATATACGAAGAGAGAGAG -3′and PeVYV-1 r: 5′-TAACCCATCACTCCTCCCAC-3′) and PeVYV-2 (PeVYV-2 f: 5′-GGACAACTGGAATTCTGCTC-3′ and PeVYV-2 r: 5′-2800bp) were obtained using the methods of KOD-plus-Neo (Toyobo) and the two products (which overlapped by 1.4kb) were cloned into the pGEM-T Easy Vector (Promega) and sequenced. The complete sequence was 6015 nt long (GenBank accession number: MT188667). Because of its distinctive features it was named pod pepper vein yellows virus (PoPeVYV). PoPeVYV has a genome organization characteristic of members of the genus Polerovirus, with seven predicted genes encoding proteins P0 to P5 and P3a [6] (Fig. 1A). Over its entire genome our isolate is most closely related (85.3% nt identity) to Tobacco vein distorting virus (TVDV, accession EF529624) and had 78.9% identity to the (Chinese) PeVYV-3 (accession KP300822). However, if the different genes are compared separately, the new isolate is slightly more closely related to all the other PeVY viruses than to TVDV over most of the genome but diverges from them strongly in the 3'-end (encoding the coat protein readthrough P5 domain). The CP-RT P5 region (nts 4252-5790) of PoPeVYV has only 43.7% nt identity to the corresponding region of PeVYV-3, but 91.1% identity to that of TVDV. This is re ected in the phylogenetic analysis of the amino acid sequences of the separate gene products [9]: the P0 of PoPeVYV is most similar to PeVYV-1/4, P1/P2/P3/P4 are closely related to other PeVYVs but the P5/RTD is most similar to TVDV (Fig. 2). Poleroviruses are prone to recombination among themselves or with viruses belonging to other genera and the relationships between PoPeVYV, the previously described PeVYV isolates and TVDV suggests that there has been a recombination event affecting the 3'-end of the genome. This was con rmed using a variety of methods on the RDP4 recombinant platform [10]. A single recombination event affecting nucleotide positions 4126-5952, with TVDV and PeVYV-3 as the respective minor and major parents was consistently identi ed using GENECONV (P  (Fig. 1C). Alignment of amino acid sequences of the PoPeVYV proteins with those of the six species of PeVYVs and TVDV also indicated a recombination event (Table. 1).
Proteins encoded by the 5' half of the genome (P0 to P4) had the highest identity to those of PeVYV-3 and PeVYV-6, whereas proteins translated from the 3' half (P5/RTD) were more closely related to those of TVDV.
These results suggest that PoPeVYV is a new recombinant polerovirus. An infectious clone of the virus was constructed for further investigation. The full-length (6015 bp) cDNA was assembled and cloned into the 35S promoter of binary vector pCB301 using two overlapping fragments (from nucleotides 1-4615 and 4596-6015) and the CloneExpress MultiS One Step Cloning Kit (Vazyme). This was transformed into Agrobacterium tumefaciens which was then delivered to C. frutescens plantlets by in ltration. There was mild upward leaf curling 45 days after inoculation (Fig. 3A), and RT-PCR using primers to detect the coat protein gene in the newlyemerged non-inoculated leaves showed that viral RNA was present and had spread systemically in all the inoculated plants (12/12) but not in the controls (Fig. 3B). Virions were puri ed from C. frutescens leaves using the method described previously [11]. Isometric particles about 25nm in diameter were observed in the puri ed preparation from the inoculated plants (Fig. 3C) but not from the controls. These results demonstrated the infectivity of full-length PoPeVYV to C. frutescens. Recombination is an important source of genetic variability in viruses, particularly for viruses possessing an RNA genome. PeVYVs have higher identities to TVDV at 5' half of genome and are considered to be recombinant from TVDV and other poleroviruses [4,7,8]. The new recombinant identi ed here has higher identity to TVDV at 3' part of genome, indicating a different recombinant event of PeVYV. Recombination poses a problem for classi cation. The currently-recommended species demarcation criteria in the family Luteoviridae suggest that different species should have >10% difference in amino acid sequence identity in any gene product from their closest relative. The P0 and minor capsid (P3-P5) proteins of PoPeVYV have respectively 14.9-22.5% and 36.8-56.2% difference in amino acid identity to those of PeVYV1-6 (

Availability of data and materials
The complete genome sequences of PoPeVYV were submitted to the GenBank (http:// www.ncbi.nlm.nih.gov/genbank/) and the accession number is MT188667.