Identiﬁcation and Molecular Characterization of a Novel Carlavirus Infecting Chrysanthemum morifolium in China

: Chrysanthemum ( Chrysanthemum morifolium ) is an important ornamental and medicinal plant suffering from many viruses and viroids worldwide. In this study, a new carlavirus, tentatively named Chinese isolate of Carya illinoinensis carlavirus 1 (CiCV1-CN), was identiﬁed from chrysanthe-mum plants in Zhejiang Province, China. The genome sequence of CiCV1-CN was 8795 nucleotides (nt) in length, with a 68-nt 5 (cid:48) -untranslated region (UTR) and a 76-nt 3 (cid:48) -UTR, which contained six predicted open reading frames (ORFs) that encode six corresponding proteins of various sizes. Phylogenetic analyses based on full-length genome and coat protein sequences revealed that CiCV1-CN is in an evolutionary branch with chrysanthemum virus R (CVR) in the Carlavirus genus. Pairwise sequence identity analysis showed that, except for CiCV1, CiCV1-CN has the highest whole-genome sequence identity of 71.3% to CVR-X6. At the amino acid level, the highest identities of predicted proteins encoded by the ORF1, ORF2, ORF3, ORF4, ORF5, and ORF6 of CiCV1-CN were 77.1% in the CVR-X21 ORF1, 80.3% in the CVR-X13 ORF2, 74.8% in the CVR-X21 ORF3, 60.9% in the CVR-BJ ORF4, 90.2% in the CVR-X6 and CVR-TX ORF5s, and 79.4% in the CVR-X21 ORF6. Furthermore, we also found a transient expression of the cysteine-rich protein (CRP) encoded by the ORF6 of CiCV1-CN in Nicotiana benthamiana plants using a potato virus X-based vector, which can result in a downward leaf curl and hypersensitive cell death over the time course. These results demonstrated that CiCV1-CN is a pathogenic virus and C. morifolium is a natural host of CiCV1


Introduction
Chrysanthemum (Chrysanthemum morifolium) is a herbaceous perennial plant in the family Asteraceae, and has important ornamental, economic, and medicinal value [1][2][3]. Chrysanthemum flowers have been made into traditional herbal remedies in China due to their potential effects on treating respiratory and cardiovascular diseases [4][5][6]. Commercially, the chrysanthemum is propagated mainly through the stem or root cuttings [7,8]. However, these reproduction methods frequently lead to the accumulation and spread of harmful pathogens [8,9], which finally cause the degradation of the chrysanthemum variety and reductions in flower yield and quality. In recent years, with the rapid expansion of chrysanthemum-growing areas, diseases caused by pathogens have become one of the most severe problems in chrysanthemum production worldwide [10][11][12][13][14].
In China, disease symptoms, such as chlorosis, mosaic, mottling, and stunting, are frequently detected in chrysanthemum plants. In 2019, the disease incidence of chrysanthemum was estimated at approximately 100% in Zhejiang Province, China, the main C. morifolium production region [15,16]. Astonishingly, viral and viroid infections cause up to

NGS and Sequence Assembly
Total RNA was extracted from infected leaf samples using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA purity and quantity were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and Nanodrop Spectrophotometer (Thermo Scientific, Waltham, MA, USA). The ribosomal RNA was removed by the Epicentre Ribo-Zero TM rRNA Removal Kit (Epicentre, Madison, WI, USA), and NGS was performed on the Illumina No-vaSeq 6000 platform (Illumina, San Diego, CA, USA) with a paired-end 150 bp set-up, as described previously [6,34,35]. After removing the adapter and low-quality sequences, the resulting clean reads were assembled using Trinity (v.2.14.0) [36]. The assembled contigs were subsequently searched against the NCBI viral (NCBI txid: 10239) sequence database

Amplification of the Full-Length Genome Sequence of CiCV1-CN
To confirm the NGS result and to understand the differences between CiCV1 isolates from different host plants, we cloned and sequenced the complete nucleotide sequence of CiCV1-CN isolated in C. morifolium from Zhejiang Province, China. To obtain the full-length genome sequence of CiCV1-CN, a rapid amplification of cDNA ends (RACE) technique was utilized [6,40]. We synthesized 5 -and 3 -RACE cDNAs using a SMARTer ® RACE 5 /3 Kit (Takara Bio Inc., Dalian, China), following the manufacturer s protocol. PCR amplification reactions were performed on a T100 PCR cycler (Bio-Rad, Pleasanton, CA, USA) using the KOD-plus DNA polymerase (Toyobo, Osaka, Japan). The primers used for the genome sequence cloning are listed in Supplementary Table S1. The obtained fulllength genome sequence of CiCV1-CN was submitted to the NCBI Genbank database (https: //www.ncbi.nlm.nih.gov/genbank/, accessed on 7 February 2023) under an accession number OQ410649.

Phylogenetic and Recombination Analyses
Multiple sequence alignment analyses of the genomes or proteins of CiCV1-CN and their closely related viruses were performed using the ClustalW program in the MEGA11 [42]. Phylogenetic trees were constructed using the MEGA11 [42] by the maximumlikelihood (ML) method with a GTR + G+I for genomes or by the neighbor-joining (NJ) method with a Jones-Taylor-Thornton (JTT) model for proteins, as described previously [6,38]. Recombination events were detected using the recombination detection program RDP4 (v4.101) [45], as described previously [13,33].

Plant Material and Growth Conditions
Wild-type Nicotiana benthamiana plants were used in this study, and they were grown in soil:vermiculite:perlite (3:3:1, v/v/v) at 25 ± 1 • C with a 16-h/8 h (light/dark) photoperiod, as described previously [46]. After 30 days of cultivation, the plants at the 5-leaf stage were used for viral inoculation.

PVX Construct and Viral Inoculation
To construct the PVX-based expression plasmids, the coding sequences of CRPs of CiCV1 and CiCV1-CN were cloned to PVX vector to generate PVX:CiCV1 CRP and PVX:CiCV1-CN CRP, respectively. After confirmation by sequencing, the constructed plasmids and empty vector were individually transferred into the Agrobacterium tumefaciens strain GV3101 by electroporation, as described previously [47,48]. The Agrobacteriummediated inoculation of N. benthamiana was performed as described previously [49,50]. Simultaneously, the PVX-based vector expressing a green fluorescent protein (GFP) (PVX:GFP) was used as a vector control.

RNA Extraction and Quantitative PCR (qPCR) Analysis
The inoculated N. benthamiana plants were photographed at 0, 7, and 14 days postinoculation (dpi); meanwhile, systemically infected leaves were sampled and frozen in liquid nitrogen. Total RNA extraction and cDNA synthesis were performed, as described previously [6]. qPCR was carried out on a CFX96 Touch Deep Well Real-Time PCR Detection System (Bio-Rad, Pleasanton, CA, USA), as described by Wang et al. [51]. N. benthamiana actin 2 (NbACT2) was used as an internal reference [46,52]. The relative viral RNA accumulation levels were calculated by the comparative C T method [53]. The reactions were performed in triplicate, and the results were averaged. The primers used for qPCR are listed in Supplementary Table S1.

Statistical Analysis
The data are presented as means ± standard deviation (SD) of three independent biological replicates. The statistical significance of differences was calculated using a Student s t-test in Microsoft Excel (v. 2021, Microsoft Corp., Redmond, WA, USA). A p-value of less than 0.05 (p < 0.05) was considered statistically significant.
To obtain the full-length genome sequence of CiCV1-CN, 5 /3 RACE technique and three-segment amplification strategy were used, and the sizes for the 5 and 3 ends and the three internal genomic fragments were 565, 990, 3753, 1861, and 2115, respectively (Figure 1b

Phylogenetic and Recombination Analyses of CiCV1-CN
To further investigate the evolutionary relationship between CiCV1-CN and other carlaviruses, we constructed phylogenetic trees at the genome and protein levels. The phylogenetic analysis of the full-length genomes of CiCV1-CN and the 31 reported carlaviruses indicated that CiCV1-CN and CiCV-1 clustered together and formed a smaller branch adjacent to the CVR subcluster ( Figure 2a). Pairwise sequence identity analysis showed that, except for CiCV1, CiCV1-CN has the highest whole-genome sequence identity of 71.3% to CVR-X6 and the lowest identity of 53.9% to cowpea mild mottle virus (CPMMV) (Supplementary Table S4). These sequence identities met the current species demarcation criteria for the Carlavirus genus [27,54,55]. These findings, therefore, suggest that CiCV1-CN is a new species of Carlavirus, which possesses a closer evolutionary relationship with Carlavirus CVR. Furthermore, the phylogenetic tree based on the CP sequences showed a similar clustering result (Figure 2b). Further amino acid sequence alignment demonstrated that CiCV1-CN CP displays high-sequence identities to CVR CPs (88.9-96.4%) ( Figure  S1a). Conserved domain analysis based on the InterPro (http://www.ebi.ac.uk/interpro/, accessed on 30 August 2022) [44] indicated that CiCV1-CN CP has a Carlavirus_coat_N domain (IPR013569, 62-112 aa) and a Pltvir_coat domain (IPR000052, 121-261 aa) in its N and C termini, respectively (Figure S1b), suggesting that CiCV1-CN CP possesses the  [27,54,55]. These findings, therefore, suggest that CiCV1-CN is a new species of Carlavirus, which possesses a closer evolutionary relationship with Carlavirus CVR. Furthermore, the phylogenetic tree based on the CP sequences showed a similar clustering result (Figure 2b). Further amino acid sequence alignment demonstrated that CiCV1-CN CP displays high-sequence identities to CVR CPs (88.9-96.4%) ( Figure S1a). Conserved domain analysis based on the InterPro (http://www.ebi.ac.uk/interpro/, accessed on 30 August 2022) [44] indicated that CiCV1-CN CP has a Carlavirus_coat_N domain (IPR013569, 62-112 aa) and a Pltvir_coat domain (IPR000052, 121-261 aa) in its N and C termini, respectively (Figure S1b), suggesting that CiCV1-CN CP possesses the typical properties of CPs of carlaviruses. Together, these results indicate that CiCV1-CN is a new virus species of Carlavirus from C. morifolium. Previously, studies have shown that RNA recombinations are frequently observed in CVB and CVR viruses [13,31,33]. Therefore, we determined whether the CiCV1-CN has RNA recombinations with other carlaviruses. However, no recombination event was detected in the genome of CiCV1-CN (Supplementary Table S5).

Sequence Identity Analysis of Carlaviruses from C. morifolium
To further examine the sequence identities of CiCV1-CN with the other carlaviruses from C. morifolium, we analyzed the nucleotide and amino acid sequences of CiCV1-CN and the 17 reported carlaviruses identified from C. morifolium. The results revealed that CiCV1-CN had sequence identities from 71.3% to 56.2% in the other carlaviruses identified from C. morifolium at the whole-genome level (Table 1). Interestingly, the 5 -UTR CiCV1-CN showed the highest identity of 84.5% in the 5 -UTRs of CVR-ZJHU1, CVR-ZJHU2, and CVR-TX, while the 3 -UTR displayed the highest identity of 88.5% only in the 3 -UTR of CVR-TX (Table 1). At the amino acid level, the highest identities of predicted proteins encoded by the ORF1, ORF2, ORF3, ORF4, ORF5, and ORF6 were 77.1% in the CVR-X21 ORF1, 80.3% in the CVR-X13 ORF2, 74.8% in the CVR-X21 ORF3, 60.9% in the CVR-BJ ORF4, 90.2% in the CVR-X6 and CVR-TX ORF5s, and 79.4% in the CVR-X21 ORF6, respectively (Table 1). Next, we performed pairwise matrix comparisons of CiCV1-CN amino acid sequences of the whole replicase-related proteins and CPs among the homologs of carlaviruses identified from C. morifolium using heat maps. As shown in Figure 3, CiCV1-CN dramatically clustered together with high-sequence identities to Carlavirus CVR at both the replicaserelated protein and the CP levels. These results further suggest that CiCV1-CN is closer to the Carlavirus CVR.
Next, we performed pairwise matrix comparisons of CiCV1-CN amino acid sequences of the whole replicase-related proteins and CPs among the homologs of carlaviruses identified from C. morifolium using heat maps. As shown in Figure 3, CiCV1-CN dramatically clustered together with high-sequence identities to Carlavirus CVR at both the replicase-related protein and the CP levels. These results further suggest that CiCV1-CN is closer to the Carlavirus CVR.

Phylogenetic and Sequence Analyses of CRP Proteins of Carlaviruses from C. morifolium
Previous studies have shown that the CRPs of the genus Carlavirus play a crucial role in causing viral symptoms [13,30,56]. We, therefore, examined the phylogenetic relationships of CiCV1-CN CRP with the other CRPs encoded by carlaviruses from C. morifolium. As expected, CiCV1-CN CRP was clustered closely with the CRPs encoded by the Carlavirus CVR (Figure 4a). This result suggests that the relationship between CiCV1-CN CRP and Carlavirus CVR CRPs is closer during the evolution. Further amino acid sequence alignment of CRPs showed that CiCV1-CN CRP had a conserved Carlavirus nucleic acid binding domain (IPR002568, 8-92 aa) (Figure 4b). Furthermore, CiCV1-CN CRP was also predicted to have an arginine-rich NLS motif ( 47 RRRR 50 ) and a ZF motif ( 57 CX2CX12CX4C 78 )

Phylogenetic and Sequence Analyses of CRP Proteins of Carlaviruses from C. morifolium
Previous studies have shown that the CRPs of the genus Carlavirus play a crucial role in causing viral symptoms [13,30,56]. We, therefore, examined the phylogenetic relationships of CiCV1-CN CRP with the other CRPs encoded by carlaviruses from C. morifolium. As expected, CiCV1-CN CRP was clustered closely with the CRPs encoded by the Carlavirus CVR (Figure 4a). This result suggests that the relationship between CiCV1-CN CRP and Carlavirus CVR CRPs is closer during the evolution. Further amino acid sequence alignment of CRPs showed that CiCV1-CN CRP had a conserved Carlavirus nucleic acid binding domain (IPR002568, 8-92 aa) (Figure 4b). Furthermore, CiCV1-CN CRP was also predicted to have an arginine-rich NLS motif ( 47 RRRR 50 ) and a ZF motif ( 57 CX 2 CX 12 CX 4 C 78 ) located adjacent to the NLS (Figure 4b). These results suggest that CiCV1-CN CRP may be a potential pathogenicity factor during viral infection. located adjacent to the NLS (Figure 4b). These results suggest that CiCV1-CN CRP may be a potential pathogenicity factor during viral infection.

CiCV1-CN CRP Is a Pathogenicity Factor of CiCV1-CN
To further determine the roles of CiCV1 CRP and CiCV1-CN CRP in the viral infection, we transiently expressed them in wild-type N. benthamiana plants using the PVX-based vector. Compared with N. benthamiana seedlings agro-inoculated with PVX:GFP, plants agro-inoculated with either PVX:CiCV1 CRP or PVX:CiCV1-CN CRP showed typical downward leaf curl at 7 dpi, especially those agro-inoculated with PVX:CiCV1-CN CRP (Figure 5a). Notably, N. benthamiana seedlings agro-inoculated with both PVX:CiCV1 CRP, and PVX:CiCV1-CN CRP exhibited a severe downward leaf curl with hypersensitive cell death at 14 dpi (Figure 5a). To verify the above observations, we determined the RNA accumulation of CRPs of CiCV1 and CiCV1-CN using qPCR. The results showed that CRP transcripts were significantly accumulated in the systemic leaves of N. benthamiana plants agro-inoculated with either PVX:CiCV1 CRP or PVX:CiCV1-CN CRP at 7 and 14 dpi (Figure 5b). Furthermore, we also found that the transcripts of PVX

CiCV1-CN CRP Is a Pathogenicity Factor of CiCV1-CN
To further determine the roles of CiCV1 CRP and CiCV1-CN CRP in the viral infection, we transiently expressed them in wild-type N. benthamiana plants using the PVXbased vector. Compared with N. benthamiana seedlings agro-inoculated with PVX:GFP, plants agro-inoculated with either PVX:CiCV1 CRP or PVX:CiCV1-CN CRP showed typical downward leaf curl at 7 dpi, especially those agro-inoculated with PVX:CiCV1-CN CRP (Figure 5a). Notably, N. benthamiana seedlings agro-inoculated with both PVX:CiCV1 CRP, and PVX:CiCV1-CN CRP exhibited a severe downward leaf curl with hypersensitive cell death at 14 dpi (Figure 5a). To verify the above observations, we determined the RNA accumulation of CRPs of CiCV1 and CiCV1-CN using qPCR. The results showed that CRP transcripts were significantly accumulated in the systemic leaves of N. benthamiana plants agro-inoculated with either PVX:CiCV1 CRP or PVX:CiCV1-CN CRP at 7 and 14 dpi (Figure 5b). Furthermore, we also found that the transcripts of PVX CP were dramatically  was used as an internal reference. The data are presented as means ± standard deviation of three biological replicates. Significant differences in expression are marked with asterisks: * p < 0.01, ** p < 0.01, or *** p < 0.001; Student s t-test. ns, not significant.

Discussion
Previous studies have shown that the chrysanthemum is one of the most susceptible plants to viral infections, and more than 20 viruses and viroids have been identified to infect the chrysanthemum plants up to now [6,13,16]. In this study, a new full-length isolate of CiCV1 with 8795 nt was identified in C. morifolium from Zhejiang Province, China, which was tentatively named the Chinese isolate of CiCV1 (CiCV1-CN). The genome of CiCV1-CN contained a 68-nt 5 -UTR and a 76-nt 3 -UTR, and encoded six putative viral proteins (Figure 1d). According to the species demarcation criteria established for the Carlavirus genus [27,54,55], CiCV1-CN is a new carlavirus species with a closer evolutionary relationship with Carlavirus CVR (Figures 2 and 3).
It has been shown that viral symptoms caused by the genus Carlavirus depend mainly on the types of viruses and host plants [26,29,60]. Previous studies have revealed that CRPs encoded by carlaviruses isolated from C. morifolium are responsible for symptom generation during viral infections [6,13,33]. For example, the CRP encoded by CVB (CVB CRP) is frequently characterized as a pathogenicity factor and a viral suppressor of RNA silencing, and the transient overexpression of CVB CRP using a PVX-based vector in N. tabacum and N. benthamiana plants can induce a hypersensitive response [30,56,61,62]. In our study, when CiCV1-CN CRP was transiently expressed in N. benthamiana using a PVXbased vector, severe downward leaf curl and hypersensitive cell death were observed in the PVX:CiCV1-CN CRP-inoculated plants ( Figure 5). These results provide new evidence that CRPs encoded by carlaviruses isolated from C. morifolium are pathogenicity factors. These findings improve our understanding of the molecular mechanisms of the transmission and pathogenesis of carlaviruses.
During the past decade, NGS technology has been extensively utilized to facilitate the discovery of new viruses and viroids [63][64][65][66][67][68]. Many new plant virus species have been identified and characterized using the NGS, including viruses that infect crops, vegetables, ornamentals, and tree plants [69][70][71]. NGS technology has also been used to identify new isolates and variants of existing plant viruses, which have emerged due to the high mutation rates and genetic diversity among plant viruses [64,66,72]. In addition, NGS-based diagnostic and screening tools have been developed to detect these new viruses quickly and accurately, facilitating their management and control in crop production [66][67][68]72]. Collectively, NGS technology has opened up new avenues for the detection, discovery, and characterization of plant viruses. The continued implementation and advancement of NGS technology in plant virology will undoubtedly lead to new discoveries and improved methods for managing plant viral diseases.

Conclusions
In this study, we reported the complete genome sequence of a Chinese isolate of CiCV1 (CiCV1-CN) from C. morifolium plants in Zhejiang Province, China, the main C. morifolium production region, with molecular properties to those of members of the Carlavirus genus. Furthermore, we confirmed that the CiCV1-CN CRP protein is a pathogenicity factor of CiCV1-CN and can elicit hypersensitive cell death in N. benthamiana plants. These results also revealed that the combination of NGS, bioinformatics, and PVX-based expression analysis is a helpful method for discovering new hosts for plant viruses.

Data Availability Statement:
The genome sequence of the Chinese isolate of Carya illinoinensis carlavirus 1 (CiCV1-CN) has been deposited in the NCBI GenBank (https://www.ncbi.nlm.nih.gov/ genbank/ (accessed on 7 February 2023)) under an accession number OQ410649. The other data presented in this study are available in this manuscript.