Analysis of Genomic Genetic Diversity of Cucurbit Aphid-borne Yellows Virus

Wei CHEN, Lina NIU, Zhengxiu CUI, Yongping LUO, Juan HE, Min CHAI, Maimaiti YUSHANJIANG

Abstract [Objectives] This study was conducted to investigate the molecular characteristics， genetic structure and evolutionary mechanism of cucurbit aphid-borne yellows virus （CABYV）， which is of great significance for clarifying the epidemic laws of the disease in the field and formulating long-term sustainable control strategies.

[Methods]120 melon leaves suspected to be infected with CABYV were randomly collected from Aksu City， Xinjiang. RT-PCR was used to detect the virus. MEGA 7.0 was used to construct a phylogenetic tree. DnaSP v5.10 was used to analyze the genetic diversity among different sequences， and RDP v.4.31 software was used to analyze possible recombination events of CABYV sequence.

[Results] The detection rate of CABYV was 31.67%. A new CABYV-2 was isolated， sequenced and cloned from these positive samples. The length of the genome is 5 497 bp， encoding six open reading frames. Compared with 23 CABYV strains isolated from different countries in NCBI database， CABYV-2 shared the highest homology with KR231942.1 and the lowest homology with JF939812.1. The results of evolutionary tree analysis showed that the 24 isolates were divided into two branches due to different geographical factors， including CABYV-2 and KR231942 1， which were clustered together and belonged to branch 1. The results of genetic diversity and neutrality tests showed that CABYV was highly variable and the population was expanding. The results of recombination analysis showed that there were two recombinants， EU636992.1 and KR231949.1， which exacerbated the variation of CABYV.

[Conclusions]The CABYV isolate from Aksu City， Xinjiang had the closest relationship with the Korean isolate KR231942.1， and the farthest relationship with the isolates from European countries. There were large genetic differences between the isolates of branch 1 and branch 2， and the recombination of CABYV in different hosts aggravated the variation of CABYV. Recombination and negative selection may be important reasons for the genetic variation of CABYV.

Key words CABYV; Genome cloning; Genetic diversity

Received： February 21， 2022  Accepted： May 23， 2022

Supported by National Natural Science Foundation of China （31760511）; Linfen Key R&D Program （2009）; 2020 National Key R&D Program-Technology Helps the Economy 2020 Key Project （2020-NK-ZL34）; Fundamental Research Program of Shanxi Province （201901D211403）.

Wei CHEN （1985-）， male， P. R. China， associate professor， devoted to research about plant viruses.

*Corresponding author. Maimaiti YUSHANJIANG （1978-）， male. P. R. China， associate professor， devoted to research about plant viruses. E-mail： yusanjan418@163.com.

Xinjiangs special geographical location and climatic characteristics make it an important melon production base in China. With the increasing planting area of melons in Xinjiang， the yield of melons is increasing day by day， and it has become an important economic growth point for agricultural production in Xinjiang. Virus diseases have always been important diseases affecting the cultivation of melons in Xinjiang. Melon plants infected by virus diseases will have various symptoms such as leaf deformity， mosaic leaves and yellowing， which will eventually lead to a serious decline in the fruit quality and yield of melons. Therefore， clarifying the occurrence， population structure and genetic diversity of cucurbit aphid-borne yellows virus （CABYV） is of great significance for the effective prevention and control of viruses. Many domestic scholars have identified the viruses infecting melon crops in Xinjiang and found that the viruses infecting melons in Xinjiang mainly include watermelon mosaic virus （WMV）， cucumber mosaic virus （CMV）， tobacco necrosis virus （TNV）， melon necrotic spot virus （MVNV） and zucchini yellow mosaic virus （ZYMV）[1-2]. Studies have confirmed that the pathogen of melon necrotic spot virus that broke out in a large area in Kashgar， Xinjiang is mainly CABYV[2]. CABYV is an important virus infecting cucurbit crops， and its hosts include sugar beet， lettuce and broad bean[3-5]. In France in 1992， CABYV was first discovered in pumpkin and cucumber plants[6]， and the occurrence of CABYV was also reported in Spain， Tunisia and Italy. The virus is a positive-sense single-stranded RNA virus （+ssRNA） belonging to the genus Polerovirus of the family Luteoviridae. It has icosahedral spherical virions with a diameter of 25-30 nm and a total length of about 5.7 kb， and is divided into two parts by a noncoding inner region （IR） of about 200 nt[7]. Open reading frames 0， 1 and 2 are expressed by genomic RNA， encoding， respectively， P0 protein， P1 protein and P1-P2 fusion protein， of which the P1-P2 fusion protein is the result of a ribosomal frameshift at the C-terminus of ORF1 during translation. Other three reading frames （ORF3， 4 and 5） are expressed from subgenomic RNAs. ORF3 encodes the P3 coat protein， which is involved in virus dissemination and virus assembly in plants， and ORF4 and ORF5 encode the movement protein P4 and a read-through protein required for virus transmission by aphids， respectively[8]. Localized to the phloem tissue of host plants， the CABYV is transmitted primarily by aphids （cotton aphids and Myzus persicae） in a cyclic and non-reproductive manner， and can cause yellowing and thickening of leaves， including initial chlorotic lesions， followed by yellowing of whole leaves and thickening of older leaves， and up to 100% of plants showing yellowing symptoms by the end of the growing season[9]， resulting in approximately 50% reduction in production. CABYV can cause severe disease symptoms on Xinjiang melon leaves. It is of great significance to detect the virus and to clarify its genetic diversity with different isolates. In this study， 120 melon leaves suspected of being infected with CABYV randomly collected in Aksu City， Xinjiang， were detected by RT-PCR to determine the incidence and clone the CABYV genome， and the molecular variation of CABYV sequences isolated from different countries and hosts was analyzed by biology information technologies. This study provides a theoretical basis for clarifying the epidemic laws of the virus disease in the field and formulating long-term sustainable prevention and control strategies.

Materials and Methods

Materials

In 2019， RT-PCR identification was performed on 120 fresh melon leaves collected in Aksu City， Xinjiang.

Primer design

The whole genome sequence of CABYV isolated from melons （accession number KR231963.1） was obtained from the NCBI website， and Primer 5.0 was used for primer design. The primer sequences and product sizes are shown in Table 1.

RNA extraction and RT-PCR amplification and sequencing

According to the method of Wang et al.[10]， total RNA extraction， RT-PCR and transformation into E. coli were performed， and positive clones were screened for sequencing.

Phylogenetic tree analysis

BLAST alignment of the whole genome sequences of CABYV was performed through the NCBI website， and 23 sequences （from different countries and hosts） similar to this study were obtained. The complete nucleotide data of 23CABYV isolates are shown in Table 2. The nucleotide sequences were aligned using DNAMAN， and a phylogenetic tree of the whole genome of CABYV was constructed by the neighbor-joining method in MEGA v.7.0， with a bootstrap value of 1 000. The branches with the phylogeny number below 50% were removed.

Genetic diversity analysis

Average pairwise nucleotide diversity （π）， number of segregating sites （S）， nonsynonymous mutation frequency （Ka）， synonymous mutation frequency （Ks） and ratio of nonsynonymous mutations to synonymous mutations （Ka/Ks） were calculated using DnaSP software. If Ka/Ks>1， there will be a positive selection effect. If Ka/Ks=1， there will be a neutral selection. If Ka/Ks<1， there will be a purification selection effect. The software was further used to perform neutrality tests of Tajimas D， Fu & Lis D and Fu & Lis F to determine the diversity faced by the population. Among them， Tajimas D test is mainly used to measure the ratios of polymorphic sites; Fu & Lis D test is to detect the quantitative difference and the difference in total number of site mutations on the basis of single site mutation; and Fu & Lis F test is to calculate the average value caused by single site mutation and pairwise sequence difference.

Recombination analysis

Twenty-four CABYV isolates were subjected to recombination analysis using the recombination detection program RDP v.4.31 software by methods including RDP， GENECONV， BootScan， MaxChi， Chimaera， SiScan and 3Seq. Recombination events with P<10-6 detected by at least five different methods were accepted as reliable results.

Results and Analysis

Identification of CABYV

Among 120 cantaloupe leaves collected from Aksu City， Xinjiang， 38 samples were positive for CABYV by RT-PCR. The 38 CABYV positive samples were sequenced and analyzed online by BLAST （https：//www.ncbi.nlm.nih.gov/）， and it was found 37 CABYV isolates had 100% nucleotide and amino acid identity to the online CABYV sequence， and one isolate （named CABYV-2） differed from the reported CABYV isolate at both the nucleotide and amino acid levels， thus confirming that CABYV-2 is a new CABYV isolate.

Genomic structure of CABYV

A new isolate （CABYV-2） was isolated from the 38 CABYV-positive samples. The whole genome of this isolate is 5 497 bp long， encoding 6 open reading frames， and the non-coding inner region （199 nt） is divided into two regions near the 5′ end and near the 3′ end （Fig. 1）. The 5′ and 3′ untranslated regions are 20 nt and 165 nt， respectively， and the ORF0 near the 5′ end encodes a 239-amino acid P0 protein， which is a gene silencing suppressor encoded by the cucurbit aphid-borne yellows virus genome， which may be involved in the inhibition of plant defense mechanism PTGS[4]. ORF1 encodes a P1 protein whose amino acid sequence is similar to that of serine proteases. ORF2 encodes a P1-P2 functional protein and has the typical amino acid motif of RNA-dependent RNA polymerase （RdRP）. The open reading frames near the 3′ end （ORF3-ORF5） encode P3 coat protein， P4 movement protein and P3-P5 read-through protein， respectively.

Phylogenetic tree analysis

The results of phylogenetic tree analysis showed that the 24 isolates were divided into 2 branches （Fig. 2）. Branch 1 contained 21 isolates， all from Asia， including 17 from Korea， 3 from China， and 1 from Japan. Among the 21 isolates， one isolate was obtained from cucumbers， gourds and pumpkins， respectively， and all other sequences were isolated from melons. The isolate CABYV-2 in this study was clustered with the isolate CY3 （KR231942.1） and belonged to branch 1. Branch 2 consists of 3 isolates from Europe （Spain and France）， all isolated from melons. It preliminarily indicated that genetic variation was related to geographical distribution， and CABYV isolates might be divided into two clusters due to different geographical factors， thus resulting in different genetic diversity.

To further analyze the diversity of these isolates， MEGA v7.0 was used to calculate the genetic distances of these 24 isolates. It showed that the genetic distances within branch 1 and branch 2 were 0.043 and 0.090， respectively， and the genetic distance between group 1 and group 2 was 2.024， which was significantly greater than those within groups.

Sequence similarity analysis

The nucleotide identity of the 24 CABYV isolates ranged from 89.72% to 99.44% （Fig. 3-A）， and the amino acid identity ranged from 81.46% to 98.70% （Fig. 3-B）， showing a high degree of identity. The isolate CABYV-2 obtained in this study has the highest nucleotide and amino acid homology with the sequence KR231942.1， and the nucleotide identity and the amino acid identity were 99.44% and 98.70%， respectively. Its nucleic acid homology with JF939812.1 was the lowest， at 89.72%.

Genetic diversity analysis

The genetic diversity of the 24 CABYV isolate sequences was analyzed by DnaSP software.  The results showed that the Ka values of various genes were significantly smaller than the Ks values， that is， ω was less than 1 （Table 3）， indicating that various genes of the CABYV isolates were affected by purification selection. In addition， the haplotype polymorphism values of different genes were all close to 1， and the π values were between 0.078 and 0.156. The gene with the highest π value was the P1 gene， indicating that the genes had greater variability. Through the three tests of Tajimas D， Fu&Lis D and Fu&Lis F， the test values of P1， P3 and P4 genes were all negative （Table 3）， indicating that the CABYV population was in an expanding stage.

Recombination analysis

To detect whether recombinants occurred in the CABYV population， the nucleotide sequences of 24 CABYV isolates were analyzed using seven methods in the RDP software. The results indicated that there were two recombinants in the 24 CABYVs （sequences or isolates） （Table 5）， and these two recombinants GS6 （Accession No.KR231949.1） and CABYV （Accession No.EU636992.1） both were located in branch 1. The isolate EU636992.1 from Xinjiang， the isolate CABYV-2 in this study and GQ221223.1 were all involved in the recombination events， and recombinant EU636992.1 was between the melon isolate CABYV-2 and the pumpkin isolate GQ221223.1， indicating that recombination of CABYV could occur between different hosts.

Conclusions and Discussion

The CABYV is important pathogen that causes yellowing symptoms of cucurbit crops. It was first detected on 9 cucurbit crops in Hebei， Henan， Shanxi and Shaanxi in China[11]， and then on cucurbit crops in 25 provinces and cities in China， including Beijing， Shandong， Zhejiang， Anhui， Hunan， Yunnan， Guangxi and Hainan[12]. In Korea， the CABYV has been confirmed to infect cucumbers and oriental melons[13]， and the virus can cause co-infection with other cucurbit-infecting viruses， increasing the probability of the emergence of recombinants[14-16].   In recent years， the virus has also been detected in cucurbit crops in China， resulting in a significant decrease in the yield and quality of watermelons， cucumbers， and pumpkins[17]. The 24 different CABYV isolates in this study were divided into two groups due to differences in geographical factors， and there were large genetic differences between the two groups. The genetic diversity analysis of various genes of different CABYV isolates showed that the nucleotide diversity value of P1 gene was the largest， at 0.237， indicating that the variability of P1 gene was greater， and the π value of CP gene was the smallest， indicating that that it had high genetic stability and it was highly conserved， which may be related to its function[18]. The results of neutrality tests were mostly negative， indicating that the CABYVA population was in an expanding state.

Geographic isolation may be an important reason for the high variability of CABYV. Based on the phylogenetic tree and nucleotide identity analysis results of the 24 isolates， different CABYV isolates were divided into 2 branches due to differences in geographical factors. Branch 1 included 21 isolates from China， Korea and Japan， and the CABYV-2 isolated in this study was clustered with CY3 （KR231942.1） and belonged to branch 1. Branch 2 included 3 isolates obtained from different countries in Europe. The different isolates of CABYV from branch 1 and branch 2 were distantly related， which may be due to the fact that the CABYV is mainly transmitted by aphids， and it is difficult to achieve long-distance transmission[2]. Shang et al.[17] analyzed the CABYV isolates from Hubei and Yunnan and different isolates at home and abroad， and found that the molecular variation of CABYV was related to the geographical distribution， which is consistent with the results of this study. The population difference test was further carried out on the two populations of group 1 and group 2. The results showed that the genetic differentiation index Fst value between the two populations was 0.913; Kst* was 0.131; Snn and Z* values were 1.00 and 4.403， respectively; and the genetic distance between branch 1 and branch 2 was 2.024. It indicated that there was large genetic differentiation between branch 1 and branch 2. The results together with the phylogenetic analysis results indicated that the genetic diversity of CABYV might be due to differences in geographical factors and genetic distances.

Recombination exacerbated CABYV variation. For single-stranded RNA viruses， inter-virus recombination is a major evolutionary method， which has a direct impact on the virus population， can expand the host range and adapt to new environments， and is an important factor causing virus mutation[19]. Recombinant CABYV strains were previously reported from Southeast Asian countries such as Thailand and Philippines[20-21]. The results of recombination analysis in this study showed that among the 24 isolates， there were two recombinants， EU636992.1 and KR231949.1 （Table 5）， from China and South Korea， respectively， and the recombination of CABYV between melon isolates and pumpkin isolates indicated that the virus may become more and more infective in the process of evolution， which should attract enough attention.

CABYV is widely distributed in China and its population is expanding continuously， and it has become a limiting factor for the production of melon plants. The CABYV isolate from Aksu City， Xinjiang had the closest relationship with the Korean isolate KR231942.1， and the farthest relationship with the isolates from European countries. There were large genetic differences between the isolates of branch 1 and branch 2， and the recombination of CABYV in different hosts aggravated the variation of CABYV. Recombination and negative selection may be important reasons for the genetic variation of CABYV.

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