Comprehensive Metatranscriptomic Analysis of Plant Viruses in Imported Frozen Cherries and Blueberries
Article information
Abstract
The possibility of new viruses emerging in various regions worldwide has increased due to a combination of factors, including climate change and the expansion of international trading. Plant viruses spread through various transmission routes, encompassing well-known avenues such as pollen, seeds, and insects. However, research on potential transmission routes beyond these known mechanisms has remained limited. To address this gap, this study employed metatranscriptomic analysis to ascertain the presence of plant viruses in imported frozen fruits, specifically cherries and blueberries. This analysis aimed to identify pathways through which plant viruses may be introduced into countries. Virome analysis revealed the presence of six species of plant viruses in frozen cherries and blueberries: cherry virus A (CVA), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), prunus virus F (PrVF), blueberry shock virus (BlShV), and blueberry latent virus (BlLV). Identifying these potential transmission routes is crucial for effectively managing and preventing the spread of plant viruses and crop protection. This study highlights the importance of robust quality control measures and monitoring systems for frozen fruits, emphasizing the need for proactive measures to mitigate the risk associated with the potential spread of plant viruses.
Global agricultural trade represents a major contributor to the spread of plant viruses. As more countries engage in agricultural trade, the movement of plant materials, seeds, and processed products with fresh produce across borders has increased significantly. This has created opportunities for plant viruses to spread to new regions and infect additional crops (Food and Agriculture Organization, 2022; Von Braun and Díaz-Bonilla, 2008). The spread of plant viruses through agricultural trade highlights the importance of effective disease surveillance, monitoring, and control measures.
Several reported foodborne virus outbreaks have been linked to imported frozen fruits. A well-known example is the 2013 hepatitis A outbreak in the United States, which was linked to frozen pomegranate seeds imported from Turkiye (Collier et al., 2014). The 2012 norovirus outbreak in Germany is another example, which was linked to imported frozen strawberries from China. These outbreaks highlight the potential for foodborne viruses to be spread through imported frozen fruits (Bernard et al., 2014). With the increasing number of frozen fruits worldwide, the spread of plant viruses through frozen fruits is possible. However, the risk of transmission can vary depending on several factors, including the type of virus and the processing methods used for freezing and storage. Some plant viruses can survive freezing temperatures and remain infectious in frozen fruits for extended periods. If infected fruits are exported and distributed globally, they could infect crops in new regions and facilitate the spread of the virus.
Black cherry (Prunus serotina), which belongs to the genus Prunus, is widely consumed due to its appealing taste, sweetness, color, and nutritional properties, such as flavonoids, vitamins, anthocyanins, and phenolic (Tricase et al., 2017; Wani et al., 2014). In addition, the increasing demand for cherries is expected to lead to a significant rise in production. According to forecasts, cherry production is predicted to increase dramatically from 220,000 tons to 4,700,000 tons the 2022/23 season (United States Department of Agriculture, 2022). Further, Chile is the largest producer among the countries in the southern hemisphere (Villacrés and Cheein, 2020). Due to the growing interest in cherries, there have been studies aimed at identifying viruses that infect cherry trees. However, most studies have focused on domestic products or fresh cherries (Jelkmann, 1995; Tahzima et al., 2019; Zong et al., 2015). Frozen cherries can also potentially serve as a vector for foreign pathogens, and viruses can survive even while being frozen in storage and preservation (de Souza Grilo et al., 2022). Therefore, effective and accurate examination and diagnosis are crucial for the management of plant viruses and for ensuring the safety of agricultural products.
Viruses are among numerous pathogens, including fungi and bacteria, that infect plants and are the primary cause of cherry disease outbreaks. This poses an economic threat as it can reduce the quality and production of cherry trees (Jones and Naidu, 2019; Thresh, 2006). Cherry virus A (CVA) belongs to the family Betaflexiviridae (genus: Capillovirus) and can be easily transmitted to cherries through grafting or propagation (Marais et al., 2011). While CVA can infect plants alone, it can also co-infect them with other viruses to produce symptoms of increased severity (Hadidi et al., 2011). Other plant viruses in the same family (Bromoviridae) and genus (Ilavirus), such as prunus necrotic ringspot virus (PNRSV) (Aparicio et al., 2010) and prune dwarf virus (PDV) (King et al., 2012), are also known to infect cherries. Additionally, prunus virus F (PrVF), a member of the genus Fabavirus in the family Secoviridae, has recently been found to cause co-infections across a wide range of hosts (Villamor et al., 2017).
Blueberry (Vaccinium spp.) crops are also produced worldwide, being cultivated in at least 39 countries, as reported in 2022 (FAOSTAT, 2022). Blueberries contain various nutritive components such as flavonoids and triterpenoids that have the potential ability to limit the incidence of cancer (Neto, 2007) and high levels of antioxidants, which have contributed to making blueberries an essential functional food (Patel, 2014). Owing to the health benefits of nutritious fruit, the successful development of processing from fresh to frozen fruit production has contributed to the continued increase in global demand for frozen and processed blueberries (Brazelton, 2013). The largest producers of blueberries in the world are the United States (Forney and Kalt, 2011), Chile (Retamales et al., 2014), and Canada (Gallardo et al., 2018).
Blueberry shock virus (BlShV) belongs to the genus Ilarvirus in the family Bromoviridae (Bujarski et al., 2019). BlShV has continued to be reported in several countries, including Canada and the United States, since its first outbreak in Washington in 1980 (Saad et al., 2021). The virus can be confused with blueberry scorch virus (BlSV) infections, as it causes blighting of both flowers and newly emerging leaves. However, BlShV-infected plants, unlike BlSV-infected plants, produce small fruit by the end of the season; they can appear normal. These properties allow BlShV-infected plants to remain infected and serve as inoculum (Bristow and Martin, 1999). The virus is not transmitted by insects (thrips, aphids) but only by bees and other pollinators through pollen (Gottula et al., 2012). Blueberry latent virus (BlLV) is a member of the genus Amalgavirus in the family Amalgaviridae. This virus can spread through seed transmission, and infected plants show asymptomatic symptoms (Saad et al., 2021).
High-throughput sequencing (HTS) has become an increasingly popular tool for identifying and characterizing plant viruses, offering a more comprehensive and accurate understanding of viral diversity and epidemiology (Adams et al., 2009; Al Rwahnih et al., 2009). This tool involves sequencing many DNA or RNA fragments in a single run, generating a vast amount of data. These data can then be analyzed to identify known or novel viruses. HTS-based approaches can detect multiple viruses in a single sample, allowing for the identification of mixed or co-infections. Currently, several HTS-based metagenomics studies have been utilized to identify plant viruses in fruit trees, including apple (Wright et al., 2020), grapevine (Czotter et al., 2018), citrus (Matsumura et al., 2017), and peach (Jo et al., 2018). Additionally, HTS can detect viruses that traditional diagnostic methods, such as serological or PCR-based assays, may have previously missed.
This study utilized comprehensive metatranscriptomic analyses using RNA sequencing to identify plant viruses present in imported frozen black cherry and blueberry samples from Chile, Canada, and the United States and determine the extent of viral inflow possibility through imported frozen fruits.
Materials and Methods
Sample collection
For this study, frozen cherries imported from Chile were purchased from a marketplace in Korea in 2022. These cherry samples were pooled, grounded in liquid nitrogen, and frozen at −80°C before later tests. Frozen blueberries were obtained from a Korean marketplace in 2023. We employed blueberries imported from three countries (Canada, Chile, and the United States) of three different company products for each country, resulting in nine samples. These blueberry samples were pooled based on each imported country, grounded in liquid nitrogen, and frozen at −80°C before later tests, similar to the frozen cherry samples.
Total RNA extraction, library preparation, and RNA sequencing
The total RNA was extracted from the frozen cherry samples using the Clear-S Total RNA Extraction kit (InVirusTech Co., Gwangju, Korea), according to the manufacturer’s instructions. The RNA quality and quantity were assessed using BioAnalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Ribosomal RNA was removed using a Ribo-zero rRNA removal kit for plants (Illumina, San Diego, CA, USA), and RNA purification was conducted using RNA Clean XP before cDNA library construction. To construct a cDNA library, TruSeq Stranded mRNA LT Sample Prep kit (Illumina) was used, and the cDNA library was sequenced on Illumina NovaSeq6000 S4 sequencer generating paired-end reads (2 × 101 bp). The quantity and quality of the library were measured using the Agilent D5000 ScreenTape system (Agilent Technologies). Sequenced data were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database.
Transcriptome assembly and virus identification
Pair-end reads (2 × 101 bp) were obtained from the RNA-Seq using Illumina NovaSeq6000 S4 sequencer, and their quality was assessed using FastQC (version 0.11.9). The quality-controlled reads were aligned to plant virus references in the NCBI database using Kallisto (version 0.46.1). Subsequently, SAMtools (version 1.14) were utilized for data sorting and indexing, involving the conversion of a pseudo bam file to a bam file format. The mapped reads in bam format were visualized and aligned to the target virus using Integrative Genomics Viewer (IGV) (version 2.10.0) and BowTie 2 (version 2.3.4.1). Finally, CAP3 assembly was employed to retrieve long contigs of the virus.
Calculation of viral proportion and viral abundance
In RNA-Seq data, transcript length biases may arise due to random RNA fragmentation and the inherent sampling nature (Oshlack and Wakefield, 2009). To standardize and quantify the relative abundance of viral contigs across each cherry and blueberry library, expression levels were determined using fragments per kilobase of transcript per million mapped reads (FPKM) (Zhao et al., 2021). This approach yields unbiased results by factoring in the sequencing depth, and the length of the mapped reads when estimating gene expression levels.
Mapping of viral sequence and genome assembly
The frozen cherry and blueberry library transcriptome sequences were subjected to BLAST analysis. Partial or complete viral genome sequences were generated, and the contig most closely resembling the virus genome in the GenBank was selected for further study. Following alignment to the reference viral genome, virus-associated reads were mapped using the ‘Map to Reference’ tool in Geneious Prime with medium sensitivity/fast settings (Kearse et al., 2012).
Phylogenetic tree construction
To conduct phylogenetic comparisons between plant virus genome sequences identified in this study and those from the NCBI GenBank, we utilized coat protein (CP) sequences from CVA, PNRSV, PDV, PrVF, and BlShV. For BlLV, we used RNA-dependent RNA polymerase (RdRp) sequences. Each virus sequence was aligned with corresponding viral genome sequences from the GenBank database using the CLUSTALW algorithm in BioEdit version 7.0.5.3. Phylogenetic trees were constructed using the maximum likelihood method with a Kimura two-parameter model and 1,000 bootstrap replicates in MEGA X (Kumar et al., 2018).
Confirmation of metagenomic analysis result by reverse transcription polymerase chain reaction
To validate the presence of identified viruses detected by RNA-Seq in frozen cherry and blueberry samples, we performed reverse transcription polymerase chain reaction (RT-PCR). Total RNA for each sample was extracted using the Clear-S Total RNA Extraction kit (InVirus Tech Co.) and pooled according to their respective countries of origin (Canada, Chile, and the United States). Virus-specific primer sets, as listed in Supplementary Table 1, were utilized. Subsequently, total RNA amplification was performed following the manufacturer’s instructions using SuPrimeScript RT-PCR Premix (GeNet Bio, Daejeon, Korea). After electrophoresis, the amplified product bands were visible on a 1.2% agarose gel. The amplicons were sequenced by Sanger sequencing.
Results
Sample collection and library construction
The obtained raw data, including sequenced reads from the library, were deposited in the SRA with accession numbers SRR23072403, SRR25316065, SRR25316064, and SRR25316063. The frozen black cherry (C-CAN) exhibited a total read base of 6,081,514,800 and 40,543,432 reads, with a GC content of 46.29%. Similarly, all frozen blueberries from three different countries (B-CAN for Canada, B-CHL for Chile, and B-USA for the United States) displayed total read bases exceeding 6,000,000,000 (6,648,966,000, 6,022,760,400, and 6,986,652,000, respectively) and total reads over 40,000,000 (44,326,440, 40,151,736 and 46,577,680, respectively). These data were obtained by trimming and de novo assembly using the CAP3 assembly program (Table 1).
Summary of frozen black cherry and frozen blueberry sequencing results
Virus identification in frozen black cherry and blueberry samples
Virus-associated reads and contigs were identified by comparing de novo transcriptome assembly results with plant virus reference sequences obtained from the NCBI using the BLASTN search function. In the frozen black cherry samples, the count of virus-associated reads ranged from 156,675 (CVA) to 614 (PDV), where the predominant virus, CVA (156,675 reads), and the subsequent one, PrVF (87,515 reads), constituted the majority. PNRSV ranked the third most prevalent virus with 1,275 reads, followed by PDV with 614 reads. Concerning virus-associated reads in the frozen blueberry samples, BlShV accounted for the highest number of viral reads, totaling 18,252 in Canada (5,718 for BlShV RNA-1, 5,267 for BlShV RNA-2, and 7,267 for BlShV RNA-3) and 19,043 in the United States (4,352 for BlShV RNA-1, 4,080 for BlShV RNA-2, and 10,611 for BlShV RNA-3). For BlLV, 14,204 viral reads were identified in Canada, 1,000 in the United States and 478 in Chile (Fig. 1A).
The number and proportion of reads and contigs for viruses identified in frozen black cherry and frozen blueberry samples. (A) The number of viral reads identified in frozen black cherry and frozen blueberry samples. (B) The number of viral contigs identified in frozen black cherry and frozen blueberry samples. The proportion of viruses identified in C-CHL (C), B-CAN (D), B-CHL (E), and B-USA (F) is based on the number of viral contigs. BILV, blueberry latent virus; BlShV, blueberry shock virus; CVA, cherry virus A; PDV, prune dwarf virus; PNRSV, prunus necrotic ringspot virus; PrVF, prunus virus F.
The number of virus-associated contigs in frozen black cherry samples ranged from 7 (PDV) to 371 (CVA), mirroring the distribution pattern observed for viral reads. The most abundant viral contigs were CVA (371), followed by PrVF (116), PNRSV (13), and PDV (7). The number of virus-associated contigs ranged from 1 (BlLV in B-Chile) to 370 (BlShV-1, 2, and 3 in B-CAN) in frozen blueberries. All viral contigs except BlLV were identified abundantly in the frozen blueberry samples, ranging from 195 (BlShV-1 in B-USA) to 370 (BlShV-1, 2, and 3 in B-CAN) (Fig. 1B).
These findings were illustrated using a circular diagram that depicted various identified viruses. The majority of the plant viruses identified in frozen black cherries were CVA, accounting for 73% (Fig. 1C). In the frozen blueberry samples, BlShV segments and BlLV were present in equal proportions in Canada (Fig. 1D); only BlLV was present in Chile (Fig. 1E); and BlShV segment 3 was dominant in the United States (Fig. 1F).
Viral proportion and viral abundance
We calculated FPKM values for the identified virus in each library to standardize and quantify the relative abundance. The proportion of viruses identified in each sample was then analyzed. In C-CHL, CVA (53.7%) and PrVF (44.7%) emerged as the most predominant (Fig. 2A). Notably, BlShV exhibited a predominant presence in blueberries (B-CAN, B-USA), while in B-CHL, only BlLV was identified. BlShV accounted for 86.5% and 86.9% of the relative abundance, respectively (Fig. 2B–D).
The proportion of identified viruses in frozen black cherry and frozen blueberry samples is based on fragments per kilobase of transcript per million mapped reads (FPKM) values. The proportion of identified viruses in C-CHL (A), B-CAN (B), B-CHL (C), and B-USA (D) is based on the FPKM values. (E) The proportion of three PNRSV RNA segments and two PrVF RNA segments identified in C-CHL is based on FPKM values. (F) The proportion of three BlShV RNA segments identified in B-CAN and B-USA is based on FPKM values. BILV, blueberry latent virus; BlShV, blueberry shock virus; CVA, cherry virus A; PDV, prune dwarf virus; PNRSV, prunus necrotic ringspot virus; PrVF, prunus virus F.
An FPKM was conducted on viruses possessing multiple RNA segments to explore viral replication patterns resulting from RNA segments and ascertain their proportions in each library. PNRSV and PrVF, identified in cherry, consisted of 3 and 2 segments, respectively. In C-CHL, PNRSV segments 1, 2, and 3 accounted for proportions ranging from 10.5% to 68.3%, with PNRSV RNA-3 displaying the highest proportion at 68.3%. PrVF, with two segments, was predominantly represented by PrVF RNA-2, comprising 75.3% of the total (Fig. 2E). Regarding blueberries, it was observed that BlShV was present at relatively similar rates in both B-CAN and B-USA. Notably, BlShV RNA-3 emerged as the most dominant, accounting for 77.7% and 77.8% in B-CAN and B-USA, respectively (Fig. 2F).
Viral genome assembly
To obtain a complete or nearly complete consensus viral genome, viral genome de novo assembly was performed, followed by mapping to corresponding reference virus complete genome sequences from frozen black cherries and blueberries. The mapping results revealed nearly identical sequences, full coverage, and superior accuracy without any gaps. In this investigation, CVA demonstrated a mapping identity of 93.1% (Figs. 3A and 4), while PNRSV exhibited identities of 98.1% for segment 1, 98.9% for segment 2, and 96.4% for segment 3 (Figs. 3B and 4). PDV was mapped with 99.0% accuracy (Figs. 3C and 4), and PrVF segments 1 and 2 were mapped with 96.1% and 95.8% identities, respectively (Figs. 3D and 4). Notably, the CVA and PrVF isolates from frozen black cherries were mapped to the complete genome with 100% coverage.
Genome assembly of identified viruses in frozen black cherry and blueberry samples. Genome mapping of CVA isolates (A), PNRSV isolates (B), PDV isolates (C), and PrVF isolates (D) from C-CHL. Genome mapping of BlLV isolates (E) from B-CHL. Genome mapping of BlShV isolates (F) and BlLV isolates (G) from B-CAN. Genome mapping of BlShV isolates (H) and BlLV isolates (I) from B-USA. BILV, blueberry latent virus; BlShV, blueberry shock virus; CVA, cherry virus A; PDV, prune dwarf virus; PNRSV, prunus necrotic ringspot virus; PrVF, prunus virus F.
Heatmap of genome mapping results of identified viruses in frozen black cherry and frozen blueberry samples. The heatmap construction utilized data including the total number of viral reads identified in each sample and the number of viral reads, coverage, and identity obtained from mapping onto the reference genomes.
Regarding the frozen blueberry samples, in B-CAN, all BlShV segments 1, 2, and 3 mapped with high identities exceeding 95% (95.8%, 95.5%, and 97.8%, respectively), while BlLV exhibited 99.4% coverage and identity (Figs. 3F, G, and 4). In B-CHL, BlLV mapped with 99.3% coverage and 99.6% identity (Figs. 3E and 4). In B-USA, BlShV segments 1, 2, and 3 mapped with identities of 96.2%, 94.8%, and 98%, respectively, and BlLV exhibited a high identity of 99.5% (Figs. 3H, I, and 4). These consensus sequences of identified viruses (CVA, PNRSV, PDV, PrVF, BlShV, and BlLV) were deposited in GenBank (Table 2).
List of identified viruses in frozen black cherry and frozen blueberry
Phylogenetic analyses of identified viruses
To investigate the phylogenetic relationships among the identified viruses from frozen black cherries and blueberries, we employed RNA-Seq alongside other isolates. Phylogenetic trees were constructed by comparing the assembled virus genomes with other isolates from the NCBI dataset. A comprehensive dataset comprising 18 CVA isolates, 26 PNRSV isolates, 26 PDV isolates, 14 PrVF isolates, and 22 BlShV isolates, all with complete CP sequences, along with 26 BlLV isolates with RdRp sequences from various regions worldwide, including isolates obtained in this study, were used for the analyses (Fig. 5).
Phylogenetic analyses for identified viruses in frozen black cherry and blueberry samples. Phylogenetic trees for CVA (A), PNRSV (B), PDV (C), and PrVF (D) identified in frozen black cherry, as well as BlShV (E) and BlLV (F) identified in frozen blueberry, were constructed with known virus isolates using MEGA X. The maximum likelihood method with a Kimura two-parameter model and 1,000 bootstrap replicates were employed. Identified virus isolates from this study are denoted by red rhombuses in the phylogenetic trees. BILV, blueberry latent virus; BlShV, blueberry shock virus; CVA, cherry virus A; PDV, prune dwarf virus; PNRSV, prunus necrotic ringspot virus; PrVF, prunus virus F.
The phylogenetic analyses of CVA, using CP sequences, revealed its division into two distinct clades. CVA CNU2 (LC LC757508) in the yellow clade exhibited close relatedness to isolates from Australia (LC523014) and Slovakia (MF048809) (Fig. 5A). In the case of PNRSV, isolates formed two distinct clades. Unlike other viruses (CVA, PDV, and PrVF) isolates from frozen black cherries, PNRSV showed a weak phylogenetic relationship with other PNRSV isolates. Within the turquoise clade, PNRSV CNU1 (LC752756) demonstrated a close relation to isolates from China (MF145103) and Bulgaria (MT178254 and MT009388) (Fig. 5B). For PDV, the phylogenetic trees also exhibited two distinct clades: Within the grey clade, PDV CNU1 (LC752757) showed close relatedness to isolates from Bulgaria (MK139687 and MT152175) and Turkey (KF718667) (Fig. 5C). The phylogenetic analysis of 14 PrVF isolates using CP sequences revealed two clades: PrVF CNU2 (LC757509) in the deep blue clade was clustered closely with most isolates used for analysis, particularly with an isolate from Belgium (MK834286) (Fig. 5D). Regarding BlShV, BlShV CAN CNU1 (LC781626) and BlShV USA CNU1 (LC781633) within the yellow clade were closely related to each other (Fig. 5E). The phylogenetic analyses of BlLV revealed two separate clades. Moreover, BlLV CAN CNU2 (LC781628), BlLV CHL CNU2 (LC781630), and BlLV USA CNU2 (LC781635) were identified within the same blue clade and showed the closet relationship with an isolate from the United States. In particular, a close relationship between BlLV CHL CNU2 and BlLV USA CNU2 was evident (Fig. 5F).
Validation of identified viruses by RT-PCR
To confirm the presence of the viruses identified by RNA-Seq, we conducted RT-PCR using specific primers designed based on conserved regions of the CP gene (PNRSV) or primer sets previously reported for diagnostic purposes for CVA, PDV, PrVF, BlShV, and BlLV (Supplementary Table 1). The viruses identified by RT-PCR were consistent with those identified by RNA-Seq. All virus amplicons (CVA, PNRSV, PDV, PrVF, BlShV, and BlLV) exhibited the expected size. For international control, nad 5 and 18s rRNA were used for both black cherries and blueberries (Fig. 6). Sanger sequencing was employed to sequence the amplicons, and confirmation of the presence of each virus was achieved by conducting a BLASTN search on the obtained sequences (data not shown).
Validation of identified viruses from frozen black cherry and frozen blueberry samples by reverse transcription polymerase chain reaction (RT-PCR). The presence of CVA, PNRSV, PDV, and PrVF from frozen black cherry and BlShV and BlLV from frozen blueberry was confirmed using specific primers in RT-PCR assays. The amplicons were visualized on 1.2% agarose gels, with M representing the DNA marker. nad5 and 18s rRNA were used as internal controls. BILV, blueberry latent virus; BlShV, blueberry shock virus; CVA, cherry virus A; PDV, prune dwarf virus; PNRSV, prunus necrotic ringspot virus; PrVF, prunus virus F.
Discussion
The exploding demand for imported frozen fruits and globalization has led to increased trade volume, production of frozen fruits (Nasheri et al., 2019), and the accompanying threat of transmitting plant viruses. Consequently, this has raised the potential threat of frozen fruits serving as vectors for transmitting plant viruses. Implementing stringent quality control measures, regular testing, and improved surveillance systems is crucial to mitigate the risks associated with the importation of frozen fruit and minimize the potential spread of viruses. Although there have been reports of several other viruses in frozen fruits, such as strawberries and cherries, particularly human and food viruses (Nasheri et al., 2019), there are currently no reports of plant viruses from imported frozen fruits worldwide.
Plant viruses are among the pathogens responsible for plant diseases, resulting in annual economic losses exceeding 30 billion (Sastry and Zitter, 2014). Viruses can damage various plants, from staple crops to fruit crops and vegetables. They are notorious for their ability to spread through assisted means, such as pollen, foliage, vegetative propagules, exudates, and soil, as well as unassisted means, including mites, insects, humans, protists, and nematodes (Jones and Naidu, 2019). These traditional modes of transmission are widely acknowledged. Recent findings suggest that viruses can be disseminated through imported pollen in several types of fruit trees (Lee and Jeong, 2022). In many countries, quarantine agencies are intensifying efforts to prevent the introduction of new pathogens and ensure that imported plants undergo thorough examination to verify pathogen-free status (Martin et al., 2016). Despite diligent quarantine efforts, the possibility of introducing exotic pathogens through imported goods remains a concern, necessitating ongoing vigilance and investigation. Presently, there is no research on the analysis and transmission of plant viruses in frozen fruits due to the diversification of global trade. Such research is deemed significant from the perspective of understanding the transmission pathways of plant viruses caused by unintended or non-biological factors.
HTS allows for the simultaneous detection of a broad range of viruses in a sample. It can identify known and novel viruses, providing a comprehensive view of the viral diversity within the sample (Maree et al., 2018). HTS does not rely on prior knowledge or specific target sequences, meaning it can identify viruses without needing specific primers or probes, enabling the discovery of new or emerging viruses. This unbiased approach is especially valuable when dealing with viral pathogens that have not been previously characterized (Fitzpatrick et al., 2021). HTS also has high sensitivity, allowing for detecting even low levels of viral genetic material in a complex sample. HTS provides quantitative data on viral abundance within a sample, which is crucial for assessing the viral load, monitoring changes in viral populations over time, and understanding the dynamics of viral infections. Moreover, HTS enables metagenomics analysis, where all genetic material in a sample, including viruses, can be sequenced. This approach allows for detecting and characterizing multiple viral species within a complex mixture, facilitating the identification of co-infections or interactions between different viruses.
In this study, we identified several species of plant viruses in imported frozen cherries and blueberries using metatranscriptomic analysis. Notably, viruses such as PDV and PNRSV, commonly found in stone fruits, have been previously reported in Chile (Fiore et al., 2016). Infections of PNSRV typically manifest as chlorotic spots, while newly sprouting leaves exhibit necrotic symptoms (Uyemoto and Scott, 1992). Meanwhile, PDV-infected trees display symptoms, including small and curled leaves and chlorotic ring spots (Sutic et al., 1999). PDV and PNRSV share a common biological trait of infecting a wide range of cherry varieties, with mixed infections often resulting in severe symptoms, such as reduced fruit bud numbers and decreased crop production (Çağlayan et al., 2011; Smith et al., 2009). Remarkably, our study revealed two correlated species of viruses exhibiting synergistic effects in one library of frozen cherries imported from Chile, highlighting the potential consequences of multiple viral infections. FPKM normalization allows for the unbiased representation of viral proposition and abundance by indicating the expression of virus-related genes within a transcript. We used FPKM values to assess the proportion and abundance of identified viruses across all libraries. In C-CHL, CVA and PrVF accounted for a significant proportion, mirroring the trend observed in the number of identified virus-associated reads. Among blueberries, BlShV emerged as the predominant plant virus in B-CAN and B-USA, except for B-CHL, where BlShV was absent. Individual viruses, particularly those with multiple segments, were analyzed using FPKM to assess the replication levels of each segment. PNRSV, PrVF, and BlShV exhibited varying degrees of confirmed expression across segments, indicating differential segment replication. Notably, gene expression levels differed among segments. In cherries, PrVF RNA-2 was predominant at 75.3%, while in blueberries, BlShV RNA-3 held the highest dominance at 77.8%. Additionally, de novo genome assembly was performed for all four libraries, yielding high coverage ranging from 96.5% to 100% and high identity ranging from 93.1% to 99.6%. Furthermore, CVA, PrVF RNA-1, and PrVF RNA-2 from C-CHL displayed complete or nearly complete sequences with 100% coverage, as reported in our study. In imported frozen blueberries, BlLV, which typically induces asymptomatic symptoms upon viral infection, was detected in all libraries (CAN, CHL, and USA). Phylogenetic analyses revealed that the three BlLV isolates identified in this study were closely related, displaying high phylogenetic affinity with isolates from the United States. This finding aligns with previously reported research indicating that BlLV exhibits minimal variability in the population structure. For instance, BILV isolates from the United States and Japan demonstrated less than 0.5% diversity (Martin et al., 2012). Furthermore, this suggests that the isolates from CHL and CAN also maintain a stable structure, as they are grouped within the same clade. Performing pathogenicity tests based on Koch’s postulates on identified plant viruses from HTS is crucial because it helps confirm whether the identified viruses are indeed responsible for the observed symptoms or diseases in the plants (Fulton, 1966). However, fruit viruses pose certain challenges for pathological tests. Unlike plant viruses, fruit viruses, including those identified in this study, require unique inoculation methods such as grafting and pollen transfer onto woody indicator plants rather than mechanical inoculation onto herbaceous indicator plants. Additionally, fruit viruses often necessitate long incubation periods for symptom development (Ahmad et al., 2023).
Similar to other food products, frozen fruits can serve as a pathway for virus transmission. For instance, noroviruses, known to cause gastroenteritis, spread through various means, such as contaminated food, person-to-person contact, water, and environmental sources. Foodborne transmission is the primary cause of norovirus outbreaks (Verhoef et al., 2015), and previous reports have detected these noroviruses in frozen fruits and berries (Rispens et al., 2020). While cases of plant virus contamination have not been identified, it remains crucial to quarantine and manage potentially tainted food while ensuring accurate diagnosis. Throughout the annals of human civilization, the dual trends of human migration and plant trade have seen a steady rise, coinciding with the observed widespread distribution of plant viruses (Jones, 2021; Santini et al., 2018). Recognizing the multifaceted nature of plant virus transmission underscores the need to remain vigilant, keeping all avenues of potential transmission under scrutiny. Consequently, conducting virome analyses on imported frozen fruits offers a promising avenue for identifying other plausible transmission routes.
The International Standards for Phytosanitary Measures No. 32 (ISPM 32) is titled “Pest risk analysis for quarantine pests and provides guidelines for conducting pest risk analysis to assess the risks associated with the introduction and spread of quarantine pests (International Plant Protection Convention, 2009). According to this standard, when importing frozen fruits, the Phytosanitary Certificate issued by the government must declare that items were frozen at −17.8°C. This ensures that the imported frozen fruits can enter other countries without undergoing on-site plant quarantine inspections, as the risk of pathogen presence is considered low. Subsequently, humans consume imported frozen fruits contaminated with exotic plant viruses. These viruses can then spread from humans to the environment, for example, through feces entering aqueous environments such as rivers. At a microscopic level, bodily excretions such as urine and feces from individual humans also present potential pathways for transmission (Mehle et al., 2018). Recent investigations have expanded the scope to include environmental elements such as rivers and rainwater (Hamza et al., 2011; Prado et al., 2022).
In conclusion, this study identified several known viruses, highlighting the potential consequences of multiple viral infections in imported frozen blackberries and blueberries by HTS. This study underscores the critical need for robust quality control measures and surveillance systems to mitigate the risks associated with the importance of frozen fruits and the potential spread of plant viruses. The increasing demand for imported frozen fruits, coupled with globalization, has heightened concerns about the transmission of plant viruses through these products. While previous reports have focused on viruses in fruit trees, this study highlights the presence of plant viruses in imported frozen fruits, minimizing the risks associated with virus transmission and ensuring the safety of global food supply chains.
Notes
Conflicts of Interest
The authors declare that they have no conflict of interest.
Acknowledgments
This work was carried out with the support of Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ014947032024) Rural Development Administration, Republic of Korea.
Electronic Supplementary Material
Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).