Actinidia Virus Research Articles

Combining Thermotherapy with Shoot Tip Culture or Cryotherapy for Improved Virus Eradication from In Vitro Actinidia macrosperma.

In recent years, kiwifruit viral diseases have become increasingly prevalent in kiwifruit-producing regions of China, significantly impacting both the yield and quality of kiwifruit. This has emerged as a significant constraint on the healthy and sustainable development of the kiwifruit industry. The use of virus-free propagation materials has been proven the most effective strategy for controlling plant viral diseases. In the present study, shoot tip culture, shoot tip cryotherapy, and their combinations with thermotherapy were established to eradicate Actinidia virus A (AcVA), Actinidia virus B (AcVB), and Actinidia chlorotic ringspot-associated virus (AcCRaV) from Actinidia macrosperma. Additionally, the impact of shoot tip size on virus eradication was evaluated. Among the three confirmed viruses, regardless of the procedure, AcVB was the easiest to eradicate, followed by AcVA and AcCRaV. Combining thermotherapy with shoot tip culture or cryotherapy resulted in a higher virus-free frequency (up to 27.3 and 50%, respectively) than shoot tip culture or cryotherapy alone (0 to 20%). Notably, the combination of thermotherapy and 0.5- to 1-mm shoot tip cryotherapy was shown to be the most effective protocol for virus eradication from A. macrosperma, which produced 50% of regenerated shoots free from all the tested viruses. To the best of our knowledge, this is the first report on virus elimination from kiwifruit infected with multiple viruses based on conventional shoot tip culture and shoot tip cryotherapy.

Occurrence and Molecular Variability of the Main Kiwifruit Viruses in the Sichuan Province of China.

Viruses cause important yield losses in kiwifruit. Here, we studied the occurrence and population structure of the major kiwifruit viruses in the Sichuan province of China. RT-PCR results showed the presence of Actinidia virus A (AcVA), Actinidia virus B (AcVB), Actinidia chlorotic ringspot-associated virus (AcCRaV), and the cucumber mosaic virus (CMV). AcCRaV was widely distributed, followed by CMV. These two viruses were often detected in co-infection with AcVA and AcVB. The virus detection rate was positively correlated with vine age. Four phylogenetic groups of AcVA and AcVB were identified, with AcVA isolates clustering mainly in subgroup I, and AcVB isolates clustering mainly in subgroups II, III, and IV. All CMV isolates clustered in subgroup II, and AcCRaV isolates clustered in subgroup IA. The genome of AcVA and AcCRaV was under negative selection pressure, while the genome of AcVB and CMV was under positive selection pressure. All the viruses, except AcVB, were in a state of expansion. The full-length genome of the most widely distributed AcCRaV isolate in kiwifruits in the Sichuan province was characterized by sequencing. Unique eight-nucleotide (TTTTTGAT) repeats were found in the 5'-terminal non-coding region of the AcCRaV RNA3 in a possible association with reduced disease symptoms. This is the first study of kiwifruit viruses in Sichuan.

Actinidia virus A

This datasheet on Actinidia virus A covers Identity, Distribution, Hosts/Species Affected, Further Information.

Establishment of Tissue Culture System of Actinidia deliciosa Cultivar “Guichang”

In order to breed virus-free plantlets of the kiwifruit cultivar “Guichang,” which belongs to Actinidia deliciosa, in this study, stem segments with buds were used as explants, the establishment of a tissue culture rapid propagation system was carried out, and then the virus status of tissue culture plantlets was detected via the real-time reverse transcription-polymerase chain reaction (RT-qPCR) method. The tissue culture rapid propagation system proved that the contamination and browning rates could be controlled below 20% and the survival rate could be exceeded by 70% when the single bud stem segment of “Guichang” kiwifruit was sterilized with 70% alcohol for 30–60 s and 15% NaClO for 15 min, respectively. Meanwhile, we screened the hormone concentration to get better results, and the appropriate medium for adventitious bud induction was MS + 6-BA (1.0 mg/L) + IBA (0.2 mg/L); for proliferation, it was MS + 6-BA (1.0 mg/L) + IBA (0.1 mg/L); and for rooting, it was 1/2 MS + IBA (0.3 mg/L), and the efficiency of induction, proliferation, and rooting could reach 74.07%, 79.63%, and 85.18%, respectively. In addition, the RT-qPCR results demonstrated that the infection rate of 9 viruses: apple stem grooving virus (ASGV), cucumber mosaic virus (CMV), Actinidia virus X (AVX), cucumber necrosis virus (CNV), ribgrass mosaic virus (RMV), citrus leaf blotch virus (CLBV), Actinidia virus B (AcVB), Pelargonium zonate spot virus (PZSV), and cherry leaf roll virus (CLRV) in the “Guichang” kiwifruit tissue culture plantlets was 0. This study could lay a foundation for the production of “Guichang” kiwifruit tissue culture seedlings, and the medium formula provided in this study was useful for the industrial rapid propagation of “Guichang” plantlets.

A novel Actinidia cytorhabdovirus characterized using genomic and viral protein interaction features.

A novel cytorhabdovirus, tentatively named Actinidia virus D (AcVD), was identified from kiwifruit (Actinidia chinensis) in China using high‐throughput sequencing technology. The genome of AcVD consists of 13,589 nucleotides and is organized into seven open reading frames (ORFs) in its antisense strand, coding for proteins in the order N‐P‐P3‐M‐G‐P6‐L. The ORFs were flanked by a 3′ leader sequence and a 5′ trailer sequence and are separated by conserved intergenic junctions. The genome sequence of AcVD was 44.6%–51.5% identical to those of reported cytorhabdoviruses. The proteins encoded by AcVD shared the highest sequence identities, ranging from 27.3% (P6) to 44.5% (L), with the respective proteins encoded by reported cytorhabdoviruses. Phylogenetic analysis revealed that AcVD clustered together with the cytorhabdovirus Wuhan insect virus 4. The subcellular locations of the viral proteins N, P, P3, M, G, and P6 in epidermal cells of Nicotiana benthamiana leaves were determined. The M protein of AcVD uniquely formed filament structures and was associated with microtubules. Bimolecular fluorescence complementation assays showed that three proteins, N, P, and M, self‐interact, protein N plays a role in the formation of cytoplasm viroplasm, and protein M recruits N, P, P3, and G to microtubules. In addition, numerous paired proteins interact in the nucleus. This study presents the first evidence of a cytorhabdovirus infecting kiwifruit plants and full location and interaction maps to gain insight into viral protein functions.

Next-Generation Sequencing Combined With Conventional Sanger Sequencing Reveals High Molecular Diversity in Actinidia Virus 1 Populations From Kiwifruit Grown in China.

Kiwifruit (Actinidia spp.) is native to China. Viral disease–like symptoms are common on kiwifruit plants. In this study, six libraries prepared from total RNA of leaf samples from 69 kiwifruit plants were subjected to next-generation sequencing (NGS). Actinidia virus 1 (AcV-1), a tentative species in the family Closteroviridae, was discovered in the six libraries. Two full-length and two near-full genome sequences of AcV-1 variants were determined by Sanger sequencing. The genome structure of these Chinese AcV-1 variants was identical to that of isolate K75 and consisted of 12 open reading frames (ORFs). Analyses of these sequences together with the NGS-derived contig sequences revealed high molecular diversity in AcV-1 populations, with the highest sequence variation occurring at ORF1a, ORF2, and ORF3, and the available variants clustered into three phylogenetic clades. For the first time, our study revealed different domain compositions in the viral ORF1a and molecular recombination events among AcV-1 variants. Specific reverse transcriptase–polymerase chain reaction assays disclosed the presence of AcV-1 in plants of four kiwifruit species and unknown Actinidia spp. in seven provinces and one city.

Characterization and complete genome sequences of two novel variants of the family Closteroviridae from Chinese kiwifruit.

Two distinct closterovirus-like genome sequences (termed AdV-1 v1 and v2) were identified in Actinidia chinensis var. deliciosa ‘Miliang-1’ that had no disease symptoms using high-throughput sequencing. Using overlapping reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends, the genomic sequences of AdV-1 v1 and v2 were confirmed as 17,646 and 18,578 nucleotides in length, respectively. The two complete genomes contained 9 and 15 open reading frames, respectively, coding for proteins having domains typical of Closteroviridae, such as RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homolog (HSP70h) and coat protein (CP). Sequence analysis showed that the amino acid sequences of RdRp, HSP70h, and CP of the two variants exhibited high similarity (> 80%), while their genomic organization was somewhat different. This suggested that the two viral genomes identified here are variants of the family Closteroviridae in a single kiwifruit host. Furthermore, phylogenetic relationship analysis revealed that the two variants had a closer relationship with the unclassified virus Persimmon virus B (PeVB) and Actinidia virus 1 (AcV-1) than with other members of the family Closteroviridae, as did their genomic organization. It is speculated that the two variants, together with PeVB and AcV-1 belong to a new subfamily of Closteroviridae.

Sequence analysis of 43‐year old samples of Plantago lanceolata show that Plantain virus X is synonymous with Actinidia virus X and is widely distributed

AbstractPlantain virus X was first recognized by the ICTV as a species in the genus Potexvirus in 1982. However, because no sequence was available for plantain virus X (PlVX), abolishing the species was proposed to the Flexiviridae working group of the ICTV in 2015. This initiated efforts to sequence the original isolates from Plantago lanceolata samples. Here we report the full‐genome sequencing of two original isolates of PlVX, which demonstrate that the virus is synonymous to Actinidia virus X, a species previously reported from kiwifruit (Actinidia sp.) and blackcurrant (Ribes nigrum). PlVX was previously noted to be widespread in the UK in P. lanceolata. This report additionally presents novel data on the distribution and diversity of PlVX, collected at the same site as the original UK isolates, and from three independent surveys, two in the Netherlands and one in Belgium. This study also includes two new host records for PlVX, Browallia americana and Capsicum annuum (sweet pepper), indicating the virus is more widespread and infects a broader range of hosts than previously reported. This stresses the importance of surveys of noncultivated species to gain insight into viral distribution and host range. This study also demonstrates the value of generating sequence data for isolates retained in virus collections. Additionally, it demonstrates the potential value in prepublication data sharing for giving context to virus detections such as the four independent studies here which, when combined, give greater clarity to the identity, diversity, distribution, and host range of plantain virus X.

Occurrence and molecular characterization of Actinidia virus C (AcVC), a novel vitivirus infecting kiwifruit (Actinidia spp.) in China

AbstractKiwifruit (Actinidia spp.) is an economically important fruit crop in the world. China is the largest producer of kiwifruit in terms of both acreage and yield. In this study, a novel virus, tentatively named Actinidia virus C (AcVC), was discovered in Actinidia deliciosa ‘Xuxiang’ grown in commercial kiwifruit fields in the Shaanxi province, a major kiwifruit‐growing region in China. Sequencing of the complete genome showed that AcVC has 7,668 nucleotides (not including the poly‐(A) sequence). Complete genome comparison and phylogenetic analysis showed that AcVC is most closely related to AcVA and then to AcVB. AcVC shares 69.2% nucleotide identity with AcVA (GenBank accession number JN427014). Based on these data, Actinidia virus C is assigned as a new tentative member of the genus Vitivirus in the family Betaflexiviridae. AcVC was found to infect A. deliciosa and A. chinesis, the two major cultivated kiwifruits. Infection frequency of AcVC varied with the kiwifruit‐growing regions and cultivars in the Shaanxi province of China.

A multiple reverse transcription PCR assay for simultaneous detection of four main viruses in kiwifruit

Kiwifruit (Actinidia spp.) is an important horticultural crop in the world. Previous studies showed that a number of viruses were found in kiwifruit around the world. To detect the multiple viruses in kiwifruit quickly and effectively, a multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) method was developed to detect four viruses simultaneously in kiwifruit: Actinidia chlorotic ringspot-associated virus (AcCRaV), Actinidia virus 1(AcV-1), Actinidia virus A (AcVA), Citrus leaf blotch virus (CLBV), respectively. Four pairs of primers were designed according to the coat protein (CP) genes of the aforementioned viruses and then were confirmed by RT-PCR assay. Actin gene (ACT1) was used as an internal control to improve reliability of the multiplex RT-PCR. Determination of detection limit of the multiplex RT-PCR showed that this method could detect four viruses at the dilution of 10−4 cDNA. Furthermore, the developed multiplex RT-PCR could detect viruses in kiwifruit field samples successfully.

First report of actinidia virus A and B infecting kiwifruit in Jiangxi province in China

First report of actinidia virus A and B infecting kiwifruit in Jiangxi province in China

Pest categorisation of non-EU viruses of Ribes L.

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses of Ribes L. determined as being either non‐EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. Alaska vitivirus 1 and Ribes virus F were excluded from categorisation because these are very poorly characterised viruses. The pest categorisation was completed for seven viruses with clear identity and for which detection methods are available. All these viruses are efficiently transmitted by vegetative propagation techniques, with plants for planting representing the major pathway for long‐distance dispersal and thus considered as the major pathway for entry. Depending on the virus, additional pathway(s) can also be Ribes seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but tomato ringspot virus (ToRSV) has a wide host range, thus extending the possible entry pathways. ToRSV meets all the criteria evaluated by EFSA to qualify as potential Union quarantine pest (QP). With the exception of impact in the EU territory, on which the Panel was unable to conclude, Actinidia virus X, blackcurrant leaf chlorosis‐associated virus, blackcurrant leafroll‐associated virus, black currant‐associated rhabdovirus, blackcurrant waikavirus A and Ribes americanum virus A satisfy all the other criteria to be considered as potential Union QPs. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically the non‐EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as potential Union regulated non‐quarantine pests.

First Report of Actinidia Virus 1 Infecting Kiwifruit in China

The family Closteroviridae comprises important viruses infecting a wide range of economically important plant species, including several species of woody fruit trees (King et al. 2012). Recently, a virus tentatively named as actinidia virus 1 (AcV-1) was identified in Actinidia chinensis grown in Italy (Blouin et al. 2013, 2018). AcV-1 has a 18,848 nucleotide (nt) genome with an organization like that of members in the family Closteroviridae. During 2013 to 2017, severe symptoms of chlorotic leaf spots, vein yellowing, and leaf deformation were continuously observed on a kiwifruit (Actinidia arguta) tree (ID: G4) grown at Wuhan city, in Hubei province in central China. Leaves collected from the symptomatic tree were subjected to small RNA sequencing on an Illumina Genome Analyzer (Biomarker Biology Technology Company, Beijing, China) as described previously (Zheng et al. 2016). The obtained clean reads within the range of 18 to 26 nt were assembled into contigs using Velvet software. BLASTn and BLASTx searches against the NCBI GenBank database showed that some contigs had the closest matches to the sequences of actinidia virus A, actinidia virus B, and actinidia chlorotic ringspot-associated emaravirus. Additionally, nine contigs coded for proteins matching partial amino acid (aa) sequences of the polymerase, HSP70h, CPm, and CP encoded by AcV-1, and showed nucleotide sequence identities of 65 to 85% with the corresponding fragments of the AcV-1 genome (GenBank accession no. KX857665), indicating a possible presence of an AcV-1 genetic variant, tentatively named AcV-1ch, in the sample. Multiple alignment showed that the polymerase of AcV-1 identified in Italy and AcV-1ch had a similar 26-aa insert located immediately after the GDD motif as compared with some other viruses in the family Closteroviridae. Two sets of primers, hsp70F/hsp70R (5′-GACGATCATAGGTATTGACTACGG-3′/5′-CCWCCRCCRAARTCGTAAACTATRT-3′) and cpF/cpR (5′-TGAGCTRGGRATAGATGTTGC-3′/5′-TCTCTCAGGGTTMGGATGAGT-3′) targeting the HSP70 and CP genes of the virus were designed based on the sequences obtained from our deep sequencing data and the KX857665 sequence. Leaf samples showing chlorotic leaf spots or mottle were collected from 12 kiwifruit trees (ID: XW1-12) grown in Jiangxi province. Total RNAs extracted from the 12 leaf samples together with the sample G4 (harboring AcV-1ch) were subjected to reverse transcription polymerase chain reaction testing for AcV-1, according to the previously described protocol (Zheng et al. 2016), except for annealing for 30 s at 53 to 57°C. Target products of 606 bp (primers hsp70F/hsp70R) and 376 bp (primers cpF/cpR) were amplified from samples G4 and XW1, and samples G4, XW1, XW4, XW5, and XW12, respectively. The 606-bp HSP70 fragments from isolates AcV-1ch and XW1 (GenBank accession nos. MH545710 and MH545709) shared 82.7% nt identity with each other and 84.7 and 81.5% nt identities with the corresponding sequence of reported AcV-1. The 376-bp CP fragments from isolates XW1, XW4, XW5, XW12, and AcV-1ch (GenBank accession nos. MH545704 to MH545708) shared 80.9 to 98.9% nt identities with each other and 81.4 to 90.7% nt identities with the corresponding sequence of AcV-1. The result further confirmed AcV-1 infection in kiwifruit plants in China, in at least two provinces, Hubei and Jiangxi. To our knowledge, this is the first report of AcV-1 in China, and the first outside of Italy. Further extensive study will be necessary for understanding the viral infection status and effect on kiwifruit production in China.

First Report of Actinidia Virus 1 Infecting Actinidia chinensis in China

China is the origin of Actinidia spp. (kiwifruit) and has largest kiwifruit production around the world. Although kiwifruit was domesticated only in the 20th century, it has become an important horticultural crop. A total of 17 viruses have been found in kiwifruit growing areas including China, Italy, and New Zealand since 2002 (Blouin et al. 2013, 2018; Veerakone et al. 2018). Sichuan Province has a unique geographical condition and climate for growing kiwifruit. It is the second largest area after Shanxi Province for commercial production of kiwifruit in China. With an increasing number of years of kiwifruit cultivation, suspect viral symptoms such as leaf mottle, chlorosis, and malformation are increasing. To confirm which viruses are infecting Actinidia spp. and to determine the extent of their distribution in Sichuan Province, a survey was conducted from April 2016 to May 2018. Fifty-six kiwifruit samples that showed virus-like symptoms such as feathery mottle, chlorotic and necrotic spots, and yellowing on leaves were collected from five different varieties of Actinidia chinensis at seven different geographic locations in Sichuan Province. Electron microscopy examination by negative staining for crude extracts of the symptomatic leaves revealed flexuous filaments of 9 to 10 nm × 1,350 to 1,400 nm. The particle size suggested a species of Closteroviridae. To identify the virus, total RNA of the respective samples was extracted with the EASYspin RNA Plant Mini Kit (Aidlab Biotechnologies Co., China) and reverse transcribed using random hexamers (Fermentas, Thermo Scientific). Actinidia virus 1 (AcV-1) coat protein specific primers, AcV1-CP F (5′- CGCGGATCCATGACTACCAAAGAAACGAA-3′) and AcV1-CP R (5′- ACGCGTCGACTCAATGATAACCTGAAGTGA-3′), were used to examine the presence of AcV-1, a new Closteroviridae virus recently identified in kiwi vines in Italy (Blouin et al. 2018). The results showed that 22 of the 56 kiwifruit samples generated bands of the expected size (732 bp) for AcV-1. One polymerase chain reaction amplicon from one sample from each of the seven different geographic locations was cloned into a pEASY-Blunt Zero vector (TransGen Biotech, China) and Sanger sequenced by Chengdu Tsingke BioTech Co. DNAMAN sequence alignment showed that across the seven clones, there were five different sequences, which shared 87.4 to 99.7% nucleotide sequence identity and 92.6 to 100% amino acid sequence identity (GenBank accession nos. MH557852 to MH557856). The variations were mainly concentrated in the middle domains of the coat protein genes. Further analysis showed that the five AcV-1 isolates shared 89.8 to 90.2% nucleotide sequence identity and 94.7 to 95.9% amino acid sequence identity to the corresponding gene of AcV-1 isolate K75 from Italy (KX857665, Blouin et al. 2018). Our results confirmed the widespread presence of AcV-1 in all kiwifruit planting areas that were investigated and in different varieties of A. chinensis (Hongyang, Donghong, Honghua, Jinguo, and Jinyan). Our sequence data also showed that there was genetic variability among different AcV-1 isolates in Sichuan Province. However, the role of AcV-1 as the causal agent for the symptoms observed in Actinidia is unknown, because plants were coinfected with other Actinidia-infecting viruses. To our knowledge, this is the first report of AcV-1 infecting A. chinensis in China. The apparent expansion of this virus is worthy of further attention.

Occurrence and Molecular Variability of Kiwifruit Viruses in Actinidia deliciosa 'Xuxiang' in the Shaanxi Province of China.

Kiwifruit (Actinidia spp.) is an economically substantial fruit crop with China the main producer. China is the primary source of wild kiwifruit and the largest producer of kiwifruit in terms of both production and planting area, and Shaanxi province is the largest kiwifruit producer in China. Previous studies reported presence of kiwifruit viruses in Actinidia chinensis. In this study, six viruses were identified in kiwifruit 'Xuxiang' (A. deliciosa) in Shaanxi, China. The incidence, distribution, and genetic diversity of these viruses were studied. The results showed that Actinidia virus A (AcVA), Actinidia virus B (AcVB), Actinidia chlorotic ringspot-associated virus (AcCRaV), cucumber mosaic virus (CMV), apple stem grooving virus (ASGV), and potato virus X (PVX) were the main viruses infecting Xuxiang kiwifruit in Shaanxi, China. Incidence of the various viruses with both single and multiple infection varied with different kiwifruit-growing counties. For single virus infection, the highest and the lowest numbers of samples infected were about 22 for AcCRaV and 0 for AcVB in Meixian out of 170 samples, 12 for AcVA and 0 for CMV in Zhouzhi out of 120 samples, 10 for AcVA and 0 for AcVB, AcCRaV, ASGV, PVX, and CMV in Yangling out of 70 samples, and 8 for AcCRaV and CMV and 0 for AcVA, AcVB, ASGV, and PVX in Hanzhong out of 80 samples, respectively. Samples which were multiply infected with two or more viruses were also detected. Analysis of the phylogenetic tree of these viruses showed some genetic variability in the AcVA, AcVB, and AcCRaV isolates of Shaanxi kiwifruit. There was no obvious molecular variation in the coat protein genes of ASGV, CMV, and PVX virus isolates from Shaanxi kiwifruit. The present study is the first large-scale survey of kiwifruit viruses in Shaanxi, China. To our knowledge, this is the first report of PVX infecting kiwifruit and the first report of molecular variability of AcVA, AcVB, and AcCRaV. These results provide important data for studying the genetic evolution of AcVA, AcVB, AcCRaV, ASGV, CMV, and PVX.

Characterization of Actinidia virus 1, a new member of the family Closteroviridae encoding a thaumatin-like protein.

A new member of the family Closteroviridae was detected in Actinidia chinensis grown in Italy, using next generation sequencing of double-stranded RNA. The virus isolate, named Actinidia virus 1 (AcV-1) has a genome of 18,848nts in length, a structure similar to the unclassified persimmon virus B (PeVB) and contains 12 open reading frames (ORFs) greater than 6KDa, one carrying two papain-like leader proteases, a methyltransferase, a helicase and an RNA-dependent RNA polymerase domain. Additional ORFs code for homologs of heat shock protein 70, heat shock protein 90 and a coat protein. Curiously, AcV-1 and PeVB genomes code for a thaumatin-like protein, a peculiarity unreported for other viruses. In phylogenetic analyses both viruses group in a distinct clade evolutionarily related to closteroviruses. The final taxonomic position of AcV-1 within the family Closteroviridae is yet to be clarified.

First Report of Actinidia virus A and B Infecting Actinidia chinensis in South Korea

First Report of <i>Actinidia virus A</i> and <i>B</i> Infecting <i>Actinidia chinensis</i> in South Korea

Complete genome sequence of a strain of Actinidia virus X detected in Ribes nigrum cv. Baldwin showing unusual symptoms.

A Ribes-infecting strain of the potexvirus Actinidia virus X (AVX-RV3124) was isolated from black currant plants (Ribes nigrum cv. Baldwin, accession 3124-03D1) showing symptoms of leaf chlorosis and deformity. This is the first description of the complete genome sequence of an isolate of this virus and the first detection of a potexvirus in Ribes. The genome of AVX-RV3124 consists of 6,888 nucleotides (nt) excluding the poly(A) tail at the 3' terminus. When AVX-RV3124 was compared to the available sequence of the AVX isolate in GenBank (accession no. KC568202), two large indel events (72 nt and 33 nt) were identified in the replicase coding region of RV3124. Evidence of recombination was detected upstream of the 3' terminus of the replicase gene of both virus isolates, providing further evidence of a common origin.

First Report of Actinidia virus A and Actinidia virus B on Kiwifruit in China.

At present, two viruses affecting kiwifruit (Actinidia spp.), Actinidia virus A (AcVA) and Actinidia virus B (AcVB), both belonging to the genus Vitivirus in the family Betaflexiviridae, have been reported from New Zealand (2). The infected trees showed leaf vein chlorosis, flecking, and ringspots. China is the largest commercial kiwifruit producer. During field investigations in the growing season of 2013, symptoms of leaf chlorosis or ringspots, similar to those caused by AcVA and AcVB (1), were observed on some kiwifruit (Actinidia chinensis) plants in Hubei Province in the central China. Leaf samples were collected from three symptomatic and two symptomless plants of two A. chinensis cultivars. Total nucleic acids were extracted from the samples using a CTAB-based protocol described by Li et al. (3) and used as template in RT-PCR for the detection of AcVA and AcVB. Each virus was detected using two sets of primers reported by Blouin et al. (1). Primer sets AcVA 1F/1R and AcVA5F/5R were used for the AcVA detection, and AcVB1F/1R and AcVB5F/Viti3'R were used for the AcVB detection. AcVA was detected in three symptomatic plants (ID: Ac-HN-1, Ac-HN-3, and Ac-HN-5), and AcVB was detected in two symptomatic plants (ID: Ac-HN-1 and Ac-HN-3) and in one symptomless plant (ID: Ac-HN-2). Neither virus was detected in the second symptomless plant (ID: Ac-HN-4). Samples Ac-HN-1 and Ac-HN-3 had mixed infection of AcVA and AcVB, and sample Ac-HN-2 had the latent infection of AcVB. The sequenced 283-bp RT-PCR amplicons of the replicase-encoding gene from AcVA isolates AC-HN-3 and AC-HN-5 using AcVA1F/1R shared 90.8% nucleotide (nt) identity with the corresponding sequence of the New Zealand AcVA isolate (GenBank Accession No. JN427014.1). The 269-bp fragments of the RNA-binding protein-encoding gene obtained by using AcVA5F/5R shared 85.5 to 85.9% nt identities with the corresponding sequence of JN427014.1. The AcVB5F/Viti3'R products of 365 to 369 bp from three AcVB isolates shared 85.5 to 88.6% nt identities with the corresponding sequence of the New Zealand AcVB isolate. The representative sequences were submitted to GenBank with accession numbers KJ696776 and KJ696777 for the 269-bp fragments of AcVA-HN-1 and AcVA-HN-3, and KJ696778 and KJ696779 for the 365-bp and 369-bp fragments of AcVB-HN-1 and AcVB-HN-2, respectively. In addition, 12 and 14 out of 42 kiwi samples (excluding HN-1 to HN-5) collected randomly were positive for AcVA and AcVB as detected by RT-PCR. Meanwhile, the sample affected by AcVA-HN-5 was subjected to deep sequencing of the small RNAs (sRNAs) for complete survey of the infecting viruses. De novo assembly of sRNAs generated four sequence contigs, with lengths ranging from 161 to 285 nt, matching to ORFs 1 to 3 of the genome of the New Zealand AcVA isolate with significant nucleotide (91 to 95%) and amino acid (80 to 94%) similarities, and some other contigs from a new virus (unpublished). The result further confirmed AcVA infection in the kiwi plant. To our knowledge, this is the first report of both AcVA and AcVB outside of New Zealand. The Chinese isolates of the two viruses are distinct from those reported from New Zealand. The results provide valuable information for improving the viral sanitary status of the kiwifruit germplasm in China.

VIRUSES OF KIWIFRUIT (ACTINIDIA SPECIES)

Kiwifruit (Actinidia deliciosa) was introduced to New Zealand more than one hundred years ago and the New Zealand-raised cv. Hayward is now the dominant cultivar grown worldwide. Further accessions of kiwifruit seed and scionwood have been sourced from China for research and breeding. In one importation consignment, the first virus naturally infecting kiwifruit, Apple stem grooving virus (ASGV), was identified following symptoms observed in quarantined plants (2003). Since that time a further 12 viruses have been identified in kiwifruit. We classify these 13 viruses into three groups. The first group comprises the non-specialist viruses and includes Alfalfa mosaic virus (AMV) and Cucumber mosaic virus (CMV) both members of the family Bromoviridae. The group also includes a further five viruses that appear to have limited effect on kiwifruit: two tobamoviruses, Ribgrass mosaic virus (RMV) and Turnip vein clearing virus (TVCV); a tombusvirus, Cucumber necrosis virus (CNV); a novel potexvirus; and Apple stem gooving virus (ASGV, genus Capillovirus). Most of the viruses classified in this first group are cosmopolitan and sometimes orchard weeds provide reservoirs for infection. The second group comprises the kiwifruit-adapted viruses. This group includes three novel viruses. i.e. two vitiviruses, Actinidia virus A (AcVA) and Actinidia virus B (AcVB), and a citrivirus closely related to Citrus leaf blotch virus (CLBV). In addition, preliminary evidence of a novel virus belonging to the Closteroviridae family has been obtained. The third group of viruses induces disease in kiwifruit. To date only two viruses have caused significant damage to kiwifruit within commercial orchards. In New Zealand, Cherry leaf roll virus (CLRV) has been detected on kiwifruit associated with symptoms including leaf spots, fruit malformation, reduction in yield, bark cracking and cane wilting. Pelargonium zonate spot virus (PZSV) has been detected in Italy associated with severe symptoms on leaves and fruit.