Genus Mandarivirus Research Articles

The Titer of Citrus Yellow Vein Clearing Virus Is Positively Associated with the Severity of Symptoms in Infected Citrus Seedlings.

Citrus yellow vein clearing virus (CYVCV), a new member of the genus Mandarivirus in the family Alphaflexiviridae, is the causal agent of citrus yellow vein clearing disease. CYVCV is transmitted to citrus by Dialeurodes citri, grafting, and contaminated knife blades, threatening citrus production. In this study, four infectious full-length complementary DNA clones of CYVCV (namely AY112, AY132, AY212, and AY221) derived from CYVCV isolate AY were obtained through yeast homologous recombination and inoculated to 'Eureka' lemon (Citrus limon Burm. f.) by Agrobacterium-mediated vacuum infiltration. Pathogenicity analysis indicated that the clones AY212 and AY221 caused more severe symptoms than AY112 and AY132. Northern blot and quantitative reverse transcription PCR analyses showed that the titers of virulent clones (AY212 and AY221) were significantly higher than those of attenuated clones (AY112 and AY132) in the infected 'Eureka' lemon seedlings. Subsequent comparative studies of viral infectivity, accumulation, and symptoms induced by AY221 in nine citrus cultivars indicated that the infectivity of AY221 varied from 25 to 100% between cultivars; 'Oota' ponkan (C. reticulata L.) showed the lowest infection rate, with mild symptoms, which might be a useful resource for CYVCY-resistance genes; and CYVCV titer was positively associated with the symptom development in infected citrus seedlings. In general, this report revealed the biological properties of CYVCV, thus laying a foundation for further investigation of pathogenic mechanisms in this virus.

Genetic diversity of the coat protein gene and biological variation of citrus yellow vein clearing virus infecting different citrus species

Citrus is one of the most important fruit crops globally and rising incidence of citrus yellow vein clearing virus (CYVCV) in citrus orchards is an increasing concern in India. CYVCV and Indian citrus ringspot virus (ICRSV) are assigned to the only two known species in the genus Mandarivirus. Both viruses naturally infect several citrus plants. ICRSV produces ringspots symptoms in Kinnow mandarin, whereas CYVCV induces vein clearing symptoms on Etrog citron and lemon. Leaf samples and bud sticks of different citrus cultivars, exhibiting diverse symptoms were collected during 2012–2014 from 18 locations of India. The genetic variability was assessed through analysis of the coat protein (CP) gene sequences. Out of the 15 CP gene sequences, 13 sequences were of 978 nucleotides (nts) and showed 96–99% sequence identity with CYVCV-Y1 isolate (JX040635.1). However, sequences from the isolates HI and DLI shared only 78% and 73% nucleotide sequence identity with CYVCV and ICRSV, respectively, and that may represent distinct CYVCV isolates in the genus Mandarivirus. In phylogenetic analysis also HI and DLI isolates clustered neither with CYVCV nor with ICRSV isolates.

Recent Advances on Citrus yellow vein clearing virus in Citrus

Citrus yellow vein clearing virus (CYVCV) is the causal agent of yellow vein clearing disease, a significant and devastating disease of most citrus species including lemon and sour orange. CYVCV, a single-strand positive-sense RNA virus containing six ORFs (Open Reading Frames), represents a new species in the genus Mandarivirus of the Alphaflexiviridae family. The virus can cause particularly serious damage, resulting in reduced tree vigor, lower yields, and decreased marketability of fruit production, and it has been found in India, Turkey, Pakistan, China and Iran. Here we described the geographical distribution of the virus, its transmission mode by grafting, mechanical inoculation, and insects, as well as currently available techniques for detection. In addition, we also discussed practical measures aimed at controlling the disease and provided theoretical guidance to prevent the acquisition and spread of the disease that is a significant step toward ensuring the health of the citrus industry.

Discovery and Survey of a New Mandarivirus Associated with Leaf Yellow Mottle Disease of Citrus in Pakistan.

During biological indexing for viruses in citrus trees, in a collection of Symons sweet orange (SSO) (Citrus sinensis L. Osbeck) graft inoculated with bark tissues of citrus trees from the Punjab Province in Pakistan, several SSO trees exhibited leaf symptoms of vein yellowing and mottle. High-throughput sequencing by Illumina of RNA preparation depleted of ribosomal RNAs from one symptomatic tree, followed by BLAST analyses, allowed identification of a novel virus, tentatively named citrus yellow mottle-associated virus (CiYMaV). Genome features of CiYMaV are typical of members of the genus Mandarivirus (family Alphaflexiviridae). Virus particles with elongated flexuous shape and size resembling those of mandariviruses were observed by transmission electron microscopy. The proteins encoded by CiYMaV share high sequence identity, conserved motifs, and phylogenetic relationships with the corresponding proteins encoded by Indian citrus ringspot virus (ICRSV) and citrus yellow vein clearing virus (CYVCV), the two current members of the genus Mandarivirus. Although CYVCV is the virus most closely related to CiYMaV, the two viruses can be serologically and biologically discriminated from each other. A reverse-transcription PCR method designed to specifically detect CiYMaV revealed high prevalence (62%) of this virus in 120 citrus trees from the Punjab Province, Pakistan, where the novel virus was found mainly in mixed infection with CYVCV and citrus tristeza virus. However, a preliminary survey on samples from 200 citrus trees from the Yunnan Province, China failed to detect CiYMaV in this region, suggesting that the molecular, serological, and biological data provided here are timely and can help to prevent the spread of this virus in citrus-producing countries.

Development of RT-PCR degenerate primers for the detection of two mandariviruses infecting citrus cultivars in India

Indian citrus ringspot virus (ICRSV) and Citrus yellow vein clearing virus (CYVCV) are the mandariviruses infecting various citrus cultivars in India and around the world. In the fields, it was observed that citrus plants infected by both the viruses and frequently expressed only ringspot symptoms. The ICRSV-specific polyclonal-antibody used in immuno-sorbent electron microscopy (ISEM) and enzyme linked immuno-sorbent assay (ELISA) could detect only ICRSV in mixed infections. Therefore, the conserved sequences of the RNA dependent RNA polymerase (RdRP) gene of the alphaflexiviruses were exploited for developing a RT-PCR based assay for detection of both the mandariviruses simultaneously, if present. A degenerate primer pair was designed to amplify a ∼435bp fragment by multiple alignments of the RdRP gene sequences of the members of genera Mandarivirus, Potexvirus and Allexivirus. The developed RT-PCR assay was validated for detecting both, CYVCV and ICRSV in mixed infections as well as in single virus-infected citrus plants. The presence of ICRSV or CYVCV or both of them together in such plants were confirmed by using primer pair specific to each of these viruses. Further, the identity of the amplicons was confirmed by sequencing and the virus species were determined with BLASTN analysis. The degenerate primers also amplified the corresponding target sequences of an allexivirus and a potexvirus from the respective infected garlic/ onion and tobacco plants. The use of the degenerate primers for the detection of these virus species of the genus Mandarivirus will be useful in citrus certification programmes.

The Coat Protein of Citrus Yellow Vein Clearing Virus Interacts with Viral Movement Proteins and Serves as an RNA Silencing Suppressor.

Citrus yellow vein clearing virus is a newly accepted member of the genus Mandarivirus in the family Alphaflexiviridae. The triple gene block proteins (TGBp1, TGBp2 and TGBp3) encoded by plant viruses in this family function on facilitating virus movement. However, the protein function of citrus yellow vein clearing virus (CYVCV) have never been explored. Here, we showed in both yeast two-hybrid (Y2H) and bimolecular fluorescence (BiFC) assays that the coat protein (CP), TGBp1 and TGBp2 of CYVCV are self-interacting. Its CP also interacts with all three TGB proteins, and TGBp1 and TGBp2 interact with each other but not with TGBp3. Furthermore, the viral CP colocalizes with TGBp1 and TGBp3 at the plasmodesmata (PD) of epidermal cells of Nicotiana benthamiana leaves, and TGBp1 can translocate TGBp2 from granular-like structures embedded within ER networks to the PD. The results suggest that these proteins could coexist at the PD of epidermal cells of N. benthamiana. Using Agrobacterium infiltration-mediated RNA silencing assays, we show that CYVCV CP is a strong RNA silencing suppressor (RSS) triggered by positive-sense green fluorescent protein (GFP) RNA. The presented results provide insights for further revealing the mechanism of the viral movement and suppression of RNA silencing.

Monoclonal antibody-based serological detection of Citrus yellow vein clearing virus in citrus groves

Citrus yellow vein clearing virus (CYVCV) is considered as the causal agent of Citrus yellow vein clearing disease and be-longs to the genus Mandarivirus in the family Alphaflexiviridae. Capsid protein (CP) of CYVCV Chongqing isolate (CYVCV-CQ) was produced using a prokaryotic expression system and used as the immunogen for monoclonal antibody (MAb) production. Four highly specific and sensitive murine MAbs and one polyclonal antibody were prepared in this study. Titers of the four MAbs in ascites fluids ranged from 10−6 to 10−7 as determined by indirect enzyme-linked immunosorbent assay (ELISA). Three serological assays, including dot enzyme-linked immunosorbent assay (dot-ELISA), tissue blot-ELISA, and double-antibody sandwich (DAS)-ELISA, were developed for quick and reliable detections of CYVCV in citrus samples. The developed dot-ELISA and DAS-ELISA methods could detect CYVCV in the infected citrus leaf crude extracts diluted at 1:2 560 and 1:10 240 (w/v, g mL−1), respectively. The detection result of 125 citrus leaf samples collected from citrus groves in Yunnan Province and Chongqing Municipality of China showed that approximately 36% samples were positive for CYVCV. This virus was, however, not detected in any sample collected from Zhejiang or Jiangxi Province, China.

OCCURRENCE OF CITRUS YELLOW VEIN CLEARING VIRUS IN CITRUS SPECIES IN IRAN

Citrus yellow vein clearing virus (CYVCV), a recently described member of the genus Mandarivirus, is associated with yellow vein clearing disease (CYVCD), a serious threat of lemon production of the world (Chen et al., 2014). The disease was first reported on its two main hosts, sour orange (Citrus aurantium) and lemon (C. limon), from Pakistan in 1988. Then it appeared in India, Turkey and China. In 2010 characteristic symptoms of the disease including leaf crinkling, vein clearing with corresponding watersoaked appearance of the lateral veins on the lower surface of the leaves, were first noticed on a sour orange seedling and a lemon tree in Mazandaran province of Iran. A large number of samples was collected from symptomatic trees of sour orange, Eureka lemon and Persian lime (C. latifolia) during surveys from citrus orchards of the North of Iran in 2016. RNA extractions by SDS-potassium acetate method (Bernad and Duran-Vila, 2006) were made from one isolate from each of these three hosts. A two-step RT-PCR was performed using Revert Aid Kit and PCR Master Mix (Fermentas) and CYVCV specific primer pair of coat protein gene (Chen et al., 2014). The resulting PCR products were sequenced directly, and multiple alignments revealed 96-98% nucleotide identity among the Iranian isolates (GenBank Accession No. KX902486 to KX902488) and also with the reference sequence of CYVCV (NC_026592), two sequences reported from Pakistan and five isolates from China. To our knowledge this is the first report of CYVCV in Iran and Persian lime as a susceptible host of the virus.

Small RNA deep sequencing reveals full-length genome of Citrus yellow vein clearing virus in Chongqing, China

To identity the potential pathogen associated with the yellow vein clearing symptom on lemon trees, the profiles of virus-derived small interfering RNAs from citrus samples were obtained and analyzed by deep sequencing method in this study. Twenty-seven contigs almost cover the full length genome of Citrus yellow vein clearing virus (CYVCV) isolate YN were obtained using the small RNA deep sequencing technology. Analysis showed that this isolate CQ shared the highest nucleotide identity with isolate Y1 (JX040635) and YN (KP313242), both of which belong to the genus Mandarivirus in the family Alphaflexiviridae. Mapping analysis of viral-derived siRNA (vsiRNA) profiles revealed an uneven distribution pattern of their generations along both positive and negative genome strands, and 22- and 21-nt vsiRNAs ranked the majority. BLAST against viroids and other viral databases confirmed that this sample was single-infected only by CYVCV, which indicated that CYVCV was the exact causal agent for the yellow clearing symptom occurring on lemon. This is the first CYVCV isolate detected in Chongqing and the second in China. This result could provide a molecular basis for the investigation of citrus viral diseases to protect citrus health in this region.

FIRST REPORT OF CITRUS YELLOW VEIN CLEARING VIRUS INFECTING NEW NATURAL HOST PLANTS IN TURKEY

In 2003, Citrus yellow vein clearing virus (CYVCV, genus Mandarivirus, family Alphaflexiviridae) (Loconsole et al., 2012) symptoms were observed in citrus lemon (Citrus limon), sour orange (C. aurantium), sweet orange (C. sinensis), mandarin (C. reticulata) and grapefruits (C. paradisi) in a collection orchard in the Cukurova region in Turkey (Onelge et al., 2011). Here we report on a two-year survey for CYVCV in the collection orchard in spring and autumn. 200 symptomless weed samples of the families Amaranthaceae, Asteraceae, Brassicaceae, Chenopodiaceae, Compositeae, Graminaceae and Solanaceae were analyzed by RT-PCR with a specific primer pair (sense: 5′-ACCTCACCGA TGGACCACGTT-3′ and antisense: CAGAAAATGGAAACTGAAAGCCTG-3′) designed in the coat protein gene (GenBank accession No. JX040635) (Loconsole et al., 2012) and total RNA extracted using the Axigen kit. A 476-bp long amplicon was obtained for three samples of Malva sylvestris, three samples of Solanum nigrum, two samples of Sinapis arvensis and two samples of Rananculus arvensis. All samples were cloned with TA Cloning kit (Invitrogen) and sequenced. BLAST analysis of all amplicons showed more than 97% identity of CYVCV isolates from weeds (Accession Nos. JX040635 and KP313241) with the corresponding region of CYVCV reference genome (Loconsole et al., 2012; Chen et al., 2014). Mechanical transmission of CYVCV from infected weeds to sour orange showed yellow vein clearing on lateral veins, leaf crinkling and deformation on young leaves of indicator plants. The presence of CYVCV was confirmed in the symptomatic sour orange plants by RT-PCR. To our knowledge this is the first report of Malva sylvestris, Solanum nigrum, Sinapis arvensis and Rananculus arvensis as hosts of CYVCV.

First Molecular Evidence of Citrus yellow vein clearing virus from Citrus in Punjab, Pakistan

Yellow vein clearing disease was first observed on lemon and sour orange trees in Pakistan in 1988 (Catara et al. 1993). It was an important vector-transmitted disease (Onelge et al. 2011) that was then found in India, Turkey, and China. Recently, a putative new member of the genus Mandarivirus, Citrus yellow vein clearing virus (CYVCV), was identified as the putative causal agent of this disease (Loconsole et al. 2012) in Turkey and then in China. However, so far, no data on a virus associated with this disease were available from Pakistan, even though the disease was originally described there. In 2009, a survey was performed to evaluate the occurrence and distribution of citrus viruses and virus-like diseases in Punjab, Pakistan. Field symptoms of distinct yellow vein clearing and leaf distortion were observed on two lemon trees in Faisalabad District. Infected budwood from one symptomatic Eureka lemon (Citrus limon) tree was grafted onto lemons budded on rough lemon rootstock as indicator plants kept in the greenhouse of Citrus Research Institute in China. Six months later, typical yellow vein clearing disease symptoms were visible on lemon seedlings. Total RNA was extracted from symptomatic lemon seedlings using Trizol. One pair of primers, CYV-627F (5′-CAAACAACCAAGCCGCTA-3′) and CYV-1254R (5′-GCCGAACTCTTTCTTTCCG-3′), was used to amplify a ∼600-bp CYVCV specific product by one-step RT-PCR (Invitrogen). The presence of CYVCV was confirmed through sequencing of the 600-bp fragment. Based on the sequences of CYVCV-Turkey, eight primer pairs were designed to amplify overlapping cDNA fragments spanning the CYVCV RNA genome. The 5′ and 3′ termini were determined using the 5′/3′ RACE kit. Each cDNA fragment was cloned with pGEM-T Easy kit and a minimum of five recombinant clones were sequenced. The sequence of the whole genome of CYVCV-Pakistan isolate (KP313241) obtained was 7,529 bp and had 97% identity to the reference sequence from Turkey. CYVCV-Pakistan had the same six open reading frames as CYVCV reported from Turkey. To confirm the virus is still in the field, samples from these two symptomatic trees in Faisalabad District and eight other symptomatic lemon trees with YVC in Sahiwal District were collected in March 2015. RT-PCR tests with primers CYV-627F and CYV-1254R confirmed CYVCV presence in all these samples. To our knowledge, this is the first molecular evidence of CYVCV occurrence in Pakistan, which confirms the strict association of CYVCV infections with lemon trees exhibiting yellow vein clearing disease, as previously reported from Turkey and China. Our finding emphasizes the need for CYVCV indexing in production and distribution of pathogen-free citrus plants in Pakistan.

Molecular characterization of a citrus yellow vein clearing virus strain from China.

The complete nucleotide sequence of an isolate of citrus yellow vein clearing virus from Yunnan, China (CYVCV-RL), was determined following whole-genome amplification by RT-PCR. The CYVCV-RL genome was 7529 nt in length, excluding the 3' poly (A) tail, and contained six open reading frames (ORFs), resembling that of viruses belonging to the genus Mandarivirus in the family Alphaflexiviridae. Sequence analysis showed that the CYVCV-RL shared the greatest nucleotide sequence identity with the CYVCV-Y1 (JX040635) isolate from Turkey for the whole genome (97.1%), 5' UTR (98.7%), 3' UTR (100.0%), and each of six ORFs (96.5% to 97.8%), suggesting that there is apparent genetic stability among CYVCV isolates of different geographic origin.

DEEP SEQUENCING OF SMALL RNAS FROM CITRUS AFFECTED BY GRAFT-TRANSMISSIBLE DISEASES OF UNKNOWN AETIOLOGY LEADS TO DISCOVERY OF TWO NOVEL VIRUSES

Diseases of unknown aetiology remain difficult or impossible to diagnose. Illumina next generation sequencing (NGS) technology was used to generate sequence datasets from citrus affected by two such diseases: citrus chlorotic dwarf (CCDD) (whitefly-transmitted) and yellow vein clearing disease (YVCD) (aphid-transmitted). Contigs from small RNAs were assembled and used to screen sequence homologies against the virus database in GenBank. DNA fragments from CCDD-affected plants were used to re-construct a circular single-stranded DNA viral genome with homologies to geminiviruses. The genome size and organization of the provisionally named Citrus chlorotic dwarf-associated virus (CCDaV) was shown to be a highly divergent member of the family Geminiviridae. Similarly, the whole genome sequence of a putative filamentous virus associated with CYVCD-affected lemon plants was reconstructed. The genome structure was found to be typical of flexiviruses and sequence data suggested that CYVCV is a new species in the genus Mandarivirus. PCR-based assays for CCDaV and CYVCV were developed that will be an important diagnostic tool for citrus disease management programs in Turkey and other citrus-producing regions.

Genomic characterization of Ambrosia asymptomatic virus 1 and evidence of other Tymovirales members in the Oklahoma tallgrass prairie revealed by sequence analysis

The Plant Virus Biodiversity and Ecology project was undertaken to better understand the nature of plant-viral interactions and the occurrence of non-pathogenic viruses. Plants from the Tallgrass Prairie Preserve (TPP), Osage County, Oklahoma, were surveyed from 2005 to 2008 for the presence of viruses, resulting in the detection, using a virus-like particle enrichment method, of the genome a novel virus, Ambrosia asymptomatic virus 1 (AAV1), from Ambrosia psilostachya DC (western ragweed). Here, we present the genomic organization and genetic variability of AAV1. The virus has a single-stranded RNA genome of about 7408 nt, which has six open reading frames (ORFs). Phylogenetic analysis of the replicase and coat protein ORFs of the virus indicates strongly that the virus should be placed in the genus Mandarivirus. No evidence of recombination was detected. We also report the detection in the TPP of two known viruses and seven other putative viruses, members of the order Tymovirales.

Identification and Characterization of Citrus yellow vein clearing virus, A Putative New Member of the Genus Mandarivirus

Molecular features and genomic organization were determined for Citrus yellow vein clearing virus (CYVCV), the putative viral causal agent of yellow vein clearing disease of lemon trees, reported in Pakistan, India, and more recently in Turkey and China. CYVCV isolate Y1 from Adana, Turkey, was used for deep sequencing analysis of the virus-induced small RNA fractions and for mechanical and graft inoculation of herbaceous and citrus indicator plants. A polyclonal antiserum was developed from CYVCV-Y1 purified from Phaseolus vulgaris and used in western blot assays to characterize the coat protein of CYVCV-Y1 and determine its serological relationship with related viruses. Contigs assembled from the Illumina sequenced short reads were used to construct the whole genome of Citrus yellow vein clearing virus (CYVCV), consisting in a positive-sense RNA of 7,529 nucleotides and containing six predicted open reading frames. The CYVCV genome organization and size resembled that of flexiviruses, and search for sequence homologies revealed that Indian citrus ringspot virus (ICRSV) (Mandarivirus, Alphaflexiviridae) is the most closely related virus. However, CYVCV had an overall nucleotide sequence identity of ≈74% with ICRSV. Although the two viruses were similar with regard to genome organization, viral particles, and herbaceous host range, CYVCV caused different symptoms in citrus and was serologically distinct from ICRSV. Primer pairs were designed and used to detect the virus by conventional and quantitative reverse transcription-polymerase chain reaction on yellow vein clearing symptomatic field trees as well as graft- and mechanically inoculated host plants. Collectively, these data suggest that CYVCV is the causal agent of yellow vein clearing disease and represents a new species in the genus Mandarivirus.

Virology Division News: The new plant virus family Flexiviridae and assessment of molecular criteria for species demarcation

The new plant virus family Flexiviridae is described. The family is named because its members have flexuous virions and it includes the existing genera Allexivirus, Capillovirus, Carlavirus, Foveavirus, Potexvirus, Trichovirus and Vitivirus, plus the new genus Mandarivirus together with some related viruses not assigned to any genus. The family is justified from phylogenetic analyses of the polymerase and coat protein (CP) sequences. To help to define suitable molecular criteria for demarcation of species, a complete set of pairwise comparisons was made using the nucleotide (nt) and amino acid (aa) sequences of each fully-sequenced gene from every available accession in the family. Based on the distributions and on inspection of the data, it was concluded that, as a general rule, distinct species have less than ca. 72% identical nt or 80% identical aa between their entire CP or replication protein genes.