Hibiscus Chlorotic Ringspot Virus Research Articles

First Report of Hibiscus Chlorotic Ringspot Virus infecting Hibiscus rosa-sinensis in Italy.

Hibiscus chlorotic ringspot virus (HCRSV) belongs to the Betacarmovirus genus of the Tombusviridae family and is a positive-sense monopartite single-stranded RNA virus. HCRSV was first described in a hibiscus variety imported into the United States from El Salvador. The HCRSV natural host range is limited to species belonging to Malvaceae family, in particular Hibiscus rosa-sinensis, H. manihot, H. diversifolius and H. syriacus. In September 2023, leaf mottle and chlorotic spots were observed on the leaves of two H. rosa-sinensis plants located in a public garden of Ercolano (Naples city, Southern Italy). Two leaves, one from each symptomatic plant, were pooled and submitted to double-stranded RNA extraction using a Viral Gene-Spin Viral DNA/RNA Extraction Kit (iNtRON, Korea), followed by cDNA library preparation with the TruSeq Stranded Total RNA (Illumina, USA). Sequencing on the Illumina NovaSeq 6000 platform (Illumina, USA) with 150-bp paired-end reads yielded 22,578,913 raw reads. 21,413,571 clean reads were obtained by quality control on the sequencing data was performed with the software FastQC (v. 0.11.5). Then low quality bases and adapters were removed with the software BBDuk in the BBTools package setting a minimum read quality of 25 and minimum read length of 35 bp. The resulted filtered reads were used to assembly viral genome by using two different algorithms (Metaspades and RNAViral) implemented in SPAdes (v. 3.15.3). Of the total contigs de novo assembled, the two algorithms implemented identified respectively 340 and 559 contigs related to viruses. BLASTn analysis of the contigs identify one coting of 3965 nt covering 97% to 100% of the whole genome sequence of nine HCRSV isolates, with percentage of identity of 87.8-95.2%. No other plant viruses or viroids have been identified during bioinformatic analysis. To confirm the result, a full-length genomic sequence of the Italian HCRSV isolate (named Ita-1), was obtained by reverse transcription polymerase chain reaction (RT-PCR) using specific primers designed on the sequence of the assembled contigs. The PCR products were sequenced using the Sanger method (Microsynth Seqlab, Germany) in both directions. The obtained full-length genomic sequence of the HCRSV isolate (Acc. No. OR981792) was 3910 nt in length. Maximum-likelihood phylogenetic trees inferred from the whole genome sequence showed that Ita-1 clustered closely with HCRSV isolates. The leaf samples were further analyzed using a HCRSV ELISA kit (Agdia, USA). Healthy H. rosa-sinensis leaves were taken as a negative control and buffer solution as a blank control. The results showed a positive reaction for the two symptomatic plants (OD = 1.345 ± 0.010 at 405 nm) relative to the negative (OD = 0.097) and blank (OD = 0.065) controls. Overall, the results of serological and molecular analyses supported that symptoms observed in H. rosa-sinensis were strictly associated to HCRSV infection. Four viruses have been reported in H. rosa-sinensis in Italy so far: eggplant mottled dwarf nucleorhabdovirus (EMDV), hibiscus latent Fort Pierce virus (HLFPV), hibiscus latent ringspot virus (HLRSV), hibiscus latent Singapore virus (HLSV). To our knowledge, this is the first report of HCRSV in H. rosa-sinensis in Italy, where probably infected plants were accidentally introduced. At present, HCRSV has been reported in three continents (Asia, Oceania and North America) and the number of countries where the virus has been detected is likely to increase rapidly as a result of increased surveillance and availability of diagnostic methods. This study will help the management of viral diseases on H. rosa-sinensis in Italy.

First report of Hibiscus soymovirus in Hibiscus rosa-sinensis in Colombia in mixed infection.

Hibiscus is native to southeast Asia but well suited to Colombia's arid soil and dry climates from the coast to the mountains of Bogotá. Viruses infecting hibiscus in Colombia are largely unexplored, with four viruses previously known: hibiscus chlorotic ringspot virus (HCRSV), hibiscus latent Fort Pierce virus (HLFPV), hibiscus latent Singapore virus (HLSV), and citrus leprosis virus C2 (CiLV-C2) (Padmanabhan et al., 2023). Mixed infections between these viruses were frequently detected. A recent virome analysis of a single hibiscus plant from Colombia revealed multiple viruses in mixed infection; : HCRSV, HLFPV, passion fruit green spot virus (PFGSV), a strain of physalis vein necrosis nepovirus, four novel carlavirus, one new potexvirus and a mitovirus. In addition, few smaller contigs of blunervirus and soymovirus were also identified in the high throughput sequencing (HTS) data, but their presence in the mixed infection could not be validated (A. Roy et al. 2023unpublish data). During Brevipalpus-transmitted virus (BTV) surveys, two asymptomatic and 15 hibiscus foliar samples showing green ringspots with central chlorotic spots in senescing areas, mosaic, and black or chlorotic spots were collected from six departments (states) in three geographical regions of Colombia: Tolima (n=4) and Cauca Valley (n=2) (Andean region), Meta (n=6) and Casanare (n=1) (Orinoquia region), and Quindío (n=1) and Risaralda (n=1) (coffee growing region). About 100 mg of 17 hibiscus leaf samples were separately processed for RNA isolation without DNase I treatment and tested for known BTVs, and for newly discovered hibiscus soymovirus (HSV; genus Soymovirus family Caulimoviridae) using PCR assays (Padmanabhan et al. 2023, Wang et al. 2023). To identify potential HSV infection in the samples, published SVF1/SVR1 and newly designed primer pairs (HSV-REP-F/-R and HSV-CPG-F/-R) were used to amplify the 430 nt transactivation (ORF-VI), 631 nt replicase (REP) and 401 nt coat protein gene (CPG), respectively (Supplementary 1). Of 17 samples tested, three from Tolima and one each from Meta and Quindío yielded all three expected size amplicons. Bi-directional sequencing followed by BLASTn analysis revealed 95-98% nt identity with the CPG, REP, and ORF-VI genes of HSV (OP757659). Ribo-depleted libraries were prepared using the RNA extracts of five HSV PCR positive samples. HTS yielded 11.6 to 50.3 million raw reads per sample library. Adapters were trimmed and filtered from the raw reads with Trimmomatic v0.39 and then assembled using SPAdes v3.15.5 (Padmanabhan et al., 2023). Contigs were blasted against the Arabidopsis proteome and a RefSeq-based viral protein database. Potential viral sequences were then blasted against the complete NCBI nr database. Assembled soymo contigs covered 99-100% of the HSV genome, with per-nucleotide read depths of 23.8 to 393. Contigs from the Tolima (Accessions; OR621030- OR621032 and Quindío samples (OR621033) covered 99-100% of the HSV genome and had >96-98% nt identity to Hawaiian isolate (OP757659) whereas the Meta sample contigs covered 78% of the genome with 9495% nt identity. HTS contigs shared >98-99% nt identities with their PCR amplicons. Along with HSV, other virus sequences (HCRSV, HLFPV, PFGSV, CiLV-C2, and mycoviruses) were variously detected from all five libraries. Due to mixed infection no symptom similarity was noticed among these 5 samples. The findings in hibiscus in Tolima, Meta and Quindío represent the first confirmed report of HSV infection in hibiscus in Colombia. The widespread distribution suggests the possibility of HSV dispersion via movement of planting material, and potential further spread to another hibiscus growing region.

First Report of Cotton Leafroll Dwarf Virus infection of Malvaviscus arboreus in China.

Cotton leafroll dwarf virus (CLRDV, genus Polerovirus, family Solemoviridae) has been reported to infect cotton in Brazil, Argentina, India, Thailand and Timor-Leste (Agrofoglio YC et al. 2017; Corrêa RL et al. 2005; Mukherjee et al. 2012; Ray et al. 2016; Sharman et al. 2015), and in the United States (Ali and Mokhtari et al. 2020; Avelar et al. 2019). It has also been recently reported to infect Cicer arietinum (chickpea) in Uzbekistan and Hibiscus syriacus in Korea (Igori et al. 2022; Kumari et al. 2020). In China, the natural infection of plants by CLRDV has not been reported previously. In August 2017, leaf samples were collected from a wild plant of Malvaviscus arboreus (Malvaceae) exhibiting symptoms including leaf yellowing and distorting in Tengchong County of Yunnan Province. Leaves were used for total RNA extraction using TRIzol Reagent (Invitrogen, USA). Small RNA library construction and deep sequencing was performed on illumina HiSeqTM 2000 platform by Novogene Bioinformatic Technology Co., Ltd (Beijing, China). A total of 11, 525, 708 raw reads were obtained and further computationally analyzed by Perl scripts. The adaptors were removed and the obtained 7, 520, 902 clean reads with size of 18- to 26-nucleotide (nt) were aligned with the GenBank virus RefSeq database using Bowtie software. These reads were mainly mapped to the genomes of hibiscus bacilliform virus (genusBadnavirus,familyCaulimoviridae), hibiscus chlorotic ringspot virus (genus Betacarmovirus, family Procedovirinae), hibiscus latent Singapore virus (genus Tobamovirus, family Virgaviridae) and CLRDV isolate ARG (accession no. GU167940). The average coverage depth of clean reads mapped to CLRDV genome was 97.76%. Contigs greater than 50 nt were used to search for similar sequences by BLASTx, and 107 contigs were annotated as homologous to CLRDV isolates. To confirm CLRDV infection, reverse transcription polymerase chain reaction (RT-PCR) was performed using the specific primer pair CLRDV-F (5'-TCCACAGGAAGTATCACGTTCG-3') and CLRDV-R (5'-CCTTGTGTGGTTTGATTCGTGA-3') designed based on two of these contigs well-aligned to the genome of CLRDV isolate ARG. An amplicon of 1095-bp size was amplified, and was sequenced by Sanger sequencing (TsingKe Biological Technology, Chengdu, China), and BLASTn search results showed a maximum nucleotide identity of 95.45% with CLRDV isolate CN-S5, an isolate obtained from soybean aphid host in China (accession no. KX588248). To obtain more information on this CLRDV isolate, four primer pairs were designed and used for RT-PCR amplification (TableS1). The amplicons with size of about 860-, 1400-, 3200- and 1100-bp, were obtained separately and assembled into a complete genome sequence up to 5, 865-nt in length (isolate YN, deposited under GenBank accession no. MN057665). BLASTn showed the highest nucleotide similarity of 94.61% with CLRDV isolate CN-S5. From 2018 to 2022, additional M. arboreus samples with leaf yellowing or curling symptoms (9 from Shapingba District in Chongqing City, 5 from Nanchong City in Sichuan Province, 9 from Kunming City and 12 from Tengchong County in Yunnan Province) were collected and tested for CLRDV by RT-PCR using primer pairs CLRDV-F/CLRDV-R. The nucleotide sequences of the CLRDV P0 gene in two samples from Tengchong County were obtained by Sanger sequencing and deposited under GenBank (CLRDV isolate TCSL1 P0 gene, accession no. OQ749809; CLRDV isolate TCSW2 P0 gene, accession no. OQ749809). To our knowledge, this is the first report of CLRDV naturally infecting Malvaviscus arboreus in China, thus extending the information on its geographical distribution and host range. Malvaviscus arboreus is a widely cultivated ornamental plant in Yunnan Province, China. The natural occurrence of CLRDV not only affects the ornamental value of Malvaviscus arboreus, but also poses a potential threat to cotton production in China. This study will assist further surveillance of CLRDV infection and future development of effective protection strategies against CLRDV in China.

Unraveling the genome and gene expression of Hibiscus chlorotic ringspot virus isolated from Hibiscus rosa-sinensis in Southwest Iran

Hibiscus chlorotic ringspot virus (HCRSV; genus Betacarmovirus, family Tombusviridae) is one of the pathogens causing disease on Hibiscus rosa-sinensis (Chinese rose), an ornamental plant grown throughout the tropic and subtropic regions of the world. In a survey on Chinese hibiscus plants showing chlorotic ring spots leaves in Khuzestan Province, Southwestern Iran, HCRSV was identified and the complete genome of the first Iranian isolate of the virus, HCRSV-IR, was determined using Illumina RNA-Seq. The genome of HCRSV-IR was 3911 nucleotides long and potentially encodes seven open reading frames (ORFs) similar to previously known HCRSV isolates. Iranian isolate of virus showed the highest nucleotide sequence identity, 94.39 %, to the Brazil isolate (MK279671.1) Hibiscus chlorotic ringspot virus (HCRSV; genus Betacarmovirus, family Tombusviridae) is one of the pathogens causing disease on Hibiscus rosa-sinensis (Chinese rose), an ornamental plant grown throughout the tropic and subtropic regions of the world. In a survey on Chinese hibiscus plants showing chlorotic ring spots leaves in Khuzestan Province, Southwestern Iran, HCRSV was identified and the complete genome of the first Iranian isolate of virus, HCRSV-IR, was determined using Illumina RNA-Seq. The genome of HCRSV-IR was 3911 nucleotides long and potentially encodes seven open reading frames (ORFs) similar to previously known HCRSV isolates. Iranian isolate of the virus showed the highest nucleotide sequence identity, 94.39 %, to Brazil isolate (MK279671.1) Chinese hibiscus plants exhibiting chlorotic ringspot symptoms were surveyed in southwestern Iran to determine the presence of Hibiscus chlorotic ringspot virus (HCRSV; genus Betacarmovirus, family Tombusviridae). The complete sequence of the Iranian isolate of the virus, HCRSV-IR, was obtained using Illumina RNA-Seq, marking the first time this sequence has been documented. HCRSV-IR (Accession No. OP779319) has a genome comprising 3911 nucleotides, which encodes seven potential open reading frames (ORFs). Comparative analysis revealed that HCRSV-IR shares the highest nucleotide sequence identity (94.39 %) with an isolate from Brazil (MK279671.1). Analysis of the gene expression profile of the HCRSV-IR population infecting hibiscus plants indicated increased expression of the coat protein gene. Additionally, seventeen single nucleotide polymorphisms (SNPs) were identified within the viral variant populations from the infected hibiscus plants. These SNPs were located in coding regions across the entire genome, implying potential evolutionary significance. To the best of our knowledge, this study represents the first investigation into the molecular characterization of the complete genome of HCRSV, specifically infecting Chinese hibiscus in Iran.

The Complete Genome Sequence, Molecular Detection, and Anatomical Analysis of Hibiscus Chlorotic Ringspot Virus Infecting Hibiscus rosa-sinensis in Peninsular Malaysia

Hibiscus rosa-sinensis is the national flower of Malaysia and is widely cultivated as landscape planting across the nation. In 1995, Hibiscus chlorotic ringspot virus (HCRSV) was reported for the first time in Malaysia. Until today, there have been no follow-up studies on the viral infection in the plant, yet the virus symptom of chlorotic spots has been observed throughout the Hibiscus population. Therefore, this study aimed to report the complete genome sequence of the HCRSV, validate a molecular detection tool for its diagnosis, and measure the impact of the virus symptom and infection in H. rosa-sinensis. This study reported the complete genome of the HCRSV through RNA sequencing. The phylogenetic analysis of the HCRSV isolated from different geographical sources and several other related viruses was performed to confirm its identity and classification. Additionally, primers based on the genome sequence were designed and used for RT-PCR to detect the presence of the virus in symptomatic leaves, further confirming the HCRSV identity and presence. Meanwhile, the impact of the virus was measured by examining the anatomical and morphological changes in the leaf structure of symptomatic samples. Light microscopy and scanning electron microscopy were used to examine potential adaptations and comparisons between the leaf structures of healthy and infected samples, especially in the leaf lamina, petiole, and midrib cells. The results confirmed the complete genome sequence of the HCRSV, its molecular detection strategy, and the impact of the viral infection on Hibiscus leaves in Peninsular Malaysia. This study is beneficial for the management strategy of the virus and for protecting an important plant in the nation.

First Detection and Genome Characterization of a New RNA Virus, Hibiscus Betacarmovirus, and a New DNA Virus, Hibiscus Soymovirus, Naturally Infecting Hibiscus spp. in Hawaii

Hibiscus (Hibiscus spp., family Malvaceae) leaves exhibiting symptoms of mosaic, ringspot, and chlorotic spots were collected in 2020 on Oahu, HI. High-throughput sequencing analysis was conducted on ribosomal RNA-depleted composite RNA samples extracted from symptomatic leaves. About 77 million paired-end reads and 161,970 contigs were generated after quality control, trimming, and de novo assembly. Contig annotation with BLASTX/BLASTN searches revealed a sequence (contig 1) resembling the RNA virus, hibiscus chlorotic ringspot virus (genus Betacarmovirus), and one (contig 2) resembling the DNA virus, peanut chlorotic streak virus (genus Soymovirus). Further bioinformatic analyses of the complete viral genome sequences indicated that these viruses, with proposed names of hibiscus betacarmovirus and hibiscus soymovirus, putatively represent new species in the genera Betacarmovirus and Soymovirus, respectively. RT-PCR using specific primers, designed based on the retrieved contigs, coupled with Sanger sequencing, further confirmed the presence of these viruses. An additional 54 hibiscus leaf samples from other locations on Oahu were examined to determine the incidence and distribution of these viruses.

Molecular modeling and interaction between Arabidopsis sulfite oxidase and the GW motif of Turnip crinkle virus coat protein

Previous study has shown that Hibiscus sulfite oxidase (SO) interacts with Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) and triggers sulfur enhanced defense (SED). In this study, we show the interaction of Arabidopsis SO (AtSO) and Turnip crinkle virus (TCV) CP in Arabidopsis thaliana plants. We identified the binding sites of TCV CP (W274) and AtSO (D223) using bioinformatics and confirmed it experimentally. Mutation of binding site W274 to A274 in TCV CP resulted in failure of TCV infection. TCV accumulation in SO over-expression (SO_OE) plants was lower than that in wild-type (WT) and SO knock-out (SO_KO) plants at 7 dpi but reached a level similar to that of WT and SO_KO plants at 10 dpi. AtSO competed with Argonaute 1 (AGO1) for TCV CP binding in vitro. AtSO may serve as an anti-viral factor through sequestering TCV CP for binding with AGO1 and confers virus resistance.

First genome sequence of an isolate of hibiscus chlorotic ringspot virus from the Western hemisphere

For the first time, the near-complete genome sequence of the betacarmovirus, hibiscus chlorotic ringspot virus (HCRSV) isolated from the Americas is disclosed. High throughput sequencing of the total RNA extract from a Hibiscus-rosa sinensis (L.) plant collected in Sao Paulo state, Brazil, revealed the genome sequence of HCRSV isolate SBO1, which is 3945 nucleotides long and shows 93.1% nucleotide sequence identity with HCRSV_Singapore (X86448), considered the type member of the species. These two viruses display a similar genomic organization and potentially encode seven open reading frames (ORFs). In addition, a phylogenetic analysis based on a fragment with 557 nts of p38, the coat protein gene, from a cohort of 14 samples collected in Brazil revealed a no clearly defined segregation of South American isolates from those previously detected in geographical areas outside this continent. The practical consequences of the use of p38 ORF-derived amplicons for detection and variability studies of HCRSV are concisely discussed.

First Report of Hibiscus Chlorotic Ringspot Virus Infecting Hibiscus in Fujian Province, China.

First Report of Hibiscus Chlorotic Ringspot Virus Infecting Hibiscus in Fujian Province, China.

In Vitro-Reassembled Plant Virus-Like Particles of Hibiscus Chlorotic Ringspot Virus (HCRSV) as Nano-Protein Cages for Drugs.

Spherical shaped plant viruses require a precise quantity, size, and shape of their coat protein subunits to assemble into virions of identical dimensions. The capsid of spherical plant virus particles typically consists of a precisely shaped protein cage, which in many cases is assembled from identical coat protein subunits. In addition to packaging the viral genome, such protein cages may have the capacity to load foreign compounds, either large molecules (e.g., polymers) or small molecules (e.g., anticancer chemotherapy drugs). Therefore, reassembled protein cages of suitable viruses can serve as carriers for cargo loading, which is what makes them an attractive platform for drug delivery. Here we describe methods to reassemble plant virus-like particles of hibiscus chlorotic ringspot virus (HCRSV) as nano-protein cages including the techniques to purify coat protein, prepare virus-like particles, and load them with foreign compounds.

First report of Hibiscus chlorotic ringspot virus in Turkey

Hibiscus rosa-sinensis (rose of China) is an ornamental plant grown throughout the tropics and subtropics, and under glass in more temperate areas. It is commonly used in landscape design; however, pathogens causing diseases on H. …

Hibiscus chlorotic ringspot virus coat protein is essential for cell-to-cell and long-distance movement but not for viral RNA replication.

Hibiscus chlorotic ringspot virus (HCRSV) is a member of the genus Carmovirus in the family Tombusviridae. In order to study its coat protein (CP) functions on virus replication and movement in kenaf (Hibiscus cannabinus L.), two HCRSV mutants, designated as p2590 (A to G) in which the first start codon ATG was replaced with GTG and p2776 (C to G) in which proline 63 was replaced with alanine, were constructed. In vitro transcripts of p2590 (A to G) were able to replicate to a similar level as wild type without CP expression in kenaf protoplasts. However, its cell-to-cell movement was not detected in the inoculated kenaf cotyledons. Structurally the proline 63 in subunit C acts as a kink for β-annulus formation during virion assembly. Progeny of transcripts derived from p2776 (C to G) was able to move from cell-to-cell in inoculated cotyledons but its long-distance movement was not detected. Virions were not observed in partially purified mutant virus samples isolated from 2776 (C to G) inoculated cotyledons. Removal of the N-terminal 77 amino acids of HCRSV CP by trypsin digestion of purified wild type HCRSV virions resulted in only T = 1 empty virus-like particles. Taken together, HCRSV CP is dispensable for viral RNA replication but essential for cell-to-cell movement, and virion is required for the virus systemic movement. The proline 63 is crucial for HCRSV virion assembly in kenaf plants and the N-terminal 77 amino acids including the β-annulus domain is required in T = 3 assembly in vitro.

Plant Growth Retardation and Conserved miRNAs Are Correlated to Hibiscus Chlorotic Ringspot Virus Infection

Virus infection may cause a multiplicity of symptoms in their host including discoloration, distortion and growth retardation. Hibiscus chlorotic ringspot virus (HCRSV) infection was studied using kenaf (Hibiscus cannabinus L.), a non-wood fiber-producing crop in this study. Infection by HCRSV reduced the fiber yield and concomitant economic value of kenaf. We investigated kenaf growth retardation and fluctuations of four selected miRNAs after HCRSV infection. Vegetative growth (including plant height, leaf size and root development) was severely retarded. From the transverse and radial sections of the mock and HCRSV-infected kenaf stem, the vascular bundles of HCRSV-infected plants were severely disrupted. In addition, four conserved plant developmental and defence related microRNAs (miRNAs) (miR165, miR167, miR168 and miR171) and their respective target genes phabulosa (PHB), auxin response factor 8 (ARF8), argonaute 1 (AGO1) and scarecrow-like protein 1 (SCL1) displayed variation in expression levels after HCRSV infection. Compared with the mock inoculated kenaf plants, miR171 and miR168 and their targets SCL1 and AGO1 showed greater fluctuations after HCRSV infection. As HCRSV upregulates plant SO transcript in kenaf and upregulated AGO1 in HCRSV-infected plants, the expression level of AGO1 transcript was further investigated under sulfite oxidase (SO) overexpression or silencing condition. Interestingly, the four selected miRNAs were also up- or down-regulated upon overexpression or silencing of SO. Plant growth retardation and fluctuation of four conserved miRNAs are correlated to HCRSV infection.

Full - Genome Sequence of Hibiscus Chlorotic Ringspot Virus from Israel

Hibiscus rosa-sinensis is one of the most prevalent ornamental plants grown in private and public gardens. Hibiscus chlorotic ringspot virus (HCRSV) is a member of the Carmovirus genus, with a positive single-strand RNA that putatively encodes seven proteins. The complete genome of the first Israeli isolate of HCRSV, HCRSV-IL, comprises 3,908 nucleotides and shows 93% nucleotide sequence identity to the Singapore isolate and 87% identity to the Taiwanese isolate.

Basic Amino Acid Mutations in the Nuclear Localization Signal of Hibiscus Chlorotic Ringspot Virus p23 Inhibit Virus Long Distance Movement

The p23 is a unique protein in the Hibiscus chlorotic ringspot virus which belongs to Family Tombusviridae Genus Carmovirus. Our previous results showed that the p23 is indispensable for host-specific replication and is localized in the nucleus with a novel nuclear localization signal. To investigate additional function(s) of p23, mutations of basic amino acids lysine (K), arginine (R) and histidine (H) that abolish its nuclear localization, were introduced into a biologically active full-length cDNA clone p223 of HCRSV for testing its effects on virus replication and virus movement in vivo. Primer-specific reverse transcription-PCR was conducted to detect gene transcript level of p23 and viral coat protein separately. Virus replication and its coat protein expression were detected by fluorescent in situ hybridization and Western blot, respectively. The effect of p23 was further confirmed by using artificial microRNA inoculation-mediated silencing. Results showed that the two mutants were able to replicate in protoplasts but unable to move from inoculated leaves to newly emerged leaves. Both the p23 and the CP genes of HCRSV were detected in the newly emerged leaves of infected plants but CP was not detected by Western blot and no symptom was observed on those leaves at 19 days post inoculation. This study demonstrates that when p23 is prevented from entering the nucleus, it results in restriction of virus long distance movement which in turn abrogates symptom expression in the newly emerged leaves. We conclude that the p23 protein of HCRSV is required for virus long distance movement.

First report of Hop stunt viroid in Hibiscus rosa‐sinensis in Italy

Hop stunt viroid (HSVd), type member of the Hostuviroid genus in the Pospiviroidae family, has a very wide host range that includes trees, shrubs and herbaceous plants. In grapevine and almond, HSVd infection may be latent, whereas in other plants it causes various specific disorders; these include stunt on hop, dapple fruit on peach and plum, fruit deformation and rugosity on apricot and plum, pale fruits on cucumber and cachexia on citrus. All these symptoms can lead to very high economic impact. In the past, Hibiscus spp. amongst ornamental plants were also reported as natural host of HSVd (Sänger, 6), but further information on this has been lacking. Recently, a survey of the phytosanitary status of ornamental hibiscus was conducted in wholesale nurseries in Central Italy (Manglli et al., 5). Plants of Hibiscus rosa-sinensis showing symptoms resembling virus infection were first examined for the presence of Hibiscus chlorotic ringspot virus (HCRSV), Hibiscus latent Singapore virus (HLSV) and Hibiscus latent Fort Pierce virus (HLFPV). However, several plants belonging to two different cultivars, showing both severe symptoms of reduction in plant growth and upward curling and deformation of leaves (Fig. 1), tested negative for these viruses. Moreover, the observed symptoms did not resemble those of known viruses in hibiscus. Consequently, the same plants were tested for HSVd using a RT-PCR protocol by Faggioli et al. (2) employing two primer pairs (VP-19 and VP-20; HSVFL1 and HSVFL2) designed respectively by Astruc et al. (1) and Kofalvi et al. (4). Out of 17 plants showing the aforementioned symptoms, four samples gave positive results. By contrast, HSVd was not detected in any of the 60 leaf samples showing only yellow spots and chlorotic mottle on leaves that tested positive for HCRSV, HLSV and/or HLFPV. The four amplification products were cloned (pGem-T vector, Promega, WI, USA) and sequenced. Sequence analysis of different clones for each isolate (GenBank Accession Nos. KC137256 to KC137266) revealed that two isolates showed the same size (296 nt) and that both had a high homology amongst clones and between the two isolates. Furthermore, both isolates were closely related to a Turkish plum isolate (EF523829, Gazel et al., 3) belonging to the Hop-group, as shown by the HSVd phylogenetic tree (Fig. 2; Kofalvi et al., 4). The sequences of the other two isolates were 299 nt long and also showed a high homology amongst clones and between the two isolates. They were practically identical to an apricot isolate (Y08437, Kofalvi et al., 4), thus grouping in the recombinant Plum-Citrus phylogenetic group (Fig. 2). These results suggest a different origin of infection in the vegetative propagation and cultivation of hibiscus plants. This study confirms that Hibiscus rosa-sinensis is a natural host of HSVd as reported by Sänger in 1988. However, to our knowledge, this is the first report of HSVd in H. rosa-sinensis in Italy.

Hibiscus chlorotic ringspot virus Coat Protein Upregulates Sulfur Metabolism Genes for Enhanced Pathogen Defense

In both Hibiscus chlorotic ringspot virus (HCRSV)-infected and HCRSV coat protein (CP) agroinfiltrated plant leaves, we showed that sulfur metabolism pathway related genes-namely, sulfite oxidase (SO), sulfite reductase, and adenosine 5'-phosphosulfate kinase-were upregulated. It led us to examine a plausible relationship between sulfur-enhanced resistance (SED) and HCRSV infection. We broadened an established method to include different concentrations of sulfur (0S, 1S, 2S, and 3S) to correlate them to symptom development of HCRSV-infected plants. We treated plants with glutathione and its inhibitor to verify the SED effect. Disease resistance was induced through elevated glutathione contents during HCRSV infection. The upregulation of SO was related to suppression of symptom development induced by sulfur treatment. In this study, we established that HCRSV-CP interacts with SO which, in turn, triggers SED and leads to enhanced plant resistance. Thus, we have discovered a new function of SO in the SED pathway. This is the first report to demonstrate that the interaction of a viral protein and host protein trigger SED in plants. It will be interesting if such interaction applies generally to other host-pathogen interactions that will lead to enhanced pathogen defense.

Identification of a Plant Viral RNA Genome in the Nucleus

Viruses contain either DNA or RNA as genomes. DNA viruses replicate within nucleus, while most RNA viruses, especially (+)-sense single-stranded RNA, replicate and are present within cytoplasm. We proposed a new thought that is contrary to the common notion that (+)-sense single-stranded RNA viruses are present only in the cytoplasm. In this study, we question whether the genome of a plant RNA virus (non-retroviral) is present in the nucleus of infected cells? Hibiscus chlorotic ringspot virus (HCRSV) RNA was detected in the nucleus of infected cells, as shown by fluorescent in situ hybridization. Western blot using anti-histone 3 and anti-phosphoenolpyruvate carboxylase showed that nuclei were highly purified from mock and HCRSV-infected kenaf (Hibiscus cannabilis L.) leaves, respectively. The p23 and HCRSV coat protein (CP) coding regions were both amplified from total RNA extracted from isolated nuclei. Viral RNA in the nucleus may be used to generate viral microRNAs (vir-miRNAs), as five putative vir-miRNAs were predicted from HCRSV using the vir-miRNAs prediction database. The vir-miRNA (hcrsv-miR-H1-5p) was detected using TaqMan® stem-loop real-time PCR, and by northern blot using DIG-end labeled probe in HCRSV-infected kenaf leaves. Finally, a novel nuclear localization signal (NLS) was discovered in p23 of HCRSV. The NLS interacts with importin α and facilitates viral RNA genome to enter nucleus. We demonstrate the presence of a (+)-sense single-stranded viral RNA within nucleus.

Hibiscus chlorotic ringspot virus upregulates plant sulfite oxidase transcripts and increases sulfate levels in kenaf (Hibiscus cannabinus L.)

Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) is required for encapsidation and virus systemic movement. To better understand the roles of HCRSV CP in virus infection and its interactions with host proteins, a cDNA library of kenaf (Hibiscus cannabinus L.) was constructed and screened by using a yeast two-hybrid system (YTHS) to identify CP-interacting proteins. One protein identified was sulfite oxidase (SO) and the interaction was confirmed in vitro and in vivo. The interaction was found to be associated with peroxisomes by immunofluorescent labelling of peroxisomes by an anti-SKL signal peptide antibody. Our YTHS results showed that only the P and S domains of CP interacted with SO from kenaf. This is probably due to the exposure of these two domains on the outer surface of the capsid. Peroxisomes were observed to aggregate in HCRSV-infected cells, and biochemical assays of total protein from kenaf leaf extracts showed that SO activity and SO-dependent H(2)O(2)-generating activity in the HCRSV-infected leaves increased compared with that in mock-inoculated kenaf plants.

Preliminary X-ray data analysis of crystalline hibiscus chlorotic ringspot virus

Hibiscus chlorotic ringspot virus (HCRSV) is a positive-sense monopartite single-stranded RNA virus that belongs to the Carmovirus genus of the Tombusviridae family, which includes carnation mottle virus (CarMV). The HCRSV virion has a 30 nm diameter icosahedral capsid with T = 3 quasi-symmetry containing 180 copies of a 38 kDa coat protein (CP) and encapsidates a full-length 3.9 kb genomic RNA. Authentic virus was harvested from infected host kenaf leaves and was purified by saturated ammonium sulfate precipitation, sucrose density-gradient centrifugation and anion-exchange chromatography. Virus crystals were grown in multiple conditions; one of the crystals diffracted to 3.2 A resolution and allowed the collection of a partial data set. The crystal belonged to space group R32, with unit-cell parameters a = b = 336.4, c = 798.5 A. Packing considerations and rotation-function analysis determined that there were three particles per unit cell, all of which have the same orientation and fixed positions, and resulted in tenfold noncrystallography symmetry for real-space averaging. The crystals used for the structure determination of southern bean mosaic virus (SBMV) have nearly identical characteristics. Together, these findings will greatly aid the high-resolution structure determination of HCRSV.