Discovery and characterization of two highly divergent variants of a novel potyvirus species infecting Madagascar periwinkle (Catharanthus roseus L.).

During summer 2022, a cluster of Madagascar periwinkle plants with white and mauve flowers were observed with foliar mild yellow mosaic symptoms on a private property in Harlingen, Cameron County, Texas. The symptoms were reproduced on mechanically inoculated periwinkle and Nicotiana benthamiana plants. Virions of 776 to 849 nm in length and 11.7 to 14.8 nm in width were observed in transmission electron microscopy of leaf dip preparations made from symptomatic periwinkle leaves. Highthroughput sequencing (HTS) analysis of total RNA extracts from symptomatic leaves revealed the occurrence of two highly divergent variants of a novel Potyvirus species as the only virus-like sequences present in the sample. The complete genomes of both variants were independently amplified via RT-PCR, cloned, and Sanger sequenced. The 5' and 3' of the genomes were acquired using RACE methodology. The assembled virus genomes were 9,936 and 9,944 nucleotides (nt) long and they shared 99.9-100% identities with the respective HTS-derived genomes. Each genome encoded hypothetical polyprotein of 3,171 amino acids (aa) (362.6 kDa) and 3,173 aa (362.7 kDa), respectively, and they shared 77.3%/84.4% nt/aa polyproteins identities, indicating that they represent highly divergent variants of the same Potyvirus species. Both genomes also shared below species threshold polyprotein identity levels with the most closely phylogenetically related known potyviruses thus indicating that they belong to a novel species. The name periwinkle mild yellow mosaic virus (PwMYMV) is given to the potyvirus with complete genomes of 9,936 nt for variant 1 (PwMYMV-1) and 9,944 nt for variant 2 (PwMYMV-2). We propose that PwMYMV be assigned into the genus Potyvirus (family Potyviridae).

Integration of Rubus yellow net virus in the raspberry genome: A story centuries in the making.

Rubus yellow net virus (RYNV) belongs to genus Badnavirus. Badnaviruses are found in plants as endogenous, inactive or activatable sequences, and/or in episomal (infectious and active) forms. To assess the state of RYNV in Rubus germplasm, we sequenced the genomes of various cultivars and mined eight raspberry whole genome datasets. Bioinformatics analysis revealed the presence of a diverse array of endogenous RYNV (endoRYNV) sequences that differ significantly in their structure; some lineages have nearly complete, yet non-functional genomes whereas others have rudimentary, short sequence fragments. We developed assays to genotype the main lineages as well as the only known episomal lineage present in the United States. This study discloses the widespread presence of endoRYNVs in commercial raspberries, likely because breeding efforts have focused on a limited pool of germplasm that harbored endoRYNVs.

Genetic Analysis of the Emerging Citrus Yellow Vein Clearing Virus Reveals a Divergent Virus Population in American Isolates.

Citrus yellow vein clearing virus is a previously reported citrus virus from Asia with widespread distribution in China. In 2022, the California Department of Food and Agriculture conducted a multipest citrus survey targeting multiple citrus pathogens including citrus yellow vein clearing virus (CYVCV). In March 2022, a lemon tree with symptoms of vein clearing, chlorosis, and mottling in a private garden in the city of Tulare, California, tested positive for CYVCV, which triggered an intensive survey in the surrounding areas. A total of 3,019 plant samples, including citrus and noncitrus species, were collected and tested for CYVCV using conventional reverse transcription polymerase chain reaction, reverse transcription quantitative polymerase chain reaction, and Sanger sequencing. Five hundred eighty-six citrus trees tested positive for CYVCV, including eight citrus species not previously recorded infected under field conditions. Comparative genomic studies were conducted using 17 complete viral genomes. Sequence analysis revealed two major phylogenetic groups. Known Asian isolates and five California isolates from this study made up the first group, whereas all other CYVCV isolates from California formed a second group, distinct from all worldwide isolates. Overall, the CYVCV population shows rapid expansion and high differentiation indicating a population bottleneck typical of a recent introduction into a new geographic area.

Plasticity of the lettuce infectious yellows virus minor coat protein (CPm) in mediating the foregut retention and transmission of a chimeric CPm mutant by whitefly vectors.

Transmission of the crinivirus, lettuce infectious yellows virus (LIYV), is determined by a minor coat protein (CPm)-mediated virion retention mechanism located in the foregut of its whitefly vector. To better understand the functions of LIYV CPm, chimeric CPm mutants engineered with different lengths of the LIYV CPm amino acid sequence and that of the crinivirus, lettuce chlorosis virus (LCV), were constructed based on bioinformatics and sequence alignment data. The 485 amino acid-long chimeric CPm of LIYV mutant, CPmP-1, contains 60 % (from position 3 to 294) of LCV CPm amino acids. The chimeric CPm of mutants CPmP-2, CPmP-3 and CPmP-4 contains 46 (position 3 to 208), 51 (position 3 to 238) and 41 % (position 261 to 442) of LCV CPm amino acids, respectively. All four mutants moved systemically, expressed the chimeric CPm and formed virus particles. However, following acquisition feeding of the virus preparations, only CPmP-1 was retained in the foreguts of a significant number of vectors and transmitted. In immuno-gold labelling transmission electron microscopy (IGL-TEM) analysis, CPmP-1 particles were distinctly labelled by antibodies directed against the LCV but not LIYV CPm. In contrast, CPmP-4 particles were not labelled by antibodies directed against the LCV or LIYV CPm, while CPmP-2 and -3 particles were weakly labelled by anti-LIYV CPm but not anti-LCV CPm antibodies. The unique antibody recognition and binding pattern of CPmP-1 was also displayed in the foreguts of whitefly vectors that fed on CPmP-1 virions. These results are consistent with the hypothesis that the chimeric CPm of CPmP-1 is incorporated into functional virions, with the LCV CPm region being potentially exposed on the surface and accessible to anti-LCV CPm antibodies.

First Report of Apricot vein clearing-associated virus Infecting flowering apricot (Prunus mume) in the United States.

Apricot vein clearing-associated virus is the type species of genus Prunevirus, family Betaflexiviridae. The virus was first discovered from an Italian apricot tree (Prunus armeniaca) showing leaf vein clearing and mottling symptoms (Elbeaino et al. 2014). Since then, apricot vein clearing-associated virus (AVCaV) has been reported in symptomatic and asymptomatic plants from other countries (Marais et al. 2015; Kinoti et al. 2017; Kubaa et al. 2014). In 2018, a domestic selection of a flowering apricot (P. mume cv. Peggy Clarke) (PC01) with no discernible foliar virus-like symptoms was received for inclusion in the Foundation Plant Services (UC-Davis) collection. The plant originated from a private Prunus collection located in California. Total nucleic acids (TNA) were isolated from PC01 leaves using MagMax Plant RNA Isolation Kit (Thermo Fisher Scientific). The TNA were analyzed for a panel of 15 Prunus-infecting viruses by reverse-transcription quantitative PCR (RT-qPCR) (Diaz-Lara et al. 2020). In addition, to screen for sap-transmissible viruses, young leaves of PC01 were homogenized in inoculation buffer and were rubbed onto leaves of herbaceous indicator plants, Chenopodium amaranticolor, C. quinoa, Cucumis sativus, and Nicotiana clevelandii (Rowhani et al. 2005). The source PC01 tested negative for the 15 screened viruses. Interestingly, vein clearing symptoms were observed on leaves of C. quinoa and C. amaranticolor plants (Figure S1). These results suggested the presence of a mechanically transmissible virus in PC01. To determine the identity of mechanically transmissible viral agent, symptomatic C. quinoa and PC01 plant were advanced for high throughput sequencing analysis. Aliquots of TNA from PC01 and C. quinoa were rRNA-depleted and used for cDNA library preparation with TruSeq Stranded Total RNA kit (Illumina). The raw reads were trimmed, de novo assembled, and subsequently were annotated using tBLASTx algorithm (Al Rwahnih et al. 2018). A total of 47,261,138 and 8,812,296 single-end reads were obtained from cDNA libraries of PC01 and C. quinoa, respectively. The de novo assembly generated near-complete contigs resembling AVCaV genome ) from both PC01 and C. quinoa, which were 99.8% identical at the nucleotide level. The longest contig (8,342 nucleotides, 73.5x coverage depth) obtained from PC01 was further completed using SMARTer RACE 5'/3' kit (Takara Bio). The complete genome sequence of AVCaV-PC01 is 8,364 nucleotides long (GenBank: MK170158). The full-length virus genome was compared with GenBank database using BLASTn, which the best hit corresponded to KY132099 with 98% identity. Additionally, AVCaV infection was confirmed in both PC01 selection and the symptomatic C. quinoa by RT-PCR as previously described (Marais et al. 2015). Lastly, symptomatic leaves of C. quinoa were used in leaf dip method to visualize virus particles by transmission electron microscope. As a result, flexuous rod-shaped virions were observed from leaf dips of symptomatic C. quinoa plants (Figure S2). Therefore, our results represent the first report of AVCaV in California, USA. Furthermore, mechanical transmission of an AVCaV isolate infecting flowering apricot to herbaceous hosts was confirmed. Field surveys and biological studies are underway to determine the prevalence of AVCaV in commercial orchards and assess its effect on tree performance.

Comparative analysis identifies amino acids critical for citrus tristeza virus (T36CA) encoded proteins involved in suppression of RNA silencing and differential systemic infection in two plant species.

Complementary (c)DNA clones corresponding to the full-length genome of T36CA (a Californian isolate of Citrus tristeza virus with the T36 genotype), which shares 99.1% identity with that of T36FL (a T36 isolate from Florida), were made into a vector system to express the green fluorescent protein (GFP). Agroinfiltration of two prototype T36CA-based vectors (pT36CA) to Nicotiana benthamiana plants resulted in local but not systemic GFP expression/viral infection. This contrasted with agroinfiltration of the T36FL-based vector (pT36FL), which resulted in both local and systemic GFP expression/viral infection. A prototype T36CA systemically infected RNA silencing-defective N. benthamiana lines, demonstrating that a genetic basis for its defective systemic infection was RNA silencing. We evaluated the in planta bioactivity of chimeric pT36CA-pT36FL constructs and the results suggested that nucleotide variants in several open reading frames of the prototype T36CA could be responsible for its defective systemic infection. A single amino acid substitution in each of two silencing suppressors, p20 (S107G) and p25 (G36D), of prototype T36CA facilitated its systemic infectivity in N. benthamiana (albeit with reduced titre relative to that of T36FL) but not in Citrus macrophylla plants. Enhanced virus accumulation and, remarkably, robust systemic infection of T36CA in N. benthamiana and C. macrophylla plants, respectively, required two additional amino acid substitutions engineered in p65 (N118S and S158L), a putative closterovirus movement protein. The availability of pT36CA provides a unique opportunity for comparative analysis to identify viral coding and noncoding nucleotides or sequences involved in functions that are vital for in planta infection.

Characterization of a New Nepovirus Infecting Grapevine.

In 2012, dormant canes of a proprietary wine grape (Vitis vinifera L.) accession were included in the collection of the University of California-Davis Foundation Plant Services. No virus-like symptoms were elicited when bud chips from propagated own-rooted canes of the accession were graft-inoculated onto a panel of biological indicators. However, chlorotic ringspot symptoms were observed on sap-inoculated Chenopodium amaranticolor Coste & A. Rein and C. quinoa Willd. plants, indicating the presence of a mechanically transmissible virus. Transmission electron microscopy of virus preparations from symptomatic C. quinoa revealed spherical, nonenveloped virions about 27 nm in diameter. Nepovirus-like haplotypes of sequence contigs were detected in both the source grape accession and symptomatic C. quinoa plants via high-throughput sequencing. A novel bipartite nepovirus-like genome was assembled from these contigs, and the termini of each RNA segment were verified by rapid amplification of complementary DNA ends assays. The RNA1 (7,186-nt) of the virus encodes a large polyprotein 1 of 231.1 kDa, and the RNA2 (4,460-nt) encodes a large polyprotein 2 of 148.9 kDa. Each of the polyadenylated RNA segments is flanked by 5'- (RNA1 = 156-nt; RNA2 = 170-nt) and 3'- (RNA1 = 834-nt; RNA2 = 261-nt) untranslated region sequences with >90% identities. Maximum likelihood phylogenetic analyses of the conserved Pro-Pol amino acid sequences revealed the clustering of the new virus within the genus Nepovirus of the family Secoviridae. Considering its biological and molecular characteristics, and based on current taxonomic criteria, we propose that the novel virus, named grapevine nepovirus A, be assigned to the genus Nepovirus.

Molecular and Biological Characterization of Tomato mottle mosaic virus and Development of RT-PCR Detection.

Tomato mottle mosaic virus (ToMMV) was first identified in 2013 as a novel tobamovirus infecting tomatoes in Mexico. In just a few years, ToMMV has been identified in several countries around the world, including the United States. In the present study, we characterized the molecular, serological, and biological properties of ToMMV and developed a species-specific RT-PCR to detect three tomato-infecting tobamoviruses: Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), and ToMMV. Previously, ToMMV has been reported in Florida and New York. In this study, we made two new reports on the occurrences of ToMMV on tomatoes in California and South Carolina. Their complete genome sequences were obtained and their genetic relationships to other tobamoviruses evaluated. In host range studies, some differential responses in host plants were also identified between ToMMV and ToMV. To alleviate cross-serological reactivity among the tomato-infecting tobamoviruses, a new multiplex RT-PCR was developed to allow for species-specific detection and identification of TMV, ToMV, and ToMMV. In addition, we observed resistance breaking by ToMMV on selected tomato cultivars that were resistant to ToMV. This has caused serious concerns to tomato growers worldwide. In conclusion, the characterization in molecular and biological properties of ToMMV would provide us with fundamental knowledge to manage this emerging virus on tomato and other solanaceous crops in the U.S. and around the world.

Distribution, Cultivar Susceptibility, and Epidemiology of Apium virus Y on Celery in Coastal California.

Apium virus Y (ApVY) is a potyvirus that was recently found to cause crop loss to celery (Apium graveolens) in California. Symptoms on leaves exhibit varying forms of chlorosis and necrosis. Depending on the cultivar, celery petioles could also exhibit extensive necrotic, sunken, elongated lesions. Severely affected plants were unmarketable. Disease incidence surveys found that a susceptible celery (cv. 414) showed 55% (2007) and 71% (2008) disease. Because it was noted that the Apiaceae weed poison hemlock (Conium maculatum) was present in almost all areas where ApVY affected celery, a 4-year survey collected overwintered hemlock from six coastal county regions and tested composite samples for ApVY using reverse transcription-polymerase chain reaction (RT-PCR) and ApVY-specific primers. These plants were consistently positive for ApVY. Seeds collected from these plants were also positive when tested with the same RTPCR method. However, when ApVY-positive hemlock seeds were germinated and the resulting seedlings tested, all results were negative. The failure of ApVY to be transmitted from hemlock seeds to seedlings was further documented by collecting newly germinated hemlock seedlings from the field and testing them with RT-PCR. All such seedlings were negative for ApVY even though large, adjacent, overwintered hemlock plants tested positive. Two crops of celery seed were produced from ApVY-positive mother plants; celery seed from these infected plants likewise tested positive for ApVY, but seedlings grown from the seed lots were negative for ApVY. Twenty-one celery and celeriac cultivars were inoculated with ApVY using viruliferous aphids, planted in a replicated field trial, and then grown to maturity. Seven cultivars remained symptomless, tested negative for ApVY, and showed signs of possible resistance. The epidemiology of disease caused by ApVY in California evidently involves poison hemlock as a common overwintering host with subsequent vectoring of the virus from hemlock to celery via aphids. ApVY was not seedborne in this weed host or in celery in our experiments. Our data suggest that growers can manage this disease by controlling poison hemlock weed populations and by planting celery cultivars that are not susceptible to ApVY.

The complete nucleotide sequence and genome organization of a novel carmovirus--honeysuckle ringspot virus isolated from honeysuckle.

A virus associated with yellow-to-purple ringspot on honeysuckle plants has been detected and tentatively named honeysuckle ringspot virus (HnRSV). The complete nucleotide sequence of HnRSV from infected honeysuckle has been determined. The genomic RNA of HnRSV is 3,956 nucleotides in length and is predicted to contain five open reading frames (ORFs). Comparisons of the amino acid sequences of the ORFs of HnRSV with those of members of the family Tombusviridae show that HnRSV is closely related to members of the genus Carmovirus. Phylogenetic analysis based on the amino acid sequences of RdRp and coat protein and nucleotide sequences of the whole genome revealed that HnRSV forms a subgroup with the carmoviruses. Together, our results support the classification of HnRSV as a member of a new species in the genus Carmovirus, family Tombusviridae.

A mutation in the Lettuce infectious yellows virus minor coat protein disrupts whitefly transmission but not in planta systemic movement.

The Lettuce infectious yellows virus (LIYV) RNA 2 mutant p1-5b was previously isolated from Bemisia tabaci-transmitted virus maintained in Chenopodium murale plants. p1-5b RNA 2 contains a single-nucleotide deletion in the minor coat protein (CPm) open reading frame (ORF) that is predicted to result in a frameshift and premature termination of the protein. Using the recently developed agroinoculation system for LIYV, we tested RNA 2 containing the p1-5b CPm mutant genotype (agro-pR6-5b) in Nicotiana benthamiana plants. We showed that plant infection triggered by agro-pR6-5b spread systemically and resulted in the formation of virions similar to those produced in p1-5b-inoculated protoplasts. However, virions derived from these mutant CPm genotypes were not transmitted by whiteflies, even though virion concentrations were above the typical transmission thresholds. In contrast, and as demonstrated for the first time, an engineered restoration mutant (agro-pR6-5bM1) was capable of both systemic movement in plants and whitefly transmission. These results provide strong molecular evidence that the full-length LIYV-encoded CPm is dispensable for systemic plant movement but is required for whitefly transmission.

Quantitative parameters determining whitefly (Bemisia tabaci) transmission of Lettuce infectious yellows virus and an engineered defective RNA.

In this study, quantitative parameters affecting in vitro acquisition and whitefly (Bemisia tabaci) transmission of Lettuce infectious yellows virus (LIYV) were examined and B. tabaci transmission of an engineered defective RNA (D-RNA) was demonstrated. Virions purified from virus- and virion RNA-inoculated Chenopodium murale plants and protoplasts of Nicotiana tabacum, respectively, were consistently transmitted to plants by B. tabaci when virion concentrations were 0.1 ng microl(-1) or greater. Transmission efficiency increased with increasing virion concentration and number of whiteflies used for inoculation. When in vitro-derived transcripts of the M5gfp D-RNA (engineered to express the green fluorescent protein, GFP) were co-inoculated to protoplasts with wild-type LIYV virion RNAs, the resulting virions were transmissible to plants. LIYV and the M5gfp D-RNA systemically invaded inoculated plants; however, GFP expression was not detected in these plants. Unlike LIYV, the M5gfp D-RNA was not subsequently transmitted by B. tabaci from the initially infected plants, but, when high concentrations of virions from plants infected by LIYV and the M5gfp D-RNA were used for in vitro acquisition by whiteflies, both were transmitted to plants. Quantitative and qualitative analyses showed that, although the M5gfp D-RNA replicated within and systemically invaded plants along with LIYV, compared with LIYV RNA 2 it was not as abundant in plants or in the resulting virions, and concentration of encapsidated RNAs is an important factor affecting transmission efficiency.

De novo generation of Lettuce infectious yellows virus defective RNAs in protoplasts.

Summary Lettuce infectious yellows virus (LIYV)-infected plants contain a heterogeneous population of defective RNAs (D RNAs) derived from LIYV genomic RNA 2. To partly address how LIYV D RNAs are generated, in vitro synthesized transcripts corresponding to the LIYV genomic RNAs 1 and 2 were inoculated to protoplasts, and these were analysed for genomic and D RNAs. De novo generated D RNAs were readily detected by 48 h post-inoculation. Furthermore, when separate aliquots from the same protoplast preparation were separately inoculated with aliquots of the same LIYV RNA 1 and RNA 2 transcript preparations, different D RNA populations were detected in each. Thus, different D RNAs arose de novo within separate protoplast samples. Nucleotide sequence analysis of some de novo LIYV D RNAs revealed that they have a similar structure to the LIYV D RNAs described previously from whole plants, and to those of other plant viruses, consisting of one large internal deletion of the LIYV genomic RNA 2, but retaining 5' and 3' terminal sequences. However, one of the LIYV D RNAs had two non-contiguous internal deletions.