The Virome of Piper nigrum: Identification, Genomic Characterization, Prevalence, and Transmission of Three New Viruses of Black Pepper in China.

Viral diseases are one of the main categories of diseases that cause substantial yield losses in black pepper. Disease symptoms in black pepper are generally complex and are often caused by both known and undescribed viruses. To identify and clarify the etiology of viral diseases in black pepper in Hainan, China, we conducted high-throughput sequencing (HTS) by targeting purified double-stranded RNA (dsRNA) and ribosomal RNA depleted total RNA (rRNA-depleted totRNA). Analysis of the data revealed the presence of one known virus, piper yellow mottle virus (PYMoV), and three newly identified viruses: black pepper virus F (BPVF) in the genus Fabavirus, black pepper virus E (BPVE) in the genus Enamovirus, and black pepper virus B (BPVB) in the genus Badnavirus. The dominant viruses in P. nigrum sampled in Hainan are PYMoV, with an incidence of 100%, followed by BPVF (84%, 133 of 158) and BPVB (66%, 105 of 158). Mechanical inoculation of sap extracts from source plants containing PYMoV, BPVF, and BPVB gave negative results on both herbaceous and woody host plants 60 days postinoculation (dpi). BPVF and PYMoV were successfully transmitted to virus-free seedlings of black pepper through bark grafting, while BPVB was experimentally undetectable up to 150 dpi. Seed transmission experiments showed that no target viruses were present in all 59 germinated seedlings. This study provides information on diagnosis, prevalence, and transmission of black-pepper-associated viruses.

Identification of a New Badnavirus in the Chinaberry (Melia azedarach) Tree and Establishment of a LAMP-LFD Assay for Its Rapid and Visual Detection.

The Chinaberry tree, a member of the Meliaceae family, is cultivated in China for use in traditional medicines. In 2020, Chinaberry trees with leaf deformation symptoms were found in Hangzhou, Zhejiang province, China. In order to identify possible pathogenic viruses, a symptomatic sample was subjected to deep sequencing of small interfering RNAs. Assembly of the resulting sequences led to the identification of a novel badnavirus, provisionally designated Chinaberry tree badnavirus 1 (ChTBV1). With the recent development of China's seedling industry and increasing online shopping platforms, the risk of tree virus transmission has increased substantially. Therefore, it is important to detect the occurrence of ChTBV1 to ensure the safety of the Chinaberry tree seedling industry. Here, we describe the development and validation of a sensitive and robust method relying on a loop-mediated isothermal amplification (LAMP) assay, targeting a 197 nt region, to detect ChTBV1 from Chinaberry tree leaves. The LAMP assay was also adapted for rapid visualization of results by a lateral flow dipstick chromatographic detection method.

Identification of Cacao Mild Mosaic Virus (CaMMV) and Cacao Yellow Vein-Banding Virus (CYVBV) in Cocoa (Theobroma cacao) Germplasm.

Cocoa, Theobroma cacao, is an important tropical perennial crop grown widely in the humid tropics. The exchange of cocoa germplasm between germplasm collections and breeding centres is vital for varietal development. Intermediate quarantine facilities, such as the International Cocoa Quarantine Centre, Reading UK (ICQC-R) play a vital role in ensuring the transfer of germplasm whilst minimising the risk of spreading pests and diseases. Current screening procedures combine visual inspection and molecular techniques, which are effective in detecting Cocoa swollen shoot virus (CSSV), a badnavirus, which causes severe losses but are restricted to West Africa. However, the detection of latent or mild virus infections that produce no visual symptoms has been a challenge. Recently two badnavirus species of cocoa producing mild symptoms, cacao mild mosaic virus (CaMMV) and cacao yellow vein-banding virus (CYVBV), have been sequenced. Here, we report new assays for the detection of these two species, for the first time in non-symptomatic accessions. Evolutionary and bioinformatic analyses of the viruses suggest their most recent source was from Trinidad, though there is historic evidence that these viruses may have their origin in South America and then become widespread globally over the last century. We also report a novel colorimetric Loop-mediated isothermal amplification (LAMP) assay for the detection of CYVBV. This simple and accurate method could be employed in field virus testing.

Extrachromosomal viral DNA produced by transcriptionally active endogenous viral elements in non-infected banana hybrids impedes quantitative PCR diagnostics of banana streak virus infections in banana hybrids.

The main edible and cultivated banana varieties are intra- and interspecific hybrids of the two main Musa species, Musa acuminata and Musa balbisiana, having diploid genomes denoted A and B, respectively. The B genome naturally hosts sequences of banana streak virus (BSV) named endogenous BSV (eBSV). Upon stress, eBSVs are identified as the origin of BSV infection for at least three BSV species, causing banana streak disease. For each of the three species, BSV and eBSV share >99.9 % sequence identity, complicating PCR-based diagnosis of viral infection in the B genome-containing bananas. Here, we designed a quantitative PCR-based method to only quantify episomal BSV particles produced, overcoming the limitation of eBSV also being detected by qPCR by using it as a 'calibrator'. However, our results revealed unexpected variation of eBSV amplification in calibrator plants composed of a clonal population of 53 replicating virus-free banana hybrids with the same AAB genotype. Our in-depth molecular analyses suggest that this calibrator variation is due to the variable abundance of non-encapsidated extrachromosomal viral DNA, likely produced via the transcription of eBSVs, followed by occasional reverse transcription. We also present evidence that accumulation of viral transcripts in AAB plants is downregulated both at post-transcriptional and transcriptional levels by an RNA interference mechanism that keeps the plants free of virus infection. Finally, we recommend that such eBSV amplification variation be taken into account to establish a quantitative viral diagnostic for banana plants with the B genome.

Complete genome sequence of aucuba ringspot virus.

A new badnavirus, aucuba ringspot virus (AuRV), was identified in plants of Aucuba japonica showing mild mosaic, vein banding, and yellow ringspot symptoms on the leaves. The complete nucleotide sequence of the AuRV genome was determined and found to be 9,092 nt in length, and the virus was found to have a genome organization typical of members of the genus Badnavirus. ORF3 was predicted to encode a polyprotein containing conserved movement protein, coat protein, aspartic protease, reverse transcriptase (RT), and RNase H domains. Phylogenetic analysis suggested that this virus is most closely related to codonopsis vein clearing virus but belongs to a distinct species, based on only 69.6% nucleotide sequence identity within the part of ORF 3 encoding the RT and RNase H domains. The vector of AuRV is unknown, but based on phylogenetic relationships, it is predicted to be a type of aphid.

Badnaviruses and banana genomes: a long association sheds light on Musa phylogeny and origin.

Badnaviruses are double-stranded DNA pararetroviruses of the family Caulimoviridae. Badnaviral sequences found in banana are distributed over three main clades of the genus Badnavirus and exhibit wide genetic diversity. Interestingly, the nuclear genome of many plants, including banana, is invaded by numerous badnaviral sequences although badnaviruses do not require an integration step to replicate, unlike animal retroviruses. Here, we confirm that banana streak viruses (BSVs) are restricted to clades 1 and 3. We also show that only BSVs from clade 3 encompassing East African viral species are not integrated into Musa genomes, unlike BSVs from clade 1. Finally, we demonstrate that sequences from clade 2 are definitively integrated into Musa genomes with no evidence of episomal counterparts; all are phylogenetically distant from BSVs known to date. Using different molecular approaches, we dissected the coevolution between badnaviral sequences of clade 2 and banana by comparing badnavirus integration patterns across a banana sampling representing major Musa speciation events. Our data suggest that primary viral integrations occurred millions of years ago in banana genomes under different possible scenarios. Endogenous badnaviral sequences can be used as powerful markers to better characterize the Musa phylogeny, narrowing down the likely geographical origin of the Musa ancestor.

First identification and molecular characterization of a new badnavirus infecting camellia.

A new badnavirus was identified in an ornamental camellia tree with yellow mottle symptom. The complete circular double-stranded DNA genome of this virus was found to consist of 8,203 bp. Its genome organization is typical of badnaviruses, containing three open reading frames (ORFs). ORFs 1 and 2 encode putative proteins with unknown functions. ORF3 encodes a large polyprotein that contains almost all of the conserved domains of badnaviruses. The virus shares 55-62% nucleotide sequence identities with other badnaviruses in the RT+RNase H region. Phylogenetic analyses placed it in group I of the genus Badnavirus. Therefore, this virus, which is tentatively named "camellia Lemon Glow virus", should represent a new species of the genus Badnavirus. This virus was found to be present in approximately a quarter of camellia trees tested.

Genomic characterisation of a newly identified badnavirus infecting ivy (Hedera helix).

High-throughput sequencing (HTS) was used to investigate ringspots on ivy (Hedera helix) leaves. De novo assembly of HTS data generated from a total RNA extract from these leaves yielded a contig with sequence similarity to viruses of the genus Badnavirus, family Caulimoviridae. The complete genome sequence of this virus consists of 8,885 nucleotides and has three open reading frames (ORFs). Genome organisation and phylogenetic analysis identifies this newly identified virus as a new member of the genus Badnavirus for which we propose the name "ivy ringspot-associated virus" (IRSaV).

Genomic characterization of cycad leaf necrosis virus, the first badnavirus identified in a gymnosperm.

A previously undescribed badnavirus was isolated from Zamia fischeri showing symptoms of chlorosis, necrosis, and ringspot. The virus has bacilliform virions 30 nm in diameter and averaging 120 nm in length. The viral genome is 9227 bp in length and contains three open reading frames characteristic of members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H. The virus is tentatively named "cycad leaf necrosis virus" (CLNV). Within the genus Badnavirus, CLNV was most closely related to sugarcane bacilliform Guadeloupe D virus (FJ439817), sharing 69% identity at the nucleotide level in the RT + RNase H region. This virus is the first badnavirus reported to infect cycads, and it has the largest genome among the currently characterized badnaviruses.

Complete genome sequence of aglaonema bacilliform virus (ABV).

Aglaonema bacilliform virus (ABV), a member of the genus Badnavirus in the family Caulimoviridae, is associated with leaf deformation and chlorosis in Aglaonema modestum. The complete genome sequence of a Minnesota isolate of ABV was determined. The ABV genome is 7,178 bp in length and similar in size and organization to those of the members of the genus Badnavirus, containing three open reading frames (ORFs) with the potential to encode three proteins of 14.92, 13.33 and 207.95 kDa, respectively. The last ORF (ORF3) encodes a putative polyprotein with conserved domains, including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H domains, in that order. Phylogenetic analysis using the amino acid sequence of the ORF3 polyprotein showed that ABV clusters with several isolates of taro bacilliform CH virus (TaBCHV). Pairwise alignment using the highly conserved RT/RNase H region reveals that ABV has the highest level of identity (71%) at the nucleotide level to a Hawaiian isolate of TaBCHV.

Spatiotemporal Spread of Cacao Swollen Shoot Virus Severe Strain 1A in Mixed Hybrid Cacao Pre-inoculated With Mild Strain N1.

The spatiotemporal spread of cocoa swollen shoot virus disease (CSSVD), which is caused by cacao swollen shoot virus (CSSV) severe strain 1A in mixed hybrid cacao pre-inoculated with CSSV mild strain N1 (CSSV-N1), was investigated during a field experiment from 2006 to 2017, at the Cocoa Research Institute of Ghana. The development of disease epidemics has been described by the use of statistical modeling. Protecting all cacao plants with CSSV-N1 reduced the rate of CSSV-1A symptom appearance by 43% (P = 0.05) compared with the nonprotected control and by 33% compared with plots where cacao plants in the outer three or five rows were protected with CSSV-N1. Similarly, creating the protective outer rings three or five rows deep reduced the rate of CSSV-1A symptoms by 14% (P = 0.05) compared with the nonprotected control. CSSV-1A epidemics increased approximately 18% faster (P = 0.05) in transects oriented from the north and east compared with those oriented from the south and west. During the last 2 years of the study, CSSVD spread decreased significantly (P = 0.05) faster in plots where all test cacao plants were inoculated with CSSV-N1 compared with other treatments. The growth of cacao did not differ significantly among the treatments over the 9-year assessment period. Similarly, differences in the cumulative yield among the treatments over the 8-year assessment period were not significant.

Molecular characterization of a new badnavirus infecting green Sichuan pepper (Zanthoxylum schinifolium).

A new virus with a circular double-stranded DNA genome was discovered in green Sichuan pepper with vein clearing symptoms. Its complete genome of 8,014 bp contains three open reading frames (ORF) on the plus strand, which is typical of members of the genus Badnavirus in the family Caulimoviridae. Sequence comparisons revealed that the new virus has the highest nucleotide sequence identity with grapevine vein-clearing virus (GVCV). In particular, the identity of the two viruses in the ORF3 RT-RNase H region is 71.9%, which is below the species demarcation cutoff of 80% for badnaviruses. Phylogenetic analysis also placed the new virus with GVCV in a cluster. The virus was tentatively named "green Sichuan pepper vein clearing-associated virus" (GSPVCaV). The geographical distribution and genetic diversity of GSPVCaV were studied. Another isolate was found to be highly divergent.

Complete genome sequence of a previously undescribed badnavirus occurring in Polyscias fruticosa L. (Ming aralia).

A previously undescribed badnavirus was identified in plants of Polyscias fruticosa (Ming aralia) showing symptoms of mild mosaic and leaf senescence. Characteristic bacilliform virions of the Polyscias badnavirus averaging 30 × 120 nm in size were observed by transmission electron microscopy in partially purified leaf tissue extracts from symptomatic but not asymptomatic plants collected in the USA and Nigeria. The isolate from the USA was complete sequenced. The genome is 7592 bp in length and contains three open reading frames with an arrangement similar to that of other members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H, in that order. The USA and Nigeria isolates of the virus had a high level (98%) of nucleotide sequence identity in the RT+RNase H region. Within the genus Badnavirus, these viruses were most closely related to schefflera ringspot virus (SRV), sharing 63% identity at the nucleotide level. Based on the ICTV species demarcation criteria for the genus Badnavirus (more than 20% nucleotide sequence divergence in the RT+RNase H region), the Polyscias virus is proposed to be a new member of the genus, and the name polyscias mosaic virus (PoMV) is proposed. The complete genome sequence was deposited in the NCBI GenBank database under accession no. MH475918.

The Previously Unidentified, Divergent Badnavirus Species Cacao red vein-banding virus is Associated with Cacao Swollen Shoot Disease in Nigeria.

Cacao swollen shoot disease (CSSD) of Theobroma cacao was reported in Nigeria in 1944; however, no badnaviral genome sequences have been found associated with the symptomatic trees. In 2017, leaf samples (n = 18) were collected from cacao trees from Osun and Oyo, Nigeria showing foliar symptoms that included red vein-banding and shoot swelling, and variable secondary mosaic, mottling, and fern-like pattern symptoms. Abutting primers designed around previously determined 500-bp intergenic region sequences were used for polymerase chain reaction (PCR) amplification. Of the 18 samples, 9 yielded an approximately 7,000-bp, apparently genome-size product. The nine genomes were sequenced and found to encode four open reading frames, and to share 86 to 99% nucleotide identity. Pairwise analysis of the Nigerian genomes with 21 previously reported CSSD badnaviruses, at the complete genome and reverse-transcription ribonuclease H (1,230 bp) sequence levels, indicated 71 to 75 and 72 to 76% nucleotide identity, respectively. Phylogenetic analysis of the nine complete genomes indicated that the closest relatives of the divergent Nigerian isolates were previously described West African CSSD badnaviruses. Based on pairwise comparisons and phylogenetic analyses, the Nigerian CSSD isolates constitute a previously unrecognized Badnavirus sp., herein named Cacao red vein-banding virus (CRVBV). Primers designed based on the CRVBV genome sequences amplified a 1,068-bp fragment from 16 of 18 field samples tested by PCR, suggesting the possible existence of additional CRVBV variants.

Complete genome sequence of a tentative new member of the genus Badnavirus identified in Codonopsis lanceolata.

High-throughput sequencing revealed a tentative new badnavirus infecting Codonopsis lanceolata, provisionally named Codonopsis vein clearing virus (CoVCV). The complete 8,112-nt CoVCV genomic DNA sequence (GenBank accession: MK044821) comprises three open reading frames (ORFs) encoding conserved domains, with typical features of badnaviruses. Additionally, BLASTn searches indicated the CoVCV genome sequence is most similar to the grapevine vein clearing virus (GVCV) genome (72% identity and 46% query coverage). Moreover, the polyprotein encoded in CoVCV ORF3 is most similar to the corresponding protein of GVCV, with 60% amino acid sequence identity (89% query coverage). These results suggest that CoVCV is a new member of the genus Badnavirus in the family Caulimoviridae.

Characterization of an Australian isolate of taro bacilliform virus and development of an infectious clone.

The badnavirus taro bacilliform virus (TaBV) has been reported to infect taro (Colocasia esculenta L.) and other edible aroids in several South Pacific island countries, but there are no published reports from Australia. Using PCR and RCA, we identified and characterized an Australian TaBV isolate. A terminally redundant cloned copy of the TaBV genome was generated and shown to be infectious in taro following agro-inoculation. This is the first report of TaBV from Australia and also the first report of an infectious clone for this virus.

Next generation sequencing elucidates cacao badnavirus diversity and reveals the existence of more than ten viral species.

Cacao swollen shoot virus is a member of the family Caulimoviridae, genus Badnavirus and is naturally transmitted to Theobroma cacao (L.) by several mealybug species. CSSV populations in West African countries are highly variable and genetically structured into several different groups based on the diversity in the first part of ORF3 which encodes the movement protein. To unravel the extent of isolate diversity and address the problems of low titer and mixed viral sequences in samples, we used Illumina MiSeq and HiSeq technology. We were able to reconstruct de novo 20 new complete genomes from cacao samples collected in the Cocoa Research Institute of Ghana (CRIG) Museum and from the field samples collected in Côte d'Ivoire or Ghana. Based on the 20% threshold of nucleotide divergence in the reverse transcriptase/ribonuclease H (RT/RNase H) region which denotes species demarcation, we conclude there exist seven new species associated with the cacao swollen shoot disease. These new species along with the three already described leads to ten, the total number of the complex of viral species associated with the disease. A sample from Sri Lanka exhibiting similar leaf symptomology to West African CSSD-affected plants was also included in the study and the corresponding sequence represents the genome of a new virus named cacao bacilliform SriLanka virus (CBSLV).

Complete genome sequence of a new member of the genus Badnavirus, Dioscorea bacilliform RT virus 3, reveals the first evidence of recombination in yam badnaviruses.

Yams (Dioscorea spp.) host a diverse range of badnaviruses (genus Badnavirus, family Caulimoviridae). The first complete genome sequence of Dioscorea bacilliform RT virus 3 (DBRTV3), which belongs to the monophyletic species group K5, is described. This virus is most closely related to Dioscorea bacilliform SN virus (DBSNV, group K4) based on a comparison of genome sequences. Recombination analysis identified a unique recombination event in DBRTV3, with DBSNV likely to be the major parent and Dioscorea bacilliform AL virus (DBALV) the minor parent, providing the first evidence for recombination in yam badnaviruses. This has important implications for yam breeding programmes globally.