Rodrigo A. Valverde

The remarkable evolutionary history of endornaviruses.

The family Endornaviridae contains several members from diverse hosts, including plants, fungi and oomycetes. They are found as large dsRNA elements with a nick in the coding strand. All members encode a conserved RNA-dependent RNA polymerase, but no other domain that is conserved among all members. Based on the conserved domain database comparison the various domains have different origins, indicating a highly modular evolutionary history. In some cases, domains with similar putative functions are found that are derived from different protein families, indicating convergent evolution for a required function.

Properties of Strains of Sweet potato feathery mottle virus and Two Newly Recognized Potyviruses Infecting Sweet Potato in the United States

Some biological and molecular properties of six potyvirus isolates (LSU-1, -2, -3, and -5; 95-2; and 95-6) from sweet potato (Ipomoea batatas) were evaluated. Isolates LSU-1 and -3 and 95-2 were transmitted by Aphis gossypii and Myzus persicae while LSU-2 and -5 were not transmitted by either aphid. The partial nucleotide sequence of the nuclear inclusion b (NIb) and the coat protein (CP) genes of these six isolates were compared with the corresponding sequences of 17 Sweet potato feathery mottle virus (SPFMV) strains and 18 other potyviruses. LSU-1 and -3 had high sequence similarity to the published sequences for Sweet potato virus G (SPVG), did not react with antisera to other known sweet potato viruses, and caused distinct symptoms. We propose to designate these two isolates as SPVG. This report documents the occurrence of this virus in the United States and provides the first characterization of its biological properties. LSU-2 and -5 were distinct in symptomatology; partial Nib, CP nucleotide, and derived amino acid sequence; and serology to other viruses. We propose to call this virus (LSU isolates 2 and 5) Ipomoea vein mosaic virus. The present study revealed a high degree of sequence similarity between 95-6 and the common strain of SPFMV, and between 95-2 and the russet crack strain of SPFMV. Results from this study suggest not only that at least two strains of SPFMV occur in the United States, but that two other potyviruses also are present.

Novel begomovirus species of recombinant nature in sweet potato (Ipomoea batatas) and Ipomoea indica: taxonomic and phylogenetic implications.

Viral diseases occur wherever sweet potato (Ipomoea batatas) is cultivated and because this crop is vegetatively propagated, accumulation and perpetuation of viruses can become a major constraint for production. Up to 90% reductions in yield have been reported in association with viral infections. About 20 officially accepted or tentative virus species have been found in sweet potato and other Ipomoea species. They include three species of begomoviruses (genus Begomovirus, family Geminiviridae) whose genomes have been fully sequenced. In this investigation, we conducted a search for begomoviruses infecting sweet potato and Ipomoea indica in Spain and characterized the complete genome of 15 isolates. In addition to sweet potato leaf curl virus (SPLCV) and Ipomoea yellowing vein virus, we identified three new begomovirus species and a novel strain of SPLCV. Our analysis also demonstrated that extensive recombination events have shaped the populations of Ipomoea-infecting begomoviruses in Spain. The increased complexity of the unique Ipomoea-infecting begomovirus group, highlighted by our results, open new horizons to understand the phylogeny and evolution of the family Geminiviridae.

Indigenous double-stranded RNA from pepper (Capsicum annuum)

Abstract A unique 12 kb linear double-stranded RNA (dsRNA) was detected in the chloroplast fraction of tissue extracts from pepper ( Capsicum annuum ). This dsRNA was detected in 10 out of 14 pepper cultivars tested. Molecular hybridization using cDNA to this dsRNA indicated that the 12 kb dsRNAs found in these pepper cultivars had nucleotide sequence similarity. This dsRNA did not hybridize to dsRNAs of similar size detected in bean and melon, nor to other dsRNAs found in non-virus inoculated plants. No evidence was obtained for a virus-like particle associated with this dsRNA.

Bell pepper endornavirus: molecular and biological properties, and occurrence in the genus Capsicum

Bell peppers (Capsicum annuum) harbour a large dsRNA virus. The linear genome (14.7 kbp) of two isolates from Japanese and USA bell pepper cultivars were completely sequenced and compared. They shared extensive sequence identity and contained a single, long ORF encoding a 4815 aa protein. This polyprotein contained conserved motifs of putative viral methyltransferase (MTR), helicase 1 (Hel-1), UDP-glycosyltransferase and RNA-dependent RNA polymerase. This unique arrangement of conserved domains has not been reported in any of the known endornaviruses. Hence this virus, for which the name Bell pepper endornavirus (BPEV) is proposed, is a distinct species in the genus Endornavirus (family Endornaviridae). The BPEV-encoded polyprotein contains a cysteine-rich region between the MTR and Hel-1 domains, with conserved CXCC motifs shared among several endornaviruses, suggesting an additional functional domain. In agreement with general endornavirus features, BPEV contains a nick in the positive-strand RNA molecule. The virus was detected in all bell pepper cultivars tested and transmitted through seed but not by graft inoculations. Analysis of dsRNA patterns and RT-PCR using degenerate primers revealed putative variants of BPEV, or closely related species, infecting other C. annuum genotypes and three other Capsicum species (C. baccatum, C. chinense and C. frutescens).

Evidence for a satellite RNA associated naturally with the U5 strain and experimentally with the U1 strain of tobacco mosaic virus

Summary Isolates of tobacco mosaic virus strain U5 (TMV-U5) from native Nicotiana glauca plants induced the accumulation of a dsRNA (mol. wt. 0.6 × 106) in infected plants that was more abundant than the replicative form (RF) dsRNA of TMV (mol. wt. 4.3 × 106). Some but not all subcultures of such a field isolate obtained from single local lesions on N. tabacum cv. Xanthi-nc, had lost the ability to induce the 0.6 × 106 mol. wt. dsRNA. Co-inoculation experiments in N. silvestris established that the dsRNA could accumulate in plants infected with TMV-U1, but to a lesser extent than when associated with TMV-U5. A ssRNA (mol. wt. 0.3 × 106) was isolated from plants containing the dsRNA. This was not infectious by itself but became so when associated with TMV-U5 or TMV-U1, and then induced the accumulation of the 0.6 × 106 dsRNA. Plants infected with TMV-U5 isolates which did or did not induce the 0.6 × 106 dsRNA had identical symptoms. The host range of the 0.3 × 106 ssRNA was the same as that of the TMV strain with which it was associated in each of the 20 experimental hosts tested. Complementary DNA transcribed from purified 0.6 × 106 dsRNA did not hybridize with RF and other dsRNAs of TMV-U5, TMV-U1, tobacco necrosis virus, potato virus X, citrus tristeza virus, and cucumber mosaic virus + CARNA 5, but the cDNA did hybridize with the 0.6 × 106 mol. wt. dsRNA and the 0.3 × 106 mol. wt. ssRNA found only in plants containing this dsRNA. The results indicate that the 0.6 × 106 dsRNA is the RF of a satellite RNA of TMV. Purified nucleoprotein from plants infected with TMV-U5 and the satellite RNA were infectious for the satellite RNA, but the nature of encapsidation of the satellite RNA has yet to be determined.

First Report of a Begomovirus Infecting Sweetpotato in Kenya

Previous surveys for viruses in sweetpotatoes (Ipomoea batatas) in Africa did not assay for the presence of begomoviruses such as Sweet potato leaf curl virus (SPLCV), which have been found recently in the Americas and Asia. Symptomatic sweetpotato plants, including some with leaf curling symptoms similar to those observed in SPLCV-infected sweet-potato plants (2), were collected from a germplasm collection plot at Kakamega Research Station in Western Kenya during February 2005. Whiteflies, the vectors for begomoviruses, were observed in the same plots. Ipomoea setosa plants graft-inoculated with scions from the symptomatic sweetpotato developed leaf curl, leaf roll, interveinal chlorosis, and stunting symptoms similar to those caused by infection with SPLCV alone or in combination with Sweet potato feathery mottle virus. Total DNA was isolated from 10 I. setosa plants using the GenElute Plant Genomic DNA Kit (Sigma-Aldrich Inc., St. Louis, MO). Sweetpotato cuttings from 39 clones, selected from the Kenyan germplasm collection for their resistance or susceptibility to sweetpotato virus disease (SPVD), were sent to the Plant Germplasm Quarantine Office of USDA-ARS. The cuttings were planted in a greenhouse. Total DNA was extracted from sweetpotato leaves 1 month later using a cetyltrimethylammoniumbromide (CTAB) extraction method (1). Degenerate primers SPG1/SPG2, developed for PCR detection of begomoviruses (1), amplified a 912-bp DNA fragment from 3 of 10 DNA extracts from I. setosa and 5 of 39 sweetpotato plants held in quarantine. The primers anneal to regions of open reading frame (ORF) AC2 and ORF AC1 that are highly conserved in begomoviruses infecting sweetpotato. SPLCV-specific primers PW285-1/PW285-2 (2) amplified a 512-bp DNA fragment of ORF AC1 from seven samples (two from I. setosa and five from I. batatas). Amplicons from three independent PCR assays of two samples and single PCR assays of four additional samples were cloned into the pGEM-T Easy vector. Clone inserts were sequenced, and compared with sequences deposited in GenBank using the basic local alignment search tool (BLAST). Sequences were closely related to SPLCV (GenBank Accession No. AF104036) with nucleotide sequence identities varying from 93% (GenBank Accession No. DQ361004) to 97% (GenBank Accession No. DQ361005). The presence of the virus poses a challenge to the dissemination of planting materials in the region because begomovirus-infected plants often do not show symptoms. To our knowledge, this is the first report of a begomovirus infecting sweetpotato in Kenya or the East African Region. References: (1) R. Li et al. Plant Dis. 88:1347, 2004. (2) P. Lotrakul et al. Plant Dis. 82:1253, 1998.

Transmission of a dsRNA in bell pepper and evidence that it consists of the genome of an endornavirus

A double-stranded (ds) RNA from bell pepper (BP-dsRNA) cv Yolo Wonder was inherited maternally and paternally after crossing Yolo Wonder with Jalapeño M or Hungarian Wax pepper. Partial sequence information was obtained from two cDNA clones derived from the BP-dsRNA and based on sequence similarity was related to members of the genus Endornavirus. Clones of the BP-dsRNA hybridized with similar dsRNAs from four other pepper cultivars, suggesting that all five dsRNAs tested are related. One of the cDNA clones contained a region that had significant similarity with UDP-glucose:glycosyltransferases from fungi, bacteria, plants, and three endornaviruses. Data presented indicate that the BP-dsRNA is the genome of a distinct species of the genus Endornavirus.

Complete Genome Sequence of a Double-Stranded RNA Virus from Avocado

ABSTRACT A number of avocado (Persea americana) cultivars are known to contain high-molecular-weight double-stranded RNA (dsRNA) molecules for which a viral nature has been suggested, although sequence data are not available. Here we report the cloning and complete sequencing of a 13.5-kbp dsRNA virus isolated from avocado and show that it corresponds to the genome of a new species of the genus Endornavirus (family Endornaviridae), tentatively named Persea americana endornavirus (PaEV).

Viral Double-Strand RNA-Binding Proteins Can Enhance Innate Immune Signaling by Toll-Like Receptor 3

Toll-like Receptor 3 (TLR3) detects double-stranded (ds) RNAs to activate innate immune responses. While poly(I:C) is an excellent agonist for TLR3 in several cell lines and in human peripheral blood mononuclear cells, viral dsRNAs tend to be poor agonists, leading to the hypothesis that additional factor(s) are likely required to allow TLR3 to respond to viral dsRNAs. TLR3 signaling was examined in a lung epithelial cell line by quantifying cytokine production and in human embryonic kidney cells by quantifying luciferase reporter levels. Recombinant 1b hepatitis C virus polymerase was found to enhance TLR3 signaling in the lung epithelial BEAS-2B cells when added to the media along with either poly(I:C) or viral dsRNAs. The polymerase from the genotype 2a JFH-1 HCV was a poor enhancer of TLR3 signaling until it was mutated to favor a conformation that could bind better to a partially duplexed RNA. The 1b polymerase also co-localizes with TLR3 in endosomes. RNA-binding capsid proteins (CPs) from two positive-strand RNA viruses and the hepadenavirus hepatitis B virus (HBV) were also potent enhancers of TLR3 signaling by poly(I:C) or viral dsRNAs. A truncated version of the HBV CP that lacked an arginine-rich RNA-binding domain was unable to enhance TLR3 signaling. These results demonstrate that several viral RNA-binding proteins can enhance the dsRNA-dependent innate immune response initiated by TLR3.