Roy French

Use of group-specific primers and the polymerase chain reaction for the detection and identification of luteoviruses

A general diagnostic assay for a number of distinct luteoviruses was developed using the polymerase chain reaction (PCR) and restriction enzyme analysis. Two minimally degenerate, group-specific primers were derived from previously published RNA sequences of three luteoviruses. This primer pair generated specific PCR fragments of about 530 bp from extracts of plants infected with potato leafroll virus, beet western yellows virus, or New York barley yellow dwarf virus (BYDV) serotypes MAV, PAV, RMV, RPV and SGV, which span much of the respective viral coat protein gene. Each virus was easily distinguished from the others by restriction enzyme analysis of the amplified DNA products. Samples from BYDV-infected oat and wheat collected in Nebraska were identified as containing PAV-like serotypes; micro-heterogeneity was detected in several samples. This method provides a rapid, sensitive and relatively inexpensive means of luteovirus detection and identification. It is the first test capable of simultaneously detecting all five BYDV serotypes.

Phylogenetic Relationships Within the Family Potyviridae: Wheat Streak Mosaic Virus and Brome Streak Mosaic Virus Are Not Members of the Genus Rymovirus

ABSTRACT The complete nucleotide sequence of wheat streak mosaic virus (WSMV) has been determined based on complementary DNA clones derived from the 9,384-nucleotide (nt) RNA of the virus. The genome of WSMV has a 130-nt 5 leader and 149-nt 3-untranslated region and is polyadenylated at the 3 end. WSMV RNA encodes a single polyprotein of 3,035 amino acid residues and has a deduced genome organization typical for a member of the family Potyviridae (5-P1/HC-Pro/P3/6K1/CI/6K2/VPg-NIa/NIb/CP-3). Because WSMV shares with ryegrass mosaic virus (RGMV) the biological property of transmission by eriophyid mites, WSMV has been assigned to the genus Rymovirus, of which RGMV is the type species. Phylogenetic analyses were conducted with complete polyprotein or NIb protein sequences of 11 members of the family Potyviridae, including viruses of monocots or dicots and viruses transmitted by aphids, whiteflies, and mites. WSMV and the monocot-infecting, mite-transmitted brome streak mosaic virus (BrSMV) are sister taxa and share a most recent common ancestor with the whitefly-transmitted sweet potato mild mottle virus, the type species of the proposed genus Ipomovirus. In contrast, RGMV shares a most recent common ancestor with aphid-transmitted species of the genus Potyvirus. These results indicate that WSMV and BrSMV should be classified within a new genus of the family Potyviridae and should not be considered species of the genus Rymovirus.

Wheat Cultivar-Specific Disease Synergism and Alteration of Virus Accumulation During Co-Infection with Wheat streak mosaic virus and Triticum mosaic virus

Triticum mosaic virus (TriMV), the type member of the newly proposed Poacevirus genus, and Wheat streak mosaic virus (WSMV), the type member of Tritimovirus genus of the family Potyviridae, infect wheat naturally in the Great Plains and are transmitted by wheat curl mites. In this study, we examined the ability of these viruses to infect selected cereal hosts, and found several differential hosts between TriMV and WSMV. Additionally, we examined the interaction between WSMV and TriMV in three wheat cultivars at two temperature regimens (19 and 20 to 26 degrees C), and quantified the virus concentration in single and double infections by real-time reverse-transcription polymerase chain reaction. Double infections in wheat cvs. Arapahoe and Tomahawk at both temperature regimens induced disease synergism with severe leaf deformation, bleaching, and stunting, with a 2.2- to 7.4-fold increase in accumulation of both viruses over single infections at 14 days postinoculation (dpi). However, at 28 dpi, in double infections at 20 to 26 degrees C, TriMV concentration was increased by 1.4- to 1.8-fold in Arapahoe and Tomahawk but WSMV concentration was decreased to 0.5-fold. WSMV or TriMV replicated poorly in Mace at 19 degrees C with no synergistic interaction whereas both viruses accumulated at moderate levels at 20 to 26 degrees C and induced mild to moderate disease synergism in doubly infected Mace compared with Arapahoe and Tomahawk. Co-infections in Mace at 20 to 26 degrees C caused increased TriMV accumulation at 14 and 28 dpi by 2.6- and 1.4-fold and WSMV accumulated at 0.5- and 1.6-fold over single infections, respectively. Our data suggest that WSMV and TriMV induced cultivar-specific disease synergism in Arapahoe, Tomahawk, and Mace, and these findings could have several implications for management of wheat viruses in the Great Plains.

Triticum mosaic virus: A Distinct Member of the Family Potyviridae with an Unusually Long Leader Sequence

The complete genome sequence of Triticum mosaic virus (TriMV), a member in the family Potyviridae, has been determined to be 10,266 nucleotides (nt) excluding the 3 polyadenylated tail. The genome encodes a large polyprotein of 3,112 amino acids with the hall-mark proteins of potyviruses, including a small overlapping gene, PIPO, in the P3 cistron. The genome of TriMV has an unusually long 5 nontranslated region of 739 nt with 12 translation initiation codons and three small open reading frames, which resemble those of the internal ribosome entry site containing 5 leader sequences of the members of Picornaviridae. Pairwise comparison of 10 putative mature proteins of TriMV with those of representative members of genera in the family Potyviridae revealed 33 to 44% amino acid identity within the highly conserved NIb protein sequence and 15 to 29% amino acid identity within the least conserved P1 protein, suggesting that TriMV is a distinct member in the family Potyviridae. In contrast, TriMV displayed 47 to 65% amino acid sequence identity with available sequences of mature proteins of Sugarcane streak mosaic virus (SCSMV), an unassigned member of the Potyviridae. Phylogenetic analyses of the complete polyprotein, NIa-Pro, NIb, and coat protein sequences of representative species of six genera and unassigned members of the family Potyviridae suggested that TriMV and SCSMV are sister taxa and share a most recent common ancestor with tritimoviruses or ipomoviruses. These results suggest that TriMV and SCSMV should be classified in a new genus, and we propose the genus Poacevirus in the family Potyviridae, with TriMV as the type member.

Phylogenetic relationships, strain diversity and biogeography of tritimoviruses

North American and Eurasian isolates of Wheat streak mosaic virus (WSMV; genus Tritimovirus) and Oat necrotic mottle virus (ONMV; genus Rymovirus) were examined. Nine WSMV isolates differentially infected oat, barley, inbred maize line SDp2 and sorghum line KS56. The WSMV isolates clustered into groups based on phylogenetic analyses of the capsid protein (CP) cistron and flanking regions. WSMV isolates from the United States (US) and Turkey were closely related, suggesting recent movement between continents. Although more divergent, WSMV from Iran (WSMV-I) also shared a most recent common ancestor with the US and Turkish isolates. Another group of WSMV isolates from central Europe and Russia may represent a distinct Eurasian population. Complete genome sequences of WSMV from the Czech Republic (WSMV-CZ) and Turkey (WSMV-TK1) were determined and comparisons based on complete sequences yielded relationships similar to those based on partial sequences. ONMV-Pp recovered from blue grass (Poa pratensis L.) in Germany displayed the same narrow host range as ONMV-Type from Canada. Western blots revealed a heterologous relationship among CP of WSMV and ONMV. Phylogenetic analyses of the capsid protein cistron and flanking genomic regions indicated that WSMV and ONMV are related species sharing 74.2-76.2% (nucleotide) and 79.2-81.0% (amino acid) identity. Thus, ONMV should be removed from the genus Rymovirus and designated a definitive member of the genus Tritimovirus. Phylogenetic analyses further suggest that Sugarcane streak mosaic virus is not a tritimovirus, and may represent a new genus within the family Potyviridae.

Plant Virus HC-Pro Is a Determinant of Eriophyid Mite Transmission

ABSTRACT The eriophyid mite transmitted Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) shares a common genome organization with aphid transmitted species of the genus Potyvirus. Although both tritimoviruses and potyviruses encode helper component-proteinase (HC-Pro) homologues (required for nonpersistent aphid transmission of potyviruses), sequence conservation is low (amino acid identity, ∼16%), and a role for HC-Pro in semipersistent transmission of WSMV by the wheat curl mite (Aceria tosichella [Keifer]) has not been investigated. Wheat curl mite transmissibility was abolished by replacement of WSMV HC-Pro with homologues of an aphid transmitted potyvirus (Turnip mosaic virus), a rymovirus (Agropyron mosaic virus) vectored by a different eriophyid mite, or a closely related tritimovirus (Oat necrotic mottle virus; ONMV) with no known vector. In contrast, both WSMV-Sidney 81 and a chimeric WSMV genome bearing HC-Pro of a divergent strain (WSMV-El Batán 3; 86% amino acid sequence identity) were efficiently transmitted by A. tosichella. Replacing portions of WSMV-Sidney 81 HC-Pro with the corresponding regions from ONMV showed that determinants of wheat curl mite transmission map to the 5′-proximal half of HC-Pro. WSMV genomes bearing HC-Pro of heterologous species retained the ability to form virions, indicating that loss of vector transmissibility was not a result of failure to encapsidate. Although titer in systemically infected leaves was reduced for all chimeric genomes relative to WSMV-Sidney 81, titer was not correlated with loss of vector transmissibility. Collectively, these results demonstrate for the first time that HC-Pro is required for virus transmission by a vector other than aphids.

Triticum mosaic poacevirus enlists P1 rather than HC-Pro to suppress RNA silencing-mediated host defense.

Triticum mosaic virus (TriMV) is the type species of the newly established Poacevirus genus in the family Potyviridae. In this study, we demonstrate that in contrast to the helper component-proteinase (HC-Pro) of Potyvirus species, the P1 proteins of TriMV and Sugarcane streak mosaic poacevirus function in suppression of RNA silencing (SRS). TriMV P1 effectively suppressed silencing induced by single- or double-stranded RNAs (ss/ds RNAs), and disrupted the systemic spread of silencing signals at a step after silencing signal production. Interestingly, contrary to enhanced SRS activity of potyviral HC-Pro by co-expression with P1, the presence of TriMV HC-Pro reduced SRS activity of TriMV P1. Furthermore, TriMV P1 suppressed systemic silencing triggered by dsRNA more efficiently than the HC-Pro of Turnip mosaic potyvirus. Furthermore, TriMV P1 enhanced the pathogenicity of a heterologous virus. Our results established poaceviral P1 as a potent RNA silencing suppressor that probably employs a novel mechanism to suppress RNA silencing-based antiviral defense.

Tritimovirus P1 functions as a suppressor of RNA silencing and an enhancer of disease symptoms.

Wheat streak mosaic virus (WSMV) is an eriophyid mite-transmitted virus of the genus Tritimovirus, family Potyviridae. Complete deletion of helper component-proteinase (HC-Pro) has no effect on WSMV virulence or disease synergism, suggesting that a different viral protein suppresses RNA silencing. RNA silencing suppression assays using Nicotiana benthamiana 16C plants expressing GFP were conducted with each WSMV protein; only P1 suppressed RNA silencing. Accumulation of GFP siRNAs was markedly reduced in leaves infiltrated with WSMV P1 at both 3 and 6 days post infiltration relative to WSMV HC-Pro and the empty vector control. On the other hand, helper component-proteinase (HC-Pro) of two species in the mite-transmitted genus Rymovirus, family Potyviridae was demonstrated to be a suppressor of RNA silencing. Symptom enhancement assays were conducted by inoculating Potato virus X (PVX) onto transgenic N. benthamiana. Symptoms produced by PVX were more severe on transgenic plants expressing WSMV P1 or potyvirus HC-Pro compared to transgenic plants expressing GFP or WSMV HC-Pro.

Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: Formation of dense fluorescent aggregates for sensitive virus tracking

A series of Wheat streak mosaic virus (WSMV)-based expression vectors were developed by engineering a cycle 3 GFP (GFP) cistron between P1 and HC-Pro cistrons with several catalytic/cleavage peptides at the C-terminus of GFP. WSMV-GFP vectors with the Foot-and-mouth disease virus 1D/2A or 2A catalytic peptides cleaved GFP from HC-Pro but expressed GFP inefficiently. WSMV-GFP vectors with homologous NIa-Pro heptapeptide cleavage sites did not release GFP from HC-Pro, but efficiently expressed GFP as dense fluorescent aggregates. However, insertion of one or two spacer amino acids on either side of NIb/CP heptapeptide cleavage site or deletion in HC-Pro cistron improved processing by NIa-Pro. WSMV-GFP vectors were remarkably stable in wheat for seven serial passages and for 120 days postinoculation. Mite transmission efficiencies of WSMV-GFP vectors correlated with the amount of free GFP produced. WSMV-GFP vectors infected the same range of cereal hosts as wild-type virus, and GFP fluorescence was detected in most wheat tissues.

Population genetics of Homalodisca vitripennis reovirus validates timing and limited introduction to California of its invasive insect host, the glassy-winged sharpshooter.

As RNA viruses evolve rapidly, we hypothesized that a virus could serve as a surrogate to discriminate recently separated populations of an invasive insect species. Homalodisca vitripennis reovirus (HoVRV) was used as a surrogate to assess population structure of glassy-winged sharpshooter (GWSS), an invasive species detected in California ~20 years ago. HoVRV nucleotide sequence polymorphism revealed a bottleneck in the introduced population, yielded population age estimates consistent with timing of GWSS discovery in California, suggested gene flow within the native range but not among native and introduced populations, and could potentially pinpoint source of the introduced population. Collectively, the data support use of a virus surrogate to define critical attributes of invasive species populations, with the caveat that life history of the surrogate must be closely linked to that of the host.