Simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduces cassava brown streak disease symptom severity and incidence.

Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive-sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome-linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E-1, eIF(iso)4E-2, novel cap-binding protein-1 (nCBP-1), and nCBP-2. Protein-protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9-mediated genome editing was employed to generate ncbp-1, ncbp-2, and ncbp-1/ncbp-2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp-1/ncbp-2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titre in storage roots relative to wild-type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.

Field-usable lateral flow immunoassay for the rapid detection of a macluravirus, large cardamom chirke virus.

A simple and rapid lateral flow immunoassay (LFIA) was developed by utilizing gold nanoparticles conjugated to a polyclonal antibody against coat protein of large cardamom chirke virus (LCCV). The LFIA based on the principle of sandwich immunoassay detected LCCV within ∼10 min and the result could be evaluated visually. The colloidal gold (CG) was made using 1% gold chloride solution. The LCCV IgG (1 µg/µl) and Mouse IgG (0.5 µg/µl) were conjugated with CG individually and coated onto a conjugate pad at 1:1 ratio. A sample extraction procedure was optimized in order to get adequate clear leaf sap of large cardamom leaf within few minutes. The sensitivity limit of the detection was 1:40 dilution of LCCV infected leaf sap. The diagnostic performance of LFIA was compared with ELISA using field samples. The LFIA was free from false positive as no visible test line was developed with healthy and potyviruses such as papaya ringspot virus and potato virus Y. The diagnostic specificity and sensitivity of LFIA was 100% and 90%, respectively. The Cohen's kappa coefficient (0.701) suggested a very good agreement between the ELISA and LFIA. Receiver operating characteristic analysis indicated that LFIA was a robust method as the area under the curve (0.950) is significantly (P <0.0001) broader.

Immunodetection of Triticum mosaic virus by DAS- and DAC-ELISA using antibodies produced against coat protein expressed in Escherichia coli: potential for high-throughput diagnostic methods.

Triticum mosaic virus (TriMV), an economically important virus infecting wheat in the Great Plains region of the USA, is the type species of the Poacevirus genus in the family Potyviridae. Sensitive and high-throughput serology-based detection methods are crucial for the management of TriMV and germplasm screening in wheat breeding programs. In this study, TriMV coat protein (CP) was expressed in Escherichia coli, and polyclonal antibodies were generated against purified soluble native form recombinant CP (rCP) in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western immuno-blot and enzyme-linked immunosorbent assays (ELISA). In direct antigen coating (DAC)-ELISA, antibodies reacted specifically, beyond 1:20,000 dilution with TriMV in crude sap, but not with healthy extracts, and antiserum at a 1:10,000 dilution detected TriMV in crude sap up to 1:4860 dilution. Notably, rabbit anti-TriMV IgG and anti-TriMV IgG-alkaline phosphatase conjugate reacted positively with native virions in crude sap in a double antibody sandwich-ELISA, suggesting that these antibodies can be used as coating antibodies which is crucial for any 'sandwich' type of assays. Finally, the recombinant antibodies reacted positively in ELISA with representative TriMV isolates collected from fields, suggesting that antibodies generated against rCP can be used for sensitive, large-scale, and broad-spectrum detection of TriMV.

Resistance to Wheat streak mosaic virus generated by expression of an artificial polycistronic microRNA in wheat.

Wheat streak mosaic virus (WSMV) is a persistent threat to wheat production, necessitating novel approaches for protection. We developed an artificial miRNA strategy against WSMV, incorporating five amiRNAs within one polycistronic amiRNA precursor. Using miRNA sequence and folding rules, we chose five amiRNAs targeting conserved regions of WSMV but avoiding off-targets in wheat. These replaced the natural miRNA in each of five arms of the polycistronic rice miR395, producing amiRNA precursor, FanGuard (FGmiR395), which was transformed into wheat behind a constitutive promoter. Splinted ligation detected all five amiRNAs being processed in transgenic leaves. Resistance was assessed over two generations. Three types of response were observed in T(1) plants of different transgenic families: completely immune; initially resistant with resistance breaking down over time; and initially susceptible followed by plant recovery. Deep sequencing of small RNAs from inoculated leaves allowed the virus sequence to be assembled from an immune transgenic, susceptible transgenic, and susceptible non-transgenic plant; the amiRNA targets were fully conserved in all three isolates, indicating virus replication on some transgenics was not a result of mutational escape by the virus. For resistant families, the resistance segregated with the transgene. Analysis in the T(2) generation confirmed the inheritance of immunity and gave further insights into the other phenotypes. Stable resistant lines developed no symptoms and no virus by ELISA; this resistance was classified as immunity when extracts failed to transmit from inoculated leaves to test plants. This study demonstrates the utility of a polycistronic amiRNA strategy in wheat against WSMV.

Tempo and mode of plant RNA virus escape from RNA interference-mediated resistance.

A biotechnological application of artificial microRNAs (amiRs) is the generation of plants that are resistant to virus infection. This resistance has proven to be highly effective and sequence specific. However, before these transgenic plants can be deployed in the field, it is important to evaluate the likelihood of the emergence of resistance-breaking mutants. Two issues are of particular interest: (i) whether such mutants can arise in nontransgenic plants that may act as reservoirs and (ii) whether a suboptimal expression level of the transgene, resulting in subinhibitory concentrations of the amiR, would favor the emergence of escape mutants. To address the first issue, we experimentally evolved independent lineages of Turnip mosaic virus (TuMV) (family Potyviridae) in fully susceptible wild-type Arabidopsis thaliana plants and then simulated the spillover of the evolving virus to fully resistant A. thaliana transgenic plants. To address the second issue, the evolution phase took place with transgenic plants that expressed the amiR at subinhibitory concentrations. Our results show that TuMV populations replicating in susceptible hosts accumulated resistance-breaking alleles that resulted in the overcoming of the resistance of fully resistant plants. The rate at which resistance was broken was 7 times higher for TuMV populations that experienced subinhibitory concentrations of the antiviral amiR. A molecular characterization of escape alleles showed that they all contained at least one nucleotide substitution in the target sequence, generally a transition of the G-to-A and C-to-U types, with many instances of convergent molecular evolution. To better understand the viral population dynamics taking place within each host, as well as to evaluate relevant population genetic parameters, we performed in silico simulations of the experiments. Together, our results contribute to the rational management of amiR-based antiviral resistance in plants.

Characterization of a filamentous virus from Bermuda grass and its molecular, serological and biological comparison with Spartina mottle virus.

Bermuda grass with mosaic symptoms have been found in many parts of Iran. No serological correlation was observed between two isolates of this filamentous virus and any of the members of the family Potyviridae that were tested. Aphid transmission was demonstrated at low efficiency for isolates of this virus, whereas no transmission through seed was observed. A DNA fragment corresponding to the 3' end of the viral genome of these two isolates from Iran and one isolate from Italy was amplified and sequenced. A BLAST search showed that these isolates are more closely related to Spartina mottle virus (SpMV) than to any other virus in the family Potyviridae. Specific serological assays confirmed the phylogenetic analysis. Sequence and phylogenetic analysis suggested that these isolates could be considered as divergent strains of SpMV in the proposed genus Sparmovirus.

Characterisation of epitopes on barley mild mosaic virus coat protein recognised by a panel of novel monoclonal antibodies.

Barley mild mosaic virus (BaMMV), a member of the family Potyviridae, genus Bymovirus, is involved in the economically important yellow mosaic disease of winter barley in East Asia and Europe. We investigated serological properties of bacterially expressed BaMMV coat protein (CP) of a German isolate. Ten mouse monoclonal antibodies were produced using purified E. coli expressed BaMMV-CP as immunogen. The reactivity of MAbs with different strains of BaMMV was analysed by several immunological methods that are frequently used in diagnostic virology: enzyme-linked immunosorbent assay (ELISA), dot-blot, Western-blotting (WB), direct tissue blotting immunoassay (DTBIA) and immunoelectron microscopy (IEM). The amino acids involved in the formation of epitopes recognised by several MAbs were mapped by using synthetic pin-bound peptides and the localisation of epitopes in assembled virus particles was determined by electron microscope studies. MAbs V29 and M1 decorated the whole virion indicating that their epitopes 6PDPI9 and 96ITDDEK101, respectively, are exposed on the surface. The MAbs V6 and V14 both interacted with 44LPEPKM49, which seems to be accessible at only one end of the virus particle. The MAbs V6, V14, V29 and M1 detected epitopes common to a wide range of BaMMV isolates and can therefore be used effectively in routine diagnostic tests for BaMMV from barley leaves. We suggest that MAbs M1, V6, V14 and V29 are most suitable for use in TAS-ELISA, V6, V14 and V29 for Western blotting and V29 and M1 for electron microscope serology.

Characterisation of a new potyvirus species infecting meadow saffron Colchicum autumnale).

The alkaloids contained in Colchicum autumnale seeds are used in numerous medicines. Good quality seeds are difficult to obtain from this undomesticated plant. Therefore, a research program was set up aiming to cultivate C. autumnale in order to improve alkaloid contents and seed yields. In this context, a collection was established in 1999 by transplanting corms from twelve different locations in Eastern France. However, serious symptoms of necrosis and decay have appeared in this collection since 2001. Electron microscopic observations of plants showing symptoms revealed the presence of filamentous particles and pinwheel-like structures characteristic of the Potyviridae family. Leaves and corms from symptomatic plants were assayed with potyvirus-specific Enzyme-Linked Immunosorbent Assay test. Positive reactions were obtained with plants from all the geographic origins, which exhibited flower breaking symptoms on petals. RT-PCR tests with family Potyviridae-specific primers confirmed the ELISA results and showed that the virus can be detected in corms, roots and flowers of symptomatic plants. The 3' region of the genome was cloned, sequenced and compared to other potyvirus species. Phylogenetic analyses suggest the presence of a new viral species tentatively named Meadow saffron breaking virus (MSBV) in C. autumnale.

Properties of a virus isolated from Vernonia amygdalina Del. in Lagos, Nigeria.

A previously uncharacterized virus tentatively named Vernonia green vein-banding virus (VGVBV) was isolated from Vernonia amygdalina Del. ("bitterleaf") from Lagos, Nigeria. The virus was mechanically transmissible but had a narrow host range restricted to Nicotiana benthamiana, Chenopodium quinoa and C. amaranticolor. It was also transmissible in a non-persistent manner by Myzus persicae. The virus was purified from N. benthamiana and about 750 nm long flexuous rod-shaped particles were observed in purified preparations as well as in leaf-dips of Vernonia sp. Inclusion bodies in the form of pinwheels and scrolls were observed in ultrathin sections of Vernonia leaves by electron microscopy. M(r), of the viral coat protein was estimated to be about 34 K. In indirect ELISA, all 20 samples from naturally infected Vernonia sp. reacted positively with a potyvirus-specific monoclonal antibody (MAb) as well as with an antiserum raised against VGVBV. Apart from the homologous antigen, the VGVBV antiserum reacted only with Plum poxvirus (PPV). The VGVBV reacted strongly with the antisera to Bean yellow mosaic virus (BYMV), Bean common mosaic virus (BCMV) and Amaranthus leaf mottle virus (AmLMV) but weakly with antisera to PPV and Cowpea aphid-borne mosaic virus (CABMV) (all members of the family Potyviridae, the genus Potyvirus) in at least one of the assays used (indirect ELISA, dot-blot immunoassay and Western blot analysis). The results of our host range, cytopathological and serological studies and the available literature indicate that a hitherto difficult to transmit VGVBV has only been reported from Nigeria. We consider VGVBV a candidate for a new potyvirus. This virus should be further investigated to collect sufficient data for a qualified proposal of VGVBV as a new potyvirus.

Development of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of sugarcane streak mosaic virus isolates from Southern India.

Sugarcane streak mosaic virus (SCSMV), causes mosaic disease of sugarcane and is thought to belong to a new undescribed genus in the family Potyviridae. The coat protein (CP) gene from the Andhra Pradesh (AP) isolate of SCSMV (SCSMV-AP) was cloned and expressed in Escherichia coli. The recombinant coat protein was used to raise high quality antiserum. The CP antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection and discrimination of SCSMV isolates in South India. The sequence of the cloned PCR products encoding 3'untranslated region (UTR) and CP regions of the virus isolates from three different locations in South India viz. Tanuku (Coastal Andhra Pradesh), Coimbatore (Tamil Nadu) and Hospet (Karnataka) was compared with that of SCSMV-AP. The analysis showed that they share 89.4, 89.5 and 90% identity respectively at the nucleotide level. This suggests that the isolates causing mosaic disease of sugarcane in South India are indeed strains of SCSMV. In addition, the sensitivity of the IC-RT-PCR was compared with direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA) and dot-blot immunobinding assays and was found to be more sensitive and hence could be used to detect the presence of virus in sugarcane breeding, germplasm centres and in quarantine programs.

Molecular and serological relationships of Spartina mottle virus (SpMV) strains from Spartina spec. and from Cynodon dactylon to other members of the Potyviridae.

Spartina mottle virus (SpMV) was first reported 1980 and classified by physical and biological properties as a tentative member of the genus Rymovirus in the Potyviridae. This genus was recently separated into two genera: Rymovirus and Tritimovirus. Now the sequence of the 3'-terminal part of the genome of SpMV was determined. Additionally a virus isolate originating from Cynodon dactylon in Italy was cloned and sequenced. This Assisi-isolate shared 87.5% amino acid sequence identity with SpMV. The high degree of identity and their close serological relationship indicate that SpMV and Assisi-isolate have to be regarded as different strains of one virus. The Assisi-isolate should be designated as SpMV-AV. Comparing the C-terminal part of the ORF of several Potyviridae the sequences of SpMV strains were more similar to those of the genera Rymovirus and Potyvirus than to the genera Tritimovirus, Macluravirus, Bymovirus and Ipomovirus. The comparisons revealed identities of less than 32% for the CP and 37% for the 3'-NIb/CP region, indicating that SpMV can not be classified to any of the established genera. The results of serological tests support a separate position of SpMV in the Potyviridae. We propose to introduce the name Sparmovirus for the new genus.

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.