One-Enzyme RTX-PCR for the Detection of RNA Viruses from Multiple Virus Genera and Crop Plants.

Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

Full genome sequence of a new mitovirus from the phytopathogenic fungus Rhizoctonia solani.

A double-stranded RNA (dsRNA) segment was identified in Rhizoctonia solani anastomosis group (AG)-2-2IIIB, the primary causal agent of Rhizoctonia crown and root rot of sugar beet. The dsRNA segment represented the genome replication intermediate of a new mitovirus that was tentatively designated as "Rhizoctonia solani mitovirus 39" (RsMV-39). The complete sequence of the dsRNA was 2805 bp in length with 61.9% A+U content. Using either the fungal mitochondrial or universal genetic code, a protein of 840 amino acids containing an RNA-dependent RNA polymerase (RdRp) domain was predicted with a molecular mass of 94.46 kDa. BLASTp analysis revealed that the RdRp domain of RsMV-39 had 43.55% to 72.96% sequence identity to viruses in the genus Mitovirus, and was the most similar (72.96% identical) to that of Ceratobasidium mitovirus A (CbMV-A). Phylogenetic analysis based on RdRp domains clearly showed that RsMV-39 is a member of a distinct species in the genus Mitovirus of the family Mitoviridae. This is the first full genome sequence of a mycovirus associated with R. solani AG-2-2IIIB.

Occurrence of putative endornaviruses in non-cultivated plant species in South Louisiana.

Extraction and electrophoretic analysis of viral dsRNA from plants has been used successfully to detect infections by RNA viruses. We used this approach as an initial tool to test non-cultivated plant species for the presence of endornaviruses. Foliar samples were collected from symptomless plants in various locations within East Baton Rouge Parish, Louisiana, USA, and tested for viral dsRNA. After testing 208 plant species belonging to 74 families, five (Geranium carolinianum, Hydrocotyle umbellata, H. prolifera, Sorghum halepense, and Sisyrinchium atlanticum) yielded dsRNAs similar in size to the dsRNAs of members of the family Endornaviridae. The endornavirus nature of the dsRNAs was confirmed by reverse-transcription PCR (RT-PCR) and sequencing the RT-PCR products. Sequence data were used to determine relationships of the putative endornaviruses to members of the family Endornaviridae. The putative endornaviruses were detected in both native and introduced plants species. This is the first survey on the occurrence of endornaviruses in non-cultivated plant species.

Phytophthora infestans RNA virus 2, a novel RNA virus from Phytophthora infestans, does not belong to any known virus group.

Phytophthora infestans is the causal agent of potato and tomato late blight. In this study, we obtained the complete genome sequence of a novel RNA virus from this plant pathogen, tentatively named "Phytophthora infestans RNA virus 2" (PiRV-2). The PiRV-2 genome is 11,170 nt in length and lacks a polyA tail. It contains a single large open reading frame (ORF) with short 5' and 3' untranslated regions. The ORF is predicted to encode a polyprotein of 3710 aa (calculated molecular weight, 410.94 kDa). This virus lacks significant similarity to any other known viruses, even in the conserved RNA-dependent RNA polymerase region. Phylogenetic analysis demonstrated that it did not cluster with any known virus group. We conclude that PiRV-2 belongs to a new virus family yet to be described. This virus was found to be faithfully transmitted through asexual reproduction.

Extracts of Polypore Mushroom Mycelia Reduce Viruses in Honey Bees.

Waves of highly infectious viruses sweeping through global honey bee populations have contributed to recent declines in honey bee health. Bees have been observed foraging on mushroom mycelium, suggesting that they may be deriving medicinal or nutritional value from fungi. Fungi are known to produce a wide array of chemicals with antimicrobial activity, including compounds active against bacteria, other fungi, or viruses. We tested extracts from the mycelium of multiple polypore fungal species known to have antiviral properties. Extracts from amadou (Fomes) and reishi (Ganoderma) fungi reduced the levels of honey bee deformed wing virus (DWV) and Lake Sinai virus (LSV) in a dose-dependent manner. In field trials, colonies fed Ganoderma resinaceum extract exhibited a 79-fold reduction in DWV and a 45,000-fold reduction in LSV compared to control colonies. These findings indicate honey bees may gain health benefits from fungi and their antimicrobial compounds.

Comparison of RNA extraction methods for the detection of BNYVV rhizomania virus from roots of sugar beet.

Rhizomania is one of serious threat to sugar beet production in Morocco and in several parts of the world. This disease led to a statistically significant decrease in the quality and yield of sugar beet plantations. Therefore, this study aimed at comparing the efficacy of six commonly used RNA extraction methods for the detection, recovery of RNA of beet necrotic yellow vein virus (BNYVV) and removal of amplification inhibitors by reverse transcription-polymerase chain reaction (RT-PCR). The efficiency of these extraction methods was then compared to that of a commercial isolation kit with high content of phenolic compounds. The results showed that the extraction with the lithium chloride technique, the commercial kit, and direct and membrane spotting crude extract methods were found effective in yielding a higher purity and a higher concentration of RNA when compared to the other tested methods. Extraction with the lithium chloride technique and the Qiagen kit (RNeasy Plant Mini Kit) allowed the most intense band, whereas the CTAB method has generated the least intense band. Furthermore, the silica capture extraction method did not yield any RNA after extraction and electrophoresis. Consequently, it was concluded that, of these six methods, the lithium chloride technique and the Qiagen kit are the most appropriate for the extraction of viral RNA from sugar beet samples prior to RT-PCR for detecting BNYVV.

Protein nutrition governs within-host race of honey bee pathogens.

Multiple infections are common in honey bees, Apis mellifera, but the possible role of nutrition in this regard is poorly understood. Microsporidian infections, which are promoted by protein-fed, can negatively correlate with virus infections, but the role of protein nutrition for the microsporidian-virus interface is unknown. Here, we challenged naturally deformed wing virus - B (DWV-B) infected adult honey bee workers fed with or without pollen ( = protein) in hoarding cages, with the microsporidian Nosema ceranae. Bee mortality was recorded for 14 days and N. ceranae spore loads and DWV-B titers were quantified. Amongst the groups inoculated with N. ceranae, more spores were counted in protein-fed bees. However, N. ceranae infected bees without protein-diet had reduced longevity compared to all other groups. N. ceranae infection had no effect on protein-fed bee's longevity, whereas bees supplied only with sugar-water showed reduced survival. Our data also support that protein-feeding can have a significant negative impact on virus infections in insects. The negative correlation between N. ceranae spore loads and DWV-B titers was stronger expressed in protein-fed hosts. Proteins not only enhance survival of infected hosts, but also significantly shape the microsporidian-virus interface, probably due to increased spore production and enhanced host immunity.

Complete nucleotide sequence of Solanum nodiflorum mottle virus.

Solanum nodiflorum mottle virus (SNMoV) was isolated from a small-flowered nightshade (Solanum nodiflorum) in Queensland, Australia. It has been included in the genus Sobemovirus based on virion morphology and serological relationships. Here, we report the sequence of the complete genome of SNMoV. Sequence analysis confirmed that SNMoV has the characteristic genome organization of sobemoviruses. Phylogenetic analysis showed that it clusters most closely with velvet tobacco mottle virus (VTMoV), another sobemovirus native to Australia. Their genomes show 56.8 % sequence identity.

Identification of internal poly(A) tracts (IPATs) of variable lengths in a novel tobacco rattle virus RNA2 in potatoes.

The nucleotide (nt) sequences of two closely related isolates (CeWF-2 and CeWGH-2) of a novel tobacco rattle virus (TRV) RNA2 were determined. The sequences of their RNA2-specific regions were almost identical and contained four open reading frames (ORFs) in an arrangement similar to that found in the previously described TRV TpO1 RNA2. Their predicted ORF 1 gene products shared 97 % amino acid sequence identity with the TpO1 coat protein, but the ORF 2 and ORF 3 gene products shared only 82 % sequence identity, and no appreciable sequence similarity was found between the CeWF-2/CeWGH-2 and TpO1 ORF 4 gene products. In the CeWGH-2 sequence, the RNA2-specific and RNA1-related regions were separated by seven adenine (A) residues. In CeWF-2, however, an internal poly(A) tract (IPAT) of variable size consisting of ca. 20 to 30 (A) residues was found. This is the first report of an IPAT occurring in a tobravirus RNA2.

Development and evaluation of a reverse transcription loop-mediated isothermal amplification assay for detection of beet necrotic yellow vein virus.

Sugar beet can be infected by many different viruses that can reduce yield; beet necrotic yellow vein virus (BNYVV) is one of the most economically important viruses of this crop plant. This report describes a new reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for identification of BNYVV. In addition, a novel immunocapture (IC) RT-LAMP assay for rapid and easy detection (without RNA extraction) of BNYVV was developed here and compared with DAS-ELISA and RT-LAMP assays. Our results show that the IC-RT-LAMP assay is a highly reliable alternative assay for identification of BNYVV.

A new virus discovered by immunocapture of double-stranded RNA, a rapid method for virus enrichment in metagenomic studies.

Next-generation sequencing technologies enable the rapid identification of viral infection of diseased organisms. However, despite a consistent decrease in sequencing costs, it is difficult to justify their use in large-scale surveys without a virus sequence enrichment technique. As the majority of plant viruses have an RNA genome, a common approach is to extract the double-stranded RNA (dsRNA) replicative form, to enrich the replicating virus genetic material over the host background. The traditional dsRNA extraction is time-consuming and labour-intensive. We present an alternative method to enrich dsRNA from plant extracts using anti-dsRNA monoclonal antibodies in a pull-down assay. The extracted dsRNA can be amplified by reverse transcriptase-polymerase chain reaction and sequenced by next-generation sequencing. In our study, we have selected three distinct plant hosts: Maori potato (Solanum tuberosum), rengarenga (Arthropodium cirratum) and broadleaved dock (Rumex obtusifolius) representing a cultivated crop, a New Zealand-native ornamental plant and a weed, respectively. Of the sequence data obtained, 31-74% of the reads were of viral origin, and we identified five viruses including Potato virus Y and Potato virus S in potato; Turnip mosaic virus in rengarenga (a new host record); and in the dock sample Cherry leaf roll virus and a novel virus belonging to the genus Macluravirus. We believe that this new assay represents a significant opportunity to upscale virus ecology studies from environmental, primary industry and/or medical samples.

Novel mitoviruses in Rhizoctonia solani AG-3PT infecting potato.

Double-stranded RNA (dsRNA) elements are ubiquitous in Rhizoctonia solani. Total dsRNA was randomly amplified from a R. solani isolate (RS002) belonging to anastomosis group-3PT (AG-3PT), associated with black scurf in potato. Assembly of resulting cDNA sequences identified a nearly complete genome of a novel virus related to the genus Mitovirus (family Narnaviridae), herein named Rhizoctonia mitovirus 1 RS002 (RMV-1-RS002). The 2797 nucleotide partial genome of RMV-1-RS002 is A-U rich (59.06 %), and can be folded into stable stem-loop structures at 5' and 3' ends. Universal and mold mitochondrial codon usages revealed a large open reading frame in the genome, putatively encoding an 826 amino acid polypeptide, which has conserved motifs for mitoviral RNA-dependent RNA polymerase. The full length putative polypeptide shared 25.6 % sequence identity with the corresponding region of Tuber excavatum mitovirus (TeMV). The partial genome of a second mitovirus (proposed name Rhizoctonia mitovirus 2 RS002 (RMV-2-RS002)) was also amplified from RS002. A nearly identical copy of RMV-1-RS002 was detected in two additional AG-3PT isolates. These data indicate that multiple mitoviruses can exist in a single isolate of R. solani AG-3PT, and that mitoviruses such as RMV-1-RS002 are probably widespread in this pathogen. The roles of mitoviruses in the biology of R. solani AG-3PT remain unknown.

Molecular characterization of a new tobacco rattle virus (TRV) RNA2 and identification of different TRV RNA1/RNA2 pairings in various potato-growing areas in Germany.

The almost complete nucleotide sequences lacking only the short primer-derived 5' and 3' ends were determined for two closely related isolates of a new tobacco rattle virus (TRV) RNA2, i.e., ByKT (Bav)-2 and ByKT (LS)-2. These isolates originated from corky-ringspot-affected potato-growing areas in southern Germany (Bavaria) and northern central Germany (Lower Saxony), respectively, where they were associated with distinct supporting TRV RNA1s. In potatoes in other parts of Germany, TRV RNA2s closely related to TRV TpO1 RNA2 were identified. They, too, were associated with distinct TRV RNA1s in different parts of the country.

Genetic diversity and population structure of beet necrotic yellow vein virus in China.

Beet necrotic yellow vein virus (BNYVV) is a serious threat to the sugar beet industry worldwide. However, little information is available regarding the genetic diversity and population structure of BNYVV in China. Here, we analyzed multiple sequences from four genomic regions (CP, RNA3, RNA4 and RNA5) of a set of Chinese isolates. Sequence analyses revealed that several isolates were mixed infections of variants with different genotypes and/or different p25 tetrad motifs. In total, 12 distinct p25 tetrads were found in the Chinese BNYVV population, of which four tetrads were newly identified. Phylogenetic analyses based on four genes (CP, RNA3-p25, RNA4-p31 and RNA5-p26) in isolates from around the world revealed the existence of two to four groups, which mostly corresponded to previously reported phylogenetic groups. Two new subgroups and a new group were identified from the Chinese isolates in p25 and p26 trees, respectively. Selection pressure analysis indicated that there was a positive selection pressure on the p25 from the Chinese isolates, but the other three proteins were under a negative selection pressure. There was frequent gene flow between geographically distant populations, which meant that BNYVV populations from different provinces were not geographically differentiated.

Diversity and evolution of potato mop-top virus.

Nearly complete sequences of RNA-CP and 3'-proximal RNA-TGB were determined for 43 samples of potato mop-top virus (PMTV) originating from potato tubers and field soil from Sweden, Denmark and the USA. The results showed limited diversity and no strict geographical grouping, suggesting only a few original introductions of PMTV from the Andes. Two distinguishable types of RNA-CP and RNA-TGB were found in the samples, but no specific combination of them correlated with spraing symptoms in tubers. Lack of positive selection in the coding sequences indicates that there is no specific molecular adaptation of PMTV to new vectors or hosts.

Genome sequence heterogeneity of Lake Sinai Virus found in honey bees and Orf1/RdRP-based polymorphisms in a single host.

Honey bees (Apis mellifera) are susceptible to a wide range of pathogens, including a broad set of viruses. Recently, next-generation sequencing has expanded the list of viruses with, for instance, two strains of Lake Sinai Virus. Soon after its discovery in the USA, LSV was also discovered in other countries and in other hosts. In the present study, we assemble four almost complete LSV genomes, and show that there is remarkable sequence heterogeneity based on the Orf1, RNA-dependent RNA polymerase and capsid protein sequences in comparison to the previously identified LSV 1 and 2 strains. Phylogenetic analyses of LSV sequences obtained from single honey bee specimens further revealed that up to three distinctive clades could be present in a single bee. Such superinfections have not previously been identified for other honey bee viruses. In a search for the putative routes of LSV transmission, we were able to demonstrate the presence of LSV in pollen pellets and in Varroa destructor mites. However, negative-strand analyses demonstrated that the virus only actively replicates in honey bees and mason bees (Osmia cornuta) and not in Varroa mites.

Complete nucleotide sequences of two closely related tobacco rattle virus RNA2s for which previously possibly only deletion/recombination mutants had been described.

The almost complete nucleotide (nt) sequences lacking only short primer-derived 5' and 3' terminal portions were determined for the RNA2s of two closely related tobacco rattle virus (TRV) isolates which were detected in recently infected roots of Nicotiana benthamiana bait plants (isolate HaB) or of field-grown potato plants (isolate HaW), respectively. The sequences of c. 1350 5' proximal nts in these RNA2s were almost identical to those in four previously described TRV isolates from potatoes in the Netherlands and Poland which had been propagated in tobacco leaves. The RNA2s of the earlier isolates, however, consist of only c. 2000 nts, whereas the HaW and HaB RNA2s comprise more than 4000 nts. In addition to the coat protein gene which is present in the RNA2s of all isolates, the HaW and HaB RNA2s contain two further open reading frames which in the previous isolates were apparently lost either entirely or of which small remnants only were left. The RNA2s of the previously described isolates and of our new ones have different RNA1-related 3' ends which apparently have been acquired from different supporting RNA1s. After transmission to tobacco leaves HaB RNA2 formed deletion/recombination mutants which--as in the previous isolates--consisted of only c. 2000 nts. They had acquired the 3' end from their supporting RNA1.

NEW BNYVV P25 VARIANTS IN BELGIUM.

Rhizomania is a widespread viral plant disease of major importance in sugar beet cropping and breeding. It is caused by the Beet necrotic yellow vein virus (BNYVV), a Benyvirus transmitted by the soil inhabiting plasmodiophorid Polymyxa betae. This vector also transmits other sugar beet virus such as Beet virus Q (BVQ) and Beet soil-borne virus (BSBV). Despite identification of resistance genes, BNYVV remains a major constraint because of resistance-breaking events as well as its ability to survive for long periods in soils in resting spores of P. betae. During the 2014 growing season, severe rhizomania symptoms were detected in Rz1 resistant beet genotypes in ten Belgian fields suggesting resistance-breaking events. Plants from these fields were sampled and total RNA was extracted from root hairs. The presence of BNYVV, BSBV, BVQ and P. betae was assessed by multiplex RT-PCR. Samples were then tested for the presence of BNYVV RNA5 and RNA3 by RT-PCR respectively targeting P26 and P25 genes. PCR products from P25 gene were then purified and sequenced. The results confirmed the presence of P. betae, BSBV and BVQ in all samples. BNYVV was detected in nine fields. Sequencing of P25 partial cDNA sequences revealed the presence of BNYVV types A and B. Two isolates possessed the amino acids motifs AYPR in the so-called tetrad region aa67-70. This motif was previously associated with resistance-breaking events. The Belgian situation will be discussed in the light of the current situation in neighbouring countries.

Molecular characterisation of an endornavirus from Rhizoctonia solani AG-3PT infecting potato.

Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is a soil-borne plant pathogenic fungus that has a broad host range, including potato. In this study, the double-stranded RNA (dsRNA) profiles were defined for 39 Rhizoctonia solani isolates representative of two different anastomosis groups (AGs) associated with black scurf of potato in New Zealand. A large dsRNA of c. 12 kb-18 kb was detected in each of the isolates, regardless of AG or virulence on potato. Characterisation of the large dsRNA from R. solani AG-3PT isolate RS002, using random amplification of total dsRNA and analyses of overlapping cDNA sequences, resulted in the assembly of a consensus sequence of 14 694 nt. A single, large open reading frame was identified on the positive strand of the assembled sequence encoding a putative polypeptide of at least 4893 amino acids, with a predicted molecular mass of 555.6 kDa. Conserved domains within this polypeptide included those for a viral methyltransferase, a viral RNA helicase 1 and an RNA-dependent RNA polymerase. The domains and their sequential organisation revealed the polyprotein was very similar to those encoded by dsRNA viruses of the genus Endornavirus, in the family Endornaviridae. This is the first report of an endornavirus in R. solani, and thus the putative virus is herein named Rhizoctonia solani endornavirus - RS002 (RsEV-RS002). Partial characterisation of the large dsRNAs in five additional AG-3PT isolates of R. solani also identified them as probable endornaviruses, suggesting this family of viruses is widespread in R. solani infecting potato. The ubiquitous nature of endornaviruses in this plant pathogen implies they may have an important, but yet uncharacterised, role in R. solani.

Complete genomic characterization of a potato mop-top virus isolate from the United States.

Potato mop-top virus (PMTV; family Virgaviridae) was reported recently in the Pacific Northwestern USA. To better understand the genetic diversity of this virus, the complete genome of an isolate from Washington State (WA), USA, was characterized. Sequence comparisons of the WA isolate with other known sequences revealed that the RNA-Rep-encoded RdRp protein and the RNA-CP-encoded coat protein displayed >99 % amino acid sequence identity to those of two Nordic (RdRp) and several European and North American isolates (CP), respectively. The RNA-TGB-encoded TGB 1 and TGB 3 protein sequences had >99 % amino acid sequence identity to the corresponding proteins of Czech and Danish isolates, whereas the TGB 2 protein is identical to those of Colombian isolates. Phylogenetic analysis of the viral genes of the WA isolate reflected the close relationship between WA and European isolates. RFLP analysis of corresponding DNA of RNA TGB and RNA CP revealed that the WA isolate has the RNA TGB-II and RNA CP-B types, which are prevalent in Europe and other parts of world. This is the first report of the complete genome characterization of PMTV from the Americas.