Virus Coat Protein Gene Research Articles

First report of Cucumber mosaic virus infecting watermelon in Greece and Bulgaria

In 2014, a total of 108 symptomatic watermelon samples were collected in Greece and analyzed by DAS-ELISA using polyclonal antisera (INRA, Montfavet) against the most common aphid-transmitted viruses. Cucumber mosaic virus (CMV) was detected in eight of 108 plants tested. Five of these plants collected at Lesvos were also infected with Watermelon mosaic virus (WMV), whereas three plants from Prohoma had single infections. Five samples with severe stunting and yellowing collected at Asenovgrad (Bulgaria) in 2013 and tested by DAS-ELISA for the above viruses hosted CMV and Cucurbit aphid-borne yellows virus (CABYV). To confirm CMV presence in watermelon, a nested RT-PCR assay was carried out first with the degenerated primers CMVup624a (5′-ATGGACAAATCTGRATC- 3′) and CMVdo1244a (5′-TGRTGCTCRAYGTCKACATGA-3′) followed by CMVup624b (5′-GGACAAA TCTGRA TCTCCCAA TGC-3′) and CMVdo1244b (5′- TGCTCRAYGTCRACATGAAG-3′) that amplify a 622 bp region from the viral coat protein gene. Total RNA (Chatzinasiou et al., 2010, method A) extracted from all ELISA- positive watermelon samples from both countries and from two healthy watermelon plants was used as template in nested RT-PCR. A product of the expected size was amplified from all serologically CMV-positive samples, but not from the healthy ones. The Greek CMV isolates from Lesvos (LN810059) and Prohoma (LN810060) showed 99% nucleotide sequence identity with potato (AB448694) and tomato (EF153734) CMV isolates from Syria and India, while the Bulgarian isolate (LN810058) showed 99% nucleotide sequence identity with an Indian cucumber isolate (JF279608). Phylogenetic analysis indicated that the Greek CMV isolate (LN810059) belongs to sub-group IA, while the Bulgarian isolate and a Greek isolate (LN810060) belong to sub-group IB. To our knowledge this is the first report of CMV infecting watermelons in Greece and Bulgaria.

Development and validation of protocols for product stewardship in transgenic white clover (Trifolium repens L.): detection of the AMV CP and npt2 transgenes in pollen, honey and honey bees

There are no current commercial releases of genetically modified white clover, but several research groups are working on traits such as virus resistance, stress tolerance and bloat safety that are likely to provide large economic benefits for livestock farmers. However, white clover pollen is a common constituent of honey produced by bees foraging white clover flowers. Therefore, there is a need to develop tools to detect the presence of genetically modified pollen in white clover honey. The results presented in this paper describe the development and application of PCR-based techniques to detect the Alfalfa mosaic virus coat protein gene (AMV CP) and the neomycin phosphotransferase 2 selectable marker gene (npt2) in genetically modified white clover pollen, whether this pollen is collected fresh, from honey bees that have been foraging white clover, or from honey. Further research and development will be required to develop ‘field-ready’ tools for the detection and quantification of these transgenes in pollen and honey products. However, this paper demonstrates prospects and principles in pollen and honey from honeybees foraging transgenic white clover.

Two Isolates of Potato Virus Y (PVY) and the Response of Different Potato Cultivars Against the Viral Infection

Potato virus Y (PVY), of the genus Potyvirus, family Potyviridae, is highly pathogenic virus, causing enormous economic losses in solanaceous crops. Based on the ELISA test for the screened areas, the virus incidence ranged from 15- 30%. Mild mosaic and/ or mottling associated with leaf deformation were observed after 21 days on N. tabacum cv (white barley and galtinosa), mechanically inoculated with PVY- Kafer El-Zayat isolate. However, only mild mosaic was observed on same plants following PVY-Komhamada challenge. Molecular analysis measures including, coat protein amplicons, RFLP, sequence and phylogenetic analysis of the viral CP genes (~900 bp), confirmed that the two PVY isolates are existed in Egypt (Borg El Arab and Kafer El Zayat isolates). These two isolates were in close relation to France (KC005883) and Germany (AJ890342) isolates with identity 95% and 90%, respectively. Our results showed that the potato cultivar Spunta was more resistant to the infection by the PVY and the viral coat protein gene exhibited a low level of expression compared to the other two cultivars, Cara and Diamont. Collectively, our study here demonstrates that the two PVY isolates were commonly distributed in the Egyptian potato fields and their origin are European and Spunta potato cultivar is the recommended cultivar for cultivation in all the examined areas

Recovery of virus-infected Dendrobium orchids by constitutive expression of the cymbidium mosaic virus coat protein gene

A common practice in the production of viral disease-resistant plant varieties is the transfer of the viral coat protein (CP) gene to a wide range of healthy plants. This is the first report on the effect of a bombarded CP transgene on the replication of cymbidium mosaic virus (CymMV) in naturally infected Dendrobium orchids. Nine months after transformation, two naturally CymMV-infected transgenic Dendrobium orchids exhibited a gradual decrease in CymMV-CP transcript and coat protein levels as detected by RT-PCR and western blot hybridization, respectively. At 18 months, the transgenic lines were completely free of endogenous CymMV contamination and were protected from CymMV re-infection following inoculation. Six weeks after inoculation with purified CymMV, these virus-free transgenic lines contained no viral coat protein transcript, replicase transcript or particle accumulation from the re-invading CymMV, as detected by RT-PCR and Immunoelectron microscopy assays, respectively. Additionally, no small RNAs derived from viral CymMV-CP were detected, but the transcribed CymMV-CP positive-strand derived from the transgene appeared in these transgenic lines.

Application of RNA interference methodology to investigate and develop SCMV resistance in maize.

Specific fragments of the sugarcane mosaic virus (SCMV) coat protein gene (cp) were amplified by reverse transcriptionpolymerase chain reaction and used to construct a marker free small interfering RNA complex expression vector against SCMV. In planta transformation was performed on maize (Zea mays) inbred line 8112 mediated by Agrobacterium tumefaciens. PCR and Southern blot analyses demonstrated successful integration of the cp segment into the 8112 genome. The in planta transformation frequency was 0.1%, and the cotransformed frequency with the cp and bar genes was 0.034%. Real-time quantitative PCR of samples from different transgenic plant organs showed that the expression of the cp gene fragment in transgenic plants was variable and that the highest expression level occurred in the tassels and leaves and the lowest expression occurred in the roots. Real-time quantitative PCR was also used to measure how gene expression in transgenic T2 generation plants inoculated with SCMV changes over time. The results showed that the hairpin RNA structure transcribed from the cp gene interfered with SCMV infection and transgenic maize lines were not equally effective in preventing SCMV infection. Our findings provide a valuable tool for controlling plant viruses using RNA interference and the posttranslational gene silencing approach.

Effect of artificial dsRNA on infection of pea plants by Pea seed-borne mosaic virus

The effect of direct application of artificial dsRNA molecules derived from the viral coat protein gene and the possibility of RNA interference induction leading to protection against Pea seed borne mosaic virus (PSbMV) have been investigated. Effectivity of virus transmission was not afected by the dsRNA treatment. Simultaneous application of dsRNA and virus inoculation as well as dsRNA application one day before PSbMV inoculation led to a significant short-term decrease in the PSbMV concentration in experimental pea plants. Either the curative effect of the artificial dsRNA biolistic application was not noticed.

Genetic Diversity of a Natural Population of Apple stem pitting virus Isolated from Apple in Korea.

Apple stem pitting virus (ASPV), of the Foveavirus genus in the family Betaflexiviridae, is one of the most common viruses of apple and pear trees. To examine variability of the coat protein (CP) gene from ASPV, eight isolates originating from 251 apple trees, which were collected from 22 apple orchards located in intensive apple growing areas of the North Gyeongsang and North Jeolla Provinces in Korea, were sequenced and compared. The nucleotide sequence identity of the CP gene of eight ASPV isolates ranged from 77.0 to 97.0%, while the amino acid sequence identity ranged from 87.7 to 98.5%. The N-terminal region of the viral CP gene was highly variable, whereas the C-terminal region was conserved. Genetic algorithm recombination detection (GARD) and single breakpoint recombination (SBP) analyses identified base substitutions between eight ASPV isolates at positions 54 and 57 and position 771, respectively. GABranch analysis was used to determine whether the eight isolates evolved due to positive selection. All values in the GABranch analysis showed a ratio of substitution rates at non-synonymous and synonymous sites (dNS/dS) below 1, suggestive of strong negative selection forces during ASPV CP history. Although negative selection dominated CP evolution in the eight ASPV isolates, SLAC and FEL tests identified four possible positive selection sites at codons 10, 22, 102, and 158. This is the first study of the ASPV genome in Korea.

Expression of symptoms, viral coat protein and silencing suppressor gene during mixed infection of a N-Wi strain of potato virus Y and an asymptomatic strain of potato virus X.

Potato virus Y (PVY) and potato virus X (PVX), the RNA viruses of two different genera results into synergistic interactions on mixed infection. In this study, a N-Wi strain of PVY and a PVX strain that is asymptomatic on potato were used to study their interactions during mixed infection in Nicotiana benthamiana and Nicotiana tabacum with reference to symptom expression, level of coat protein (CP) using ELISA and suppressor gene using real time PCR under high temperature (26-40°C) and low temperature (5-25°C) conditions. Both mixed and single infection caused severe necrosis and death of N. benthamiana plants. Single infection of these viruses in N. tabacum showed mild symptoms but mixed infection caused more severe symptoms. Synergistic symptoms were more pronounced under low temperature conditions than at high temperature. In low temperature conditions, the CP level of PVX in N. benthamiana was twofold higher than PVY and both the viruses reached at peak at 28 dpi in single virus infection. When PVY and PVX inoculated together, the CP levels of both the viruses increased and reached to the peak earlier (within 7-14days) than that in the single virus inoculation. Although, the CP level of PVX was higher than PVY in mixed infection, at later stage (28dpi) both the CP level declined to the similar level. The level of p25 suppressor gene was higher than HC-Pro in single inoculation. However, under mixed inoculation of PVY and PVX, expression of p25 was declined to the level of HC-Pro when the CP levels of both the virus also were observed to decline. The expression pattern of CP and suppressor gene was different in plants when mixed infection was created by inoculation of one virus followed by the other. This study showed the level of CP and suppressor gene of specific strain of PVY and PVX during their mixed infection in tobacco.

Tombusvirus-based vector systems to permit over-expression of genes or that serve as sensors of antiviral RNA silencing in plants

A next generation Tomato bushy stunt virus (TBSV) coat protein gene replacement vector system is described that can be applied by either RNA inoculation or through agroinfiltration. A vector expressing GFP rapidly yields high levels of transient gene expression in inoculated leaves of various plant species, as illustrated for Nicotiana benthamiana, cowpea, tomato, pepper, and lettuce. A start-codon mutation to down-regulate the dose of the P19 silencing suppressor reduces GFP accumulation, whereas mutations that result in undetectable levels of P19 trigger rapid silencing of GFP. Compared to existing virus vectors the TBSV system has a unique combination of a very broad host range, rapid and high levels of replication and gene expression, and the ability to regulate its suppressor. These features are attractive for quick transient assays in numerous plant species for over-expression of genes of interest, or as a sensor to monitor the efficacy of antiviral RNA silencing.

Molecular characterization of barley yellow dwarf virus in Tunisia.

Barley yellow dwarf disease is a worldwide ubiquitous virus disease of cereal crops. In order to characterize the B/CYDV isolates occurring in Tunisia, 240 barley leaves were randomly sampled from 6 fields following a North-South trend and analyzed by serological and molecular tests. DAS-ELISA results showed 40 positive samples with a prevalence of barley yellow dwarf virus (BYDV)-PAV (77.5%), followed by cereal yellow dwarf virus (CYDV)-RPV (25%) and BYDV-MAV (15%). Studies of the geographic distribution showed a high incidence of B/CYDV in the Tunisian Southern provinces. RT-PCR assays were performed to amplify the viral coat protein gene (CP) and sequence analyses revealed six BYDV-PAV haplotypes named PAV-TN1 to PAV-TN6. Phylogenetic analysis showed that the six Tunisian haplotypes were close to BYDV-PAV-II subspecies and had a strong similarity with Moroccan, Czech, French and German haplotypes. Although PAV-TN2 and PAV-TN5 showed up to 10% divergence from BYDV-PAV-II at the amino acid level, it seems to belong to the same subspecies but in a separated cluster. Our results will be important in developing appropriate control measures against BYDV disease in Tunisia.

Genetic variation analysis of apple chlorotic leaf spot virus coat protein reveals a new phylogenetic type and two recombinants in China

In this study, we generated sequences of the apple chlorotic leaf spot virus (ACLSV) coat protein (CP) gene. Genetic variation and phylogenetic analyses were carried out on these sequences along with others reported previously. ACLSV populations clustered into four types: in three of the four types, combinations of three amino acids at positions of 40, 75 and 79 were conserved. The fourth phylogenetic type, newly identified here, was characterized by co-variation of Ser(40)-Tyr(75)-Ser(79). Statistically significant genetic differentiation and infrequent gene flow were detected among the four types. Two natural recombinants were detected for the first time among ACLSV isolates/genotypes from China.

First report of Grapevine virus E from grapevines in China.

A survey to assess the presence and incidence of Grapevine virus E (GVE) in China was carried out in 2011 and 2012 by testing dormant canes of 192 grapevine samples collected from the Chinese provinces of Liaoning (183 samples), Xinjiang (8 samples), and Jilin (1 sample). Total RNA was extracted from cortical scrapings according to Foissac et al., (2001) and submitted to RT-PCR using GVE-specific primers designed on the viral movement protein (GVE-MP1 5’-TGTGGGGTGCATAGTCATAGGTTT-3’ and GVE-MP2 5’-GCTTTTGACTCCATTGGCTTTCTC-3’) and coat protein (GVE-CP1 5’-GTGGGTGAACCACTCAAGGT-3’ and GVE-CP2 5’-AGACCACTTGCGGCTCTTTA-3’) genes. Ten of 192 tested samples (5.2%) resulted positive with both primer sets. Considering that Grapevine virus A and B (GVA and GVB) infected 4.7% and 1.6% of the samples, respectively, GVE resulted the most common vitivirus in our survey. The 992-bp and 478-bp long PCR products amplified from a Chinese table grape cultivar (Guifeimeigui) were cloned and sequenced. These sequences comprised the complete CP and putative nucleic acid binding protein genes, as well as the partial sequence of the MP gene. Comparisons with GenBank sequences revealed 99% identity of thie Chinese GVE isolate (GenBank accession No. JX570675 and JX570674) with the South African strain (GU903012). Further molecular analysis revealed that all GVE-infected vines hosted also one or two other viruses, including Grapevine leafroll associated virus -1, -2, -3 and Grapevine rupestris stem pitting associated virus (GRSPaV). The presence of mixed infections did not allow to establish a direct relationship between GVE and disease symptoms, except for an observed aggravation of leafroll symptoms, that needs further studies. To the best of our knowledge, this is the first report of GVE in China.

Influence Factors Analysis of Transgenic Shoot Regeneration Frequency for Sugar Beet Explants

Beet necrotic yellow vein virus coat protein (BNYVV CP) gene cloned from BNYVV in Inner Mongolia of China was transferred to sugar beet explants using agrobacterium tumefaciens-mediated method. Transgenic regeneration shoots were induced from petioles and cotyledons tissues of sugar beet. Three sugar beet lines that were fit for genetic transformation experiment were screened, which were Sumfull8-1, Sumfull8-3 and 9718. The experiment results showed that plant regeneration frequency was influenced by many factors, which include hormone compositions and concentration in shoots regeneration medium, culture conditions, sugar beet genotype, original explant types and culture methods. Influence factors of transgenic shoot regeneration frequency for sugar beet explants were discussed in this paper. Efficient genetic transformation system and transgenic shoots regeneration system of sugar beet were established.

Production of marker-free and RSV-resistant transgenic rice using a twin T-DNA system and RNAi

A twin T-DNA system is a convenient strategy for creating selectable marker-free transgenic plants. The standard transformation plasmid, pCAMBIA 1300, was modified into a binary vector consisting of two separate T-DNAs, one of which contained the hygromycin phosphotransferase (hpt) marker gene. Using this binary vector, we constructed two vectors that expressed inverted-repeat (IR) structures targeting the rice stripe virus (RSV) coat protein (CP) gene and the special-disease protein (SP) gene. Transgenic rice lines were obtained via Agrobacterium-mediated transformation. Seven independent clones harbouring both the hpt marker gene and the target genes (RSV CP or SP) were obtained in the primary transformants of pDTRSVCP and pDTRSVSP, respectively. The segregation frequencies of the target gene and the marker gene in the T1 plants were 8.72 percent for pDTRSVCP and 12.33 percent for pDTRSVSP. Two of the pDTRSVCP lines and three pDTRSVSP lines harbouring the homozygous target gene, but not the hpt gene, were strongly resistant to RSV. A molecular analysis of the resistant transgenic plants confirmed the stable integration and expression of the target genes. The resistant transgenic plants displayed lower levels of the transgene transcripts and specific small interfering RNAs, suggesting that RNAi induced the viral resistance.

Erratum to: Genomic classification of betanodavirus by molecular phylogenetic analysis of the coat protein gene

The classification of betanodavirus into four species was reviewed including newer and well-characterised isolates. Six major clusters were identified, four of which were similar to the classic species. Two single isolate clusters were worth consideration as new species. Betanodaviruses are small (37 nm average) icosahedral viruses with two positive-stranded non-polyadenylated RNAs as its genome. RNA1 (3.1 kb) codes for the viral RNA-dependent RNA polymerase (protein A), while RNA2 (1.4 kb) codes for the coat protein [16]. A subgenomic RNA3 synthesized from RNA1 during early viral replication codes for protein B2 [17], which functions against the host RNA interference system [3, 8]. These viruses cause viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) in fin fishes. They were first identified as picorna-like virus particles [5] and later described as members of the family Nodaviridae [12]. Taxonomically, these viruses belong to the genus Betanodavirus of family Nodaviridae, with four species recognised so far [16] based on similarities in the variable region of the viral coat protein (CP) gene (nt 604-1030) [14]. The currently recognized species are Redspotted grouper nervous necrosis virus (RGNNV), Striped jack nervous necrosis virus (SJNNV), Tiger puffer nervous necrosis virus (TPNNV) and Barfin flounder nervous necrosis virus (BFNNV), with SJNNV as the type species. The preferred temperature ranges of members of these species are 25-30 C, 20-25 C, 20 C and 15-20 C, respectively. Over 40 fish species have been reported to be susceptible to betanodaviruses, and the number is steadily increasing [10, 13]. Since the first description 15 years ago, the taxonomy of betanodavirus has remained more or less the same irrespective of the huge number of virus isolates being reported from different geographical environments and from different fish hosts. Some modifications were proposed to the system [4, 11, 15, 19], but these have been largely restricted to the geographical area of the researcher. The study presented here is an attempt to analyze the global phylogenetic relationships among the viruses by comparing all isolates for which full-length coat protein (CP) gene ORF sequence information is available in the NCBI GenBank database. A short variable region in the CP gene is shown to be sufficient for determining the phylogenetics of these viruses [14], but the full-length CP gene ORF is used in this study to obtain more reliable results. Sequences of the full-length coat protein gene ORF of betanodaviruses were retrieved from the NCBI GenBank database with due care to include members of the species described in Eighth Report of the International Committee on Taxonomy of Viruses [16]. A total of 78 sequences were retrieved from the database. The sequences were labelled with their geographical location of isolation for ease of compilation for analysing the distribution pattern of the isolates included in this study. Betanodaviruses show high temperature specificity [6] and low host specificity [19], and this is expected to be reflected in their geographical distribution. The nucleotide and deduced amino acid sequences were aligned using the MUSCLE program [2] in MEGA software [18]. Neighbour-joining trees were C. P. Binesh (&) Sacred Heart College, Thevara, Cochin 682013, Kerala, India e-mail: bineshkanayi@gmail.com C. Greeshma Department of Life Sciences, Calicut University, Thenjippalam, Malappauram, Kerala, India e-mail: greeshmakongatta@gmail.com 123 Arch Virol (2013) 158:1589–1594 DOI 10.1007/s00705-012-1549-7

Visual detection of Potato Leafroll virus by loop-mediated isothermal amplification of DNA with the GeneFinder™ dye

The most common virus affecting potatoes in the field worldwide is Potato Leafroll virus (PLRV), belonging to the family Luteoviridae, genius Plerovirus. There are several molecular methods to detect PLRV including polymerase chain reaction (PCR), Multiplex AmpliDet RNA and double antibody sandwich ELISA (DAS-ELISA). But these techniques take a long time for 3h to two days, requiring sophisticated tools. The aim of this study was to reduce the time required to detect PLRV, using a newly designed loop-mediated isothermal amplification (LAMP) technique requiring only an ordinary water bath or thermoblock.PLRV RNA was extracted from overall 80 infected naturally potato leaves. A set of six novel primers for the LAMP reaction was designed according to the highly conserved sequence of the viral coat protein (CP) gene. LAMP was carried out under isothermal conditions, applying the Bst DNA polymerase enzyme; the LAMP products were detected visually using the GeneFinder™ florescence dye.A positive result using the GeneFinder™ dye was a color change from the original orange to green. Results confirmed LAMP with GeneFinder™ provides a rapid and safe assay for detection of PLRV. Since with other molecular methods, equipping laboratories with a thermocycler or expensive detector systems is unavoidable, this assay was found to be a simple, cost-effective molecular method that has the potential to replace other diagnostic methods in primary laboratories without the need for expensive equipment or specialized techniques. It can also be considered as a reliable alternative viral detection system in further investigations.

Rapid detection of tobacco viruses by reverse transcription loop-mediated isothermal amplification

Tobacco viruses may cause a wide range of diseases that heavily reduce tobacco quality and yield worldwide. In order to detect viral diseases in tobacco fields, a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was established. Nucleotide amplification could be observed clearly after adding SYBR Green I, within 60 min under isothermal conditions, at 63-65 °C with a set of primers targeting the viral coat protein (CP) genes of tobacco viruses including cucumber mosaic virus (CMV), potato virus Y (PVY), tobacco etch virus (TEV), tobacco mosaic virus (TMV) and tobacco vein banding mosaic virus (TVBMV). This method has high specificity and sensitivity. The sensitivity of the RT-LAMP was 10 to 100 times higher than that of the conventional RT-PCR method. The RT-LAMP assay was proven reliable for virus diagnosis of tobacco samples from the field.

Multivalent Ligand Displayed on Plant Virus Induces Rapid Onset of Bone Differentiation

Viruses are monodispersed biomacromolecules with well-defined 3-D structures at the nanometer level. The relative ease to manipulate viral coat protein gene to display numerous functional groups affords an attractive feature for these nanomaterials, and the inability of plant viruses to infect mammalian hosts poses little or no cytotoxic concerns. As such, these nanosized molecular tools serve as powerful templates for many pharmacological applications ranging as multifunctional theranostic agents with tissue targeting motifs and imaging agents, potent vaccine scaffolds to induce cellular immunity and for probing cellular functions as synthetic biomaterials. The results herein show that combination of serum-free, chemically defined media with genetically modified plant virus induces rapid onset of key bone differentiation markers for bone marrow derived mesenchymal stem cells within two days. The xeno-free culture is often a key step toward development of ex vivo implants, and the early onset of osteocalcin, BMP-2 and calcium sequestration are some of the key molecular markers in the progression toward bone formation. The results herein will provide some key insights to engineering functional materials for rapid bone repair.

Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation

Wei, C. B., Wu, Q. Y., Yang, Y. and Liu, C. Y. 2012. Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation. Can. J. Plant Sci. 92: 495–500. Fritillaria anhuiensis is a valued traditional Chinese medicinal plant. Thunberg fritillary mosaic virus (TFMV) was found to cause mosaic symptoms. Virus particles of the Lu'an isolate were purified and its RNA genome was extracted. cDNA was synthesized from the RNA and specific PCR primers were used to amplify the virus coat protein (CP) gene. It shared 96.8% nucleotide identity and 97.6% amino acid identity with the sequences of CPs from the other TFMV isolates. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys S strain. The expressed protein was first purified by 12% (wt/vol) SDS-PAGE and subsequently by 5–20% (wt/vol) gradient SDS-PAGE. Antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis suggesting that it may be useful for detecting the virus.

Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation

Wei, C. B., Wu, Q. Y., Yang, Y. and Liu, C. Y. 2012. Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation. Can. J. Plant Sci. 92: 495–500. Fritillaria anhuiensis is a valued traditional Chinese medicinal plant. Thunberg fritillary mosaic virus (TFMV) was found to cause mosaic symptoms. Virus particles of the Lu'an isolate were purified and its RNA genome was extracted. cDNA was synthesized from the RNA and specific PCR primers were used to amplify the virus coat protein (CP) gene. It shared 96.8% nucleotide identity and 97.6% amino acid identity with the sequences of CPs from the other TFMV isolates. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys S strain. The expressed protein was first purified by 12% (wt/vol) SDS-PAGE and subsequently by 5–20% (wt/vol) gradient SDS-PAGE. Antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis suggesting that it may be useful for detecting the...