Full-length cDNA Clone Research Articles

Infectious clones of the crinivirus cucurbit chlorotic yellows virus are competent for plant systemic infection and vector transmission.

Cucurbit chlorotic yellows virus (CCYV), a recently identified bipartite crinivirus, causes economic losses in cucurbit plants. CCYV is naturally transmitted only by whitefly Bemisia tabaci. Here we constructed full-length cDNA clones of CCYV (RNA1 and RNA2) fused to the T7 RNA polymerase promoter and the cauliflower mosaic virus 35S promoter. CCYV replicated and accumulated efficiently in Cucumis sativus protoplasts transfected with in vitro transcripts. Without RNA2, RNA1 replicated efficiently in C. sativus protoplasts. Agroinoculation with the infectious cDNA clones of CCYV resulted in systemic infection in the host plants of C. sativus and Nicotiana benthamiana. Virus derived from the infectious clones could be transmitted between cucumber plants by vector whiteflies. This system will greatly enhance the reverse genetic studies of CCYV gene functions.

A novel dengue virus serotype 1 vaccine candidate based on Japanese encephalitis virus vaccine strain SA14-14-2 as the backbone.

To develop a potential dengue vaccine candidate, a full-length cDNA clone of a novel chimeric virus was constructed using recombinant DNA technology, with Japanese encephalitis virus (JEV) vaccine strain SA14-14-2 as the backbone, with its premembrane (prM) and envelope (E) genes substituted by their counterparts from dengue virus type 1 (DENV1). The chimeric virus (JEV/DENV1) was successfully recovered from primary hamster kidney (PHK) cells by transfection with the in vitro transcription products of JEV/DENV1 cDNA and was identified by complete genome sequencing and immunofluorescent staining. No neuroinvasiveness of this chimeric virus was observed in mice inoculated by the subcutaneous route (s.c.) or by the intraperitoneal route (i.p.), while some neurovirulence was displayed in mice that were inoculated directly by the intracerebral route (i.c.). The chimeric virus was able to stimulate high-titer production of antibodies against DENV1 and provided protection against lethal challenge with neuroadapted dengue virus in mice. These results suggest that the chimeric virus is a promising dengue vaccine candidate.

Identification of two genes encoding microsomal oleate desaturases (FAD2) from the biodiesel plant Pongamia pinnata L.

The current study dissect microsomal oleate desaturase genes having differential expression pattern with respect to temperature from the seeds of Pongamia pinnata, and are grouped with other legumes and biofuel plants. Biofuel often is available as plant oil or products derived thereafter, such as biodiesel. In view of the anticipated fossil fuel shortage, the biochemical and genetic basis of vegetable oil biosynthesis is vital. We are focusing on the versatile oil-yielding legume tree Pongamia pinnata. Microsomal oleate desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in non-photosynthetic tissues. This work reports on the isolation of two full length cDNA clones, tissue expression and copy number detection of Pongamia FAD2 genes, and also on the transcriptional regulation at low and high temperatures. The deduced amino acid sequences of both PpFAD2 proteins share 83 % identity and display three typical histidine boxes that are characteristics of all membrane-bound microsomal oleate desaturases. Both sequences possess aromatic amino acid containing sequence motifs at the C-terminal end necessary for maintaining endoplasmic reticulum (ER) localization. Southern blot analysis is consistent with the presence of at least two copies of FAD2 in the pongamia genome. Quantitative real-time PCR analysis showed that PpFAD2-1 is expressed strongly in developing seeds and, showing very low levels of expression in vegetative tissues, whereas PpFAD2-2 is constitutively expressed in both vegetative tissues and the developing seeds, with higher transcript levels in roots, stems and leaves. In response to low temperature stress PpFAD2-1 and PpFAD2-2 were differentially expressed in various tissues. The PpFAD2-2 transcript dramatically decreased in roots, stems and leaves under low temperatures, whereas PpFAD2-1 showed a significant increase in these tissues.

Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection.

The potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a Canadian Plum pox virus (PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. The trans expression of 6K1 or the cis expression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage. Potyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically processed into 11 mature proteins, with the majority of them having been at least partially functionally characterized. However, the functional role of a small protein named 6K1 remains obscure. In this study, we showed that deletion of 6K1 or a short motif/region of 6K1 in the full-length cDNA clones of plum pox virus abolishes viral replication and that mutation of the N- or C-terminal cleavage sites of 6K1 to prevent its release from the polyprotein greatly attenuates or completely inhibits viral replication, suggesting its important role in potyviral infection. We report that 6K1 forms punctate structures and targets the replication vesicles in PPV-infected plant leaf cells at the early infection stage. Our data reveal that 6K1 is an important viral protein of the potyviral replication complex.

Molecular and biological characterization of highly infectious transcripts from full-length cDNA clones of broad bean wilt virus 1

Broad bean wilt virus 1 (BBWV-1), genus Fabavirus, has a genome composed of two single-stranded positive-sense RNAs of ∼5.8 (RNA1) and 3.4kb (RNA2). Full-length cDNA clones of both genomic RNAs (pBenR1 and pBenR2) from BBWV-1 isolate Ben were constructed under the control of the T7 promoter. In vitro derived capped transcripts were infectious in Nicotiana benthamiana, Chenopodium quinoa and Vicia faba plants. The biological activity of viral transcripts was not affected by extra bases at the 5′-terminus introduced during in vitro transcription. Virions derived from the infectious cDNA clones displayed similar viral infectivity and accumulation, as well as symptom induction as the wild-type BBWV-1 isolate.

Establishment of a Simple and Rapid Gene Delivery System for Cucurbits by Using Engineered of Zucchini yellow mosaic virus.

The infectious full-length cDNA clone of zucchini yellow mosaic virus (ZYMV) isolate PA (pZYMV-PA), which was isolated from pumpkin, was constructed by utilizing viral transcription and processing signals to produce infectious in vivo transcripts. Simple rub-inoculation of plasmid DNAs of pZYMV-PA was successful to cause infection of zucchini plants (Cucurbita pepo L.). We further engineered this infectious cDNA clone of ZYMV as a viral vector for systemic expression of heterologous proteins in cucurbits. We successfully expressed two reporter genes including gfp and bar in zucchini plants by simple rub-inoculation of plasmid DNAs of the ZYMV-based expression constructs. Our method of the ZYMV-based viral vector in association with the simple rub-inoculation provides an easy and rapid approach for introduction and evaluation of heterologous genes in cucurbits.

Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A.

ABSTRACTPorcine epidemic diarrhea virus (PEDV) is a highly pathogenic alphacoronavirus. In the United States, highly virulent PEDV strains cause between 80 and 100% mortality in suckling piglets and are rapidly transmitted between animals and farms. To study the genetic factors that regulate pathogenesis and transmission, we developed a molecular clone of PEDV strain PC22A. The infectious-clone-derived PEDV (icPEDV) replicated as efficiently as the parental virus in cell culture and in pigs, resulting in lethal disease in vivo. Importantly, recombinant PEDV was rapidly transmitted to uninoculated pigs via indirect contact, demonstrating virulence and efficient transmission while replicating phenotypes seen in the wild-type virus. Using reverse genetics, we removed open reading frame 3 (ORF3) and replaced this region with a red fluorescent protein (RFP) gene to generate icPEDV-ΔORF3-RFP. icPEDV-ΔORF3-RFP replicated efficiently in vitro and in vivo, was efficiently transmitted among pigs, and produced lethal disease outcomes. However, the diarrheic scores in icPEDV-ΔORF3-RFP-infected pigs were lower than those in wild-type-virus- or icPEDV-infected pigs, and the virus formed smaller plaques than those of PC22A. Together, these data describe the development of a robust reverse-genetics platform for identifying genetic factors that regulate pathogenic outcomes and transmission efficiency in vivo, providing key infrastructural developments for developing and evaluating the efficacy of live attenuated vaccines and therapeutics in a clinical setting.

Molecular Cloning and Characterization of Full-Length cDNA of Calmodulin Gene from Pacific Oyster Crassostrea gigas.

The shell of the pearl oyster (Pinctada fucata) mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas) is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM) is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM) and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization.

Generation and Characterization of a Chimeric Tick-Borne Encephalitis Virus Attenuated Strain ChinTBEV.

Tick-borne encephalitis (TBE), caused by TBE virus (TBEV), is one of the most serious human viral diseases endemic in Europe and East Asia. No effective treatment for TBEV infection exists and the primary preventive measure is vaccination. Although several inactivated vaccines have been licensed, the development of novel and more effective vaccines remains a high priority especially in disease-endemic countries. Here we describe a universal vaccine design approach to construct a live chimeric recombinant TBEV attenuated strain ChinTBEV based on the infectious full-length cDNA clone of Japanese encephalitis virus using standard reverse genetic technology. The in vitro and in vivo characterization of the ChinTBEV is also presented here.

Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A

Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A

Development and characterization of a synthetic infectious cDNA clone of the virulent Bucyrus strain of equine arteritis virus expressing mCherry (red fluorescent protein).

Strains of equine arteritis virus (EAV) differ in their virulence phenotypes, causing anywhere from subclinical infections to severe disease in horses. Here, we describe the in silico design and de novo synthesis of a full-length infectious cDNA clone of the horse-adapted virulent Bucyrus strain (VBS) of EAV encoding mCherry along with in vitro characterization of the progeny virions (EAV sVBSmCherry) in terms of host-cell tropism, replicative capacity and stability of the mCherry coding sequences following sequential passage in cell culture. The relative stability of the mCherry sequence during sequential cell culture passage coupled with a comparable host-cell range phenotype (equine endothelial cells, CD3(+) T cells and CD14(+) monocytes) to parental EAV VBS suggest that EAV-sVBSmCherry-derived virus could become a valuable research tool for identification of host-cell tropism determinants and for characterization of the viral proteins involved in virus attachment and entry into different subpopulations of peripheral blood mononuclear cells. Furthermore, this study demonstrates that advances in nucleic acid synthesis technology permit synthesis of complex viral genomes with overlapping genes like those of arteriviruses, thereby circumventing the need for complicated molecular cloning techniques. In summary, de novo nucleic acid synthesis technology facilitates innovative viral vector design without the tedium and risks posed by more-conventional laboratory techniques.

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion® Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

Establishment of an Agrobacterium-mediated Inoculation System for Cucumber Green Mottle Mosaic Virus.

The infectious full-length cDNA clones of Cucumber green mottle mosaic virus (CGMMV) isolates KW and KOM, which were isolated from watermelon and oriental melon, respectively, were constructed under the control of the cauliflower mosaic virus 35S promoter. We successfully inoculated Nicotiana benthamiana with the cloned CGMMV isolates KW and KOM by Agrobacterium-mediated infiltration. Virulence and symptomatic characteristics of the cloned CGMMV isolates KW and KOM were tested on several indicator plants. No obvious differences between two cloned isolates in disease development were observed on the tested indicator plants. We also determined full genome sequences of the cloned CGMMV isolates KW and KOM. Sequence comparison revealed that only four amino acids (at positions 228, 699, 1212, and 1238 of the replicase protein region) differ between the cloned isolates KW and KOM. A previous study reported that the isolate KOM could not infect Chenopodium amaranticolor, but the cloned KOM induced chlorotic spots on the inoculated leaves. When compared with the previously reported sequence of the original KOM isolate, the cloned KOM contained one amino acid mutation (Ala to Thr) at position 228 of the replicase protein, suggesting that this mutation might be responsible for induction of chlorotic spots on the inoculated leaves of C. amaranticolor.

Expression of the Vacuolar Na+/H+ Antiporter Gene (NHX1) in Three Plantago Species Differing in Salt Tolerance

We have isolated full-length cDNA clones of the NHX1 gene - encoding the vacuolar Na + /H + antiporter - from three Plantago species ( P. crassifolia , P. coronopus and P. major ) differing in salt tolerance. A phylogenetic tree of their deduced protein sequences and those of NHX1 antiporters previously isolated from other species suggested the presence of some structural features characteristic of the proteins of salt tolerant taxa. The expression patterns of the NHX1 genes in the presence of salt correlated with the relative degree of salt tolerance of the three species: no induction was detected in the least tolerant P. major - and in the glycophyte A. thaliana - and the highest and quickest induction was observed in the most tolerant P. crassifolia . These data support the hypothesis that the NHX1 antiporters play a functional role in the mechanisms of salt tolerance in plants

First Report of Iresine viroid 1 in Ornamental Plants in Italy and of Celosia cristata as a Novel Natural Host

HomePlant DiseaseVol. 99, No. 11First Report of Iresine viroid 1 in Ornamental Plants in Italy and of Celosia cristata as a Novel Natural Host PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Iresine viroid 1 in Ornamental Plants in Italy and of Celosia cristata as a Novel Natural HostR. Sorrentino, M. Minutolo, D. Alioto, E.M. Torchetti, and F. Di SerioR. SorrentinoSearch for more papers by this author, M. MinutoloSearch for more papers by this author, D. AliotoSearch for more papers by this author, E.M. TorchettiSearch for more papers by this author, and F. Di SerioSearch for more papers by this authorAffiliationsAuthors and Affiliations R. Sorrentino M. Minutolo D. Alioto , Dipartimento di Agraria, Sezione di Biologia e Protezione dei Sistemi Agricoli e Forestali, Università degli Studi di Napoli Federico II, 80055 Portici, Italy E.M. Torchetti F. Di Serio , Istituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche, 70122 Bari, Italy. Published Online:2 Sep 2015https://doi.org/10.1094/PDIS-03-15-0361-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Iresine viroid 1 (IrVd-1) (Spieker 1996) is a member of the genus Pospiviroid. Occurrence of IrVd-1 has so far been reported in Canada, Germany, India, the Netherlands, and Slovenia. We have previously developed a polyprobe (POSPIprobe) that in hybridization assays efficiently detects eight pospiviroids and that was predicted to detect also IrVd-1 (Torchetti et al. 2012), although in the absence of an IrVd-1 isolate in our collection, we could not put to test this prediction. In a survey carried out in the spring of 2014 in the frame of the project URCOFI (Campania region, Southern Italy) a total of 60 ornamental species (Euphorbia spp., Begonia spp., Baucarmea recurvata, Portulaca spp., Celosia cristata, and C. plumosa) were tested by tissue printing hybridization (TPH) with the POSPIprobe as reported previously (Lolic et al. 2007). Most samples tested negative, except nine asymptomatic plants of the species Portulaca (3 out of 4 tested plants), C. cristata (1 out of 5 tested plants), and C. plumosa (5 out of 11 tested plants). Northern-blot hybridization assays, performed according to Hajizadeh et al. 2012 using the POSPIprobe, showed the presence, in the TPH-positive plants, of a circular RNA with a molecular size of about 370 nt and its corresponding linear form, confirming that those plants were infected by a pospiviroid. When the same RNA preparations were examined by RT-PCR using generic primers designed to detect most pospiviroids (Bostan et al. 2004), an amplicon of about 220 bp was generated, the sequence of which was highly similar to that of IrVd-1 (NC003613). Based on this information, we corroborated the presence of IrVd-1 in Portulaca spp, C. plumosa, and C. cristata by designing two sets of adjacent and complementary primers (Ir1F: 5′-GGAGCTCGTCTCCTTCCTTTC-3′ and Ir2R: 5′-TCCTGTTTCTTCCGCCGCG-3′) and (Ir5R: 5′-CCAGGTTTCCCCGGGGATC-3′ and Ir6F: 5′-GGAGCGAACTCGGCAAGGAGG-3′) specific for this viroid that were used, separately, for amplifying, cloning, and sequencing full-length cDNAs from one representative plant of the three different host species. Collectively, a total of 22 full-length cDNA clones were sequenced, which showed sequences almost identical to each other (97 to 100%) and to the reference IrVd-1 variant (96 to 97%). A total of five IrVd-1 new variants were deposited in GenBank (KR020037 to KR020041). No significant clustering according to the geographic origin or the host species was observed when a phylogenetic analysis was performed using all IrVd-1 sequence variants reported in this and previous studies. To our knowledge, this is the first report of IrVd-1 in Italy and of C. cristata as a natural host of this viroid. Additionally, this study confirms the ability of the POSPIprobe to detect IrVd-1, expanding to nine pospiviroids the range of this polyprobe, and supports the usefulness of TPH as a preliminary approach for viroid detection.

Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination

To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector.

In Vitro Transcripts of Wild-Type and Fluorescent Protein-Tagged Triticum mosaic virus (Family Potyviridae) are Biologically Active in Wheat.

Triticum mosaic virus (TriMV) (genus Poacevirus, family Potyviridae) is a recently described eriophyid mite-transmitted wheat virus. In vitro RNA transcripts generated from full-length cDNA clones of TriMV proved infectious on wheat. Wheat seedlings inoculated with in vitro transcripts elicited mosaic and mottling symptoms similar to the wild-type virus, and the progeny virus was efficiently transmitted by wheat curl mites, indicating that the cloned virus retained pathogenicity, movement, and wheat curl mite transmission characteristics. A series of TriMV-based expression vectors was constructed by engineering a green fluorescent protein (GFP) or red fluorescent protein (RFP) open reading frame with homologous NIa-Pro cleavage peptides between the P1 and HC-Pro cistrons. We found that GFP-tagged TriMV with seven or nine amino acid cleavage peptides efficiently processed GFP from HC-Pro. TriMV-GFP vectors were stable in wheat for more than 120 days and for six serial passages at 14-day intervals by mechanical inoculation and were transmitted by wheat curl mites similarly to the wild-type virus. Fluorescent protein-tagged TriMV was observed in wheat leaves, stems, and crowns. The availability of fluorescent protein-tagged TriMV will facilitate the examination of virus movement and distribution in cereal hosts and the mechanisms of cross protection and synergistic interactions between TriMV and Wheat streak mosaic virus.

Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction.

RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from a moderately virulent isolate. In addition to here being used as a model for RNA viruses generally, CSFV has high socioeconomic importance and remains a threat to animal welfare and pig production. We find that the majority of the investigated genomes are non-functional and only 12% produced infectious RNA transcripts. Full length sequencing of cDNA clones and deep sequencing of the parental population identified substitutions important for the observed phenotypes. The investigated cDNA clones were furthermore used as the basis for inferring the sequence of functional viruses. Since each unique clone must necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants.

Molecular cloning and nutritional regulation of putative ∆6 desaturase mRNA from striped catfish (Pangasianodon hypophthalmus)

Here we report the full-length cDNA cloning and nutritional regulation of a putative Δ6 fatty acyl desaturase (Δ6 fads) gene from striped catfish, Pangasianodon hypophthalmus. The 5′ and 3′ RACE analysis revealed a cDNA sequence of 1896 base pairs (bp) comprising a 5′ UTR of 49bp, 3′ UTR of 509bp and an open reading frame (ORF) of 1338bp specifying a protein of 445 amino acids. Bioinformatics analysis showed that the deduced peptide sequence included all the characteristic features of microsomal fatty acyl desaturases, including N-terminal cytochrome b5 domain containing the haem binding motif H-P-G-G, fatty acyl (FA) desaturase domain, three histidine boxes and transmembrane regions. P. hypophthalmus ∆6 desaturase peptide sequence shares high similarity with Pangasius larnaudii (95%) and Clarias macrocephalus (90%). The predicted secondary and tertiary protein structures revealed that desaturase protein contains mostly α-helix followed by random coils and strands. Tissue distribution analysis showed that Δ6 fads is mainly expressed in brain followed by liver, heart, gill and muscle. Since α-linolenic acid is a substrate for the Δ6 FADS enzyme, various concentrations of this fatty acid (0.5, 1, 1.5 and 2gkg−1) were administered to experimental fish through feed to find out its effect on the expression of Δ6 fads. Results showed that the liver Δ6 fads gene expression is up-regulated in fish fed with experimental diets (p<0.05). Statement of relevanceThe fatty acid composition of fish depends on both dietary fatty acid input and endogenous HUFA biosynthesis capacity. Fatty acyl desaturases (fads) introduce double bonds in selected positions of the acyl chains. Particularly, Δ6 FADS are the first enzymes involved in the biosynthesis of long-chain HUFA from the C18 n-3 and n-6 precursors, which is a rate limiting step. The extent to which fish can desaturate/elongate C18 FAs to HUFAs varies with species. Hence it is important to study the relationships between LC-PUFA biosynthesis pathways and the habitat and feeding habit of fish species, which will provide the basis for understanding the regulatory mechanisms controlling LC-PUFA biosynthesis pathways. The present study reports the full-length cDNA cloning, tissue distribution and nutritional regulation of Δ6 fads-like gene from freshwater catfish, P. hypophthalmus. This is the first report on cloning and expression analysis of Δ6 fads-like gene from any freshwater catfish.

A determinant of disease symptom severity is located in RNA2 of broad bean wilt virus 2

Broad bean wilt virus 2 (BBWV2), which belongs to the genus Fabavirus, is a destructive pathogen of many economically important horticultural and ornamental crops. In this study, we constructed infectious full-length cDNA clones of two distinct isolates of BBWV2 under control of the cauliflower mosaic virus 35S promoter. BBWV2-PAP1 isolated from paprika (Capsicum annuum var. gulosum) induces severe disease symptoms in various pepper varieties, whereas BBWV2-RP1 isolated from red pepper (Capsicum annuum L.) causes mild symptoms. Agrobacterium-mediated inoculation of the infectious cDNA clones of BBWV2-PAP1 and RP1 resulted in the same symptoms as the original virus isolates. The infectious cDNA clones of BBWV2-PAP1 and RP1 were used to examine the symptoms induced by pseudorecombinants between the two isolates to localize in which of the two genomic RNAs are the symptom severity determinants in BBWV2. The pseudorecombinant of RP1-RNA1 and PAP1-RNA2 induced severe symptoms, similar to those caused by the parental isolate PAP1, whereas the pseudorecombinant of PAP1-RNA1 and RP1-RNA2 induced mild symptoms, similar to those caused by the parental isolate RP1. Our results suggest that BBWV2 RNA2 contains a symptom determinant(s) capable of enhancing symptom severity.