Virus Coat Protein Gene Research Articles

Watermelon transformation with Zucchini yellow mosaic virus coat protein gene and comparison with parental cultivar

The objective of this work was to transfer Zucchini yellow mosaic virus coat protein (ZYMV-CP) and neomycin phosphotransferase II (NPT II) genes to the watermelon 'Crimson Sweet'(CS) genome, and to compare the transgenic progenies T1 and T2 with the nontransformed parental cultivar for morphological, pomological, growth and yield characteristics. The ZYMV-CP gene was transferred by Agrobacterium tumefaciens. The presence of the gene in transgenic T0, T1 and T2 plants was determined by polymerase chain reaction, and the results were confirmed by Southern blot. Two experiments were performed, one in the winter-spring and the other in the summer-autumn. In both experiments, the hypocotyl length of transgenic seedlings was significantly higher than that of nontransgenic parental ones. In the second experiment, the differences between transgenic and nontransgenic individuals were significant concerning fruit rind thickness, flesh firmness, fruit peduncle length, size of pistil scar, and a* values for fruit stripe or flesh color. Transferring ZYMV-CP gene to CS genome affected only a few characteristics from the 80 evaluated ones. The changes in rind thickness, flesh firmness and flesh color a* values are favorable, while the increase in the size of pistil scar is undesirable. The transgenic watermelon line having ZYMV-CP gene and the parental cultivar CS are very similar.

Development of transgenic watermelon resistant to Cucumber mosaic virus and Watermelon mosaic virus by using a single chimeric transgene construct

Watermelon, an important fruit crop worldwide, is prone to attack by several viruses that often results in destructive yield loss. To develop a transgenic watermelon resistant to multiple virus infection, a single chimeric transgene comprising a silencer DNA from the partial N gene of Watermelon silver mottle virus (WSMoV) fused to the partial coat protein (CP) gene sequences of Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV) and Watermelon mosaic virus (WMV) was constructed and transformed into watermelon (cv. Feeling) via Agrobacterium-mediated transformation. Single or multiple transgene copies randomly inserted into various locations in the genome were confirmed by Southern blot analysis. Transgenic watermelon R(0) plants were individually challenged with CMV, CGMMV or WMV, or with a mixture of these three viruses for resistance evaluation. Two lines were identified to exhibit resistance to CMV, CGMMV, WMV individually, and a mixed inoculation of the three viruses. The R(1) progeny of the two resistant R(0) lines showed resistance to CMV and WMV, but not to CGMMV. Low level accumulation of transgene transcripts in resistant plants and small interfering (si) RNAs specific to CMV and WMV were readily detected in the resistant R(1) plants by northern blot analysis, indicating that the resistance was established via RNA-mediated post-transcriptional gene silencing (PTGS). Loss of the CGMMV CP-transgene fragment in R1 progeny might be the reason for the failure to resistant CGMMV infection, as shown by the absence of a hybridization signal and no detectable siRNA specific to CGMMV in Southern and northern blot analyses. In summary, this study demonstrated that fusion of different viral CP gene fragments in transgenic watermelon contributed to multiple virus resistance via PTGS. The construct and resistant watermelon lines developed in this study could be used in a watermelon breeding program for resistance to multiple viruses.

Do transgenic chili pepper plants producing viral coat protein affect the structure of a soil microbial community?

We used field trials to study the potential non-target effects of transgenic chili pepper on the composition of a rhizosphere microbial community. These plants expressed a viral coat protein (CP) gene that confers resistance to cucumber mosaic virus (CMV). For comparison, we included a non-transgenic parental cultivar (wild type) and another non-transgenic commercial variety. Soil samples were collected at the time of flowering (June) and twice during the fruit-ripening stage (August and September). The microbial community was characterized by analyzing levels of phospholipid fatty acids (PLFAs) as well as the terminal restriction fragment length polymorphism (T-RFLP) of bacterial 16S-rRNA and the fungal internal transcribed spacer (ITS) region. Although the amounts of PLFAs and their diversity indices did not differ among pepper lines, they did vary significantly over the growing season. We also used non-metric multidimensional scaling (NMDS) and permutational multivariate analysis of variance (PerMANOVA) to evaluate microbial assemblages from these plants. Overall, no differences were found among pepper lines. However, a significant difference was found between wild type and transgenic peppers, with regard to their bacterial composition based on the T-RFLP profile from HhaI digest. We conclude that the impact of CMV-resistant transgenic pepper on soil microbial assemblages is weak and minor, compared with the dominating natural variations associated with plant growth stages.

Molecular breeding of transgenic white clover (Trifolium repens L.) with field resistance to Alfalfa mosaic virus through the expression of its coat protein gene

Viral diseases, such as Alfalfa mosaic virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mitotic and meiotic stability of the transgene, were confirmed by phenotypic evaluation of the transgenic plants at two sites within Australia. The T(0) and T(1) generations of transgenic plants showed immunity to infection by AMV under glasshouse and field conditions, while the T(4) generation in an agronomically elite 'Grasslands Sustain' genetic background, showed a very high level of resistance to AMV in the field. An extensive biochemical study of the T(4) generation of transgenic plants, aiming to evaluate the level and composition of natural toxicants and key nutritional parameters, showed that the composition of the transgenic plants was within the range of variation seen in non-transgenic populations.

A high-throughput transformation system allows the regeneration of marker-free plum plants (Prunus domestica)

A high-throughput transformation system previously developed in our laboratory was used for the regeneration of transgenic plum plants without the use of antibiotic selection. The system was first tested with two experimental constructs, pGA482GGi and pCAMBIAgfp94(35S) that contain selective marker and reporter genes. Transformation was monitored by GUS detection, and estimated transformation efficiencies were 5.7% and 17.7% for pGA482GGi and pCAMBIAgfp94(35S), respectively. Subsequently, an intron-hairpin-RNA (ihpRNA) construct, carrying the Plum Pox Virus coat protein (ppv-cp) gene, without selectable or reporter marker genes was designed. Five transgenic lines were regenerated as confirmed by DNA blot analysis. We believe that this is the first report on the production of marker-free plants transformed with a potential agronomically important trait in a Prunus species.

Strategy for a generic resistance to geminiviruses infecting tomato and papaya through in silico siRNA search

Use of siRNA is a powerful methodology to particularly knockdown the targeted genes in a sequence specific manner. The potential of siRNA can be harnessed for silencing specific geminiviral genes in papaya and tomato plant hosts, thus making them resistant to the respective viruses. The challenge is in designing exogenous siRNA which can trigger silencing of viral genes irrespective of the genetic variability in different viral isolates and at the same time the selected siRNA does not target any plant gene (off target silencing). In this study, we have designed siRNA from the most conserved regions of viral coat protein (AV1) and replicase (AC1) genes retrieved from different isolates of geminiviruses infecting papaya (PLCV), and tomato (TLCV & TLCV, Northern India), so as to give a broad spectrum resistance and efficient silencing as it is highly homology-dependent strategy. Software siRNA finder (Ambion) was used on the selected conserved sequences in order to select only those putative siRNA oligonucleotides which fulfill all the basic criteria required as per the algorithm. Finally, a cross search using BLAST was performed to confirm that the designed siRNAs do not have any homology to plant genome sequences. The putative siRNA sequences thus designed to target essential genes of geminiviruses and introduced into the plants may facilitate developing papaya and tomato crops with generic resistance to geminiviruses.

RNA2 of TRV SYM breaks the rules for tobravirus genome structure

Currently, all of the RNA2 molecules described for all of the more than thirty sequenced isolates of the three tobraviruses, Tobacco rattle virus (TRV), Pea early-browning virus (PEBV) and Pepper ringspot virus (PepRSV), have the virus coat protein (CP) gene located in the 5′ proximal position. However, sequencing of the RNA2 of the SYM isolate of TRV revealed that this isolate has a unique genome structure in which the virus CP gene is located in the central region of RNA2 downstream of three completely novel open reading frames (ORFN1, ORFN2 and ORFN3). An infectious clone of SYM RNA2 was constructed and mutations were introduced separately into each of the novel genes to interrupt their translation. However, none of the mutations resulted in any noticeable change in the ability of TRV RNA1 or RNA2 to replicate and move systemically in the leaves or roots of infected plants. In addition, individual expression of the novel ORFs either from a Potato virus X (PVX) vector or from a binary plasmid in Agrobacterium tumefaciens did not reveal any potential function.

MOLECULAR IDENTIFICATION OF POTYVIRUSES INFECTING BULBOUS ORNAMENTALS BY THE ANALYSIS OF COAT PROTEIN (CP) SEQUENCES

Potyviruses (genus Potyvirus, family Potyviridae) are transmitted by aphids in a non-persistent manner and cause significant losses in many crops including bulbous ornamentals. Host range, symptoms, physical and biochemical properties of many potyviruses in bulbous ornamentals are reported, but, especially for viruses infecting ornamentals of minor economical importance, sequence data are still lacking. We used molecular techniques for the identification, characterization and detection of these viruses. Leaf material of several ornamental crops showing virus-like symptoms were tested in indirect ELISA, using monoclonal antibodies specific for potyviruses. Generic potyvirus primers were used in an RT-PCR to amplify the 3' terminal region of these viruses. The fragments encode the viral coat protein (CP) gene and comprise the 3'-untranslated region (3'-UTR). Nucleotide sequences of the obtained fragments were determined and compared with potyvirus sequences present in the NCBI database using the BLAST algorithm. We have characterized some potyviruses previously accepted by the International Committee on Taxonomy of Viruses (ICTV), including Freesia mosaic virus (FreMV), Gloriosa stripe mosaic virus (GSMV), Hippeastrum mosaic virus (HiMV), Hyacinth mosaic virus (HyaMV), Iris mild mosaic virus (IMMV) and Nerine yellow stripe virus (NeYSV). Additionally, the identities of other potyviruses infecting ornamentals such as Anemone, Galtonia, Muscari, Ornithogalum, Allium, Stenomesson and Veltheimia have been determined. These viruses, however, have not yet been reported by the ICTV. The virus-specific sequence information generated in this research project can subsequently be used to develop PCR-based detection methods.

OVERVIEW OF THE INVESTIGATIONS OF TRANSGENIC PLUMS IN ROMANIA

Transgenic plums of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were the subjects of three experiments undertaken in Romania. In the first experiment PPV-CP transgenic clones C2, C3, C4, C5, C6 and PT3 were evaluated for Sharka resistance under high natural infection pressure. Transgenic clone C5, subsequently named HoneySweet, showed high resistance to PPV. None of the C5 trees became naturally infected by aphids for more than ten years. The resistance of C5 was based on post-transcriptional gene silencing (PTGS). In the second experiment we assessed the effect of two heterologous viruses (Prunus necrotic ringspot virus and Prune dwarf virus) on the efficacy and stability of PTGS - mediated resistance to PPV displayed by the C5 plum. The engineered resistance to PPV in C5 transgenic plums was stable and was not suppressed by the presence of the assayed heterologous viruses. Some PPV-CP transgenic plum clones that are susceptible to PPV including C2, C3, C4 and PT3 display a constitutive transcription of PPV-CP sequence. In the third experiment we used these plants to assess the environmental safety issues related to potential hazards concerning the emergence of PPV variants. The serological and molecular variability of PPV detected in transgenic and in conventional plums revealed that the transgenic plums do not affect the diversity of indigenous PPV populations. We suggest that the safety and efficacy data developed in Romania and other European countries and in the United States of America over the last 15 years justifies an expansion of the field tests in Romania and in other countries that are experiencing the damages to plum production caused by PPV.

Sequence diversity and potential recombination events in the coat protein gene of Apple stem pitting virus

The variability of the Apple stem pitting virus (ASPV) coat protein (CP) gene was investigated. The CP gene of ten virus isolates from apple and pear trees was sequenced. Comparison of all sequenced virus isolates revealed high diversity of the CP gene (70.7–93.5% at the nucleotide level and 77.8–98.7% at the amino acid level). Additionally, one or two deletions in the N-terminal part of the coat protein gene of the studied virus isolates were identified. The ratio of nonsynonymous to synonymous polymorphic sites indicated that purifying selection has acted to eliminate deleterious mutations in coding sites. Moreover, the evidences for recombination in analyzed sequences were provided. It is likely that recombination, along with selection, enhances the speed of elimination of deleterious mutations in ASPV, following the mutational deterministic hypothesis of Kondrashov.

First Report of Tomato yellow leaf curl virus Infecting Cowpea in China

Tomato yellow leaf curl virus (TYLCV) is a devastating pathogen of tomato that causes significant yield losses in many tropical and subtropical regions (1). In China, this virus was first found in 2006 on tomato in Shanghai (2). In October 2008, chlorotic yellow leaves of cowpea (Vigna sinensis) were observed in Qingpu, Shanghai, China with 15 to 20% incidence in plants in high tunnels. Large populations of whiteflies were observed in association with the diseased cowpea. The disease agent was transmitted to cowpea (and tomato) by whiteflies, which resulted in chlorotic yellow leaves on cowpea (yellow leaf curl symptoms on tomato) that were identical to those observed in the field. On the basis of the suspected insect vector, symptomology, and severe epidemics of tomato yellow leaf curl disease (TYLCD) in Shanghai in recent years, Tomato yellow leaf curl virus was suspected as the causal agent. Total DNA was extracted from four symptomatic cowpea samples. PCR was performed with specific primers V416 (5'-CAAGGCACAAACAAGCGACG-3') and C1287 (5'-CTCAACTTCCGAATTTGGACGAC-3') to amplify a 872-bp DNA fragment of the viral coat protein (CP) gene and an amplicon of the expected size was obtained in all four samples but not from healthy leaf samples. The PCR products were sequenced and the sequences were identical among samples. Primers TYLCV-F (5'-CAGGAGGCAGCCAAGTATGAG-3') and TYLCV-R (5'-ACTAATGCCTGTTCYTTCATTCC-3') (Y = C or T/U) were designed on the basis of the sequence (Accession No. HM804856) and reported (Accession No. FM163463) CP gene to amplify the full-length viral DNA of cowpea isolate (CN:SH:Cowpea:08). The sequence was determined to be 2,781 nucleotides long (Accession No. GU434143). A comparison of the sequence with those in GenBank shows that the cowpea isolate has the highest nucleotide sequence identity (99%) with TYLCV isolate XH2 from tomato in Xinghua, Jiangsu, China (Accession No. GU111505). To our knowledge, this is the first report of TYLCV infecting cowpea in China and also the first report in the world.

The Barley stripe mosaic virus system used for virus-induced gene silencing in cereals differentially affects susceptibility to fungal pathogens in wheat

Barley stripe mosaic virus (BSMV) has emerged as a vector for virus-induced gene silencing (VIGS) in cereals, having been used to study a number of genes involved in resistance in both wheat and barley. However, the effects of the BSMV vector on plant physiology and disease resistance in plants remains unexplored. The BSMV inoculation control vector, BSMV:GFP was shown to cause severe viral symptoms in wheat, displaying chlorosis, leaf curling and growth inhibition typical of the symptoms seen in BSMV-infected barley. These viral symptoms were accompanied by induction of genes implicated in defense against pathogens, namely PR1, PR4, PR5, PR10 and PAL. Subsequent inoculation of BSMV:GFP-infected wheat with a wheat pathotype of Magnaporthe oryzae, the blast pathogen, resulted in decreased susceptibility. Penetration of epidermal cells and subsequent multiple cell colonization by M. oryzae was significantly reduced. This increased restriction of pathogen growth observed for BSMV:GFP infections with and without the viral coat protein gene. However, prior infection with BSMV:GFP had no effect on the development of a compatible isolate of Blumeria graminis f. sp. tritici, the causal agent of powdery mildew.

Construction of banana bunchy top nanovirus-DNA-3 encoding the coat protein gene and its introducing into banana plants cv. Williams

Banana ( Musa sp.) is considered as one of the most important fruit crops worldwide as well as in Egypt. The main goal of this study was to construct the open reading frame (ORF) of banana bunchy top nanovirus (BBTV)-DNA-3 that encodes the viral coat protein ( cp ) gene for banana transformation. The previously sequenced BBTV-G-DNA-3-ORF that cloned into plasmid pH1 was used as a template for PCR amplification using two specific primers containing Bam H1 site. A new plasmid called pRHA1 containing the amplified ORF under the control of maize polyubiquitin ( ubi ) promoter was created. The bar gene (herbicide-resistance gene as a selectable marker) cassette ( bar gene, Cauliflower mosaic caulimovirus (CaMV) 35S promoter and nos terminator) was released from plasmid pAB6 using Hind III-digestion and subcloned into the Hind III-digested plasmid pRHA1 to create the plasmid pRHA2 via the microprojectile bombardment transformation system. The plasmid pRHA2 was successfully introduced in the applied banana cultivar. Leaf painting test was conducted to confirm the expression of the bar gene in the putative transformed banana lines. The presence and expression of BBTV-G- cp gene were also detected using some molecular (polymerase chain reaction and dot blot using a cold DNA probe) and serological (ELISA and western blot) techniques, respectively, in the obtained transgenic banana lines.

Characterization of cucumber mosaic virus originating from cucurbits in Serbia

Cucumber mosaic virus (CMV) is considered one of the most economically important plant viruses and has a worldwide distribution and a very wide host range including plants from family Cucurbitaceae. In Serbia, on cucurbits CMV was detected in single and mixed infections with Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV). Viruses, including CMV, are constantly present in cucurbit crops, but their frequency changes by year and locality. Surveys and sample collections were conducted in cucurbit crops in the period from 2008 to 2009 at 15 localities in Vojvodina province, and sample testing was carried out using the DAS-ELISA method and commercially available antisera for six economically most important cucurbit viruses. In 2008, a total of 51 samples were collected from 13 cucurbit crops of oilseed pumpkin Olinka variety, squash, and bottle gourd and CMV was detected in a total of 55% of tested samples with symptoms of viral infection. The most common infectious type was mixed infection with ZYMV and WMV (35.3%), and then mixed infection with ZYMV (17.7%) and WMV (2%). A total of 599 symptomatic samples of oilseed pumpkin Olinka variety, zucchini squash varieties Beogradska and Tosca, squash, and winter squash were collected in 15 cucurbits crops in 2009. CMV was present in 4.4% of total collected samples, in single infections in 1.3%, and in mixed with WMV or ZYMV in 1.3%, and 1.8%. Five CMV isolates were obtained by mechanical inoculations of N. glutinosa and one of them was selected for further biological characterization. Test plants which were described to be hosts of CMV expressed symptoms characteristic for those caused by CMV after inoculations by isolate 115-08. CMV specific primers Au1u/Au2d were used to amplify an 850 bp fragment using RT-PCR method. Amplified fragment encodes the entire viral coat protein (CP) gene and partial 5? and 3? UTRs of two selected CMV isolates. Amplified fragments were sequenced and deposited in the NCBI, where they were assigned accession numbers, HM065510 (115-08) and HM065509 (151-08). The sequences of CMV isolates from Serbia shared the highest nucleotide and amino acid identity with isolates from subgroup IA, from 99.5 to 97.4% and 99.1 to 97.4%, and the lowest identities were with the subgroup II isolates from 66.9 to 64, 5%, from 75.8 to 74.1%.

Overview of the Investigations of Transgenic Plums in Romania

Transgenic plums of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were the subjects of three experiments undertaken in Romania. In the first experiment PPV-CP transgenic clones C2, C3, C4, C5, C6 and PT3 were evaluated for Sharka resistance under high natural infection pressure. Transgenic clone C5, subsequently named „HoneySweet‟, showed high resistance to PPV. None of the C5 trees became naturally infected by aphids for more than ten years. The resistance of C5 was based on post-transcriptional gene silencing (PTGS). In the second experiment we assessed the effect of two heterologous viruses (Prunus necrotic ringspot virus and Prune dwarf virus) on the efficacy and stability of PTGS - mediated resistance to PPV displayed by the C5 plum. The engineered resistance to PPV in C5 transgenic plums was stable and was not suppressed by the presence of the assayed heterologous viruses. Some PPV-CP transgenic plum clones that are susceptible to PPV including C2, C3, C4 and PT3 display a constitutive transcription of PPV-CP sequence. In the third experiment we used these plants to assess the environmental safety issues related to potential hazards concerning the emergence of PPV variants. The serological and molecular variability of PPV detected in transgenic and in conventional plums revealed that the transgenic plums do not affect the diversity of indigenous PPV populations. We suggest that the safety and efficacy data developed in Romania and other European countries and in the United States of America over the last 15 years justifies an expansion of the field tests in Romania and in other countries that are experiencing the damages to plum production caused by PPV.

Five phylogenetic groups identified in the coat protein gene of grapevine leafroll-associated virus 3 obtained from Portuguese grapevine varieties

The genetic variability and population structure of grapevine leafroll-associated virus 3 (GLRaV-3) variants were updated by examining the diversity within the viral coat protein (CP) gene among 174 isolates belonging to a collection of Vitis vinifera representing most of the Portuguese varieties. Phylogenetic analysis revealed the existence of five well-defined clusters. Three of these correspond to previously defined groups, another corresponds to variants from Chile for which only one sequence has been previously identified, and an additional new group includes only Portuguese variants. A typing tool based on asymmetric PCR-ELISA (APET) was developed within the frame of this population structure. This tool was used to assess the prevalence of each phylogenetic group among the infected grapevine varieties. Although most of the isolates harbour variants from groups 1 and 2, variants from the remaining three groups exist in a number of varieties, reinforcing the notion that they are genuine genomic variants and are not isolated, atypical cases.

Characterization of Pepper yellow leaf curl virus, a tentative new Polerovirus species causing a yellowing disease of pepper

Pepper (Capsicum annuum) is an important crop worldwide. In Israel, approximately 2,500 ha are grown all year round for the local and export markets. Herein, we report the identification of a viral pathogen causing a new devastating disease in pepper crops. The disease syndrome includes shortening of stem internodes, interveinal yellowing, and upward rolling of the leaf blade, accompanied by fruit discoloration and size reduction. Virus purification from infected plants yielded isometric particles, 25 nm in diameter. The causal agent of the disease was tentatively named Pepper yellow leaf curl virus (PYLCV). The virus cross-reacted in DAS ELISA with antisera against Cucurbit aphid-borne yellows virus and Potato leafroll virus, members of the Polerovirus genus. The partial nucleotide sequence obtained from the cloned viral coat protein and movement protein genes indicated 92% identity at the amino acid level with Tobacco vein distortion virus (TVDV), another member of the Polerovirus genus. However, the host range of PYLCV is significantly different from the host range described for TVDV. Based on our findings, the taxonomic status of PYLCV is discussed.

Mapping the subgenomic RNA promoter of the Citrus leaf blotch virus coat protein gene by Agrobacterium-mediated inoculation

Citrus leaf blotch virus has a single-stranded positive-sense genomic RNA (gRNA) of 8747 nt organized in three open reading frames (ORFs). The ORF1, encoding a polyprotein involved in replication, is translated directly from the gRNA, whereas ORFs encoding the movement (MP) and coat (CP) proteins are expressed via 3' coterminal subgenomic RNAs (sgRNAs). We characterized the minimal promoter region critical for the CP-sgRNA expression in infected cells by deletion analyses using Agrobacterium-mediated infection of Nicotiana benthamiana plants. The minimal CP-sgRNA promoter was mapped between nucleotides −67 and + 50 nt around the transcription start site. Surprisingly, larger deletions in the region between the CP-sgRNA transcription start site and the CP translation initiation codon resulted in increased CP-sgRNA accumulation, suggesting that this sequence could modulate the CP-sgRNA transcription. Site-specific mutational analysis of the transcription start site revealed that the + 1 guanylate and the + 2 adenylate are important for CP-sgRNA synthesis.

Mass mortality of hatchery-produced larvae of Asian seabass, Lates calcarifer (Bloch), associated with viral nervous necrosis in Sabah, Malaysia

Culture of Asian seabass, Lates calcarifer (Bloch) is a popular aquaculture activity in Malaysia. This fish is in high demand and fetches a good price in the local market. The seed for this fish is commercially produced by induced spawning in hatcheries. However, the seed supply is affected by frequent mass mortality of larvae aged between 15 and 60 dph. The clinical signs shown by the affected larvae include lethargy, loss of appetite, uncoordinated swimming, unusual spiral movement pattern and dark coloration. Histological examination of brain and eye of the affected specimens revealed extensive cell vacuolation in larvae aged 15–25 dph. Partial nucleotide sequence of the nervous necrosis virus coat protein gene of the affected larvae showed 94.0–96.1% homology to the nucleotide sequences of coat protein gene from nervous necrosis virus isolated from other countries in the Southeast Asia and Australia. This study provides scientific evidence based on molecular technique that many episodes of mass mortality in seabass larvae in Sabah is associated with the viral nervous necrosis. Because no effective treatment has been reported for this infection, stringent biosecurity measures must be adopted for exclusion of the pathogen from the culture system.

Detection of three major RNA viruses infecting sugarcane by multiplex reverse transcription–polymerase chain reaction (multiplex-RT-PCR)

A multiplex reverse transcription-polymerase chain reaction (multiplex-RT-PCR) was developed for the detection of Sugarcane mosaic virus (SCMV), Sugarcane streak mosaic virus (SCSMV) and Sugarcane yellow leaf virus (SCYLV), three of the major RNA viruses widely prevailing in the sugarcane-growing regions around the world. Primers designed from the coat protein genes of the respective viruses amplified fragments of ∼860 bp (SCMV), ∼690 bp (SCSMV) and ∼615 bp (SCYLV) in multiplex-RT-PCR. In this study, we could specifically detect the target viruses in diseasesuspected sugarcane cultivars. Of nine samples tested, four were found infected with all three targeted viruses, two had infections of SCMV and SCSMV and the remaining three samples had the viruses found separately. Furthermore, multiplex-RT-PCR was found to be equally efficient to uniplex-RT-PCR to amplify the target viruses from sugarcane. This is the first report on detecting three viruses together in a single PCR reaction in sugarcane and this will have immense application in sugarcane quarantine programs.