Molecular and Biological Characterization of Two Very Virulent Infectious Bursal Disease Virus Isolates, Upm94/273 and Upm97/61

An atypical very virulent (w) strain (UPM94/273) and typical vv strain (UPM97/61) of infectious bursal disease virus (IBOV) isolated in Malaysia, were characterized both in vivo and at the molecular level. Comparison of the deduced amino acid sequences with other serotype 1 and 2 sequences revealed 16 amino acid residues, which were conserved only in the vvIBDV. Among the 16 unique amino acid differences, 8 were in VP1 (146 Asp, 147 Asn, 242 Glu, 390 Met, 393 Asp, 562 Pro, 687 Pro and 695 Arg), 3 were in VP2 (222 Ala, 256 lie and 294 lie), 2 were in VP3 (990 Val and 1005 Ala) and 3 were in VP4 (685 Asn, 715 Ser and 751 Asp). The importance of these unique amino acid residues is not known but they could affect the virulence of vvIBOV. The UPM94/273 also demonstrated 6 unique amino acid residues at segment A at positions Ser254, Glu270, Lys588, Ser745, Phe838 and Lys863 and 8 unique amino acid residues at segment B at positions Ala92, Ser100, Va1208, Asp253, Asp560, Asn565, Gly750 and Gly876. In addition, these amino acid substitutions have not been reported before in vvlBDV and were found only on variant, classical and/or serotype 2 strains. However, the VP5 region of both vvlBDV strains was conserved. The UPM97/6 1 demonstrated 7 unique amino acid substitutions at segment A and 4 unique amino acid substitutions at segment B. However, none of the amino acids changes have been reported elsewhere in other IBDV strains. Although the actual functions of the amino acid substitutions are not know, the unusual amino acid substitutions at segment A and/or B of both isolates may be important in virus virulence. Alignments of the nucleic acid and amino acid sequences of segment A and B followed by distance analysis allowed the generation of phylogenetic trees. Phylogenetic analysis based on segment A and B revealed that all the vvlBDV strains including U PM94/273 isolate can be clustered in a group that is distinct from classical, variant, attenuated and serotype 2 strains. However, the tree branching patterns were quite different between segment A and segment B. In addition, the vvlBDV strains showed several conserved amino acid substitutions at segment B as found in the Australian 002-73 and serotype 2 strains. These findings indicate that probably a genetic reassortment may have play an important role in the emergence of vvIBDV. Flow cytometry and real time peR assays, indicated that chickens infected with UPM97/61 induced higher percentages of apoptotic cells but lower level of viral load whereas UPM94/273 induced lower percentages of apoptotic cells but higher level of viral load, suggesting a negative correlation between viral load and apoptosis. These results indicated that U PM97/61 was more virulent than UPM94/273

Development of Real-Time Polymerase Chain Reaction Assays for the Detection and Differentiation of Infectious Bursal Disease Virus Subtypes

Two different real-time polymerase chain reaction (PCR) detection approaches based on SYBR Green I dye and Taqman probe based assays were developed for the detection and differentiation of infectious bursal disease virus (IBDV) subtypes. Both approaches were able to detect and differentiate IBDV subtypes based on the use of subtype-specific primers or subtype-specific probes where the primers were designed based on single nucleotide polymorphism (SNP) concept. After optimization of the primer combinations and PCR parameters, very virulent-specific primer, IF & IVIR, and classical-specific primer, IF & RCLA were used in the SYBR Green I real-time RT-PCR assay. Plasmid DNA carrying the VP4 gene of the references IBDV strains: very virulent strain UPM94/273 and classical strain D78 were established and used as positive controls in the real time RT-PCR. The developed assay had a dynamic detection limit which spans over 5 log10 concentration range for very virulent and spans over 7 log10 concentration range for classical strain, respectively. The correlation coefficient for amplification of very virulent and classical strain was R2 = 0.9918 and R2 = 0.9977, respectively. No amplification was found when the subtype-specific primers were used to amplify other avian RNA viruses. The performance of the SYBR Green I based assay was tested on various IBDV isolates including 10 previously characterized IBDV and 11 commercial vaccine strains. The very virulent-specific primer only detected and amplified the very virulent IBDV with threshold cycle (CT) ranged from 14.93 to 21.52 and melting temperature (Tm) between 85.6°C to 88.0°C. The classical-specific primer was only able to amplify the classical IBDV with CT value ranged from 11.99 to 20.89 and Tm between 85.6°C to 86.8°C. The diagnostic efficacy of the developed assay was also evaluated using bursal samples obtained from experimentally infected chickens. Bursal samples collected from D78 vaccine infected chickens at day 3 and 5 p.i were positive for IBDV with average CT of 23.05+1.31, Tm of 85.8+0.17°C and average CT 21.82+1.42, Tm of 86.0+0.28°C , respectively. Bursal samples collected at day 10 p.i from this group were also found positive for IBDV with average CT of 24.42+1.20 and Tm of 85.9+0.18°C. On the other hand, only bursal samples collected at day 3 and 5 p.i were found positive for yery virulent IBDV with average CT 19.39+0.72, Tm of 86.6+0.14°C and average CT 23.55+1.39, Tm of 86.5+0.19°C, respectively. In the case of samples from dual infection with different IBDV subtypes, viral RNA was detectable only on day 3 and 5 p.i. In general, majority of the bursal samples have higher very virulent virus with an average CT value ranged from 21.24+0.68 to 22.19+0.97 compared to vaccine virus with Ct value ranged from 23.88+0.74 to 25.36+1.19. The performance of the developed SYBR Green I based assay was analyzed with other standard diagnostic methods. In the uninfected control group, no obvious microscopic lesions were found in the bursa and the lesions score was less than 1.0. However, mild bursal lesions without signs of inflammation with lesions score less than 3.0 was detected from bursal tissue obtained from chickens inoculated with vaccine strain D78. Based on the lesion score, it was clear that bursal pathology developed rapidly, with complete loss of tissue architecture by day 3 p.i. when the chickens were infected with virulent IBDV. The correlation between ELISA antibody titers and real-time CT values were inversely related, where the lower titers of antibodies associated with higher level of viral RNA as found in chickens infected with very virulent strain UPM94/273. On the other hand, vaccine strain D78 induced higher detectable antibody titers than UPM94/273, which indirectly support less virus replication with late positive amplification in real-time RT-PCR. Thus, the level of viral RNA in bursal samples obtained from D78 infected chickens was lower than UPM94/273 infected chickens. A total of 37 bursal samples from IBD suspected field cases were collected and then tested on the developed assay. The developed SYBR Green I based PCR assay was able to detect 9 samples positive for very virulent, 4 positive for classical IBDV and 12 samples positive for both very virulent and vaccine strains of IBDV. Sequence analysis of the hypervariable region of the VP2 gene of the IBDV samples revealed that the residues involved in determining the virulence of VV IBDV and CL IBDV were highly conserved. For the Taqman based duplex real-time PCR assay development, a new set of primers FWDC and RVSC were designed from the conserved region of VP4 of both very virulent and classical strains. A dual-labeled fluorescent probe each specific for very virulent IBDV (ProVV) and vaccine IBDV (ProCL) were designed The performance of the developed Taqman assay was compared with other PCR methods namely conventional RT-PCR and previously developed SYBR Green I assay. The Taqman assay was found far more superior in terms of turn around time and sensitivity. With the aid of β-actin gene, the Taqman assay was also used to determine the viral load fold changes in bursal samples that were positive for both vaccine and very virulent IBDV. Majority of these samples have higher viral load fold change in very virulent than the classical strain except for three samples MB078/04, MB001/05 and MB033/05 which showed higher fold change in classical strain than very virulent strain. In conclusion, this study has successfully developed SYBR Green I based and Taqman based one-step real-time PCR assays for rapid detection and differentiation of IBDV subtypes in particular very virulent and classical IBDV strains

Detection and Characterization of Cucumber mosaic virus Infecting Ginger (Zingiber officinale Roscoe) in Malaysia

Ginger (Zingiber officinale Rosc.) is herbaceous crop belonging to the family Zingiberaceae and cultivated for its medicinal properties and as marketable spice. A study was carried out to investigate Cucumber mosaic virus (CMV) infection in Malaysian ginger plants, showing conspicuous symptoms of mosaic, stripping and yellowing. A total of 45 symptomatic and 15 non symptomatic ginger samples were collected from three States in Malaysia. Reverse-transcription polymerase chain reaction (RT-PCR) analysis using CMV specific primers showed 14 out of 60 samples were positive for CMV. Cloning and sequence analyses of the 500 bp amplicons revealed that the CMV isolates obtained were closely related to CMV isolates from China (tomato) and Thailand (cucumber) with 95% to 96% sequence similarity respectively. Phylogenetic analysis placed the Malaysian CMV isolates into Subgroup IB.  This is the first report of CMV infecting ginger in Malaysi

Citrus Bent Leaf Viroid

Citrus bent leaf viroid (CBLVd) from genus Apscaviroid, is one of the widely distributed viroids among the seven citrus viroids. It is comprised of three variants: Citrus viroid-Ia (CVd-Ia) (327 - 329 nucleotides), Citrus viroid-Ib (CVd-Ib) (315 - 319 nucleotides) and Citrus viroid-I-low sequence similarity (CVd-I-LSS) (325 - 330 nucleotides). Virulence of CBLVd totally expressed on citrus plants. Etrog citron (Citrus medica (L.)) coinfected with CBLVd, Citrus exocortis viroid (CEVd), Citrus viroid-III (CVd-III) and Citrus viroid-V (CVd-V) showed epinasty, leaf rolling, and stunting. CBLVd has been reported to reduce the canopy proportion and fruit production of citrus trees inserted on trifoliate orange rootstock. Moreover, citrus tree infected with singly CBLVd or in combinations with CEVd, Hop stunt viroid (CVd-II) and CVd-III induced dwarfing have been associated with poor development of the root system. Reverse-transcriptase polymerase chain reaction (RT-PCR) amplification and multiplex reverse-transcriptase polymerase chain reaction (MRT-PCR) amplification have been widely used to detect citrus viroids including CBLVd. As citrus viroids are emerging threats in citrus groves, therefore, this review covers the evolution, geographical distribution and epidemiology, economic impact and symptomatology, host range and transmission, detection, and management will be helpful in formulating the integrated management strategies for CBLVd

Detection of Candidatus Phytoplasma Asteris’ (16srI) Associated with Bitter Gourd Leaf and Floral Malformations in Malaysia

Bitter gourd vines (Momordica charantia) exhibiting symptoms of leaf and floral malformations including reduced leaf and flower size and shortened internodes were observed in farmer’s fields in Selangor, Malaysia. The causal agent was detected by nested and semi nested Polymerase Chain Reaction (PCR) using phytoplasma universal primers based on 16SrRNA and SecA gene sequences. Sequence analysis of 1.2 kb and 480 bp amplicons of the 16SrRNA and SecA gene respectively confirmed the presence of phytoplasma DNA associated with Candidatus phytoplasma asteris (Group16SrI) in the symptomatic bitter gourd samples. Phylogenetic analysis of the 16SrDNA and SecA sequences placed the bitter gourd phytoplasma in the 16SrI phytoplasma group.  This is the first report of phytoplasma infection in bitter gourd in Malaysia

Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60. min at 60. °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd

Development of SYBR green I based one-step real-time RT-PCR assay for the detection and differentiation of very virulent and classical strains of infectious bursal disease virus

A SYBR Green I based one-step real-time reverse transcriptase polymerase chain reaction was developed for the detection and differentiation of very virulent (vv) and classical strains of infectious bursal disease virus (IBDV). The assay showed high PCR efficiency >93% and high reproducibility with coefficient of variation less than 0.5%.Whentested on characterized IBDVstrains, the very virulent and classical-specific primers detected accurately only vvIBDV and classical IBDV strains, respectively. The diagnostic efficacy of the assay was also tested on 140 bursal samples from experimental infection and 37 bursal samples from cases suspected of IBD. The assay was able to detect IBDV from bursal samples collected at days 3 and 5 post-infection with the vvIBDV strain UPM94/273 and the classical IBDV strain D78. The assay was also able to detect bursal samples infected dually with D78 and UPM94/273. The melting temperature values of the amplification products from the classical and very virulent viral infection were statistically significant (P < 0.05). The specificity of the assay for detecting IBDV from suspected cases was confirmed by sequence analysis of the VP2 gene. The assay showed high sensitivity since bursal samples whichwere negative for IBDV were confirmed by virus isolation and PCR amplification. Hence, the new assay offers an attractive method for rapid detection of strains of IBDV

'Candidatus Phytoplasma wodyetiae’, a new taxon associated with yellow decline disease of foxtail palm (Wodyetia bifurcata) in Malaysia

Landscape-grown foxtail palm (Wodyetia bifurcata A. K. Irvine) trees displaying symptoms of severe foliar chlorosis, stunting, general decline and mortality reminiscent of coconut yellow decline disease were observed in Bangi, Malaysia, during 2012. DNA samples from foliage tissues of 15 symptomatic palms were analysed by employing a nested PCR assay primed by phytoplasma universal ribosomal RNA operon primer pairs, P1/P7 followed by R16F2n/R2. The assay yielded amplicons of a single band of 1.25 kb from DNA samples of 11 symptomatic palms. Results from cloning and sequence analysis of the PCR-amplified 16S rRNA gene segments revealed that, in three palms, three mutually distinct phytoplasmas comprising strains related to 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma cynodontis', as well as a novel phytoplasma, were present as triple infections. The 16S rRNA gene sequence derived from the novel phytoplasma shared less than 96 % nucleotide sequence identity with that of each previously describedspecies of the provisional genus 'Ca. Phytoplasma', justifying its recognition as the reference strain of a new taxon, 'Candidatus Phytoplasma wodyetiae'. Virtual RFLP profiles of the R16F2n/R2 portion of the 16S rRNA gene and the pattern similarity coefficient value (0.74) supported the delineation of 'Ca. Phytoplasma wodyetiae' as the sole representative subgroup A member of a new phytoplasma ribosomal group, 16SrXXXVI

Constructions of expression vectors of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and transient expression of transgenes in immature oil palm embryos.

Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome