Methods: The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones.
Results: Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing.
Conclusion: NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.
OBJECTIVES: The plant virus Cowpea Mosaic Virus (CPMV) has been innovatively used as a nanoscaffold. Utilization of the internal cavity of empty Virus-Like Particles (VLPs) for the inclusion of therapeutics within the capsid has opened many opportunities in drug delivery and imaging applications.
METHODS: The encapsidation of magnetic materials and anticancer drugs was achieved. SuperscriptCPMV denotes molecules attached to the external surface of CPMV and CPMVSubscript denotes molecules within the interior of the capsid.
RESULTS: Here, the generation of novel VLPs incorporating iron-platinum nanoparticles TCPMVFePt and cisplatin (Cis) (TCPMVCis) is reported. TCPMVCis exhibited a cytotoxic IC50 of TCPMVCis on both A549 and MDA-MB-231 cell lines of 1.8 μM and 3.9 μM, respectively after 72 hours of incubation. The TCPMVFePt were prepared as potential MRI contrast agents.
CONCLUSION: Cisplatin loaded VLP (TCPMVCis) is shown to enhance cisplatin cytotoxicity in cancer cell lines with its potency increased by 2.3-folds.
Materials and Methods: A batch of newly hatched hybrid grouper fry (Epinephelus fuscoguttatus × Epinephelus lanceolatus) were followed from the larval stage to market size. Samples of the hybrid groupers, water, live feed, and artificial fish pellets were collected periodically from day 0 to 180 in the hybrid grouper hatchery. Reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR amplifications were carried out on VNN-related sequences. The phylogenetic tree including the sampled causative agent of VNN was inferred from the coat protein genes from all known Betanodavirus species using Molecular Evolutionary Genetics Analysis (MEGA). Pearson's correlation coefficient values were calculated to determine the strength of the correlation between the presence of VNN in hybrid grouper samples and its associated risk factors.
Results: A total of 113 out of 146 pooled and individual samples, including hybrid grouper, water, and artificial fish pellet samples, demonstrated positive results in tests for the presence of VNN-associated viruses. The clinical signs of infection observed in the samples included darkened skin, deformation of the backbone, abdominal distension, skin lesions, and fin erosion. VNN was present throughout the life stages of the hybrid groupers, with the first detection occurring at day 10. VNN-associated risk factors included water temperature, dissolved oxygen content, salinity, ammonia level, fish size (adults more at risk than younger stages), and life stage (age). Detection of VNN-associated viruses in water samples demonstrated evidence of horizontal transmission of the disease. All the nucleotide sequences found in this study had high nucleotide identities of 88% to 100% to each other, striped jack nervous necrosis virus (SJNNV), and the reassortant strain red-spotted grouper NNV/SJNNV (RGNNV/SJNNV) isolate 430.2004 (GenBank accession number JN189932.1) (n=26). The phylogenetic analysis showed that quasispecies was present in each VNN-causing virus-positive sample, which differed based on the type of sample and life stage.
Conclusion: This study was the first to confirm the existence of a reassortant strain (RGNNV/SJNNV) in hybrid groupers from Malaysia and Southeast Asia. However, the association between the mode of transmission and the risk factors of this virus needs to be investigated further to understand the evolution and potential new host species of the reassortant strain.
METHODS: A total of 1917 samples with adequate volume for RT-PCR analysis were collected from patients hospitalised with HFMD throughout Vietnam and 637 were positive for EV71. VP1 gene (n=87) and complete genome (n=9) sequencing was performed. Maximum-likelihood phylogenetic analysis was performed to characterise the B5, C4 and C5 strains detected.
RESULTS: Sequence analyses revealed that the dominant subgenogroup associated with the 2012 outbreak was C4, with B5 and C5 strains representing a small proportion of these cases.
CONCLUSIONS: Numerous countries in the region including Malaysia, Taiwan and China have a large influence on strain diversity in Vietnam and understanding the transmission of EV71 throughout Southeast Asia is vital to inform preventative public health measures and vaccine development efforts.