Genome analysis and experimental evidence of horizontal transmission of Nervous Necrosis Virus (NNV) infecting hatchery stocks of marine fish

Benny Obrain Anak Joseph Manin, (2011) Genome analysis and experimental evidence of horizontal transmission of Nervous Necrosis Virus (NNV) infecting hatchery stocks of marine fish. Masters thesis, University Malaysia Sabah.

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Abstract

High stocking density in larviculture is important for successful production of marine fish species in hatchery. However, mass mortality due to nervous necrosis virus (NNV) often occurred in newly hatched fish larvae. This reduces the marine fish seed production in hatcheries throughout the country. Hence, this study was conducted to analyze the genomes of NNV and examine the possibility of horizontal transmission of the virus in fish larvae through viral-contaminated fish. In the first experiment, NNV was detected using RT-PCR and histopathological methods from twenty sampling sets of fish larvae collected from different hatcheries and aquaculture farms. The collected fish specimens represented the four most cultured marine fish species in the South East Asian region. The RT-PCR analysis revealed that 60.98% of the fish specimens were infected by NNV. The designed PCR primers in this study were suitably used for the purpose of detection of NNV in marine fish species. The histopathological study showed that cell vacuolation has been observed in brain and retina tissues of infected fish. In the second experiment, the complete coding sequence of both genomes (RdRp and Cp genes) in NNV was successfully PCR amplified and sequenced. The complete coding sequence of RdRp gene consisted of 3024 nucleotides and 982 amino acids. Meanwhile the complete coding sequence for Cp gene consisted of 1363 nucleotides and 338 amino acids. The analysis of nucleotide sequences revealed that both RdRp and Cp genes in NNV isolates in Malaysia had 94.5 - 99.7% and 95.9 - 99.8% similarity to RGNNV genotype, respectively. This shows that only RGNNV genotype of the virus exists in Malaysia. In the third experiment, amplified fragments of RdRp and Cp genes of NNV were restricted using six different restriction enzymes. The result showed that RdRp and Cp genes produced five and seven different RFLP-PCR profiles, respectively. The phylogenetic tree further showed that the RGNNV genotype in Malaysia could be clustered into five main clusters based on RdRp gene and seven main clusters based on Cp gene. In the last experiment, clinically healthy Asian seabass, Lates calcarifer and tiger grouper, Eplnephelus fuscoguttatus larvae were exposed to pure culture of GPNNV and tissue homogenates of infected fish. The transmission of NNV was evaluated using RT-PCR, histopathological, RFLP-PCR and DNA sequencing methods. The results showed that horizontal transmission of NNV has successfully occurred in the exposed fish groups except for groups D2 and E2, respectively. The exposed fish specimens showed cell vacuolations in brain and retina tissues similar to those observed in naturally infected fish. The cDNA of Cp gene in NNV isolated from the exposed fish showed 100% similarity in RFLP-PCR profiles and nucleotide sequence to the viral sources. Overall, this study has provided Scientific evidence that aquaculture industry in Malaysia is threatened by NNV infection. This requires holistic management approaches to be adopted in both hatcheries and aquaculture farms to prevent spreading of the pathogen thus ensuring the sustainability of sea food security in the country.

Item Type:Thesis (Masters)
Uncontrolled Keywords:nervous necrosis virus (NNV), hatchery, marine fish, RT-PCR, histopathological method, genome analysis, horizontal transmission
Subjects:Q Science > QL Zoology
Divisions:SCHOOL > Borneo Marine Research Institute
ID Code:8969
Deposited By:IR Admin
Deposited On:08 May 2014 15:43
Last Modified:08 May 2014 15:43

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