| Abstract |
In Southeast Asia, the intensification of aquaculture industry has led to
the occurrence of various infectious diseases. Virus diseases in
particular, affecting especially the larval and juvenile stages, are the
most suppressing factors for sustainable production of high value fish
species such as seabass (Lates ca/carifer). Inevitable, because of lack of
sensitive methods that could be used for diagnosis and the inadequacy of
pathogen control, the aquaculture industry will continue to be plagued by
these diseases. Currently, viral nervous necrosis (VNN) is one of fish
diseases listed by the Office International des Epizooties (OIE) as a
notifiable disease in the production of marine fish worldwide. Thus the
main objective of the present study was to determine the role of VNN virus
(VNNv) as a pathogen in the Malaysian aquaculture industry, with emphasis
on virus isolation in susceptible cell line, virus identification using
polymerase chain reaction (PCR) and identification of characteristic
microscopy lesions in VNNv infections. A new cell line designated as Asian
Seabass Brain (ASBB-1) was derived from the brain tissue of seabass
cultured in Malaysia. This cell line was maintained in Leibovitz L-15
media supplemented with 10% fetal bovine serum (FBS). The ASBB-1 cell line
was sub-cultured more than 60 times over a period of 15 months. This cell
line consists predominantly of fibroblastic-like cells, which are able to
grow at temperatures between 20C and 30C with an optimum temperature of
25 Celsius. At 25C, the growth rate of these cells increased as the
proportion of FBS increased from 5 to 20% with optimum growth at 15% or
10% FBS. The ASBB-1 cell line was characterized by karyotyping, and
chromosome number distributions were different with subcultures P20 and
P50 producing 48 and 46 chromosome peaks respectively. The ASBB-1 cells
showed 90% viability after recovery from one year storage in liquid
nitrogen. The ASBB-1 cells up on characterization by peR using primer sets
of microsatellite markers of Asian seabass (Lates calcarifer) were shown
to be similar to the cells from tissues of seabass at 250 bp. The VNNv
isolate derived from seabass was tested positive for VNN by the 102000 Kit.
The isolate designated UPM08-1 M was used in the cell line infectivity
study. The susceptibilities of ASBB-1 cell line isolate and the highly
permissive commercial SSN-1 cells to UPM08-1 M were compared. The results
showed that ASBB-1 cell line was susceptible to VNNv (RGNNV genotype) with
typical cytopathic effect (CPE) manifesting mainly as rounding-up at1 day
post infection (dpi), severe vacuolation within 3-5dpi and complete
detachment within 7dpi. The VNNv-induced CPE was further elucidated by
electron microscopy (EM). Under EM, the ASBB-1cells exhibited vacuolated
degeneration with presence of viral inclusion-like bodies. The CPE of VNNv
on ASBB-1cells was producedat a virus titer of 109.5 TCID50/ml. This
indicated that the ASBB-1 cell line is highly susceptible for use in the
isolation of VNNv. Biophysical and biochemical characterization of VNNv
isolate was determined by heat treatment, UV irradiation and the stability
under effect of chemical disinfectants. The VNNv isolate showed
susceptibility to heat treatment at 60 Celsius within 30 minutes with no
viable virus after 1 hour. UV irradiation at an intensity of 440 mWrcm2
resulted in a reduction in virus titer after 8 minutes. The virus appeared
relatively resistant to changes in pH ranging between 2 to 11 after 1 hour
incubation at 25 Celsius. Treatment with 2% formalin was not totally
effective even after 6 hours. Iodine did not inactivate the virus.
Molecular characterization of the tissue culture-propagated virus using
RT-PCR and nested PCR, showed positive amplifications of genome size of 460
bp and 220 bp respectively. The 220 bp nucleotides from the T4 region of
the coat protein gene was sequenced and the phylogenetic analysis results
showed close resemblance between the UPM08-1 M strain and SBNNV and GPNNV2
strains of Malaysian isolates. The local isolate of UPM08-1 M also showed
phylogenetic similarity with other strains from South East Asia, while
showed relatively distant similarity with other VNNv strains from the
Middle East. Cold water VNNv strains isolated from Norway, Canada and USA
were distant phylogenetic relations to the UPM08-1 M strain. Experimental
infectivity study was also performed using fresh water guppy fish
(Poecilia reticulata) as a model. Guppies were intranasally infected with
0.1 ml of the UPM08-1M strain at a titre of 109.5 TCID5010.1ml. Although
the artificial infection resulted in slight mortality (11%) occurring
within 14 days post inoculation, the VNN-specific lesions such as necrosis
and vacuolation in the target organs of brain and retina were evident. The
presence of virus in infected brain and retina tissues was confirmed by
transmission electron microscopy (TEM). The VNNv from experimentally
infected guppy was successfully reisolated in ASBB-1 cells inoculated with
homogenate of brain and retinas of the survivors. There was gradual
increment of virus titer in the guppy throughout the experimental period.
The results indicated that guppy fish could be infected with VNNv isolated
from diseased seabass. In summary, the study successfully isolated VNNv
using the ASBB-1 cell line. Complete CPE of this cell line was observed
within 5 -7 dpi after infection with VNNv at producing considerable higher
titer of 1095TCID5010.1ml. Thus ASBB-1 cells can be recommended for VNNv
(RGNNV genotype) isolation and will be an important tool for future
conduct of fish health assurance programs in the aquaculture industry. The
study also showed that RT-PCR followed by nested-PCR is a sensitive
technique for identification of VNNv isolated from sea bass. It was also
shown that guppy fish could be experimentally infected with VNNv, thus can
be used as a virus-infection model.
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