| Summary: | 碩士 === 國立中興大學 === 獸醫學系 === 85 === AbstractThe aims of this investigation are to study the entry
and distribution of aquatic birnavirus in tilapia (Tilapia
aureus) by the single-tube reverse-transcription polymerase
chain reaction (single tube RT-PCR) and virus isolation
techniques. The principle of the developed method for the
detection of aquatic birnavirus RNA in fish tissues is to
combine both steps in one reaction tube with a wax interface
which prevented the inhibitory effect of RTase on Taq
polymerase.It could shorten the time of RT-PCR to separate the
reverse transcription of viral RNA into cDNA and subsequent
amplification of polymerase chain reacitons. The results of the
specificity of the single-tube RT-PCR technique revealed that it
was sensitive enough to differentiate aquatic birnavirus from
infectious bursal disease virus (IBDV) belonged to the same
Birnaviridae and infectious heamotopoietic necrosis virus (IHNV)
caused a serve diseases in fish. The results of the sensitivity
showed that our method could detect the viral amount of the RNA
to 15 pg that is equal to about 200 viral particles.In order to
understand the entry and distribution of aquatic birnavirus in
fish, the Taiwan isolate, 3372, was used to infect tilapia in
vitro and in vivo then the viral titers in organs and tissues
from infected fish were studied. The results of in vitro
infection revealed that existences of aquatic birnavirus were
demonstrated in all collected organs and tissues including skin,
fin, gill, liver, kidney, spleen, and muscle and in the tissue
culture medium by the single-tube RT-PCR and virus isolation
techniques. The highest titers were found in skin and muscle but
gill had the lowest titer. There were no remarkable evolution in
all inoculated organs and tissues during the experiment days.
However, viral titers in the tissue culture medium significantly
increased within everyday after infection. The lowest titer
showed in fin but the highest titer were found in kidney, liver,
and muscle. Because the tissues had post-mortem change in the
4th day post infection, the virus isolation technique was not
suitable for virus detection. At this moment, our method showed
that it is efficient to detect the viral RNA from the post-
mortem changed tissues. The results of the in vivo infection
experiment suggested that viruses could be recovered in
intestine from infected fish by immersion. The viral titers
could persistently detected in the organs up to the 3rd day post
infection. Instead of infection by feeding, viruses could be
found in intestine only. At the same situation, viruses were
detected in most tissues from infected fish which were
inoculated viruses by intraperitoneal injection; it suggested
that viruses were spreaded rapidly by the circulatory system to
all organs. Viruses were able to present in fish even at the
35th day after injection. The recovered time of the longest and
the shortest were in intestine and in gill, respectedly.
Intestine , kidney and spleen had the highest titers but the
lowest titers was found in gill. After the 1st day post
infection, the viral titers in all collected organs increased
and proceeded up to the 7th day then they decreased.
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