| Summary: | 博士 === 國立中興大學 === 獸醫學系 === 88 === ABSTRACT
Liver tissues from animals that were suspected to have died of rabbit hemorrhagic disease (RHD) were used for isolation and characterization of the causative agent. Three strains of RHD virus were isolated as the supernatants of liver homogenate reacted positively in hemagglutination assays and were infective for rabbits after second passage in animals. Following extraction of liver homogenates from animals infected with each of three isolates, each virus strain was purified by CsCl density gradient ultracentrifugation for further characterization. In negative-stained preparations, the purified virions were icosahedral, measured approximately 40 nm in diameter, and nonenveloped. Morphologically, three isolates were similar. By immunoblotting, a protein with a molecular weight of 60,000 was identified as the major structural protein for each isolate. Furthermore, two sets of primer framed two different regions within RHD virus genome could amplify two fragments of the expected size, respectively, from each isolate, whereas, none were obtained from unifected control samples. The identity of the amplified products was further confirmed by using different restriction endonuclease digestion. Among three isolates of RHD virus, neither protein migration patterns of the virions nor cleavage patterns of the amplified product by restriction enzymes were found to differ. Amplified fragment with a length of 511 bp was detected in liver extracts of 4-month-old rabbits infected with each three strains of RHDV, whereas, none was obtained from mock-infected liver extracts. Furthermore, amplified fragment was also detected in tissue specimens prepared from liver, bile, spleen, blood, mesenteric lymph node, kidney, thymus, lung, muscle, and urine of rabbits experimentally infected with RHDV-R9, but not from fecal sample. Viral RNA extracted from purified virions of RHDV-R9 was used for testing the sensitivity of RT-PCR. The detection limit was 300 fg in ethidium bromide stained gel and could be further enhanced to 30 fg by hybridization after southern transfer. By using the RT-PCR test and sequencing, 99.9﹪homology was demonstrated in the capsid protein (VP60) of three isolates from Taiwan. Nucleotide sequence and phylogenetic analysis reveal these viruses a low rate （2.3-8.2）of change.
Adult and 4-to-5 week old rabbits were inoculated subcutaneously with rabbit hemorrhagic disease virus（RHDV）. Samples were prepared from various tissues at intervals postinoculation （PI）for the detection of viral RNA and antigens. Using a hemagglutination test (HAT), viral antigens were detected in the liver, bile, and spleen of the adult rabbit at and after 36 h PI. The reverse transcription-polymerase chain reaction (RT-PCR) showed that RHDV RNA was present in the liver, bile, and spleen as early as 18 h PI, whereas lung, kidney, thymus, mesenteric lymph node, and buffy coat were found to be positive after more than 26 h PI. In addition, viral RNA in urine and faeces showed a variable positivity at and after 36 h PI. In the young rabbit, RT-PCR showed that RHDV RNA was present as early as 1 day PI in the liver, bile, spleen, and buffy coat; whereas lung, kidney, thymus, mesenteric lymph node, and faeces were found to be positive at and after 2 days PI. Bile and spleen were the only samples in which viral RNA was detected throughout the length of the experiment. Virus was not reactivated in six recovered virus inoculated rabbits treated with dexamethasome or a classical swine fever virus vaccine. Using a hemagglutination inhibition test and an ELISA, antibody titers increased rapidly from one week PI onwards, peaked at approximately three weeks of age, and were maintained throughout the length of the experiment.
The possibility of the transmission of rabbit hemorrhagic disease virus (RHDV) to swine through lapinized hog cholera virus (HCV) vaccine exists. To investigate the infectivity of RHDV in swine, 16 four- to six-week old piglets were inoculated subcutaneously with RHDV and samples of liver, lung, spleen, kidney, bile, adrenal gland, tonsil, mesenteric lymph node, thymus, urine, buffy coat, and feces were collected from each of 2 animals on days 0, 1, 2, 3, 5, 7, 14, and 28 postinfection. Using reverse transcription-polymerase chain reaction, viral RNA was detected in most tissues by day 3 and was absent after day 5, except in lung and liver tissues in which viral RNA was detected up to day 14. Viral RNA was not detected in kidney, urine, feces or bile. Antibody responses, as detected by hemagglutination inhibition, were of low titer and short duration, and were similar in animals inoculated with viable RHD and in those given formalin-inactivated RHDV (n=2). Neither viral RNA nor antibody were detected in the negative control or in the uninfected, in-contact animals.
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