Viral hepatitis are classified as A, B, C, D, and E type on the basis of antigenic specificity of etiologic viruses. Although several serological diagnostic methods have been applied, there are still many cases in which the etiologic viruses
could
not
be defined. Therefore many workers have strongly suggested that there are new hepatitis viruses other than described above. In this study to define a new hepatitis virus, to extract some viral nucleic acids from serum obtained from a patient with
chronic persistent hepatitis was used. Although etiological virus was not detected from this serum by conventional serological methods, the patient was diagnosed as chronic hepatitis by liver biopsy. After packaging the nucleic acid extraction
from
serum into Lambda gt11(λgt11), DNA and cDNA libraries were into E. coli and clones contained exogenous sequences were selected by immunoblotting methods. Exogenous proteins which were reacted with patient's serum were detected from selected gt11
plaques. After 3rd immunoscreening, 49 recombinants were selected. And human and E. coli genes were excluded by dot blot hybridization using probes of human and E. coli genomic DNA. Among them, 9 clones were excluded. Selected recombinants were
confirmed by Southern hybridization. Finally, three clones were selected. Two of them were derived from cDNA libraries and one was originated from DNA libraries. DNAs of three clones were amplified by PCR, Ec RI digested insertion sequences were
electrophoresed and eluted. Three. clones were subcloned into TA(pCR) vector and sequences of three clones were analysed. Clone 1 was composed 189 base pairs and clone 2, and clone 3 were 258, and 161 base pairs, respectively. The sequences was
compared
with Gene Bank Software. But all of three clones didn't match with them. Therefore, genomic PCR were performed. Primers were designed from sequences of each clones. Templates of genomic PCR were human, E. coli, and yeast genomic DNA. Clone 1 and
3
matched with human genomic DNA were excluded.
For final confirmation, serum PCR for clone 2 were performed using a primer designed from the sequence of clone 2 and a template obtained from cloning source. PCR products having the same size with insertion of clone 2 were found in serum PCR.
The
internal oligonucleotide of clone 2 sequence were used as the probe for Southern hybridization. Internal oligonucleotides probe reacted with product of serum PCR for clone 2.
In conclusion, exogenous DNA was found from the serum of patient with chronic viral hepatitis.
|