The culture systems of Hantaan virus can be devided largely into two categories. That is, one is the use of cultured cell lines and the other is the use of competent animals. In the former case, A549 and Vero-E6 cell lines have been used widely
in
vitro
culture and isolation as well, and also in case of the latter the suckling mice were used frequently for brain inoculation.
It has been known that when Hantaan virus cultured in Vero-E6 cells was inoculated intracerebally into suckling mice, not only the fatality of the mice was increased but also the periods required for the paralysis were shortern gradually at some
degrees
during increasing number of the passage. In addition to theses facts, the ELISA titer of viral antigen in brain is increased gradually at 2-4 folds through the each passage steps upto 1 : 8,000 and a sudden increase at particular level is not
detected.
These facts have implied that certain genetic changes might occur in virus during the continous brain passage.
To investigate above observation at the level of nucleotide sequence, a part of G2 glycoprotein coding region(1970-2317) of viral cDNAs which are prepared from the virus of each passage were amplifed by RT-PCR and the viral cDNA fragments was
sequenced
using Sequenase Ver2.0. The result of sequencing showed that the percent heterology between amplifed regions of cell and brain passed virus was 3%. In amino acid level, 3 changes among 116 amino acid were detected. Effect of this change to
neutralizing
epitopes was checked by plaque reduction neutralization(PRN) test of two viruses to the same anti-Hantaan antibody and there was no differences in PRN titers between tow viruses. Considering all of the above results, though a small part of viral
genome
was compared, the changes of nucleotide sequence in G2 region suggest that nucleotide sequence variation of another region of genome can also be occurred and such variation might be able to evoke above phenomena such as shortern incubation pariod
and
increasing virus titer, although the sequence variation observed observed through brain passage is not great enough to alter the neutralizing epitopes.
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