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Abstract
Various factors using cell lines can effect kinds and frequencies of infectious viruses obtained in the detection tests on various water samples. We tried to find out technical problems for the maximum virus isolations from water samples and characterize the virus isolates from waters in nature and in various purification stages. Fourteen viruses were isolated from 169 water samples by virus monitoring protocol for the information collection requirements rule, US EPA. The morphological changes caused by viruses and mycoplasma infections were compared with for increasing the specificity of tests employed. Cytopathic effects of slow growing viruses were found very similar with those by toxic effects in water samples and mycoplasma infections. Five of 6 stream water samples tested (83.33%) showed virus contaminations with the range of 1.03 to 5.75 MPNs/100 liter. Eight of 24 source water samples (33.35%) showed viral contaminations. One water sample of 24 water samples during precipitation stages was shown to include infectious viruses. It was confirmed that infectious viruses were significantly decreased by purification stages from streams. The titers (TCID50) of virus isolates were ranged as 10–6.8 ∼ 10–6.925/ml. The virus isolates were identified by immune fluorescent antibody (IFA) method using virus specific immune sera and serotyped using serotype specific reference sera. Of 14 virus isolates, 7 samples were identified as poliovirus and the other 7 were identified as coxsakie virus. Of 7 polioviruses, one was serotyped as type I, 3 viruses as type II and another 3 as type III. Conclusively, BGM cell lines must be free of mycoplasma for the strict examination of infectious viruses in water and highly sensitive for mainly enteroviruses. In addition, most of infectious viruses showing typical cytopathic effect from water samples were confirmed as coxsackie B and live attenuated vaccine strains of 3 polio types when BGM cells were used for virus isolations.
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Figure 1.
Cytopathic effects (CPE) shown by poliovirus type 1 isolate from a stream water. CPEs were usually appeared 3 or 4 days after the water samples were tried to inoculate to BGM cells for the first time. However, the CPEs were appeared more rapidly when the positive samples were passaged in BGM Cell line culture. The poliovirus showed typical CPE of round cells and separated from cell culture flask walls completely in few days.
Figure 2.
Decrease of virus isolation rates by water flow stages. One case of virus isolation from precipitation area is interpreted insignificant because of only one case in rainy season. The detailed numbers of isolates and water samples for virus isolations were described in the result section of the theses and table 2.
Figure 3.
Results of virus identifications tests on virus isolate cultures by direct immuno fluorescence antibody (IFA) test using polio antibody labelled with fluorescein isothiocyanate conjugate. (A) shows typical intra-nuclear positive antigen presence of blue green colour by polio specific reference immune serum, whereas (B) shows no particular colour reaction by IFA on BGM cell culture not inoculated by virus isolate.
Figure 4.
Cytopathic effect similar to morphological changes by mycoplasma infection on BGM cells and CPE by reovirus infection. CPE or morpholocal changes on BGM cells by mycoplasma and reovirus were appeared 6∼14 days after inoculation of samples into cell culture. (A) showed morphological changes by mycoplasma strains, wheras (B) showed CPE by reovirus reference adapted strain.
Figure 5.
Results of universal PCR on 6 cell culture supernatants. The samples showed bands of 1.6 kbp in positive reaction. Lanes (1) to (2)s are undiluted sample of morphologically normal cell supernatants. (3) and (4) are 10–1 and 10–2 diluted PCR results of morphologically normal BGM cells. (5) and (6) are undiluted samples of positive samples No 1 and 2 respectively. (7) and (8) are 10–1 and 10–2 diluted PCR results of positive samples No 1 and 2, respectively. (9) and (10) are undiluted of positive samples 3 and 4, respectively. (11) and (12) are 10–1 and 10–2 diluted PCR results of positive samples 3 and 4, (13) and (14) are 10–4 diluted PCR results of positive samples 3 and 4, respectively. (15) is positive control by M. pneumoniae, wheres (16) and (17) are negative controls.
Figure 6.
Culture and antigen tests on Mycoplasma isolates from BGM tissue culture supernatants: (A) shows the typical fried egg colony morphology of the strain identified as Mycoplasma fermentans. (B) shows the colour reactions of 6 cell culture supernatants by Mycoplasma specific ELISA tests. Negative 1 and 2 were culture supernatants morphological normal cells, whereas Positive 3, 4, 5 and 6 were morphologically abnormal cell samples.
Table 1.
Origins of virus isolated water samples
| Kind of water samples | Number | Percentage to whole samples (%) |
|---|---|---|
| Stream waters | 5 | 35.8 |
| Source waters | 8 | 57.1 |
| Settled water | 1 | 7.1 |
| Total | 14 | 100 |
Table 2.
Percentage of water samples to virus isolations from which was isolated
| Kind of water samples | Numbers of virus tested | Numbers of virus Isolated | Percentage to whole samples (%) |
|---|---|---|---|
| Stream waters | 6 | 5 | 83.3 |
| Source waters | 24 | 8 | 33.3 |
| Settled water | 20 | 1 | 5.0∗ |
| Total | 50 | 14 | 28.0 |
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