Propagation of Asian isolates of canine distemper virus (CDV) in hamster cell lines
© Sultan et al; licensee BioMed Central Ltd. 2009
Received: 6 July 2009
Accepted: 16 October 2009
Published: 16 October 2009
The aim of this study was to confirm the propagation of various canine distemper viruses (CDV) in hamster cell lines of HmLu and BHK, since only a little is known about the possibility of propagation of CDV in rodent cells irrespective of their epidemiological importance.
The growth of CDV in hamster cell lines was monitored by titration using Vero.dogSLAMtag (Vero-DST) cells that had been proven to be susceptible to almost all field isolates of CDV, with the preparations of cell-free and cell-associated virus from the cultures infected with recent Asian isolates of CDV (13 strains) and by observing the development of cytopathic effect (CPE) in infected cultures of hamster cell lines.
Eleven of 13 strains grew in HmLu cells, and 12 of 13 strains grew in BHK cells with apparent CPE of cell fusion in the late stage of infection. Two strains and a strain of Asia 1 group could not grow in HmLu cells and BHK cells, respectively.
The present study demonstrates at the first time that hamster cell lines can propagate the majority of Asian field isolates of CDV. The usage of two hamster cell lines suggested to be useful to characterize the field isolates biologically.
Canine distemper virus (CDV) is a negative-strand RNA virus that belongs to the genus Morbillivirus in the family Paramyxoviridae. The CDV induce distemper in dogs, raccoons  and ferrets . Outbreaks of distemper in seals on Lake Baikal , in leopards and other feline animals in zoos , and in lions in the Serengeti National Park , indicate additional animal hosts other than the generally accepted hosts.
CDV has been isolated by a few cell lines other than the cells derived from dog as follows. Vero  and B95a B-cell line  were derived from monkey, and MV1Lu lung cells were derived from mink . Recently, the gene of dog signaling lymphocyte activation molecule (also known as dog SLAM or CD150) , was introduced into Vero cells and Vero.dogSLAMtag (Vero-DST) cells were established . By Vero-DST cells, various field strains were isolated [10–12]. Thus there are only a few cell lines derived from other than generally accepted hosts.
During a study on the molecular characterization of the hemagglutinin (H) and fusion (F) protein genes using mouse retrovirus vectors, the hamster HmLu cell line was suggested to be susceptible to CDV infection. Hamsters are not generally accepted hosts and there is little knowledge of the propagation of CDV in rodent cells.
Cells and Viruses
Vero-DST cells were established as described above . HmLu cells were derived from hamster lung cells . BHK cells were derived from baby hamster kidney cells . All cells were passaged and maintained in Dulbecco's modified Eagle's medium (D-MEM; autoclavable; Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 10% fetal bovine serum in a CO2 incubator at 37°C.
Thirteen strains of CDV were isolated and propagated, one or a few times, in Vero-DST cells and stored at -80°C until use. Specimens were collected from diseased dogs in Japan except for strain Th12, which was sampled in Thailand. Nine strains were newly isolated except for strains of 007 Lm, 009L, 011C , and Ac96I . The phylogenetic relationship of nine new strains were determined by the sequencing of their (H) genes  where strains 55L, M25CR, 50Con and 50Cbl were assigned as members of Asia 2 group, while strains Th12, 50Sc, 81ND, 82Con and 83mLN were assigned as members of Asia 1 group. The vaccine strain of Onderstepoort (Ond) was used as a control.
Vero-DST cells were seeded on 24-well multi-plates (Sumitomo-Bakelite, Tokyo, Japan) at a concentration of 1 × 105 cells per well and incubated overnight. After the removal of media, the cultures were overlaid with 0.1 ml of each serial 10-fold-diluted virus suspensions in duplicate manner, and incubated for 1 h with rocking at 15-min intervals. After virus adsorption, the suspensions were removed by suction and the cultures were washed once with culture media. The cultures were then overlaid with medium (1 ml) containing 1% methyl cellulose-4,000 (Nacalai Tesque, Kyoto, Japan; methyl cellulose media), incubated for 3 days, then fixed with formalin, stained with crystal violet solution, and plaques were counted. Virus titers were shown as mean value after two independent titrations which were carried out in a duplicate form.
Passage of HmLu and BHK cells infected with various CDV strains
Sub-confluent cultures of HmLu or BHK cells were infected with strains Ond, 007Lm, 55L, 009L, M25CR, 011C, 50Con, 50Cbl, Ac96I, Th12, 50Sc, 81ND, 82Con, or 83mLN of CDV at a multiplicity of infection (MOI) = 1 in culture bottles. When the cultures became confluent (usually after 3-day incubation) the supernatant media was harvested, centrifuged at 3,000 rpm for 10 min to remove cell debris, aliquotted, and stored at -80°C until use as cell-free virus. The residual cells were liberated by trypsinization, and one tenth of the liberated cells underwent further cultivation, while the residual nine-tenths were used to liberate the cell-associated virus by freezing at -80°C, thawing under flowing water, and centrifugation at 3,000 rpm for 10 min to remove cell debris. The supernatant fraction was aliquotted and stored at -80°C until use as cell-associated virus. The cycle of passage was repeated, and all cell-free and cell-associated virus samples were titrated on Vero-DST cells.
Propagation of CDV strains in HmLu and BHK cells
The recovered virus titer from HmLu and BHK cells after several passages.
Titer (PFU/ml) recovered from
HmLu (7P) a)
BHK (5P) a)
3.4 × 103
6.3 × 104
6.0 × 105
1.2 × 106
2.5 × 105
2.2 × 106(7P)b)
1.6 × 105
2.4 × 105
1.0 × 105
9.2 × 105(7P)b)
3.0 × 105
4.0 × 105
2.0 × 103
8.0 × 105
4.0 × 105
2.0 × 105
5.0 × 103
2.0 × 104
0.6 × 105
1.6 × 105
3.0 × 104
2.5 × 105
0.5 × 105
3.0 × 101
2.2 × 102
1.0 × 104
7.0 × 103 (3P)b)
1.0 × 104
8.0 × 103 (3P)b)
1.0 × 105
1.9 × 105(7P)b)
1.1 × 105
1.9 × 105
1.5 × 104
2.5 × 104(7P)b)
5.5 × 103
1.5 × 104 (3P)b)
1.2 × 106
1.2 × 106(4P)b)
4.5 × 105
5.5 × 105(4P)b)
2.0 × 105
1.0 × 105(7P)b)
1.3 × 105
1.5 × 105
Characterization of the CPE induced in hamster cells infected with CDV isolates
Strains 55L, 009L, M25CR, 011C, 50Con, 50Cb1, 50Sc, 81ND, 82Con, and 83mLN induced similar CPE of giant cell formation in the infected HmLu culture at the middle and late stage of infection except at the first infection. Similar CPE of giant cell formation were also induced in the culture of BHK cells infected with every Asian isolate except for strain Ac96I.
BHK cells had a tendency to propagate field CDV strains more efficiently than HmLu cells as shown in the cases of strains 50Cbl, Th12 and 81ND (Table 1) and strain 007Lm. Fujita et al.  reported that BHK cells showed a low susceptibility to recombinant Yanaka virus but showed a high level CDV transcription. This high level CDV transcription might correlate to the rapid destruction of BHK cultures infected with strains 007Lm, 50Cbl, Th12 and 81ND when compared with the infected cultures of HmLu cells. However, strain 82Con grew in HmLu cells as rapidly as in BHK cells. Strain Ac96I could not grow in both HmLu and BHK cells and Th12 could grow in BHK cells but not in HmLu cells although it is not known what factor controls the growth of CDV in hamster cells at present. Thus, each virus strain behaved differently against the two hamster cell lines (Table 1). These results indicate that the usage of two hamster cell lines is useful to characterize various field strains by their biological nature.
In this report, all of Asian isolates induced no CPE at the initial stage of infection in the hamster cell lines of both HmLu and BHK, and this result is coincided with the reports [11, 17] that both groups of Asia 1 and Asia 2 showed no CPE in Vero cells at the first infection. However, repeated passage of infected hamster cells resulted in the fact that even Asia 1 isolates except for Ac96I, could grow in hamster cell lines with apparent CPE of giant cell formation. Thus, it is necessary to characterize the infection to a cell line not only at the initial stage but also at the late stage of infection.
The HmLu cells infected with 007Lm had a high passage level of 15 passages, whereas the HmLu cells infected with Ond strain induced massive detachment of giant cells after the 1st passage and were unable to be transferred further. These observations indicated that the development of CPE of giant cell formation in the 007Lm-infected HmLu cells was very slow. The slow development of CPE enabled the observation of various types of colonies at the 8th passage as shown in Fig. 3. Giant cells seemed to be produced gradually from the infected cells with normal shape by cell fusion. Once giant cells were formed, they had a tendency to detach from the surface of the dish and turn into the floating giant cells.
This report demonstrated at the first time that the majority of Asian field isolates tested were propagated in hamster cell lines of HmLu and BHK. Each virus strain behaved differently against the two cell lines. Since there is no effective method to characterize the field isolates of CDV biologically, the usage of two hamster cell lines can be useful.
Serageldeen Sultan received financial support in the form of a PhD degree scholarship from the Egyptian government.
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