Research in Veterinary Science 1992, 53, 11-18
Improved isolation of rinderpest virus in transformed bovine T lymphoblast cell lines P. B. ROSSITER, K. A. J. HERNIMAN, H. M. WAMWAYI, Division of Virology, National
Veterinary Research Centre, Kenya Agricultural Research Institute, Muguga, PO Box 32, Kikuyu, Kenya are most frequently used for isolation at present. Although these are sufficiently sensitive for use with most virulent strains of the virus (Plowright and Ferfis 1962) the authors have recently found them comparatively insensitive for some mild field strains (Wamwayi et al 1992) and they are non-permissive for most caprinised and lapinised vaccine strains (Isogai 1961, Plowright and Ferris 1962). In an earlier study it was shown that continuous lines of bovine lymphoblasts transformed by the haemoprotozoan parasite Theileriaparva are permissive for the virus (Rossiter et al 1988). Further investigation has shown that lines bearing alpha/beta T cell markers allow the development of large syncytia that are easily visible microscopically whereas infected lines of B or gamma/delta T cell phenotype do not develop syncytia (Rossiter and others 1992). Consequently it was decided to see whether alpha/beta T (T) lymphoblasts offered an advantage over BKand Vero monolayers for rinderpest virus isolation. The opportunity was also taken to see whether bovine lymphoblasts would support the growth of other morbilliviruses.
Bovine T lymphoblast cell fines transformed by the protozoan Theileriaparva were compared with bovine kidney (BK) and Veto cells for their ability to isolate various strains of rinderpest virus from tissues and infected secretions. All of the strains of rinderpest virus that were tested, including attenuated cell-culture, caprinised and lapiulsed vaccines, and both mild and virulent pathogenic strains, readily induced syncytial cytopathic effect (cpe) in T lymphohlasts. The cpe could often be detected within one day of inoculation of lymphoblasts, whereas it took three to 14 days to appear in Veto and BK cells. Using lymphoblasts it was possible to reisolate rinderpest virus from nine of 42 swabs collected from three cattle experimentally infected with an isolate from a recent outbreak of mild disease whereas the same swabs yielded only one reisolate on BKcells. It was also possible using the lympboblasts to detect infectious virus in the ocular, nasal and oral secretions of goats and rabbits infected with caprinised and lapinised virus, respectively. Peste des petits ruminants virus appeared to grow as rapidly as rinderpest virus in the lympboblasts whereas canine distemper virus readily induced cpe on first passage but less readily on subsequent passage. Measles virus induced relatively fittle cpe when inoculated into lymphoblasts and did not appear to passage in these cells. The lymphoblasts are easy to maintain in culture and since they rapidly recovered 11 isolates from 37 diagnostic samples could prove useful in laboratories carrying out rinderpest diagnosis.
Materials and methods
Cell cultures Tube monolayers of secondary BK or Vero cells were prepared from trypsin and ethylene diamine tetra-acetic acid (EDTA) dispersed cell suspensions, and used for virus isolation when at least 50 per cent confluent. They were grown and maintained in Eagle's minimum essential medium (MEM)supplemented with antibiotics and 10 per cent or 2 per cent rinderpest virus antibody free ox serum, respectively.
ISOLATION of rinderpest virus is a useful aid to diagnosis and will become more important as current eradication campaigns attempt to gather field strains for epidemiological 'typing'. Monolayers of bovine kidney (BK) or Vero cells 11
12
P. B. Rossiter, K. A. J. Herniman, 1t. M. Wamwayi
One lymphoblast fine, D409.T1, which has the alpha/beta CD4 + phenotype was used throughout the study. It had been generated by in vitro infection of bovine lymphoblasts, from animal 409, with Theileria parva sporozoites and phenotyped with monoclonal antibodies (Baldwin et al 1988, Goddeeris et a11990). It was grown in RPM1 1640 medium supplemented with 2mM L-glutamine and 5xl0-SmM 2-mercaptoethanol and cultured in loose capped 25 or 75 era2 plastic flasks or 4 ml test tubes (Sterilin, uI~)in 5 per cent carbon dioxide in air. Under these conditions the lymphoblasts grew rapidly and were sprit approximately 1:4 every two or three days.
Virus and samples
(Caniffa; Rhone-Merieux) were obtained as live vaccines said to contain at least 103 TCIDS0ml-1. Peste des petits ruminants virus was the Nig 75/1 strain (Taylor and Abegunde 1979) which had been passaged 44 times in Veto cells, and contained 10s TCIDS0ml-k
Experiment 1: comparison of lymphoblasts, Vero and BK cells for isolation of virulent rinderpest virus Rinderpest virus strain RBO~B~4 was titrated six times in parallel on BK, Veto and lymphoblast cultures. The titrations were made using 0.5 logl0 dilution intervals and five tubes per dilution. Monolayer cultures were decanted and then incubated for one to two hours with 0.2 ml of virus after which 2.0 ml of medium was added. Approximately lxl06 to 0-5x106 lymphoblasts were pelleted by gentle centrifugation in loose capped plastic tubes (Sterilin) and the supernatant medium removed. The lymphoblasts were resuspended in 0.2 ml of virus dilution for one hour at 37°C and then 2.0 ml of medium added to each tube, after which they were cultured at 37°C in 5 per cent carbon dioxide in air. All cultures were examined daily for seven days by inverted microscopy to detect cpe, and infectivity titres determined by the Spearrnan-Karber formula. Cytocentrifuge smear preparations were prepared from selected cultures, stained by MayGrunwald-Giemsa and examined by transmitted light microscopy for inclusion bodies and syncytia, ffimilarly, some cytocentrifugepreparations were fixed with acetone and stained with a direct fluorescent antibody conjugate to rinderpest virus (Rossiter and Jesset 1982).
Freeze dried bovine spleen containing rinderpest virus strain RBOI~that had been previously passaged four times in BI~ cells, was used as virulent virus in the first experiment. Two milder strains of virus were each used to infect three cattle; the RBT/1 strain was isolated from clinically affected calves in Tanzania in 1960 (Plowright 1963) and the RB~Kiambu/88/1 strain from a cow in Kenya in 1988 (Wamwayi et al 1992). Ocular and nasal secretions were swabbed daily from each animal for three weeks after inoculation. Ocular, nasal and oral swabs and whole blood samples in Et~TAwere also collected from 28 suspected bovine field cases of rinderpest occurring in the same outbreak from which RB~Kiambu/ 88/1 had been originally isolated. Goat adapted virus was the Kabete attenuated goat (KAG) vaccine strain (Daubney 1949). Live virus was inoculated into four goats which were killed three or four days later and 20 per cent suspensions prepared from their spleens, mesenExperiment 2: comparison of lymphoblasts and teric and prescapular lymph nodes. Four rabbits Vero cells for isolation of mild strains of rinderwere inoculated with lapinised virus derived in pest virus from experimentally infected cattle Kenya from the Nakamura III strain Ocular and nasal swabs (unsterilised commer(Brotherston 1951). Two or three days later the rabbits were killed and 20 per cent suspensions cial cotton buds) from cattle infected with the prepared from their spleens, mesenteric lymph RBT/1 or RBK/Kiambu/88/1 strains of rinderpest nodes and a pool of gut-associated lymphoid tis- virus were shaken together in 2-0 ml of Eagle's sues (6ALT) comprising Peyer's patches, MEM containing double normal strength antibiappendix, caecal tonsil and saeculus rotundus. otics and I0 per cent serum and stored at -70°C Combined ocular/nasal/oral swabs were collected until used. After thawing, the transport medium was lightly centrifuged to remove gross debris from the rabbits and from two of the goats. Measles virus Schwartz strain (Rouvac; and 0.2 ml absorbed onto each of five Vero cell Institut Merieux) and canine distemper virus tubes. Lymphoblasts were cultured in 25 cm2
Isolation of rinderpest virus loose capped culture bottles. Approximately 2x106 cells were incubated in 0-5 ml of medium with 1-0 ml of swab sample for one hour and then fresh medium added to give a volume of 5 ml. The cultures were maintained and examined as in experiment 1.
Experiment 3." isolation of caprinised and lapinised vaccine virus in lymphoblasts Tenfold dilutions of infected goat and rabbit tissues were prepared in maintenance medium, and 1 ml of each dilution added to each of two bottles of lymphoblasts. The oculo/nasal/oral swab samples were processed as in experiment 2 and all 2 ml of medium inoculated undiluted without being frozen. The cultures were maintained and examined as in experiment 1. If cpe developed an attempt was made to passage the effect to fresh cultures with cell-free fluid.
Experiment 4: the growth of peste des petits ruminants, measles and canine distemper viruses in bovine lymphoblasts 25 cm 2 flasks containing lxl06 to 2×106 lymphoblasts in approximately 1.0 ml of medium were inoculated with 1.0 ml of either peste des petits ruminants or measles or canine distemper virus containing approximately 103 TODS0 and then maintained and examined as described in experiment 2. Clarified medium from cultures which developed cpe was passaged to uninfected lymphoblasts.
Isolation in lymphoblasts of Rv from diagnostic samples Swab samples were inoculated into 25 cm 2 flasks oflymphoblasts as described in experiment 2. Buffy coat samples were prepared from l0 ml of whole blood, washed once in medium with EDTA and resuspended in approximately 1-0 ml of medium. 0,5 ml was inoculated into 25 cm 2 flasks of lymphoblasts. Results
Experiment l Specific viral cpe developed in all three types of cell. The cpe in lymphoblasts was characterised by large ballooning syncytia which were easily visible even when the blasts tended to clump
13
TABLE 1 : Titres* of rinderpest virus strain RBOK(virulent) detected daily after the start of titration in T lymphoblasts, Vero and BK cells
Cel~type
1
2
T lymphoblast Vero BK
2 7 4 1 3"86
Improved isolation of rinderpest virus in transformed bovine T lymphoblast cell lines P. B. ROSSITER, K. A. J. HERNIMAN, H. M. WAMWAYI, Division of Virology, National
Veterinary Research Centre, Kenya Agricultural Research Institute, Muguga, PO Box 32, Kikuyu, Kenya are most frequently used for isolation at present. Although these are sufficiently sensitive for use with most virulent strains of the virus (Plowright and Ferfis 1962) the authors have recently found them comparatively insensitive for some mild field strains (Wamwayi et al 1992) and they are non-permissive for most caprinised and lapinised vaccine strains (Isogai 1961, Plowright and Ferris 1962). In an earlier study it was shown that continuous lines of bovine lymphoblasts transformed by the haemoprotozoan parasite Theileriaparva are permissive for the virus (Rossiter et al 1988). Further investigation has shown that lines bearing alpha/beta T cell markers allow the development of large syncytia that are easily visible microscopically whereas infected lines of B or gamma/delta T cell phenotype do not develop syncytia (Rossiter and others 1992). Consequently it was decided to see whether alpha/beta T (T) lymphoblasts offered an advantage over BKand Vero monolayers for rinderpest virus isolation. The opportunity was also taken to see whether bovine lymphoblasts would support the growth of other morbilliviruses.
Bovine T lymphoblast cell fines transformed by the protozoan Theileriaparva were compared with bovine kidney (BK) and Veto cells for their ability to isolate various strains of rinderpest virus from tissues and infected secretions. All of the strains of rinderpest virus that were tested, including attenuated cell-culture, caprinised and lapiulsed vaccines, and both mild and virulent pathogenic strains, readily induced syncytial cytopathic effect (cpe) in T lymphohlasts. The cpe could often be detected within one day of inoculation of lymphoblasts, whereas it took three to 14 days to appear in Veto and BK cells. Using lymphoblasts it was possible to reisolate rinderpest virus from nine of 42 swabs collected from three cattle experimentally infected with an isolate from a recent outbreak of mild disease whereas the same swabs yielded only one reisolate on BKcells. It was also possible using the lympboblasts to detect infectious virus in the ocular, nasal and oral secretions of goats and rabbits infected with caprinised and lapinised virus, respectively. Peste des petits ruminants virus appeared to grow as rapidly as rinderpest virus in the lympboblasts whereas canine distemper virus readily induced cpe on first passage but less readily on subsequent passage. Measles virus induced relatively fittle cpe when inoculated into lymphoblasts and did not appear to passage in these cells. The lymphoblasts are easy to maintain in culture and since they rapidly recovered 11 isolates from 37 diagnostic samples could prove useful in laboratories carrying out rinderpest diagnosis.
Materials and methods
Cell cultures Tube monolayers of secondary BK or Vero cells were prepared from trypsin and ethylene diamine tetra-acetic acid (EDTA) dispersed cell suspensions, and used for virus isolation when at least 50 per cent confluent. They were grown and maintained in Eagle's minimum essential medium (MEM)supplemented with antibiotics and 10 per cent or 2 per cent rinderpest virus antibody free ox serum, respectively.
ISOLATION of rinderpest virus is a useful aid to diagnosis and will become more important as current eradication campaigns attempt to gather field strains for epidemiological 'typing'. Monolayers of bovine kidney (BK) or Vero cells 11
12
P. B. Rossiter, K. A. J. Herniman, 1t. M. Wamwayi
One lymphoblast fine, D409.T1, which has the alpha/beta CD4 + phenotype was used throughout the study. It had been generated by in vitro infection of bovine lymphoblasts, from animal 409, with Theileria parva sporozoites and phenotyped with monoclonal antibodies (Baldwin et al 1988, Goddeeris et a11990). It was grown in RPM1 1640 medium supplemented with 2mM L-glutamine and 5xl0-SmM 2-mercaptoethanol and cultured in loose capped 25 or 75 era2 plastic flasks or 4 ml test tubes (Sterilin, uI~)in 5 per cent carbon dioxide in air. Under these conditions the lymphoblasts grew rapidly and were sprit approximately 1:4 every two or three days.
Virus and samples
(Caniffa; Rhone-Merieux) were obtained as live vaccines said to contain at least 103 TCIDS0ml-1. Peste des petits ruminants virus was the Nig 75/1 strain (Taylor and Abegunde 1979) which had been passaged 44 times in Veto cells, and contained 10s TCIDS0ml-k
Experiment 1: comparison of lymphoblasts, Vero and BK cells for isolation of virulent rinderpest virus Rinderpest virus strain RBO~B~4 was titrated six times in parallel on BK, Veto and lymphoblast cultures. The titrations were made using 0.5 logl0 dilution intervals and five tubes per dilution. Monolayer cultures were decanted and then incubated for one to two hours with 0.2 ml of virus after which 2.0 ml of medium was added. Approximately lxl06 to 0-5x106 lymphoblasts were pelleted by gentle centrifugation in loose capped plastic tubes (Sterilin) and the supernatant medium removed. The lymphoblasts were resuspended in 0.2 ml of virus dilution for one hour at 37°C and then 2.0 ml of medium added to each tube, after which they were cultured at 37°C in 5 per cent carbon dioxide in air. All cultures were examined daily for seven days by inverted microscopy to detect cpe, and infectivity titres determined by the Spearrnan-Karber formula. Cytocentrifuge smear preparations were prepared from selected cultures, stained by MayGrunwald-Giemsa and examined by transmitted light microscopy for inclusion bodies and syncytia, ffimilarly, some cytocentrifugepreparations were fixed with acetone and stained with a direct fluorescent antibody conjugate to rinderpest virus (Rossiter and Jesset 1982).
Freeze dried bovine spleen containing rinderpest virus strain RBOI~that had been previously passaged four times in BI~ cells, was used as virulent virus in the first experiment. Two milder strains of virus were each used to infect three cattle; the RBT/1 strain was isolated from clinically affected calves in Tanzania in 1960 (Plowright 1963) and the RB~Kiambu/88/1 strain from a cow in Kenya in 1988 (Wamwayi et al 1992). Ocular and nasal secretions were swabbed daily from each animal for three weeks after inoculation. Ocular, nasal and oral swabs and whole blood samples in Et~TAwere also collected from 28 suspected bovine field cases of rinderpest occurring in the same outbreak from which RB~Kiambu/ 88/1 had been originally isolated. Goat adapted virus was the Kabete attenuated goat (KAG) vaccine strain (Daubney 1949). Live virus was inoculated into four goats which were killed three or four days later and 20 per cent suspensions prepared from their spleens, mesenExperiment 2: comparison of lymphoblasts and teric and prescapular lymph nodes. Four rabbits Vero cells for isolation of mild strains of rinderwere inoculated with lapinised virus derived in pest virus from experimentally infected cattle Kenya from the Nakamura III strain Ocular and nasal swabs (unsterilised commer(Brotherston 1951). Two or three days later the rabbits were killed and 20 per cent suspensions cial cotton buds) from cattle infected with the prepared from their spleens, mesenteric lymph RBT/1 or RBK/Kiambu/88/1 strains of rinderpest nodes and a pool of gut-associated lymphoid tis- virus were shaken together in 2-0 ml of Eagle's sues (6ALT) comprising Peyer's patches, MEM containing double normal strength antibiappendix, caecal tonsil and saeculus rotundus. otics and I0 per cent serum and stored at -70°C Combined ocular/nasal/oral swabs were collected until used. After thawing, the transport medium was lightly centrifuged to remove gross debris from the rabbits and from two of the goats. Measles virus Schwartz strain (Rouvac; and 0.2 ml absorbed onto each of five Vero cell Institut Merieux) and canine distemper virus tubes. Lymphoblasts were cultured in 25 cm2
Isolation of rinderpest virus loose capped culture bottles. Approximately 2x106 cells were incubated in 0-5 ml of medium with 1-0 ml of swab sample for one hour and then fresh medium added to give a volume of 5 ml. The cultures were maintained and examined as in experiment 1.
Experiment 3." isolation of caprinised and lapinised vaccine virus in lymphoblasts Tenfold dilutions of infected goat and rabbit tissues were prepared in maintenance medium, and 1 ml of each dilution added to each of two bottles of lymphoblasts. The oculo/nasal/oral swab samples were processed as in experiment 2 and all 2 ml of medium inoculated undiluted without being frozen. The cultures were maintained and examined as in experiment 1. If cpe developed an attempt was made to passage the effect to fresh cultures with cell-free fluid.
Experiment 4: the growth of peste des petits ruminants, measles and canine distemper viruses in bovine lymphoblasts 25 cm 2 flasks containing lxl06 to 2×106 lymphoblasts in approximately 1.0 ml of medium were inoculated with 1.0 ml of either peste des petits ruminants or measles or canine distemper virus containing approximately 103 TODS0 and then maintained and examined as described in experiment 2. Clarified medium from cultures which developed cpe was passaged to uninfected lymphoblasts.
Isolation in lymphoblasts of Rv from diagnostic samples Swab samples were inoculated into 25 cm 2 flasks oflymphoblasts as described in experiment 2. Buffy coat samples were prepared from l0 ml of whole blood, washed once in medium with EDTA and resuspended in approximately 1-0 ml of medium. 0,5 ml was inoculated into 25 cm 2 flasks of lymphoblasts. Results
Experiment l Specific viral cpe developed in all three types of cell. The cpe in lymphoblasts was characterised by large ballooning syncytia which were easily visible even when the blasts tended to clump
13
TABLE 1 : Titres* of rinderpest virus strain RBOK(virulent) detected daily after the start of titration in T lymphoblasts, Vero and BK cells
Cel~type
1
2
T lymphoblast Vero BK
2 7 4 1 3"86
