Studies on Isolation, Serum-free Cultivation and Manufacture of Mink Enteritis Virus Optimized for Vaccine Preparation

827674—BIOL 25/1 (ISSUE)—MS 0019

Biologicals (1997) 25, 103–111

Studies on Isolation, Serum-free Cultivation and Manufacture of Mink Enteritis Virus Optimized for Vaccine Preparation De-Li Zhang Chinese Army Laboratory of Molecular Virology & Genetic Engineering, Institute of Preventive Medicine & Veterinary Science in Beijing Military Area of Chinese PLA, No. 42, Donghuochang Road, Fengtai District, Beijing City 100071, P.R. China

Abstract. Optimum conditions have been determined for growth of mink enteritis virus (MEV) in the feline kidney cell (FKC) lines. The S18 and S36 strains of MEV which grow to titres of 8192 to 32 786 haemagglutination (HA) units or 109.7–10.3 TCID50 by alternate cultivation for 22 passage in FKC with very different susceptibility. Mink with typical clinical symptoms of mink viral enteritis (MVE) eliminated intestinal mucosal cylinders from which the L12 strain of MEV was isolated, which is capable of replication to 8192 HA units or 109.7 TCID50 . The S18 , L12 and S36 strains of MEV grow to high titres and the inactivated virus vaccine induced a good immune response in mink. The S18 and L12 strains of MEV are the best strains for vaccine preparation and the L12 strain is the most virulent for challenge tests. = 1997 The International Association of Biological Standardization

Introduction Mink enteritas virus (MEV), feline panleukopenia virus (FPLV), canine parvovirus(CPV) and raccoon parvovirus (RPV) are antigenically related,1–6 are genetically similar members of the parvovirus genus2,7–8 and are four subspecies of the feline parvovirus (FPV) species.3,6,8–11 Parvovirus enteritis in weasels, cats, dogs and raccoons has a high morbidity and mortality, exhibits a rapid clinical course and is highly contagious. Growth of MEV in tissue culture usually produces infectious titres of TCID50 107⋅6 and HA titres of 32.1–3,5–6,10,12–15 For vaccine virus production, the use of newborn calf serum (CS) or fetal calf serum (FCS) in the culture media is a logistical and economic problem and the growth of parvovirus in such tissue culture is low. The purpose of these studies is to improve the reproductive titre of MEV without serum in the maintenance medium.

Materials and methods Viral strains and cell lines The S11 , S18 , S36 , S37 and Q41 strains of MEV were isolated and identified by Yu Yong-Ren et al. 16 1045–1056/97/010103 + 9 $25.00/0/bg960065

The Lgold and L12 strains of MEV were isolated from intestinal mucosa shed by mink exhibiting symptoms of MEV during an outbreak at a fur ranch in Dalian City in 1986. The American standard strain of MEV was supplied by the University of Washington, U.S.A. The feline kidney cell (FKC) line C81 originating from the ATCC was supplied by the Harbin Veterinary Institute of the Chinese Academy of Agricultural Sciences. The FKC line F81 strain from the ATCC was provided by the Chinese Supervisory Institute of Veterinary Bioproducts and Pharmaceuticals and the Animal Quarantine Institute of the Agricultural Ministry, respectively. Growth medium used was Eagle’s MEM supplemented with 16% newborn calf serum, 0⋅3 mg/ml glutamine, 0⋅1 mg/ml sodium pyruvate, 1⋅5 mg/ml NaHCO3 , 100 units penicillin and 100 mg/ml streptomycin. Maintenance media were similar, except that the serum concentration was reduced to 0–5% (depending on the experiment) and NaHCO3 was increased to 2⋅25 mg/ml. Using a sub-cultivation ratio of 1:3–1:4, large quantities of cell suspension were prepared by gradually increasing from 30, 100, 500, 1000, 2000 ml square flasks to 10 000 ml roller bottles. 7 1997 The International Association of Biological Standardization

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Virus culture

Results

Virus was serially propagated in cultures of C81 or F81 FKC inoculated with a 10−1 or 10−2 dilution of MEV from the last passage. Growth medium containing 10% calf serum (CS) was changed to maintenance medium which contained 1–5% CS or without CS at 24 h after inoculation of static or spinner cultures with MEV. After incubation of 3–7 days at 37°C, cytopathic effect (CPE) appeared and the cell-cultured MEV fluids were harvested. The virus containing cell culture fluids were frozen and thawed three times and assayed for HA activity and infectious virus. Optimum conditions for the static and spinner cultivation of MEV were determined for each cell line and virus in terms of virus inoculation dose, culture medium, and time of virus harvest. The best strains of MEV for vaccine preparation were selected by alternate cultivation of MEV in C81 or F81 FKC cell lines having very different susceptibility to MEV infection.

Selection and cultivation results of super reproductive MEV strains for vaccine preparation

Virus assays The micro-test for HA activity were performed in 8 × 12-well plastic V-plates using 0⋅5% glutaraldehyde-fixed porcine red blood cells (GluPRBC). A suspension of 0⋅5% GluPRBC was used routinely for convenience in place of fresh macaque red blood cells (FMRBC), both of which have the same sensitivity and specificity to MEV determined by the author at pH 6⋅25 and at a temperature of 4°C. Virus suspensions were diluted in a PBS solution of pH 6⋅25 starting from an initial 1:2 dilution, and equal volume of 0⋅5% ice cold GluPRBC was added to each well and the plates were incubated at 4°C. The test results were read after RBC controls had settled completely, usually after 2–4 h. Micro-TCID50 tests were performed in 4 × 10-well plastic culture U-plates, using the F81 line of FKC cells. Serial 10-fold dilutions of the MEV samples in growth media were mixed with the cell suspensions. One hundred microlitres of each dilution was added to each of the microtitre wells, and incubated for 24 h at 37°C in a 5%-CO2 humidified atmosphere. A complete cell sheet formed and growth media containing 10% CS was replaced with serum-free maintenance media. After incubation for an additional 120 h, CPE was observed and recorded, and the TCID50 titre calculated by the method of Reed and Muench.17

The S18 , S36 , L12 and American strains of MEV were cultured in FKC/F81 cells. All of the MEV strains reproduced well by static or spinner cultivation for 20–30 passages. Table 1A shows that most MEV strains replicated in FKC/F81 cells with HA titres of up to 256. In contrast, virus HA titre declined when MEV strains were cultivated in FKC/C81 cells (Table 1B). The highest titres for S18 or S36 virus produced in the FKC/C81 cells were HA titres of 32–128. Lgold and American strains of MEV did not grow in FKC/C81 cells after 3–5 successive passages of serial cultivation of virus or three blind passages of infected cells, at which time cells appeared normal and HA titres were negative. These data indicate that the FKC/F81 cell line is susceptible to serial propagation of MEV, FKC/C81 cell line is not very susceptible. The reproductive HA titres of six strains of MEV by cultivation of 7 passages (from 5th to 11th passages) in FKC/F81 cells were examined and the results are shown in Table 1. The MEV strains in decreasing order of geometric average value of HA titre (in log 2 HA units) were Q41 (4⋅9), S18 (4⋅8), S36 (4⋅0), S11 (3⋅7), American (3⋅6) and S37 (2⋅3) strains, S11 and S37 strains of MEV were not further cultivated for vaccine development. The use of t-test of the HA data in Table 1 showed that the reproductive ability of S18 strain of MEV was significantly greater than that of S36 strain (P Q 0⋅01), and the ability of S36 strain was significantly greater than that of American strain (P Q 0⋅01). Improvement of MEV reproduction was achieved by alternate cultivation in cell lines with very different susceptibility. Vaccine candidates for MEV S18 , S36 and American strains were selected by cultivation in FKC/F81 cells for four passages followed by cultivation of seven passages in susceptible FKC/C81 cells followed by cultivation in susceptible FKC/F81 cells for nine passages (Fig. 1). After cultivation for an additional 5–6 passages in susceptible FKC/F81 cells, the reproductive HA titres of S18 , S36 and American strains of MEV increased to 8192–32 768 during 21–22 passages (Table 1C). Table 1A shows that the reproductive HA titre of 5 strains of MEV cultured in susceptible FKC/F81 cells during 9–11 passages was significantly higher than the titre during 5–8 passages (P Q 0⋅05). Thus, the reproductive titre of MEV increased slowly with

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Table 1. Detection of replication by haemagglutination (log2 HA) of MEV strains after cultivation in cell lines with different susceptibility to MEV infections

FKC line (A) F81 Cells

(B) C81 Cells

Form of MEV propagation

Passage of MEV cultivation

Normal propagation

5 6 7 8 9 10 11 9 10 11 12 13 14 14 15 15 16 17 18 19 20 21 20 21 22

Normal propagation

Forced-infection propagation (C) F81 Cells

Normal propagation

S11

S18

S36

S37

Q41

American

4 1 1 3

3 3 1 6

4 1 2 5

3 1 1 4

4 2 2 5

3 3 1 — 3 3 6 6 7 4 3 2 1 1 1 2 — — — —

4 4* 5 6 5

5 5 4 4 6 6 6 6 8 4 7 5 4 3 4 4 3 3 4 4 2 1 2 2 1 1 1 1 — — — — 5 3 4 2 1 1 1 1 1 1 1 1 r8† r8 6 6 r11 r11 12 7 9 6 r11 r11 10 9 r15 r15 r14 r14 9 13 8 8 13 7

5 6 6 6 7 5

r8 5 8 7 6

Lgold

6 4 4 2 — — 2 1 —

4 2 1 — — — 1 —

r 11

11 8 8 8 6

*Second number is result from duplicate culture of another square flask. †Means that HA endpoint was not detected at higher dilution tested. the cultivation passage increasing. Further, the reproductive HA titre of three strains of MEV cultured in susceptible FKC/F81 cells during 20–22 passages was significantly higher than the titre during 16–19 passages (t = 2⋅2221, df = 31; t0⋅05 = 2⋅042, df = 30; P Q 0⋅05). Thus, the reproductive titre of MEV clearly increased with increasing cultivation passage. Optimization of virus inoculation To determine optimum virus inoculation dose, cell cultures of FKC/F81 were inoculated with 10−1 through 10−6 dilutions of MEV virus strains S18 , S36 and American which had HA titres of 128 at the 23rd passage. The reproductive HA titres of MEV from 10−1 , 10−2 , and 10−3 inoculation doses were 256, 8 and 4, respectively, and the reproductive

HA titre from 10−4 , 10−5 and 10−6 inoculation doses were all less than that detectable. It can be seen from Table 2 that cultures of FKC/F81 cells inoculated with 10−2 dilutions of MEV stocks with HA titres of 64–4096, and incubated in serum-free culture medium produced virus suspensions which contained 256–32 768 HA units. Therefore, 10−2 dilutions were employed thereafter. When S18 , S36 , and Lgold strains of MEV were propagated in FKC/C81 cells, little replication was observed (HA titres were 2) whether maintenance medium was added at 30 or 47 h. However, HA titres were slightly higher for those MEV strains grown in FKC/F81 cells when maintenance medium was added at 24 h (HA titres r256) compared to no medium change (HA titres of 64–128). By rank

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FKC/F81 P5

better compared with the MEV inoculation after 4 h cell culture but the difference was not significant (0⋅05 Q P Q 0⋅15). However, the simultaneous inoculation of MEV with cell suspension was simple and convenient compared with MEV inoculation after 4–12 h of cell cultivation. Therefore, subsequent studies used simultaneous inoculation of MEV.

FKC/F81 P8

FKC/F81 P11

FKC/C81 P13

FKC/C81 P15

FKC/F81 P22

Figure 1. A flow chart outlining the procedure of alternative cultivation of the S18 and S36 strains of MEV in cell lines of high and low susceptibility.

analysis of paired data, this difference was statistically significant (P Q 0⋅05). After culture for 4, 8 and 12 h, respectively, cultures of FKC/F81 cells were inoculated with a 10−2 dilution of a mixture of S18 , S36 and American strains of MEV at the 22nd passage (or with a 10−2 dilution of L12 strain at the 3rd passage). MEV HA titres of resulting harvest fluids were 256–1024, similar to levels found in undiluted inoculum. In subsequent pair design tests, FKC/F81 cells were cultured for 0 or 4 h and then were inoculated with a mixture of MEV fluids of S18 , S36 and American strains at the 22nd passage. The MEV HA titre of fluid harvested after 48–211 h of cultivation was examined and the results are shown in Table 3B. It was shown by t-test of the pair data that the simultaneous inoculation of MEV may be slightly

Optimization of virus harvesting The HA titre and CPE of virus-infected cell cultures during 24–211 h after the propagation of mixed MEV of S18 , S36 and American strains at the 23rd passage in FKC/F81 cells and MEV of S36 strain at the 11th passage in FKC/C81 cells were measured. The results are shown in Table 3A. There is no simple relationship between HA titre, CPE and time. Method for serum-free static cultivation of MEV in 30–2000 ml square flasks In complete pair design test, S18 , S36 , Lgold and American strains of MEV were cultured for six passages with serum-free or serum containing maintenance medium in FKC/C81 cells. MEV HA titres of the same four strains of MEV respectively harvested from serum-free and serum-containing cell cultures are shown in Table 4. By t-test of pair data, the difference in the HA titres of MEV fluids harvested respectively from serum-free and serum-containing cell cultures was not significant (P q 0⋅05). Serum-free culture of MEV in FKC line of F81 strain The result of the t-test of non-pair data showed that the HA titre of MEV (S18 , S36 and Lgold strains)

Table 2. The detection results of the reproductive titre (log2 HA) of MEV fluids harvested from serum-free cell cultures of FKC line of F81 strain inoculated synchronously with MEV fluids S18 strain passage 19

MEV passage 20 10 passage 21 passage 22

r15* r14*

(12) 10 (9) r15* (r11*) 13

S36 strain passage 19 10

9

q15*

r15*

13

r14*

(7) 9 (6) r15* (r11*) 9 8

American strain passage 15 9

11

r15*

8

8

8

7

(7) 11 (6) 8 (r11*) 8

11 8 6

Note: 3 strains of MEV were statically cultured in FKC line of F81 strain in square flasks (capacity 500 ml). The number in parentheses is (log2 HA) value of MEV inoculum fluid before dilution. *Means that HA endpoint was not detected at higher dilution tested.

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0/0 7 12/0 9 25/12 9 12/0 6 12/0 9 12/0 5 25/0 7 37/25 9 50/37 10 50/12 7 8 100/100 4 100/87 4 75/75 4 0 h culture (B) C81

4 h culture

Note: CPE is presented as the specific value of dead cells + active cells divided by active cells. The inoculation dosage of MEV fluid was all 10 − 2.

50/37 9 37/12 8 75/50 6 50/37 10 75/75 6 100/87 6 50/25 11 37/25 8 87/75 4 87/87 9 87/75 5

CPE log2 HA CPE log2 HA CPE log2 HA 0 h culture (A) F81

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during 20–22 passages harvested from serum-free culture fluid was greater than the titre of MEV (the same 3 strains) during 16–19 passages from serum-containing culture fluid (P Q 0⋅05), as shown in Table 1. The selection of this method of propagation seems to be responsible for the increasing of HA titre of three strains of MEV. However, when L12 strain of MEV from one to four passages was cultured in FKC/F81 cells, the MEV HA titre was basically the same no matter whether serum was present in the maintenance medium.

37/25 9 50/25 7

168 144 132 120 108 96 72 48 FKC line

Parameter

24

Time after MEV inoculations (h)

Timing of MEV inoculation

Table 3. The cytopathogenic effect (CPE) and haemagglutination (HA) titre of virus-infected cell-cultures

192

211

Development of mink parvovirus vaccines

Optimization of serum-free spinner cultivation of MEV in 10 litre roller bottles The optimum conditions for the static cultivation of MEV in square flasks were implemented for the spinner cultivation of MEV in large roller bottles, and the results showed that the optimum conditions for the spinner and static cultivation of MEV were identical and the optimum spinner velocity of the roller bottles was 0⋅13–0⋅16 rpm. Results from optimization studies showed that the HA titre of MEV harvested from spinner cell cultures with the growth medium replaced by serum-free or serum-containing maintenance medium after 24 h of simultaneous cultivation of L12 , S18 , S36 , American strains of MEV in FKC/F81 cells was up to three times greater than the HA titre of the four strains of MEV without replacing the growth medium. No difference in HA titre was observed between cultures maintained in serum-free or serum-containing medium. Stability of the reproductive capacity and titre of S18 , S36 , and L12 strains of MEV from serum-free propagation Mixed MEV fluid of S18 , S36 and American strains at the 22nd passage with HA titre of 8192 (or TCID50 109⋅7 ) and L12 strain MEV fluid at the 2nd passage with the same titre were stored at −30°C. After 8 months, the titre of them all decreased to HA titre of 512 (or TCID50 108⋅8 ), and then the mixed cultures of MEV (S18 , S36 and American strains) and MEV of L12 strain were continuously propagated for three passages in FKC/F81 cells, with the reproductive HA titre of 1024–2048 (or TCID50 108⋅8–9⋅1 ). After preservation for 14–15 months at −30°C, S18 strain MEV at the 22nd passage and L12 strain MEV at the 2nd passage were used for the production of serum-free MEV antigens in batch spinner cultures. The reproductive HA titre of both strains were 2048–4096 (or TCID50 109⋅1–9⋅4 ) in the first two passages. But after the spinner cultivation of

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Table 4. Comparison of the reproductive titre (log2 HA) of MEV fluids harvested respectively from serum-free and serum-positive cell cultures in FKC/C81 cells in pair design tests MEV passage

10 14 15 15 10 11 11 11 12 12 13 11 10 10 12 13 14 14

Seed MEV strain CS in maintaining medium 0⋅5% GluPRBC*

1% GluPRBC

0⋅5% FMRBC† 1% FMRBC

S18 strain passage 9

S36 strain passage 9

− 4 4 3 4 2

+ 3 5 2 3 3

− 4 4 2 4 3

+ 5 5 2 2 3

1 2 1 2 4 3 2 1 4 3

1 2 1 2 3 3 2 — 6 3

2 1 1 2 4 3 2 1 5 4

2 3 1 2 5 4 2 — 7 4

American strain passage 9

Lgold strain passage 1

− 4

+ 4

− 3

+ 2

3 3 1 3 — — — 1 4 3 — — — 2

3 3 4 4 — — — 2 4 3 — — — 1

2 2 1 3 —

2 2 1 2 —

3 2 — — —

2 2 — — —

*GluPRBC represents glutaraldehyde-fixed porcine red blood cell. †FMRBC represents fresh macaque red blood cell. additional 10–20 passages, the reproductive titres of S18 and L12 strains of MEV all decreased to HA titre of 64–512 (or TCID50 107⋅6–8⋅5 ). S18 and S36 strains of MEV respectively mixed from 10 to 15 passages cultured in FKC line of C81 strain

were stored at −30°C. After preservation for 16–17 months, both strains of MEV were used for three passages of the static and spinner cultivation in FKC line of F81 strain, with the reproductive HA titre of 128–512.

Table 5. The reproductive titre (log2 HA) of three strains of MEV by forced-infection cultivation in FKC line of C81 strain Seed MEV strain The passage of MEV by forced-infection cultivation

S18 strain passage 13

S36 strain passage 13

Lgold strain passage 4

14 14 15 15 15 15 16 16

5 3 1 1 1 1 — —

4 2 1 1 1 1 — —

2 — 1 1 — — — —

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After preservation for 21 months, S18 strain of MEV at the 22nd passage (newly selected by alternate cultivation) and L12 strain of MEV at the 2nd passage (newly isolated and identified) were used for the static cultivation in square flasks (capacity 500–1000 ml each) of two passages, and the reproductive HA titres of both strains of MEV were 128 at the 1st passage and 512 at the 2nd passage. In the two years from 1986 to 1988, the stability results of reproductive rate of S18 and L12 strains of MEV in pair design test showed that the two strains of MEV have consistent stability. More than 17 000 litres of serum-free cell-cultured L12 MEV virus has been produced in batches, and the reproductive titre of L12 strain of MEV at the 2nd passage (newly isolated and identified) and S18 and S36 strains of MEV at the 22nd passage (newly selected by alternate cultivation) by the spinner and static cultivation of additional 20–30 passages or so again has been HA titres of 64–8192 (or TCID50 107⋅6–9⋅7 ) or can be generally stable up to HA titres of 256–1024 (or TCID50 108⋅2–8⋅8 ), which is even higher than the previously observed highest reproductive HA titre of 32 (or TCID50 107⋅0 ) of MEV. Discussion Several advances in the technological process for the production of MEV antigens are reported. First, S18 and S36 strains of MEV with excellent HA production capability for vaccine preparation were successfully selected by alternate cultivation in cell lines with very different susceptibility. Second, an L12 strain of MEV with excellent HA production capability for vaccine preparation was successfully isolated from a small number of infected mink with typical clinical symptoms of MVE from the intestine in the field. Third, the optimum reproduction conditions for spinner and static cultivation of MEV were determined, which have been successfully used for the production of MEV antigens in batches on a large scale. Fourth, the serum-free maintenance medium for the cultivation of MEV has been used for large-scale production of MEV antigens successfully since 1986. Fifth, MEV vaccines for S18 , L12 and S36 strains prepared by these methods have been stable so far. When the optimum nutritive media and the optimum conditions for serum-free culture of MEV determined by the author were used for CPV propagation, the reproductive HA titre of CPV in FKC line of F81 strain increased from 128–256

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to 1024–2048, and the HA titre of CPV in canine kidney cell (CKC) line of MDCK strain increased from 128–512 to 1024–2048, even up to 2048–16 384. In addition, laboratory rabbits were vaccinated with live CPV immunogens of high HA titre with Freund’s adjuvant, and lgG antibodies of anti-CPV serum with higher HI titre were successfully prepared. When these optimized conditions with serum-free maintenance medium for MEV propagation were used for the cultivation of FPLV, the reproductive titre of FPLV was also improved markedly. (Q. M. Zhou and X. Z. Xia, personal communication). MEV, FPLV, CPV and RPV have strong similarities in antigenic structure, physicochemical properties, serological response, genomes, pathogenicity and immunity.3–6,8,11,18–25 The use of these culture conditions and media optimized for MEV propagation to improve the cultivation of CPV and FPLV is consistent with the close relationship of these parvoviruses. Perhaps this selection of FPLV, CPV and RPV by alternate cultivation in cell lines with very different susceptibility could also have significant effect on increasing the reproductive capacity of FPLV, CPV and RPV, respectively. The permanent cell line FKC/F81 strain is an optimally susceptible cell strain for MEV cultivation, and FKC/C81 line is not very susceptible to MEV. These facts were utilized to select MEV strains for vaccine preparation by alternate cultivation in cell lines with very different susceptibility. These selected MEV strains propagated efficiently with high HA and TCID50 titres. A set of optimum conditions for the static and spinner cultivation of MEV in FKC/F81 cells was determined by systematic testing. Using these conditions improved the culture titre of MEV, and of CPV and FPLV as well. The serum-free maintenance media can be successfully used for continuous cultivation of MEV without loss of titre over a long period of time, which can be successfully used for cultivation of CPV and FPLV also. The L12 strain of MEV (newly isolated and identified) and S18 strain of MEV (newly selected by alternate cultivation) are the best strains for vaccine preparation. Typical harvest titre by the spinner and static cultivation are r4096–8192 units (or TCID50 Lg9⋅4–9⋅7 ). Production cost of MEV antigens for preparation of inactivated vaccines have been reduced at least 10-fold, and the MEV antigens can be manufactured in batches on a large scale.

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As described in the accompanying paper,26 all the mink over 5 weeks of age, whether healthy or with typical clinical symptoms of MVE, could be inoculated with inactivated vaccines formulated with mineral oil or Al (OH)3 gel adjuvant from serum-free cell culture of MEV. More than 70 litres MEV vaccine with mineral oil adjuvant and more than 18 000 litres MEV vaccine with Al (OH)3 gel adjuvant (about 1700 batches) have been produced since October 1986 and have been used for immunization of animals at mink, raccoon dog, fox, dog and cat farms in 15 provinces and 7 cities of China affected by FPV enteritis. When stored at 2–8°C for up to 6–9 months, both vaccine formulations with titres of HA 64–8192 units or 107⋅6–9⋅7 TCID50 were used at 0⋅3–1-ml doses. Significant decrease of morbidity and mortality of the animals was seen in 3–10 days and no FPV enteritis occurred during the subsequent 6–12 months observation period with the immune protective rate of the vaccines 100% (P Q 0⋅01).15 An optimized means for large-scale cell culture production of MEV using serum-free cell-cultured medium and superior seed viruses has been developed and established. This lays a foundation for the successful preparation of inactivated vaccines from cell cultures of MEV which are very safe and highly effective and also opening up a good way for refinement of FPV vaccines as well. References 1. Parrish C, Carmichael L, Antczak D. Antigenic relationship between canine parvovirus type 2, feline panleukopenia virus and mink enteritis virus using conventional antisera and monoclonal antibodies. Arch Virol 1982; 72: 267–278. 2. Tratschin JD, McMaster GK, Kronauer G, Siegel G. Canine parvovirus: relationship to wild-type and vaccine strains of feline panleukopenia virus and mink enteritis virus. J Gen Virol 1982; 61: 33–41. 3. Barker IK, Povey RC, Voigt DR. Response of mink, skunk, red fox and raccoon to inoculation with mink virus enteritis, feline panleukopenia and canine parvovirus and prevalence of antibody to parvovirus in wild carnivores in Ontario. Can J Comp Med 1983; 47: 188–197. 4. Carman PS, Povey RC. Comparison on viral proteins among canine parvovirus type-2, mink enteritis virus and feline panleukopenia virus. Vet Microbiol 1983; 8: 423–435. 5. Goto H et al. Comparison on biological and physical characteristics among mink enteritis virus, feline panleukopenia virus and canine parvovirus. Jpn J Vet Sci 1986; 48: 1025–1028. 6. Parrish CR, Leather CW, Pearson R et al. Comparison of feline panleukopenia virus, canine parvovirus,

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parvoviruses: a monoclonal antibody study. Vet Microbiol 1988; 16: 219–230. 25. Parrish CR. Mapping specific functions in the capsid structure of canine parvovirus and feline panleukopenia virus using infectious plasmid clones. Virology 1991; 183: 195–205.

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Received for publication 6 February 1996; accepted 11 November 1996