Effect of Heparin on Infection of Cells by Equine Arteritis Virus.

Abstract

ABSTRACT. The number of plaques formed by equine arteritis virus (EAV) and Aujesky's disease virus (ADV) was reduced to 14% and 5% of the untreated control(100%), respectively, by 10 U/ml of heparin, but could not be reduced below to 13 and 4%, respectively, by use of concentration up to 100 U/ml. An inhibitory effect of heparin, at concentration up to 100 U/ml, was not observed on parainfluenza virus 3 (PIV-3). Heparinase treatment of RK 13 cells reduced the number of EAV-, as well as ADV-induced plaques. On the other hand, the number of PIV-3 induced plaques did not decrease after treatment of RK 13 cells with heparinase. -KEY WORDS: equine arteritis virus, heparin, inhibitory effect.J. Vet. Med. Sci. 59(8): 727-728, 1997 incubator at 37°C for 4 days for EAV and ADV, and 6 days for PIV-3, the cultures were fixed by addition of 10% formalin in phosphate buffered saline solution (PBS, pH 7.2) and stained with 0.5% crystal violet solution for counting plaque-forming units (PFU Each virus (containing approximately 200 PFU) of EAV, ADV or PIV-3, was mixed with an equal volume of 0.02, 0.2, 2, 20, and 200 U/ml of heparin of PBS containing 1% BSA (PBS-BSA; pH 7.2), respectively. The mixtures were immediately inoculated onto monolayers of RK 13 cells in plastic dishes. The monolayers were incubated at 37°C for 1 hr. They were then washed twice with PBS and flooded with the overlay medium. Controls using PBS instead of heparin were prepared, inoculated, and assayed in a similar manner.Heparin inhibited plaque formation of RK 13 cells by EAV as well as did ADV (Fig. 1). Both viruses were affected by heparin treatment, but EAV was less sensitive to heparin than was ADV. The number of plaques formed by EAV was reduced to 21 and 14% of the untreated control (100%), by a final concentration of 1 and 10 U/ml of heparin, respectively, but could not be reduced below 13% even with concentration up to 100 U/ml, whereas the number of plaques formed by ADV was reduced to 15 and 5% of the untreated control value by 1 and 10 U/ml of heparin, respectively, and was reduced to 4% by 100 U/ml of heparin. At heparin concentration of 1 to 10 U/ml, a dose-dependent inhibition of plaque formation was observed for both viruses. To the contrary, an inhibitory effect of heparin on PIV-3, even at concentration up to 100 U/ml, was not observed.When RK 13 cells were treated with heparinase before virus inoculation, the increased concentration of heparinase resulted in the decreased number of plaques formed by EAV and ADV (Fig. 2). However, the decrease of plaque number was not remarkable, compared with that of plaque number by the heparin-treated virus. This finding suggested that all

Effect of Heparin on Hemagglutination by Equine Arteritis Virus.

“…These results do appear to corroborate our findings that growth of EAV is inhibited by heparin [1], therefore, it is possible that the cellular receptor for EAV hemagglutinin is a heparin-like molecule.…”

“…Because all our findings, together with our previous results [1], demonstrated that EAV infection was inhibited by heparin, heparinized blood must be regarded as not an ideal specimen for the virus isolation attempts.…”

“…Recently, we also found that the growth of EAV was inhibited by heparin [1]. It was, therefore, of interest to test the effect of heparin on EAV hemagglutination.…”

Hemagglutination with Equine Arteritis Virus.

“…The cells were grown in Eagle's minimum essential medium (MEM) supplemented with 10% tryptose phosphate broth, 5% heat-inactivated calf serum, 0.12% NaHCO 3 , and 100 units of penicillin, 100 µg of Kanamycin, and 2 µg of fungizone/ml. The maintenance medium (MM) was MEM supplemented with 0.5% lactalbumin hydrolysate, 0.2% bovine serum albumin(BSA), 0.12% NaHCO 3 , and antibiotics as described previously [1].…”

Equine viral arteritis: Current status and prevention

“…Heparinized blood is not suitable for virus isolation because of the inhibitory effect of heparin on the isolation of EAV in cell culture [71]. Collection of semen containing the sperm rich fraction of the ejaculate is optimal for virus isolation from equine semen samples [15].…”