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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 RK13 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 RK13 cells with heparinase.

Heparin inhibited haemagglutination by porcine reproductive and respiratory syndrome virus (PRRSV) and by Aujesky's disease virus, but failed to inhibit haemagglutination by parainfluenza virus type 3. The minimal inhibitory concentration of heparin required to inhibit 8 HA U of PRRSV haemagglutinin ranged from 0.1 to 1 U ml-1. Mouse erythrocytes failed to combine with the haemagglutination inhibitory factor of heparin. However, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by PRRSV. The formation of a haemagglutinin-heparin complex could be observed by sedimenting heparin with the haemagglutinin. All these findings suggest that a heparin-like molecule on the surface of mouse erythrocytes serves as the virus-cell receptor.

Objective: To investigate whether heparin has any effect on the growth of porcine reproductive and respiratory syndrome virus (PRRSV)., Sample Population: 2 isolates of PRRSV, and as control viruses, 1 isolate of pseudorabies virus (PRV) and 1 isolate of parainfluenza 3 virus (PIV-3)., Procedures: Plaque assays, using a continuous cell line (MARC-145) derived from African green monkey kidney cell line (MA104), were performed for determination of inhibitory effect of heparin on PRRSV, PRV, and PIV-3. The effect of various doses of heparin and heparinase on the growth of PRRSV, PRV, and PIV-3 was evaluated and compared. In each experiment, value were expressed as the mean value for duplicate samples., Results: The number of plaques formed by PRRSV and PRV was reduced to 24 to 25 and 15% of the untreated control (100%), respectively, by 1 U of heparin/ml, but could not be reduced below 6 to 7 and 3%, respectively, by use of concentrations up to 50 U/ml. An inhibitory effect of heparin, at a concentration up to 50 U/ml, was not observed on PIV-3. Delaying addition of heparin for 30 minutes after the addition of PRRSV and PRV reduced plaque formation by 48 to 51 and 68%, respectively, compared with 91 to 92 and 95%, respectively, if heparin was added at the time of infection. In addition, most PRRSV added was retained by heparin beads, as was PRV. Heparinase treatment of MARC-145 cells reduced the number of PRRSV-, as well as PRV-induced plaques. On the other hand, the number of PIV-3-induced plaques did not decrease after treatment of MARC-145 cells with heparinase., Conclusions: Addition of heparin to PRRSV or to the MARC-145 cells before virus inoculation and treatment of the cells with heparinase prevented the virus from infecting the cells.

Porcine reproductive and respiratory syndrome virus hemagglutinin (HAin) was readily adsorbed on mouse erythrocytes at 4, 22, or 37 degrees C, but not on goose erythrocytes. The adsorbed HAin could not be eluted from the cells by resuspending in phosphate buffered saline, by incubating at 37 or 50 degrees C, or by incubating in the presence of neuraminidase. The hemagglutinating activity was not dependent on the pH and NaCl molarity tested. The receptor of mouse erythrocytes for the HAin was relatively stable to trypsin, neuraminidase, sodium deoxycholate (DOC), potassium periodate (KIO4), dithiothreitol (DTT), 2-mercaptoethanol (2-ME) and formalin treatments. The HAin was inactivated by 2-ME and was gradually inactivated by pepsin, formalin and DTT, but not by beta-glucosidase, trypsin, alpha-amylase, papain, phospholipase C, neuraminidase, KIO4, and ethylendiamine tetraacetic acid (EDTA) treatments. The HAin was stable at 37 degrees C or lower temperatures, but not at 56 degrees C or higher. The HAin was relatively resistant to ultraviolet irradiation and sonication. In the equilibrium centrifugation of the HAin preparation on a CsCl density gradient, the HAin activity showed a sharp peak at 1.17 g/cm2. In the SDS-PAGE analysis, the structural polypeptide of HAin in the peak fraction seems to be the nucleocapsid (N) polypeptide with molecular weight of 15 kDa.

Various conditions were evaluated and modified to enhance the sensitivity of the neutralization (NT) test for detecting antibody in swine infected with porcine reproductive and respiratory syndrome (PRRS) virus. Higher NT antibody titers were consistently obtained by the addition of 10% (v/v) complement, fresh guinea pig serum, to the virus diluent and by the incubation of serum-virus mixture at 4 degrees C for 24 hr. The appearance and persistence of antibodies detected by the modified NT test showed a similar pattern to those of antibodies detected by the indirect fluorescent antibody (IFA) assay, although the antibody titers obtained by the former method were consistently lower than those obtained by the latter method. Slow-reacting complement-requiring NT antibody was detected in sera from pig 2 weeks after infection with PRRS virus. The slow-reacting complement-requiring NT antibody in the early serum samples was sensitive to 2-mercaptoethanol (2-ME), whereas the slow-reacting complement-requiring NT antibody in the late serum samples was resistant to 2-ME. The initial phase may represent the IgM response and the later phase a change to IgG. A NT test was developed in which serum-virus mixtures were incubated at 4 degrees C for 24 hr with complement; this gave an improved sensitivity over the previous incubation at 37 degrees C for 60 min.

Porcine reproductive and respiratory syndrome (PRRS) virus grown on MARC-145 cell cultures was tested for hemagglutination (HA) with erythrocytes from a variety of species at 4 degrees C, room temperature and 37 degrees C. HA was observed at all temperatures with mouse erythrocytes but not with cattle, sheep, goat, horse, swine, guinea pig, mongolian gerbil, goose and chicken erythrocytes. The HA activity was enhanced by treatment of virus materials with Tween 80 followed by treatment with ether. The HA titer and HA pattern of virus materials treated with Tween 80 and ether (TE) were 4- to 8-fold higher and more clear than those of the virus materials without TE treatment. The optimum conditions consisted in pretreatment of virus material with Tween 80 at a final concentration of 0.06-0.125% (v/ v) for 15-60 min followed by treatment with ether at a concentration of 50% (v/v) for 5-15 min in ice bath with continuous shaking. The curve of active virus production in intra- and extracellular virus samples resembled that of HA production although it rose somewhat earlier in intracellular virus samples. The HA reaction was inhibited by specific antiserum. HI antibody titers of individual pig sera showed a significant positive correlation with their neutralizing antibody titers.

Heparin inhibited the hemagglutinin activity of Akabane and Aino viruses. The minimal inhibitory concentration of heparin required to inhibit 8 hemagglutination (HA) U of Akabane and Aino viruses was 10 U/ml. Goose erythrocytes failed to combine with the HA inhibitory factor of heparin. On the other hand, goose erythrocytes treated with heparinase had greatly reduced agglutinability by Akabane virus. Virus-heparin complex formation was observed by sedimenting heparin with the virus particles.

Kasba virus grown in BHK21-WI2 cells was tested for hemagglutination (HA) with erythrocytes of a variety of species at 4 degrees C, 25 degrees C and 37 degrees C. HA was observed at all temperatures with cattle, sheep and goat but not with swine, chicken, and goose erythrocytes. The HA was dependent on not only the NaCl concentration but also the pH of the diluent. The HA titer significantly improved by increasing the NaCl molarity to 0.6 M and standardizing pH to 7.5. The HA titer was 16- or 32-fold higher in 0.4 M or 0.6 M solution than in 0.2 M solution of not only NaCl but also several other salts. The HA reaction was inhibited by specific antibody.