Characterization of Porcine Reproductive and Respiratory Syndrome Virus Hemagglutinin.

Abstract

ABSTRACT. Porcine reproductive and respiratory syndrome virus hemagglutinin (HAin) was readily adsorbed on mouse erythrocytes at 4, 22, or 37°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°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 (KIO 4 ), 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 β-glucosidase, trypsin, α-amylase, papain, phospholipase C, neuraminidase, KIO 4 , and ethylendiamine tetraacetic acid (EDTA) treatments. The HAin was stable at 37°C or lower temperatures, but not at 56°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/cm 3 . 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. bovine serum albumin (BSA; Fraction V) and antibiotics as above. Preparation of HAin:The HAin was prepared by the method described previously [4], with slight modification. Briefly, the infected culture fluid of MARC-145 cells was concentrated by adding 2% of 1 M zinc acetate solution and adjusted to pH 7.2 with 1 M NaOH. The mixture was stirred constantly at 4°C for 1 hr. After low-speed centrifugation the pellet was suspended in 1/50 the original volume of saturated solution of ethylenediamine tetraacetic acid (EDTA), adjusted to pH 7.2 by the addition of solid Tris (hydroxymethyl) aminomethane (Wako, Japan). The resulting solution was clarified by low-speed centrifugation and the supernatant was layered on 20% (w/v) sucrose solution and centrifuged at 100,000 × g for 4 hr (SW40Ti rotor Beckman, U.S.A.). The pellet was resuspended in 1/ 100 the original volume of PBS (0.15 M NaCl, 0.02 M phosphate buffer, pH 7.2) containing 0.2% BSA (PBS-BA) and then pretreated with 0.06% (v/v) of Tween 80 followed by 50% (v/v) of ether (Diethylether, Wako, Japan). The aqueous phase was used as HAin after the remaining of ether was sacked off and discarded. The HAin was stored at 4°C or 80°C before used.Preparation of pig immune serum: A 13-day-old pig obtained from the specific-pathogen-free farm was inoculated intranasally with 10 3.5 TCID 50 of the EDRD-1 strain of PRRSV grown on primary porcine alveolar macrophages. Serum samples were taken immediately before inoculation and at 6 weeks after inoculation for immunoblotting assay.HA test: HA test was carried out by microtiter method using ddY mouse erythrocytes as described previously [4].

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

“…These findings, together with our previous findings with PRRSV [3], indicated no evidence that heparin interacted with RK 13 cells, suggesting that heparin inhibits the growth of EAV by direct interaction with its virions, rather than by interaction with RK 13 cells. In addition, our results indicated that heparinase treatment of RK 13 cells reduced the binding of EAV to the cells and rendered the cells resistant to plaque formation.…”

“…Recently, we reported that the growth of bovine ephemeral fever, Kimberley, and Berrimah viruses of the family Rhabdoviridae, genus Ephemerovirus, Akabane virus of the family Bunyaviridae, genus Bunyavirus, and porcine reproductive and respiratory syndrome virus (PRRSV), a member of the genus Arterivirus [5] were inhibited by heparin [3,7].…”

“…Although heparin has been known to inhibit infection of cultured cells by herpesviruses [4,6,[8][9][10], some arboviruses, including ephemeroviruses and bunyaviruses [3,7], and PRRSV [3], its inhibitory activity on EAV has not been reported to the authors' knowledge. In this study, EAV was also inhibited by heparin in RK 13 cell cultures when the virus was mixed with heparin before its inoculation.…”

Label‐Free Quantitative Proteomic Analysis of Differentially Expressed Membrane Proteins of Pulmonary Alveolar Macrophages Infected with Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus and Its Attenuated Strain

“…String network analysis was used to elucidate interactions among differentially expressed proteins. The essential factors and receptors involved in the entry of PRRSV are reportedly CD163 (Q2VL90), nmHC II‐A (MYH9, F1SKJ1), sialoadhesin (SN, A7LCJ3), CD151 (http://F1RYZ1), and vimentin (VIM) (P02543) . We assessed the involvement of these MPs together with proteins detected by string analysis on viral entry.…”

Development of an epitope-based marker system for use in categorizing porcine reproductive and respiratory syndrome viruses

“…Pretreatment of the virus with Tween 80 and ether greatly increased hemagglutination. Moreover, Jusa et al (1997a) found that the hemagglutinin activity peaked at 1.17 g /cm^ in cesium chloride gradient centrifiigation. The hemagglutinin peak fraction was demonstrated in the SDS-PAGE to be composed of the N protein.…”