key: cord-342151-1e6x589e authors: Talbot, Pierre J. title: Hemagglutination by Murine Hepatitis Viruses date: 2008-07-29 journal: Intervirology DOI: 10.1159/000150083 sha: doc_id: 342151 cord_uid: 1e6x589e Erythrocytes from twelve mammalian and avian sources in ten different buffers at three incubation temperatures could not be hemagglutinated with murine hepatitis virus (MHV) strains 3, A59, or S grown on DBT cells. Viral antigen preparation in the absence of fetal calf serum, partial virus purification, or various concentrations of red blood cells still failed to yield detectable hemagglutinating activity. Thus, the newly described MHV-DVIM remains the only hemagglutinating strain of murine coronavirus. Hemagglutination represents a useful model for virus adsorption to cells, a very convenient assay for the presence of virus in biological samples, and may be relevant to viral pathogenesis. Several members of the Coronaviridae family of enveloped RNA vi ruses were shown to possess such a hemag glutinating activity: namely bovine coronavi rus, hemagglutinating encephalomyelitis vi rus of pigs, human coronavirus strain OC43, rabbit enteric coronavirus, and avian in-fectious bronchitis virus [1] . Recently, a hem agglutinin was demonstrated on transmissi ble gastroenteritis virus [2] . However, hemag glutination by murine coronavirus had not been reported until Sugiyama and Amano [3] identified a new enteric strain of murine hep atitis virus (MHV): the diarrhea virus of in fant mice (DVIM). Like bovine coronavirus [4] and strain OC43 of human coronavirus [5] , the hemagglutinating activity of MHV-DVIM appeared to correlate with the pres ence of a structural glycoprotein of 140 kD, comprised of 2 subunits of 65-69 kD linked by disulfide bridges [6] , This presumed hem agglutinin appears distinct from the peplomer glycoprotein (named E2, S, or P) of 180-190 kD [5, 6] and its proteolytically cleaved monomers of 100-120 kD [4, 5] . On * * Buffers as described in the text and incubation at 4,22, or 37 °. The same result was obtained at any of these temperatures. b Prepared as described in the text and used at 0.3 or0.5%(v/v). The same result was obtained at any of these concentrations. c Grown in serum-free medium and concentrated 90-fold with polyethylene glycol. d Not done. c Grown with or without serum and used as clarified medium. ' Semipurified, as described in the text. the other hand, the hemagglutinating activity of infectious bronchitis virus [7, 8] and trans missible gastroenteritis virus [2] was local ized on the unique peplomer glycoprotein. Thus, the preliminary report of hemaggluti nation by another strain of MHV (MHV-3; grown in murine liver and semipurified on sucrose gradient) [9] , which lacks structural proteins of 140 and 65-69 kD [ The origin and culture of MHV strains 3, A59, and S and of DBT cells were described previously [14] . For use as antigen for hemag glutination assays, MHV-3 was grown in the absence of fetal calf serum (FCS) and har vested from clarified medium by precipita tion with 10% (w/v) polyethylene glycol in 0.5 M NaCl. After centrifugation at 10,000 g for 30 min, the pellet was resuspended and dialyzed against TMEN buffer: 50 mM Tris-HCi (pH 6.2), 0.1 M NaCl, 1 m M EDTA. MHV-A59, which replicates in vitro more efficiently than any other strain of M H V, was grown with or without 1% (v/v) FCS and used mouse CDI C57B1/6 BALB/C day-old chick rooster -- directly in the form of medium freed of cell debris by centrifugation at f0,000 g for 20 min. Finally, MHV-S was grown in me dium containing 2% (v/v) FCS, concentrated as described above, and partially purified on a discontinuous 10 and 50% (w/v) Nyco-denz® (Nyegaard, Oslo, Norway) gradient centrifuged for 4 h at 25,000 rpm in an SW28 rotor (Beckman Instruments, Fullerton, Cal if., USA). The virus band was dialyzed against TMEN buffer. All viral antigens had infectious titers of 6.0-7.3 logm PFU/ml. Red blood cells from various mammalian and avian sources were collected and washed in Alsever buffer [15] and resuspended in the appropriate buffers. Ten different buffers were tested: (v) phos phate-buffered saline, pH 7.4 [17] ; and (vi). phosphate-buffered saline with 1% kaolintreated FCS [18] , and (7-10) BABS, pH 5.75, 6.0, 6.2, or 6.4, respectively [16] , The hemag glutination assay was performed as de scribed previously [18] , except that various buffers were used, red blood cells were made up to 0.3 or 0.5% (v/v) in the appropriate buffer, and microtiter plates were incubated at 4, 22, or 37°. Control hemagglutinating antigens were either rabbit enteric coronavirus (titer 1/64 with rabbit red blood cells ) or pneumonia virus of mice (titer 1/320 with CDI mouse erythrocytes). Both antigens were kindly provided by Dr. Jean-Paul Descoteaux (Institut Armand-Frappier). As shown in table 1, all tested combina tions of buffer, source or concentration of erythrocytes, antigen, or incubation temper atures yielded negative hemagglutination ti ters. One antigen (MHV-A59) was prepared in the presence or absence of FCS to monitor for the possible presence of hemagglutina tion inhibitors in serum [16] , However, simi lar results were obtained with either antigen (table 1). Concentrated or partially purified MHV-3 and MHV-S antigens, respectively, were used to both increase viral infectivity titers and prevent reported inconsistent hem agglutination by crude antigen [9] . Even though not all possible combinations of anti gen, erythrocyte, and buffer were tested, the results presented here show that hemaggluti nation by these three strains of MHV grown on DBT cells is at least not an obvious biolog ical activity, unlike MHV-DVIM [3] . It is possible that the previously reported hemag-glutinating activity of MHV-3 [9] was de pendent on the different host used for virus growth or the virus purification method. Fi nally, it can be concluded that the 65-kD structural protein observed under reducing conditions in polyacrylamide gels of purified MHV-S [6,12; PJT, unpubl. data] apparently is not a viral hemagglutinin. The molecular biology ofcoronaviruses Hemagglu tination with transmissible gastroenteritis virus Hemagglutination and structural polypeptides of a new Coronavirus as sociated with diarrhea in infant mice Bovine Coronavirus hemagglutinin protein Structural proteins of hu man respiratory Coronavirus OC43 Structural polypeptides of the murine Coronavirus DVIM Coronaviruses IBV: Virus retaining spike glycopolypeptide S2 but not SI is unable to induce virusneutralizing or haemagglutination-inhibiting an tibody or induce chicken tracheal protection Coronavirus IBV: Remov al of spike glycopolypeptide SI by urea abolishes infectivity and haemagglutination but not attach ment to cells Haemagglutination by mouse hepatitis virus type 3. Ann Virol (Inst Pas teur Comparison of polypeptides of two strains of murine hepatitis virus RNA and polypeptide homology among murine coronaviruses Comparative analysis of RNA genomes of mouse hepatitis viruses Analysis and localization of mouse hepatitis virus 3 (M H V-3)polypeptides Physico-chemical properties of murine hepatitis virus, strain A59 Effect of several salts on rubella virus hemaggluti nation Techniques for haemagglu tination inhibition with arthropod-borne viruses Plaque formation and isola tion of pure lines with poliomyelitis virus Hemag glutination activity with bovine herpesvirus type I The expert technical assistance of Francine Lam bert, manuscript preparation by Lucie Summerside, and red blood cell preparations by Michel Boivin and Claude Duhamel are gratefully acknowledged. This work was supported by a grant (U0387) and a scholar ship from the Natural Sciences and Engineering Re search Council of Canada.