key: cord-0004899-ido065mh authors: Nagy, Éva; Lomniczi, B. title: Polypeptide patterns of infectious bronchitis virus serotypes fall into two categories date: 1979 journal: Arch Virol DOI: 10.1007/bf01315022 sha: b8285bbe914cc646d376053fa2eb50d5bf27c91b doc_id: 4899 cord_uid: ido065mh Molecular weights of six major polypeptides of infectious bronchitis virus (IBV) are: 1. 75,000; 2. 50,000; 3. 45,000; 4. 35,000; 5. 28,000 or 24,000, and 6. 22,000 dalton. According to the mobility of protein 5 the polypeptide patterns of IBV serotypes fall into two main categories. Medical Institute, Helsinki; strain L E D (Connecticut serotype) and strain PV (Massachusetts serotype) were isolated in H u n g a r y (8) . Virus strains were propagated in 10-day-old embryonated eggs and purified at 4 ° C as follows. Virus was clarified at 10,000 × g for 30 minutes and the supernatant pelleted at 59,000 × g for 3 hours in a Type 19 rotor of Beckman L 2 -6 5 B ultracentrifuge. The pellet was gently resuspended in N T E buffer (NaC1 i00 raM, Tris-HC] p H 6.8 10 mM and E D T A 1 mM). After further clarification the virus suspension was pelleted through 30 per cent (w/v) sucrose made up in NTE-buffer in a SW-27 rotor with 100,000 × g for 1 hour. R, esuspended and clarified virus was subjected to a velocity gradient centrifugation for I hour into 10 to 40 per cent (w/v) sucrose. The band (or double band) containing at least half of the virus applied was further purified on a 20 to 60 per cent (w/v) sucrose gradien~ at 80,000 × g for 14 hours. Equilibrium centrifugation alone resulted in virus preparations of insufficient purity as described (15) . Virus peak was recovered b y sedimentation and taken up in sample buffer (50 mI~{ Tris-HC1 p H 6.8, 10 per cent glycerol and 0.001 per cent bromphenol blue). Virus was dissolved in 2 per cent sodium dodecyl sulphate (SDS) and 0,5 per cent 2-mercaptoethanol in a boifing water bath for 3 minutes. Samples were run in 10 per cent slab gels (30:0.4 b y weight acrylamide bis-acrylamide) using a discontinuous buffer system (5) for 14 hours at 50 volt. Gels were fixed in a mixture of methanol, acetic acid and water (5 : 1 : 5) and stained in 0.25 per cent Coomassie blue. Molecular weights were calculated by using the following markers: myoglobin (17,200), ovalbumin (43,000), bovine serum albumin (68,000), Newcastle disease virus (12) and Sindbis virus proteins (13) . All IBV strains examined showed one of two protein patterns with polypeptides having apparent molecular weights in the range of 20,000 and 110,000 dalton (Fig. 1) . Six major polypeptides were observed in all strains: VP75 (virion polypeptide of 75,000 dalton), VP50, VP45, VP35, VP28 or VP24 (depending on the serotype) and VP22. Minor polypeptides were also noted (VP 110, VP48) but their viral origin is uncertain. The viral origin of VP45 is also doubtful for two reasons: a) there was a tendency of reduction of the relative amount of VP45 when velocity gradient ccntrifugation was combined with equilibrium gradient purification, h) this protein is equally present in preparations of egg-grown Sindbis virus which have been purified only by pelleting the virus under a 30 per cent sucrose layer, which also suggest that it may be a cellular contaminant (Fig. 1) . According to the variation in the mobility of a "broad" polypeptide at least two basic polypeptide patterns were recognized (Fig. 2) . Pattern M (named after Massachusetts) includes strains from two serotypes, Massachusetts and Georgia (SE 17), and here the "broad" protein has an apparent molecular weight of 28,000 dalton. Four strains (Beaudette, M41, PV, and Lerida) belonging to the Massachusetts serotype all showed identical M-pattern (PV is not shown). Pattern C (named after Connecticut) is characterized by a smaller "broad" protein of an apparent molecular weight of 24,000 dalton, and includes the five remaining serotypes studied: Connecticut, Delaware (Gray), Iowa 97, Iowa 609 and Australia T. ]~VA NA.GY and B. LO~IOZI: Three Connecticut strains (one prototype strain from Weybridge, another from Helsinki and LED) also exhibited identical C-pattern. Apart from variation in the mobility of the "broad" protein it was found that in strain Gray and LED VP22 was prominent relative to the other strains. Whether this suggests a closer relationship of the two strains, or an artifact, is presently unknown. It is to be noted, that there was no difference between the polypeptide patterns of heated and unheated samples. The polypeptide patterns presented here classify IBV scrotypes into at least two main categories but no known biological properties common in serotypes belonging to a particular pattern can so far be identified. Our results seem to be at variance with those of formerly published investigations (10) in two respects: a) molecular weights of the majority of the polypeptides in that study fall in the range of 33,000 to 130,000 dalton, although using harsher reducing conditions resulted in polypeptides smaller than 33,000 dalton; b) no significant difference was found between the protein pattern of strain Beaudette and Connecticut. The polypeptide patterns of IBV presented here, however, show a partial similarity to that published by COLLI~S et el. (2) , and a remarkable similarity to that obtained after a different purification method by LASCER and I-IowAgD (6) . The similarity with pattern obtained in the latter works resides in the presence of smaller than 33,000 dalton proteins in the virions of IBV. The polypeptide composition of avian infectious bronchitis virus Heterogeneity of infectious bronchitis virus grown in eggs Antigenic variation in strains of avian infectious bronchitis virus Serological comparison of strains of infectious bronchitis virus using plaque-purified isolants Cleavage of structural proteins during the assembly of bacteriophage T4 Characterization of infectious bronchitis virus Serologic differences between strains of infectious bronchitis virus from New ZeMand, Australia, and the United States Isolation of the virus of infectious bronchitis Genome of infectious bronchitis virus The polypeptide composition of avian infectious bronchitis virus particles Coronaviruses: a comparative review Characterization of the strueturM proteins of different strains of Newcastle disease virus Reproduction of togaviruses Presence of infectious polyadenylated I%NA in the eoronavirus avian bronchitis virus The evaluation of AKR leukemia virus purity: The requirement for both velocity and isopycnie eentrifugation methods The skillful technical assistance of Mrs. Sz. Sz6esy is appreciated.Authors' address: Dr. ]~vA NAGY, 1581 Budapest, P.F. 18, Hungary.1%eeeived February 26, 1979