key: cord-0010209-z70fxibe authors: Shibuta, Hiroshi; Kanda, Tadahito; Adachi, Akio; Yogo, Yoshiyuki title: Characterization of Bovine Parainfluenza Virus Type 3 date: 2013-11-14 journal: Microbiol Immunol DOI: 10.1111/j.1348-0421.1979.tb00502.x sha: 0fd7f743bb31fcf944f2cd0ba4f6969fc61532d6 doc_id: 10209 cord_uid: z70fxibe Bovine parainfluenza virus type 3 (PIV‐3) has a buoyant density of 1.197. The RNA of PIV‐3, like that of Sendai virus, is a single continuous chain which lacks polyadenylic acid sequences and tends to self‐anneal to a marked extent. It has a sedimentation coefficient of 42S and a molecular weight of 4.5 × 10(6), being slightly smaller than Sendai virus RNA (47S, 5.3 × 10(6)). PIV‐3 has 5 main structural proteins, of which 2 are glycoproteins. The molecular weights of protein(1), protein(2), protein(3), glycoprotein(1), and glycoprotein(2) were estimated to be 79,000, 68,000, 35,000, 69,000, and 55,000, respectively. Protein(2) was suggested to be nucleocapsid protein. and proteins (6, 8, 16, 18, 19, 22, 23, 31) , and therefore, have evoked much interest in researches of other remaining members of the paramyxovirus group. This communication describes a number of similarities between bovine parainfluenza virus type 3 (PIV-3), another member of the paramyxovirus group, and Sendai virus in their RNA and structural proteins. The most marked difference was that PIV -3, unlike Sendai virus, could form plaques in Vero and MDBK cells without the aid of trypsin incorporated into agar overlay. Viruses. Four strains of PIV-3, passaged several times in primary bovine kidney cell cultures, were kindly supplied by Dr. Y. Inaba, National Institute of Animal Health, Kodaira, Tokyo. These strains grew well and formed plaques in Vero cells, a stable cell line derived from an African green monkey kidney, and Mardin-Darby bovine kidney (MDBK) cells. A small amount of trypsin in agar overlay, an essential factor for Sendai virus plaquing in Vero and MDBK cells, did not favor the plaquing of PIV-3 but decreased the number and size of plaques to about one half. Among the 4 strains, Shimane 910N showed homogeneous plaques and therefore was used as a representative strain in this study. The virus received serial passages in ET Buoyant Density of PIV -3 PIV -3 labeled with 3H-leucine was concentrated from culture fluids by polyethylenglycol precipitation, as described in Materials and Methods, and sedimented through a linear sucrose density gradient (20 to 55% w/v in NTE buffer) at 25,000 rpm for 16 hr at 4 C in a Spinco SW 27 -1 rotor. As presented in Fig. 1, a sharp peak of infectivity coincided with the peak of hemagglutinating activity and that of TCA-insoluble radioactivity, being located at the density of 1.197 which was very similar to the value reported by Tsai and Thomson (32 ) . An electron microscopic study of the peak revealed roughly spherical virus particles carrying spikes on the surface, sharing common features with other viruses of the paramyxovirus group (Fig. 2) and resembling closely the electron micrographs of PIV -3 presented by Waterson et al (33 ) . The size of particles ranged from 80 nm to 300 nm in diameter with a mean value of 130 to 140 nm. The virus in the peak formed a band at the top of the 47% sucrose layer in the discontinuous sucrose density gradient used for virus purification. Occasionally PIV -3 formed another minor band between the 47% and 52% sucrose layers (lower band), depending on each virus preparation. The lower band and 3H-glucosamine in another gel. These figures show that PIV-3 has at least 5 proteins, of which 2 are glycoprotein. We designated the PIV-3 proteins as P1, P2, P3, glycoproteini (GP1), and GP2. Analysis of Triton X-100-treated samples electrophoresed in other slots of the gel identical to that of Fig. 6a revealed that the radioactivity of 3H-glucosamine of GP1 remained entirely in Triton-sup (see Materials and Methods) (Fig. 6b) and did not sediment with Triton-ppt (Fig. 6c) , whereas Triton-ppt showed a single sharp peak of 14C-radioactivity of P2 (Fig. 6c) . These facts suggested that GP1 was an envelope-associated glycoprotein and P2 nucleocapsid protein (22 ) . The insufficient recovery of GP2, P1, and P3 after Triton X-100 treatment could not be explained but was supposed to be due to loss of these proteins dur- ing repeated washing with solvents. Periodic acid-Schiff (PAS) staining of gels confirmed that GP1 and GP2 were glycoproteins whereas Pi, P2, and P3 were not (not shown). From the results, the molecular weights of PIV-3 structural proteins were calculated, comparing their migration rates with those of Sendai virus proteins of known molecular weights (8) . Thus, P1, P2, P3, GP1, and GP2 of PIV-3 were estimated to be 79,000, 68,000, 35,000, 69,000, and 55,000 daltons, respectively. in vitro RNA synthesis that Poly (A ) of the poliovirus genome is genetically coded On the classification bovine parainfluenza 3 viruses Trypsin action on the growth of Sendai virus in tissue culture cells. I. Restoration of the infectivity for L cells by direct action of typsin on L-cell borne Sendai virus Trypsin action on the growth of Sendai virus in tissue culture cells. II. Restoration of the hemolytic activity of L cell-borne Sendai virus by trypsin Trypsin action on the growth of Sendai virus in tissue culture cells. III. Structural difference of Sendai viruses grown in eggs and tissue culture cells Restoration of the fusion activity of L cell-borne Sendai virus by trypsin A proposal for designation of Sendai virus protein Molecular weight determination of Sendai virus RNA by dimethylsulfoxide gradient sedimentation Self-annealing of Sendai virus RNA Molecular weight determination of Sendai and Newcastle disease virus RNA Brain tumors induced with Rous sarcoma virus, Schmidt-Ruppin strain. I. Induction of brain tumor in adult mice with Rous chicken sarcoma cells Molecular weights of ribosomal RNA in relation to evolution The polypeptide of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of type 2, 7A and 12 1975. Negative strand viruses Measles virus and its associated diseases Effect of trypsin on reproduction of type 4 parainfluenza in Vero cell cultures under fluid overlay Proteins and glycoproteins of paramyxoviruses : a comparison of simian virus 5, Newcastle disease virus, and Sendai virus Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus Complementary RNAs in paramyxovirions and paramyxovirus-infected cells Self-annealing of subgroup 2 myxovirus RNAs Isolation of paramyxovirus glycoprotein. Association of both hemagglutinating and neuraminidase activities with the larger SV5 glycoprotein Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity by proteolytic cleavage of an inactive precursor protein of Sendai virus Replication of Vesicular stomatitis virus. II. Separation and characterization of virus-specific RNA species Animal RNA viruses : genome structure and function Detection of polyadenylic acid sequences in viral and eukariotic RNA Plaque formation by Sendai virus of parainfluenza virus group, type 1 on Monkey, calf kidney and chick embryo cell monolayers Effect of trypsin on the infectivity of Sendai virus grown in several host cells Experimental parainfluenza infection. I. Hydrocephalus of mice due to infection with parainfluenza virus type 1 and type 3 Requirement of 3'-terminal poly (adenylic acid) for the infectivity of poliovirus RNA Structural components of Sendai virus. Serological and physicochemical characterization of hemagglutinin subunit associated with neuraminidase activity Bovine parainfluenza type 3 virus infection: Virus replication in bovine embryo cell cultures and virion separation by rate-zonal centrifugation The structure of parainfluenza 3 virus Polyadenylate in the virion RNA of mouse hepatitis virus