R. Rott

Activation of influenza A viruses by trypsin treatment

Abstract A comparative analysis has been carried out on the infectivity of virus of several influenza A strains grown in different host systems. Strains A/swine/Shope/31 (Hsw1N1), A/PR/8/34 (HON1), A/FM/1 (H1N1), A/Singapore/1/57 (H2N2), A/equine/Miami/1/63 (Heq2Neq2), and A/chick/Germany/49 (Hav2Neq1) exhibit host-dependent differences in infectivity. Virions grown in embryonated eggs and cultures of chorioallantoic membrane cells are highly infectious, whereas virions grown in cultures of chick embryo cells have a low infectivity that significantly increases after treatment in vitro with trypsin. In contrast, fowl plague viruses do not show host-dependent variations in infectivity. Virions grown in all host systems tested are highly infectious, and the infectivity of virions grown in chick embryo cells cannot be enhanced by trypsin treatment. The activation of virus particles appears to be based on the cleavage of hemagglutinin glycoprotein HA. This concept is supported by the following observations: (i) In virions of low infectivity only uncleaved glycoprotein HA can be detected. Virions of high infectivity exhibit complete or at least partial cleavage of the hemagglutinin. (ii) The activation of virions by trypsin treatment is always paralleled by cleavage of HA. (iii) Cleavage of HA is the only effect which can be detected after trypsin treatment. The neuraminidase is neither inactivated nor removed from the virion. (iv) Studies on recombinants of virus N and fowl plague virus (Rostock) show that host-dependent variation of infectivity and activation by trypsin, features specific for parent virus N, are found only with recombinant N(H)-FPV/Ro(N) but not with recombinant FPV/Ro(H)-N(N). Efficient plaque formation and serial passages are possible only if highly infectious particles are formed in a given host system. Thus, all strains analyzed undergo, in the absence of trypsin, successive growth cycles in eggs and chorioallantoic membrane cells and form plaques in chorioallantoic membrane cells. In contrast, in chick embryo cells only viruses containing the fowl plague virus hemagglutinin produce plaques and replicate under multiple cycle conditions without the addition of trypsin. The data show that cleavage of HA is not a precondition for virus assembly and hemagglutinating activity, but that it is necessary for infectivity. These findings are compatible with the hypothesis that, in addition to its role in adsorption, the hemagglutinin has another function in the infection process and cleavage is required for this function.

On the origin of the human influenza virus subtypes H2N2 and H3N2

The RNA of the human influenza virus Singapore (H2N2) strain has been labeled in vivo by phosphorus-32 and separated by polyacrylamide gel electrophoresis into eight segments, which were correlated to the corresponding gene functions and/or proteins. The base sequence homology between the individual genes (segments) of the H2N2 virus and those of different influenza A strains has been determined by molecular hybridization. Segments 1, 5, 7, and 8 of the Singapore strain exhibit a base sequence homology of almost 100% as compared to the FM1 strain (HlNl), while the homology between the other segments is significantly lower (24–76%). For the Singapore and Hong Kong (H3N2) strains all segments except that coding for the hemagglutinin (HA, 24%) exhibit a homology close to 100%. The 32P-labeled segment 4 (HA-gene) of the avian influenza A strain duck Ukraine (Hav7Neg2) shows a homology of 92% to Hong Kong, while the homology of at least two other segments is significantly lower. These results are taken as an indication that the H2N2 subtype is derived from the HlN1 subtype by a recombination event retaining four H1N1 segments, while the other four segments are gained from another yet unknown strain. The H3N2 subtype is presumably derived from a H2N2 subtype, retaining seven segments of the H2N2 subtype, while the gene coding for the HA is obtained from the duck Ukraine or another highly related strain.

Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus.

Abstract In search of a molecular basis underlying the variations in virulence observed with different strains of Newcastle disease virus, a comparative study has been carried out on the biosynthesis and function of the viral glycoproteins. Five virulent (Italien, Herts, Field Pheasant, Texas, Warwick) and five avirulent strains (La Sota, B 1 , F, Queensland, Ulster) have been analyzed. They were grown in five different host systems (embryonated eggs, cultures of BHK21-F, MDBK, chick embryo, and chick chorioallantoic membrane cells). Glycoprotein F (MW 56,000) which is responsible for hemolysis and cell fusion has been found with all strains to be derived by proteolytic cleavage from the precursor glycoprotein F o (MW 68,000). With strains Queensland and Ulster, in addition, a precursor glycoprotein HN o (MW 82,000) has been identified which is converted, again by proteolytic cleavage, into the hemagglutinin-neuraminidase glycoprotein HN (MW 74,000). Cleavage of F o is necessary for the expression of cell fusing and hemolytic activity, and the available evidence suggests that cleavage of HN o is paralleled by an enhancement of hemagglutinating and neuraminidase activity. However, activation of the glycoproteins is not required for virus assembly. Thus, virus particles containing the precursor F o may be formed which have a reduced infectivity. Infectivity is even lower, if both glycoproteins are present in the uncleaved form. After in vitro treatment with trypsin, such particles display full biological activity. Whether the glycoproteins are cleaved in vivo depends on the virus strain and on the host cell. With virulent strains, cleavage occurs in all host systems analyzed, and the virions formed contain HN and F. With avirulent strains, however, this is the case only in the embryonated egg and in cultures of chorioallantoic membrane cells. All other cells produce particles containing uncleaved glycoproteins. From these observations the following conclusions can be drawn: only a few host systems are permissive for avirulent strains, i.e., they produce highly infectious virus; other systems are nonpermissive for these strains, i.e., they produce defective virus; in contrast, all host systems studied are permissive for virulent strains. This concept is supported by the finding that multiple replication cycles and plaque formation occur only in permissive cells or in a nonpermissive culture after substitution of trypsin. Thus, plaque assays are now available for avirulent strains, either by the use of MDBK and chick embryo cells in the presence of trypsin or by the use of chorioallantoic membrane cells. These observations demonstrate striking differences in host range between virulent and avirulent strains which are determined by the susceptibility of the envelope glycoproteins to proteolytic cleavage. It is suggested that these differences account at least in part for the variations in the virulence of Newcastle disease virus.

Proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between HA1 and HA2 determines proteolytic cleavability and pathogenicity of avian influenza viruses

The structural basis for the different proteolytic cleavability of influenza virus hemagglutinin (HA) was investigated with a group of pathogenic and nonpathogenic avian influenza viruses belonging to the antigenic subtype H7 (Hav1). Infected cel lysates or lystates of purified virus particles were subjected to two-dimensional gel electrophoresis. The first dimension, isoelectric focusing,- was done under nonreducing conditions, the second dimension, SDS-PAGE, under reducing conditions. The results obtained permit the following conclusions: The amino acid sequence of the connecting peptide between HA1 and HA2 determines proteolytic cleavability by a trypsin-like cellular enzyme. Upon proteolytic cleavage of HA of pathogenic strains, peptides of differing positive charge were eliminated. These HAs have, however, significantly more basic connecting peptides than HAs of nonpathogenic viruses. HAs of nonpathogenic H7 strains appear to have a connecting peptide similar to the human influenza viruses, since treatment of these viruses with trypsin results in a similar small charge shift which probably corresponds to the elimination of one basic amino acid. Thus, the primary structure of the connecting peptide determines biological activation and thereby pathogenicity of these viruses.

The Molecular Biology of Influenza Virus Pathogenicity

Publisher Summary It is an accepted concept that the pathogenicity of a virus is of polygenic nature. Because of their segmented genome, influenza viruses provide a suitable system to prove this concept. The studies employing virus mutants and reassortants have indicated that the pathogenicity depends on the functional integrity of each gene and on a gene constellation optimal for the infection of a given host. As a consequence, virtually every gene product of influenza virus has been reported to contribute to pathogenicity, but evidence is steadily growing that a key role has to be assigned to hemagglutinin. As the initiator of infection, hemagglutinin has a double function: (1) promotion of adsorption of the virus to the cell surface, and (2) penetration of the viral genome through a fusion process among viral and cellular membranes. Adsorption is based on the binding to neuraminic acid-containing receptors, and different virus strains display a distinct preference for specific oligosaccharides. Fusion capacity depends on proteolytic cleavage by host proteases, and variations in amino acid sequence at the cleavage site determine whether hemagglutinin is activated in a given cell. Differences in cleavability and presumably also in receptor specificity are important determinants for host tropism, spread of infection, and pathogenicity. The concept that proteolytic activation is a determinant for pathogenicity was originally derived from studies on avian influenza viruses, but there is now evidence that it may also be relevant for the disease in humans because bacterial proteases have been found to promote the development of influenza pneumonia in mammals.

The structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses

Abstract Comparative studies on naturally occurring avian influenza viruses have been carried out in order to investigate the determinant(s) for pathogenicity for chickens. At least one virus isolate from each of the nine different hemagglutinin (HA) subtypes was included. The polypeptides of these viruses were studied by analyzing infected cell extracts on SDS-polyacrylamide gels. Both viral glycoproteins, HA and neuraminidase, showed remarkable variation in their electrophoretic mobility even among serologically closely related viruses. Pulse-chase experiments revealed that most avian influenza virus strains had an HA which was not susceptible to proteolytic cleavage in MDCK, turkey (TEC), and chicken embryo cells (CEC). Only viruses belonging to the subtype Hav5 and some strains of the subtype Hav1 possessed a cleaved HA in these cells. Only the virus strains with cleaved HA were produced in infectious form in MDCK, CEC, TEC, as well as in duck embryo cells (DEC) and quail embryo cells (QEC). The other virus strains produced plaques in these cells only in the presence of trypsin. There was a strict correlation between the cleavability of the HA, the potential of the virus to be produced in infectious form in a wide range of host cells, and their pathogenicity for chickens. No evidence was obtained for an involvement of the neuraminidase in determining pathogenicity. For the nonpathogenic viruses it could be shown that they can replicate and produce infectious progeny in some organs of the chicken. The results obtained permit the conclusion that in naturally occurring avian influenza viruses the structure of the hemagglutinin, that is its susceptibility to proteolytic cleavage in a broad spectrum of host cells, is the determining factor for pathogenicity.

Natural and Experimental Borna Disease in Animals

Borna disease (BD) is a transmissible, progressive polioencephalomyelitis of horses and sheep, which are the main natural hosts. It occurs sporadically in endemic areas of Germany and Switzerland, while its presence in other countries has not been fully substantiated.

Influenza viruses cause hemolysis and fusion of cells.

Abstract Influenza viruses have been found to cause extensive hemolysis and fusion of cells. It can be shown that an optimal pH exists for each virus strain at which hemolysis and fusion activities become most apparent and for these activities proteolytic activation of hemagglutinin is required. Furthermore, it can be demonstrated that the hemolytic activity differs among virus strains and that under optimal conditions influenza viruses hemolyse more efficiently than paramyxoviruses.

Replication of Borna disease virus in cell cultures

Borna disease (BD) virus from infected brain tissue of horses or rabbits could be grown in embryonic brain cells from rabbits or rats with high virus yields. The cells became persistently infected and could be subcultivated without loss of infectivity.Cocultivation of infected rabbit brain (ERB) cells with GMK-, Vero-, or MDCK-cells led to persistently infected cell lines. BD virus grown in MDCK cells after cocultivation became adapted to this cell type and could be used directly for further infection of MDCK cells.

Functional significance of sialidase during influenza virus multiplication

Abstract Highly specific and active antiserum against fowl plague virus sialidase has been prepared. This antiserum is devoid of demonstrable hemagglutinating inhibiting and virus neutralizing capacity. It strongly inhibits the sialidase of fowl plague virus, and when it is added to monolayers infected with fowl plague virus, no hemagglutinin, no infective virus, nor any sialidase activity is released from the infected cells. These cells synthesized the normal amount of complement-fixing antigen and approximately one-third of the normal number of infective units. Thus, the results strongly suggest that one of the most important functions of sialidase may be for the release of virion from infected cells.