Ruben, Jon Michael Simon

2018-05-14T10:15:51Z

2018-05-14T10:15:51Z

1977

The properties of strains of NDV occurring in persistently infected cultures of ox (BK pi), pig (PK pi) and sheep (OK pi) kidney cells were compared with those of lentogenic and virulent strains of NDV, grown in fertile hens* eggs and cell cultures.

The results obtained indicate that a number of changes have occurred in the carrier cell-lines during prolonged serial sub-culture. The most important of these include, a greater release of infectious virus and an increased percentage of cells capable of haemadsorption, despite lower titres of released haemagglutinins.

Detailed studies of the biological activities and infectivity of the persistent virus show these to be generally reduced compared with 'wild-type* strains of NDV grown in fertile hens' eggs. However, the characteristics of 'wild-type' strains of NDV released from mammalian cell-lines were also defective. In addition the enzyme phosphodiesterase which has not been previously described in NDV, was characterised and shown to be associated with the small viral glycoprotein. It was found to be of reduced activity in the virus released from the carrier cell-lines.

Polyacrylamide gel electrophoresis (PAGE) of purified virions revealed that virus released from persistently infected cells or from control cell cultures infected with 'wild-type' strains of NDV, (xiv) NDV, contained large amounts of a protein of 70,000 daltons which was not found in virus grown in fertile hens' eggs. This protein was not seen when electrophoresis was carried out under non-reduced conditions and its disappearance was apparently related to an increase in the quantities of the nucleocapsid and haemagglutinin proteins observed. An abnormal protein of 62,OOO daltons was also found when virus released from cell cultures were examined by PAGE, and this was shown to be an inactive form of the small glycoprotein. Haemolysin activity and normal molecular weight were restored to this protein by treatment with 4 p.p.m. of trypsin. The large glycoprotein of NDV was not present in the virus released from the persistently infected BK pi cells and its absence was associated with an extremely low haemagglutinin activity in this strain of virus.

The synthesis of viral proteins was studied in the BK pi cell-line and in a healthy bovine kidney cell-line (MDBK) infected with the B1 strain of NDV. Virus-associated proteins were identified with the following molecular weights: 180,000; 75,OOO; 70,OCX); 62,OOO; 55,OOO; 53,OOO; 49,OOO; 42,000 and 38,OOO daltons. All but three of these corresponded to the structural proteins of wild-type strains of NDV, grown in fertile hens' eggs. The exceptions were the 70,000 and 62,OOO dalton proteins which are found in virus released from mammalian cells and also the 38,OOO dalton protein which is thought to be a a precursor of one of the structural proteins.

Growth of the carrier cell-lines under conditions that slowed the rate of cellular metabolism, including low levels of nutrition and low temperatures of incubation, allowed the release of greater quantities of virus, without increasing its infectivity.

Virus from all three persistently infected celllines was shown to be incapable of replication at 4l°C but was able to persist without synthesising viral antigen for periods of over two months at this non-permissive temperature. Viral protein synthesis was also inhibited by actinomycin D but the infectivity of the released virus was increased. These two phenomena may be related to the ability of the persistent virus to utilise a replicative pathway involving DNA

The carrier cell-lines were capable of forming colonies' in semi-solid agar. This is characteristic of cellular transformation and may be related to the fact that BK pi cells were able to adsorb erythrocytes to areas of the cell membrane from which budding virus was absent.

Infection of fertile hens' eggs and 5-week-old chickens with aliquots of concentrated virus obtained from the carrier cell-lines resulted in the release of highly virulent strains of NDV.

There was also evidence that::

1) the replicative cycle of NDV in the healthy mammalian cell-lines is generally abnormal and defective virus virus particles are formed with low infectivity. Reduction in their biological activities is probably due to abnormalities in the synthesis of viral proteins and in the assembly of mature virions.

2) persistence of virus in a non-permissive cellsystem requires the mutation of the virus, giving rise to a form that is capable of employing the host cell machinery for RNA synthesis by means of a DNA transcription of the viral genome.

3) this mutation was accompanied by other changes in the virus, including increased temperature-sensitivity and decreased biological activities.

4) cells persistently infected with NDV undergo partial transformation because of the presence of the viral proteins .

http://hdl.handle.net/1842/29975

The University of Edinburgh

Annexe Thesis Digitisation Project 2018 Block 18

Already catalogued

Studies on defective strains of Newcastle disease virus

Thesis or Dissertation

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PhD Doctor of Philosophy