Numerical simulation of laser melting of alloys.

Abstract

Thesis title: The nucleocapsid protein N and the haemagglutinin protein H of rinderpest virus: Production, characterization of monoclonal antibodies and application to the study of longterm persistence of the virus in vitro. Rinderpest virus (RPV) belongs to the morbillivirus subgroup of the family Paramyxoviridae, the other members being measles virus, canine distemper virus, and peste des petits ruminant virus. Studies on the viral proteins and genome of morbilliviruses have intensified because of their involvement in chronic disorders of the central nervous system. Measles virus and canine distemper virus, in particular, have been implicated in subacute sclerosing panencephalitis (SSPE) in humans and olddog encephalitis (ODE) in dogs. These disorders are associated with persistent viral infections in their natural hosts. The fundamental mechanisms underlying persistent viral infections are poorly understood at both cellular and molecular levels. However, persistent infections can be established in several cell lines, providing an excellent system to investigate complex virus-host interactions in vitro. Epitopic changes occurring in viral proteins during persistence can be examined in detail using monospecific antibodies. Monoclonal antibodies enable fine structural and functional characterization of individual viral components and have revealed high frequencies of spontaneous and induced antigenic variation even in viruses considered monotypic. The aim of this investigation was to prepare and characterize monoclonal antibodies against the nucleocapsid protein (N) and haemagglutinin protein (H) of the RBOK vaccine strain of RPV, and to use these as immunological probes to study:

The synthesis and fate of virusspecific proteins in acutely infected cells and in a persistently infected Vero cell line (Pi2), developed earlier in this laboratory. Antigenic variation in viral proteins during persistent infection.

One of the monoclonal antibodies produced was also used to purify the H protein from infected cell extracts.

Chapter I Provides a comprehensive review of morbillivirus structural proteins, their purification strategies, monoclonal antibody generation, and mechanisms of viral persistence in paramyxoviruses, especially morbilliviruses.

Chapter II - Production and Characterization of Monoclonal Antibodies Monoclonal antibodies were produced against purified N and H proteins of RPV. Antigenic mapping of these two proteins was carried out using competitive binding assays. Key findings:

H protein:

Three epitopes were identified. Sites I and II overlap; Site III is distinct. Two monoclonal antibodies showed virusneutralization activity. All nine antibodies inhibited measles haemagglutination to varying extents.

N protein:

Two epitopes were identified; both overlapped. Epitope homology across morbilliviruses was high for N protein but varied considerably for H protein.

The antigenic sites on measles virus, canine distemper virus, and a sheep RPV field isolate were compared with those on RPV (RBOK) using ELISA. Greater antigenic diversity was observed in H than N protein.

Chapter III - Purification of H and F Proteins H and fusion (F) proteins copurified on lentillectin Sepharose, and further separation was not successful. Specific antibodies against H and F proteins were raised.

F protein was purified via an antipeptide antibody affinity column targeting the Famino terminus. H protein was purified using an antiH monoclonal antibody affinity column.

Amino acid composition analysis showed the H protein exists as two forms:

Unglycosylated form (Mr 66,000) Mature glycosylated form

The unglycosylated form was confirmed by detecting a similar protein in tunicamycintreated infected cells.

Chapter IV - Synthesis and Turnover of Viral Proteins Using monoclonal and polyclonal antibodies, synthesis and turnover of H, F, M, and N proteins were compared in acute and persistent infection (Pi2 cells). Acute vs. persistent infection (Pi2 cells):

H, F, N proteins: Reduced steadystate levels in Pi2 cells M protein: Undetectable in Pi2 cells

Pulselabeling experiments showed:

Reduced synthesis of F and N Unprocessed H was undetectable initially, but trace amounts appeared during chase Transport kinetics of newly synthesized H protein to the surface remained normal, but the quantity was reduced

Epitope analysis revealed antigenic variation in H and N proteins in Pi2 cells, more pronounced in H. Immunofluorescence studies showed:

Reduced intracellular fluorescence with N and H monoclonals Strong reduction in cellsurface H protein

These findings suggest that mutations in H and particularly M protein genes may have accumulated during persistent infection, affecting their synthesis.