The gene order of morbilliviruses was established by a combination of hybrid arrest translation and cloning of the sixth and largest (L) gene of measles virus (MV). The order appeared to be identical to that of Sendai virus. Clones for the fusion (F) and haemagglutinin (H) genes of MV and canine distemper virus (CDV) were identified and sequenced in collaboration with other groups.
The gene sequences of the glycoproteins F and H of a number of MV strains were analyzed in order to identify areas of high variability to give an indication where antigenic pressure may have led to sequence changes. The sequences of the F genes revealed too little variation for this approach to be feasible. The sequences of the H genes of the Edmonston strains, the P9 variant, strain IP3CA, strains BAB and MCI JM and CM, BAB and CAM as well as of 4' monoclonal antibody (MAb) escape mutants of CAM were determined. Analysis indicated 1 major immunogenic region of the H protein of MV, as well as interesting correlations between sequence variations and the levels of neurovirulence associated with glycosylation sites. In 1 case the only difference between the CAM/RB strain, neurovirulent in intracerebral injection of Lewis rates, and a nonvirulent MAb escape mutant was the presence of a glycosylation site.
The sequences of matrix (M) genes of 14 different strains of MV were determined, bringing the total number of sequences how known to 24. Comparison of the sequences showed that defects in the M gene occurred frequently in cases of subacute sclerosing panencephalitis (SSPE) or measles inclusion body encephalitis (MICE). Thus M proteins of brain associated viruses often show truncations due to premature stop codons or show the phenomenon of biased hypermutation. The analysis did not reveal the presence of hypervariable regions in the M protein of MV. Particularly, 1 stretch of 15 amino acids which represents the only major sequence difference between MV and CDV did not appear to be varia ble to any great extent between MV strains.
A comparison was made between the gene sequences of the phosphoproteins (P) of 9 strains of MV, rinderpest virus (RPV), CDV and phocine distemper virus (PDV) in order to identify the constant and variable regions of the P protein, as well as of the 2 nonstructural proteins C and V. The C protein is encoded by an overlapping reading frame and the V protein of the morbilliviruses is expressed after cotranscriptional editing of the messanger ribonucleic acid (mRNA). The roles of C and V are unknown and comparison of the identified constant regions with the sequence databanks have not indicated a function as no homologue have been found.
The gene sequences of the nucleocapsid (N) proteins of 7 strains of MV have been determined. Comparison of the 9 sequences now known shows that the carboxyterminal 150 animo acid residues of the protein represent the most variable part of any of the MV proteins. The sequence of this variable domain has been determined for 36 strains and isolates of MV revealing a number of cocirculating MV lineages.
Expression of all MV proteins except L has been achieved in bacterial systems as fusion proteins. These have been used to make preliminary analyses of the localization of B cell and T cell epitopes. Recombinant vaccinia viruses (VV) expressing the N, P, V, C, L, F and H genes of MV have also been constructed (or obtained from other groups). The N gene clones have been used to generate Baculovirus recombinants that express the virus N protein which has been purified in large quantities. This has proved useful in diagnostic enzyme linked immunosorbent assay (ELISA).
The ability to protect Lewis rats from fatal measles encephalitis by injection with vaccinia recombinant viruses expressing the various proteins of MV has been assessed. The vaccinia recombinants which contain the F (vvF), H or N genes of MV protected to animals fully. No protection was observed with recombinants expressing th e viral V protein (a humoral responsedeveloped but no CD8 positive cells were detected). Partial protection was obtained with the vvM and vvP recombinants. Adoptive transfer experiments showed these protective effects to be related to MV specific CD4 positive lymphocytes.
The cell mediated immune responses to MV glycoproteins and other structural proteins in mice which develop a measles encephalitis were examined. The ability to generate cytotoxic T lymphocytes (CTLs) appears to correlate with resistance or susceptibility to MV infection. Mice with low susceptibilities generate good CTL responses while those strains with high susceptibility reveal very poor CTL responses. MV induced CTLS were usually CDS positive and generation of these cells was independent of route of time postinfection. CD4 positive T cells were generally only weakly lytic. The N protein was a major target antigen for CTLs in Balb/c mice although in some experiments the H protein was also recognized.
Hypermutation events in SSPE brain material were studied. A detailed analysis of sequences in various brain areas from 1 SSPE case and analysis of the same areas by in situ hybridization with sequence variant specific oligonucleotide probes has led to the conclusion that hypermutation is a frequent event in the central nervous system and that clonal expansion of the mutated virus occurs. The clones appear to spread from 1 brain region to another, possibly without the need for formation of infectious virus.
A data bank has been established containing all known morbillivirus sequences. Alignments of all the paramyxovirus and morbillivirus protein sequences as well as the nudeotide sequences of all genes of various MV strains are being maintained.
It will be a prime objective of this application to generate full length clones of gene 4 gene 5 for MV and CDV. Two approaches will be taken. First, reverse transcription of genomic RNA (a mixture of positive and negative strands of about 15kb length) will be carried out using either the existing clones or using the consensus sequence. ATTATAAAAAACTTAG or its inverse complement as primer nucleotides. The consensus sequence is found at the intergenic boundaries. A second approach would be to pursue reverse transcription of poly-adenylated RNA extracted form infected cells using the existing clones as primers or the using the sequence 5'TTTTTTATAAT3' as a specific oligo nucleotide primer for reverse transcription of MV and CDV specific mRNAs. Clones will be prepared using standard cDNA cloning procedures involving either double stranded DNA synthesis or direct cloning of RNA/DNA hybrids as successfully used by us in earlier work. The cloning vectors used will be the -gt-11 or pUC8 systems. Selection of recombinant DNA clones of interest will be made using the inserts of existing clones as probes. These will be made radioactive by copying of denatured DNA with the klenow fragment of DNA polymerase I primed by random primers. Alternatively, probes are made by in vitro transcription. Full length clones representing the coding sequence of genes 4 and gene 5 will be prepared by joining together partial cDNA clones or they will be obtained directly from the above cloning strategies.
Funding SchemeCSC - Cost-sharing contracts