Biomolecular characterization of mumps virus genotypes with varying neurovirulence

Abstract: Mumps virus is an RNA virus, which belongs to the Rubula virus genus of the family Paramyxoviridae. Mumps is a common childhood infection. Classically, mumps infection presents as parotitis although it is frequently asymptomatic in young children. Complications of mumps include pancreatitis, orchitis and meningitis, which occur more frequently in older children and adults. The nucleotide sequence of the small hydrophobic (SH) protein gene, is highly variable between strains and has been used to distinguish vaccine and wild type viruses and to establish genetic lineages between mumps virus isolates. Phylogenetic comparisons of the nucleotide sequences of the SH gene from mumps virus isolates have shown the existence of ten genotypes. The SH protein of 57 amino acids has been identified immunologically but its function is not yet known. The fusion (F) glycoprotein mediates the fusion of lipid membranes at neutral pH, and is required for the penetration of the viral nucleocapsid into the host cell. Both the F and the haemagglutinin-neuraminidase (HN) glycoproteins are needed to effectuate an efficient cell-to cell-fusion process. The aim of this thesis was (i) genotyping virus strains circulating in the Stockholm area and Denmark over a long time- period by nucleotide sequencing of the SH gene to follow the epidemiological situation. (ii) developing an easy method for genotyping by use of PCR with specific primers. (iii) to investigate differences in the primary structure of the F protein of different mumps genotypes and to study the antigenic relationship between the F proteins by the use of monoclonal antibodies. (iv) to investigate if mumps virus neutralizing antobodies protect against reinfection with a heterologous mumps virus genotype. (v) to genotype the virus strains responsible for the mumps epidemic in Lithuania. (vi) to compare individual genotypes with respect to clinical symptoms in patients. Nineteen virus isolates and 22 scrum samples collected between 1971 and 1997 from patients with mumps were genotyped by PCR with specific primer pairs for A, C and D. All serum samples were subjected to nucleotide sequence analysis. In the Stockholm area, co-circulation of genotypes A, C and D at different times was found. Genotypes C and D were associated with meningitis, and in some cases parotitis, whereas infection with genotype A most often resulted in parotitis but seldom in meningitis. The F proteins of 12 mumps virus strains belonging to the A, C and D genotypes were sequenced and phylogenetic analysis showed a similar clustering of the genotypes as for the SH gene. An antigenic comparison between the F proteins of the different strains was performed with ten monoclonal antibodies directed against the F protein of genotype A. It was concluded that the structure and antigenicity of the F protein is well conserved both intra- and intergenotypically over long periods of time. Serological testing and molecular analysis was performed on five consecutive serum samples from a patient with chronic mumps virus infection. Mumps virus RNA of genotype A was demonstrated in three serum samples collected during the course of clinical disease. A preinfection serum sample could neutralize a heterologous genotype D strain but was unable to neutralize the homologous genotype A virus. The finding may offer an explanation of a mechanism behind previously observed vaccine failures and the occurrence of reinfection. Twenty-nine Danish virus isolates and 14 serum samples from patients with mumps collected between 1979 to 1999 from different parts of Denmark were genotyped and compared with previously published sequences. Four neurovirulent genotypes of C, D, H and a new genotype, designated J, were found. However, from 1996 the D genotype is predominant, in contrast to Sweden where only the A genotype has been found since 1985. Virus samples were collected from 80 patients treated at the hospital of Kaunas during the epidemic of mumps in Lithuania. Twenty-seven samples were genotyped. Twenty-five belonged to the C genotype and two were of the D genotype. Two variants were found within the C genotype, Cl virus strains causing meningitis and C2 virus strains associated with parotitis. When virus strains belonging to the same genotype were compared intra-genotypically, and a variation between two and four per cent was recorded. The level of inter-genotypical variation was higher and was found to be within the range of eight to eighteen percent. These results allowed the conclusion that the establishment of a new genotype should be founded on a nucleotide variation of the SH gene of six per cent or more in relation to previously described genotypes.