Robert Rydbeck

Hemagglutinin-neuraminidase (HN) amino acid alterations in neutralization escape mutants of Kilham mumps virus

The hemagglutinin-neuraminidase genes of the Kilham strain of mumps virus and three neutralization escape mutants (M11, M12 and M13) of this strain (Löve et al., 1985a) were sequenced using their genomes as template. The predicted amino acid sequences were compared. While one mutant had only one amino acid substitution the other two mutants had four and five respectively. A putative region for the epitope of the selected neutralizing monoclonal antibody was identified in a hydrophilic region encompassing amino acids 352-360, since the single amino acid substitution of one mutant occurred in this region and the other two mutants showed non-conserved amino acid changes in this part of the protein. The previously sequenced prototype strain RW, which lacks capacity to react with the selected neutralizing monoclonal antibody also has one non-conserved amino acid change in the region of the proposed neutralizing epitope. The three mutants showed different biological characteristics. These particular characteristics were therefore interpreted to be primarily associated with strain-specific amino acid changes outside the region of the presumed neutralizing epitope. The decrease in molecular weight in one mutant (M11) was shown to be due to a substitution in position 329 of an asparagine for an aspartic acid, leading to abolishment of a potential N-linked glycosylation site. In the other mutants, one substitution in position 239 of a lysine for a methionine was correlated with an increased neuraminidase activity of strain M12, while a substitution in position 360 of an arginine for a cysteine appeared to represent the most likely explanation for the reduced neurovirulence of strain M13.

Characterization of four parainfluenza virus type 3 proteins by use of monoclonal antibodies.

Monoclonal antibodies directed against four structural components of the ATCC strain C243 of parainfluenza virus type 3 were produced. The specific reaction of the antibodies with individual structural components was determined by radioimmune precipitation assay. In the collection of monoclonal antibodies, 21 reacted with the haemagglutinin-neuraminidase (HN) glycoprotein (mol. wt. 72,000), eight with the fusion (F) glycoprotein (mol. wt. 64,000), 27 with the nucleocapsid (NP) protein (mol. wt. 69,000) and 24 with the matrix (M) protein (mol. wt. 40,000). The F-specific monoclonal antibodies precipitated two proteins which were interpreted to represent intact F protein and the large cleavage product F1 (mol. wt. 52,000). The numbers of epitopes were determined in a competition ELISA with the monoclonal antibodies. The epitopes found were six for the HN, two for the F, six for the NP and six for the M protein. The six groups of antibodies reacting with different epitopes on the HN molecule showed varying capacities to inhibit biological activities. Two exhibited high neutralization (NT), haemagglutination inhibition (HI) and haemolysis inhibition (HLI) activity. Three groups had somewhat lower NT, lower HI and no detectable HLI activity. One group showed no activity in these tests. Of the eight monoclonal antibodies directed to the F protein two had demonstrable HLI activity.

Antigenic Variation of Envelope and Internal Proteins of Mumps Virus Strains Detected with Monoclonal Antibodies

Antigenic characteristics of nine mumps virus strains were determined by immunofluorescence and radioimmunoprecipitation assay (RIPA) using a collection of 44 monoclonal antibodies. These antibodies were directed against five different structural components of mumps virus, the haemagglutinin-neuraminidase (HN), fusion (F), matrix (M), phospho- (P) and nucleocapsid (NP) proteins. The nine mumps virus strains could be divided into two groups according to their antigenic characteristics. One group included two strains isolated more than a decade ago and the Jeryl Lynn vaccine strain. These three strains reacted with a wider range of monoclonal antibodies than the second group of six recently isolated strains of different geographical origin. In the F, M and P proteins variations were only found in single antigenic determinants. In the HN and NP components, RIPA revealed variations in three and seven determinants respectively. The Jeryl Lynn vaccine strain showed a unique lack of reaction with one anti-HN antibody clone in the RIPA.

Antigenic variation of human and bovine parainfluenza virus type 3 strains.

Three human and six bovine parainfluenza virus type 3 (PIV3) strains were examined by the use of 60 monoclonal antibodies (MAbs). Fifty-three MAbs to the human C243 strain were directed against six, four, nine and seven epitopes of the haemagglutinin-neuraminidase (HN), fusion (F), nucleocapsid (N) and matrix proteins, respectively. Seven MAbs to the bovine strain were directed against three epitopes of the HN protein and three epitopes of the F protein. Each strain was characterized in ELISA and immunofluorescence tests with all MAbs and in a haemagglutination inhibition assay with the anti-HN MAbs. There were marked differences between human and bovine viruses, primarily in the HN protein where five epitopes differed. One epitope of the F and one of the N protein also differed. Bovine PIV3 was found to be a homogeneous subtype and distinct from human PIV3.

Antigenic Analysis of Human and Bovine Parainfluenza Virus Type 3 Strains with Monoclonal Antibodies

The antigenic characteristics of eight human strains and two bovine strains, one of which is represented by two plaque variants, of parainfluenza virus type 3 were analysed. The strains and variants were compared using 52 monoclonal antibodies against five, two, six and six epitopes of the haemagglutinin-neuraminidase (HN), fusion, nucleocapsid and matrix viral proteins respectively, employing radioimmuno-precipitation and immunofluorescence assays. The human strains, seven of which were isolated over 6 years at different geographical locations and the eighth one representing an older prototype strain, showed very little antigenic variation. Extensive differences were detected in all four proteins examined between the human strains and the two strains of bovine origin. Two bovine variants were less effectively neutralized than the prototype human strain with a series of monoclonal antibodies against the HN protein.

Immunological relationships between mumps virus and parainfluenza viruses studied with monoclonal antibodies.

The immunological relationships between mumps virus and parainfluenza viruses were investigated with 74, 78 and 80 previously developed monoclonal antibodies directed against five major structural proteins of mumps virus, Sendai virus (a murine parainfluenza type 1 virus) and parainfluenza type 3 virus. These monoclonal antibodies were reacted with the three viruses, with parainfluenza type 2 virus and with Newcastle disease virus (NDV) in ELISA and immunofluorescence (IF) tests. In addition, immunoprecipitation tests with [35S]methionine-labelled extracellular virions were carried out with cross-reacting monoclonal antibodies. None of all 232 monoclonal antibodies against the three viruses cross-reacted with either parainfluenza type 2 virus or NDV in ELISA and IF tests. In the collection of 74 mumps virus monoclonal antibodies, three directed against the nucleocapsid (NP) protein, polymerase protein, and fusion protein cross-reacted with Sendai virus. Two Sendai virus monoclonal antibodies directed against two different epitopes of the haemagglutinin-neuraminidase (HN) protein cross-reacted with parainfluenza type 3 virus. Six other Sendai virus monoclonal antibodies directed against four different epitopes of the HN protein and one directed against the NP protein cross-reacted with mumps virus. Eight out of 80 monoclonal antibodies directed against parainfluenza type 3 virus cross-reacted with Sendai virus. One was directed against the HN protein, four were directed against a minimum of two epitopes of the matrix protein and three were directed against three different epitopes of the NP protein. The different cross-reactions found show that Sendai virus is antigenically related to both mumps virus and parainfluenza type 3 virus. In contrast, no antigenic relationship could be demonstrated between mumps virus and parainfluenza type 3 virus.

Parainfluenza virus type 2 haemagglutinin-neuraminidase glycoprotein characterized with monoclonal antibodies.

Thirteen monoclonal antibodies (MAbs) were prepared against human parainfluenza virus type 2 (PIV2). These MAbs reacted with the haemagglutinin-neuraminidase glycoprotein with an Mr of 84K. The MAbs defined one antigenic site which could be divided into five epitopes. A correlation between haemagglutination inhibition (HI) and neutralization activity could be seen although one MAb, which recognized a distinct epitope, showed neutralization and no HI activity to PIV2. The reactivity of the MAbs was tested against Sendai virus, parainfluenza virus type 3, simian virus 5 (SV5), mumps virus, Newcastle disease virus, measles virus and canine distemper virus. Only one MAb showed any cross-reaction with a low HI titre to SV5.

Selection of mutants of mumps virus with altered structure and pathogenicity by passage in vivo.

The neurotropic Kilham strain of mumps virus was serially passaged in newborn hamster brains in order to assess possible changes in viral characteristics. Two modes of passage were employed, one with a 4-5 day interval between inoculation and harvest and the other with a 10-12 day interval. After 10 and 8 passages, respectively, two viral variants were isolated which differed in antigen characteristics and in pathogenicity. In Vero cell cultures the variant derived from the short-term passage, designated as RK, showed much greater fusion capacity than the other, designated as SK. The highly fusing variant was highly lethal and caused much more extensive necrosis and grew to higher titers in the brain. With a series of monoclonal antibodies directed against the structural proteins of mumps virus marked differences between the variants could be detected in the nucleocapsid (NP) protein and also slight changes in the hemagglutinin-neuraminidase (HN) and phospho- (P) proteins. Differences were found in the preference of the viral variants to infect various regions of the brain. The RK variant heavily infected the caudate nucleus whereas the SK variant did not. This study demonstrates that different modes of passage can affect characteristics of virion components and disease pattern.

Protective effects of monoclonal antibodies against parainfluenza virus type 3-induced brain infection in hamsters.

A collection of monoclonal antibodies (MAbs) against the haemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of parainfluenza virus type 3 (PIV3) was used for passive immunization of intracerebrally infected newborn hamsters. A significant degree of protection was obtained with MAbs against both the HN and F viral proteins, but no individual antibody conferred complete protection against disease. MAbs against different epitopes provided different degrees of protection. A MAb to the Sendai virus HN protein was found to confer protection against PIV3 brain infection.

Developmental Disturbances in the Hamster Retina Caused by a Mutant of Mumps Virus

Newborn hamsters were inoculated intracerebrally with either the neurovirulent Kilham strain of mumps virus or a mutant (M13) strain of this virus. The M13 strain has an alteration in the haemagglutinin-neuraminidase protein of its envelope and causes a low-grade infection of the brain. Both strains spread consistently to the retina where the Kilham strain caused an extensive necrotizing infection. In contrast, the M13 strain predominantly caused an infection of the retinal pigment epithelium (RPE) with the involvement of scattered neurons in the retina. Only minimal degenerative or inflammatory changes were seen, but at 12 days of age developmental alterations were seen in all eyes. These included stretches with failure of photoreceptor segment development and the formation of folds in the outer nuclear layer. The former changes occurred in areas with loss of RPE cells and the latter generally in connection with displaced pigment-loaded cells from the RPE layer. It is suggested that these retinal alterations are mainly secondary to the RPE infection with the M13 strain.