Claes Örvell

Two distinct subtypes of human respiratory syncytial virus.

Antigenic variation of human respiratory syncytial (RS) virus strains was analysed using a collection of nine, six, six, nine and one monoclonal antibodies respectively directed against the large glycoprotein (G), fusion protein (F), matrix protein (M), nucleoprotein (NP) and phosphoprotein (P) components of the Long strain of RS virus. A comparison was made with seven other strains isolated during different years in radioimmune precipitation analyses and immune fluorescence tests. Two different subtypes of the virus were demonstrable. Subtype A included the prototype strains Long and A2 and virus isolates from 1973, 1983 and 1984; subtype B included four virus strains isolated in successive years from 1979 to 1982. Subtype A viruses reacted with all the antibodies, whereas subtype B viruses showed different epitope characteristics in four structural components. The number of altered epitopes were 5/6, 1/2, 2/6 and 1/6 in the G, F, M and NP components, respectively. It is concluded that the two subtypes have evolved separately. The finding of two subtypes may explain previously observed strain variations in neutralization tests, and gives a new perspective on the immunobiology of RS virus.

Measles virus phosphoprotein retains the nucleocapsid protein in the cytoplasm

Measles virus (MV) proteins were efficiently expressed in COS and Vero cells from vectors based on the strong cytomegalovirus enhancer-promoter and the simian virus 40 origin of replication. When expressed alone, nucleocapsid protein (N) migrates predominantly into the nucleus whereas phosphoprotein (P) is located in the cytoplasm. Coexpression of N and P proteins results in retention of the N protein in the cytoplasm, as seen also in infected cells. The retention of N protein is due to specific interactions with the P protein since coexpression of N with either the matrix or the hemagglutinin protein had no effect. Mapping of the regions of N-P interactions on P protein revealed that the carboxy-terminal 40% of P was sufficient for specific binding to N; however, the carboxy-terminal 60% of P was required for retention of N in the cytoplasm. Thus, the V and C proteins encoded within the first half of the P gene are not involved in the cytoplasmic retention of N protein. N protein might be fortuitously targeted to the nucleus as a result of its many basic amino acids, presumably destined to interact with the MV genome. However, this set of experiments has allowed to analyze in vivo the interactions between the N and P proteins.

Preparation and Characterization of Monoclonal Antibodies Directed against Five Structural Components of Human Respiratory Syncytial Virus Subgroup B

Mouse hybridomas producing antibodies against the structural proteins of strain WV4843, a subgroup B strain of respiratory syncytial (RS) virus, were produced by fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with purified preparations of the virus. After immunoprecipitation test with [35S]methionine-labelled extracellular virions, 35 clones found to produce antibodies against the fusion (F) protein, six against the member (M) protein, 21 against the nucleocapsid (NP) and eight against the phospho- (P) protein were further characterized. Immunoprecipitation with [3H]glucosamine-labelled intracellular virus polypeptides detected nine hybridoma cell lines producing antibodies against the large glyco- (G) protein of the virus. By competitive binding ELISA tests with monoclonal antibodies against each of the structural components, a minimum of two, 24, four, 15 and three epitopes were detected on the G, F, M, NP and P proteins, respectively. Eleven monoclonal antibodies directed against nine epitopes of the F protein could neutralize the infectivity of the virus. In contrast, none of the nine monoclonal antibodies against G could neutralize the infectivity of the virus. In order to find out more about the antigenic relationship between human and bovine RS virus strains all monoclonal antibodies were reacted with subgroup A RS virus and also with three different strains of bovine RS virus and one strain of caprine RS virus in immunofluorescence, ELISA and immunoprecipitation tests. In addition, 31 previously developed monoclonal antibodies against subgroup A virus were reacted with the bovine and caprine strains. The numbers of monoclonal antibodies of subgroup B specific for the B type of the two human subgroups were 9/9, 3/35, 0/6, 0/21, 0/8, for the G, F, M, NP and P proteins, respectively. No antigenic variations were found between the three bovine strains and the caprine strain. They did not react with the nine monoclonal antibodies against the G protein of subgroup B, nor did they react with nine monoclonal antibodies against subgroup A. Most but not all of the monoclonal antibodies against the other structural proteins of the two human RS virus subgroups reacted with the four strains. All 11 monoclonal antibodies against the F protein of subgroup B that could neutralize the infectivity of subgroup B also reacted with the bovine strains and neutralized their infectivity. It is concluded that although the bovine strains share many epitopes with the two human subgroups, they are antigenically distinct from the human viruses.

Studies on manifestations of canine distemper virus infection in an urban dog population.

An upsurge of canine distemper was recognized at the beginning of 1991 in the urban dog population of the Copenhagen area. The outbreak had the characteristics of a virulent morbillivirus introduction in a partly immune population, where the disease primarily was manifested in young individuals. Testing of single serum samples for the presence of canine distemper virus (CDV) IgM antibodies using an IgM ELISA confirmed current and recent CDV infections in an urban dog population, where the use of attenuated CDV vaccines was widespread. In 49 out of 66 sera from clinical cases suspected of canine distemper we detected CDV IgM antibodies, as compared to the detection of viral antigen by indirect immunofluorescence in 27 of 65 specimens of conjunctival cells. The antigenic make-up of isolates from acute and subacute clinical cases was investigated with a panel of 51 monoclonal antibodies directed against CDV and the related phocine distemper virus. The isolates exhibited an homogeneous reaction pattern and shared overall antigenic characteristics of the CDV prototype. The majority of cases were diagnosed among unvaccinated dogs and individuals with unknown or obscure vaccination record. However, severe clinical cases were also diagnosed in vaccinated individuals.

Mumps virus neutralizing antibodies do not protect against reinfection with a heterologous mumps virus genotype

In April 1999, a previously healthy 22-year-old woman was taken ill with fever and bilateral swelling of the parotid glands. A chronic course of disease extending from April to December was found with swelling of the parotid glands, fatigue, low grade fever, episodes of tachycardia and nightswetting. Mumps virus RNA of genotype A character based on the SH (small hydrophobic) protein gene classification was demonstrated in three serum samples collected during the course of clinical disease. Different criteria for reinfection were fulfilled including demonstration of IgG antibodies by ELISA in a preinfection serum sample. The preinfection serum sample of the patient was able to efficiently neutralize the infectivity of a heterologous genotype D strain but was unable to neutralize the homologous genotype A virus. The findings in the present study may offer an explanation of a mechanism behind previously observed vaccine failures and the occurrence of reinfection with heterologous mumps virus strains.

Characterization of morbilliviruses isolated from dolphins and porpoises in Europe

A previously unidentified morbillivirus was isolated from two harbour porpoises (Phocoena phocoena) that had died in the Dutch Waddensea (North Sea) in 1990. This porpoise morbillivirus (PMV) and a dolphin morbillivirus (DMV), which had recently caused a heavy mortality in Mediterranean striped dolphins (Stenella coeruleoalba), were compared antigenically with other members of the genus Morbillivirus, including the newly recognized phocine distemper virus type 1. DMV and PMV proved to be similar but distinct morbillivurses, closely related to rinderpest virus and peste-des-petitsruminants virus. Cell cultures of cetacean, pinniped, ruminant and canine origin showed a different pattern of susceptibility to DMV and PMV infection. Ruminants and dogs proved to be susceptible to experimental infection with DMV and PMV, which both caused a transient leukopenia most pronounced in the ruminants. Pre-exposure of dogs to DMV and PMV protected them from developing CDV viraemia and clinical signs upon challenge infection with virulent CDV. A serological survey among stranded animals of different cetacean species in Europe indicated that infections with DMV- and PMV-like morbilliviruses are not uncommon among these aquatic mammals.

The antigenic relationship between measles, canine distemper and rinderpest viruses studied with monoclonal antibodies

Monoclonal antibodies (MAbs) were used to delineate the antigenic relationship between the three morbillivirus types: measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). Panels of six to 31 MAbs against the haemagglutinin (H), fusion (F), nucleocapsid protein (NP), phosphoprotein (P) and matrix (M) proteins of MV and the H, F, NP and P proteins of CDV were employed. Nine strains of MV, three strains of CDV and four strains of RPV were examined by radioimmunoprecipitation assay and immune fluorescence for reactivity with the heterologous MAbs. Overall, the NP and in particular the F proteins of the morbilliviruses showed a high degree of epitopic homology; the P and M proteins showed a partial epitopic homology, with the greatest variation between the M proteins of CDV and MV; the H proteins showed a low degree of epitopic homology and then only between MV and RPV. These data indicate that the major cross-protecting antigen in heterotypic vaccination amongst morbilliviruses is the F antigen. The epitopic relationships found between morbilliviruses as identified by the MAbs were classified as follows. (i) Group-specific epitopes were present on all strains of the three morbillivirus types. (ii) Group-cross-reactive epitopes were present on only some of the strains from each morbillivirus type (these epitopes identified the presence of intratypic strain variation in all proteins of all three virus types). (iii) Type-specific epitopes, i.e. MV unique or CDV unique, were found only on the homologous morbillivirus type. (iv) CDV-RPV intertypic and MV-RPV intertypic epitopes were, respectively, epitopes shared by CDV and RPV but not with any MV strain, and epitopes shared by MV and RPV but not with any CDV strain. These cross-reactivities and type-specific reactions were obtained with the internal viral proteins (M, P and NP). The epitopes of the F proteins were mainly group-specific and no CDV-RPV or MV-RPV intertypic epitopes were found. The epitopes of the H protein were either type-specific or MV-RPV intertypic. These data support the proposed evolutionary relationship between the morbilliviruses.

Preparation and Characterization of Monoclonal Antibodies Directed against Four Structural Components of Canine Distemper Virus

Mouse hybridomas producing antibodies against structural proteins of canine distemper virus (CDV) were produced by fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with purified preparations of Vero cell-grown CDV. Ascites fluids collected after intraperitoneal inoculation with 149 CDV antibody-producing hybridoma cell lines were characterized by different serological tests. By immune precipitation tests with [35S]methionine-labelled extracellular virions and intracellular virus polypeptides, 57 clones were found to produce antibodies against the nucleocapsid protein (NP), 22 against the polymerase (P) protein, 10 against the fusion (F) protein and nine against the large uncleaved glycoprotein (named H in analogy with measles virus). By competitive binding enzyme-linked immunosorbent assay (ELISA) tests with monoclonal antibodies against each structural component, a minimum of 18, six, three and seven separate antigenic determinants were identified on the NP, P, F and H proteins, respectively. The reactions of clones directed against F and H surface components of the virus were tested for their ability to inhibit the infectivity of both CDV and measles virus in the absence and presence of anti-gamma-globulin. In addition, the inhibitory activity of the clones on measles haemagglutinating (HA) and haemolysis (HL) activity were examined. Monoclonal antibodies against six of the seven antigenic determinants of the H protein could neutralize the infectivity of the virus. After addition of anti-gamma-globulin to the test, increases of titres varying from twofold to several hundredfold were observed with the different clones. None of all the clones against H could block measles virus infectivity, HA or HL activity. The 10 clones directed against the F protein could not neutralize the infectivity of CDV even in the presence of anti-gamma-globulin. Further, the antibodies could not inhibit measles HA and HL activity in the absence of anti-gamma-globulin. However, after the addition of anti-gamma-globulin, antibodies against two of the three sites were found to block measles virus HL activity. The reactions of all clones were tested in immune fluorescence, ELISA and immune precipitation tests with three strains of CDV. Each strain had a few unique antigenic sites. Variation was found in four, one and three different antigenic sites of the NP, P and H proteins, respectively.

Immunological relationships between phocid and canine distemper virus studied with monoclonal antibodies

The immunological relationships between distemper viruses, isolated from a seal and mink in Denmark and from a dog in Greenland, were investigated with 39 previously developed monoclonal antibodies (MAbs) directed against four major structural proteins of canine distemper virus (CDV). They were also investigated with 16 newly developed MAbs directed against the fusion (F) and large glycoprotein (named H in analogy with measles virus) of phocid distemper virus (PDV) isolated from a harbour seal (Phoca vitulina). These MAbs were reacted with the three different isolated viruses and with the LEC strain of measles virus, in ELISA and immunofluorescence tests. In addition, immunoprecipitation tests were carried out with some of the cross-reacting antibodies. All 55 MAbs reacted identically with distemper virus isolated from seals or mink. When the MAbs produced against CDV were tested, 37 of 39 antibodies reacted with a virus isolated from a sled dog diseased in an outbreak of distemper in Greenland prior to the epizootic among seals in the North Sea. Of the 39 antibodies, 25 reacted with PDV and distemper virus isolated from mink. Of these antibodies, only three of the nine antibodies directed against the H protein of CDV cross-reacted with PDV and distemper virus from mink. Eleven MAbs, reacting with six epitopes of the H protein of PDV, were produced. All 11 antibodies reacted with distemper virus from mink, two of the antibodies reacted with CDV and none reacted with measles virus. All five antibodies reacting with three different epitopes of the F protein of PDV reacted with distemper virus from mink and CDV. Of these five antibodies three, directed against two epitopes, reacted with measles virus. Of the two envelope proteins, the H protein shows pronounced immunological differences between PDV and CDV. In contrast, immunologically the F protein appears to be well conserved among morbilliviruses. It is concluded that the virus causing the epizootic in seals in the North Sea in 1988 may have infected mink on land, or, alternatively, the virus in the sea may have originated from virus-infected mink.

Is Rinderpest Virus the Archevirus of the Morbillivirus Genus

Groups of 6-39 monoclonal antibodies identifying 3-18 distinct epitopes on the nucleoprotein (NP), polymerase (P), hemagglutinin (H; equivalent in canine distemper and rinderpest viruses), and fusion (F) components of measles and canine distemper viruses were characterized in immunofluorescence tests with fixed Vero cell cultures infected with measles, canine distemper and rinderpest viruses. The majority of NP-specific monoclonal antibodies reacted with all three viruses, but one-third of the antibodies only reacted with the homologous virus. A few antibodies detected epitopes uniquely shared between either measles and rinderpest viruses or canine distemper and rinderpest viruses. Of the P-specific antibodies, two-thirds only reacted with the homologous virus, one antibody detected an epitope shared between canine distemper and rinderpest viruses, and the rest reacted with all three viruses. Also, the majority of antibodies against the H component were type-specific, but four antibodies reacted both with measles and rinderpest viruses. In contrast, the F component was antigenically highly conserved. 17 of 21 antibodies against this component reacted with all three viruses; one antibody reacted only with measles and rinderpest virus F components, and three antibodies reacted only with the homologous virus. No monoclonal antibody of any specificity selectively reacted with only measles and canine distemper viruses. Furthermore, the measles virus H component appeared to be more closely related to the equivalent rinderpest virus component than to the canine distemper virus component. Thus, it is proposed that rinderpest virus is the archevirus of the morbillivirus group from which canine distemper virus was first to evolve and, more recently (perhaps about 5,000 years ago), measles virus.