Production and use of monoclonal antibodies for the characterization of avian reoviruses and Nelson Bay virus

Monoclonal antibodies (MAbs) of murine origin were produced against virion proteins of Nelson Bay virus (NBV) and the RAM-1 strain of avian reovirus. The NBV MAbs reacted with individual viral proteins of molecular weights 122 and 42 kilodaltons (KDa) as determined by radioimmunoprecipitation (RIP) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two additional NBV MAbs co-precipitated proteins of 75 and 72 KDa. The avian reovirus MAbs reacted with individual viral proteins of 124, 76, 72, 45 and 39 KDa. Other avian reovirus MAbs co-precipitated virion proteins of 130 and 105 KDa. and 74 and 72 KDa. The MAbs were used to determine by immunoelectron microscopy the location in the virus particles of the reactive proteins, and to characterise the proteins involved in neutralisation of virus and virus-induced cell fusion. The NBV MAbs reactive with the 122 KDa protein and the co-precipitated 75 and 72 KDa proteins caused clumping of virus indicating the reactive proteins were exposed on the surface of the virus particle. The MAbs against the 122 KDa protein also neutralised virus infectivity, and this protein was considered analogous to the λ2 or spike protein of mammalian reoviruses. The MAbs reactive with the co-precipitated 75 and 72 KDa proteins also neutralised virus infectivity and inhibited virus-induced fusion of cells. These MAbs against the 75 and 72 KDa proteins also co-precipitated a 39 KDa protein under certain conditions. The results suggested that the 72 KDa protein was a cleavage product of the 75 KDa protein and these proteins were analogous to the μ1/ μ1C proteins of mammalian reovirus, and that the 72 and 39 KDa proteins were analogous to the μ1C- σ3 complex of mammalian reovirus which form the outer shell capsomers. In NBV, MAbs against 75/72 protein complex neutralized virus infectivity and inhibited virus-induced cell fusion, whereas in mammalian reovirus MAbs against the σ3 protein but not the μ1/ μ1C complex have been detected which neutralise virus infectivity. The avian reovirus MAbs against the 124 KDa protein, the 130 and 105 KDa protein complex, the 74 and 72 KDa protein complex, and the 72 and 39 KDa proteins caused clumping of virus indicating these proteins were exposed on the surface of the virus particle. The MAbs reactive with the 124 KDa (λ b) protein neutralised virus infectivity and this protein appeared to be analogous to the 122 KDa protein of NBV and the λ2 or spike protein of mammalian reovirus. One MAb which consistently co-precipitated the 130 KDa (λ a) and 105 KDa (λ c) proteins also neutralised virus infectivity. These results indicated there are two λ -proteins (λ b and either λ a or λ c) exposed on the surface of the avian reovirus particle and that both are involved in neutralisation of virus infectivity; in mammalian reovirus only one λ -protein is exposed on the surface of the virus particle. From immunoprecipitation studies there was evidence that the 72 KDa protein was a cleavage product of the 74 KDa protein, and analogous to the μ1 and μ1C proteins of mammalian reovirus and the 75 and 72 KDa proteins of NBV. The 74 and 72 KDa proteins were co-precipitated with the 43 KDa protein under certain conditions, which suggested that the 72 and 43 KDa proteins of avian reovirus were analogous to the μ1C- σ3 as complex in mammalian reovirus and the 72 and 39 KDa proteins of NBV, and probably form the outer shell capsomers. As in mammalian reovirus, but unlike the apparently analogous proteins in NBV, antibodies to the 72 KDa protein of avian reovirus did not neutralise virus infectivity. The 76 KDa (μ) protein precipitated from avian reovirus-infected cell lysates by one MAb was not precipitated by polyclonal antisera and could not be identified but was possibly analogous to the 75 KDa (μ ns) protein previously reported in extracts of virusinfected cells. The 39 KDa (σ c) protein of avian reovirus, located on the surface of the virus, was involved in virus neutralisation and virus-induced cell fusion. The neutralisation activity of the MAbs against the 39 KDa (σ c) protein was serotype-specific. The fusioninhibition activity of the MAbs against the 39 KDa protein was also serotype-specific. The neutralising MAbs recognized three epitopes on the 39 KDa protein of RAM-1 virus: one involved in neutralisation and fusion in chick kidney (CK) cells but only fusion in Vero cells, one involved in neutralisation and fusion in Vero cells and only neutralisation in CK cells, and a third epitope which appeared to be sterically involved in virus neutralisation and cell fusion activity in both CK and Vero cells. The 39 KDa protein was detected at the surface of virus-infected cells nine hours post infection. Nine avian reovirus isolates of Australian origin, previously classified into three serogroups, were re-classified into five serogroups on the basis of plaque reduction neutralisation tests using RAM-1-specific neutralising MAbs. The 45 KDa (σA) protein of avian reovirus which is located in the interior of the virus was shown to be the most antigenically conserved of the six viral proteins investigated in the current study. This protein was antigenically related to the 42 KDa (σ1) protein of NBV as shown by both IF and RIP tests. Monoclonal antibodies to the 42 KDa protein of NBV also reacted with the HEV strain of mammalian reovirus type 3 by indirect IF tests