Orf virus is a worldwide cause of a significant disease of sheep and goats, and in many
countries there is a need to differentiate orf virus infections from those caused by other
poxviruses. Twenty-five monoclonal antibodies (mabs) against orf virus, a parapoxvirus
(ppv), were produced following the immunisation of mice with a lysate of cells infected with
orf -11 virus. These mabs, together with 2 others recloned from an earlier fusion, were
identified by ELISA and IFT and characterised. No neutralising activity was shown by any of
the mabs. The size of orf proteins detected by the mabs was measured using western blotting
and radioimmunoprecipitation (RIP). Western blotting was conducted with two types of orf11 antigen preparations:- gradient purified virus or a lysate of orf-11 infected cells. Five
mabs detected a protein of approximately 40 kDa with both purified virus and infected lysate
antigens. Three mabs detected a protein approximately 65 kDa in size, but only with infected
cell lysate antigen. In RIP studies, 21 mabs produced bands 13 of which were against the 65
kDa protein, 7 against the 40 kDa protein while one was against a 50 kDa protein. Twentyone of the 27 mabs reacted with at least two of 18 vaccinia virus orf virus (VVOV)
recombinants expressing a library of orf genome fragments of the NZ-2 virus strain. Four of
the mabs which had identified the native 40kDa protein reacted with 2 overlapping
recombinants (245 and 247). Seventeen of the mabs, 16 of which had identified the native
65 kDa protein recognised three recombinants 79, 285 and 286 all of which contain different
inserts from the same region of the orf virus genome. Subsequent sequencing of the
overlapping site between recombinants 245 and 247 by New Zealand collaborators has
identified a new orf gene, designated F1F which has been shown to be analogous to the H3F
vaccinia virus gene which codes for an immunodominant 35 kDa envelope protein. Cells
infected with a new VVOV recombinant expressing only the F1F orf gene showed positive
fluorescence with 3 of the 4 mabs which reacted with the 245 and 247 recombinants,
confirming the target of these mabs is the product of the F1F gene.Cross-reactivity of the 27 mabs against eight parapoxviruses (ppvs) clearly differentiated the
6 ruminant ppvs from ppvs isolated from a seal and a squirrel. In IFT, 11 mabs reacted with
the 4 sheep and 2 cattle ppvs; seven mabs recognised the 4 sheep and 1 or other of the cattle
ppvs; four mabs detected the 6 ruminant ppvs and the seal ppv; two mabs reacted with the
sheep isolates only; one reacted with the 6 ruminant ppvs and the squirrel ppv and one mab
(1C7) detected all 8 ppvs. None of the mabs reacted with vaccinia virus but one of the 27,
1C7 has been shown to react with capripoxvirus. The panel of mabs has therefore,
demonstrated its ability to discriminate isolates of ruminant ppvs from each other and from
capripoxvirus.On the basis of their protein reactivity and relatively high ELISA ODs, eight mabs were
chosen for competition studies with a view to developing a capture ELISA for the rapid
detection of orf virus from clinical specimens. The 4 selected mabs against the 40 kDa
protein recognised 3 epitopes as did the 4 selected mabs against the 65 kDa protein. A double
mab ELISA for detecting orf virus was developed and evaluated using suspensions of cell
culture grown virus, detergent-treated infected cell cultures, virus purified from skin scabs
and crude detergent-treated skin scabs. The ELISA detected as few as 100 TCID50 of cell
culture grown virus and was reproducible for all the orf virus preparations except the crude
detergent-treated skin scab. An ELISA which detects orf virus in crude scabs was developed
using a polyclonal antibody capture phase and a mab detector which after further optimisation
may be of advantage in field situations
