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Interferons (IFNs) are important mediators of the immune system. Their antiviral activity is an integral part of the innate immune defence, but all IFNs have immune regulatory functions also. Besides rec.eq.IFN-beta detailed descriptions on other rec.IFNs were lacking and none of the proteins was available. To compare the equine IFNs and allow detailed studies on proteins and bioactivity, we performed the expression cloning of rec.eq.IFN-alpha, -beta and -gamma. To achieve maximal expression, a bacterial expression system was chosen. Additionally, rec.eq.IFN-beta and -gamma were expressed in mouse B-cells. The antiviral activity was characterised using different cell lines and equine viruses. The results demonstrate a broad antiviral activity of rec.eq.IFN-alpha being active against all viruses tested, including the equine herpesviruses EHV-1 and -4, while rec.eq.IFN-beta was only active using primary horse cells. Protection depended on viruses, cell lines, infectious doses, amount and time of IFN action prior to infection. While rec.eq.IFN-gamma did not act antivirally, it was effective as an immune modulator of monocytes in vitro. The implications of our findings on clinical immunology and virology, including therapeutic applications of equine IFNs will be discussed.