T-helper cells express CD4 as a co-receptor that binds to major histocompatibility complex class II to synchronize the immune response against upcoming threats via mediating several cytokines. We have previously reported the presence of CD4 homologues in brown trout. The study of cellular immune responses in brown trout is limited by the availability of specific antibodies. We here describe the generation of a polyclonal antibody against CD4-1 that allows for the investigation of CD4⁺ cells. We used this novel tool to study CD4⁺ cells in different tissues during viral haemorrhagic septicaemia infection (VHSV) using flow cytometric technique. Flow cytometric analyses revealed an enhanced level of surface CD4-1 expression in the infected group in major lymphoid organs and in the intestine. These results suggest an important role for the T-helper cells within the immune response against viruses, comparable to the immune response in higher vertebrates.