We report on the cloning and sequencing of two Tranosema rostrale ichnovirus (TrIV) genes, and assess their relatedness to TrV1, the gene encoding the most abundant TrIV transcript in last-instar Choristoneura fumiferana larvae parasitized by T. rostrale. One of the two newly isolated genes, TrV2, features an organization similar to that of TrV1, with one intron flanked by two exons; it encodes a 102 amino acid protein showing 79% similarity to TrV1. The third gene, TrV4, encodes a larger protein (143 aa) displaying similarity to the other two only over the first approximately 50 amino acid residues of its sequence; the remaining portion contains an imperfect octad repeat. Although the TrV4 gene contains only one exon, it has an intron similar in size and sequence to that of TrV1 and TrV2; in fact, the non-coding regions of all three genes show higher sequence identity than the coding regions, pointing to their common origin. Southern analysis suggests that each gene maps to a different TrIV genome segment, with homologous sequences apparently present on other segments. TrV1 and TrV4 transcription in penultimate (5th) instar hosts, parasitized shortly after the molt, was strong for both genes 1 and 2 days p.p., with transcript abundance decreasing after the final molt; thus, neither of these genes is upregulated during induction of developmental arrest in last-instar hosts. Cf-124T cells inoculated with T. rostrale calyx fluid showed significant levels of apoptosis 24-72 h p.i.; TrV1 was detected in the culture medium, suggesting that this and/or other TrIV-encoded proteins may be responsible for the observed cytopathic effect. Southern and Northern analyses, using DNA and RNA extracted from infected Cf-124T cells, revealed the presence of both TrV1- and TrV4-carrying genome segments and transcripts, but neither DNA, at least in episomal form, nor mRNA persisted for more than a few days p.i. This in vitro system may provide a suitable starting point for the study of TrIV gene functions.