Student's t test was used to determine statistical significance of experimental results when indicated (p#0.05). Bioinformatics analysis of rabbit haemorrhagic disease virus genome BACKGROUND: Rabbit haemorrhagic disease virus (RHDV), as the pathogeny of Rabbit haemorrhagic disease, can cause a highly infectious and often fatal disease only affecting wild and domestic rabbits. Recent researches revealed that it, as one number of the Caliciviridae, has some specialties in its genome, its reproduction and so on. RESULTS: In this report, we firstly analyzed its genome and two open reading frameworks (ORFs) from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 (encoding by ORF1) and VP10 (encoding by ORF2). Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. Synonymous codons are not used randomly [1] . The variation of codon usage among ORFs in different organisms is accounted by mutational pressure and translational selection as two main factors [2, 3] . Levels and causes of codon usage bias are available to understand viral evolution and the interplay between viruses and the immune response [4] . Thus, many organisms such as bacteria, yeast, Drosophila, and mammals, have been studied in great detail up on codon usage bias and nucleotide composition [5] . However, same researches in viruses, especially in animal viruses, have been less studied. It has been observed that codon usage bias in human RNA viruses is related to mutational pressure, G +C content, the segmented nature of the genome and the route of transmission of the virus [6] . For some vertebrate DNA viruses, genome-wide mutational pressure is regarded as the main determinant of codon usage rather than natural selection for specific coding triplets [4] . Analysis of the bovine papillomavirus type 1 (BPV1) late genes has revealed a relationship between codon usage and tRNA availability [7] . In the mammalian papillomaviruses, it has been proposed that differences from the average codon usage frequencies in the host genome strongly influence both viral replication and gene expression [8] . Codon usage may play a key role in regulating latent versus productive infection in Epstein-Barr virus [9] . Recently, it was reported that codon usage is an important driving force in the evolution of astroviruses and small DNA viruses [10, 11] . Clearly, studies of synonymous codon usage in viruses can reveal much about the molecular evolution of viruses or individual genes. Such information would be relevant in understanding the regulation of viral gene expression.