In summary, our findings indicate that APMV serotypes 2-9 were avirulent in both chickens and ducks as well as in standard international assays. Each of the APMV serotypes replicated to low-to-moderate titers in the trachea of chickens, with APMV-3 having the highest titers. Among APMV types 2-9, only APMV3 replicated systemically and was neuroinvasive and neurotropic, although not neurovirulent. The sequence of the F protein cleavage site was not a reliable predictor of pathogenicity of APMVs in chickens, indicating incongruity with the well-known APMV-1 paradigm. For example, the systemic replication and neurotropism of APMV-3 is inconsistent with its lack of a furin cleavage site and dependence on exogenous trypsin for replication in cell culture. As another example, several of the APMV serotypes, including types 2, 4, 6, and 7, lacked a furin site, but replication of these viruses in cell culture was not improved by the addition of exogenous trypsin. As yet another example, APMV-5 was the only one of serotypes 2-9 to have a furin site, and yet it replicated to only moderate levels in cell culture, and replication in chickens could be detected only by seroconversion. Thus, the paradigm from studies with APMV-1 that virulence and systemic spread correlates with the presence of a furin site generally was not observed with APMV serotypes 2-9. Reverse genetics for different pathotypes of APMV-1 and for prototype strains of APMV-2, 3, and -7 have been developed and used for studying determinants of virus pathogenesis [38, [43] [44] [45] . Further characterization of virus replication and pathogenicity using reverse genetics will enhance our understanding of overall APMV pathogenesis. Patterns and influencing factor of synonymous codon usage in porcine circovirus BACKGROUND: Analysis of codon usage can reveal much about the molecular evolution of the viruses. Nevertheless, little information about synonymous codon usage pattern of porcine circovirus (PCV) genome in the process of its evolution is available. In this study, to give a new understanding on the evolutionary characteristics of PCV and the effects of natural selection from its host on the codon usage pattern of the virus, Patterns and the key determinants of codon usage in PCV were examined. METHODS: We carried out comprehensive analysis on codon usage pattern in the PCV genome, by calculating relative synonymous codon usage (RSCU), effective number of codons (ENC), dinucleotides and nucleic acid content of the PCV genome. RESULTS: PCV genomes have relatively much lower content of GC and codon preference, this result shows that nucleotide constraints have a major impact on its synonymous codon usage. The results of the correspondence analysis indicate codon usage patterns of PCV of various genotypes, various subgenotypes changed greatly, and significant differences in codon usage patterns of Each virus of Circoviridae.There is much comparability between PCV and its host in their synonymous codon usage, suggesting that the natural selection pressure from the host factor also affect the codon usage patterns of PCV. In particular, PCV genotype II is in synonymous codon usage more similar to pig than to PCV genotype I, which may be one of the most important molecular mechanisms of PCV genotype II to cause disease. The calculations results of the relative abundance of dinucleotides indicate that the composition of dinucleotides also plays a key role in the variation found in synonymous codon usage in PCV. Furthermore, geographic factors, the general average hydrophobicity and the aromaticity may be related to the formation of codon usage patterns of PCV. CONCLUSION: The results of these studies suggest that synonymous codon usage pattern of PCV genome are the result of interaction between mutation pressure and natural selection from its host. The information from this study may not only have theoretical value in understanding the characteristics of synonymous codon usage in PCV genomes, but also have significant value for the molecular evolution of PCV. Genetic information is transmitted from mRNA to protein in a mode of triplet codon. Each amino acid matches with at least one codon, at most six codons. The codons encoding the same amino acid is called synonymous codon. During biosynthesis of protein, usage probability of those synonymous codons is different. Some species or some genes are usually prone to use one or several particular synonymous codons. These codons are called preferable codons, which is called as codon bias. Usage bias of codons from various species has been studied, and it is found that during protein biosynthesis synonymous codons encoding amino acid is not used randomly [1] [2] [3] . Many studies have indicated that obvious bias exists between different genes from different species or the same species [4] [5] [6] . Usage bias of codons is influenced mainly by mutation bias, translation selection, secondary protein structure, replication and selective transcription, hydrophobia and hydrophilia of protein, and external environment [7] [8] [9] [10] [11] [12] [13] .