1. Introduction
Porcine circovirus is the smallest virus known to replicate autonomously in mammalian cells (Breitbart et al., 2017). Now, according to the antigenicity of porcine circovirus and the difference of its genome sequence, porcine circovirus is divided into three serotypes, porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3).  PCV2 is the main pathogenof post-weaning multisystemic wasting syndrome (PMWS) and nephrotic syndrome (PDNS) (An et al., 2007). Similar to PCV2,PCV3 may cause reproduction disorder in sows and PDNS in adult pigs (Palinski et al., 2017).
Currently, PCV2 is divided into five different genotypes: PCV2a, PCV2b, PCV2c, PCV2d and PCV2e. PCV2a, PCV2b and PCV2d are the major genotypes. Before 2001, the most clinically prevalent genotype affected pigs was PCV2a. Since 2003, PCV2b genotype has essentially replaced the previously predominant PCV2a genotype in global. Recently, the worldwide prevalence of PCV2d has increased, and seems a tendency to replace PCV2b (Franzo et al., 2018; Wang et al., 2020).
Rep protein is the key protein for viral replication. Some special structures are essential to maintain Rep protein function, and mutations or deletions of related genes affect virus replication (Mankertz et al., 2001). Studies have shown that mutations in Cap protein from proline to alanine in 110th and from arginine to serine in 191th could enhance the replication capacity of PCV2 in vitro and reduce toxicity in vivo (Fenaux et al., 2004). It had been shown that altering the amino acid sequence at the Rep protein also altered the replication ability of the virus (Shi et al., 2015). However, information regarding the differential site mutation of different genotypes PCV2 Rep protein, as well as the correlation between the mutation and replication, remains limited.
In order to analyze the effect of the key sites on viral replication, we analyzed 1000 PCV2 genome, the alignment result showed the differences between PCV2b and PCV2d in 6th, 34th, 77th amino acids. Moreover, for the PCV2b and PCV2d, the mutation frequency of the 6th is 1.75% and 26.17%, respectively. Here we used site-directed mutagenesis to mutate the 6th aa of Rep between PCV2b and PCV2d infectious clone to assess the effect of key sites on viral replication.