Serine 105 and 120 are important phosphorylation sites for porcine reproductive and respiratory syndrome virus N protein function.

The nucleocapsid (N) protein is the most abundant protein of porcine reproductive and respiratory syndrome virus (PRRSV). It has been shown to be multiphosphorylated. However, the phosphorylation sites are still unknown. In this study, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to analyze the phosphorylation sites of N protein expressed in Sf9 cells. The results showed that N protein contains two phosphorylation sites. Since N protein can regulate IL-10, which may facilitate PRRSV replication, we constructed four plasmids (pCA-XH-GD, pCA-A105, pCA-A120 and pCA-A105-120) and transfected them into Pig alveolar macrophages (PAMs,3D4/2). The qPCR results showed that mutations at residues 105 and 120 were associated with down-regulation of the IL-10 mRNA level, potentially decreasing the viral growth ability. Then, we mutated the phosphorylation sites (S105A and S120A) and rescued three mutated viruses, namely, A105, A120 and A105-120. Compared with wild-type virus titers, the titers of the mutated viruses at 48 hpi were significantly decreased. The Nsp(non-structural protein) 9 qPCR results were consistent with the multistep growth kinetics results. The infected PAMs (primary PAMs) results were similar with Marc-145.The findings indicated that the mutations impaired the viral replication ability. The confocal microscopy results suggested that mutations to residues 105 and 120 did not affect N protein distribution. Whether the mutations affected other functions of N protein and what the underlying mechanisms are need further investigation. In conclusion, our results show that residues 105 and 120 are phosphorylation sites and are important for N protein function and for viral replication ability.

First identification of porcine parvovirus 7 in China.

Porcine parvovirus (PPV) are small, non-enveloped and single-stranded DNA viruses, taxonomically classifiable within the family Parvoviridae. Seven PPV genotypes (PPV1 to PPV7) have been identified to date. PPV7, the most recently discovered PPV genotype, was first reported in US pigs in 2016. To explore PPV7 status in Chinese pig populations a total of 64 serum samples collected from two commercial farms in Guangdong province in 2014 were analyzed. PPV7 DNA was detected in 32.8% (21/64) of tested samples. On the porcine circovirus type 2 (PCV2) positive farm, the prevalence rate of PPV7 was 65.5% (19/29) which was significantly higher than that on the PCV2 negative farm (2/35, 5.7%), indicating a possible association between PCV2 and PPV7 infections. The sequences of three PPV7 strains were determined. Phylogenetic analysis revealed that the identified PPV7 strains circulating in China shared 98.7%-99.7% nucleotide homology with the US strain. Further sequence comparison analysis indicated that GD-2014-2 and GD-2014-3 possess a consecutive 9-nt deletion in the VP gene. This is the first report of the existence of PPV7 in China and this finding will strengthen understanding of the epidemiology of porcine parvovirus in Chinese pigs.