摘要：
Targeted integration of exogenous genes into so-called safe harbors/friend sites, offers the advantages of expressing normal levels of target genes and preventing potentially adverse effects on endogenous genes. However, the ideal genomic loci for this purpose remain limited. Additionally, due to the inherent and unresolved issues with the current genome editing tools, traditional embryonic stem (ES) cell-based targeted transgenesis technology is still preferred in practical applications. Here, we report that a high and repeatable homologous recombination (HR) frequency (>95%) is achieved when an approximate 6kb DNA sequence flanking the MYH9 gene exon 2 site is used to create the homology arms for the knockout/knock-in of diverse nonmuscle myosin II (NM II) isoforms in mouse ES cells. The easily obtained ES clones greatly facilitated the generation of multiple NM II genetic replacement mouse models, as characterized previously. Further investigation demonstrated that though the targeted integration site for exogenous genes is shifted to MYH9 intron 2 (about 500bp downstream exon 2), the high HR efficiency and the endogenous MYH9 gene integrity are not only preserved, but the expected expression of the inserted gene(s) is observed in a pre-designed set of experiments conducted in mouse ES cells. Importantly, we confirmed that the expression and normal function of the endogenous MYH9 gene is not affected by the insertion of the exogenous gene in these cases. Therefore, these findings suggest that like the commonly used ROSA26 site, the MYH9 gene locus may be considered a new safe harbor for high-efficiency targeted transgenesis and for biomedical applications.
摘要：
A newly emerging porcine circovirus, designated PCV3, has been reported in various countries (USA, Poland, South Korea and China) since 2017. Its presence may be associated with porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, and multisystem inflammation. In this study, we report identification of PCV3 in cases of reproductive failure in various regions in Hunan, China. From January 2015 to December 2016, sera were collected from 190 sows from seven farms with reproductive problems. Specifically, 85 samples were from sows with a history of reproductive failure, whereas the remaining 105 were from healthy sows. The PCV3-positive rate was significantly higher in sows with reproductive failure (45.9%) than in healthy sows (21.9%), based on quantitative PCR (qPCR) assays. Although phylogenetic analysis based on the cap gene suggested that these PCV3 isolates belonged to the clade PCV3a, amino acid sequence variations in the Cap protein still occurred among these isolates, and these might have contributed to antigenic alterations of the Cap protein, based on the Jameson-Wolf antigenic index. Finally, we concluded that PCV3 was circulating in sows in Hunan province, China. However, the association of PCV3 with reproductive failure in sows and its potential for vertical transmission need to be studied further.
通讯机构：
[Yang, Yi] H;[Jiang, Ping] N;Hunan Agr Univ, LFP, Coll Vet Med, Changsha 410128, Hunan, Peoples R China.;Hunan Agr Univ, RCRV, Coll Vet Med, Changsha 410128, Hunan, Peoples R China.;Nanjing Agr Univ, Key Lab Anim Dis Diagnost & Immunol, Minist Agr, Coll Vet Med, Nanjing 210095, Jiangsu, Peoples R China.
关键词：
Porcine circovirus type 2 (PCV2);Virus-like particles (VLPs);Osmolytes;Stability
摘要：
Although porcine circovirus type 2 (PCV2) virus-like particles (VLPs) have been successfully harvested from various protein expression systems, conditions to promote their stability and integrity during long-term storage have not been well defined since only the intact VLPs, instead of the monomeric capsid protein (Cap), can induce neutralizing antibodies in pigs in previous studies. In this study, freshly prepared PCV2 VLPs were stored in several media (various concentrations of NaCl, sorbitol, sucrose and trehalose) at three temperatures (4 °C, −20 °C and −80 °C) and their stability and integration were evaluated after 7 month. Addition of 15% trehalose in storage buffer promoted long-term preservation of PCV2 VLPs. In contrast, storage buffer with 5% osmolytes (sucrose, trehalose and sorbitol) did not confer stabilization for long-term storage. These refined storage conditions for stabilization of PCV2 VLPs should enhance their use in vaccines.
摘要：
Porcine circovirus associated diseases (PCVAD) caused by PCV2 are responsible for severe economic losses in the swine industry. The mechanism of PCV2 replication has not been fully elucidated yet. PCV2 may be successfully rescued by means of either an infectious DNA clone containing the full length of the viral genomic DNA, or from PCV2-infected clinical tissues in PK15 cell culture. However, viruses harvested by both methods have low titres. In this study, PCV2 was prepared with a higher titre from PK15 cells infected by recombinant baculoviruses containing 1PCV2 (one stem-loop structure) or 1.1PCV2 (two stem-loop structure) genomic DNA copy. In addition, infectious DNA clones containing two stem-loop structures in either plasmid or baculovirus backbones are capable of generating a higher virus titre than the DNA clones with only one copy of stem-loop structure.
期刊：
Archives of Virology,2017年162(7):2015-2020 ISSN：0304-8608
通讯作者：
Wang, Aibing
作者机构：
[Hu, Yi; Liu, Tanbin; Liu, Wei; Wang, Aibing; Lei, Hongyu] Hunan Agr Univ, Coll Vet Med, Res Ctr Reverse Vaccinol RCRV, Lab Anim Models & Funct Genom LAMFG, Changsha 410128, Hunan, Peoples R China.;[Zhan, Yang; Xie, Xiaohong; Wang, Naidong; Wang, Dongliang; Deng, Zhibang; Yang, Yi] Hunan Agr Univ, Coll Vet Med, Res Ctr Reverse Vaccinol RCRV, Lab Funct Prote LFP, Changsha 410128, Hunan, Peoples R China.
通讯机构：
[Wang, Aibing] H;Hunan Agr Univ, Coll Vet Med, Res Ctr Reverse Vaccinol RCRV, Lab Anim Models & Funct Genom LAMFG, Changsha 410128, Hunan, Peoples R China.
关键词：
PCV2 Strain;Major Open Reading Frame;PCV2 Genome;PCV2 Genotype;Porcine Circoviruses
摘要：
Porcine circovirus type 2 (PCV2) is the causative pathogen of porcine circovirus-associated diseases (PCVAD). This virus evolves mostly through point mutations and genome recombination between different PCV2 genotypes (e.g. PCV2a and PCV2b), as has been confirmed in swine herds. In the current work, the complete PCV2 genome sequences of 69 clones derived from various tissues (lymph node, spleen and lung,) of an infected individual, were subjected to phylogenetic and alignment analyses. The results not only demonstrate co-infection with distinct PCV2b subtypes (e.g. 1B and 1C) in the same animal, but also highlight another mechanism of evolution - diverse point mutations acquired during immune evasion by this virus.
摘要：
Cellular DNA damage response (DDR) triggered by infection of DNA viruses mediate cell cycle checkpoint activation, DNA repair, or apoptosis induction. In the present study, infection of porcine circovirus type 2 (PCV2), which serves as a major etiological agent of PCV2-associated diseases (PCVAD), was found to elicit a DNA damage response (DDR) as observed by the phosphorylation of H2AX and RPA32 following infection. The response requires active viral replication, and all the ATM (ataxia telangiectasia-mutated kinase), ATR (ATM- and Rad3-related kinase), and DNA-PK (DNA-dependent protein kinase) are the transducers of the DDR signaling events in the PCV2-infected cells as demonstrated by the phosphorylation of ATM, ATR, and DNA-PK signalings as well as reductions in their activations after treatment with specific kinase inhibitors. Inhibitions of ATM, ATR, and DNA-PK activations block viral replication and prevent apoptotic responses as observed by decreases in cleaved poly-ADP ribose polymerase (PARP) and caspase-3 as well as fragmented DNA following PCV2 infection. These results reveal that PCV2 is able to exploit the cellular DNA damage response machinery for its own efficient replication and for apoptosis induction, further extending our understanding for the molecular mechanism of PCV2 infection.
作者机构：
[Yang, Yi] Hunan Agr Univ, LFP, Coll Vet Med, Changsha 410128, Hunan, Peoples R China.;Hunan Agr Univ, RCRV, Coll Vet Med, Changsha 410128, Hunan, Peoples R China.
通讯机构：
[Yang, Yi] H;Hunan Agr Univ, LFP, Coll Vet Med, Changsha 410128, Hunan, Peoples R China.
关键词：
Chimeric virus-like particles;Immune responses;Porcine circovirus type 2;Porcine reproductive and respiratory syndrome virus GP5 epitope B
摘要：
Virus-like particles (VLPs) can be used as transfer vehicles carrying foreign proteins or antigen epitopes to produce chimeric VLPs for bivalent or multivalent vaccines. Based on the crystal structure of porcine circovirus type 2 (PCV2) capsid protein (Cap), in addition to alignment of the Cap sequences collected from various isolates of PCV2 and PCV1, we predicted that Loop CD of the PCV2 Cap should tolerate insertion of foreign epitopes, and furthermore that such an insertion could be presented on the surface of PCV2 VLPs. To validate this, the GP5 epitope B of porcine reproductive and respiratory syndrome virus (PRRSV) was inserted into Loop CD of the PCV2 Cap. The 3D structure of the recombinant PCV2 Cap (rCap) was simulated by homology modeling; it appeared that the GP5 epitope B was folded as a relatively independent unit, separated from the PCV2 Cap backbone. Furthermore, based on transmission electron microscopy, the purified PCV2 rCap self-assembled into chimeric VLPs which entered PK-15 cells. In addition, PCV2 chimeric VLPs induced strong humoral (neutralizing antibodies against PCV2 and PRRSV) and cellular immune responses in mice. We concluded that the identified insertion site in the PCV2 Cap had great potential to develop PCV2 VLPs-based bivalent or multivalent vaccines; furthermore, it would also facilitate development of a nano-device to present a functional peptide on the surface of the VLPs that could be used for therapeutic purposes.
期刊：
JOURNAL OF GENERAL VIROLOGY,2016年97(12):3331-3344 ISSN：0022-1317
通讯作者：
Yang, Yi
作者机构：
[Wang, Naidong; Zhan, Yang; Wang, Aibing; Yang, Yi] Hunan Agr Univ, LFP, Coll Vet Med, Changsha, Hunan, Peoples R China.;[Wang, Naidong; Zhan, Yang; Wang, Aibing; Yang, Yi] Hunan Agr Univ, RCRV, Coll Vet Med, Changsha, Hunan, Peoples R China.;[Zhang, Lijie] Beijing Comp Ctr, Beijing, Peoples R China.;[Khayat, Reza] CUNY City Coll, Dept Chem, New York, NY 10031 USA.;[Khayat, Reza] CUNY, Grad Ctr, PhD Program Biochem, New York, NY USA.
通讯机构：
[Yang, Yi] H;Hunan Agr Univ, LFP, Coll Vet Med, Changsha, Hunan, Peoples R China.;Hunan Agr Univ, RCRV, Coll Vet Med, Changsha, Hunan, Peoples R China.
