Naidong Wang

In silico analyses of antigenicity and surface structure variation of an emerging porcine circovirus genotype 2b mutant, prevalent in southern China from 2013 to 2015.

Porcine circovirus type 2 (PCV2) is the pivotal pathogen causing porcine circovirus-associated diseases. In this study, 62 PCV2 isolates were identified from seven farms in southern China from 2013 to 2015 and phylogenetic trees were reconstructed based on whole-genome sequences or the cap gene. In this investigation, PCV2b was the main genotype in circulation throughout these farms. Furthermore, an emerging mutant (PCV2b-1C), isolated from PCV2-vaccinated farms, was the predominant strain prevalent on these farms. In addition, we isolated a new cluster that may represent evolution of the virus through recombination of PCV2b-1A/1B and PCV2b-1C. Finally, we discuss evidence that antigenicity and surface structure variation of the capsid resulted from mutation of the C-terminal loop (Loop CT) of the PCV2b-1C Cap in silico.

Generation and immunogenicity of porcine circovirus type 2 chimeric virus-like particles displaying porcine reproductive and respiratory syndrome virus GP5 epitope B.

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.

In silico analysis of surface structure variation of PCV2 capsid resulting from loop mutations of its capsid protein (Cap).

Outbreaks of porcine circovirus (PCV) type 2 (PCV2)-associated diseases have caused substantial economic losses worldwide in the last 20 years. The PCV capsid protein (Cap) is the sole structural protein and main antigenic determinant of this virus. In this study, not only were phylogenetic trees reconstructed, but variations of surface structure of the PCV capsid were analysed in the course of evolution. Unique surface patterns of the icosahedral fivefold axes of the PCV2 capsid were identified and characterized, all of which were absent in PCV type 1 (PCV1). Icosahedral fivefold axes, decorated with Loops BC, HI and DE, were distinctly different between PCV2 and PCV1. Loops BC, determining the outermost surface around the fivefold axes of PCV capsids, had limited homology between Caps of PCV1 and PCV2. A conserved tyrosine phosphorylation motif in Loop HI that might be recognized by non-receptor tyrosine kinase(s) in vivo was present only in PCV2. Particularly, the concurrent presence of 60 pairs of the conserved tyrosine and a canonical PXXP motif on the PCV2 capsid surface could be a mechanism for PXXP motif binding to and activation of an SH3-domain-containing tyrosine kinase in host cells. Additionally, a conserved cysteine in Loop DE of the PCV2 Cap was substituted by an arginine in PCV1, indicating potentially distinct assembly mechanisms of the capsid in vitro between PCV1 and PCV2. Therefore, these unique patterns on the PCV2 capsid surface, absent in PCV1 isolates, might be related to cell entry, virus function and pathogenesis.

Optimized conditions for preserving stability and integrity of porcine circovirus type2 virus-like particles during long-term storage

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.

Complete Genome Sequence of Porcine Circovirus Strain 102 with a Novel Mutation, Isolated from Hunan Province, China

ABSTRACT Porcine circovirus 2 (PCV2) strain 102 belongs to the PCV2b-1C subtype, and its sole structural protein (Cap) exhibits high homology with that of other PCV2b isolates reported in South Korea, China, and the United States. Strain 102 contains a new mutation (R37H) in the domain of the nuclear localization signal (NLS) of the Cap.

Identification and characterization of MYH9 locus for high efficient gene knock-in and stable expression in mouse embryonic stem cells

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.

Evidence of natural co-infection with PCV2b subtypes in vivo

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.

The protective effects of DL-Selenomethionine against T-2/HT-2 toxins-induced cytotoxicity and oxidative stress in broiler hepatocytes

T-2 and HT-2 toxins can cause cytotoxicity and oxidative stress in animals, while DL-Selenomethionine plays an important role in preventing oxidative stress and improving cell viability. However, the role of DL-Selenomethionine in T-2/HT-2 toxins-induced cell damage is still unknown. In this study, we investigated whether DL-Selenomethionine plays a protective role against T-2/HT-2-induced cytotoxicity and oxidative stress in primary hepatocytes. Our results demonstrated that T-2/HT-2 toxins-exposed broiler hepatocytes exhibited significantly decreased cell viability and intracellular glutathione (GSH) concentration while increased Lacate dehydrogenase (LDH) leakage, intracellular reactive oxygen species (ROS), glutathione peroxidase (GSH-PX), malondialdehyde (MDA) and catalase (CAT) levels, as well as elevated expression levels of genes related to oxidative stress, in a toxin dose-dependent manner (P < 0.05). However, the application of DL-Selenomethionine into T-2/HT-2 treated hepatocytes effectively alleviated the adverse effects of T-2/HT-2, as demonstrated by increased cell viability, decreased LDH leakage, declined intracellular ROS and MDA levels, increased expression of oxidative stress-related genes, as well as accordingly enhanced activities of GSH, GSH-PX, SOD and CAT as compared to the control groups (P < 0.05). Therefore, our in vitro data demonstrate that DL-Selenomethionine can function as an effectively protective agent against T-2/HT-2-induced cytotoxicity and oxidative stress.

Structure-Based Design of Porcine Circovirus Type 2 Chimeric VLPs (cVLPs) Displays Foreign Peptides on the Capsid Surface

Although porcine circovirus-like particles can function as a vector to carry foreign peptides into host cells, displaying foreign peptides on the surface of virus-like particles (VLPs) remains challenging. In this study, a plateau, consisting of the middle portion of Loop CD (MP-Lcd) from two neighboring subunits of PCV2 capsid protein (Cap), was identified as an ideal site to insert various foreign peptides or epitopes and display them on the surface of PCV2 VLPs. One of the goals of this work is to determine if the surface pattern of this plateau can be altered without compromising the neutralizing activity against PCV2 infections. Therefore, biological roles of MP-Lcd regarding VLPs assembly, cell entry, and antigenicity were investigated to determine whether this was a universal site for insertion of foreign functional peptides. Three-dimensional (3D) structure simulations and mutation assays revealed MP-Lcd was dispensable for PCV2 Cap assembly into VLPs and their entry into host cells. Notably, substitution of MP-Lcd with a foreign peptide, caused surface pattern changes around two-fold axes of PCV2 VLPs based on 3D structure simulation, but was not detrimental to VLPs assembly and cell entry. Moreover, this substitution had no adverse effect on eliciting neutralizing antibodies (NAbs) against PCV2 infection in pigs. In conclusion, MP-Lcd of the PCV2 Cap was a promising site to accommodate and display foreign epitopes or functional peptides on the surface of PCV2 VLPs. Furthermore, chimeric VLPs (cVLPs) would have potential as bivalent or multivalent vaccines and carriers to deliver functional peptides to target cells.

Generation of PCV2 in PK15 cells transfected with recombinant baculovirus containing a 1.1 copy of the PCV2 genome

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.