The present study was conducted to develop an effective method for establishment of porcine parthenogenetic embryonic stem cells (ppESCs) from parthenogenetically activated oocyte-derived blastocysts. The addition of 10% fetal bovine serum (FBS) to the medium on the 3rd day of oocyte culturing improved the development of blastocysts, attachment of inner cell masses (ICMs) onto feeder cells, and formation of primitive ppESC colonies. ICM attachment was further enhanced by basic fibroblast growth factor, stem cell factor, and leukemia inhibitory factor. From these attached ICMs, seven ppESC lines were established. ppESC pluripotency was verified by strong enzymatic alkaline phosphatase activity and the expression of pluripotent markers OCT3/4, Nanog, and SSEA4. Moreover, the ppESCs were induced to form an embryoid body and teratoma. Differentiation into three germ layers (ectoderm, mesoderm, and endoderm) was confirmed by the expression of specific markers for the layers and histological analysis. In conclusion, data from the present study suggested that our modified culture conditions using FBS and cytokines are highly useful for improving the generation of pluripotent ppESCs.
Animals ; Blastocyst/*cytology ; Cell Culture Techniques/*veterinary ; *Cell Differentiation ; Cytokines/metabolism ; Embryonic Stem Cells/*cytology ; Parthenogenesis ; Pluripotent Stem Cells/*cytology ; Swine/*physiology

Porcine epidemic diarrhea virus (PEDV) LJB/03 strain was isolated from the feces of piglets suspected to be suffering from a severe diarrhea in Heilongjiang Province, and was identified by immunofluorescence test, immunelectronmicroscopy, RT-PCR and indirect ELISA assay. Characteristics of the virus culture and the methods of improvement of virus titer were explored. The results showed that the virus had the typical appearance of the coronavirus. Analysis of the nucleotide sequences of RT-PCR products revealed 98% homology with the reference strains. Indirect immunofluorescence assay showed a significant presence of green fluorescence, and an average P/N ratio of 7.6 by indirect ELISA assay. Taken together, these tests showed positive isolation of PEDV. Using the virus plaque purification cloning methods established in the test, the purified PEDV large plaque and small plaque were obtained, and the large plaque and small plaque titers were measured with significant difference. These results provide potential for the application of PEDV on the basis of the biological features of isolated virus.
Animals ; Cell Culture Techniques ; China ; epidemiology ; Coronavirus Infections ; epidemiology ; veterinary ; virology ; Epidemics ; Feces ; virology ; Porcine epidemic diarrhea virus ; genetics ; growth & development ; isolation & purification ; Swine ; Swine Diseases ; epidemiology ; virology ; Virus Cultivation

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.
Animals ; Antigens, Viral/genetics/metabolism ; Caliciviridae Infections/prevention & control/*veterinary/virology ; Capsid Proteins/*genetics/metabolism ; Cell Culture Techniques/*methods ; Codon/genetics/metabolism ; Enzyme-Linked Immunosorbent Assay/veterinary ; *Gene Expression Regulation, Viral ; Hemorrhagic Disease Virus, Rabbit/*genetics/immunology ; *Rabbits ; Recombinant Proteins/genetics/metabolism ; Sf9 Cells ; Spodoptera ; Viral Structural Proteins/*genetics/metabolism ; Viral Vaccines/genetics/immunology