• KSBB Journal
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• v.26 no.2
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• pp.107-111
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• 2011

The essential operating parameters in virus vaccine production are multiplicity of infection (MOI), harvest time, and infection time. Stimulating agents also can be applied in order to improve vaccine productivity further. We investigated the optimum operating conditions in porcine transmissible gastroenteritis virus (TGEV) vaccine production and the applicability of sodium butyrate (NaBu) as a stimulating agents for the improvement of vaccine productivity. The optimum MOI, infection time, and harvest time for high production of TGEV by swine testicle (ST) cells were found to be 0.0001 pfu/cell, 3 day after cell inoculation, and 24 hpi, respectively. NaBu is known as a histone deacetylase inhibitor that has been widely used for the high expression of recombinant protein using mammalian cells and for the enhancement of virus propagation. So we tried to examine the potential of NaBu as a stimulating agent and to determine the optimum concentration by comparing TGEV titers with different range of NaBu concentration. TGEV titer with 5 mM NaBu was 1.5 times higher than control. Therefore, we concluded that NaBu can be a promising agent for stimulating various vaccine production including TGEV and the optimum NaBu concentration for TGEV production was determined to be 5 mM.

• KSBB Journal
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• v.9 no.3
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• pp.272-278
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• 1994

Attenuated varicella-zoster virus(VZV) was cultured in human embryonic lung cells. The effects of serum type and its concentration on the production of VZV were studied. Regardless of cell culture conditions, VZV yield was increased with multiplicity of infection, and the total cell concentration was decreased after virus infection. The newborn calf serum, calf serum, or horse serum was not as good as the fetal bovine serum or calf serum supplemented with iron for the propagation of VZV in the human embryonic lung cells. The yields of total VZV(cell-associated virus and cell-free virus) in the medium with calf serum containing iron were comparable to those in the medium supplemented with fetal bovine serum. Furthermore, some components of serum appear to be important for the maintenance of VZV infectivity.

• KSBB Journal
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• v.10 no.3
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• pp.292-297
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• 1995

Studies on the production of Newcastle disease virus(NDV) were carried out to optimize culture conditions such as initial pH, temperature, serum concentration, multiplicity of infection(M.O.I.) as well as the addition of polycation, antioxidant, and DMSO. Initial pH from 7.2 to 8.1 showed little difference on NDV production but the initial pH below 6.8 resulted in the negative effect. The highest NDV titer was obtained at 0.1 M.O.I. In addition, the maximum production of virus was achieved at 2% FBS and optimum temperature was found to be $34^{\circ}C$. Treatment of polycoation increased the virus production. When ascorbic acid was added as an antioxidant, NDV production was also enhanced. Utilization of DMSO, a well-known permeabilizing agent, showed an inhibitory effect on the propagation of NDV.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.281-284
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• 2002

Specific pathogen free (SPF) eggs have been used to produce live vaccines. however, their application causes many problems such as cost, space and waste disposal. The substitution of mammalian cells for SPF eggs offers a desirable system of vaccine production. In this study, mammalian cells were tested for the infection of Newcastle disease virus (NDV). As a result, DF-I and MDBK cells showed high virus productivity compared to the other mammalian cells. For the highest productivity of NDV, the optimal multiplicity of infection (M.O.I.) in DF-I or MDBK cells was determined to be 0.2 or 0.5 M.O.I., respectively.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.203-206
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• 2005

TGE (Transmissible Gastroenteritis) caused by a virus belonging to family coronavirus, results in an acute infection of the small intestine of the pig. The optimum operation variables such as multiplicity of infection (MOI), infection time and harvest time were investigated for TGE vaccine production by immobilized ST(swine testicle) cells. In the culture supplemented with 5% serum, maximum virus titer of $1.2{\times}10^6pfu/ml$ was obtained at the conditions of 0.01 MOI, 2day infection time, and 1 day harvest time. Serum is a potential source of bacterial, mycoplasmal and viral contamination, and it has a possibility of the introduction of serum proteins, prion and pyrogens into the final product. For these reasons, much attention has been focused on the development of serum-free media. A new serum-free media (SFM) has been developed in order to produce TGE vaccine of high quality with low cost. The performance of SFM developed was compared with other commercially available serum-free media and serum supplemented media in terms of virus productivity. The cultures with serum-free media showed higher titer than that with serum supplemented media. Among various serum-free media tested, CHO-S-SFMII showed highest virus titer.

• Korean Journal of Veterinary Service
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• v.37 no.1
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• pp.1-9
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• 2014

Although it has been generally accepted that porcine reproductive and respiratory syndrome virus (PRRSV) induces weak and delayed protective immunity after infection, it is unclear that the same immunological features can be applicable to all PRRS viruses because huge genetic variation exists even among the same genotypes of PRRSV (Type 1 and 2). In the current study, two genetically distinct type 2 PRRSV strains (VR-2332 and JA142) which showed approximately 90% nucleotide homology based on ORF5 sequences were characterized by both in vitro and in vivo assessments to determine the immunological features of the viruses. For in vitro assessment, porcine alveolar macrophages (PAM) were infected with the viruses at $10^{-3}$ multiplicity of infection (MOI) and then supernatants and cells were collected separately at 36 hrs post infection to determine the relative expression levels of IL-$1{\alpha}$, IL-12, TNF-${\alpha}$ and INF-${\alpha}/{\beta}$ by quantitative RT-PCR. In addition, five PRRSV-free pigs were inoculated with either of JA142 or VR2332 for in vivo assessment. Serum samples were collected every week until 6 weeks post challenge. The serum samples were analyzed for the levels of viremia, PRRSV nucleocapsid-specific antibody and virus neutralizing antibody. Based on those assessments, the two viruses showed different patterns of cytokine expression in PAM and immune responses in pigs after infection. These results indicate that genetically distinct PRRSV strains have different immunological features, which might be criteria for virus classification and selection of candidate virus strains for vaccine development in the future.

• International Journal of Industrial Entomology and Biomaterials
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• v.29 no.2
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• pp.207-213
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• 2014

The structural proteins of classical swine fever virus (CSFV) consist of nucleocapsid protein C and envelope glycoprotein $E^{rns}$ (E0), E1 and E2. Among them, E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. In this study, to determine the optimal expression conditions of glycoprotein E2 using baculovirus system, we investigated the influence of insect cells and media to the expression of recombinant E2. Recombinant virus containing glycoprotein E2 coding gene was constructed with bApGOZA DNA. Expression of the glycoprotein E2 was analyzed by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. Expression of glycoprotein E2 in Sf21 cells was first observed after 3 days and reached a maximum on the 5th day after infection. Furthermore, the highest levels of glycoprotein E2 expression were observed at multiplicity of infection (MOI) of 5. When three different insect cell lines (Sf21, High-Five and Se301) were tested, High-Five cells showed the highest production. In addition, four different serum-free and serum-supplemented media, respectively, were tested for the expression of glycoprotein E2 and the budded virus (BV) titers. As a result, serum-supplemented medium provided the best conditions for protein production and the BV yield.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.05a
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• pp.23-36
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• 2000

Japanese encephalitis virus (JEV) is the causative agent of a mosquito-borne encephalitis and is transmitted to human via persistently infected mosquito vectors. Although the virus is known to cause only acute infection, there were reports that showed neurological sequelae, latent infection in peripheral mononuclear cells, and recurrence of the disease after acute encephalitis. Innate resistance of certain cell lines, abnormal SN1 expression of the virus, and anti-apoptotic effect of cullular bcl-2 have been suggested as probable causes of JEV persistence even in the absence of defective interfering (DI) particles. Although possible involvement of DI particles in JEV persistence was suggested, neither has a direct evidence for DI presence nor its molecular characterization been made. Two questions asked in this study are whether the DI virus plays any role in JEV persistent infection if it is associated with and what type of change(s) can be made in persistently infected cells to avoid apoptosis even with the continuous virus replication, DI-free standard stock of JEV was infected in BHK-21, Vero, and SW13 cells and serial high multiplicity passages were performed in order to generate DI particles. There different-sized DI RNA species which were defective in both structural and nonstructural protein coding genes. Rescued ORFs of the DI genome maintained in-frame and the presence of replicative intermediate or replicative form RNA of the DI particles confirmed their replication competence. On the other hand, several clones with JEV persistent infection were established from the cells survived acute infections during the passages. Timing of the DI virus generation during the passages seemed coincide to the appearance of persistently infected cells. The DI RNAs were identified in most of persistently infected cells and were observed throughout the cell maintenance. One of the cloned cell line maintained the viral persistence without DI RNA coreplication. The cells with viral persistence released the reduced but continuous infectious JEV particle for up to 9 months and were refractory to homologous virus superinfection but not to heterologous challenges. Unlike the cells with acute infection these cells were devoid of characteristic DNA fragmentation and JEV-induced apoptosis with or without homologous superinfection. Therefore, the DI RNA generated during JEV undiluted serial passage on mammalian cells was shown to be biologically active and it seemed to be responsible, at least in part, for the establishment and maintenance of the JEV persistence in mammalian cells. Viral persistence without DI RNA coreplication, as in one of the cell clones, supports that JEV persistent infection could be maintained with or without the presence of DI particles. In addition, the fact that the cells with JEV persistence were resistant against homologous virus superinfection, but not against heterologous one, suggests that different viruses have their own and independent pathway for cytopathogenesis even if viral cytopathic effect could be converged to an apoptosis after all.

• Korean journal of applied entomology
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• v.46 no.2
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• pp.319-324
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• 2007

To determine the optimal concentration of fetal bovine serum (FBS) on the growth of insect cells and the multiplicity of viruses, the growth of cells (Sf21 and Bm5) and viruses were examined on the various concentrations of FBS. In view of the viability, growth speed, proliferation of cells and the amount of FBS, the most proper concentration for the cell culture were 7% and 5% for Sf21 and Bm5, respectively. The multiplicity of viruses at the various concentrations of FBS was similar in both cell lines at 5 days post-infection (p.i.). However, it differed significantly at 2 and 3 days p.i. The proper concentration of FBS were 10% and 3% for Sf21 at 2 and 3 days p.i., respectively, and 5% for Bm5 at both 2 and 3 days p.i. These results suggested that the optimal concentration of FBS should be determined according to the used cell lines and viruses for their optimum production.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.221-229
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• 2002

Japanese encephalitis virus (JEV) persistence was established and maintained in Vero cell culture for over 1 year. Eleven clones of plaque morphology mutant JEV, with large and small plaque sizes, were obtained from the cell culture supernatant. Genomic RNA replication efficiency of the mutants in naive Vero cell appeared to correspond to their different plaque sizes. No significant changes in envelop protein ORF or in non-coding regions at both ends of the RNA genome suggested that there could be an unidentified factor(s) playing role in JEV attenuation. Unlike to the replication of wild-type JEV, the mutants did not induce severe degree of cytopathic effect in Vero cells upon infection. While obvious decrease of Bcl-2 and its mRNA expression and sharp increase of p53 in naive Vero cells infected with either wild-type JEV or the large plaque-forming mutant, those changes were not observed with the small plaque-forming one. Together with these observation, internucleosomal DNA fragmentation and chromosomal DNA profile in the Vero cells infected with the mutants suggest that an overall changes in cytopathic effect in the plaque morphology mutants-infected cells should be primarily due to the reduced genomic RNA replication and the compromised degree of p53-independent apoptosis by the virus infection at least in part.