The cultured cells were derived from rabbit corneal stroma by explant technique following microdissection and serial passage. The ultrastructural features of fourteenth-passage keratocytes were examined with both scanning and transmission electron microscope. The cells can be divided into activated, intermediate and old cell according to the differences in electron density and surface microvillous pattern. The morphologic characteristics of the cultured keratocytes partially resemble those shown in corneal keratocytes in vivo.
Cells, Cultured ; Corneal Keratocytes* ; Corneal Stroma ; Microdissection ; Serial Passage

Porcine deltacoronavirus (PDCoV) has emerged in several pig-raising countries and has been a causative pathogen associated with diarrheal diseases in South Korea since 2014. In the present study, we were able to isolate and cultivate a Korean PDCoV strain (KNU16-07) in cell culture and investigate its pathogenicity. PDCoV-inoculated piglets showed watery diarrhea accompanied by acute enteritis in the natural host. Sequencing analysis demonstrated the genetic stability of KNU16-07 for at least thirty serial passages.
Cell Culture Techniques ; Diarrhea ; Enteritis ; Korea ; Serial Passage ; Virulence

Cell cultures of adults bovine retinal pigment epithelium(RPE) were propagated from central and peripheral regions of the same eyes to study the topographical differences in cell growth and to compare the differences in growth rate between two areas. The results obtained were as follows: A regional variation in the morpholgy was observed between the RPE from central and that from peripheral regions. Retinal pigment epithelium from central region attached to culture dish more slowly(average 4 days) than those from peripheral region(average 3.5 days) The growth rate of retinal pigment epithelium declined with serial passage in culture. The growth rate of retinal pigment epithelium from peripheral region at the first generation was highest. And there was a statistical difference in growth rate with passing in generation(P<0.05). This study reveals that growth rate and cell activity of RPE from central region are lower than from peripheral region.
Adult ; Cell Culture Techniques ; Humans ; Macular Degeneration ; Retinal Pigment Epithelium* ; Retinaldehyde* ; Serial Passage

OBJECTIVETo investigate the genetic stability during the serial passages of non-replicating recombinant adenoviruses based on the novel AdEasy system.

METHODSFour non-replicating adenoviruses expressing rotavirus antigens, which were generated by the AdEasy system, were used as models and continuously propagated on 293 cells for 20 passages. Samples of the infected cells were collected at every 5 passages for the PCR analysis of the inserted rotavirus genes, replication-competent adenoviruses (RCAs) as well as the Western blot examination of the expression of the certain rotavirus genes.

RESULTSThe adenoviruses can be stably propagated on 293 cells. The existence and the stable expression of inserted rotavirus genes were able to be detected during the serial passage. The RCAs were not found within the first 10 passages, but the rvAdG2VP7(o) at passage 15 and the rvAdG2VP7(o) and rvAdG1VP7(o) at passage 20.

CONCLUSIONThe non-replicating adenoviruses showed promising genetic stability during the continuous passage on 293 cells. The RCAs normally will not be detectable within 10 continuous passages. The results indicated the potential of such recombinant adenoviruses in the research and development of the gene therapies and adenoviral-vectored vaccines.

Adenoviridae ; genetics ; isolation & purification ; physiology ; Cell Line ; DNA, Recombinant ; genetics ; Humans ; Serial Passage ; Transgenes ; Virus Replication

Although trivalent subunit vaccine has been available, the influenza vaccine has been under-utilized because of cumbersome route of vaccination and low level of protection. Therefore, there has always been a great need to develop live attenuated influenza vaccine which can be administered through nasal route and elicit better immunogenicity. Through conventional repeated passage at low temperature, a live influenza vaccine carrier could be established. By reassortant formation between the 'cold- adapted' vaccine carrier and virulent strains, a prototype of trivalent live influenza vaccine is developed. Influenza A virus was adapted to replicate at low temperature. Serial passage at progressively lower temperature (30degrees C, 27degrees C and 24degrees C)resulted in the generation of cold-adapted (ca), temperature-sensitive (ts) mutant and attenuation (att) phenotype. This strain was evaluated for their ability to protect mice from challenge with same subtype and different subtype of influenza A virus. The study showed that vaccination of mice with live attenuated influenza virus provided complete protection against homologous and heterologous virus challenge. We also evaluated therapeutic potential of ca influenza virus. The mice infected with ca virus before challenge with wild type viruses or infected with simultaneously showed reduced clinical symptoms suggesting potential therapeutic effects.
Animals ; Influenza A virus ; Influenza Vaccines ; Mice ; Orthomyxoviridae ; Phenotype ; Serial Passage ; Vaccination

Although trivalent subunit vaccine has been available, the influenza vaccine has been under-utilized because of cumbersome route of vaccination and low level of protection. Therefore, there has always been a great need to develop live attenuated influenza vaccine which can be administered through nasal route and elicit better immunogenicity. Through conventional repeated passage at low temperature, a live influenza vaccine carrier could be established. By reassortant formation between the 'cold- adapted' vaccine carrier and virulent strains, a prototype of trivalent live influenza vaccine is developed. Influenza A virus was adapted to replicate at low temperature. Serial passage at progressively lower temperature (30degrees C, 27degrees C and 24degrees C)resulted in the generation of cold-adapted (ca), temperature-sensitive (ts) mutant and attenuation (att) phenotype. This strain was evaluated for their ability to protect mice from challenge with same subtype and different subtype of influenza A virus. The study showed that vaccination of mice with live attenuated influenza virus provided complete protection against homologous and heterologous virus challenge. We also evaluated therapeutic potential of ca influenza virus. The mice infected with ca virus before challenge with wild type viruses or infected with simultaneously showed reduced clinical symptoms suggesting potential therapeutic effects.
Animals ; Influenza A virus ; Influenza Vaccines ; Mice ; Orthomyxoviridae ; Phenotype ; Serial Passage ; Vaccination

Due to the increased frequency of interspecies transmission of avian influenza viruses, studies designed to identify the molecular determinants that could lead to an expansion of the host range have been increased. A variety of mouse-based mammalian-adaptation studies of avian influenza viruses have provided insight into the genetic alterations of various avian influenza subtypes that may contribute to the generation of a pandemic virus. To date, the studies have focused on avian influenza subtypes H5, H6, H7, H9, and H10 which have recently caused human infection. Although mice cannot fully reflect the course of human infection with avian influenza, these mouse studies can be a useful method for investigating potential mammalian adaptive markers against newly emerging avian influenza viruses. In addition, due to the lack of appropriate vaccines against the diverse emerging influenza viruses, the generation of mouse-adapted lethal variants could contribute to the development of effective vaccines or therapeutic agents. Within this review, we will summarize studies that have demonstrated adaptations of avian influenza viruses that result in an altered pathogenicity in mice which may suggest the potential application of mouse-lethal strains in the development of influenza vaccines and/or therapeutics in preclinical studies.
Animals ; Host Specificity ; Humans ; Influenza A virus ; Influenza in Birds* ; Influenza Vaccines ; Methods ; Mice ; Orthomyxoviridae ; Pandemics ; Serial Passage ; Vaccination ; Vaccines* ; Virulence

We have reported RPS-Vax system by introducing multiple cloning site (MCS) and 3C-protease cutting site at the N-terminal end of the poliovirus Sabin 1 cDNA. Potential vaccine genes can be easily introduced into recombinant polioviral genome and expressed during the viral replication as a part of virus polyprotein and subsequently processed from the mature viral protein by the poliovirus-specific 3C-protease. However, these poliovirus vector-mediated chimeric viral vaccine was not efficient to induce the cell-mediated immunity because of its rapid cytolytic capacity. In order to make CTL-inducing vaccine vector, we integrated a protein transduction domain (PTD) into the pRPS-Vax vector system right ahead of the MCS, named RPS-Vax/PTD. We have incorporated the HCV core (N-terminal 100aa) antigen into the MCS of pRPSvax-PTD vector, followed by production of chimeric virus, named RPSvax-PTD/HCVc. The chimeric virus was genetically stable during the serial passages. Replication capacity of the RPSvax-PTD/HCVc was 1~2 log lower than that of RPS-Vax control virus. These chimeric virus was very efficient to inducing antigen-specific IgG2a in the immunized mice, implying that the recombinant virus has a capacity to induce HCV-specific Th1 type immunity in the immunized animals or humans.
Animals ; Clone Cells ; Cloning, Organism ; DNA, Complementary ; Genome ; Hepatitis C* ; Hepatitis* ; Humans ; Immunity, Cellular ; Immunoglobulin G ; Mice ; Poliovirus ; Serial Passage

We have reported RPS-Vax system by introducing multiple cloning site (MCS) and 3C-protease cutting site at the N-terminal end of the poliovirus Sabin 1 cDNA. Potential vaccine genes can be easily introduced into recombinant polioviral genome and expressed during the viral replication as a part of virus polyprotein and subsequently processed from the mature viral protein by the poliovirus-specific 3C-protease. However, these poliovirus vector-mediated chimeric viral vaccine was not efficient to induce the cell-mediated immunity because of its rapid cytolytic capacity. In order to make CTL-inducing vaccine vector, we integrated a protein transduction domain (PTD) into the pRPS-Vax vector system right ahead of the MCS, named RPS-Vax/PTD. We have incorporated the HCV core (N-terminal 100aa) antigen into the MCS of pRPSvax-PTD vector, followed by production of chimeric virus, named RPSvax-PTD/HCVc. The chimeric virus was genetically stable during the serial passages. Replication capacity of the RPSvax-PTD/HCVc was 1~2 log lower than that of RPS-Vax control virus. These chimeric virus was very efficient to inducing antigen-specific IgG2a in the immunized mice, implying that the recombinant virus has a capacity to induce HCV-specific Th1 type immunity in the immunized animals or humans.
Animals ; Clone Cells ; Cloning, Organism ; DNA, Complementary ; Genome ; Hepatitis C* ; Hepatitis* ; Humans ; Immunity, Cellular ; Immunoglobulin G ; Mice ; Poliovirus ; Serial Passage

To character a novel chimera(1b/2a) hepatitis C virus cell culture (HCVcc) system carrying envelope E1E2 coding gene from Hebei strain of China, chimera HCVcc (cHCVcc) was developed from Huh7.5-CD81 cells after transfection with in vitro transcribed full-length 1b/2a chimera RNA, which carrying envelope E1E2 coding gene from Hebei strain of China. Then the replication, expression and infectious titer of serial passage HCVcc were assessed by Real Time RT-PCR, indirect immunofluorescence assay (IFA) and Western blotting (WB). In addition, chimeric envelope gene from HCVcc was sequenced after serial passage. We found that the number of HCV positive focus increased gradually in cell post-transfection with chimera HCVcc (1b/2a) RNA and reach a peak platform (80% to 90%) at 41 days post-transfection; the expression of HCV protein was also confirmed by WAB during serial passage. At meantime, HCV RNA copy number in the supernatant peaked at 10(4)-10(7) copies/mL and the highest infectious titer of this 1b/2a cHCVcc reinfection were tested as 10(4) ffu/mL. Sequence analysis indicated 6 of adaptive amino acid substitutes occur among chimeric envelope E1E2 during serial passages. We con:luded that a novel 1b/2a chimera HCVcc carrying envelope E1E2 coding gene from Hebei strain of China was developed and its infectious titer increased after serial passage of HCVcc. This novel cHCVcc will be an effective tool for further evaluation of anti-virus drugs and immune effects against the major genotype from Chinese.
Cell Line ; China ; Hepacivirus ; genetics ; growth & development ; metabolism ; Hepatitis C ; virology ; Humans ; Serial Passage ; Viral Envelope Proteins ; genetics ; metabolism