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PubMed1989

Development and serial passage of persistent lymphocytosis associated with bovine leukemia virus infection in cattle.

Two calves each were inoculated with 1.5 x 10(8) or 5 x 10(9) lymphocytes collected from each one cow which had persistent lymphocytosis (PL) and antibodies to bovine leukemia virus (BLV). A sudden increase in the number of peripheral blood lymphocytes (PBL) was observed 14 and 23 days, respectively, after inoculation and the maximum number reached 29,000 and 52,000/microliters 72 and 57 days after inoculation. Although the degree of PL decreased gradually in these cattle, it continued until 14 and 44 months after inoculation when one animal was sacrificed and the other died of lymphosarcoma. The PL was passaged in cattle by inoculation of a large number of PBL obtained from cattle at the stage of PL (PLL). The degree of PL was severer in cattle inoculated with a larger number of PLL. PL was not caused by inoculation of PBL obtained from either BLV-infected non-PL cattle or cattle free of BLV. The PL was also caused by inoculation of PLL into BLV-infected non-PL cattle. On the other hand, it was not observed after inoculation of a large amount of cell-free virus obtained from short-term cultures of PLL. Antibodies to BLV developed earlier and to higher levels in cattle inoculated with PLL than in those inoculated with cell-free virus. These facts show that infection with BLV was established more effectively by PLL than by cell-free virus, the infection may occur by lymphocyte to lymphocyte interaction and the actual number of infected BLV may have an important role in development of PL.

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PubMed2004

Propagation of adult rat bone marrow-derived hepatocyte-like cells by serial passages in vitro.

Previously, we found that hepatocyte growth factor receptor (c-Met)-and alpha-fetoprotein (AFP)-expressing cells were present in adult rat bone marrow, and that these cells also expressed hematopoietic stem cell markers, such as CD34, Thy-1, and c-Kit. When bone marrow cells were cultured in a hepatocyte growth medium (HGM) with HGF and EGF, colonies composed of polygonal cells resembling mature hepatocytes appeared by 2 weeks and grew very slowly because of overgrowth of stromal cells. At days 34-41, 2-mm2 sheets of hepatocyte-like cells were cut out of their colonies by scratching with an injection needle under observation with a phase contrast microscope, transferred into wells of 24-well plates, and cultured in the HGM medium in the presence or absence of HGF and EGF. When cells reached confluence, cells were detached with trypsin and EDTA and transferred step by step into bigger culture vessels. Thus, hepatocyte-like cells were expanded 1000-fold during less than 4 months. These cells were immunocytochemically stained for albumin and also for AFP and the hematopoietic stem cell markers described above, showing characteristics of oval cells. By RT-PCR, we detected mRNAs of tryptophan-2,3-dioxygenase and tyrosine aminotransferase, markers of hepatocytes at a terminal differentiation stage. The present culture system may be useful for supply of hepatocyte resources for cell transplantation therapy.

Animals↗
PubMed1984

Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. II. Attributes of virus cloned at different dog kidney passage levels.

Uncloned dengue (DEN) 4 (H-241) which had been passaged 15, 30 and 50 times in primary dog kidney (PDK) cells were subjected to two successive terminal dilution procedures. In the first (3Cl), virus was diluted in 10-fold steps in 10 replicate tubes. An infected tube from a dilution row with three or fewer virus-infected tubes was selected for two further passages. In the second (TD3), virus was triple terminal diluted using 2-fold dilution steps and selecting one positive tube out of 10. Both procedures selected virus population which differed from antecedents. Plaque size of PDK 15 was medium, PDK 30, small and PDK 50, pin-point. PDK 19-3Cl were medium and 56-3Cl, 24-TD3, 35-TD3 and 61-TD3 were all small. All cloned virus replication was completely shut-off at 38.5 degrees C; PDK 15 and 30 continued to replicate at this temperature. Uncloned viruses showed a graduated decrease in monkey virulence with PDK passage; cloned viruses were either avirulent for monkeys (19-3Cl, 56-31Cl, 24-TD3 and 35-TD3) or produced revertant large plaque parental-type viremia (35-3Cl and 61-TD3). Those cloned viruses which exhibited temperature sensitivity, reduced monkey virulence and stability after monkey passage may be suitable as vaccine candidates for evaluation in human beings.

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PubMed1984

Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. IV. Characterization of a vaccine candidate in fetal rhesus lung cells.

A strain of primary dog kidney (PDK)-passaged dengue (DEN) 4 (H-241) virus cloned by terminal dilution (PDK 35-TD3) was propagated in large volumes in fetal rhesus lung (FRhL) cells to produce a candidate vaccine for evaluation in man. Production seed (FRhL p2) and candidate vaccine (FRhL p3) were subjected to rigorous safety tests to exclude contaminating microbial agents. There was no significant monkey neurovirulence of parental or PDK-passaged DEN-4 virus or of control fluid cultures. FRhL-passaged viruses retained the phenotypic characteristics: small (occasional medium) plaque; temperature sensitivity at 38.5 degrees C; and absence of plaque formation in African green monkey kidney cells, cytopathic effect in LLC-MK2 cells, and viral growth in human monocytes. FRhL p2 virus displayed low virulence for monkeys; only one of four animals was viremic and three of four developed low-titered antibody. FRhL p3 virus produced viremia in three monkeys and moderate to high hemagglutination-inhibition and neutralizing antibody titers in all animals. Virus at both passages in FRhL exhibited reduced neurovirulence in suckling mice as compared to parental DEN-4. Because of its safety and desirable monkey virulence attributes PDK 35-TD3 FRhL p3 is recommended for human phase I trial.

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PubMed1984

Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. V. Human response to immunization with a candidate vaccine prepared in fetal rhesus lung cells.

A dengue 4 (strain H241, PDK35-TD3 FRhL p3) vaccine attenuated by passage in primary dog kidney cells followed by passage and final vaccine preparation in DBS-FRhL-2 cells was tested in five yellow fever-immune volunteers. Only two volunteers seroconverted by producing hemagglutination-inhibiting and neutralizing antibodies. Mild illness, compatible with dengue infection was found only in the individuals who later developed antibodies. Both volunteers developed a rash by the 8th day following vaccination, coinciding with a slight elevation in temperature and leukopenia. Additionally, several serum enzymes were elevated during the observation period. Dengue 4 virus was isolated from the blood of the two infected volunteers starting as early as day 5 post vaccination. During the viremic period, which lasted 5 days, phenotypically-changed virus was recovered, indicating genetic instability of the vaccine virus. The clinical disease and immune response in the two infected individuals was probably related to replication of the variant virus. Further testing of this vaccine in its present form is not indicated.

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PubMedDate not supplied

Infectious bursal disease live vaccines: changes in the virus population during serial passage in chickens and chicken embryo fibroblast cells.

Two attenuated infectious bursal disease virus strains used as commercial live vaccine were passaged five successive times in specific-pathogen-free chickens and chicken embryo fibroblast (CEF) cells. Both attenuated strains increased in virulence during the passage in susceptible chickens as evidenced by the decrease in bursa/body weight ratios. A direct nucleotide sequence analysis of the VP2 hypervariable domain amplified by the reverse transcription-polymerase chain reaction revealed that the nucleotide at position 890 (T) in both strains was A after the passage in chicken. In addition, the nucleotide at position 890 (A) was T or C after the subsequent passage in CEF cells. Because of the nucleotide differences, the amino acid residue at position 253 (His) in both vaccines was Gln after the passage in chickens, and the amino acid residue Gln was changed back to His during the subsequent passage in CEF cells. The digestion of the amplified fragment with restriction endonucleases Stul and Ncol, which recognize the sequence difference at position 890, showed that the population of the virus that had amino acid Gln at position 253 was gradually increased during the passage in chickens. Conversely, the population of the virus that had amino acid His at position 253 was gradually increased during the subsequent passage in CEF cells.

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PubMedDate not supplied

Genetic stability of the VP2 hypervariable region of four infectious bursal disease virus isolates after serial passage in specific-pathogen-free chicken embryos.

Infectious bursal disease virus strains U2, 586, L1, and Q2 were isolated from pooled bursal samples collected from commercially reared broilers. These viruses were propagated in specific-pathogen-free (SPF) embryonated chicken eggs for 24 or 25 passages. Nucleotide sequences of a 743-bp reverse transcription (RT)/polymerase chain reaction (PCR) product containing the VP2 hypervariable region were compared before and after passage of the viruses in embryonated chicken eggs. To determine the genetic stability of the viruses, each isolate was compared with its egg-passed ancestor; virus isolates were not compared with each other. When the restriction enzymes BstNI and MboI were used, no differences were observed in the restriction fragment length polymorphism profiles of the RT/ PCR products after embryo passage. After embryo passage, six nucleotide changes were identified in the viruses. Among the four viruses examined, these nucleotide changes resulted in a total of five amino acid changes. The amino acid changes were S-222-L in virus 586, K-249-N in viruses U2, L1, and Q2, and G-281-V in virus Q2. Three of the five amino acid changes occurred at residue 249. The convergent nature of this residue shift in three of four of the chick embryo-passed viruses suggests the occurrence of a functional, as opposed to random, mutation. The original isolates caused typical signs of infectious bursal disease in 3-wk-old SPF chicks. Their embryo-passed ancestors also produced typical signs of infectious bursal disease in 3-wk-old SPF chicks, suggesting the amino acid mutations observed did not affect virulence of the viruses.

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