The production of foot-and-mouth disease virus from BHK 21 C 13 cells grown on the surface of DEAE sephadex A50 beads.

Methods are described which make possible the production of foot-and-mouth disease (FMD) virus from BHK 21 C13 monolayer cells which have been grown on the surface of serum coated DEAE Sephadex A50 beads. The yield of cells and their susceptibility to infection by FMD virus are equivalent to conventional Roux monolayer systems. The potential for the commercial application of the DEAE Sephadex A50 system is discussed in relation to other unit process monolayer systems and in particular to the system in which cells are cultured in a deep bed of small glass spheres.     

Typing foot-and-mouth disease virus by fluorescent antibody technique.

Typing of foot-and-mouth disease (FMD) virus was performed by the direct fluorescent antibody (FA) technique. Type-specific FA was prepared from the following two sorts of procedures: (1) FA against live virus (FA-live) was prepared from hyperimmune serum taken from guinea pigs having received live FMD virus. Then it was adsorbed with concentrated heterotype antigen. (2) FA against inactivated virus (FA-Inact) was prepared from antiserum taken from guinea pigs immunized with purified FMD virus inactivated with acetylethyleneimine. Seventeen strains of FMD virus (seven strains of type A, seven strains of type O, and three strains of thpe C) were used. Type-specific FMD virus antigen was detected distinctly from the monolayer of BHK cells infected with each type of virus and fixed in acetone, in spite of negative results obtained from the cells fixed in methyl alcohol. All the 17 strains were typed successfully by the implementation of these two FA methods.     

Preparation of FMD type A87/IRN inactivated vaccine by gamma irradiation and the immune response on guinea pig

FMD is one of the most economically damaging diseases that affect livestock animals. In this study FMD Virus type A87/IRN was multiplied on BHK21 cells. The virus was titrated by TCID50 method, it was 10^7.5/ml. The FMD virus samples were inactivated by gamma ray from 60Co source at −20°C. Safety test was done by IBRS2 monolayer cell culture method, also antigenicity of irradiated and un-irradiated virus samples were studied by Complement Fixation Test. The Dose/Survival curve for irradiated FMD Virus was drawn, the optimum dose range for inactivation of FMDV type A87/IRN and unaltered antigenicity was obtained 40–44 kGy. The inactivated virus samples by irradiation and ethyleneimine (EI) were formulated respectively as vaccine with Al(OH)3 gel and other substances. The vaccines were inoculated to Guinea pigs and the results of Serum Neutralization Test for the normal vaccine and radio-vaccine showed protective titer after 8 months. The potency test of the inactivated vaccines was done, PD50 Value of the vaccines were calculated 7.06 and 5.6 for inactivated vaccine by EI and gamma irradiation respectively.     

Monolayer cultures of animal cells with the gyrogen equipped with tubes

The gyrogen with tubes has been the object of repeated trials carried out over a period of some years. The aim of these trials was essentially to multiply BHK cells in monolayer and to reproduce the FMD virus in one operation. At the laboratory stage, the gyrogen represented three roller flasks; at the pilot stage, 100 roller flasks; and at the industrial stage, 500 flasks. Culturing cells on a glass support, is not in itself new, but the difficulty appeared when replicating the virus in order to obtain the high parameters necessary for vaccine production. Everything was accomplished so that finally the cell culture and viral multiplication could be carried out in situ in the same apparatus.     

The scale-up from 0.1 to 100 liter of a unit process system based on 3-mm-diameter glass spheres for the production of four strains of FMDV from BHK monolayer cells

This paper describes the scale-up from 0.1 to 100 liter of the unit process based on 3-mm-diameter glass spheres for the growth of BHK monolayer cells. The production of four strains of FMD virus at the 0.1-, 10-, and 100-liter scales was examined. Cell growth was estimated from measurements of the concentration of glucose in the growth medium, while the release of virus was inferred from measuring the concentration of LDH in the culture supernatant fluid. The yields of virus at 0.1-, 1-, and 10-liter scales were similar but that from the 100-liter version was somewhat lower. The reason for this lower yield and the method used to overcome it over outlined.     

Selection of a stable clone of the MVPK-1 fetal porcine kidney cell for assays of foot-and-month disease virus.

The MVPK-1 cell line, derived from fetal porcine kidney cells, supports the replication of foot-and-mouth disease (FMD) virus. The cell line was adapted to grow in medium containing 5% bovine serum. The susceptibility of the adapted cells decreased as they aged at 37 degrees C. Various clones were isolated from the adapted cells and their growth characteristics and sustained susceptibility to FMD virus were compared. Clone 7 maintained uniform susceptibility to FMD virus over a 3-day period at 37 degrees C and proved superior to other clones in the characteristics studied. The clone has maintained satisfactory susceptibility to FMD virus through 40 subcultures. Clone 7 can replace primary bovine kidney cells for routine viral assays, but cannot detect as much FMD virus in animal specimens as primary bovine kidney, bovine thyroid, or swine kidney cells.     

The improvement and standardization of the simplified process for the production of foot and mouth disease virus from BHK suspension cells

Improvements have been made to the methodology for the production of foot and mouth disease (FMD) virus from BHK 21 clone 13 suspension cells by the simplified process. Data derived from some 600 individual 8-1 cultures covering all seven types of FMD virus has been analysed. The production of virus was shown to (1) have no direct relationship to cell passage level and (2) to be inversely related to the cell multiplication factor observed during the cell growth cycle immediately prior to infection.     

Engineering Foot-and-Mouth Disease Viruses with Improved Growth Properties for Vaccine Development

Background

No licensed vaccine is currently available against serotype A foot-and-mouth disease (FMD) in China, despite the isolation of A/WH/CHA/09 in 2009, partly because this strain does not replicate well in baby hamster kidney (BHK) cells.

Methodology/Principal Findings

A novel plasmid-based reverse genetics system was used to construct a chimeric strain by replacing the P1 gene in the vaccine strain O/CHA/99 with that from the epidemic stain A/WH/CHA/09. The chimeric virus displayed growth kinetics similar to those of O/CHA/99 and was selected for use as a candidate vaccine strain after 12 passages in BHK cells. Cattle were vaccinated with the inactivated vaccine and humoral immune responses were induced in most of the animals on day 7. A challenge infection with A/WH/CHA/09 on day 28 indicated that the group given a 4-µg dose was fully protected and neither developed viremia nor seroconverted to a 3ABC antigen.

Conclusions/Significance

Our data demonstrate that the chimeric virus not only propagates well in BHK cells and has excellent antigenic matching against serotype A FMD, but is also a potential marker vaccine to distinguish infection from vaccination. These results suggest that reverse genetics technology is a useful tool for engineering vaccines for the prevention and control of FMD.     

Growth of the IB-RS-2 Pig Kidney Cell Line in Suspension Culture and Its Susceptibility to Foot-and-Mouth Disease Virus

The adaptation of the pig kidney cell line IB-RS-2, clone 60, to growth in suspension culture is described. When fully adapted, an approximate threefold increase in viable cells was obtained within 72 hr from initial cell concentrations of 5 x 10(5) per ml in culture volumes up to 1,500 ml. The monolayer cells (99th passage level) used to initiate the suspension cultures and the fully adapted suspension cells were shown to have an aneuploid chromosome karyotype, whereas earlier monolayer cultures (32nd passage level) had a pseudodiploid karyotype. Replicate virus titrations in monolayers prepared from suspension-adapted cells, IB-RS-2 monolayer cells, BHK monolayer cells, and in suckling mice showed that the suspension cells had retained sensitivity to foot-and-mouth disease virus. The geometric mean peak infectivity of seven strains of foot-and-mouth disease virus grown in IB-RS-2 suspension cells was 10(8.2) plaque-forming units per ml, with a mean complement-fixing activity of approximately 135 complement-fixing units per ml. These preliminary results indicate that submerged cultures of these cells on an industrial scale may be useful for commercial foot-and-mouth disease vaccine production.     

In vitro tumoricidal activity of macrophages against virus-transformed lines with temperature-dependent transformed phenotypic characteristics.

The susceptibility of targets to destruction by tumoricidal rat and mouse macrophages was studied with virus-transformed cell lines in which various elements of the transformed phenotype are only expressed at specific temperatures. BHK cells transformed by the ts3 mutant of polyoma virus, rat embryo 3Y1 cells transformed by a temperature-sensitive A cistron mutant of simian virus 40 (SV40) and the ts-H6-15 temperature-sensitive line of SV40-transformed mouse 3T3 cells were killed in vitro by macrophages at both the permissive (33 °C) or nonpermissive (39 °C) temperatures for expression of the transformed phenotype. 3T3, 3Y1 and BHK cells transformed by wild-type SV40 or polyoma virus were also destroyed by tumoricidal macrophages at both 33 and 39 °C, but untransformed 3T3, 3Y1, and BHK cells were not. Thus, transformed cells are killed by macrophages regardless of whether or not they express cell surface LETS protein or Forssman antigen, display surface changes which permit agglutination by low doses of plant lectins, express SV40 T antigen, have a low saturation density, or exhibit density-dependent inhibition of DNA synthesis.     

Hexokinase activity as marker to assess time of harvest of foot-and-mouth disease virus in BHK21 Cl13 cell cultures

Hexokinase (B.C. 2.7.1.1) activity as a marker enzyme during FMD viral infection has been observed spectrophotometrically in a system coupled with glucose-6-phosphate dehydrogenase, in supernatants of BHK(21)Cl(13) suspension as well as anchored cell culture at a minimum of 10(4) infective virus particles/ml. Specific activity increased with virus concentration in culture supernatants and abruptly decreased with a fall in virus titer, as has been noted by TCID/50,146 S concentration, and enzyme-linked immunosorbent assay (ELISA) readings.     

Reversible susceptibility to natural cell-mediated cytotoxicity observed in altered BHK cells resistant to HVJ (Sendai virus) infection

Altered baby hamster kidney (BHK-R) cells which were subcultured in the continuous presence of HVJ (hemagglutinating virus of Japan--the Sendai strain of parainfluenza 1 virus) showed a high susceptibility to natural cell-mediated cytotoxicity, although BHK-R cells are not transiently or persistently infected with HVJ but contain the restricted amount of sialic acid. By repeated subcultivation of BHK-R cells in growth medium free of HVJ, the sensitivity to natural killer cytotoxicity decreased to the level of normal BHK cells with a counter increase of cellular sialic acid, and the subsequent treatment of the cells with neuraminidase caused a loss of proper sialic acid residues, once again resulting in a significant enhancement of lysis by natural killer cells. In the BHK-R cell system which exhibits a reversible resistance to the interferon action, the enhancing effect induced by interferon on target cell susceptibility to natural killer activity became more pronounced in accord with the recovery of sensitivity to the antiviral action of interferon.     

Growth and immunogenicity of foot-and-mouth disease virus in baby hamster kidney cells adapted to and continuously grown in a serum-free chemically defined media

The production of foot-and-mouth disease (FMD) virus in baby hamster kidney (BHK) suspension cells grown in serum-free media for subsequent use in vaccines was attempted because of the limited availability of serum in quantities sufficient for propagation of large amounts of cells, as well as the possible presence of mycoplasma, viral contaminants, and interfering antibodies in sera. Suspension cultures (50 to 600 ml) of BHK-21 cells adapted to and continually passed in a glutamine-free autoclavable, chemically defined medium (BHK-S system) were infected with all seven types of FMD virus. Cells were infected at multiplicities of infection (MOI) ranging from 10^?1 to 10^?7 plaque-forming units per cell (PFU/cell). The time course of infectious virus release and the amount of complement-fixing (CF) antigen produced were then followed. Peak harvest infectivities of approximately 10^8.5 PFU/ml were obtained from 12 to 24 hr after inoculation, depending on input MOI, and were apparently independent of cell concentration over the range 1.5 to 4.0 million cells/ml; the CF endpoint dilutions increased from 1:12 at the lower cell concentrations to 1:48 at the highest cell concentration. Monovalent and trivalent vaccines have been produced using viruses from the BHK-S system, inactivated with acetylethyleneimine and emulsified in oil, and the results of tests in steers and guinea pigs are presented.     

Plasmid DNA encoding replicating foot-and-mouth disease virus genomes induces antiviral immune responses in swine.

DNA vaccine candidates for foot-and-mouth disease (FMD) were engineered to produce FMD virus (FMDV) particles that were noninfectious in cell culture or animals. The prototype plasmid, pWRM, contains a cytomegalovirus immediate-early promoter-driven genome-length type A12 cDNA followed by the bovine growth hormone polyadenylation site. BHK cells transfected with this plasmid produced virus, but the specific infectivity of pWRM was much lower than that achieved with in vitro-generated RNA genomes. To improve the infectivity of the plasmid, a cDNA encoding the hepatitis delta virus ribozyme was added to the 3' end of the FMDV cDNA. The resulting plasmid, pWRMH, exhibited slightly increased infectivity in cell culture and produced virus when inoculated into suckling mice. A third plasmid, pWRMHX, was created by removal of the sequences encoding the cell binding site found in capsid protein VP1 of pWRMH. Although cells transfected with pWRMHX produced viral capsids, this plasmid was not lethal in suckling mice, indicating that particles lacking the cell binding site were not able to initiate secondary infectious cycles. Swine inoculated with pWRMHX did not show any signs of disease and produced neutralizing antibodies to FMDV, and 20% of the vaccinated animals were protected from challenge. A derivative of pWRMHX, pWRMHX-pol-, harboring a mutation designed to inactivate the viral polymerase was much less immunogenic, indicating that immunogenicity of pWRMHX resulted, in part, from amplification of the viral genome in the animal.     

Failure of an Influenza Virus to Initiate Infection in Enucleate BHK cells

Studies employing indirect immunofluorescent staining, acrylamide gel electrophoresis of [(35)S]methionine-labeled cellular polypeptides, and RNA-RNA hybridization of [(3)H]uridine-labeled cellular RNA, failed to detect evidence of fowl plague virus infection of BHK cells enucleated with cytochalasin B, although virus-specific polypeptide and RNA synthesis was detected in nucleate BHK cells. The enucleate cells permitted the synthesis of Newcastle disease and vaccinia virus structural proteins. We conclude that influenza virus fails to initiate macromolecular synthesis in enucleate BHK cells, and the role of the nucleus in influenza virus replication is discussed.     

Propagation of MM Virus in Continuous Cell Lines

Baby hamster kidney (BHK), McCoy, and L cell lines were found to be suitable for isolation of MM virus from infected mouse brain tissue. The virus was recovered in high titer in the first passage in BHK and McCoy cells, with concomitant cytopathic effect (CPE). In L cells, virus yield was lower than in the other two cell lines and CPE was incomplete. Adaptation of the virus to BHK and McCoy cells by serial passages was evidenced by accelerated development of the CPE and increase in the virus titer. Plaques were obtained in all three cell lines when inoculated with infected mouse brain or with the tissue culture-propagated virus. In the BHK cells, the virus release preceded the appearance of CPE and maximal yield of virus was obtained after 1 to 3 days of incubation, depending on the size of inoculum. The BHK-propagated virus had the same lethality for mice as did the mouse brain-propagated stock, and there was no difference in the course of the disease caused by the two preparations.     

Soluble Receptor Potentiates Receptor-Independent Infection by Murine Coronavirus

Mouse hepatitis virus (MHV) infection spreads from MHV-infected DBT cells, which express the MHV receptor CEACAM1 (MHVR), to BHK cells, which are devoid of the receptor, by intercellular membrane fusion (MHVR-independent fusion). This mode of infection is a property of wild-type (wt) JHMV cl-2 virus but is not seen in cultures infected with the mutant virus JHMV srr7. In this study, we show that soluble MHVR (soMHVR) potentiates MHVR-independent fusion in JHMV srr7-infected cultures. Thus, in the presence of soMHVR, JHMV srr7-infected DBT cells overlaid onto BHK cells induce BHK cell syncytia and the spread of JHMV srr7 infection. This does not occur in the absence of soMHVR. soMHVR also enhanced wt virus MHVR-independent fusion. These effects were dependent on the concentration of soMHVR in the culture and were specifically blocked by the anti-MHVR monoclonal antibody CC1. Together with these observations, direct binding of soMHVR to the virus spike (S) glycoprotein as revealed by coimmunoprecipitation demonstrated that the effect is mediated by the binding of soMHVR to the S protein. Furthermore, fusion of BHK cells expressing the JHMV srr7 S protein was also induced by soMHVR. These results indicated that the binding of soMHVR to the S protein expressed on the DBT cell surface potentiates the fusion of MHV-infected DBT cells with nonpermissive BHK cells. We conclude that the binding of soMHVR to the S protein converts the S protein to a fusion-active form competent to mediate cell-cell fusion, in a fashion similar to the fusion of virus and cell membranes.     

Establishment and maintenance of persistent infection by Sindbis virus in BHK cells.

We have established a persistent infection of BHK cells with a preparation of Sindbis virus heavily enriched in defective interfering (DI) particles. The small fraction of cells that survived the initial infection grew out to form a stable population of cells [BHK(Sin-1) cells], most of which synthesized viral RNA and viral antigens. The presence of DI particles in this virus stock was required to establish this persistent state. BHK(Sin-1) cells released a small-plaque, temperature-sensitive virus (Sin-1 virus) as well as DI particles containing DI RNAs larger than those present in the original stock used to establish the persistent state. A cloned stock of Sin-1 virus, free of detectable DI particles, was able to initiate a persistent infection more quickly and with greater cell survival than the original stock of Sindbis virus containing DI particles. About 2 weeks after the Sin-1 virus-infected cells were cultured, DI RNAs arose and soon became the dominant viral RNA species produced by these cells.     

Propagation of MM Virus in L Cells

MM virus (mouse-brain stock) replicated to a limited extent in L cells without cytopathic effects; the average yield was less than 1 plaque-forming unit/cell. Passage in BHK-21 cells resulted in MM virus [MM(BHK)] which replicated to high titers (200 to 300 plaque-forming units/cell) in L cells with complete cytopathic effects. Appearance of mature MM(BHK) virus in L-cell cultures begins 4 hr after infection and is completed by 8 hr. Release of mature virus was slow (less than 1% at 8 hr) but was completed by 24 hr.     

我国D2-43病毒株PrM-E基因的重组SFV的制备及生物学鉴定

 为构建含PrM E基因的重组SFV病毒 ,将含我国登革 2型病毒PrM E基因的SFV表达载体和辅助载体DNA线性化后 ,进行体外转录 .然后将获得的 2种RNA转录物共转染BHK2 1细胞 ,以RT PCR法证明转染后的细胞培养上清中含有重组SFV .进一步将该重组病毒经蛋白酶激活后可使BHK2 1细胞产生细胞病变 ,并且以间接免疫荧光法在感染细胞内可检测到登革 2型病毒所特有的蛋白 .含我国登革 2型病毒PrM E基因的重组SFV的获得 ,为进一步观察该重组病毒的免疫原性奠定了基础 ,从而为登革新型疫苗的研制提供新途径