One-step Growth Kinetics Research Articles

Recombinant polioviruses expressing hepatitis B virus-specific cytotoxic T-lymphocyte epitopes

T-cell immune response to recombinant poliovirus expressing foreign antigens has not been elucidated well. In order to investigate the potential use of poliovirus as a T-cell vaccine vector, we constructed recombinant polioviruses expressing HBV-derived CTL epitopes, HBsAg 28–39 (L d-restricted; IPQSLDSWWTSL) and HBc 93–100 (K b-restricted; MGLKFRQL) at the junction between the P2 and P3 regions, designated V3CDs and V3CDc, respectively. The V3CDs and V3CDc recombinant viruses replicated efficiently in HeLa cells and showed a similar infection profile to that of the wild-type Mahoney strain in one-step growth kinetics. Genetic stability analysis showed that V3CDc retained the foreign insert over twelve successive passages examined, whereas V3CDs lost part of the foreign insert after four passages. Our results indicated that the stability of the inserted foreign sequences was rather affected by their nucleotide sequence than by their length when located between the P2 and P3 regions. The junction between these nonstructural protein-coding regions is a novel site for the construction of replication-competent recombinant poliovirus. Immunization of BALB/c (H-2 d) and C57BL/6 mice (H-2 b) with V3CDs and V3CDc, respectively, elicited significant antigen-specific CTL responses to HBsAg 28–39 but not to HBcAg 93–100.

Construction and characterization of a glycoprotein E gene-deleted bovine herpesvirus type 1 recombinant

Abstract Objective To construct and characterize a recombinant glycoprotein (g)E gene-deleted bovine herpesvirus (BHV) type 1 (BHV-1). Procedure The BHV-1 gE gene-coding region and the flanking upstream and downstream sequences were cloned. The aforementioned cloned DNA was digested with suitable enzymes to release the amino terminal two thirds of that region, and was ligated to the β-galactosidase (β-gal) gene. The resulting plasmid DNA was cotransfected with DNA from full-length, wild-type (WT), BHV-1 Cooper strain of the virus. Recombinant viruses expressing β-gal (blue plaques) were plaque purified and assayed further by blot hybridization for genetic characterization and by immunoblotting for reactivity against BHV-1 gE peptide-specific rabbit polyclonal antibody. One recombinant virus, gEΔ3.1IBR, was characterized in vitro and in vivo. The ability of the recombinant virus to induce BHV-1 neutralizing antibodies in infected calves was investigated by plaque-reduction tests. Results and Conclusions The gEΔ3.1IBR virus contained a deletion in the viral gE gene-coding sequences where a stable chimeric reporter (β-gal) gene was inserted. One-step growth kinetics and virus yield of the recombinant and parent viruses were similar, but early after infection, the recombinant virus yield was comparatively less. After intranasal inoculation, the recombinant gEΔ3.1IBR virus replicated in the upper respiratory tract of calves, but the amount of progeny viruses produced was hundredsfold reduced, and duration of virus shedding was shorter. Results of in vivo calf experiments and serum neutralization tests indicated that deleting the gE gene has little effect on inducing neutralizing antibodies against BHV-1, but is sufficient to reduce BHV-1 virulence in calves. (Am J Vet Res 1999; 60:227-232)

Construction and characterization of an attenuated bovine herpesvirus type 1 (BHV-1) recombinant virus

A recombinant bovine herpesvirus type 1 (BHV-1) virus (Gal-TK) has been constructed. The Gal-TK virus contains a chimeric reporter/marker gene coding for bacterial β-galactosidase (β-gal gene) that was inserted stably within the viral TK gene. This resulted in inactivation of the TK gene. The β-gal gene is under the regulation of a strong, human cytomegalovirus-immediate early (HCMV-IE) promoter and is expressed as an authentic viral-coded gene. Even though the one-step growth kinetics of the recombinant and parent viruses were similar, the recombinant virus yielded less than the parent virus on Madin-Darby bovine kidney cells. After intranasal inoculation, the engineered virus was virtually avirulent for colostrum-deprived new-born calves. Similar to the parent virus, the recombinant virus replicated in the upper respiratory tract of calves, but the amount of progeny viruses produced was reduced significantly. The progeny viruses recovered from nasal swabs of animals inoculated with the recombinant and the Cooper strains of BHV-1 were easily distinguishable based on the β-gal marker.

Characterization of o393-A2, a bacteriophage that infects Lactobacillus casei

Bacteriophage ø393-A2, isolated from an artisanal cheese whey sample, is a temperate phage able to generate stable lysogens through integration of its DNA into the bacterial genome. One-step growth kinetics of its lytic development revealed eclipse and latent periods of 100 and 140 min, respectively, with a burst size of about 200 p.f.u. per infected cell. ϕ393-A2 virions have an isometric head and a long, non-contractile tail terminating in a baseplate. The capsid is composed of two major and at least nine minor structural polypeptides. The phage genome consists of a double-stranded DNA molecule of 44 kbp bearing 3′-protruding cohesive ends. A physical map of the phage DNA has been constructed for six restriction enzymes. The whole ϕ393-A2 genome has been cloned in Escherichiacoli using plasmid- and phage-derived cloning vectors.

Proteolytic cleavage of bovine herpesvirus 1 (BHV-1) glycoprotein gB is not necessary for its function in BHV-1 or pseudorabies virus.

Glycoprotein B homologs represent the most highly conserved group of herpesvirus glycoproteins. They exist in oligomeric forms based on a dimeric structure. Despite the high degree of sequence and structural conservation, differences in posttranslational processing are observed. Whereas gB of herpes simplex virus is not proteolytically processed after oligomerization, most other gB homologs are cleaved by a cellular protease into subunits that remain linked via disulfide bonds. Proteolytic cleavage is common for activation of viral fusion proteins, and it has been shown that herpesvirus gB homologs are essential for membrane fusion events during infection, e.g., virus penetration and direct viral cell-to-cell spread. To analyze the importance of proteolytic cleavage for the function of gB homologs, we isolated a mutant bovine herpesvirus 1 (BHV-1) expressing a BHV-1 gB that is no longer proteolytically processed because of a deletion of the proteolytic cleavage site and analyzed its phenotype in cell culture. We showed previously that BHV-1 gB can functionally substitute for the homologous glycoprotein in pseudorabies virus (PrV), based on the isolation of a PrV gB-negative PrV recombinant that expresses BHV-1 gB (A. Kopp and T. C. Mettenleiter, J. Virol, 66:2754-2762, 1992). Therefore, we also isolated a mutant PrV lacking PrV gB but expressing a noncleavable BHV-1 gB. Our results show that cleavage of BHV-1 gB is not essential for its function in either a BHV-1 or a PrV background. Compared with the PrV recombinant expressing cleavable BHV-1 gB, deletion of the cleavage site in the recombinant PrV did not detectably alter the viral phenotype, as analyzed by plaque assays, one-step growth kinetics, and penetration kinetics. In the BHV-1 mutant, the uncleaved BHV-1 gB was functionally equivalent to the wild-type protein with regard to penetration and showed only slightly delayed one-step growth kinetics compared with parental wild-type BHV-1. However, the resulting plaques were significantly smaller, indicating a role for proteolytic cleavage of BHV-1 gB in cell-to-cell spread of BHV-1.

Characterization of a virulent Lactobacillus sake phage PWH2

A virulent bacteriophage PWH2 was isolated from fermented sausage. Out of 14 strains of Lactobacillus sake and 5 strains of L. curvatus tested as potential hosts, only L. sake Ls2 was sensitive. The plaques had a diameter of 0.5-1 mm. One-step growth kinetics of bacteriophage PWH2 revealed the following characteristics: a latent period of 1.5 h, a rise period of 1 h and a burst size of 110 (+− 10) phages per cell. From electron micrographs it was deduced that bacteriophage PWH2 has an icosahedral head, a long non-contractile tail and a complex base plate. The genome is linear and 35 kbp in length. The structural proteins consist of three major and two minor proteins. After an infection of L. sake Ls2, isolates were obtained that were resistant to PWH2. By treatment with mitomycin C, isolate R4a was found to be lysogenic. DNA/DNA hybridisation proved homology of phage PWH2 with the chromosomal DNA of strain R4a.

Characterization of Lactobacillus bulgaricus Bacteriophage ch2

Bacteriophage ch2, a virulent bacteriophage of Lactobacillus bulgaricus CH2, was characterized according to its morphology, genome size, structural proteins, and growth kinetics. Electron micrographs revealed that bacteriophage ch2 has an icosahedral head of 50-nm diameter and a long tail of 170 nm. Its genome is linear and 35 kilobases in length, and its structural proteins consist of two major and eight minor proteins. One-step growth kinetics of bacteriophage ch2 under optimal conditions (45 degrees C in MRS medium [Oxoid Ltd.]) showed that the latent time was 40 min, the rise period was 15 min, and the burst size was 130 bacteriophages per cell. To monitor the effects of bacteriophage infection on host growth and beta-galactosidase production, the absorbance of the culture and the beta-galactosidase activity were followed during the infection cycle. Before lysis the infected culture continued to grow and produce beta-galactosidase at the same rate as the uninfected culture.

Factors affecting the intracellular parasitic growth of Bdellovibrio bacteriovorus developing within Escherichia coli.

A procedure for one-step growth experiments on Bdellovibrio bacteriovorus growing parasitically in Escherichia coli B was developed. The resulting one-step growth curves showed that, under defined conditions at 30 C, each singly infected E. coli host cell, on the average, gave rise to 5.7 Bdellovibrio cells. This value was confirmed by single-burst experiments and by microscopic observations. In the temperature range of 25 to 38 C, the average burst size and the duration of the latent period were inversely proportional to the temperature. The effect of hydrogen ion concentration on the one-step growth kinetics in this system indicated a broad pH optimum, ranging from neutrality to slightly alkaline pH values. After Bdellovibrio cells and host cells were mixed, there was always a delay (the so-called "lag phase") before the parasite titer increased in terms of plaque-forming units. Phase-contrast microscopic observations indicated that this delay stems in part from the polyphasic nature of the Bdellovibrio life cycle. We propose the following five terms to make explicit the sequence of events in this life cycle: "attachment," "penetration," "elongation," "fragmentation," and "burst." Nutritional experiments revealed that Bdellovibrio obtains a major fraction of its cellular components from host-cell material. Infection of E. coli by Bdellovibrio without added Mg(++) or Ca(++) (0.003 m Mg(++), 0.002 m Ca(++)) resulted in partial or total lysis of the host cell soon after infection. Protoplast integrity was necessary for the normal completion of the intracellular growth phase of Bdellovibrio in E. coli; normal development of the parasite took place only in the presence of Mg(++) or Ca(++).