Kinetics of baculovirus replication and release using real-time quantitative polymerase chain reaction

The study of viral-based processes is hampered by (a) their complex, transient nature, (b) the instability of products, and (c) the lack of accurate diagnostic assays. Here, we describe the use of real-time quantitative polymerase chain reaction to characterize baculoviral infection. Baculovirus DNA content doubles every 1.7 h from 6 h post-infection until replication is halted at the onset of budding. No dynamic equilibrium exists between replication and release, and the kinetics are independent of the cell density at the time of infection. No more than 16% of the intracellular virus copies bud from the cell. (C) 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 476-480, 2002; DOI 10.1002/bit.10126.

Identification of a new cell line permissive to porcine reproductive and resporatory syndrome virus replication

Le syndrome reproducteur et respiratoire porcin (SRRP) est une des maladies les plus dévastatrices économiquement pour l'industrie mondiale du porc. L'agent étiologique du SRRP est le virus du SRRP (VSRRP) lequel est connu pour avoir une spécificité d'hôte très restreinte et pour sa transmission par voie aerosol. Les antigènes et les ARN du VSRRP ont été trouvés dans des cellules épithéliales du tractus respiratoire de porcs infectés par le virus. L’interaction entre les macrophages alvéolaires porcins (PAMs) et le VSRRP a été démontrée comme jouant un rôle important dans l’infection causée par le virus. Malgré cela, l’interaction prenant place entre les cellules épithéliales du tractus respiratoire porcin et le virus ne devrait pas être négligée. Jusqu’à présent, la réplication du VSRRP in vitro dans des cellules épithéliales du tractus respiratoire porcin n’a pas été conduite avec succès et les tentatives pour le faire ont échoué. Une nouvelle lignée de cellules épithéliales de poumon de porc (SJPL) est maintenant disponible et sera utilisée dans cette étude afin de déterminer si elle est permissive à la réplication du VSRRP et si elle peut être un modèle approprié pour l’étude de la pathogénèse virale du VSRRP. L’expérimentation a démontré que cette nouvelle lignée cellulaire était permissive à l’infection et à la réplication du VSRRP. Afin de corroborer ces résultats, la cinétique de réplication du virus à été effectuée avec les cellules MARC-145 et SJPL. Aucune différence significative dans la production virale totale n’a été trouvée entre les deux lignées cellulaires. Les cellules SJPL ont permis la réplication de plusieurs souches Nord-Américaines du VSRRP, quoiqu’elles sont légèrement moins efficaces que les cellules MARC-145 pour l’isolement du virus. De plus, les cellules SJPL sont phénotypiquement différentes des cellules MARC-145. Plus précisément, les cellules SJPL sont plus sensibles à l’activation par le VSRRP des pro-caspases 3/7 et plusieurs inducteurs apoptotiques. Elles ont également montré de 8 à 16 fois plus de sensibilité à l’effet antiviral causé par l’IFN-α sur la réplication du virus contrairement aux cellules MARC-145. Ces résultats démontrent que les cellules SJPL pourraient représenter un substitut intéressant aux cellules MARC-145 pour la production d’antigènes pour un vaccin anti-VSRRP. Également, dû à leurs origines (poumon de l’hôte naturel), elles pourraient s’avérer être un modèle in vitro plus approprié pour l’étude de la pathogénèse du VSRRP.

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.

INFLUENCE OF TYPE 2 BOVINE VIRAL DIARRHEA VIRUS NPRO ON ENHANCEMENT OF BOVINE RESPIRATORY SYNCYTIAL VIRUS REPLICATION MEDIATED BY ANTAGONISM OF HOST CELL INTERFERON TYPE I RESPONSES

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, Family Flaviviridae. The virus can infect many species of animals of the order Artiodactyla. The BVDV genome encodes an auto protease, Npro, that degrades interferon regulatory factor-3 (IRF-3) reducing type I interferon (IFN-I) production from host cells. Bovine respiratory syncytial virus (BRSV) is a member of the genus Pneumovirus, Family Paramyxoviridae. Concurrent infection with BVDV and BRSV causes more severe respiratory and enteric disease than infection with either virus alone. Our hypothesis was that Npro modulates the innate immune responses to BVDV infection and enhances replication of BVDV or BRSV co-infection. The noncytopathic BVDV2 viruses NY93/c N- Npro 18 EGFP (a mutant with modified Npro fused with enhanced green fluorescent protein), NY93 infectious clone (NY93/c), wild-type NY93-BVDV2 (NY93-wt), and BRSV were evaluated in this study. The objectives of this study were: (1) to characterize the replication kinetics and IFN-I induction in Madin-Darby bovine kidney (MDBK) cells following infection with each of the BVDV isolates, and (2) to characterize the influence of BVDV-mediated IFN-I antagonism on enhancement of BRSV replication in bovine turbinate (BT) cells. NY93/c N- Npro 18 EGFP replicated 0.4 – 1.6 TCID50 logs lower than NY93-wt in MDBK cells. NY93/c N- Npro 18 EGFP-infected MDBK cells synthesized IFN-I significantly higher than NY93/c- and NY93-wt-infected MDBK cells. BT cells co-infected with NY93/c N- Npro 18 EGFP/BRSV or NY93-wt/BRSV were evaluated to determine the effects of co-infection on BRSV replication and IFN-I induction in BT cells. BRSV RNA levels in NY93-wt/BRSV co-infected BT cells were 2.49, 2.79, and 2.89 copy number logs significantly greater than in NY93/c N- Npro 18 EGFP/BRSV co-infected BT cells on days 5, 7, and 9 post-infection, respectively. BVDV RNA levels in NY93/c N- Npro 18 EGFP-infected BT cells were 1.64 – 4.38 copy number logs lower than in NY93-wt-infected BT cells. NY93/c N- Npro 18 EGFP single and co-infected BT cells synthesized IFN-I significantly higher than NY93-wt single and co-infected BT cells. In summary, these findings suggest: (1) NY93/c N- Npro 18 EGFP BVDV2 induced higher levels of IFN-I than BVDV2-wt and may be useful as a safer, replicating BVDV vaccine, and (2) Enhancement of BRSV infection by BVDV co-infection is mediated by antagonism of IFN-I.

Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins.

The broad host range plasmid RK2 replicates and regulates its copy number in a wide range of Gram-negative bacteria. The plasmid-encoded trans-acting replication protein TrfA and the origin of replication oriV are sufficient for controlled replication of the plasmid in all Gram-negative bacteria tested. The TrfA protein binds specifically to direct repeat sequences (iterons) at the origin of replication. A replication control model, designated handcuffing or coupling, has been proposed whereby the formation of coupled TrfA-oriV complexes between plasmid molecules results in hindrance of origin activity and, consequently, a shut-down of plasmid replication under conditions of higher than normal copy number. Therefore, according to this model, the coupling activity of an initiation protein is essential for copy number control and a copy-up initiation protein mutant should have reduced ability to form coupled complexes. To test this model for plasmid RK2, two previously characterized copy-up TrfA mutations, trfA-254D and trfA-267L, were combined and the resulting copy-up double mutant TFrfA protein TrfA-254D/267L was characterized. Despite initiating runaway (uncontrolled) replication in vivo, the copy-up double-mutant TrfA protein exhibited replication kinetics similar to the wild-type protein in vitro. Purified TrfA-254D, TrfA-267L, and TrfA-254D/267L proteins were then examined for binding to the iterons and for coupling activity using an in vitro ligase-catalyzed multimerization assay. It was found that both single and double TrfA mutant proteins exhibited substantially reduced (single mutants) or barely detectable (double mutant) levels of coupling activity while not being diminished in their capacity to bind to the origin of replication. These observations provide direct evidence in support of the coupling model of replication control.

Human immunodeficiency virus type 1 fitness is a determining factor in viral rebound and set point in chronic infection.

Human immunodeficiency virus type 1 (HIV-1) isolates from 20 chronically infected patients who participated in a structured treatment interruption (STI) trial were studied to determine whether viral fitness influences reestablishment of viremia. Viruses derived from individuals who spontaneously controlled viremia had significantly lower in vitro replication capacities than viruses derived from individuals that did not control viremia after interruption of antiretroviral therapy (ART), and replication capacities correlated with pre-ART and post-STI viral set points. Of note, no clinically relevant improvement of viral loads upon STI occurred. Virus isolates from controlling and noncontrolling patients were indistinguishable in terms of coreceptor usage, genetic subtype, and sensitivity to neutralizing antibodies. In contrast, viruses from controlling patients exhibited increased sensitivity to inhibition by chemokines. Sensitivity to inhibition by RANTES correlated strongly with slower replication kinetics of the virus isolates, suggesting a marked dependency of these virus isolates on high coreceptor densities on the target cells. In summary, our data indicate that viral fitness is a driving factor in determining the magnitude of viral rebound and viral set point in chronic HIV-1 infection, and thus fitness should be considered as a parameter influencing the outcome of therapeutic intervention in chronic infection.

Equid herpesvirus type-1 exhibits neurotropism and neurovirulence in a mouse model

Intranasal inoculation of equid herpesvirus type-1 (EHV-1) Brazilian strains A4/72 and A9/92 induced an acute and lethal infection in four different inbred mouse strains. Clinical and neurological signs appeared between the 2nd and 3rd day post inoculation (dpi) and included weight loss, ruffled fur, a hunched posture, crouching in corners, nasal and ocular discharges, dyspnoea, dehydration and increased salivation. These signs were followed by increased reactivity to external stimulation, seizures, recumbency and death. The virus was recovered consistently from the brain and viscera of all mice with neurological signs. Histopathological changes consisted of leptomeningitis, focal haemorrhage, ventriculitis, neuronal degeneration and necrosis, neuronophagia, non-suppurative inflammation, multifocal gliosis and perivascular infiltration of polymorphonuclear and mononuclear cells. Immunohistochemical examination demonstrated that EHV-1 strains A4/72 and A9/92 replicated in neurons of the olfactory bulb, the cortex and the hippocampus. In contrast, mice inoculated with the EHV-1 Brazilian strain A3/97 showed neither weight loss nor apparent clinical or neurological signs; however, the virus was recovered consistently from their lungs at 3 dpi. These three EHV-1 strains showed distinct degrees of virulence and tissue tropism in mice. EHV-1 strains A4/72 and A9/92 exhibited a high degree of central nervous system tropism with neuroinvasion and neurovirulence. EHV-1 strain A3/97 was not neurovirulent despite being detected in the brains of infected BALB/c nude mice. These findings indicate that several inbred mouse strains are susceptible to neuropathogenic EHV-1 strains and should be useful models for studying the pathogenesis and mechanisms contributing to EHV-induced myeloencephalopathy in horses. (C) 2011 Elsevier Ltd. All rights reserved.

The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.

Long-term antiretroviral treatment initiated at primary HIV-1 infection affects the size, composition, and decay kinetics of the reservoir of HIV-1-infected CD4 T cells.

Initiation of antiretroviral therapy during the earliest stages of HIV-1 infection may limit the seeding of a long-lasting viral reservoir, but long-term effects of early antiretroviral treatment initiation remain unknown. Here, we analyzed immunological and virological characteristics of nine patients who started antiretroviral therapy at primary HIV-1 infection and remained on suppressive treatment for >10 years; patients with similar treatment duration but initiation of suppressive therapy during chronic HIV-1 infection served as controls. We observed that independently of the timing of treatment initiation, HIV-1 DNA in CD4 T cells decayed primarily during the initial 3 to 4 years of treatment. However, in patients who started antiretroviral therapy in early infection, this decay occurred faster and was more pronounced, leading to substantially lower levels of cell-associated HIV-1 DNA after long-term treatment. Despite this smaller size, the viral CD4 T cell reservoir in persons with early treatment initiation consisted more dominantly of the long-lasting central-memory and T memory stem cells. HIV-1-specific T cell responses remained continuously detectable during antiretroviral therapy, independently of the timing of treatment initiation. Together, these data suggest that early HIV-1 treatment initiation, even when continued for >10 years, is unlikely to lead to viral eradication, but the presence of low viral reservoirs and durable HIV-1 T cell responses may make such patients good candidates for future interventional studies aiming at HIV-1 eradication and cure. IMPORTANCE: Antiretroviral therapy can effectively suppress HIV-1 replication to undetectable levels; however, HIV-1 can persist despite treatment, and viral replication rapidly rebounds when treatment is discontinued. This is mainly due to the presence of latently infected CD4 T cells, which are not susceptible to antiretroviral drugs. Starting treatment in the earliest stages of HIV-1 infection can limit the number of these latently infected cells, raising the possibility that these viral reservoirs are naturally eliminated if suppressive antiretroviral treatment is continued for extremely long periods of time. Here, we analyzed nine patients who started on antiretroviral therapy within the earliest weeks of the disease and continued treatment for more than 10 years. Our data show that early treatment accelerated the decay of infected CD4 T cells and led to very low residual levels of detectable HIV-1 after long-term therapy, levels that were otherwise detectable in patients who are able to maintain a spontaneous, drug-free control of HIV-1 replication. Thus, long-term antiretroviral treatment started during early infection cannot eliminate HIV-1, but the reduced reservoirs of HIV-1 infected cells in such patients may increase their chances to respond to clinical interventions aiming at inducing a drug-free remission of HIV-1 infection.

A quasispecies approach to the evolution of sexual replication in unicellular organisms

This study develops a simplified model describing the evolutionary dynamics of a population composed of obligate sexually and asexually reproducing, unicellular organisms. The model assumes that the organisms have diploid genomes consisting of two chromosomes, and that the sexual organisms replicate by first dividing into haploid intermediates, which then combine with other haploids, followed by the normal mitotic division of the resulting diploid into two new daughter cells. We assume that the fitness landscape of the diploids is analogous to the single-fitness-peak approach often used in single-chromosome studies. That is, we assume a master chromosome that becomes defective with just one point mutation. The diploid fitness then depends on whether the genome has zero, one, or two copies of the master chromosome. We also assume that only pairs of haploids with a master chromosome are capable of combining so as to produce sexual diploid cells, and that this process is described by second-order kinetics. We find that, in a range of intermediate values of the replication fidelity, sexually reproducing cells can outcompete asexual ones, provided the initial abundance of sexual cells is above some threshold value. The range of values where sexual reproduction outcompetes asexual reproduction increases with decreasing replication rate and increasing population density. We critically evaluate a common approach, based on a group selection perspective, used to study the competition between populations and show its flaws in addressing the evolution of sex problem.

The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos

The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere- (NT-B) and fibroblast- (NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p < 0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p < 0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.

Kinetics of ocular and systemic antigen-specific T-cell responses elicited during murine cytomegalovirus retinitis

Cytomegalovirus (CMV) reactivation in the retina of immunocompromized patients is a cause of significant morbidity as it can lead to blindness. The adaptive immune response is critical in controlling murine CMV (MCMV) infection in MCMV-susceptible mouse strains. CD8(+) T cells limit systemic viral replication in the acute phase of infection and are essential to contain latent virus. In this study, we provide the first evaluation of the kinetics of anti-viral T-cell responses after subretinal infection with MCMV. The acute response was characterized by a rapid expansion phase, with infiltration of CD8(+) T cells into the infected retina, followed by a contraction phase. MCMV-specific T cells displayed biphasic kinetics with a first peak at day 12 and contraction by day 18 followed by sustained recruitment of these cells into the retina at later time points post-infection. MCMV-specific CD8(+) T cells were also observed in the draining cervical lymph nodes and the spleen. Presentation of viral epitopes and activation of CD8(+) T cells was widespread and could be detected in the spleen and the draining lymph nodes, but not in the retina or iris. Moreover, after intraocular infection, antigen-specific cytotoxic activity was detectable and exhibited kinetics equivalent to those observed after intraperitoneal infection with the same viral dose. These data provide novel insights of how and where immune responses are initiated when viral antigen is present in the subretinal space.

Effects of potentised substances on growth kinetics of Saccharomyces cerevisiae and Schizosaccharomyces pombe

BACKGROUND: Homeopathic potencies are used as specific remedies in complementary medicine. Since the mode of action is unknown, the presumed specificity is discussed controversially. OBJECTIVE: This study investigated the effects of potentised substances on two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe, in a stable and reliable test system with systematic negative controls. MATERIALS AND METHODS: Yeast cells were cultivated in either potentised substances or water controls in microplates and their growth kinetics were measured photometrically. Water control runs were performed repeatedly to investigate the stability of the experimental set-up (systematic negative controls). RESULTS: 4 out of 14 screened substances seem to have affected the growth curve parameters slope or yield. Out of these substances, azoxystrobin and phosphorus were chosen for 8 further replication experiments, which partly confirmed the results of the screening. On the average of all experiments, azoxystrobin affected the slope of the growth curve of Saccharomyces cerevisiae (p < 0.05), and phosphorus affected the slope of the growth curve of Schizosaccharomyces pombe (p < 0.05). No effects were seen in the water control runs. In addition, significant interactions between treatment with potentised substances and experiment number were observed in all experiments with potentised substances (p < 0.01), but not in the water control runs. CONCLUSIONS: Both yeast species reacted to certain potentised substances by changing their growth kinetics. However, the interactions found point to additional factors of still unknown nature, that modulate the effects of potentised substances. This stable test system with yeasts may be suitable for further studies regarding the efficacy of homeopathic potencies.

Modeling the control of DNA replication in fission yeast

A central event in the eukaryotic cell cycle is the decision to commence DNA replication (S phase). Strict controls normally operate to prevent repeated rounds of DNA replication without intervening mitoses (“endoreplication”) or initiation of mitosis before DNA is fully replicated (“mitotic catastrophe”). Some of the genetic interactions involved in these controls have recently been identified in yeast. From this evidence we propose a molecular mechanism of “Start” control in Schizosaccharomyces pombe. Using established principles of biochemical kinetics, we compare the properties of this model in detail with the observed behavior of various mutant strains of fission yeast: wee1− (size control at Start), cdc13Δ and rum1OP (endoreplication), and wee1− rum1Δ (rapid division cycles of diminishing cell size). We discuss essential features of the mechanism that are responsible for characteristic properties of Start control in fission yeast, to expose our proposal to crucial experimental tests.

Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells

Increasing evidence suggests that HIV-1-specific cytotoxic T lymphocytes (CTLs) are a key host immune response to HIV-1 infection. Generation of CTL responses for prevention or therapy of HIV-1 infection has several intrinsic technical barriers such as antigen expression and presentation, the varying HLA restrictions between different individuals, and the potential for viral escape by sequence variation or surface molecule alteration on infected cells. A strategy to circumvent these limitations is the construction of a chimeric T cell receptor containing human CD4 or HIV-1-specific Ig sequences linked to the signaling domain of the T cell receptor ζ chain (universal T cell receptor). CD8+ CTLs transduced with this universal receptor can then bind and lyse infected cells that express surface HIV-1 gp120. We evaluated the ability of universal-receptor-bearing CD8+ cells from a seronegative donor to lyse acutely infected cells and inhibit HIV-1 replication in vitro. The kinetics of lysis and efficiency of inhibition were comparable to that of naturally occurring HIV-1-specific CTL clones isolated from infected individuals. Further study will be required to determine the utility of these cells as a therapeutic strategy in vivo.