ABSTRACT
Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a slower doubling time in both two-dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.
Subject(s)
Cell Culture Techniques/methods , Culture Media, Serum-Free , Vero Cells , Virus Cultivation/methods , Animals , Chlorocebus aethiops , Culture Media, Serum-Free/chemistry , Culture Media, Serum-Free/metabolism , Plant Preparations , Recombinant Proteins , Vero Cells/cytology , Vero Cells/metabolism , Viral Plaque AssayABSTRACT
The maintenance of terminally differentiated cells, especially hepatocytes, in vitro has proven challenging. Here we demonstrated the long-term in vitro maintenance of primary human hepatocytes (PHHs) by modulating cell signaling pathways with a combination of five chemicals (5C). 5C-cultured PHHs showed global gene expression profiles and hepatocyte-specific functions resembling those of freshly isolated counterparts. Furthermore, these cells efficiently recapitulated the entire course of hepatitis B virus (HBV) infection over 4 weeks with the production of infectious viral particles and formation of HBV covalently closed circular DNA. Our study demonstrates that, with a chemical approach, functional maintenance of PHHs supports long-term HBV infection in vitro, providing an efficient platform for investigating HBV cell biology and antiviral drug screening.
Subject(s)
Hepatitis B virus/growth & development , Hepatocytes/physiology , Hepatocytes/virology , Primary Cell Culture/methods , Virus Cultivation/methods , Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , DNA, Circular/biosynthesis , DNA, Circular/isolation & purification , DNA, Viral/biosynthesis , DNA, Viral/isolation & purification , Drug Evaluation, Preclinical , Hepatitis B virus/drug effects , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Transcriptome , Virion/drug effects , Virion/growth & developmentABSTRACT
Treatments with poxvirus vectors can have long-lasting immunological impact in the host, and thus they have been extensively studied to treat diseases and for vaccine development. More importantly, the oncolytic properties of poxviruses have led to their development as cancer therapeutics. Two poxviruses, vaccinia virus (VACV) and myxoma virus (MYXV), have been extensively studied as virotherapeutics with promising results. Vaccinia virus vectors have advanced to the clinic and have been tested as oncolytic therapeutics for several cancer types with successes in phase I/II clinical trials. In addition to oncolytic applications, MYXV has been explored for additional applications including immunotherapeutics, purging of cancer progenitor cells, and treatments for graft-versus-host diseases. These novel therapeutic applications have encouraged its advancement into clinical trials. To meet the demands of different treatment needs, VACV and MYXV can be genetically engineered to express therapeutic transgenes. The engineering process used in poxvirus vectors can be very different from that of other DNA virus vectors (e.g., the herpesviruses). This chapter is intended to serve as a guide to those wishing to engineer poxvirus vectors for therapeutic transgene expression and to produce viral preparations for preclinical studies.
Subject(s)
Genetic Engineering/methods , Genetic Vectors/genetics , Poxviridae/growth & development , Virus Cultivation/methods , Animals , Cell Line , Chlorocebus aethiops , Drug Evaluation, Preclinical , Genetic Therapy , Humans , Poxviridae/genetics , Transgenes , Vero CellsABSTRACT
The six major epidemiologically important hepatitis C virus (HCV) genotypes differ in global distribution and antiviral responses. Full-length infectious cell-culture adapted clones, the gold standard for HCV studies in vitro, are missing for genotypes 4 and 5. To address this challenge for genotype 5, we constructed a consensus full-length clone of strain SA13 (SA13fl), which was found non-viable in Huh7.5 cells. Step-wise adaptation of SA13fl-based recombinants, beginning with a virus encoding the NS5B-thumb domain and 3´UTR of JFH1 (SA13/JF372-X), resulted in a high-titer SA13 virus with only 41 JFH1-encoded NS5B-thumb residues (SA13/JF470-510cc); this required sixteen cell-culture adaptive substitutions within the SA13fl polyprotein and two 3´UTR-changes. SA13/JF372-X and SA13/JF470-510cc were equally sensitive to nucleoside polymerase inhibitors, including sofosbuvir, but showed differential sensitivity to inhibitors targeting the NS5B palm or thumb. SA13/JF470-510cc represents a model to elucidate the influence of HCV RNA elements on viral replication and map determinants of sensitivity to polymerase inhibitors.
Subject(s)
Antiviral Agents/isolation & purification , Drug Evaluation, Preclinical/methods , Hepacivirus/growth & development , Hepacivirus/genetics , Hepatocytes/virology , Viral Nonstructural Proteins/genetics , Virus Cultivation/methods , Antiviral Agents/pharmacology , Cell Culture Techniques/methods , Cell Line , Genotype , Hepacivirus/classification , HumansABSTRACT
The human Hepatitis Delta Virus (HDV) is unique among all viral pathogens. Encoding only one protein (Hepatitis Delta Antigen; HDAg) within its viroid-like self-complementary RNA, HDV constitutes the smallest known virus in the animal kingdom. To disseminate in its host, HDV depends on a helper virus, the human Hepatitis B virus (HBV), which provides the envelope proteins required for HDV assembly. HDV affects an estimated 15-20 million out of the 240 million chronic HBV-carriers and disperses unequally in disparate geographical regions of the world. The disease it causes (chronic Hepatitis D) presents as the most severe form of viral hepatitis, leading to accelerated progression of liver dysfunction including cirrhosis and hepatocellular carcinoma and a high mortality rate. The lack of approved drugs interfering with specific steps of HDV replication poses a high burden for gaining insights into the molecular biology of the virus and, consequently, the development of specific novel medications that resiliently control HDV replication or, in the best case, functionally cure HDV infection or HBV/HDV co-infection. This review summarizes our current knowledge of HBV molecular biology, presents an update on novel cell culture and animal models to study the virus and provides updates on the clinical development of the three developmental drugs Lonafarnib, REP2139-Ca and Myrcludex B.
Subject(s)
Antiviral Agents/therapeutic use , Hepatitis D, Chronic/drug therapy , Hepatitis Delta Virus/physiology , Virus Replication , Animals , Antiviral Agents/isolation & purification , Disease Models, Animal , Drug Evaluation, Preclinical , Humans , Virus Cultivation/methodsABSTRACT
Feline herpesvirus type-1 (FHV-1) is the most common viral cause of ocular surface disease in cats. Many antiviral drugs are used to treat FHV-1, but require frequent topical application and most lack well-controlled in vivo studies to justify their clinical use. Therefore, better validation of current and novel treatment options are urgently needed. Here, we report on the development of a feline whole corneal explant model that supports FHV-1 replication and thus can be used as a novel model system to evaluate the efficacy of antiviral drugs. The anti-herpes nucleoside analogues cidofovir and acyclovir, which are used clinically to treat ocular herpesvirus infection in cats and have previously been evaluated in traditional two-dimensional feline cell cultures in vitro, were evaluated in this explant model. Both drugs suppressed FHV-1 replication when given every 12 h, with cidofovir showing greater efficacy. In addition, the potential efficacy of the retroviral integrase inhibitor raltegravir against FHV-1 was evaluated in cell culture as well as in the explant model. Raltegravir was not toxic to feline cells or corneas, and most significantly, inhibited FHV-1 replication at 500 µM in both systems. Importantly, this drug was effective when given only once every 24 h. Taken together, our data indicate that the feline whole corneal explant model is a useful tool for the evaluation of antiviral drugs and, furthermore, that raltegravir appears a promising novel antiviral drug to treat ocular herpesvirus infection in cats.
Subject(s)
Antiviral Agents/pharmacology , Cornea/virology , Drug Evaluation, Preclinical/methods , Organ Culture Techniques/methods , Varicellovirus/drug effects , Virus Cultivation/methods , Acyclovir/pharmacology , Animals , Cats , Cidofovir , Cytosine/analogs & derivatives , Cytosine/pharmacology , Organophosphonates/pharmacologyABSTRACT
The world-wide HIV epidemic is characterized by increasing genetic diversity with multiple viral subtypes, circulating recombinant forms (CRFs) and unique recombinant forms (URFs). Antiretroviral drug design and basic virology studies have largely focused on HIV-1 subtype B. There have been few direct comparisons by subtype, perhaps due to the lack of uniform and standardized culture systems for the in vitro propagation of diverse HIV-1 subtypes. Although peripheral blood mononuclear cells (PBMCs) are major targets and reservoirs of HIV, PBMCs culturing is relatively difficult and not always reproducible. In addition, long-term experiments cannot be performed because PBMCs are short-lived cells. We faced these problems during the in vitro testing of an experimental RNA interference (RNAi) based gene therapy. Therefore, many T cell lines that support HIV-1 infection were tested and compared for replication of HIV-1 isolates, including viruses that use different receptors and diverse subtypes. The PM1 T cell line was comparable to PBMCs for culturing of any of the HIV-1 strains and subtypes. The advantage of PM1 cells in long-term cultures for testing the safety and efficacy of an RNAi-based gene therapy was demonstrated. PM1 may thus provide a valuable research tool for studying new anti-HIV therapies.
Subject(s)
Anti-HIV Agents/isolation & purification , Anti-HIV Agents/pharmacology , Drug Evaluation, Preclinical/methods , HIV-1/drug effects , HIV-1/physiology , T-Lymphocytes/virology , Viral Tropism , Cell Line , Humans , Virus Cultivation/methodsABSTRACT
The adeno-associated virus (AAV) is one of the most useful viral vectors for gene delivery for both in vivo and in vitro applications. A variety of methods have been established to produce and characterize recombinant AAV (rAAV) vectors; however most methods are quite cumbersome and obtaining consistently high titer can be problematic. This protocol describes a triple-plasmid co-transfection approach with 25 kDa linear polyethylenimine (PEI) in 293 T cells for the production of AAV serotype 2. Seventy-two hours post-transfection, supernatant and cells were harvested and purified by a discontinuous iodixanol density gradient ultracentrifugation, then dialyzed and concentrated with an Amicon 15 100,000 MWCO concentration unit. To optimize the protocol for AAV2 production using PEI, various N/P ratios and DNA amounts were compared. We found that an N/P ratio of 40 coupled with 1.05 µg DNA per ml of media (21 µg DNA/15 cm dish) was found to produce the highest yields for viral replication and assembly measured multiple ways. The infectious units, as determined by serial dilution, were between 1×10(8) and 2×10(9) IU/ml. The genomic titer of the viral stock was determined by qPCR and ranged from 2×10(12) to 6×10(13) VG/ml. These viral vectors showed high expression both in vivo within the brain and in vitro in cell culture. The use of linear 25 kDa polyethylenamine PEI as a transfection reagent is a simple, more cost-effective, and stable means of high-throughput production of high-titer AAV serotype 2. The use of PEI also eliminates the need to change cell medium post-transfection, lowering cost and workload, while producing high-titer, efficacious AAV2 vectors for routine gene transfer.
Subject(s)
Dependovirus/growth & development , Dependovirus/isolation & purification , Polyethyleneimine/chemistry , Transfection , Cell Line , Centrifugation, Density Gradient , Dialysis , Humans , Nitrogen/analysis , Phosphorus/analysis , Polyethyleneimine/metabolism , Viral Load , Virus Cultivation/methodsABSTRACT
BACKGROUND: Avian H5N1 influenza has caused human infections globally and has a high mortality rate. Rapid production of effective vaccines is needed. METHODS: A phase 1, randomized, observer-blinded clinical trial was conducted to examine the safety and immunogenicity of an inactivated whole virion vaccine against the influenza A/H5N1 virus produced from the Madin-Darby canine kidney (MDCK) cell line. Participants were randomized to four groups and administered two intramuscular doses of vaccine containing 3 µg hemagglutinin (HA), 3 µg HA with 300 µg aluminum phosphate (AlPO4), 6 µg HA, and 6 µg HA with 300 µg AlPO4, respectively, at two visits, 21 days apart. Serum hemagglutination inhibition (HAI) and neutralizing antibody levels were determined at baseline and on Days 21 and 42. RESULTS: Sixty healthy individuals were enrolled. The neutralization assay showed a significant immune response in the 6 µg with ALPO4 group on Day 42 compared to pre-vaccination levels (11.32±9.77 vs. 4.00±0, p=0.02). The adjuvant effect in neutralization assay was also significant on Day 42 in the 6 µg group (4.52±1.94 without adjuvant vs. 11.32±9.77 with adjuvant, p=0.02). HAI assay also showed an aluminum adjuvant-induced increasing trend in HAI geometric mean titer on Day 42 in the 3 µg and 6 µg groups (6.02 versus 8.20, p=0.05 and 5.74 versus 8.21, p=0.14). The most frequent adverse event was local pain (20% to 60%). There were no vaccine-related severe adverse effects. CONCLUSION: MDCK cell line-derived H5N1 vaccine was well tolerated. It is necessary to investigate further the immunogenicity of higher antigen doses and the role of aluminum adjuvant in augmenting the effect of the vaccine.
Subject(s)
Influenza A Virus, H5N1 Subtype/immunology , Influenza Vaccines/adverse effects , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Adjuvants, Immunologic/administration & dosage , Adult , Aluminum Compounds/administration & dosage , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Dogs , Drug-Related Side Effects and Adverse Reactions/epidemiology , Drug-Related Side Effects and Adverse Reactions/pathology , Female , Healthy Volunteers , Hemagglutination Inhibition Tests , Humans , Influenza Vaccines/administration & dosage , Influenza Vaccines/isolation & purification , Injections, Intramuscular , Madin Darby Canine Kidney Cells , Male , Middle Aged , Neutralization Tests , Phosphates/administration & dosage , Prospective Studies , Single-Blind Method , Taiwan , Vaccination/methods , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/adverse effects , Vaccines, Inactivated/immunology , Vaccines, Inactivated/isolation & purification , Virus Cultivation/methods , Young AdultABSTRACT
Cranberry juice contains high molecular weight non-dialyzable material (NDM) which was found to inhibit hemagglutination induced by the influenza virus (IV) as well as to neutralize the cytotoxicity of IV in cell cultures. Because influenza virus surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) are involved in viral replication and in the infectious process, we sought in the present study to examine the effect of NDM on neuraminidases which are the target of most anti-influenza drugs today. NDM inhibited the NA enzymatic activity of influenza A and B strains as well as that of Streptococcus pneumoniae. This finding is of importance considering the emergence of influenza isolates resistant to antiviral drugs, reaching 90 % in some places. The anti-NA activity of NDM, evaluated by the MUNANA method and expressed as the concentration required for 50 % inhibition (IC50), was most potent against N1 (IC50, 192 µg/mL), less active against BN and N2 (IC50, 509 µg/mL and 1128 µg/mL, respectively), and moderately active against Streptococcus pneumoniae NA (IC50, 594 µg/mL). The in vitro findings of the present study suggest that cranberry constituents may have a therapeutic potential against both A and B influenza virus infections and might also interfere with the development of secondary bacterial complications.
Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Influenza A Virus, H1N1 Subtype/drug effects , Influenza B virus/drug effects , Plant Extracts/pharmacology , Vaccinium macrocarpon/chemistry , Animals , Anti-Bacterial Agents/pharmacology , Cell Line , Dogs , Drug Evaluation, Preclinical , Enzyme Activation , Erythrocytes , Hemagglutination Tests/methods , Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H3N2 Subtype/drug effects , Influenza A Virus, H3N2 Subtype/enzymology , Influenza B virus/enzymology , Inhibitory Concentration 50 , Microbial Sensitivity Tests/methods , Molecular Weight , Neuraminidase/antagonists & inhibitors , Plant Extracts/chemistry , Streptococcus/drug effects , Streptococcus/enzymology , Viral Proteins/antagonists & inhibitors , Virus Cultivation/methodsABSTRACT
Our previous report showed that quercetin 3-rhamnoside (Q3R) possessed antiviral activity against influenza A/WS/33 virus in vitro. The present study evaluated the effect of Q3R on influenza A/WS/33 virus infected mice. Mice orally treated with Q3R (6.25 mg/kg per dose) at 2 h before and once daily for 6 days after influenza virus infection showed significant decreases in weight loss, and decreased mortality. Lung virus titers of mice killed at 6 days after infection were about 2000 times lower than that of the placebo-treated control mice and about two times lower than that for the oseltamivir-treated mice. Furthermore, histological evaluation showed that administration of Q3R delayed the development and progression of pulmonary lesions. Therefore, Q3R could be an attractive lead for the development of antiviral agents against influenza virus.
Subject(s)
Antiviral Agents/therapeutic use , Influenza A virus/drug effects , Orthomyxoviridae Infections/drug therapy , Quercetin/analogs & derivatives , Quercetin/therapeutic use , Animals , Antiviral Agents/administration & dosage , Cell Line , Dogs , Influenza A virus/pathogenicity , Lung/pathology , Lung/virology , Male , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/virology , Oseltamivir/pharmacology , Phytotherapy , Quercetin/administration & dosage , Viral Load , Virus Cultivation/methods , Weight LossABSTRACT
Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, dual envelope pseudovirion (DEP) assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes.
Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays/methods , Severe acute respiratory syndrome-related coronavirus/drug effects , Virus Internalization/drug effects , Drug Evaluation, Preclinical/standards , High-Throughput Screening Assays/standards , Humans , Microbial Sensitivity Tests/methods , Microbial Sensitivity Tests/standards , Virus Cultivation/methods , Virus Cultivation/standardsABSTRACT
Replication of infectious hepatitis C virus in Huh7 cells, a human hepatocyte cell line, has become possible due to the unique properties of the JFH1 isolate. Developing reporter virus systems for a simple titration has been attempted by integrating heterologous reporter genes into the JFH1 genome, resulting in a big infectivity reduction that limits the usefulness of such reporter systems. To overcome this problem, JFH1-infected Huh7 cells were cultured continuously for 2 years to obtain Huh7-adapted JFH1 variants capable of yielding up to 1000-fold higher titers. Sequence analysis of variant genome RNA suggested that this adapted population consisted mainly of two variants. By joining the 5'-half of the obtained representative viral complementary DNA (cDNA) fragments of the variants with the 3'-half of the wild-type's, two prototype clones, A/WT and B/WT, were constructed. Replication of A/WT and B/WT viruses in Huh7 cells showed up to 100-1000-fold higher titers than the wild-type. A Renilla luciferase cDNA was inserted into the Nonstructural Protein 5A region of the A/WT and B/WT cDNA to generate A/WT-Rluc and B/WT-Rluc, respectively. Transfection of Huh7 cells with in vitro-transcribed A/WT-Rluc and B/WT-Rluc RNA resulted in production of infectious viruses with approximately 15- and 25-fold higher titers, respectively, than the wild-type RNA. The replication of A/WT-Rluc and B/WT-Rluc viruses was more vigorous than the wild-type even with insertion of the luciferase cDNA showing a good correlation of luciferase activities with infectious titers. Furthermore, interferon-alpha inhibited the replication of A/WT-Rluc and B/WT-Rluc viruses in a dose-dependent manner as determined by a luciferase assay. These results imply that our system is potentially a tool useful for screening anti-hepatitis C virus drugs in a simple and time/cost-saving manner.
Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , Hepacivirus/drug effects , Hepacivirus/physiology , Virus Replication/drug effects , Adaptation, Biological , Cell Line , Genes, Reporter , Hepatocytes/virology , Humans , Interferon-alpha/pharmacology , Luciferases, Renilla/genetics , Luciferases, Renilla/metabolism , Microbial Sensitivity Tests/methods , RNA, Viral/genetics , Sequence Analysis, DNA , Serial Passage , Staining and Labeling/methods , Virus Cultivation/methodsABSTRACT
Although the smallpox virus was eradicated over 20 years ago, its potential release through bioterrorism has generated renewed interest in vaccination. To develop a modern smallpox vaccine, we have adapted vaccinia virus that was derived from the existing Dryvax vaccine for growth in a human diploid cell line. We characterized six cloned and one uncloned vaccine candidates. One clone, designated ACAM1000, was chosen for development based on its comparability to Dryvax when tested in mice, rabbits and monkeys for virulence and immunogenicity. By most measures, ACAM1000 was less virulent than Dryvax. We compared ACAM1000 and Dryvax in a randomized, double-blind human clinical study. The vaccines were equivalent in their ability to produce major cutaneous reactions ('takes') and to induce neutralizing antibody and cell-mediated immunity against vaccinia virus.
Subject(s)
Smallpox Vaccine/immunology , Smallpox Vaccine/pharmacology , Vaccinia virus/immunology , Animals , Bioterrorism , Cell Line/virology , Clone Cells , Double-Blind Method , Drug Evaluation, Preclinical/methods , Humans , Macaca mulatta , Mice , Mice, Inbred BALB C , Neutralization Tests , Rabbits , Vaccinia virus/pathogenicity , Virus Cultivation/methodsABSTRACT
The North American strain of viral hemorrhagic septicemia virus (NA-VHSV) could be recovered for up to 40 h in natural filtered seawater (27 ppt) with a 50% loss of infectivity after approximately 10 h at 15 degrees C. Addition of 10 ppb North Slope crude oil to the seawater had no effect on virus survival. However, when various concentrations of teleost ovarian fluid were added to seawater, virus could be recovered after 72 h at 0.01% ovarian fluid and after 96 h at 1.0%. When cell culture medium supplemented with 10% fetal bovine serum was added to the seawater, 100% of the virus could be recovered for the first 15 d and 60% of the virus remained after 36 d. These findings quantify NA-VHSV infectivity in natural seawater and demonstrate that ovarian fluid, which occurs naturally during spawning events, significantly prolongs the survival and infectivity of the virus. The extended stabilization of virus in culture medium supplemented with serum allows for low titer field samples to be collected and transported in an unfrozen state without significant loss of virus titer.
Subject(s)
Culture Media/chemistry , Fish Diseases/virology , Petroleum/analysis , Rhabdoviridae Infections/veterinary , Rhabdoviridae/isolation & purification , Seawater/virology , Virus Cultivation/veterinary , Water Pollution, Chemical , Animals , Female , Filtration , Fishes , Ovary , Virus Cultivation/methodsABSTRACT
Influenza viruses for vaccine production are currently grown on embryonated eggs. This manufacturing system conveys many major drawbacks such as inflexibility, cumbersome down stream processing, inability of some strains to replicate on eggs to high enough yields, and selection of receptor-binding variants with reduced antigenicity. These limitations emphasize the need for a cell line-based production system that could replace eggs in the production of influenza virus vaccines in a pandemic proof fashion. Here we present the efficient propagation of influenza A and B viruses on the fully characterized and standardized human cell line PER.C6.
Subject(s)
Influenza Vaccines/isolation & purification , Virus Cultivation/methods , Animals , Antigens, Viral/isolation & purification , Bioreactors , Cell Line , Chickens , Edetic Acid , Hemagglutinins, Viral/isolation & purification , Humans , Influenza Vaccines/immunology , Orthomyxoviridae/immunology , Orthomyxoviridae/physiology , Trypsin , Virus ReplicationABSTRACT
Hepatitis A virus (HAV) is closely related to the genus enterovirus. HAV is very stable and resistant to acid pH and elevated temperature, as well as to chemicals and environmental influences. Human poliovirus is still one of the model viruses for testing disinfectants but there are discussions about changing to hepatitis A virus. The purpose of this study was to develop a method for using adapted hepatitis A virus to test hand disinfectants. Using HAV strains HM175/24a and FRhK-4 cytopathic effects were visible rarely, and not before 14 days. To verify virus growth in cells a RT-PCR was developed. Two disinfectants tested did not show the required virucidal activity to satisfy current German guidelines.
Subject(s)
2-Propanol/pharmacology , Anti-Infective Agents, Local/pharmacology , Disinfectants/pharmacology , Drug Evaluation, Preclinical/methods , Ethanol/pharmacology , Hand Disinfection/methods , Hepatitis A virus/drug effects , Virus Cultivation/methods , Animals , Cytopathogenic Effect, Viral/drug effects , Drug Evaluation, Preclinical/standards , Enzyme-Linked Immunosorbent Assay , Guidelines as Topic , Hand Disinfection/standards , Hepatitis A virus/classification , Hepatitis A virus/pathogenicity , Humans , Hydrogen-Ion Concentration , Macaca mulatta , Reverse Transcriptase Polymerase Chain Reaction , Temperature , Virus Cultivation/standardsABSTRACT
All tissue culture systems for propagating HBV employed so far make use of tandemly arranged HBV genomes usually under the control of strong foreign promoters. Thus these systems are helpful for virus production but are of limited value in the investigation of the regulation of HBV replication or of the extent to which the expression of viral genes might be influenced by cellular signal transduction pathways. To overcome this barrier we established an HBV-producing cell line (HepG2-4A5) by stably transfecting HepG2 cells with a replication-competent, terminally redundant HBV plasmid (pSPT1.2 xHBV) that contains each of the four major HBV-ORFs only once and exclusively under the control of their own regulatory elements. HepG2-4A5 cells contain a single, nonrearranged, chromosomally integrated, replication-competent HBV genome. In the cytoplasm of HepG2-4A5 cells, all typical viral mRNAs were detectable, but no other viral transcripts were found. Furthermore, all viral gene products are synthesized in a balanced ratio, as close as possible to that found in an in vivo infection. Dane-like particles released from HepG2-4A5 cells were indistinguishable from virions synthesized in vivo, by all physical (electron microscopy, buoyant density) and biochemical (endogenous polymerase reaction, immunogenic behaviour) criteria. Because of the autologous genome organization in this system, the HepG2-4A5 cell line allows studies on the function of the HBV gene products with respect to their involvement in regulating HBV replication under conditions imitating as closely as possible the situation in vivo. Furthermore, this cell line might be a helpful tool in screening antiviral drugs and in studying their effect on regulating HBV replication.
Subject(s)
Hepatitis B virus/physiology , Tumor Cells, Cultured , Virus Cultivation/methods , Virus Replication , Antiviral Agents/pharmacology , DNA, Viral , Drug Evaluation, Preclinical/methods , Hepatitis B Surface Antigens/analysis , Hepatitis B e Antigens/analysis , Hepatitis B virus/drug effects , Hepatitis B virus/genetics , HumansABSTRACT
The human colonic epithelial cell line HT-29 can be productively infected with various HIV-1 and HIV-2 isolates that are highly cytopathic for T lymphocytes. In each case, a chronically infected HT-29 cell line can be established, and progeny viruses retain their original properties including high cytopathogenicity for T cells. Inasmuch as AIDS vaccines should include viral isolates capable of infecting mucosal epithelial cells, it may be useful to produce these isolates in such cells at a large scale. We describe here a microcarrier-based culture system allowing the production of infectious viruses from HT-29 cells grown in a chemically defined serum-free medium (Dulbecco's modified Eagle's medium/F12, HEPES 15 mM, pH 7.4, transferrin 5 micrograms/ml, selenium 10 ng/ml). The yield of HIV-1 from microcarrier cultures (275 ng of p24gag/ml) was greater than the yield from conventional culture flasks (122 ng of p24gag/ml). This virus, produced in serum-free medium, can be used either as a viral stock or as a source for HIV-1 proteins.