Preparation Of Stocks Research Articles

Propagating and banking genetically diverse human sapovirus strains using a human duodenal cell line: investigating antigenic differences between strains.

The control of human sapovirus, which causes acute gastroenteritis in individuals of all ages, is challenging because of its association with outbreaks similar to those caused by human norovirus. The establishment of conditions for efficient viral propagation of various viral strains is essential for understanding the infection mechanism and identifying potential control methods. In this study, two critical factors for human sapovirus propagation in a conventional human duodenal cell line were identified, and 15 strains of different genotypes that differed at the genetic and antigenic levels were isolated and used to prepare virus stocks. The preparation of virus stocks has not been successful for noroviruses, which belong to the same family as sapoviruses. Securing virus stocks of multiple human sapovirus strains represents a significant advance toward establishing a reliable experimental system that does not depend on limited virus-positive fecal material.

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers.

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

A Sensitive and Quantitative Assay to Enumerate and Measure Mycobacterium leprae Viability in Clinical and Experimental Specimens.

Mycobacterium leprae, the etiologic agent of leprosy, cannot be cultured on artificial media. This characteristic, coupled with its long generation time, presents a number of unique challenges to studying this pathogen. One of the difficulties facing both researchers and clinicians is the absence of a rapid test to measure the viability of M. leprae in clinical or experimental specimens. The lack of such a tool limits the understanding of M. leprae immunopathogenesis and makes determining the efficacy of drug treatments difficult. With this in mind, we developed a robust two-step molecular viability assay (MVA) that first enumerates the M. leprae in the tissue; then, this data is used to normalize bacterial RNA quantities for the second step, in which the expression of M. leprae esxA and hsp18 are measured. This assay is specific and sensitive enough to be used on most clinical samples. This protocol describes the steps required to extract DNA and RNA from M. leprae-infected tissue, enumerate M. leprae, and measure M. leprae viability based on the normalized expression of two M. leprae-specific genes (hsp18 and esxA). This protocol also outlines an optimal laboratory design and workflow for performing this assay. © 2022 The Leprosy Mission Nepal. Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: DNA and RNA P purification from M. leprae-infected tissue Basic Protocol 2: Enumeration of M. leprae by RLEP qPCR on the DNA fraction Basic Protocol 3: Calculation of M. leprae per tissue and normalization of RNA Basic Protocol 4: Reverse-transcription of normalized RNA to generate cDNA Basic Protocol 5: Determination of M. leprae viability using HSP18 and ESXA qPCR on the cDNA Support Protocol 1: M. leprae qPCR primer/probe stock preparation Support Protocol 2: Preparation of plasmid stocks and standard curves.

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium.

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver disease worldwide due to its relationship with obesity, diabetes type 2, and dyslipidemia. Hepatic steatosis, the accumulation of lipid droplets in the liver parenchyma, is a key feature of the disease preceding the inflammation observed in steatohepatitis, fibrosis, and end-stage liver disease. Lipid accumulation in hepatocytes might interfere with proper metabolism of xenobiotics and endogenous molecules, as well as to induce cellular processes leading to the advance of the disease. Although the experimental study of steatosis can be performed in vivo, in vitro approaches to the study of steatosis are complementary tools with different advantages. Hepatocyte culture in lipid overload-conditioned medium is an excellent reproducible option for the study of hepatic steatosis allowing the identification of cellular processes related to lipid accumulation, such as oxidative and reticular stresses, autophagia, proliferation, cell death, etcetera, as well as other testing including drug effectiveness, and toxicological testing, among many other possible applications. Here, it was aimed to describe the methodology of hepatocyte cell culture in lipid overload-conditioned medium. HepG2 cells were cultured in RMPI 1640 medium conditioned with sodium palmitate and sodium oleate. Importantly, the ratio of these two lipids is crucial to favor lipid droplet accumulation, while maintaining cell proliferation and a moderate mortality rate, as occurs in the liver during the disease. The methodology, from the preparation of the lipid solution stocks, mixture, addition to the medium, and hepatocyte culture is shown. With this approach, it is possible to identify lipid droplets in the hepatocytes that are readily observable by Oil-red O staining, as well as curves of proliferation/mortality rates.

Agrobacterium tumefaciens-Mediated Transformation of Three Milkweed Species (Asclepias hallii, A. syriaca, and A. tuberosa: Apocynaceae).

Milkweeds have ecological significance for insect herbivores that rely on them as hosts for either part of or the entirety of their life cycles. Interesting interactions, some of which are not completely understood, have evolved over time. To develop these species as models to elucidate the interplay with insect herbivores, we established Agrobacterium tumefaciens-mediated transformation approaches for Asclepias hallii (Hall's milkweed), A. syriaca (common milkweed), and A. tuberosa (butterflyweed). The method is based on infection of stem internodal explants, which were more amenable to transformation than leaf explants. We found that addition of freshly prepared dithiothreitol was critical to prevent browning of stem explants. Depending on the species, the time from infection to the regeneration of transgenic lines ranges from 2 to 4 months. Transformation efficiency for A. hallii was 9%, whereas efficiencies for A. syriaca and A. tuberosa were 6% and 13%, respectively. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Agrobacterium tumefaciens-mediated transformation of Asclepias internodal stem explants Basic Protocol 2: Preparation of Agrobacterium glycerol stocks containing gene constructs.

Preparation of Lubricant Base Stocks with High Viscosity Index through 1-Decene Oligomerization Catalyzed by Alkylaluminum Chloride Promoted by Metal Chloride

In this paper, poly-α-olefins oil base stocks with medium kinematic viscosity and high viscosity index were prepared by 1-decene oligomerization catalyzed using the alkylaluminum chloride catalyst ...

Quantifying HIV-1-Mediated Gut CD4+ T Cell Deathin the Lamina Propria Aggregate Culture (LPAC) Model.

Gut CD4 T cells are major targets of HIV-1 and are massively depleted early during infection. To better understand the mechanisms governing HIV-1-mediated CD4 T cell death, we developed the physiologically-relevant Lamina Propria Aggregate Culture (LPAC) model. The LPAC model is ideal for studying CD4 T cell death induced by clinically-relevant Transmitted/Founder (TF) HIV-1 strains and is also suitable for studying how enteric microbes and soluble factors (e.g., Type I Interferons) impact LP CD4 T cell death and function. Here, we detail the protocol to establish LP CD4 T cell infection using a process of spinoculation, the subsequent evaluation of infection levels using multicolor flow cytometry and the determination of overall LP CD4 T cell death using absolute LP CD4 T cell counts. We also describe the preparation of virus stocks of Transmitted/Founder (TF) HIV-1 infectious molecular clones that were successfully used in the LPAC model.

Change of status of stems indicators in the process of healing of stock after hemp seeds harvesting with a grain harvester for different technologies

The aim is to study the indicators of changes in the state of stems in the process of stocks retting after hemp seeds harvesting with a combine harvester using various technologies. A brief description of the work performed. With two methods of hemp seeds harvesting by a combine harvester by cutting the seed part and combing it with subsequent threshing, it was investigated that during the preparation of the stocks, the state of all constituent stems, namely, left standing, after the harvesting process is carried out, is in the wheel area and threshed in the thresher for option with a cut. It was established that during the preparation of hemp stocks after harvesting the seeds with a combine harvester, the color of the stems changes from yellow-green (corresponds to straw) to gray with slight brown shades, which corresponds to the stock for all components. The color change of the stems during the preparation of the stock can be controlled by the indicator of the intensity of the reflected light flux from the test surface, which decreases and for straw corresponds to 46-54 lux, the beginning of the formation of the stock, when on the first stems the fiber begins to separate from the wood - 30-32 lux and matted stock - 25-28 lux. Conclusions. Indicators of changes in the state of stems in the process of maturing stocks after hemp seeds harvesting with a combine harvester using different technologies are determined.

Generation and characterization of a stable cell line persistently replicating and secreting the human hepatitis delta virus

Human hepatitis delta virus (HDV) causes the most severe form of viral hepatitis. Approximately 15–25 million people are chronically infected with HDV. As a satellite virus of the human hepatitis B virus (HBV), HDV uses the HBV-encoded envelope proteins for egress from and de novo entry into hepatocytes. So far, in vitro production of HDV particles is restricted to co-transfection of cells with HDV/HBV encoding cDNAs. This approach has several limitations. In this study, we established HuH7-END cells, which continuously secrete infectious HDV virions. The cell line was generated through stepwise stable integration of the cDNA of the HDV antigenome, the genes for the HBV envelope proteins and the HBV/HDV receptor NTCP. We found that HuH7-END cells release infectious HDV particles up to 400 million copies/milliliter and support virus spread to co-cultured cells. Due to the expression of NTCP, HuH7-END cells are also susceptible to de novo HDV entry. Virus production is stable for >16 passages and can be scaled up for preparation of large HDV virus stocks. Finally, HuH7-END cells are suitable for screening of antiviral drugs targeting HDV replication. In summary, the HuH7-END cell line provides a novel tool to study HDV replication in vitro.

Growth-Adaptive Mutations in the Ebola Virus Makona Glycoprotein Alter Different Steps in the Virus Entry Pathway.

The Zaire ebolavirus (EBOV) glycoprotein (GP) is cleaved into two subunits (GP1 and GP2) that are both required for virus attachment and entry into cells. Sequence changes in the GP have been proposed to increase pathogenesis and to alter virus growth properties. Mutations in GP acquired during EBOV tissue culture passage have also been reported to change virus growth properties. Here, we report the isolation of six amino acid mutations in EBOV GP that spontaneously appeared during recovery and passage of an EBOV-Makona GP-pseudotyped vesicular stomatitis virus (VSV), two of which also occur during passage of EBOV clinical isolates in tissue culture. Each of the six mutations resulted in increased virus growth in monkey and human cell lines. All mutations are located in the GP2 fusion subunit and increase entry kinetics of EBOV virus-like particles (VLPs). The gain-of-entry function mapped to two mechanistic phenotypes. Mutations in heptad repeat 1 (HR1) decreased the requirement for cathepsin B activity for viral infection. Mutations directly within the fusion loop increased entry kinetics without altering the cathepsin B dependence. Several mutations in the fusion loop were substitutions of residues present in other ebolavirus glycoproteins, illustrating the evolutionary paths for maintaining an optimally functioning fusion loop under selection pressure.IMPORTANCEZaire ebolavirus (EBOV) is the causative agent of the highly lethal Ebola virus disease and poses a significant threat to the global health community. Approved antivirals against EBOV are lacking; however, promising therapies targeting the EBOV glycoprotein are being developed. Efficacy testing of these candidate therapeutics relies on EBOV laboratory stocks, which when grown in tissue culture may acquire mutations in the glycoprotein. These mutations can produce inaccurate results in therapeutic testing. Until recently, distinguishing between tissue culture mutations and naturally occurring polymorphisms in EBOV GP was difficult in the absence of consensus clinical GP sequences. Here, we utilize recombinant VSV (rVSV) pseudotyped with the consensus clinical EBOV Makona GP to identify several mutations that have emerged or have potential to emerge in EBOV GP during tissue culture passage. Identifying these mutations informs the EBOV research community as to which mutations may arise during preparation of laboratory virus stocks.

Pengembangan Aplikasi Perhitungan Prediksi Stock Motor Menggunakan Algoritma C 4.5 Sebagai Bagian dari Sistem Pengambilan Keputusan (Studi Kasus di Saudara Motor)

Limited company resources must be managed effectively and efficiently in order to guarantee corporate tax information technology information that will assist management within the company for every necessary process and reliable information. C 4.5 can be used to predict motor stock at Brothers Motor. the learning process that existed in the algorithm C 4.5 is preparing the data traning, calculate the root of the tree, and the partition process of the decision. The decision process will stop when all the records are in the N node that gets the same class, there are no attributes in the record that are partitioned again, there is no record in the empty network. The result of the average calculation algorithm algorithm C 4. Then the results of the calculation can be used for data processing. Data Mining uses the C4.5 algorithm then obtains a decision tree. Produce data obtained 12 rules (rules) in determining the prediction of preparation of motor stocks at Brothers Motor dealers.Key word: dss, algortm c 5.4,motorbike, dealer

Propagation and Titration of Influenza Viruses.

Influenza viruses are constantly circulating among humans, in which they cause seasonal epidemics of severe respiratory disease. Additionally, these zoonotic viruses infect different mammals and birds, from which new antigenic variants are occasionally transmitted to humans leading to devastating global pandemics. Surveillance programs, in which viruses from the main reservoir (waterfowl), intermediate hosts (like pigs and other farm animals), and other affected species are isolated and characterized, are crucial for the global influenza prevention strategy. This chapter gives an overview of the most commonly used methods for the propagation and titration of influenza viruses, which are key steps in surveillance procedures, as well as in vaccine development and basic research. Depending on the host and the viral strain, primary isolates are obtained from biological samples of different origin and subsequently amplified in embryonated chicken eggs or cell cultures. These propagation procedures are the focus of the first part of this chapter. Once the initial isolates have been amplified, virus titration methods based on particular characteristics of influenza viruses, such as their ability to agglutinate red blood cells (RBCs) or to induce cytopathic effects (CPE) in cell monolayers, are used to estimate the amount of viral particles. Such approaches, like the hemagglutination assay (HA assay), 50% tissue culture infectious dose (TCID50), or plaque assay, are included in the second part of this chapter. Although they are simple and cost-effective, some of these techniques have been partially replaced by faster and more sensitive methods based on the quantification of viral genomes, such as the quantitative real-time reverse transcription PCR (RT-qPCR), which is presented at the end of this section. The different protocols are explained in detail in order to facilitate the preparation and quantification of infectious virus stocks.

Isolation, Culture, and Cryopreservation of Adult Rodent Schwann Cells Derived from Immediately Dissociated Teased Fibers.

Adult Schwann cell (SC) cultures are usually derived from nerves subjected to a lengthy step of pre-degeneration to facilitate enzymatic digestion and recovery of viable cells. To overcome the need for pre-degeneration, we developed a method that allows the isolation of adult rat sciatic nerve SCs immediately after tissue harvesting. This method combines the advantages of implementing a rapid enzymatic dissociation of the nerve fibers and a straightforward separation of cells versus myelin that improves both cell yield and viability. Essentially, the method consists of (1) acute dissociation with collagenase and dispase immediately after removal of the epineurium layer and extensive teasing of the nerve fibers, (2) removal of myelin debris by selective attachment of the cells to a highly adhesive poly-L-lysine/laminin substrate, (3) expansion of the initial SC population in medium containing chemical mitogens, and (4) preparation of cryogenic stocks for transfer or delayed experimentation. This protocol allows for the procurement of homogeneous SC cultures deprived of myelin and fibroblast growth as soon as 3-4days after nerve tissue dissection. SC cultures can be used as such for experimentation or subjected to consecutive rounds of expansion prior to use, purification, or cryopreservation.

Preparation of Cell Cultures and Vaccinia Virus Stocks.

The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included. © 2017 by John Wiley & Sons, Inc.

Preparation of Cell Cultures and Vaccinia Virus Stocks.

The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included. © 2017 by John Wiley & Sons, Inc.

Phage on tap-a quick and efficient protocol for the preparation of bacteriophage laboratory stocks.

A major limitation with traditional phage preparations is the variability in titer, salts, and bacterial contaminants between successive propagations. Here we introduce the Phage On Tap (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) stocks. This method produces homogenous, laboratory-scale, high titer (up to 1010–11 PFU·ml−1), endotoxin reduced phage banks that can be used to eliminate the variability between phage propagations and improve the molecular characterizations of phage. The method consists of five major parts, including phage propagation, phage clean up by 0.22 μm filtering and chloroform treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and storage of phage banks for continuous laboratory use. From a starting liquid lysate of > 100 mL, the PoT protocol generated a clean, homogenous, laboratory phage bank with a phage recovery efficiency of 85% within just two days. In contrast, the traditional method took upwards of five days to produce a high titer, but lower volume phage stock with a recovery efficiency of only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing endotoxin concentrations by over 3,000-fold while maintaining phage titer. The PoT protocol focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated to sufficient titer, producing homogenous, high titer phage banks that are applicable for molecular and cellular assays.

Microbiological investigation into the cause of Equine Grass Sickness

A role for bacteriophage therapy was envisaged earlylast century; however, due to discovery of the antimicrobials, it fell out of research interest. Currently, bacteriophages are resurfacing as an alternative to antimicrobials in order to overcome the increasing incidence of antimicrobial resistance. Here, we report isolation of bacteriophages against Escherichia coli, Shigella spp., Aeromonas hydrophila and Citrobacter sedlakii isolates of equine origin. Phages were isolated from equine farm soil and sewage samples. For enrichment, sample aliquot was incubated overnight with host bacteria at 37 C with vigorous shaking. The crude lysate obtained was centrifuged and filtered and the presence of any phage in the suspension was detected by agar overlay technique. Appropriate dilutions of enriched samples were plated to obtain individual plaques and the most dominant plaque was transferred into SM buffer, serially diluted and plated for plaque re-isolation three times to ensure purity, followed by large scale preparation of phage stocks. Any host nucleic acids was degraded using pancreatic DNaseI and RNase and bacteriophage particles were precipitated using PEG8000. Phage titre was determined by plaque assay and phage concentrates were accessioned in the Veterinary Type Culture Collection (VTCC) repository. The phage concentrates were visualized by transmission electron microscopy (TEM). The temperature stability of bacteriophages was checked after incubating phage concentrates over the range of 4 C-8 0C temperature for one hour. A clear single plaque was obtained on nutrient agar against Shigella spp. and after purification and concentration, its analysis by electron microscopy revealed presence of multiple phages belonging to families Myoviridae, Siphoviridae and Podoviridae. However in case of Citrobacter sedlakii, a Siphoviridae phage (VTCCBPA61) with dimensions: 60 nm x 650 nm was observed. Against a pathogenic A. hydrophila isolate of equine origin (expressing aerolysin gene), a Myoviridae phage (VTCCBPA6) was isolated with dimensions: 62 nm x 138 nm. Against E. coli of equine origin, a Myoviridae phage (VTCCBPA9) of dimensions: 86 nm x 100 nm was obtained. Bacteriophage VTCCPBA61 against C. sedlakii was found to completely lose its biological activity at 65 C in vitro however the group of phages against Shigella spp. were found to be stable upto temperature as high as 80C. Thus we demonstrated the basic biological characteristics of phages, and some novel ones (such as against A. hyrophila and C. sedlakii) bacterial isolates of equine originwhich have never been reported till now. These lytic phages could find potential in phage therapy, as biocides, in biosensors and in phage ligand technology and are being explored by us further to depict their therapeutic value in small animal model. As more studies are reporting safety, tolerance and efficacy of phage therapy in humans and animals, their use in phage therapy has a promising future as an emerging alternative to chemical agents.

Isolation and characterization of bacteriophages against equine pathogens-novel phages revealed as phage therapy candidates

A role for bacteriophage therapy was envisaged early last century; however, due to discovery of the antimicrobials, it fell out of research interest. Currently, bacteriophages are resurfacing as an alternative to antimicrobials in order to overcome the increasing incidence of antimicrobial resistance. Here, we report isolation of bacteriophages against Escherichia coli, Shigella spp., Aeromonas hydrophila and Citrobacter sedlakii isolates of equine origin. Phages were isolated from equine farm soil and sewage samples. For enrichment, sample aliquot was incubated overnight with host bacteria at 37 C with vigorous shaking. The crude lysate obtained was centrifuged and filtered and the presence of any phage in the suspension was detected by agar overlay technique. Appropriate dilutions of enriched samples were plated to obtain individual plaques and themost dominant plaquewas transferred into SM buffer, serially diluted and plated for plaque re-isolation three times to ensure purity, followed by large scale preparation of phage stocks. Any host nucleic acids was degraded using pancreatic DNaseI and RNase and bacteriophage particles were precipitated using PEG8000. Phage titre was determined by plaque assay and phage concentrates were accessioned in the Veterinary Type Culture Collection (VTCC) repository. The phage concentrates were visualized by transmission electron microscopy (TEM). The temperature stability of bacteriophages was checked after incubating phage concentrates over the range of 4 C 80 C temperature for one hour. A clear single plaque was obtained on nutrient agar against Shigella spp. and after purification and concentration, its analysis by electron microscopy revealed presence of multiple phages belonging to families Myoviridae, Siphoviridae and Podoviridae. However in case of Citrobacter sedlakii, a Siphoviridae phage (VTCCBPA61) with dimensions: 60 nm x 650 nm was observed. Against a pathogenic A. hydrophila isolate of equine origin (expressing aerolysin gene), a Myoviridae phage (VTCCBPA6) was isolated with dimensions: 62 nm x 138 nm. Against E. coli of equine origin, a Myoviridae phage (VTCCBPA9) of dimensions: 86 nm x 100 nm was obtained. Bacteriophage VTCCPBA61 against C. sedlakiiwas found to completely lose its biological activity at 65 C in vitro however the group of phages against Shigella spp. were found to be stable upto temperature as high as 80 C. Thus we demonstrated the basic biological characteristics of phages, and some novel ones (such as against A. hyrophila and C. sedlakii) bacterial isolates of equine originwhich have never been reported till now. These lytic phages could find potential in phage therapy, as biocides, in biosensors and in phage ligand technology and are being explored by us further to depict their therapeutic value in small animal model. As more studies are reporting safety, tolerance and efficacy of phage therapy in humans and animals, their use in phage therapy has a promising future as an emerging alternative to chemical agents. Posters 006 Microbiological investigation into the cause of Equine Grass Sickness

Preparation of Respiratory Syncytial Virus with High or Low Content of Defective Viral Particles and Their Purification from Viral Stocks.

Respiratory syncytial virus (RSV) belongs to the paramyxovirus family that includes many clinically relevant viruses, such as the human metapneumovirus and measles. RSV infection can cause severe disease in infants, the elderly, and some immunocompromised adults. During RSV replication, a series of truncated forms of the viral genome are generated. These truncated viral genomes are known as defective viral genomes (DVGs) and are generated by many viruses (Lazzarini, et al., 1981; Rao and Huang, 1982; Prince et al., 1996; Sun et al., 2015; Tapia et al., 2013). DVGs can restrict the replication of the full-length virus and are the primary natural triggers of the innate immune response to RSV (Sun et al., 2015; Tapia et al., 2013). Here we discuss in detail how to prepare RSV stocks with a high or low content of DVGs, and how to purify defective viral particles containing DVGs from a RSV stock enriched in defective viral particles. These procedures are useful for the preparation of viral stocks and defective viral particles necessary for laboratory research. In brief, the different RSV stocks are produced in HEp2 cells, which are commonly used to amplify this virus in the laboratory. To generate an RSV stock with a high content of DVGs, HEp2 cells are sequentially infected with a high multiplicity of infection (MOI) multiple times followed by purification of the viral particles containing DVGs using gradient centrifugation. The procedure describe here has four parts: 1. Amplification of seed RSV stock with a low DVG content (RSV-LD), 2. Generation of a stock with a high DVG content (RSV-HD), 3. Purification of DVGs by gradient centrifugation, 4. Characterization of purified DVGs.

Preparation of Cell Cultures and Vaccinia Virus Stocks.

The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included.