Authors would like to thank past and present members in the laboratories of Drs. Peter Palese and Adolfo Garc&iacute;a-Sastre for the development of NDV reverse genetics techniques and for technical assistance. Research in Newcastle disease virus in AG-S laboratory is partially funded by NIAD grant R01AI088770 and by the Department of Homeland Security Science &amp; Technology Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD, award number 2010-ST-061-AG001). Research in LM-S laboratory is funded by the NIH grants RO1 AI077719, R21NS075611-01, R03AI099681-01A1, the NIAID Centers of Excellence for Influenza Research and Surveillance (HHSN266200700008C), and The University of Rochester Center for Biodefense Immune Modeling (HHSN272201000055C).

1. Preparation of Mammalian Cells (Figure 4A, Day 1)
Split HEp-2 or A549 cells the day before transfection in 6-well plates. Density of the cells should reach 80-90% confluence the following day. Usually, a confluent 100 mm dish can be split into 8 wells (around 1 x 106 cells per well). For each virus to be rescued, 2-4 different wells should be included, as well as 2 extra wells for the controls pCAGGs-GFP and pCITE-GFP18, aimed to monitor transfection and MVA-T7 infection efficiencies, respectively.
2. Infection of Mammalian Cells with the Recombinant Modified Vaccinia Ankara Virus Expressing the Bacteriophage T7 RNA Polymerase (MVA-T7) (Figure 4A, Day 2)
<ol>
	<li>Warm up PBS 1x/BA/PS and media at 37 &#176;C.</li>
	<li>Wash cells, twice, with 1 ml of PBS 1x/BA/PS.</li>
	<li>Infect the cultured mammalian cells with the MVA-T7 virus at a multiplicity of infection (moi) of 1 pfu/cell in a final volume of 200 &#956;l in PBS/BA/PS for 1 hr at room temperature.</li>
	<li>During viral infection incubation, prepare the transfection mix as described in 3.</li>
</ol>
3. Transfection of Mammalian Cells (Figure 4A, Day 2)
<ol>
	<li>Preparation of Lipofectamine: Mix 1-2 &#956;g of LPF2000 with 250 &#956;l of OptiMEM per rescue attempt and incubate for 5 min at room temperature.</li>
	<li>Preparation of DNA: Make a plasmid cocktail for each rescue in 50 &#956;l of OptiMEM. Add 0.4 &#956;g of pTM1-NP, 0.2 &#956;g of pTM1-P, and 0.2 &#956;g of pTM1-L per tube. Add 1 &#956;g of the full-length cDNA clone of NDV to be rescued to each tube. Also prepare two control transfections, one with 2 &#956;g of pCAGGs-GFP (to check transfection efficiency) and the other with 2 &#956;g of pCITE-GFP (to check T7 polymerase driven expression by the MVA-T7).</li>
	<li>After the LPF2000-OptiMEM solution has been preincubated for 5 min (step 3.1), add 250 &#956;l of the solution to the DNA tubes (step 3.2) and incubate for 20-30 min at room temperature.</li>
	<li>After incubation with MVA-T7, remove the virus inoculum by aspiration and replace with the DNA-LPF2000 transfection mix (step 3.3).</li>
	<li>Add 1 ml of DMEM 10% FBS/PS to each dish.</li>
	<li>Incubate the infected/transfected cells for 6-8 hr (or overnight) at 37 &#176;C, 5% CO2 and then replace the transfection media with 1.5 ml of fresh DMEM 10% FBS/PS.</li>
	<li>At 24 hr post-transfection, control wells can be observed under a fluorescence microscope to assess transfection efficiency (pCAGGs-GFP) and T7 polymerase activity (pCITE-GFP) (Figure 5A). Proceed with the co-culture of the infected/transfected cells with avian fibroblasts as described below.</li>
</ol>
4. Co-culture of Mammalian Cells with Avian Cells (Figure 4A, Day 3)
Usually, a confluent 100 mm tissue culture dish of chicken (CEFs) or duck (DEFs) embryo fibroblasts is used per two transfected wells. Be sure to prepare, in advance, enough 100 mm tissue culture dishes of avian cells per all rescue attempts. For efficient rescue of the virus, DMEM 10% FBS/PS media is supplemented with 5% of allantoic fluid and 30 mM MgCl2. At this point, 24 hr p.i., mammalian cells may start showing cytopathic effect (CPE) due to MVA-T7 infection, asevident in Figure 5A.
<ol>
	<li>Warm up PBS 1x and media to 37 &#176;C.</li>
	<li>Wash mammalian cells, 2x, with 1 ml of PBS 1x.</li>
	<li>Trypsinize mammalian cells with 0.2 ml of EDTA-trypsin until they detached. Resuspend the cells in 1 ml of DMEM 10% FBS/PS, 5% allantoic fluid, 30 mM MgCl2 and transfer to a 100 mm tissue culture dish. Add 3 ml of same media.</li>
	<li>Wash avian cells, twice, with 4 ml of PBS 1x.</li>
	<li>Trypsinize avian cells with 1 ml of EDTA-trypsin and incubate for 1-2 min at 37 &#176;C until cells detach. Resuspend the cells adding 8 ml of DMEM 10% FBS/PS, 5% allantoic fluid, 30 mM MgCl2 and add 4 ml of the trypsinized cells to the mammalian cells in the co-cultured 100 mm tissue culture dish for a total volume of 8 ml (4 ml mammalian, 4 ml avian cells).</li>
	<li>Gently shake by hand the co-cultured cells in the 100 mm dish in order to have a uniform distribution and then place them in the incubator at 37 &#176;C for 3-4 days. The criteria for infecting eggs 3 or 4 days after co-culture of mammalian and avian cells depends on how much cytopathic effect (cell rounding, death, detachment from the surface, etc.) is observed in the MVA-T7 viral infection.</li>
</ol>
5. Infection of Chicken Embryonated Eggs (Figure 4A, Days 6-7)
<ol>
	<li>After 3-4 days of co-culture of mammalian and avian cells, remove 1 ml of the tissue culture supernatant and add to an Eppendorf tube.</li>
	<li>Centrifuge the tissue culture supernatant for 1 min at 12,000 rpm in a bench top centrifuge to remove cellular debris.</li>
	<li>Candle the eggs using a light candling box to see the interface between the air sac and the allantoic cavity. Make a mark on the interface border avoiding blood vessel localization. Spray 70% ethanol over the eggs to establish sterile conditions. Gently make a hole in the eggshell on the marked spot and inoculate 500 &#956;l of the supernatant using an insulin (1 ml) syringe, aiming the needle vertically and directly into the allantoic cavity. (Figure 4B).</li>
	<li>Seal the nick in the eggshell with melted wax or paraffin.</li>
	<li>Incubate the eggs for 2-3 days at 37 &#176;C.</li>
</ol>
6. Harvest of Allantoic Fluid from Infected Chicken Embryonated Eggs (Figure 4A, Days 8-10)
<ol>
	<li>Incubate the infected eggs for 2-4 hr or overnight at 4 &#176;C in order to kill the chicken embryo and coagulate the blood.</li>
	<li>Spray the eggs with 70% ethanol (to maintain sterile conditions).</li>
	<li>Carefully tap the apical section of the egg, over the air cavity, with a spoon. Once the eggshell is cracked, remove the fragments with forceps and fully expose the allantoic membrane.</li>
	<li>Expose the allantoic cavity by excising the allantoic membrane with forceps and scissors, avoiding damage to the blood vessels and the yolk.</li>
	<li>Carefully push down the embryo with a spatula and collect the upflowing allantoic fluid (8-12 ml per egg) with a 10 ml pipette. Avoid damaging the yolk membrane. Transfer the allantoic fluid to a 15 ml centrifuge tube on ice (use one tube per egg).</li>
	<li>Clarify the allantoic fluid by centrifuging for 5 min at 1,500 rpm. Transfer the supernatant to a fresh tube without disturbing the pellet.</li>
	<li>Store samples at 4 &#176;C for up to 1 week, until checking them for presence of NDV by hemagglutination assay.</li>
</ol>
7. Hemagglutination (HA) Assay
The presence of virus in the allantoic fluid from infected eggs can be determined macroscopically by their ability to hemagglutinate turkey red blood cells (RBC). In the case of NDV, approximately 106 plaque forming units (pfu) per ml are required to give a positive signal in the HA assay. HA assays are carried out in V-bottom 96-well plates. Negative (PBS 1x, uninfected allantoic fluid) as well as positive (allantoic fluid from any NDV virus) control samples should always be included in any HA assay to validate it. To perform an HA assay:
<ol>
	<li>Pipette 50 &#956;l of PBS 1x per well in a V-bottom 96-well plate.</li>
	<li>Pipette 50 &#956;l of the allantoic fluid samples into the wells on the first column of the plate. Perform 2-fold serial dilutions through the rest of the plate and discard the last, extra 50 &#956;l from wells in the last column.</li>
	<li>Dispense 50 &#956;l of 0.5% turkey RBC in PBS 1x per well. Gently shake the plate by tapping.</li>
	<li>Incubate the plate at 4 &#176;C (or ice) for 30-45 min or until a clear pellet is formed in the negative control wells.</li>
</ol>

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Adolfo Garc&iacute;a-Sastre is an inventor of patents on recombinant Newcastle disease viruses that are owned by the Icahn School of Medicine at Mount Sinai.

Several factors are to be considered to achieve good results while rescuing NDV. First, the full-length cDNA construct to be used needs to be designed to allow the functional incorporation of the new transgenes/modifications into the NDV genome. This means, as stated above, that (i) appropriate gene end (GE), intergenic (IG) and gene start (GS) sequences are to be added if required; (ii) there are no putative GE or GS sequences into the foreign gene, and (iii) the full recombinant genome follows the &#34;rule of six&#34;...

Newcastle disease virus (NDV), an avian paramyxovirus belonging to the Avulavirus genus1, is an economically relevant and thus widely researched and surveilled zoonotic agent, which can severely affect poultry farming all around the world. Although not a human pathogen, NDV has also been thoroughly studied beyond the veterinarian field both as a model paramyxovirus and due to its highly interesting, natural oncolytic properties6. Research on NDV greatly benefited from the development of reverse genetics techniques for single-stranded, non-segmented negative-sense RNA viruses, first described for rabies virus by Conzelmann and coleagues2. A variety of genetically modified NDVs, carrying foreign genes or modifications of their wild type genome have been widely studied ever since. Work with these recombinant viruses has been critical to characterize different virulence factors not only of NDV but also of other relevant human pathogens such as influenza A virus7 - or the emergent Nipah virus8. Furthermore, a number of different studies have explored the use of these techniques to improve the innate antitumoral activity of NDV6,9,10, mostly by enhancing the immunostimulatory properties of the virus. Other relevant area of research on recombinant NDVs has been the generation of vaccine candidates against other viral diseases such as influenza5,11,12, HIV13, measles14, SARS15, or that caused by the respiratory sincytial virus (RSV)16. Amongst the various noteworthy advantages provided by NDV are the lack of preexisting immunity in human populations, the stability of the foreign genetic inserts, a lack of recombinatory activity and overall a high safety profile combined with the aforementioned natural immunostimulatory properties17. It&#39;s also noteworthy the potential use of recombinant bivalent vaccines in poultry, protective against both NDV and highly pathogenic avian influenza viruses11,12. This may be an excellent way to decrease the chances of the latter spreading from wild to domesticated animals, thus also helping to prevent a possible inter-specific jump of the dreaded avian influenza to humans. Finally, reporter-expressing NDV have been used for the evaluation of innate immune responses as well as the identification of interferon antagonist encoded by multiple viruses18-27.
The rescue process of a recombinant, non-segmented, negative-stranded RNA virus basically consists on artificially forcing a viral replication cycle in a producing cell by transfecting cDNA encoding the minimal infective molecular machinery, known as ribonucleoprotein or RNP (Figure 2). The RNPs consist of the viral polymerase (P and L proteins), the nucleoprotein (NP) and the full-length antigenomic RNA of the virus. This RNA+ antigenome is the template required for the generation of the complementary RNA- genomes, which, also associated with the rest of the proteins of the viral RNP, recapitulates the same infectious complex that a natural virus would release on the cytoplasm of the cell upon infection (Figure 2A). From this step onwards, the viral cycle can proceed naturally and recombinant virions, encapsidating the modified genomes, will be generated (Figure 2B). Remarkably, transfection of the genomic cDNA instead of the antigenomic cDNA greatly impairs or completely abolishes rescue efficiency2,28-30. Even when antigenomic cDNA is transfected, the efficiency of the encapsidation of the recombinant RNA into RNPs in transfected cells is probably very low. Because of this, rescue protocols for NDV often include different steps for the amplification of the few viral particles released from the originally transfected cells by coculturing them with permissive cells and/or by the infection of embryonated eggs.
Prior to the rescue, the cDNA can be manipulated by standard cloning procedures in order to generate the desired modifications. While specific mutations of the different gene products and regulatory sequences of the virus can be straightforwardly achieved this way, many of the published work involving recombinant NDV has required the addition of a new transcriptional unit into the NDV genome. Like other members of the paramyxovirus family, the NDV genome encodes eight different proteins into six transcriptional units, which are differentially expressed depending on their location respect to the 3&#39; end in a decreasing gradient critical for the viral life cycle1. Because of this, the location of the new transcriptional unit within the genome must be carefully chosen to achieve a balance between expression of the transgene and impairment of viral replication. Insertion between P and M genes has been used the most, though other sites have also been tested13,31.
Whatever the insert, the cloning into NDV cDNA needs to follow some rules to generate a rescuable construct: (i) any new gene to be included into the NDV genome has to be under control of the appropriate signals for the viral RNA-dependent-RNA polymerase. These sequences must be added upstream of the new open reading frame (ORF) so the polymerase can recognize the end of the previous gene (GE) and the beginning of the new transgene (GS), spaced by a single nucleotide intergenic sequence (IG). Addition of a valid Kozak (K) sequence to improve eukaryotic ribosomal translation is also recommended for better foreign protein expression32; (ii) efficient replication of NDV, as for most members of the Paramyxoviridae family, is dependent on the genome length being multiple of six33; therefore, any insertion into the NDV has to follow this &#34;rule of six&#34;. If necessary, required additional nucleotides can be added downstream the new ORF; and (iii) the sequence of the transgene should be checked to find possible GE and GS like sequences which could impair rescue efficiency, transgene expression and/or virus viability. If present, these sequences must be removed by silent mutagenesis. The generation of recombinant full length cDNA following aforementioned rules is the first step in order to efficiently produce a genetically modified NDV as detailed here.
In the system all DNA constructs are under control of the T7 RNA polymerase promoter (Figure 3). This cytoplasmic polymerase is provided in trans by coinfection with a recombinant modified vaccinia Ankara virus (MVA-T7)34. Figure 3A show the pNDV-B1 plasmid, which encodes the full-length antigenomic cDNA5. Figure 3B shows pTM1 plasmids encoding NP, P and L ORFs. Plasmids pCITE-GFP, which encodes, under the T7 promoter, the Green Fluorescent Protein (GFP), and pCAGGs GFP18, which encodes the same ORF under the chicken beta actin promoter 35, are used as controls. In this protocol we show the procedure to rescue recombinant NDV from the cDNA of the lentogenic NDV strain Hitchner B15 (Figure 4).

<table border="1">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>CORNING Cellgro</td>
			<td>10-013-CV</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>GIBCO</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (LPF2000)</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>35% Bovine Albumin (BA)</td>
			<td>Sigma</td>
			<td>232-936-2</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>CORNING Cellgro</td>
			<td>25-052-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (PS) 100x</td>
			<td>CORNING Cellgro</td>
			<td>30-002-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			Cell lines 
			A549 cells (catalogue number CRL-185), HEp-2 cells (catalogue number CRL-185), chicken embryo fibroblasts (catalogue number CRL-12203) and duck embryo fibroblasts (catalogue number CCL-141) are available from the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA. 20110-2209 USA). All cell lines are maintained in a 37 &#176;C incubator with 5% CO2 in DMEM 10% FBS, 1% PS. 
			Embryonated chicken eggs 
			Embryonated chicken eggs can be obtained from Charles River Laboratories, Specific Pathogen Fee Avian Supply (SPAFAS) Avian Products and Services (Franklin Commons, 106 Route 32, North Franklin, CT 06254, USA) and are maintained at 37 &#176;C. Viability of the embryos is assessed with an egg candler. Eggs are infected when they reach 8-10 days old. Both infection and harvest of the allantoic fluid takes place under sterile conditions. All eggs are autoclaved and discarded following standard laboratory biosafety protocols. 
			Turkey red blood cells (RBC) 
			Turkey RBC can be purchased from Truslow Farms (201 Valley Road, Chestertown, Md 21620, USA)and stored at 4 &#176;C. To prepare RBC for HA assay, wash 5 ml of the commercial stock with 45 ml of PBS 1x in a 50 ml conical tube. Centrifuge for 5 min at 1,000 rpm and carefully discard the supernatant. Dilute pelleted RBC 1:1,000 in PBS 1x for a final 0.5-1.0% concentration. Washed RBC can be stored at 4 &#176;C for several days. 
			Plasmids 
			Plasmid preparations are obtained with any commercially available maxiprep kit following manufacturer&#39;s instructions, diluted in ddH20 to a final concentration of 1 &#956;g/&#956;l and stored at -20 &#176;C. DNA concentration and purity are assessed by spectrophotometry at 260 and 280 nm. Preparations with a 260/280 ratio higher than 1.8 are considered of acceptable quality for the rescue. Plasmid DNA quality is also routinely double-checked by agarose gel chromatography. 
			Viruses 
			The described protocol for rescue of the lentogenic NDV strain Hitchner B1 can be performed under biosafety level (BSL) 2 conditions. The Modified Vaccinia Ankara expressing the T7 RNA polymerase (MVA-T7) was described34 and obtained from Dr. Bernard Moss. This virus is growth in confluent monolayers of chicken embryo fibroblasts and titrated in mammalian (A549 or HEp-2) cells. NDV stocks are grown in embryonated chicken eggs and titrated by IFA using polyclonal serum raised against purified virions. All contaminated material should be safely sterilized and disposed according to standard biosafety procedures. 
			Tissue culture media and solutions: 
			DMEM 10% FBS 1% PS: Dulbecco&#39;s modified Eagle&#39;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Penicillin/Streptomycin (P/S): 445 ml of DMEM, 50 ml of heat-inactivated FBS, 5 ml of 100x commercial P/S solution. Store at 4 &#176;C. 
			10x Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 L. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature. 
			1x PBS: Dilute 10x PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature. 
			100x Ca/Mg : 1.327 g CaCl2.2H2O, 2.133 g MgCl2&#8226;6H2O and add ddH2O up to 100 ml. Autoclave and store at room temperature. 
			1x PBS/BA/PS: 50 ml of 10x Phosphate buffered saline (PBS) in 437 ml ddH2O. Autoclave and when cooled down to room temperature, add 5 ml 100x Penicillin/Streptomycin 3 ml 35% Bovine and 5 ml of 100x Ca/Mg. Store at 4&#176;C.
			</td>
		</tr>
	</tbody>
</table>

rescue of recombinant newcastle disease virus from cdna

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Rescue of NDV is a well-established procedure, routinely performed in the laboratories that have access to the complete cDNA of the virus. However, the intrinsic stochastic nature of the method makes it difficult to achieve 100% rescue efficiency. Monitoring the early steps of the process, specially the transfection efficiency and the infection with MVA-T7, helps identifying possible problems. Figure 5A shows standard transfection and transfection/infection efficiencies that are enough for a successful N...

Watch this Scientific Journal Video about Rescue of Recombinant Newcastle Disease Virus from cDNA at JoVE.com

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

rescue-of-recombinant-newcastle-disease-virus-from-cdna

Research

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Immunology and Infection

19.2K Views.  Icahn School of Medicine at Mount Sinai. Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

Video: Rescue of Recombinant Newcastle Disease Virus from cDNA

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...