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Construction of the pXJ41-FL13 infectious clone. (a) Illustration of the plasmid pXJ41-AP-FragmA. The pXJ41 vector is modified to include two restriction sites, AscI and PacI. Fragment A containing the PRRSV genomic sequence from 1 to 2546 is amplified by PCR and two restriction sequences AscI and PacI are added at the 5′-and 3′-termini, respectively. AscI-PacI double-digested fragment A and pXJ41-AP are ligated together to generate plasmid pXJ41-AP-FragmA. The unique SpeI site in the PRRSV genome at position 2532 is included in this plasmid. (b) Digest the PRRSV infectious clone pFL12 with SpeI and PacI to obtain fragment B. Purified fragment B then is ligated with SpeI-PacI-digested pXJ41-AP-FragmA
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Reverse genetics is the process of generating an RNA virus from a cDNA copy. Reverse genetics systems have truly transformed our ability to manipulate and study negative-strand RNA viruses. Plasmid-based reverse genetics approaches for influenza viruses provide a better understanding of virulence, transmission, mechanisms of antiviral resistance, a...
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... all ten reovirus gene segments. Viable virus was recoverable within 48 h post-transfection [15]. Longer incubation times permitted amplification of rescued virus and yielded higher recovery titers. To increase rescue efficiency, a second-generation system employed baby hamster kidney cells that stably express T7 RNA polymerase (BHK-T7 cells) (Fig. 2) [19]. Use of BHK-T7 cells enhances the efficiency of reovirus recovery by ensuring that T7 RNA polymerase is expressed in every cell that receives plasmids. The second-generation system also uses plasmids that encode multiple reovirus gene segments to further enhance rescue efficiency by reducing the number of plasmids that must be ...
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... reovirus 4-plasmid system 10-plasmid system L1 L2 L3 M1 M2 M3 S1 S2 S3 S4 Fig. 2 Reverse genetics for recombinant reovirus rescue. Using the ten-or fourplasmid system, BHK-T7 cells are transfected with plasmids containing reovirus cDNA. The cells are incubated at 37 °C for 2-4 days and then lysed by multiple freeze/thaw cycles to harvest recombinant reovirus 2. Spinner-adapted mouse L929 ...
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... pXJ41-FL13 cDNA clone consists of two large overlapping fragments (fragment A and fragment B) from the viral genome in addition to the backbone sequence of vector, pXJ41-AP, as described below (Fig. ...
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... A, comprising from the nucleotide positions 1-2550 of the PRRSV genome, was amplified by PCR using pFL12 as the template. Restriction enzyme sites AscI and PacI were designed at the 5′-and 3′-termini of the fragment, respectively (Fig. 2a). Also, the SpeI site unique for full-length genomic sequence was included in the fragment ...
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... vector pXJ41 was modified to include the restriction enzyme sites AscI and PacI (Fig. 2). The TATA box sequence was added to the upstream of AscI. This modified vector was designated pXJ41-AP. The vector pXJ41-AP and fragment A were digested with restriction enzymes AscI and PacI, and subjected to ligation to generate ...
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... Place 150 μL of undiluted samples into wells of columns 1 and 7, and put 135 μL of diluent into remaining wells (Fig. 2). Alternatively, put 135 μL of diluent into all wells and then put 15 μL of undiluted virus samples into wells of columns 1 and 7, making −1 dilution. Mix the samples thoroughly and be consistent among ...
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... Assemble the whole genome following Fig. 2 2. Incubate at 37 °C water bath for 2 h. Analyze by agar gel electrophoresis with 1 μL ...
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... breadth of the Coronaviridae family, including pathogenic viruses from groups 1a and 1b of the alphacoronaviruses and groups 2a, 2b, and 2c of the betacoronaviruses (Fig. 1). The entire CoV fragments are joined by type IIS or IIG restriction sites (e.g., BglI, SapI, and BsaI) that support directional, seamless ligation into full-length genome (Fig. 2). For type IIS (e.g., SapI) restriction enzymes, the recognition sequences are separated from its cleavage site by one or more variable nucleotides, leaving three to four nucleotide unique overhangs (Fig. 2). Thus, these enzymes leave 64-256 unique ends, providing directionality during multi-segment assembly. Moreover, the recognition ...
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... joined by type IIS or IIG restriction sites (e.g., BglI, SapI, and BsaI) that support directional, seamless ligation into full-length genome (Fig. 2). For type IIS (e.g., SapI) restriction enzymes, the recognition sequences are separated from its cleavage site by one or more variable nucleotides, leaving three to four nucleotide unique overhangs (Fig. 2). Thus, these enzymes leave 64-256 unique ends, providing directionality during multi-segment assembly. Moreover, the recognition site is not palindromic, allowing for seamless assembly of component cDNA clones into full-length genes and genomes. By orienting the Adam S. Cockrell et al. Organization of coronavirus genomes and infections ...
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... lost, seamlessly joining the cDNAs, while preserving ORF integrity and sequence authenticity. A second approach uses type IIG (e.g., BglI) restriction endonucleases, which has a palindrome restriction site, bisected by a variable domain of five nucleotides and also leaves 64 different overhangs for directional assembly of large genome molecules (Fig. 2). In this instance, the restriction site is retained in the assembled product. As coronavirus genomes are unstable in bacteria, junctions are oriented within toxic sequence domains, thereby bisecting region toxicity and increasing component clone stability. Plasmids are digested with type IIS or IIG restriction enzymes to isolate each ...
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... the restriction site is retained in the assembled product. As coronavirus genomes are unstable in bacteria, junctions are oriented within toxic sequence domains, thereby bisecting region toxicity and increasing component clone stability. Plasmids are digested with type IIS or IIG restriction enzymes to isolate each fragment of the CoV genome (Fig. 2). Fragments are then resolved on an agarose gel, purified, and ligated (Fig. 2). Following ligation, the coronavirus genome-length mRNA is in vitro transcribed from a T7 promoter added at the 5′ end of the 5′ UTR. In some instances, strong T7 stop sites are mutated to promote full-length transcript synthesis in in vitro transcription ...
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... genomes are unstable in bacteria, junctions are oriented within toxic sequence domains, thereby bisecting region toxicity and increasing component clone stability. Plasmids are digested with type IIS or IIG restriction enzymes to isolate each fragment of the CoV genome (Fig. 2). Fragments are then resolved on an agarose gel, purified, and ligated (Fig. 2). Following ligation, the coronavirus genome-length mRNA is in vitro transcribed from a T7 promoter added at the 5′ end of the 5′ UTR. In some instances, strong T7 stop sites are mutated to promote full-length transcript synthesis in in vitro transcription reactions [20]. Resulting genomelength mRNA is electroporated into a permissive ...
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... ligation, the coronavirus genome-length mRNA is in vitro transcribed from a T7 promoter added at the 5′ end of the 5′ UTR. In some instances, strong T7 stop sites are mutated to promote full-length transcript synthesis in in vitro transcription reactions [20]. Resulting genomelength mRNA is electroporated into a permissive mammalian cell line (Fig. 2). Cloning success and viral fitness can be measured by plaque assay and growth curves (Fig. 3). During viral replication Adam S. Cockrell et ...
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... a mouse permissive to MERS-CoV infection (transgenic mouse lung), but not in a nonpermissive, wild-type mouse lung. Both of these methods can be used to confirm productive CoV replication following RGS clone generation Partitioning the SARS-CoV genome allows for efficient handling of genomic fragments under standard BSL1/2 containment conditions (Fig. 2). For reconstruction of full-length genomes encoding CoVs restricted to BSL3 containment (SARS-CoV, MERS-CoV, preemergent bat CoVs), fragment ligation and all subsequent steps are executed under BSL3 conditions, including recovery of recombinant viruses (Fig. ...
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... efficient handling of genomic fragments under standard BSL1/2 containment conditions (Fig. 2). For reconstruction of full-length genomes encoding CoVs restricted to BSL3 containment (SARS-CoV, MERS-CoV, preemergent bat CoVs), fragment ligation and all subsequent steps are executed under BSL3 conditions, including recovery of recombinant viruses (Fig. ...
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... advantage of a segmented molecular clone design is that mutagenesis can occur in parallel on multiple fragments, and that the individual fragments can be "reassorted" to make larger panels of derivative mutants encoding mutation subsets (Fig. 2). For example, an early application included the introduction of over 27 mutations into the SARS-CoV genome at 9 different genome transcription regulatory sequences, thereby demonstrating for the first time that the transcription regulatory circuit of a virus could be rewired [33]. The applicability of the RGS was ratified in ensuing ...
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... Select a replicate from the plasmid library with the correct insert size and sequence for each MERS-CoV fragment (Fig. ...
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... the second step, this complete plasmid sequence is integrated into the IBV sequence within the vaccinia virus genome (Fig. 2). This occurs as a result of a single crossover event involving homologous recombination between the IBV cDNA in the plasmid and the IBV cDNA sequence in the vaccinia virus genome. The resulting recombinant vaccinia viruses (rVV) are highly unstable due to the presence of duplicate sequences and are only maintained by the selective ...
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... day 2, transfected cells that have received the transfected constructs should express mKate2 protein (emission wavelength of 633 nm) and be visible under a far-red channel such as Texas Red (Fig. 2) [13]. However, it will remain difficult to identify virus-infected cells by bright field at this time (see Note 8). The cell density of the BSR cells in the six-well plate should now appear over-confluent and requires passage into a dish or ...
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... Depending on the RSV mutant's growth characteristics, the virus infection will progress from small syncytia to larger syncytia and to rounded syncytial masses (see Note 9, Fig. ...
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... At the time of harvesting, CPE should be evident throughout the flask with at least half of the monolayer involved in syncytial masses (Fig. 2d). Approximately five cryovial tubes should be labeled for harvesting of the virus ...
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... Between 3 and 6 days post-transfection, active syncytia should begin to grow and the virus infection should become apparent. The monolayer should go through phases beginning with small syncytia, then larger syncytia, then syncytia that begin to round up, and lastly detachment from the monolayer (Fig. ...
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... delta virus (HDV) ribozyme, which is positioned at the 3′ end of the genome. The exact 5′ end of the transcript is determined by positioning the site of the T7 promoter as close to the terminus of the initial transcript as possible resulting in transcription by T7 RNA polymerase immediately adjacent to the T7 polymerase promoter sequence (Fig. 2). To increase transcription from the T7 Fig. 1 Schematic representation of the HMPV replication cycle. Upon attachment of the virion to the plasma membrane and subsequent fusion of the viral and plasma membranes, the virion is uncoated and the RNP (containing the negative-sense genome) is released in the cytoplasm. Here, the ...
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... Site-Directed Mutagenesis (Fig. 2) 10. Inoculate each colony into separate 15 mL sterile conical tubes or round-bottom culture tubes containing 5 mL of LB broth plus 100 μg/mL of ...
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... leader RNA coding sequences and subsequent Sanger sequencing of the resultant PCR product. RNA circulation involves removal of the 5′-phosphates at the viral end by a RNA 5′-Pyrophosphohydrolase (RppH) and ligation by T4 RNA Ligase. Both reactions can be done in two subsequent reactions described elsewhere [5] or as a single tube reaction (see Fig. 2b) described here. Both methods can be efficiently used for circularization and subsequent sequencing (Fig. ...
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... involves removal of the 5′-phosphates at the viral end by a RNA 5′-Pyrophosphohydrolase (RppH) and ligation by T4 RNA Ligase. Both reactions can be done in two subsequent reactions described elsewhere [5] or as a single tube reaction (see Fig. 2b) described here. Both methods can be efficiently used for circularization and subsequent sequencing (Fig. ...
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... amplification of complete virus genome cDNAs, the 12 kb RABV RNA is reverse transcribed by a terminal positive sense primer (Table 1) designed according to the individual virus endsequences (see Fig. 2c). Together with a reverse negative sense primer that hybridizes to the opposite genome terminus, a PCR reaction is formed at conditions optimized for long-range ...
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... gene transcription is mediated by the viral promoters located within the untranslated regions (UTRs) at the 3′ termini of viral RNA (vRNA) and complementary RNA species (cRNA) [1] (Fig. 2a). NP and L proteins, located at the 3′-end of the S and L viral segments, respectively, are translated from mRNAs with vRNA expression plasmids under the control of the mouse polymerase I (mPol-I) promoter (gray arrow) and terminator (gray box) sequences direct the synthesis of LASV vRNA S (top) and L (middle) segments. For the ...
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... segments. For the minigenome (MG) assays, a mPol-I vcRNA plasmid S where reporter genes substitute the viral NP (GFP, green) and GP (Gluc, yellow) is used (bottom), mPol-I MG Luis Martínez-Sobrido et al. antigenomic sense polarity transcribed directly from the vRNAs and, therefore, are the first arenaviral proteins encoded upon infection [1] (Fig. 2b). The 3′-ends of the non-polyadenylated viral mRNAs have been mapped to a predicted a stem-loop structure within the noncoding intergenic region (IGR) found between the two viral genes in each vRNA segment [1] (Fig. 2b). GP and Z proteins, on the other hand, are located, respectively, at the 5′-end of the S and L viral segments and are ...
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... sense polarity transcribed directly from the vRNAs and, therefore, are the first arenaviral proteins encoded upon infection [1] (Fig. 2b). The 3′-ends of the non-polyadenylated viral mRNAs have been mapped to a predicted a stem-loop structure within the noncoding intergenic region (IGR) found between the two viral genes in each vRNA segment [1] (Fig. 2b). GP and Z proteins, on the other hand, are located, respectively, at the 5′-end of the S and L viral segments and are not translated directly from mRNA derived from the vRNAs but from antigenome complementary RNA species after replication of the vRNAs [1] (Fig. 2b). Complementary RNA segments also serve as templates for the synthesis ...
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... intergenic region (IGR) found between the two viral genes in each vRNA segment [1] (Fig. 2b). GP and Z proteins, on the other hand, are located, respectively, at the 5′-end of the S and L viral segments and are not translated directly from mRNA derived from the vRNAs but from antigenome complementary RNA species after replication of the vRNAs [1] (Fig. 2b). Complementary RNA segments also serve as templates for the synthesis of nascent vRNAs [1] (Fig. 2b). Newly synthesized vRNAs are encapsidated by the viral NP to form the vRNP complexes and are packaged into progeny infectious virions by interaction of the viral Z [16]. Arenavirus assembly involves the interaction of the new vRNP ...
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... Z proteins, on the other hand, are located, respectively, at the 5′-end of the S and L viral segments and are not translated directly from mRNA derived from the vRNAs but from antigenome complementary RNA species after replication of the vRNAs [1] (Fig. 2b). Complementary RNA segments also serve as templates for the synthesis of nascent vRNAs [1] (Fig. 2b). Newly synthesized vRNAs are encapsidated by the viral NP to form the vRNP complexes and are packaged into progeny infectious virions by interaction of the viral Z [16]. Arenavirus assembly involves the interaction of the new vRNP complexes with the GP1/GP2 complexes present in the membrane of infected cells, a process mediated by ...
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... have developed an RNA mPol-I/Pol-II reverse genetics system for the rescue of a molecular clone of the highly pathogenic Josiah strain of LASV lineage IV [37]. Plasmids for the generation of recombinant LASV (rLASV) (Fig. 2) can be grown in DH5α-competent bacteria (Invitrogen) using Luria broth (LB) media in the presence of 100 μg/mL of Ampicillin (Fisher Scientific) at 37 °C for 16-18 h, with the exception of the mPol-I L plasmid (see Notes 1 and 2) [37]. Plasmids can be purified using commercial maxiprep kits (e.g., Qiagen) following manufacturer's ...
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... plasmid contains the chicken polymerase II (Pol-II)-driven β-actin promoter and the rabbit β-globin polyadenylation (pA) signal sequence to produce LASV NP and L, which are the minimum components for LASV replication and transcription ( Fig. 2a) required to evaluate LASV genome replication and gene transcription using the minigenome (MG) approach (Fig. 3) and for the generation of recombinant rLASV (Fig. 4) [37]. A pCAGGS plasmid encoding ostracod Cypridina noctiluca luciferase (Cluc) secreted luciferase is used in the MG assay (Fig. 3) to normalize transfection efficiencies ...
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... mPol-I vRNA expression plasmids: LASV reverse genetics use the mouse RNA polymerase I (mPol-I) promoter to direct intracellular synthesis of S and L genome or antigenome, RNA species [37], that is species specificity [29]; and the mPol-I terminator to obtain authentic LASV vRNA 3′-ends [37] ( Fig. 2b) (see Notes 2 and 7). LASV RNA replication and gene transcription can be evaluated using a LASV vRNA-like MG plasmid (mPol-I MG) (Fig. 2b) [15, 16, 28-31, 34, 35] where LASV GP and NP open reading frames (ORFs) are replaced by reporter genes. In our case, we replaced GP for Gaussia luciferase (Gluc) and NP for the green fluorescent ...
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... I (mPol-I) promoter to direct intracellular synthesis of S and L genome or antigenome, RNA species [37], that is species specificity [29]; and the mPol-I terminator to obtain authentic LASV vRNA 3′-ends [37] ( Fig. 2b) (see Notes 2 and 7). LASV RNA replication and gene transcription can be evaluated using a LASV vRNA-like MG plasmid (mPol-I MG) (Fig. 2b) [15, 16, 28-31, 34, 35] where LASV GP and NP open reading frames (ORFs) are replaced by reporter genes. In our case, we replaced GP for Gaussia luciferase (Gluc) and NP for the green fluorescent protein (GFP) [15, 16, 28-31, 34, 35, 37] (Fig. 3) (see Notes 8 and 9). For the generation of rLASV (Fig. 4), two mPol-I plasmids expressing ...
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... are replaced by reporter genes. In our case, we replaced GP for Gaussia luciferase (Gluc) and NP for the green fluorescent protein (GFP) [15, 16, 28-31, 34, 35, 37] (Fig. 3) (see Notes 8 and 9). For the generation of rLASV (Fig. 4), two mPol-I plasmids expressing the S (mPol-I S) and L (mPol-I L) viral genomic or antigenomic RNAs are used [37] (Fig. 2b) (see Note ...
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... experimental procedures used for the development of LASV MG systems and rescue of infectious rLASV described here are based on the use of the mPol-I promoter to direct intracellular synthesis of the appropriate MG vRNA-like (Figs. 2b and 3), ...
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... S and L genome or antigenome RNA species (Figs. 2b and 4), respectively [37] (see Note 7). The activity of the mPol-I promoter exhibits species specificity and therefore is restricted to rodent cells [29]. We use baby hamster kidney (BHK-21) cells since they are easy to maintain, have high transfection efficiencies, and are able to produce high viral titers [29,30,33,34] Fig. 3 LASV ...
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... contains eight single-stranded, negative-sense, viral (v)RNA segments (PB2, PB1, PA, HA, NP, NA, M, and NS) encapsidated by the viral nucleoprotein (NP). Associated with the eight vRNP complexes are the viral PB2, PB1, and PA polymerase subunits that, together with the viral NP, are involved in viral genome replication and gene transcription (Fig. 2b). For the development of reverse genetics techniques, influenza B vRNAs are cloned into bidirectional plasmids. Transfection of the eight ambisense plasmids into permissible cells leads to rescue of recombinant influenza B virus. The eight influenza B virus genes (gray boxes) are represented in 3′ to 5′ negative sense. Alternative or ...
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... regions (NCR) at the 3′ and 5′ ends of each viral segment. The eight ambisense plasmids contain the influenza B viral cDNAs (gray boxes), the human polymerase I (hPol-I) promoter (black arrow), the mouse Pol-I terminator (black box), a polymerase II-dependent promoter (white arrow), and a polyadenylation sequence (white box). See main text and Fig. 2a for more information Influenza B Reverse Genetics dperez1@uga.edu 11. Laboratory equipment: Microcentrifuge with a rotor capable of reaching up to 12,000 × g, a water bath or heat block, an electrophoresis system, a power supply, a NanoDrop (or similar spectrophotometer), petri dishes, a microbiological incubator and microcentrifuge ...
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... pDP-2002 (Fig. 2a) is a derivative of pHW2000 [48]. This bidirectional plasmid contains two transcription units in opposite orientation. The first unit is the human polymerase I (hPol-I) promoter and a murine Pol-I transcription terminator (TI) for the expression of influenza B vRNAs. Expression from the hPol-I cassette generates vRNAs without additional ...
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... terminator (TI) for the expression of influenza B vRNAs. Expression from the hPol-I cassette generates vRNAs without additional nucleotides at the 3′ and 5′ end of the viral genome that are recognized by the influenza B viral NP and polymerase complex (PB2, PB1, and PA) for viral genome replication (cRNA) and gene transcription (mRNA) (Fig. 2b). The second unit is the polymerase II-driven cytomegalovirus promoter (pCMV) and the bovine growth hormone polyadenylation signal (aBGH) to express mRNAs (Fig. 2b). The use of this combined RNA Pol-I/II approach allows vRNA and mRNA syntheses from the same vector, eliminating the need for separate vRNA and mRNA plasmids and, therefore, ...
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... 5′ end of the viral genome that are recognized by the influenza B viral NP and polymerase complex (PB2, PB1, and PA) for viral genome replication (cRNA) and gene transcription (mRNA) (Fig. 2b). The second unit is the polymerase II-driven cytomegalovirus promoter (pCMV) and the bovine growth hormone polyadenylation signal (aBGH) to express mRNAs (Fig. 2b). The use of this combined RNA Pol-I/II approach allows vRNA and mRNA syntheses from the same vector, eliminating the need for separate vRNA and mRNA plasmids and, therefore, reducing the number of plasmids to eight for the efficient rescue of influenza B viruses [32,42] (see Note 4). Influenza B viral cDNAs are cloned into the pDP-2002 ...
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... from the same vector, eliminating the need for separate vRNA and mRNA plasmids and, therefore, reducing the number of plasmids to eight for the efficient rescue of influenza B viruses [32,42] (see Note 4). Influenza B viral cDNAs are cloned into the pDP-2002 plasmid using two BsmBI restriction sites located between both transcription cassettes (Fig. 2a). The bidirectional pDP-2002 plasmid contains a spacer sequence of 444 nucleotides between the two BsmBI restriction sites that can be used to monitor BsmBI digestion efficiency using agarose gel ...
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... generate influenza B virus entirely from cloned cDNA, the eight individual viral genomic segments should be cloned into a bidirectional rescue plasmid [31,40,41]. Here we described the cloning of influenza B/Brisbane/60/2008 into the ambisense pDP-2002 ( Fig. 2a) (see Note 4). In this plasmid, influenza B viral cDNAs are inserted between the human RNA polymerase I promoter (hPol-I) and the mouse terminator (TI) sequences (Fig. 2a) (see Note 19). This polymerase I transcription/terminator cassette is flanked by an RNA polymerase II-dependent (Pol-II) cytomegalovirus promoter (pCMV) and a ...
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... genomic segments should be cloned into a bidirectional rescue plasmid [31,40,41]. Here we described the cloning of influenza B/Brisbane/60/2008 into the ambisense pDP-2002 ( Fig. 2a) (see Note 4). In this plasmid, influenza B viral cDNAs are inserted between the human RNA polymerase I promoter (hPol-I) and the mouse terminator (TI) sequences (Fig. 2a) (see Note 19). This polymerase I transcription/terminator cassette is flanked by an RNA polymerase II-dependent (Pol-II) cytomegalovirus promoter (pCMV) and a polyadenylation site (aBGH) (Fig. 2a). The orientation of the two transcription units allows the synthesis of negative-sense vRNA from the hPol-I cassette, and positive-sense ...
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... 4). In this plasmid, influenza B viral cDNAs are inserted between the human RNA polymerase I promoter (hPol-I) and the mouse terminator (TI) sequences (Fig. 2a) (see Note 19). This polymerase I transcription/terminator cassette is flanked by an RNA polymerase II-dependent (Pol-II) cytomegalovirus promoter (pCMV) and a polyadenylation site (aBGH) (Fig. 2a). The orientation of the two transcription units allows the synthesis of negative-sense vRNA from the hPol-I cassette, and positive-sense mRNA from the Pol-II unit, from one viral cDNA template (Fig. 2a). Preparation of influenza B cDNA inserts and pDP-2002 ...
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... cassette is flanked by an RNA polymerase II-dependent (Pol-II) cytomegalovirus promoter (pCMV) and a polyadenylation site (aBGH) (Fig. 2a). The orientation of the two transcription units allows the synthesis of negative-sense vRNA from the hPol-I cassette, and positive-sense mRNA from the Pol-II unit, from one viral cDNA template (Fig. 2a). Preparation of influenza B cDNA inserts and pDP-2002 ...
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... to that of an RNA polymerase I promoter [11]. In addition, to avoid the generation of vRNA molecules containing additional nucleotides in both RNA ends that affect the correct interaction between the viral genome and the viral polymerase, the hammerhead and the hepatitis δ virus ribozymes flank the 5′ and the 3′ end of each vRNA, respectively (Fig. 2). Finally, to ensure the completion of transcription, the sequence of rabbit β-globin terminator was added to complete the design of the cassette [12]. With the strategic incorporation of all these elements into the plasmid system, the Atlantic salmon kidney (ASK) cells did not require any element in trans to generate recombinant ISAV. ...
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... (NA), are the main antigenic determinants of the virus to which neutralizing antibodies are elicited. IAVs are classified by the antigenic properties of the HA and NA. Currently, 18 HA and 11 NA subtypes have been described [2,3]. Wild aquatic birds are considered the primary hosts for IAVs in which 16 HA and 9 NA subtypes have been found (Fig. ...
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... the use of a T7 RNA polymerase promoter directly upstream of viral cDNA cloned in the negative sense [16]. The 3′ end of the vRNA is formed by the hepatitis delta ribozyme cleavage sequenced cloned immediately downstream. Transcription of the eight vRNAs, together with the four protein expression plasmids responsible for viral replication and Fig. 2 Influenza A virus wheel. Wild aquatic birds of the world are considered the natural reservoir of all influenza viruses. There is extensive evidence for transmission of influenza viruses between wild ducks and other species. Two-way transmission events between pigs and humans have been extensively documented and led to the emergence of ...
Citations
... The recombinant influenza virus FluCoV-N was constructed using the reverse genetics method [31,32]. Plasmid pHW-PR8-NN_c contained a chimeric ns gene encoding the truncated NS 1-124 protein of influenza virus and the N-terminal part of the N 1-209 protein of SARS-CoV-2. ...
BACKGROUND: The challenge of vaccine effectiveness against viruses that undergo constant antigenic changes during evolution is currently being addressed by updating vaccine formulations to match circulating strains. However, this approach proves ineffective if a virus undergoes antigenic drift and shift, or if a new virus, such as SARS-CoV-2, emerges and enters circulation. Hence, there is a pressing need to develop universal vaccines that elicit a T-cell immune response targeting conserved antigenic determinants of pathogens.
OBJECTIVE: To develop a vaccine candidate against influenza virus and coronavirus based on an attenuated influenza vector.
METHODS: In pursuit of this objective, we developed a recombinant influenza vector named FluCoV-N. It incorporates attenuating modifications in the ns1 and nep genes and expresses the N-terminal half of the N protein (N 1-209 ) of the SARS-CoV-2 virus. To assess the vector’s protective efficacy against influenza, ferrets were infected with heterologous influenza A/Austria/1516645/2022 (H3N2) virus on the 25th day after a single immunization with 9.4 log 10 EID 50 of the studied vector. To test protection against coronavirus, hamsters were immunized once with the vector at a dose of 8.2 log 10 EID 50 and challenged with SARS-CoV-2 virus 21 days later.
RESULTS: As a result of modifications to the NS genomic segment, the constructed vector acquired a temperature-sensitive (ts) phenotype and demonstrated a heightened ability to induce type 1 interferons. It was harmless to animals when administered intranasally at high doses exceeding 8.0 log 10 EID 50 . In ferrets, a single intranasal immunization with FluCoV-N accelerated the resolution of infection caused by heterologous influenza H3N2 virus. Similar immunization in hamsters led to a 10,000-fold reduction in SARS-CoV-2 viral titers in the lungs on the second day after challenge and reduced pathology in the lungs of animals.
CONCLUSION: A single intranasal immunization with the FluCoV-N vector protected from heterologous influenza or SARS-CoV-2 viruses in ferrets and hamsters.
... Конструирование рекомбинантного вируса гриппа FluCoV-N осуществляли с помощью метода обратной генетики [31,32]. Были использованы следующие плазмиды, полученные синтетическим путем для заражения 10-дневных куриных эмбрионов и культивировали двое суток при температуре 34°C. ...
ПРЕДПОСЫЛКИ: Проблема эффективности вакцинации в отношении вирусов, претерпевающих постоянные антигенные изменения в процессе эволюции, в настоящее время решается за счет обновления состава вакцин для обеспечения соответствия циркулирующим штаммам. В случае внезапного появления в популяции вируса, значительно отличающегося от циркулирующего (антигенный шифт или дрейф), или нового вируса, каким стал SARS-CoV-2, этот подход не работает. В связи с этим существует необходимость создания универсальной вакцины, индуцирующей Т-клеточный иммунный ответ к консервативным антигенным детерминантам вируса.
ЦЕЛЬ ИССЛЕДОВАНИЯ: Получение вакцинного кандидата на основе аттенуированного гриппозного вектора для иммунизации с целью индукции защиты от гетерологичного вируса гриппа и коронавируса.
МЕТОДЫ: На основе вируса гриппа A/Puerto Rico/8/1934 (H1N1) (PR/8/34) был сконструирован рекомбинантный вектор FluCoV-N, содержащий аттенуирующие мутации в генах ns1 и nep и экспрессирующий N-концевую последовательность белка N (N 1-209 ) вируса SARS-CoV-2. Для моделирования защиты хорьков от гриппа через 24 дня после однократной вакцинации вектором в дозе 9.4 log 10 ЭИД 50 их заражали гетерологичным штаммом вируса гриппа A/Austria/1516645/2022 (H3N2). Для проверки защиты хомяков от коронавируса проводили их однократную иммунизацию вектором в дозе 8.2 log 10 ЭИД 50 , а через 21 день их инфицировали SARS-CoV-2.
РЕЗУЛЬТАТЫ: Полученный вектор характеризовался температурочувствительностью (ts), способностью к стимуляции системы интерферонов I типа (IFN I) и безвредностью для животных при интраназальном применении в высокой дозе. Однократная интраназальная иммунизация хорьков FluCoV-N приводила к ускоренному разрешению инфекции, вызванной гетерологичным вирусом гриппа H3N2. Аналогичная иммунизация хомяков обеспечивала снижение титров вируса SARS-CoV-2 в легких в 10000 раз на второй день после инфекции и уменьшала патологию в легких животных.
ЗАКЛЮЧЕНИЕ: Однократная интраназальная иммунизация хорьков или хомяков гриппозным вектором FluCoV-N защищала животных, ослабляя развитие заболевания, вызванного гетерологичным вирусом гриппа или SARS-CoV-2 соответственно.
... However, further studies are needed to understand which of the individual mutations and reassorted genes were the critical changes that allowed cross-species infection and subsequent sustained transmission in pigs. (Table 1) were generated by reverse genetics (rg) using an 8-plasmid system as previously described [46,47] and previously tested for pathogenesis and transmission in vivo [8]. Briefly, plasmids were generated in the bidirectional plasmid vector pDP2002 for all genes of two parental viruses, a human-origin swine H3N1 virus of the 2010.1 lineage (A/swine/Missouri/A01410819/2014; sw/MO/14) detected through the USDA IAV in swine surveillance system and a seasonal human H3N2 virus (A/Victoria/361/2011; A/VIC/11) with similar HA ancestry. ...
The current diversity of influenza A viruses (IAV) circulating in swine is largely a consequence of human-to-swine transmission events and consequent evolution in pigs. However, little is known about the requirements for human IAVs to transmit to and subsequently adapt in pigs. Novel human-like H3 viruses were detected in swine herds in the U.S. in 2012 and have continued to circulate and evolve in swine. We evaluated the contributions of gene segments on the ability of these viruses to infect pigs by using a series of in vitro models. For this purpose, reassortant viruses were generated by reverse genetics (rg) swapping the surface genes (hemagglutinin-HA and neuraminidase-NA) and internal gene segment backbones between a human-like H3N1 isolated from swine and a seasonal human H3N2 virus with common HA ancestry. Virus growth kinetics in porcine intestinal epithelial cells (SD-PJEC) and in ex-vivo porcine trachea explants were significantly reduced by replacing the swine-adapted HA with the human seasonal HA. Unlike the human HA, the swine-adapted HA demonstrated more abundant attachment to epithelial cells throughout the swine respiratory tract by virus histochemistry and increased entry into SD-PJEC swine cells. The human seasonal internal gene segments improved replication of the swine-adapted HA at 33 °C, but decreased replication at 40 °C. Although the HA was crucial for the infectivity in pigs and swine tissues, these results suggest that the adaptation of human seasonal H3 viruses to swine is multigenic and that the swine-adapted HA alone was not sufficient to confer the full phenotype of the wild-type swine-adapted virus.
... Answering these questions required an improved understanding of the virus components, its evolutionary dynamics, and its infection epidemiology. After several unsuccessful attempts by different laboratories around the world, Neumann et al. [18] succeeded in completely assembling a replication-competent IV from cDNA in 1999, which was further developed and later employed to seek answers to the aforementioned questions [19]. An expert group of researchers 'revived' the virus strain from formalin-fixed lung samples of 1918 victims, sequenced its genome, recreated the whole virus in highly contained BSL-3 laboratories at the CDC, ultimately characterizing its biological features [20,21]. ...
Research in infection biology aims to understand the complex nature of host-pathogen interactions. While this knowledge facilitates strategies for preventing and treating diseases, it can also be intentionally misused to cause harm. Such dual-use risk is potentially high for highly pathogenic microbes such as Risk Group-3 (RG3) bacteria and RG4 viruses, which could be used in bioterrorism attacks. However, other pathogens such as influenza virus (IV) and enterohaemorrhagic Escherichia coli (EHEC), usually classified as RG2 pathogens, also demonstrate high dual-use risk. As the currently-approved therapeutics against these pathogens are not satisfactorily effective, previous outbreaks of these pathogens caused enormous public fear, media attention, and economic burden. In this interdisciplinary review, we summarize the current perspectives of dual-use research on IV and EHEC, and further highlight the dual-use risk associated with evolutionary experiments with these infectious pathogens. We support the need to carry-out experiments pertaining to pathogen evolution, including to gain predictive insights on their evolutionary trajectories, which cannot be otherwise achieved with stand-alone theoretical models and epidemiological data. However, we also advocate for increased awareness and assessment strategies to better quantify the risks-versus-benefits associated with such evolutionary experiments. In addition to building public trust in dual-use research, we propose that these approaches can be extended to other pathogens currently classified as low risk, but bearing high dual-use potential, given the particular pressing nature of their rapid evolutionary potential.
... Reverse genetics systems are available for generating recombinant influenza viruses in the laboratory (207,208), and are the basis for use as a vaccine vector. IAV infection can induce CD4+ andCD8+ T cell responses to produce cytokines (209,210). ...
Influenza A virus is one of the most important zoonotic pathogens that can cause severe symptoms and has the potential to cause high number of deaths and great economic loss. Vaccination is still the best option to prevent influenza virus infection. Different types of influenza vaccines, including live attenuated virus vaccines, inactivated whole virus vaccines, virosome vaccines, split-virion vaccines and subunit vaccines have been developed. However, they have several limitations, such as the relatively high manufacturing cost and long production time, moderate efficacy of some of the vaccines in certain populations, and lack of cross-reactivity. These are some of the problems that need to be solved. Here, we summarized recent advances in the development and application of different types of influenza vaccines, including the recent development of viral vectored influenza vaccines. We also described the construction of other vaccines that are based on recombinant influenza viruses as viral vectors. Information provided in this review article might lead to the development of safe and highly effective novel influenza vaccines.
... Reverse genetics, a technology that generates genetically defined RNA viruses from a cDNA copy, serves as the cornerstone for modern studies investigating influenza replication, pathogenesis and transmission (Perez et al., 2020). Plasmid-based reverse genetic systems for influenza viruses require two sources of RNA: 1) negative sense, genomic viral RNA copies of each segment (vRNA), and 2) messenger RNA copies of the most relevant structural components of the virus, particularly the RNA-dependent RNA polymerase complex (RdRp) (Perez et al., 2020). ...
... Reverse genetics, a technology that generates genetically defined RNA viruses from a cDNA copy, serves as the cornerstone for modern studies investigating influenza replication, pathogenesis and transmission (Perez et al., 2020). Plasmid-based reverse genetic systems for influenza viruses require two sources of RNA: 1) negative sense, genomic viral RNA copies of each segment (vRNA), and 2) messenger RNA copies of the most relevant structural components of the virus, particularly the RNA-dependent RNA polymerase complex (RdRp) (Perez et al., 2020). It has been noted that the number of plasmids required to recover virus could be consolidated by arranging the promoters for viral mRNA and vRNA transcription in opposite orientations around the cloned cDNA, which would then serve as template for both RNA species (Hoffmann et al., 2000). ...
... To generate the pPIG-vGluc(NS) plasmid, the hpol1 promoter of pDP-Gluc(NS) was replaced with a minimal spol1 promoter corresponding to nucleotide positions 372-539 of Sus scrofa 45S ribosomal RNA promoter region ( Fig. 1B). Subsequently, the spol1-vGluc(NS) cassette was subcloned between a pCMV promoter and a bovine growth hormone polyadenylation signal (aBGH) of pDP2002 to produce the bidirectional vector pPIG2012_Gluc(NS) (Perez et al., 2020). To produce the reverse genetics vector pPIG2012, the pPIG2012_Gluc(NS) plasmid was amplified by inverse PCR with the primer set 5 ′ -ATATCGTCTCGTCCCCC CCAACTTCGGAGGTCG-3 ′ and 5 ′ -TATTCGTCTCGATCTACCTGGTG ACAGAAAAGG-3 ′ and digested with BsmBI (NEB, Ipswich, MA) to remove the vGluc(NS) cassette. ...
Influenza viruses are among the most significant pathogens of humans and animals. Reverse genetics allows for the study of molecular attributes that modulate virus host range, virulence and transmission. The most common reverse genetics methods use bi-directional vectors containing a host RNA polymerase (pol) I promoter to produce virus-like RNAs and a host RNA pol II promoter to direct the synthesis of viral proteins. Given the species-dependency of the pol I promoter and virus-host interactions that influence replication of animal-origin influenza viruses in human-derived cells, we explored the potential of using the swine RNA pol I promoter (spol1) in a bi-directional vector for rescuing type A and B influenza viruses (IAV and IBV, respectively) in swine and human cells. The spol1-based bi-directional plasmid vector led to efficient rescue of IAVs of different origins (human, swine, and avian) as well as IBV in both swine- and human-origin tissue culture cells. In addition, virus rescue was successful using a recombinant bacmid containing all eight segments of a swine origin IAV. In conclusion, the spol1-based reverse genetics system is a new platform to study influenza viruses and produce swine influenza vaccines with increased transfection efficiency.
Influenza viruses are considered prominent pathogens of humans and animals that are extensively investigated because of public health importance. Plasmid-based reverse genetics is a fundamental tool that facilitates the generation of genetically modified viruses from a cDNA copy. The ability to rescue viruses enables researchers to understand different biological characteristics including IV replication, pathogenesis, and transmission. Furthermore, understanding the biology and ability to manipulate different aspects of the virus can aid in providing a better understanding of the mechanisms of antiviral resistance and development of alternative vaccination strategies. This chapter describes the process of cloning cDNA copies of IAV and IBV RNA segments into a swine polymerase-driven reverse genetics plasmid vector, successful generation of recombinant IVs in swine cells, and propagation of virus in cells or eggs. The swine polymerase reverse genetics system was previously shown to be efficient for de novo rescue of human-, swine-, and avian-origin IAVs and IBV in swine and human origin cell lines utilizing the same protocols discussed in this chapter.
Influenza A viruses are constantly evolving. Crucial steps in the infection cycle, such as sialic acid receptor binding on the host cell surface, can either promote or hamper the emergence of new variants. We previously assessed the relative fitness in Japanese quail of H9N2 variant viruses differing at a single amino acid position, residue 216 in the hemagglutinin (HA) viral surface protein. This site is known to modulate sialic acid recognition. Our prior study generated a valuable set of longitudinal samples from quail transmission groups where the inoculum comprised different mixed populations of HA 216 variant viruses. Here, we leveraged these samples to examine the evolutionary dynamics of viral populations within and between inoculated and naïve contact quails. We found that positive selection dominated HA gene evolution, but fixation of the fittest variant depended on the competition mixture. Analysis of the whole genome revealed further evidence of positive selection acting both within and between hosts. Positive selection drove fixation of variants in non-HA segments within inoculated and contact quails. Importantly, transmission bottlenecks were modulated by the molecular signature at HA 216, revealing viral receptor usage as a determinant of transmitted diversity. Overall, we show that selection strongly shaped the evolutionary dynamics within and between quails. These findings support the notion that selective processes act effectively on influenza A virus populations in poultry hosts, facilitating rapid viral evolution in this ecological niche.
Astroviruses are one of the main causes of gastroenteritis of medical and veterinary relevance worldwide. Recently, these viruses were associated with neurological disease in mammals, including humans. Reverse genetics systems are the most powerful tool to improve our understanding of the virus replication, and eventually to develop safe vaccine candidates. In the present review, it is summarized the current knowledge on the different strategies used to develop reverse genetics systems for mamastroviruses and avastroviruses, and some of the biological answers that have provided are discussed.
Vaccination of hens against influenza leads to the transfer of protective maternally-derived antibodies (MDA) to hatchlings. However, little is known about the transfer of H7N3 vaccine-induced MDA. Here, we evaluated transfer, duration, and protective effect of MDA in chickens against H7N3 HPAIV. To generate chickens with MDA (MDA (+)), 15-week-old White Leghorn hens were vaccinated and boosted twice with an inactivated H7N3 low pathogenic avian influenza virus vaccine, adjuvanted with Montanide ISA 71 VG. One week after the final boost, eggs were hatched. Eggs from non-vaccinated hens were hatched for chickens without MDA (MDA (−)). Both MDA (+) and MDA (−) hatchlings were monitored weekly for antibody levels. Anti-HA MDA were detected by hemagglutination inhibition assay mostly until day 7 post-hatch. However, anti-nucleoprotein MDA were still detected three weeks post-hatch. Three weeks post-hatch, chickens were challenged with 106 EID50/bird of Mexican-origin H7N3 HPAIV. Interestingly, while 0% of the MDA (−) chickens survived the challenge, 95% of the MDA (+) chickens survived. Furthermore, virus shedding was significantly reduced by day 5 post-challenge in the MDA (+) group. In conclusion, MDA confers partial protection against mortality upon challenge with H7N3 HPAIV, as far as three weeks post-hatch, even in the absence of detectable anti-HA antibodies, and reduce virus shedding after challenge.
