Partial and Full PCR-Based Reverse Genetics Strategy for Influenza Viruses

<div><p>Since 1999, plasmid-based reverse genetics (RG) systems have revolutionized the way influenza viruses are studied. However, it is not unusual to encounter cloning difficulties for one or more influenza genes while attempting to recover virus <em>de novo</em>. To overcome some of these shortcomings we sought to develop partial or full plasmid-free RG systems. The influenza gene of choice is assembled into a RG competent unit by virtue of overlapping PCR reactions containing a cDNA copy of the viral gene segment under the control of RNA polymerase I promoter (pol1) and termination (t1) signals – herein referred to as Flu PCR amplicons. Transfection of tissue culture cells with either HA or NA Flu PCR amplicons and 7 plasmids encoding the remaining influenza RG units, resulted in efficient virus rescue. Likewise, transfections including both HA and NA Flu PCR amplicons and 6 RG plasmids also resulted in efficient virus rescue. In addition, influenza viruses were recovered from a full set of Flu PCR amplicons without the use of plasmids.</p> </div

Virus rescue with Flu PCR amplicons in 293T/MDCK co-cultured cells.

a<p>utr: Viruses obtained from Flu PCR amplicons lacking a t1 termination signal.</p>b<p>UTR: No viruses obtained when Flu PCR amplicons lacked the pol 1 promoter sequence.</p

Overlapping PCR strategy and reconstitution of HA and NA PCR amplicons.

<p>The strategy to produce full-length HA and NA PCR amplicons was based on amplification of the pol1 promoter from the pDP2002 vector, subtype specific internal primers for HA and NA and, depending on the product, primers containing a t1 termination signal. A) The HA_pdm was amplified with the following overlapping fragments: 1, fragment spanning the primer set <i>pT1FragFwd</i> and <i>SwHA-931R</i> (which incorporates the <i>t1</i> signal), 2, fragment spanning from the <i>SwHA-752F</i> and <i>polFragRev</i>, and 3, <i>polI</i> promoter fragment amplified using <i>polF</i> and <i>hPol1Rev</i> primers. The three PCR products above were purified by agarose gel electrophoresis and combined in equal proportions (10 ng each) to create the full length <i>pol1HA_pdmt1</i> PCR amplicon using the primer pair <i>pT1FragFwd</i> and <i>hPol1Rev</i>. B) An overlapping PCR strategy to produce an H5 <i>HA</i> segment in which the polybasic cleavage site from the chicken/North Sumatra strain was removed and replaced by the sequence of low pathogenic virus following the strategy described above. C) and D) Depict the strategies used for generation of full length N1 PCR products from the H1N1_pdm and 072 H5N1 strains. E) Alternative PCR products were generated to serve as controls for PCR-based reverse genetics. Agarose gel shows the expected size for lane 1, <i>hpol1</i> PCR amplicon; lane 2, GeneRuler™ 1 kb plus DNA Ladder; lane 3, <i>pol1HA_pdmt1</i>; lane 4, <i>pol1HA_pdmutr</i>; lane 5, <i>pol1NA_pdmt1</i>; lane 6, <i>pol1NA_pdmutr</i>; lane 7, <i>UTRHA_pdmutr</i>; and lane 8, <i>UTRNA_pdmutr</i>. F) <i>polHA</i>_Δ<i>072</i><i>t1</i> (lane 1) and <i>polNA</i>_Δ<i>072</i><i>t1</i> (lane 3) amplicons were prepared following the strategy described above and depicted in B) and D), respectively. Lane 2 corresponds to GeneRuler™ 1 kb Plus DNA Ladder (75–20,000 bp) from Fermentas Inc (lane 2). All the PCR products were amplified here and in the supplemental information.</p

PCR-based reverse genetics.

<p>A) PCR-based Flu reporter replicon encoding GFP: PCR amplification was performed using primers spanning the pol1 to t1 sequences and pHW72EGFP. After agarose gel purification and testing to show that the PCR product is devoid of plasmid DNA contamination, the Flu GFP amplicon is transfected into 293T cells along with four expression plasmids encoding the polymerase complex of influenza virus. Expression of GFP reflects influenza polymerase activity on a vRNA Flu GFP replicon generated from pol1 transcription of the Flu GFP amplicon. Variations to this these are described in the main text and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046378#pone-0046378-g002" target="_blank">Fig. 2</a>. B) Starting with a influenza virus candidate, vRNA, cDNA and reconstitution of a full-length Flu PCR amplicon (in this case, the HA and NA PCR amplicons are depicted) is performed. Transfection of Flu PCR amplicons along with appropriate complementary RG plasmids into susceptible cells leads to the generation of recombinant influenza viruses with the desired gene constellation. The strategy speeds up the reverse genetics process by obviating a classical cloning step, which is currently part of the plasmid-based RG system.</p

Flu PCR amplicons rescued with the VN1203 backbone in MDCK cells.

<p>Flu PCR amplicons rescued with the VN1203 backbone in MDCK cells.</p

Identification of virus reassortants obtained using Flu PCR amplicons.

<p>Viruses recovered by reverse genetics were used to infect MDCK cells at a MOI of 0.001. At 36 hpi, cells were fixed with 4% Paraformaldehyde in PBS solution (Santa Cruz) and viral antigen detected by IFA using subtype specific monoclonal antibodies and FITC-labeled antimouse antibodies (green). Counterstaining was performed with propidium iodide (red). A) mAb 3B2 specific for the HA H1N1_pdm virus has no reaction with the HA of PR8 strain (only cell nuclei can be seen in red reacting with propidium iodide) (X10). B) and C) Positive viral antigen green signal detected using mAb 3B2 in cells infected with either the H1N1_pdmutr:6PR8 or H1N1_pdm:6PR8 viruses, respectively (X10). D) mAb DPJY01 specific to H5N1 virus has no reaction with PR8-infected cells (X20). E) Positive reaction of mAb DPJY01 cells infected with H5Δ₀₇₂N1:6PR8 (X20). F) Sequence of HA cleavage site of H5Δ₀₇₂N1:6PR8 virus indicates the presence of a low pathogenic pattern (TETR), which was created by overlapping PCR as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046378#s4" target="_blank">materials and methods</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046378#pone-0046378-g003" target="_blank">Fig. 3B</a>. G) Plaque assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046378#s4" target="_blank">materials and methods</a>. The PR8 and H5Δ₀₇₂:7PR8 viruses form plaques in agarose plates in the presence of 1 µg/mL TPCK-Trypsin but not in its absence.</p

Virus rescue with H1N1_pdm PCR amplicons in Vero cells.

<p>Virus rescue with H1N1_pdm PCR amplicons in Vero cells.</p

Enhanced inflammation in New Zealand white rabbits when MERS-CoV reinfection occurs in the absence of neutralizing antibody

<div><p>The Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that was first detected in humans in 2012 as a cause of severe acute respiratory disease. As of July 28, 2017, there have been 2,040 confirmed cases with 712 reported deaths. While many infections have been fatal, there have also been a large number of mild or asymptomatic cases discovered through monitoring and contact tracing. New Zealand white rabbits are a possible model for asymptomatic infection with MERS-CoV. In order to discover more about non-lethal infections and to learn whether a single infection with MERS-CoV would protect against reinfection, we inoculated rabbits with MERS-CoV and monitored the antibody and inflammatory response. Following intranasal infection, rabbits developed a transient dose-dependent pulmonary infection with moderately high levels of viral RNA, viral antigen, and perivascular inflammation in multiple lung lobes that was not associated with clinical signs. The rabbits developed antibodies against viral proteins that lacked neutralizing activity and the animals were not protected from reinfection. In fact, reinfection resulted in enhanced pulmonary inflammation, without an associated increase in viral RNA titers. Interestingly, passive transfer of serum from previously infected rabbits to naïve rabbits was associated with enhanced inflammation upon infection. We further found this inflammation was accompanied by increased recruitment of complement proteins compared to primary infection. However, reinfection elicited neutralizing antibodies that protected rabbits from subsequent viral challenge. Our data from the rabbit model suggests that people exposed to MERS-CoV who fail to develop a neutralizing antibody response, or persons whose neutralizing antibody titers have waned, may be at risk for severe lung disease on re-exposure to MERS-CoV.</p></div

CD3+ cells in the lungs following primary infection and reinfection.

<p>DAB images from primary infection (A) and reinfection (B). Immunofluorescence (IF) image of CD3 (green) and virus antigen (red) within the same perivascular region following reinfection (C). DAB images from day 3 post-infection at 10x, bar equivalent to 100μm. IF images at 40x, bar equivalent to 20μm.</p

Histopathology in the lungs following primary infection with EMC/2012 strain of MERS-CoV.

<p>Images show H&E (left) and IHC against the MERS-CoV N protein (right) following infection with 10⁵ TCID₅₀ (A,D), 10³ TCID₅₀ (B,E), or a media only control (C,F). All images at 10x, (bar equivalent to 100μm) with 40x insets (bar equivalent to 20μm). Images shown are from day 3 post-infection for all groups.</p