53 Leyi Wang (ed.), Animal Coronaviruses, Springer Protocols Handbooks, DOI 10.1007/978-1-4939-3414-0_6, © Springer Science+Business Media New York 2016 Chapter 6 Reverse Genetics of Avian Coronavirus Infectious Bronchitis Virus Sarah M. Keep , Erica Bickerton , and Paul Britton Abstract We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifi cations can be introduced into the IBV cDNA using homologous recombina- tion, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifi cations into the IBV cDNA; that has been successfully used for the substitution of specifi c nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modifi ed IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA dependant RNA polymerase. Key words Transient dominant selection (TDS) , Vaccinia virus , Infectious bronchitis virus (IBV) , Coronavirus , Avian , Reverse genetics , Nidovirus , Fowlpox virus , T7 RNA polymerase 1 Introduction Avian infectious bronchitis virus (IBV) is a gammacoronavirus that is the etiological agent of infectious bronchitis (IB); an acute and high contagious disease of poultry characterized by nasal discharge, snicking, tracheal ciliostasis and rales [ 1]. IBV replicates primarily in the respiratory tract but also in many other epithelial surfaces including oviducts, enteric surfaces and kidneys [ 2– 5]. Following infection with IBV, egg production and quality may be impaired in layers and weight gain in broilers is reduced [ 6]. Infected birds are predisposed to secondary bacterial infections such as colibaccilosis and mortality in young chicks is not uncommon. Fecal excretion of the virus is a consequence of replication in the intestinal tract; however, this does not normally result in clinical disease. Infectious bronchitis was fi rst described in the USA in the 1930s [ 7– 9] and is prevalent in poultry farming across the world due to 54 the intensive nature of poultry production, estimated to involve the global production of 55 billion chickens (50 billion broilers and 5 billion layers) on an annual basis. In a report, commissioned by Defra in 2005 [ 10], IBV was indicated as a major cause of ill health amongst chickens and was implicated as being responsible for more economic loss in the UK poultry industry than any other disease [ 11, 12]; IBV was estimated to cost the UK economy nearly £19 million per year, mainly due to loss of egg production, with serious implications for animal welfare. The cost of control through vacci- nation is approximately £5 million per year in the UK. Coronaviruses are enveloped viruses which replicate in the cell cytoplasm. Coronavirus genomes consist of single stranded posi- tive sense RNA, and are the largest of all the RNA viruses ranging from approximately 27 to 32 kb; the genome of IBV is 27.6 kb. Molecular analysis of the role of individual genes in the pathogen- esis of RNA viruses has been advanced by the availability of full- length cDNAs, for the generation of infectious RNA transcripts that can replicate and result in infectious viruses. The assembly of full-length coronavirus cDNAs was hampered due to regions from the replicase gene being unstable in bacteria. We therefore devised a reverse genetics strategy for IBV involving the insertion of a full- length cDNA copy of the IBV genome, under the control of a T7 RNA promoter, into the vaccinia virus genome in place of the thy- midine kinase (TK) gene. A hepatitis δ ribozyme (HδR) is located downstream of the coronavirus poly(A) tail followed by a T7 ter- mination sequence. IBV infectious RNA is generated from the T7 promoter immediately adjacent to the 5′ end of the IBV cDNA using T7 RNA polymerase and terminates at the T7 termination sequence downstream of the HδR sequence, which autocleaves itself and the T7-termination sequence at the end of the poly(A) sequence, resulting in an authentic IBV genomic RNA copy. Infectious IBV is recovered in situ in cells both transfected with vaccinia virus DNA and infected with a recombinant fowlpox virus expressing T7 RNA polymerase [ 13]. One of the main advantages of using vaccinia virus as a vector for IBV cDNA is its ability to accept large quantities of foreign DNA without loss of integrity and stability [ 14]. A second and equally important advantage is the ability to modify the IBV cDNA within the vaccinia virus vector through transient dominant selec- tion ( TDS ), a method taking advantage of recombinant events between homologous sequences [ 15, 16]. The TDS method relies on a three-step procedure. In the fi rst step, the modifi ed IBV cDNA is inserted into a plasmid containing a selective marker under the control of a vaccinia virus promoter. In our case we use a plasmid, pGPTNEB193 (Fig. 1; [ 17]), which contains a domi- nant selective marker gene, Escherichia coli guanine phosphoribosyl- transferase ( Ecogpt; [ 18]), under the control of the vaccinia virus P7.5K early/late promoter. Sarah M. Keep et al. 55 In the second step, this complete plasmid sequence is inte- grated 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 plas- mid and the IBV cDNA sequence in the vaccinia virus genome. The resulting recombinant vaccinia viruses (rVV) are highly unsta- ble due to the presence of duplicate sequences and are only main- tained by the selective pressure of the Ecogpt gene, which confers resistance to mycophenolic acid (MPA) in the presence of xanthine and hypoxanthine [ 15]. In the third step, the MPA-resistant rVVs are grown in the absence of MPA selection, resulting in the loss of the Ecogpt gene due to a second single homologous recombination event between the duplicated sequences (Fig. 3). During this third step two recombination events can occur; one event will result in the generation of the original (unmodifi ed) IBV sequence and the other in the generation of an IBV cDNA containing the desired modifi cation (i.e., the modifi cation within the plasmid sequence). In theory these two events will occur at equal frequency however in practice this is not necessarily the case. Ecogpt VV P7.5k promoter pGPTNEB193 cloning sites Ascl BamHl Pacl Xbal Hincl Sall Pstl Sphl Hindlll Fig. 1 Schematic diagram of the recombination vector for insertion of genes into a vaccinia virus genome using TDS . Plasmid pGPTNEB193 contains the Ecogpt selection gene under the control of the vaccinia virus early/late P 7.5K promoter, a multiple cloning region for the insertion of the sequence to be incorporated into the vaccinia virus genome and the bla gene (not shown) for ampicillin selection of the plasmid in E. coli . For modifi cation of the IBV genome, a sequence corre- sponding to the region being modifi ed, plus fl anking regions of 500–800 nucleo- tides for recombination purposes is inserted into the multiple cloning sites using an appropriate restriction endonuclease. The plasmid is purifi ed from E. coli and transfected into Vero cells previously infected with a recombinant vaccinia virus containing a full-length cDNA copy of the IBV genome Reverse genetics of IBV 56 To recover infectious rIBVs from the rVV vector, rVV DNA is transfected into primary chick kidney (CK) cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase (rFPV-T7; [ 19]). In addition, a plasmid, pCi-Nuc [ 13, 20], expressing the IBV nucleoprotein (N), under the control of both the cytomegalovirus (CMV) RNA polymerase II promoter and the T7 RNA promoter, is co-transfected into the CK cells. Expression of T7 RNA polymerase in the presence of the IBV N protein and the rVV DNA, containing the full-length IBV cDNA under the control of a T7 promoter, results in the generation of infectious IBV RNA, which in turn results in the production of infectious rIBVs (Fig. 4). Primary CK cells are refractory for growth of most IBV isolates; therefore rIBVs expressing S glycoproteins from such isolates cannot be recovered using CK cells. In order to recover gpt gpt pGPT-vector with Modified S gene In situ recombination event vNotl-IBVFL Replicase Replicase Modified region of S gene Modified region of S gene gene 3 gene 3 gene 5 S M N S M N gene 5 Single cross-over event resulting in integration of complete plasmid DNA with GPT gene Fig. 2 Schematic diagram demonstrating the TDS method for integrating a modifi ed IBV sequence into the full- length IBV cDNA within the genome of a recombinant vaccinia virus (vNotI-IBVFL). The diagram shows a potential fi rst single-step recombination event between the modifi ed IBV sequence within pGPTNEB193 and the IBV cDNA within vNotI-IBVFL. In order to guarantee a single-step recombination event any potential recom- binant vaccinia viruses are selected in the presence of MPA; only vaccinia viruses expressing the Ecogpt gene are selected. The main IBV genes are indicated, the replicase, spike (S), membrane (M) and nucleocapsid (N) genes. The IBV gene 3 and 5 gene clusters that express three and two gene products, respectively, are also indicated. In the example shown a modifi ed region of the S gene is being introduced into the IBV genome Sarah M. Keep et al. 57 such rIBVs, the supernatants from the transfected CK cells are used to infect 10-day-old embryonated hen’s eggs. Allantoic fl uid is collected and any potential virus passed a further three times in 10-day-old embryos. RNA is extracted from the allantoic fl uid of infected eggs and RT-PCR followed by sequencing is used to con- fi rm the identity of the rIBV. The overall procedure is a multi-step process which can be divided into two parts; the generation of an rVV containing the modifi ed IBV cDNA (Fig. 5) and the recovery of infectious rIBV from the rVV vector (Fig. 4). The generation of the Ecogpt plas- mids, based on pGPTNEB193, containing the modifi ed IBV cDNA, is by standard E. coli cloning methods [ 21, 22] and is not described here. General methods for growing vaccinia virus have been published by Mackett et al. [ 23] and for using the TDS method for modifying the vaccinia virus genome by Smith [ 24]. Unstable Intermediate rVV gene 3 gene 3 Replicase Replicase S M N S M N gpt gene 5 gene 5 Second cross-over events resulting in loss of plasmid DNA and GPT gene II) Integration of modified IBV sequence I) Back to original IBV cDNA sequence as in vNotl-IBVFL Recombinant vaccinia virus with modified IBV cDNA Fig. 3 Schematic diagram demonstrating the second step of the TDS method. Integration of the complete pGPTNEB193 plasmid into the vaccinia virus genome results in an unstable intermediate because of the pres- ence of tandem repeat sequences, in this example the 3′ end of the replicase gene, the S gene and the 5′ end of gene 3. The second single-step recombination event is induced in the absence of MPA; loss of selection allows the unstable intermediate to lose one of the tandem repeat sequences including the Ecogpt gene. The second step recombination event can result in either (I) the original sequence of the input vaccinia virus IBV cDNA sequence, in this case shown as a recombination event between the two copies of the 3′ end of the replicase gene which results in loss of the modifi ed S gene sequence along with Ecogpt gene; or (II) retention of the modifi ed S gene sequence and loss of the original S gene sequence and Ecogpt gene as a result of a potential recombination event between the two copies of the 5′ end of the S gene sequence. This event results in a modifi ed S gene sequence within the IBV cDNA in a recombinant vaccinia virus Reverse genetics of IBV 58 CK cell CK cells CK cells CK cells pCl-Nuc DNA Transfection Transfection DNA from rVV with modified IBV cDNA IBV N mRNA IBV N protein T7 RNA Pol rVV DNA Infectious T7-derived IBV gRNA Replication, transcription & translation Production of IBV Release FPV-T7 rVV DNA pCi-Nuc Filter media to remove FPV-T7 b a FPV-T7 Virus rlBV P0 P1 P2 Fig. 4 A schematic representation of the recovery process for obtaining rIBV from DNA isolated from a recombinant vaccinia virus containing a full-length IBV cDNA under the control of a T7 promoter. ( a ) In addition to the vaccinia virus DNA containing the full-length IBV cDNA under the control of a T7 promoter a plasmid, pCi-Nuc, expressing the IBV nucleoprotein, required for successful rescue of IBV, is transfected into CK cells previously infected with a recombinant fowl pox virus, FPV-T7, expressing T7 RNA polymerase . The T7 RNA polymerase results in the synthesis of an infectious RNA from the vaccinia virus DNA that consequently leads to the generation of infectious IBV being released from the cell. ( b ) Any recovered rIBV present in the media of P 0 CK cells is used to infect P 1 CK cells. The media is fi ltered through a 0.22 μm fi lter to remove any FPV-T7 virus. IBV- induced CPE is normally observed in the P 1 CK cells following a successful recov- ery experiment. Any rIBV is passaged a further two times, P 2 and P 3 , in CK cells. Total RNA is extracted from the P 1 to P 3 CK cells and the IBV-derived RNA ana- lyzed by RT-PCR for the presence of the required modifi cation Sarah M. Keep et al. 59 2 Materials 1. Vero cells . 2. PBSa: 172 mM NaCl, 3 mM KCl, 10 mM Na 2HPO 4 and 2 mM KH 2PO 4, adjusted to pH 7.2 with HCl. 3. 1× Eagle’s Minimum Essential Medium (E-MEM) with Earle’s salts, 2 mM L-glutamine, and 2.2 g/l sodium bicarbonate. 2.1 Homologous Recombination and Transient Dominant Selection in Vero Cells Three rounds of plaque purification of the rVVs in presence of selection agents Three rounds of plaque purification in absence of selection agents Small stocks of rVV grown from individual plaques Screen sequence of rVVs. 50% will contain the desired modification. 50% will revert back to the original sequence Selection agents: MPA Xanthine Hypoxanthine Vero cells GPT plasmid Vaccinia Virus Fig. 5 Schematic detailing the multistep process of constructing a recombinant vaccinia virus. Vero cells are infected with rVV containing IBV cDNA and then transfected with a plasmid containing the IBV sequence to be inserted and the selective marker gene Ecogpt . Homologous recombination occurs and the complete plasmid sequence is inserted into the rVV. The Ecogpt gene allows positive selection of these rVV as it confers resis- tance to MPA in the presence of xanthine and hypoxanthine. The viruses are plaque purifi ed three times in the presence of selection agents ensuring no wild type VV is present. The removal of the selection agents results in a second recombination event with the loss of the Ecogpt gene. Plaque purifi cation in the absence of selec- tion agents not only ensures the loss of the GPT gene but also ensures the maintenance of a single viral popu- lation. Small stocks of rVV are grown from individual plaques which are screened through PCR for the desired modifi cation; this is found in theoretically 50 % of rVVs Reverse genetics of IBV 60 4. BES medium: 1× E-MEM, 0.3 % tryptose phosphate broth (TPB), 0.2 % bovine serum albumin (BSA), 20 mM N, N - Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 0.21 % sodium bicarbonate, 2 mM L-glutamine, 250 U/ml nystatin, 100 U/ml penicillin, and 100 U/ml streptomycin. 5. Opti-MEM 1 with GlutaMAX-1 (Life Technologies). 6. Lipofectin (Life Technologies). 7. Mycophenolic acid (MPA): 10 mg/min 0.1 M NaOH (30 mM); 400× concentrated. 8. Xanthine: 10 mg/ml in 0.1 M NaOH (66 mM); 40× concen- trated. Heat at 37 °C to dissolve. 9. Hypoxanthine: 10 mg/ml in 0.1 M NaOH (73 mM); 667× concentrated. 10. Screw-top 1.5 ml microfuge tubes with gasket. 11. Cup form sonicator. 12. 2× E-MEM: 2× E-MEM, 10 % fetal calf serum, 0.35 % sodium bicarbonate, 4 mM L-glutamine, 1000 U/ml nystatin, 200 U/ ml penicillin, and 200 U/ml streptomycin. 13. 2 % agar. 14. Ecogpt selection medium: 1× E-MEM, 75 μM MPA, 1.65 mM xanthine, 109 μM hypoxanthine, 1 % agar ( see Note 1). 15. Overlay medium: 1× E-MEM, 1 % agar. 16. 1 % Neutral red solution (H 2O). 1. 20 mg/ml proteinase K. 2. 2× proteinase K buffer: 200 mM Tris–HCl pH 7.5, 10 mM EDTA, 0.4 % SDS, 400 mM NaCl. 3. Phenol–chloroform–isoamyl alcohol (25:24:1). 4. Chloroform. 5. Absolute ethanol. 6. 70 % ethanol. 7. QlAamp DNA mini kit (QIAGEN). 8. 3 M sodium acetate. 1. BHK-21 maintenance medium: Glasgow-Modifi ed Eagle’s Medium (G-MEM), 2 mM L-glutamine, 0.275 % sodium bicar- bonate, 1 % fetal calf serum, 0.3 % TPB, 500 U/ml nystatin, 100 U/ml penicillin, and 100 U/ml streptomycin. 2. TE buffer: 10 mM Tris–HCl pH 9, 1 mM EDTA. 3. BHK-21 cells. 4. 50 ml Falcon tubes. 2.2 Extraction of DNA from Recombinant Vaccinia Virus 2.3 Production of Large Stocks of Vaccinia Virus Sarah M. Keep et al. 61 1. 30 % sucrose (w/v) in 1 mM Tris–HCl pH 9, fi ltered through 0.22 μm. 2. Superspin 630 rotor and Sorvall OTD65B ultracentrifuge or equivalent. 1. 10× TBE buffer: 1 M Tris, 0.9 M boric acid pH 8, and 10 mM EDTA. 2. Pulsed fi eld certifi ed ultrapure DNA grade agarose. 3. DNA markers (e.g., 8–48 kb markers, Bio-Rad). 4. 0.5 mg/ml ethidium bromide. 5. CHEF-DR ® II pulsed fi eld gel electrophoresis ( PFGE ) appara- tus (Bio-Rad) or equivalent. 6. 6× sample loading buffer: 62.5 % glycerol, 62.5 mM Tris–HCl pH 8, 125 mM EDTA, and 0.06 % bromophenol blue. 1. Chicken embryo fi broblast (CEF) cells. 2. CEF maintenance medium: 1× 199 Medium with Earle’s Salts, 0.3 % TPB, 2 % newborn calf serum (NBCS), 0.225 % sodium bicarbonate, 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ ml streptomycin, and 500 U/ml nystatin. 1. Chick kidney (CK) cells. 2. Stock of rFPV-T7 virus. 3. The rVV DNA prepared from large partially purifi ed stocks of rVV. 4. Plasmid pCi-Nuc which contains IBV nucleoprotein under the control of the CMV and T7 promoters. 5. 0.22 μm syringe driven fi lters. 6. 5 ml syringes. 3 Methods 1. Freeze-thaw the vaccinia virus containing the full-length IBV cDNA genome to be modifi ed three times (37 °C/dry ice) and sonicate for 2 min using a cup form sonicator, continuous pulse at 70 % duty cycle, seven output control ( see Notes 2– 5). 2. Infect 6-well plates of 40 % confl uent monolayers of Vero cells with the rVV at a multiplicity of infection (MOI) of 0.2. Use two independent wells per recombination ( see Notes 2– 5). 3. Incubate at 37 °C 5 % CO 2 for 2 h to allow the virus to infect the cells. 4. After 1 h of incubation, prepare the following solutions for transfection : 2.4 Vaccinia Virus Partial Purifi cation 2.5 Analysis of Vaccinia Virus DNA by Pulse Field Agarose Gel Electrophoresis 2.6 Preparation of rFPV-T7 Stock Virus 2.7 Recovery of rIBV and Serial Passage on CK Cells 3.1 Infection / Transfection of Vero Cells with Vaccinia Virus Reverse genetics of IBV 62 Solution A: For each transfection : Dilute 5 μg of modifi ed pGPTNEB193 (containing the modifi ed IBV cDNA) in 1.5 ml of Opti-MEM medium. Solution B: Dilute 12 μl of Lipofectin in 1.5 ml of Opti- MEM for each transfection . 5. Incubate solutions A and B separately for 30 min at room tem- perature, then mix the two solutions together and incubate the mixture at room temperature for 15 min. 6. During the 15 min incubation, remove the inoculum from the vaccinia virus infected cells and wash the cells twice with Opti-MEM. 7. Add 3 ml of the transfection mixture (prepared in step 5) to each well. 8. Incubate for 60–90 min at 37 °C 5 % CO 2 ( see Note 6). 9. Remove the transfection mixture from each well and replace it with 5 ml of BES medium. 10. Incubate the transfected cells overnight at 37 °C, 5 % CO 2. 11. The following morning add the MXH selection components, MPA 12.5 μl, xanthine 125 μl, and hypoxanthine 7.4 μl, directly to each well ( see Note 7). 12. Incubate the cells at 37 °C 5 % CO 2 until they display extensive vaccinia virus induced cytopathic effect (CPE) (normally 2 days). 13. Harvest the infected/transfected cells into the cell medium of the wells and centrifuge for 3–4 min at 300 × g. Discard super- natant and resuspend the pellet in 400 μl 1× E-MEM and store at −20 °C. 1. Freeze-thaw the vaccinia virus produced from Sect. 3.1 three times and sonicate as described in the previous section (Sect. 3.1 step 1). 2. Remove the medium from confl uent Vero cells in 6-well plates and wash the cells once with PBSa. 3. Prepare 10 −1 to 10 −3 serial dilutions of the recombinant vac- cinia virus in 1× E-MEM. 4. Remove the PBSa from the Vero cells and add 500 μl of the diluted virus per well. 5. Incubate for 1–2 h at 37 °C 5 % CO 2. 6. Remove the inoculum and add 3 ml of the Ecogpt selection medium ( see Note 1). 7. Incubate for 3–4 days at 37 °C 5 % CO 2 and stain the cells by adding 2 ml of 1× E-MEM containing 1 % agar and 0.01 % neutral red. 8. Incubate the cells at 37 °C 5 % CO 2 for 6–24 h and pick 2–3 well isolated plaques for each recombinant, by taking a plug of 3.2 Plaque Purifi cation in the Presence of GPT Selection Agents: Selection of MPA Resistant Recombinant Vaccinia Viruses (GPT+ Phenotype) Sarah M. Keep et al.