Construction of a Novel Infectious Clone of Recombinant Herpesvirus of Turkey Fc-126 Expressing VP2 of IBDV

The increased virulence of infectious bursal disease virus (IBDV) is a threat to the chicken industry. The construction of novel herpesvirus of turkey-vectored (HVT) vaccines expressing VP2 of virulent IBDV may be a promising vaccine candidate for controlling this serious disease in chickens. We generated a novel infectious clone of HVT Fc-126 by inserting mini-F sequences in lieu of the glycoprotein C (gC) gene. Based on this bacterial artificial chromosome (BAC), a VP2 expression cassette containing the pMCMV IE promoter and a VP2 sequence from the virulent IBDV NJ09 strain was inserted into the noncoding area between the UL55 and UL56 genes to generate the HVT vector VP2 recombinant, named HVT-VP2-09. The recovered vectored mutant HVT-VP2-09 exhibited higher titers (p = 0.0202 at 36 h) or similar growth kinetics to the parental virus HVT Fc-126 (p = 0.1181 at 48 h and p = 0.1296 at 64 h). The high reactivation ability and strong expression of VP2 by HVT-VP2-09 in chicken embryo fibroblasts (CEFs) were confirmed by indirect immunofluorescence (IFA) and Western blotting. The AGP antibodies against IBDV were detected beginning at 3 weeks post-inoculation (P.I.) of HVT-VP2-09 in 1-day-old SPF chickens. Seven of ten chickens immunized with HVT-VP2-09 were protected post-challenge (P.C.) with the virulent IBDV NJ09 strain. In contrast, all chickens in the challenge control group showed typical IBD lesions in bursals, and eight of ten died P.C. In this study, we demonstrated that (i) a novel HVT BAC with the whole genome of the Fc-126 strain was obtained with the insertion of mini-F sequences in lieu of the gC gene; (ii) HVT-VP2-09 harboring the VP2 expression cassette from virulent IBDV exhibited in vitro growth properties similar to those of the parental HVT virus in CEF cells; and (iii) HVT-VP2-09 can provide efficient protection against the IBDV NJ09 strain.


Introduction
Herpesvirus of turkeys (HVT) is Gallid herpesvirus 1 or nonpathogenic Marek's disease virus (MDV) (serotype 3) originally isolated from domestic turkeys and widely used as a vaccine to prevent Marek's disease (MD) in commercial poultry [1,2]. To date, HVT has been used as a potential recombinant vector for other poultry infections, such as Newcastle disease and avian influenza [3][4][5][6]. HVT vectors are promising avian vaccine vehicles for several reasons. HVT has a large genome with multiple nonessential and noncoding regions that are suitable for the insertion of multiple or large antigens [7]. HVT induces both humoral and cellular immune responses and provides long-term protection against pathogens induced in chickens [8].
Infectious bursal disease (IBD) causes long-lasting immunosuppression in poultry [9][10][11], resulting in tremendous economic losses due to vaccine failures and increased susceptibility to opportunistic pathogens [12]. The pathogen of IBD is the infectious bursal disease virus (IBDV), which is highly resistant to many disinfectants and is very difficult to remove from contaminated poultry premises. IBDV encodes five proteins known as VP1, VP2, VP3, VP4, and VP5 [13]. The VP2 protein is considered a major protective antigen that elicits neutralizing antibodies to protect chickens from virulent IBDV [14]. Therefore, the VP2 protein is often used as an immunogenicity antigen for recombinant genetically engineered vaccines [15][16][17][18][19].
HVT is already used as a vector for expressing VP2 of IBDV and other protective antigens of avian pathogens, such as Newcastle disease virus (NDV) [5], Eimeria acervuline [20], avian influenza virus (AIV) [21][22][23], and infectious laryngotracheitis virus (ILTV) [24]. Such recombinant HVT-based vaccines confer excellent and long-lasting simultaneous protective immunity in chickens against pathogens [4,25]. However, most of these HVT recombinants were constructed through conventional homologous recombination techniques, which are extremely time consuming and labor intensive. It is difficult to recover purified recombinant viruses through plaque selection methods [26]. The establishment of herpesvirus as a bacterial artificial chromosome (BAC) [27,28] and its related EN PASSANT protocol has facilitated the generation of herpesvirus vector vaccines [29,30]. However, most alpha herpesviruses can grow in cell culture in the absence of various glycoproteins [31]. This differential requirement of glycoproteins is attributed to the strict cell-associated nature. It has been proven that the deletion of glycoprotein B (gB) interferes with the cell-to-cell spread of HVT [32]. However, glycoprotein C (gC) is a nonessential gene, and its deletion does not interfere with the rescue of recombinant HVT. The recombinant virus (rHVT-pmpD-N) was recovered from primary chicken embryo fibroblast (CEF) cells by the replacement of the UL44-UL46 gene of HVT [33]. Therefore, gC is a suitable insertion for an exogenous gene.
In this study, we generated a novel infectious clone of the HVT Fc-126 strain by the insertion of miniF sequences in lieu of the gC gene, and based on the clone, recombinant HVT-VP2-09 was constructed as a bivalent vaccine vector by insertion of the expression cassette of the VP2 gene of a very virulent IBDV field isolate NJ09 strain. The expression of heterologous VP2 by HVT-VP2-09 was demonstrated in CEF cells. We also demonstrated that the HVT-VP2-09 bivalent vaccine administered to one-day-old chicks induced VP2specific antibodies and conferred protection in vaccinated chicks subsequently challenged with IBDV.

Materials and Methods
The experiment was approved by the Institutional Animal Care and Use Committee at the Jiangsu Academy of Agriculture Sciences and was performed strictly according to the guidelines provided by the Institutional Biosafety Committee.

Viruses and Plasmids
IBDV (NJ09) was identified and preserved in our lab. The BAC transfer vector plasmid was kindly provided by Professor Nikolaus Osterrieder from the Free University of Berlin [34]. The chicken embryo fibroblasts (CEFs) cells were prepared from SPF chicken embryo eggs from Beijing Merial Vital Laboratories Animal Technology Company Limited and prepared by standard method. All HVT strains were propagated on primary or secondary CEFs. Virus stocks were prepared from CEF cultures, which were infected with viruses at a multiplicity of infection (MOI) of 0.01 and cultured for 72 h in CEFs. PFU titers were determined on CEFs according to the standard titration method [35].
The VP2 expression cassette containing a pMCMV IE promoter and VP2 gene of IBDV was cloned into T-Vector pMD19 (Takara, Japan) with slight modification to generate the plasmid pHVT-VP2-09 (at the UL55-UL56 region). The promoter pMCMV IE included a sequence complementary to the sequence between sites 184,336 and 182,946 in the MCMV genome (GenBank: GU305914.1) followed by a Kozak sequence. The plasmid pHVT-VP2-09-KAN containing the VP2 expression cassette and a kanamycin resistance gene inserted at the EcoR I restriction site was constructed by cutting and ligating for further En Passant recombination [36].

Cells, Viral DNA Extraction, and Transfection
CEFs were propagated in Earle's minimal essential medium (EMEM; Gibco, Los Angeles, CA, USA) supplemented with 10% newborn calf serum (NBCS; Gibco, Los Angeles, CA, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 • C under a 5% CO 2 atmosphere. Viral DNA was purified from infected cells by sodium dodecyl sulfate (SDS)proteinase K extraction described previously [37]. Transfection of DNA from plasmids, viruses, or BACs was achieved by calcium phosphate precipitation [38]. Briefly, approximately 200 ng DNA was mixed with water, and then 62 µL of 2 M CaCl 2 was added dropwise to a total volume of 500 µL. The transfection mixture was incubated overnight at 4 • C followed by adding 500 µL cold 2 × HEPES-buffered saline (HBS) solution dropwise. The medium was replaced with 500 µL of fresh EMEM without NBCS or antibiotics and incubated with the transfection mixture at 37 • C for 3-4 h. Media were discarded, and washed twice with PBS. Then, 1.5 mL of 15% glycerol HBS solution was added, and incubated at 37 • C for 2 min. The transfection solution was replaced with EMEM supplemented with 10% NBCS and antibiotics after washing twice with PBS for culture at 37 • C in an incubator with 5% CO 2 .

Construction of an Infectious Clone of the HVT Fc-126 Strain
The HVT Fc-126 infectious clone was generated with the method modified from that used for the generation of BAC [32]. Briefly, cotransfection of DNA of HVT Fc-126 and pUC19-H1-H2-miniF was conducted on primary CEFs (24 h) to allow for insertion of mini-F sequences in lieu of glycoprotein C (gC). After green plaques were observed under UV light (488 nm), a homogeneous population of mini-F recombinant HVT-miniF was obtained by 6 rounds of picking and plating on CEF cultures in a medium containing mycophenolic acid, xanthine, and hypoxanthine as described previously [34]. Genomic DNA extracted from these HVT-miniF CEF cultures was electroporated into E. coli DH10B competent cells (Invitrogen). Positive clones with chloramphenicol resistance were examined through RFLP with EcoR I and BamH I to select a clone of the HVT complete genome. The infectivity of HVT-BAC DNA was tested after transfection into CEFs using Lipofectamine 3000 (Invitrogen).

Cloning of the IBDV VP2 Gene and VP2 Cassette
The gene cDNA corresponding to the IBDV-NJ09 VP 2 open reading frame (1359 nucleotides) was PCR-amplified as described previously [36], by using specific primers (Table 1). Briefly, the PCR products of cDNA VP 2 were digested with Hind III and Sal I and cloned into the pMCMV IE vector (Clontech, Tokyo, Japan). The VP 2 cassette containing the MCMV IE promoter and the SV40 polyadenylation signal was PCR-amplified using primers containing homologous sequences of the HVT UL55-UL56 insertion site. Table 1. PCR amplification using specific primers.

Construct
Sequence

Construction of HVT BAC with Insertion of VP2 Expression Cassette
The VP2 expression cassette was inserted into the noncoding area between UL55 and UL56 in the pHVT-BAC genome through the En Passant method ( Figure 1). Briefly, PCR was performed using plasmid pHVT-VP2-09-KAN in DNA as a template and a pair of primers (HVT ins VP2 casse F and HVT VP2 casse UL55 R) to amplify the HA cassette with 40 bp homologous sequences flanking both terminals. After digestion with Dpn I to eliminate possible plasmid contamination, the PCR product was electroporated via the first red recombination method [36] into competent pHVT BAC cells to generate the first recombination with the cassette at the indicated sites. The target recombinant pHVT-VP2-09 BAC was generated by deletion of the kanamycin resistance gene by the second recombination ( Figure 1C). To insert a kanamycin resistance gene into plasmid pHVT-VP2-09, a pair of specific primers (Kan ins' VP2 F and Kan ins' VP2 R) ( Table 1) were designed with the help of VectorNTI and prime 3 software with two Sac I restriction sites added to both terminals for cutting and ligation. The construct was examined by gene sequencing to check the correct insertion of the kanamycin resistance gene. Another pair of primers (HVT ins VP 2 casse UL55 F and HVT ins VP 2 casse UL55 R) ( Table 1) was used to insert the VP 2 cassette into the HVT BAC clone through the En Passant protocol. To repair the gC genes of the gC-negative virus, a pair of primers (HVT gC flanking F and HVT gC flanking R) ( Table 1) were used to amplify a fragment that included the gC gene and two homologous 1 kbp flanking sequences of gC. The BACs and mutants were subjected to RFLP analysis with EcoR I.

One-Step Growth Kinetics
The growth characteristics of viruses were tested on primary or secondary CEFs with an MOI of 0.01, as described previously [39], with a slight modification. Briefly, the virus titers of the cell-associated viruses were checked at 16, 24, 36, 48, 60, 72, 84, and 96 h postinfection (h.p.i.) for parental viruses and mutants. Virus titers were tested following the standard plaque-forming unit (pfu) titration method [34]. The growth kinetics curve was established based on data from three independent experiments [39].

One-Step Growth Kinetics
The growth characteristics of viruses were tested on primary or secondary CEFs with an MOI of 0.01, as described previously [39], with a slight modification. Briefly, the virus titers of the cell-associated viruses were checked at 16, 24, 36, 48, 60, 72, 84, and 96 h post-infection (h.p.i.) for parental viruses and mutants. Virus titers were tested following the standard plaque-forming unit (pfu) titration method [34]. The growth kinetics curve was established based on data from three independent experiments [39].

Indirect Immunofluorescence Assay (IFA) and Western Blot Analysis
For the IFA test, chicken embryo fibroblast monolayers (80-90% confluent) were infected with HVT-VP2-09 or HVT, and washed 3 times with phosphate-buffered saline (PBS) and fixed with ice-cold ethanol for 15 min following the appearance of CPE. The cells were overlaid with polyclonal rabbit antibodies produced by vaccination with the IBDV VP2 protein (1:200) and incubated at 37 • C for 1 h. Washed 3 times with PBS and incubated with goat anti-rabbit IgG-FITC antibody (1:1000) (Abcam) at 37 • C for 1 h. The wells were then washed, dried, and analyzed by inversion fluorescence microscopy (Zeiss, SM-33TCI).
For Western-blot analysis, CEFs were infected with HVT-VP2-09 viruses at an MOI of 0.01. Infected cells were lysed with radio immunoprecipitation assay (RIPA) lysis buffer (Biosharp Life Sciences, Beijing, China) and 1% phenyl methyl sulfonyl fluoride (PMSF) protease inhibitor (Solarbio, Beijing, China) for 5 min on ice, and cell lysates were denatured by heating at 95 • C for 10 min. Proteins were separated by SDS-10% polyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membranes (Merck) as described previously [39]. A mixture of VP2 polyclonal rabbit antibodies was used as the primary antibody for Western blotting, and a 1:10,000 dilution of goat anti-rabbit IgG (Abcam) was used as the secondary antibody. HVT-infected CEFs were used as a control. Samples were detected with enhanced chemiluminescence (Sigma-Aldrich) [40].

Immunization Experiments
For the IBDV protection test, 40 one-day-old SPF chickens were randomly divided into four groups ( Table 2). Chickens in group A were inoculated subcutaneously with HVT Fc-126 virus, and chickens in group B were inoculated subcutaneously with HVT-VP2-09 at a dose of 3000 PFU. Chickens in groups C and D were inoculated subcutaneously with 0.2 mL of PBS as controls. After inoculation, sera were sequentially collected from each animal, and antibody titers were determined by the AGP test. Seven weeks after inoculation, all chickens were challenged with the very virulent IBDV field strain NJ09 at a dose of 100 LD 50 and observed daily for 84 h to analyze morbidity and mortality.

Generation of Bacterial Artificial Chromosomes of the HVT Fc-126 Strain
Colonies with resistance to chloramphenicol were obtained after electroporating DNA of HVT Fc-126 harboring mini-F sequences into E. coli DH10B competent cells. Then, the Midi-prep DNA of the selected colonies was electroporated into E. coli GS1783 competent cells to generate the infectious clone BAC HVT-G for further VP2 cassette insertion with the En Passant method. RFLP of BAC HVT-G with BamH I and EcoR I showed suspected patterns compared to in silico predictions based on the published whole genome of the HVT Fc-126 strain (GebBank: AF291866) ( Figure 3B).

Construction of the Recombinant HVT BAC with the Expression Cassette of IBDV VP2
Following the En Passant protocol, we sought to insert the expression cassette of VP2 from IBDV (NJ09) (Figure 1). The insertion was targeted in lieu of the gC gene of the HVT genome. With the first Red recombination, approximately 300 ng of PCR product of the expression cassette of VP2 was gel-purified with a DNA gel purification kit (Shanghai Generay Biotech Co. Ltd., Shanghai, China) and electroporated into BAC HVT-G . Several colonies resistant to kanamycin and chloramphenicol were obtained 2 days after plating. One colony terminated BAC HVT-VP2-09 K+ was selected for further deletion of the kanamycin resistance marker gene by the 2nd Red recombination, resulting in colonies with resistance to chloramphenicol but not kanamycin. One of the final recombinant BACs containing the VP2 expression cassette, termed BAC HVT-VP2-09 , was selected for PCR and RFLP analysis. PCR results showed bands of 450 bp and 3757 bp for BAC HVT and BAC HVT-VP2 , respectively. These results were exactly the same as suspected, and subsequent sequencing proved the correct insertion ( Figure 3A). RFLP patterns corresponded well with the predicted patterns after digestion with BamH I and EcoR I ( Figure 3B

Generation of Bacterial Artificial Chromosomes of the HVT Fc-126 Strain
Colonies with resistance to chloramphenicol were obtained after electroporating DNA of HVT Fc-126 harboring mini-F sequences into E. coli DH10B competent cells. Then, the Midi-prep DNA of the selected colonies was electroporated into E. coli GS1783 competent cells to generate the infectious clone BAC HVT-G for further VP2 cassette insertion with the En Passant method. RFLP of BAC HVT-G with BamH I and EcoR I showed suspected patterns compared to in silico predictions based on the published whole genome of the HVT Fc-126 strain (GebBank: AF291866) ( Figure 3B).  (Figure 3B). RFLP, PCR, and sequencing results showed correct insertion of the VP2 expression cassette and deletion of the kanamycin resistance selection marker, even though the RFLP pattern was slightly different from the expected pattern, which might be due to the different passages of HVT Fc-126 used in this study and the reference strain genome.

Construction of the Recombinant HVT BAC with the Expression Cassette of IBDV VP2
Following the En Passant protocol, we sought to insert the expression cassette of VP2 from IBDV (NJ09) (Figure 1). The insertion was targeted in lieu of the gC gene of the HVT genome. With the first Red recombination, approximately 300 ng of PCR product of the expression cassette of VP2 was gel-purified with a DNA gel purification kit (Shanghai Generay Biotech Co., Ltd., Shanghai, China) and electroporated into BAC HVT-G . Several colonies resistant to kanamycin and chloramphenicol were obtained 2 days after plating. One colony terminated BAC HVT-VP2-09 K+ was selected for further deletion of the kanamycin resistance marker gene by the 2nd Red recombination, resulting in colonies with resistance to chloramphenicol but not kanamycin. One of the final recombinant BACs containing the VP2 expression cassette, termed BAC HVT-VP2-09 , was selected for PCR and RFLP analysis. PCR results showed bands of 450 bp and 3757 bp for BAC HVT and BAC HVT-VP2 , respectively. These results were exactly the same as suspected, and subsequent sequencing proved the correct insertion ( Figure 3A). RFLP patterns corresponded well with the predicted patterns after digestion with BamH I and EcoR I ( Figure 3B Figure 3B). RFLP, PCR, and sequencing results showed correct insertion of the VP2 expression cassette and deletion of the kanamycin resistance selection marker, even though the RFLP pattern was slightly different from the expected pattern, which might be due to the different passages of HVT Fc-126 used in this study and the reference strain genome.   (Table 1).

Growth Kinetics of HVT Fc-126 Vectored VP2
The growth properties of the engineered mutant (HVT-VP2-09 (NJ09)) were pared with those of the parental virus (HVT Fc-126) in three independent experim Virus titers were tested on fresh CEF cells after trypsinization of virus-infected cells out freeze-thawing. The virus titers determined at 0 h, 16 Figure 5). results showed that the BAC constructed in this study was the whole genome clone HVT Fc-126 strain and the insertion of the VP2 cassette in the noncoding area be UL55 and UL56 did not affect the growth ability of the mutant virus, at least in CEF   (Table 1).

Growth Kinetics of HVT Fc-126 Vectored VP2
The growth properties of the engineered mutant (HVT-VP2-09 (NJ09)) were compared with those of the parental virus (HVT Fc-126) in three independent experiments. Virus titers were tested on fresh CEF cells after trypsinization of virus-infected cells without freeze-thawing. The virus titers determined at 0 h, 16 Figure 5). These results showed that the BAC constructed in this study was the whole genome clone of the HVT Fc-126 strain and the insertion of the VP2 cassette in the noncoding area between UL55 and UL56 did not affect the growth ability of the mutant virus, at least in CEF cells.
Vaccines 2022, 10, x FOR PEER REVIEW 9 of 14 suspected ( Figure 4B). Subsequent sequencing results proved the correct recovery of the gC gene as in the parental virus and confirmed the correct insertion of the VP2 cassette in the noncoding area between UL55 and UL56.  (Table 1).

Growth Kinetics of HVT Fc-126 Vectored VP2
The growth properties of the engineered mutant (HVT-VP2-09 (NJ09)) were compared with those of the parental virus (HVT Fc-126) in three independent experiments. Virus titers were tested on fresh CEF cells after trypsinization of virus-infected cells without freeze-thawing. The virus titers determined at 0 h, 16 Figure 5). These results showed that the BAC constructed in this study was the whole genome clone of the HVT Fc-126 strain and the insertion of the VP2 cassette in the noncoding area between UL55 and UL56 did not affect the growth ability of the mutant virus, at least in CEF cells.

Expression of IBDV VP2 by Recombinant HVT Fc-126 Vectored VP2
In Western blotting, while proteins with molecular masses of approximately 48 kilodalton (KDa) were observed in lysates of CEFs infected with HVT Fc-126 vector VP2, no protein band was detected in CEFs infected with HVT Fc-126 ( Figure 6). The sizes of the detected bands were in accordance with the theoretical results we expected. An indirect immunofluorescence test was performed with the first antibody against VP2 of IBDV obtained from rabbits. Strong signals were observed on HVT-VP2-09 (NJ09) plaques using goat anti-rabbit IgG antibodies. In contrast, no signals were observed on parental HVT Fc-126 strain plaques under the same treatment conditions (Figure 7). These results showed that the mutant HVT-VP2-09 (NJ09) could express VP2 with high reactivation and that VP2 expression of HVT-VP2-09 (NJ09) was strong and stable in vitro on CEFs.

Expression of IBDV VP2 by Recombinant HVT Fc-126 Vectored VP2
In Western blotting, while proteins with molecular masses of approximately 48 kilodalton (KDa) were observed in lysates of CEFs infected with HVT Fc-126 vector VP2, no protein band was detected in CEFs infected with HVT Fc-126 ( Figure 6). The sizes of the detected bands were in accordance with the theoretical results we expected. An indirect immunofluorescence test was performed with the first antibody against VP2 of IBDV obtained from rabbits. Strong signals were observed on HVT-VP2-09 (NJ09) plaques using goat anti-rabbit IgG antibodies. In contrast, no signals were observed on parental HVT Fc-126 strain plaques under the same treatment conditions (Figure 7). These results showed that the mutant HVT-VP2-09 (NJ09) could express VP2 with high reactivation and that VP2 expression of HVT-VP2-09 (NJ09) was strong and stable in vitro on CEFs.

Immunogenicity of HVT Fc-126 Vectored VP2 in Chickens
All chickens were healthy post-inoculation, and no AGP antibodies against IBDV were detected in chickens in groups A, C, or D. AGP antibodies were detected in the chickens of group B at 21, 28, 35, and 42 days post-inoculation (Figure 8).

Immunogenicity of HVT Fc-126 Vectored VP2 in Chickens
All chickens were healthy post-inoculation, and no AGP antibodies against IBDV were detected in chickens in groups A, C, or D. AGP antibodies were detected in the chickens of group B at 21, 28, 35, and 42 days post-inoculation (Figure 8). All chickens other than group D were challenged with virulent NJ09 at seven weeks post-inoculation and observed daily for 84 h. At 84 h post-challenge (P.C.), eight chickens died in group C, and for chickens in group A, a total of seven chickens died in the 84 h P.C. Meanwhile, no chickens died in group B P.C. (Table 2). All dead chickens showed edema in bursals. No chickens died in group D. All surviving chickens were euthanized to check lesions in bursals and muscles. All the chickens in groups A and C showed edema in bursals and/or hemorrhage in the keratin between the glandular stomach and gizzard. Three chickens in group B showed edema in bursal.
The chickens in group D showed normal bursal and keratin between the glandular stomach and gizzard (data not shown) (Figure 9) ( Table 2). These results showed that HVT-VP2-09 (NJ09) could induce efficient protection against virulent IBDV NJ09 strain challenge, indicating the successful construction of the infectious clone of the whole genome of the HVT Fc-126 strain and the promising prospect of HVT-VP2-09 (NJ09) to serve as a vectored vaccine candidate. Further research on the protection against the MDV challenge is needed.
The chickens in group D showed normal bursal and keratin between the glandular stomach and gizzard (data not shown) (Figure 9) ( Table 2). These results showed that HVT-VP2-09 (NJ09) could induce efficient protection against virulent IBDV NJ09 strain challenge, indicating the successful construction of the infectious clone of the whole genome of the HVT Fc-126 strain and the promising prospect of HVT-VP2-09 (NJ09) to serve as a vectored vaccine candidate. Further research on the protection against the MDV challenge is needed. Figure 9. The histologic appearance of the bursa of Fabricius in chicks after IBDV (NJ09) challenge Chickens in group A were inoculated subcutaneously with HVT Fc-126 virus, and those in group B were inoculated subcutaneously with HVT-VP2-09 at a dose of 3000 PFU. Chickens in groups C and D were inoculated subcutaneously with 0.2 mL of PBS as controls. Six weeks after inoculation, the chickens of groups A-C were challenged with virulent NJ09 at a dose of 100 LD50 and observed daily for 84 h to analyze the histologic appearance changes.

Conclusions
In this study, a novel bacterial artificial chromosome of the HVT Fc-126 strain was generated with the insertion of mini-F sequences in lieu of the gC gene. The HVT vector HVT-VP2-09 serves as an infectious clone of the HVT Fc-126 strain's entire genome. In conclusion, the vectored vaccine was successfully constructed using the En Passant protocol using BAC with the insertion of a VP2 expression cassette of the highly contagious IBDV field isolate NJ09 strain. Vaccinated 1-day-old SPF chickens can produce significant antibodies as early as the third week and can resist the attack of virulent IBDV strains as late as the sixth week, indicating a very strong immune protection effect. This will be of great significance for the construction of a multivalent turkey herpesvirus live vector vaccine. To our knowledge, the HVT Fc-126 strain has never been used as a vaccine candidate before, and the HVT-VP-09 can be used as a potential vaccine against IBDV strain NJ09 in chicken. Figure 9. The histologic appearance of the bursa of Fabricius in chicks after IBDV (NJ09) challenge Chickens in group A were inoculated subcutaneously with HVT Fc-126 virus, and those in group B were inoculated subcutaneously with HVT-VP2-09 at a dose of 3000 PFU. Chickens in groups C and D were inoculated subcutaneously with 0.2 mL of PBS as controls. Six weeks after inoculation, the chickens of groups A-C were challenged with virulent NJ09 at a dose of 100 LD 50 and observed daily for 84 h to analyze the histologic appearance changes.

Conclusions
In this study, a novel bacterial artificial chromosome of the HVT Fc-126 strain was generated with the insertion of mini-F sequences in lieu of the gC gene. The HVT vector HVT-VP2-09 serves as an infectious clone of the HVT Fc-126 strain's entire genome. In conclusion, the vectored vaccine was successfully constructed using the En Passant protocol using BAC with the insertion of a VP2 expression cassette of the highly contagious IBDV field isolate NJ09 strain. Vaccinated 1-day-old SPF chickens can produce significant antibodies as early as the third week and can resist the attack of virulent IBDV strains as late as the sixth week, indicating a very strong immune protection effect. This will be of great significance for the construction of a multivalent turkey herpesvirus live vector vaccine. To our knowledge, the HVT Fc-126 strain has never been used as a vaccine candidate before, and the HVT-VP-09 can be used as a potential vaccine against IBDV strain NJ09 in chicken.