Primary chicken embryo fibroblast (CEF) cells were prepared from 10-day-old specific-pathogen-free embryos (Vital Merial Experimental Animal Co., Ltd., Beijing, China) (15). The HD11 chicken macrophage cell line was maintained in RPMI-1640 medium with 10% FBS (v/v). The DNA of C. psittaci strain CB7 was extracted and stored in our lad as reported previously (10).

The recombinant HVT was generated as described previously (10). The recombinant HVT bacterial artificial chromosome (BAC) was based on EF-1α promoter, which was from the vector of pEF6/V5-His (Invitrogen, Carlsbad, CA, USA). A pair of primers used to amplify the expression cassette with the EF-1α promoter at the 5′ end and the BGH polyadenylation (poly A) site at the 3′ end was designed using Oligo 7 (Molecular Biology Insights, USA). The forward primer was 5′-GGTTAATTAACCTTCTAGGTCTTGAAAGGAGTGGGA-3′, and the reverse primer was 5′-GGTTAATTAAGCCTCAGAAGCCATAGAGCCCACC-3′. Both primers contained a PacI restriction site at their 5′ termini. The recombinant virus was designated as rHVT-EF-pmpD.

The plaque size, morphology, and plaque-forming unit (PFU) of rHVT-EF-pmpD were compared with those of the same passage of rHVT-CMV-pmpD by the immunohistochemical assay as recorded previously (15). Briefly, recombinant viruses were 10-fold diluted and inoculated into the CEF cells, which were seeded in six-well plates. Four days later, ice-clod acetone was added into each well to fix the cells. Cells were washed by phosphate-buffered saline (PBS) and then blocked by blocking buffer (PBS containing 0.1% BSA). Cells were incubated with the wild type HVT-specific polyclonal antibodies raised in chickens (diluted 1:100), and subsequently reacted with horseradish peroxidase-labeled goat-anti-chicken IgG (1:4,000) (Sigma-Aldrich, Shanghai, China). Cells containing the conjugated antibody were identified by incubation at 37°C for 1 h in developing solution containing 1% (w/v) 3-amino-9-ethylcarbazole (Sigma) and 0.02% (v/v) H₂O₂ in 0.1 M sodium acetate (pH 4.8). Plaques were counted using an inverted microscope. Moreover, the growth rates of the recombinant viruses were studied on CEF cells by calculating the virus plaques at 12, 24, 48, 72, 96, and 120 h postinfection (16).

Immunoblot analysis was carried out as described previously (10). Briefly, 3,000 PFU of rHVT-CMV-pmpD, rHVT-EF-pmpD, and parental HVT were used to infect CEF cells seeded in T25 flasks. The cells were washed twice with PBS when the cytopathic effect occurred. Lysates of cells were subjected to 12% SDS-PAGE and electroblotted to polyvinylidene fluoride membranes, followed by 0.25% trypsin for 2 min. Membranes were incubated with the C. psittaci strain 6BC-specific polyclonal antibodies (diluted 1:100) previously prepared by our own lab, and then reacted with horseradish peroxidase-labeled goat-anti-chicken IgG (1:4,000) (Sigma-Aldrich, Shanghai, China). The PmpD-N glycoprotein bands were visualized using the enhanced DAB reagents (Tiangen, Beijing, China) according to the manufacturer’s instructions.

The expression and distribution of PmpD-N were determined in rHVT-CMV-pmpD-infected cells and rHVT-EF-pmpD-infected cells by immunofluorescence. First, CEF cells were infected with 3,000 PFU rHVT-CMV-pmpD and rHVT-EF-pmpD, respectively. The cells were probed with mouse anti-PmpD-N polyclonal serum of C. psittaci (previously prepared in our lab) and anti-HVT polyclonal serum (IVDC, Beijing, China) at a dilution of 1:100, and then overlaid with a mixture of goat anti-mouse IgG labeled with Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) and goat anti-chicken IgY labeled with Alexa Fluor 568 (1:600 dilution) (Invitrogen, Carlsbad, CA, USA). Moreover, cell nuclei were stained with 1:10,000 dilution of 4,6-diamidino-2-phenylindole (DAPI) for 1 min or longer. Finally, the rinsed cover slips were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and examined using a confocal microscope (Nikon, Tokyo, Japan).

HD11 cells were infected by 400 PFU rHVT-CMV-pmpD and rHVT-EF-pmpD, respectively. Cell culture supernatants were collected on day 1, 2, and 4 postinfection. OD values of supernatants were measured at 540 nm. NO production by activated HD11 cells was assessed as nitrite content in conditioned media using Griess reagent as illustrated previously (19). Sodium nitrite was used as the standard.

The infected cells treated above were also collected on day 1, 2, and 4 after inoculation. The cells were washed twice with PBS. Total RNA of cells on different day’s postinfection was extracted. The quantity of IL-6 was measured by relative quantitative real time RT-PCR, and the internal control gene was GAPDH as described previously (20).

Data were analyzed using the one-way ANOVA. A P-value of less than 0.05 was considered statistically significant.

The pmpD-N gene with EF-1α promoter was successfully inserted into the HVT BAC region between UL45 and UL46. The recombinant HVT BAC DNA based on EF-1α promoter was transfected into CEF cells. Four days after transfection, typical plaques appeared in the CEF monolayers, these plaques being similar in size and morphology compared with those formed by rHVT-CMV-pmpD (Figure 1). The viral titration by staining plaques revealed no significant difference between rHVT-EF-pmpD and rHVT-CMV-pmpD.

To confirm the expression of the PmpD-N protein, CEF cells infected with rHVT-EF-pmpD, rHVT-CMV-pmpD, and parental HVT were analyzed by immunoblot and immunofluorescence. A 43-kDa band corresponding to the PmpD-N polypeptide was identified with polyclonal antibodies generated against C. psittaci 6BC strain by immunoblot analysis (Figure 2A). Under immunofluorescence microscopy, PmpD-N protein of rHVT-EF-pmpD and rHVT-CMV-pmpD was both expressed in the cytoplasm and on the cell surface (Figures 2B,C).

Growth trends of both recombinant viruses were similar as revealed by calculating the virus plaques at various times postinfection (Figure 3). Growth rates displayed a significant increase between 24 and 96 h, and a sharp decrease from 96 to 120 h. Except for 96 h postinfection, the growth rate of the rHVT-EF-pmpD was consistently higher than that of rHVT-CMV-pmpD, with a consequential difference appearing on 48, 72, and 120 h postinfection.

The quantity of NO and IL-6 on HD11 cells infected by rHVT-CMV-pmpD and rHVT-EF-pmpD was assayed on days 1, 2, and 4. The NO production of rHVT-EF-pmpD was significantly higher than those of rHVT-CMV-pmpD on day 2 and day 4 (P < 0.05) (Figure 4). The IL-6 level of rHVT-EF-pmpD was apparently higher than that of rHVT-CMV-pmpD on day 4 (Figure 5).