Influenza virus WSN-HK that contained the NA gene from A/Hong Kong/1/68 (H3N2) and all other genes from A/WSN/33 (H1N1), was obtained from Dr. Yoshi Kawaoka and used to rescue the mutant WSN NA gene. The Madin-Darby bovine kidney (MDBK) cell line was cultured in minimal essential medium containing 10% fetal bovine serum. Madin-Darby canine kidney (MDCK) cells were cultured in the same conditions as were MDBK cells.

Viral cores, nucleoprotein protein (NP) and polymerase protein (P) were isolated by glycerol and glycerol-cesium chloride (CsCl) gradient centrifugation from Influenza A virus, strain X-31, a reassortant of A/HK/68 and A/PR/8/34, after viral particle disruption with detergents (100 mM Tris-HCl [pH 7.4], 100 mM NaCl, 5 mM MgCl₂, 1 mM dithiothreitol, 5% glycerol, 1% NP40). Viral core fraction were separated from sodium dodecyl sulfate polyacrylamide gel electrophoresis and treated with micrococcal nuclease (Sigma, St. Louis, MO, USA) to degrade RNA [13]. Final core proteins were stored at -20℃ for further experiment.

A plasmid pT3WSN (NA15) containing the WSN NA gene flanked by the Ksp632I site and T3 RNA polymerase promoter sequence, was obtained from Dr. Peter Palese (Mount Sinai School of Medicine, New York, NY, USA) [15]. A second plasmid, pT3WSN-NAmut which had additional restriction enzyme sites, SacI and KpnI in the stalk region was made by polymerase chain reaction. Plasmids for the generation of recombinant influenza A viruses were constructed from pT3WSN-NAmut by inserting nucleotides derived from HBV surface antigen between 139-147 or pre-surface antigen in the region encoding the NA stalk respectively. An NA ribonucleoprotein (RNP) complex was prepared by in vitro transcription of pT3WSN-NAmut with T3 RNA polymerase in the presence of the NP and P for the generation of mutant influenza viruses after these plasmids were digested with Ksp632I and filled in with Klenow fragment.

The NA RNP complex was transfected into 70% to 80% confluent MDBK cells infected 1 hour before transfection with WSN-HK at a multiplicity of infection (MOI) of 1. Eighteen hours after transfection, transfectants in the supernatant were plaqued on MDBK cells and then plaque purified three times in MDBK cells.

The ability of recombinant viruses to grow in MDBK and MDCK cells was determined by analyzing multiple replication cycles. Confluent MDBK and MDCK cell monolayers grown on 6 well plates were washed with phosphate-buffered saline, overlaid with 0.5 mL of diluted virus suspension (to achieve a MOI of 0.01 plaque-forming unit [PFU]/cell), and incubated at room temperature (RT) for 40 minutes. The virus suspension was then removed by aspiration, and 3 mL of minimal essential medium was added. Sample supernatants were collected at 12-hour intervals until 72 hours after inoculation. The virus titer of the sample was determined by plaque assay on MDBK and MDCK cells.

Recombinant influenza viruses expressing HBV epitopes at 80 hemagglutination unit (HAU)/50 µL concentration with two fold serial dilution were used as a coating antigen on 96-well microtiter plates. After incubation for 1 hour at RT, plates were washed once with phosphate buffered saline (PBS) and then incubated at 37℃ for 1 hour in 0.1% bovine serum albumin-PBS to block the remaining binding sites. After removal of the blocking buffer and washing, HBV polyclonal antibody at 2 µg/mL was added in each well of microtiter plates and the plates were incubated at 37℃ for 1 hour. The plates were washed again (3 times with PBST), a 1:1,000 dilution of alkaline phosphatase conjugated solution for 1 hour at RT. This was followed by removal of the conjugate, washing and addition of p-nitrophenyl phosphate as a substrate. Finally, absorbance was read at 405 nm.

Recombinant influenza A viruses were generated by a RNP transfection based reverse genetic system. In previous study, Seong and Brownlee [13] used RNA transcribed in vitro and micrococcal nuclease-treated virus cores proteins (purified NP and P) for genetic rescue into influenza virus. Using the same procedure, we attempted to rescue recombinant influenza A viruses which contained additional amino acid peptides in the stalk region derived from the surface and pre-surface protein of HBV. The rescue system depends on the fact that the NA gene of WSN (H1N1) virus is responsible for plaque formation of the virus in MDBK cells without trypsin protease. Hence, the WSN-HK reassortant virus, which contains only the NA gene from A/Hong Kong/1/68 (H3N2) virus and the rest of the genes from WSN virus, can be used as a helper virus to rescue the WSN NA gene (Fig. 1). Because the WSN-HK virus does not plaque on MDBK cells, the rescue of the WSN NA gene allows the virus to plaque in these cells. In this study, we generated two chimeric influenza A viruses rIV-BVS which has additional 22 amino acids derived from HBV surface antigen and rIV-BVPreS2 which has same number of epitope sequences derived from HBV pre-S2 genome in the same place in stalk region (Fig. 2) and wild type virus using wild type NA gene from WSN virus as a control. These recombinant viruses were harvested at 18 hours after transfection of RNP complex in the cell supernatants and then plaque purified three times in MDBK cells.

To access the effect of the replacement of the NA gene segment in WSN virus on subsequent virus amplification in cells, we infected triplicate samples of near confluent MDBK cells with rIV-BVS, rIV-BVPreS2 and WSN virus at a low MOI (0.5) and monitored virus replication by assaying culture supernatants. Both rIV-BVS and rIV-BVPreS2 had maximal titers (7.7 and 7.9 log₁₀ PFU/mL, respectively) similar to wild type WSN virus (7.4 log₁₀ PFU/mL) (Fig. 3A). Although the peak titers of all three viruses were similar, rIV-BVS reach early to the maximum titer at 36 hours compare to WSN wt virus at 72 hours. Additionally, multiple cycle growth curves were performed using MDCK cells. Both rIV-BVS, rIV-BVPreS2 reached maximal titers of 8.3 log₁₀ PFU/mL, at 72 hours (Fig. 3B). On the other hand, WSN wt virus showed lower maximal titer of 7.5 log₁₀ PFU/mL at 36 hours then recombinant viruses (p<0.05) and the viral titer slightly decreased until 72 hours (Fig. 3B).

To determine whether the HBV epitopes that expressed in the NA stalk are exposed on the surface of the recombinant influenza viruses, we tested the reactivity of the anti-HBV polyclonal antibody to rIV-BVS and rIV-BVPreS2. In an ELISA, the anti-HBV polyclonal antibody reacted with the rIV-BVS and rIV-BVPreS2 but the WSN virus that had wild type NA protein showed only background level in the lowest diluted fraction (Fig. 4). A polyclonal antibody to the HBV reacted with both rIV-BVS and rIV-BVPreS2 but the intensity of the reaction with the rIV-BVPreS2 was weaker than the intensity of the reaction with the rIV-BVS.