Avian influenza virus (H5N1) isolates from Thailand were selected and the nucleotide sequences of hemagglutinin (HA), neuraminidase (NA), matrix (M), and non-structural (NS) genes were identified as H5 and N1 with the accession number: A/Thailand/HA20/2005 (DQ885618), A/Thailand/M38/2005 (DQ885619Q), A/Thailand/NA60/2005 (DQ885620), and A/Thailand/NS49/2005 (DQ885621), respectively. All viruses were grown in MDCK cell line and processed in biosafety level 3 containment by trained lab technicians. Viral RNA was extracted from culture supernatant by using QiaAamp viral RNA mini kit (Qiagen, Germany).
Cloning of avian influenza virus genes (HA, NA, NS, M)
After cDNA was amplified from viral RNA lysate with universal primer (5'-AGCAAAAGCAGG-3') by RT-PCR using Superscript III One step RT PCR (Invitrogen, USA). PCR was used to amplify HA gene with forward primer (5'-CTC GAG GAT ATC CAA AAG CAG GGG TCC GAT CT-3') and reverse primer (5'-AAG CTT GCG GCC GCC AAT GAC CCA TTG GAA CA-3'), NA gene with forward primer (5'-CTG CAG AAG CTT AGC AAA AGC AGG AGT-3') and reverse primer (5'-GAA TTC GCG GCC GCG TAC TTG TCA ATG GTG A-3'), M gene with forward primer (5'-GAG CTC GAT ATC ATG AGT CTT CTA ACC GAG GTC-3') and reverse primer (5'-GAA TTC GCG GCC GCC TTG AAT CGC TGC ATT TGC AC-3'), and NS gene with forward primer (5'-CTC GAG GAT ATC AGC AAA AGC AGG GTG-3') and reverse primer (5'-GAA TTC GCG GCC GCC CAT CTT ATC TCT TGA-3'). The expected amplified size of HA, NA, M, and NS1 genes are 1778 bps, 1413 bps, 1027 bps, and 890 bps, respectively.
PCR was performed for 3 cycles, each consisted of 94°C denaturation step for 1 min (6 min for first cycle), 55°C annealing step for 1 min, and 72°C extension step for 1 min, followed by 31 cycles of 94°C for 15 sec, 55°C for 45 sec, 72°C for 90 sec and the final extension at 72°C for 10 min in both of first round and second round PCR. The amplified products were cloned into vector pGEM-T (Promega, USA) and subcloned into pBAD/His C vector (Invitrogen, USA.) and used to transform LMG194 competent E. coli cells. All colonies of E. coli containing pBAD/His-HA, pBAD/His-NA, pBAD/His-M, pBAD/His-NS1, were checked for positive clones containing insert fragment of and by digestion plasmid DNA with restriction enzymes, Pst I and EcoRI.
To construct the mammalian expression vector, pSecTag2/Hygro C (Invitrogen, USA.) for HA, NA, M, and NS1 protein expression in COS-7 cell line, the XhoI/ApaI digested DNA from pBAD/His-HA or pBAD/His-NA or pBAD/His-NS or pBAD/His-M was subcloned into digested pSecTag2/Hygro C vector to produce pSec-His-HA, pSec-His-NS1 and pSec-His-NA. The pBAD/His-HA, pBAD/His-NA, pBAD/His-NS1, pBAD/His-M DNA was used for transformation into DH5α competent E. Coli cells and pSec-His-HA, pSec-His-NS1 and pSec-His-NA DNA to transform COS-7 cell line by using polyfect transfection system (Qiagen, USA).
Recombinant protein expression and purification [7, 8]
Overnight culture of E. coli strain LMG containing pBAD/His-HA or pBAD/His-NA or pBAD/His-NS1 or pBAD/His-M was added to a final of 0.2% to induce the production of polyhistidine tagged protein and the recombinant protein was extracted and purified by metal affinity column, MagneHis™ protein purification system (Promega, USA), to purify the polyhistidine tagged protein.
The stably expressed pSec-His-HA, pSec-His-NS1, pSec-His-M and pSec-His-NA in COS-7 cell lines in medium containing 200 μg/ml of hygromycin B were lysed with lysozyme. The cell lysate was used to purify recombinant protein with MagneHis™ protein purification system (Promega, USA). The recombinant HA, NA, NS, M proteins were detected by SDS-PAGE analysis (3.85% stacking gel and 10% separating gel with a constant voltage of 150 volts for 1 hour) and followed by Western blot analysis against mouse anti-Xpress serum (Invitrogen, USA) as previously described .
Immunization of mice
Animal usage in this study was performed according to the national guidelines and instructional policies. Mice were purchased from the National Laboratory Animal Center, Thailand. Six to eight-week-old pathogen-free, female Balb/c mice were used for vaccination. The animals were housed in a temperature controlled environment at 22–24°C with 12 h day-night cycles, and received food and water ad libitum. Mice were immunized three times intramuscularly (IM) at 1-week interval with 200 μl doses of 50, 100, 150, 200, and 250 μg of rHA, rNA, rNS1, and rM protein produced in E. coli and COS-7 cells plus an emulsion prepared with Alum adjuvant. Two mice were injected with pSecHis/HygroC vector protein as control. Boosts were given at 2 and 3 weeks after the first immunization. One week after the last boosting, mice were sacrificed and whole blood was collected for immunogenicity analysis. Then, 250 μg of rHA, rNA, rNS1, and rM protein produced in E. coli and COS-7 cells were selected to immunize 5 groups of mice (5 mice/group) for each protein and boosted as described. Serum was prepared by centrifugation of clotted blood at 1800 × g for 5 min, stored at -80°C until used.
Detection of H5N1 specific antibody from immunized mice sera 
The presence of serum anti-HA, -NA, -NS1, -M specific immunoglobulins was determined by an enzyme linked immunosorbent assay (ELISA). Briefly, 500 μl of purified HA or NA, or NS or M proteins were incubated with 50 μl of MagnaHis bead (Promega, USA) and 5 μg/ml of bead solution was added to each 96-well plates. Diluted mice sera in blocking solution (PBS/Tween 20 containing 5% skim milk) were added after 5 times washing. The horseradish peroxidase-labeled goat anti-mouse Ig(G+M+A) diluted 1:1000 in blocking solution and 100 μl of TMB substrate were used for ELISA. Reactions were stopped by adding 100 μl of 1 N H2SO4. The absorbance was measured at 450 nm with an ELISA microplate reader. The cut off value of absorbance was calculated as formula: cut off = 0.124 [(X+3SD) × 2], X = mean of all negative samples absorbance +3 standard deviation) × 2. ELISA index (EI = Absorbance/cut off) is a ratio of absorbance value of any sera and cut off value. EI of any area is less than 1, interpretation is negative, and EI ≥ 1 means positive result.
Micro neutralization assay 
The H5N1 virus, A/Thailand/RPNP/2005 (DQ885616) with tissue infectious dosage 50 (TCID50) per ml were incubated with diluted mice serum samples twofold in medium, from 1:4 to 1:2560. Mixtures of virus and serum were transferred to monolayers of MDCK cells and incubated for one hour at 37°C in 5% CO2 for 3 days. After three days, cell medium was incubated with 100 μl of monoclonal antibodies directed against the influenza type A or type B nucleocapsid antigen (Chemicon Europe, Hampshire, UK). After incubation for 60 min at 37°C and washing, affinity-purified peroxidase-conjugated, goat anti-mouse IgG (Jackson ImmunoResearch Europe, Cambridgeshire, UK), was added and the plates were incubated at room temperature for 120 min. After being washed, 100 μl substrate (orthophenylenediamine) was added, and the enzyme reaction was stopped after 30 min with 100 μl 2.5 M sulfuric acid. The reaction was quantified by measuring the OD at wavelength 492 nm. The neutralization (NT) titers were defined as the inverted value of the serum dilution giving ≥ 50% OD reduction compared to the virus control.