The pathogenic IBV strain Massachusetts 41(M41) used in this study was purchased from China Institute of Veterinary Drug Control(Beijing, China) and propagated in 10-day-old specific pathogen-free(SPF)chicken embryos(Beijing Merial Vital Laboratory Animal Technology Co., Ltd). The infected allantoic fluid was harvested at 72 h postinoculation, confirmed to lack other pathogens as analyzed by reverse transcription polymerase chain reaction(RT-PCR) and bacterial culture, titrated in 10-day-old SPF chicken embryos, and finally stored at –80 ℃ before animal infection.
The SPF white leghorn chicks used in this study were self-hatched. The chicks were raised in a sterile, quiet, comfortable animal room of the Institute of Animal Science and Technology of Guangxi University until reaching 14 days of age. Sterile food and water were provided ad libitum.
Forty-eight 2-week-old chicks were r and omly allocated to two groups of 24 chicks each and raised separately in two isolation rooms with individual ventilation. Chicks from group A were inoculated oculonasally with 0.2 mL of 106 EID50 of IBV M41 allantoic fluid per chick. Chicks from group B were inoculated with 0.2 mL sterilized negative allantoic fluid in the same manner. Clinical symptoms were observed and recorded daily. At 1, 3, 5, 8, 11, 14, 21, and 28 days post inoculation(dpi), three birds in each group were bled before euthanasia and necropsy. The trachea and kidney tissues were collected aseptically. Whole blood was collected, and sera were separated enzyme-linked-immunosorbent assay(ELISA). All samples were stored appropriately and immediately.
After washing in phosphate-buffered saline(PBS), trachea and kidney tissues were fixed in 4% paraformaldehyde solution for 1 day, embedded in paraffin wax, cut into 3-μm-thick sections(Leica RM4450, Germany), and stained with hematoxylin and eosin using st and ard histological procedure. The slides were examined by light microscopy(Nikon, Japan).
Equiponderant trachea and kidney tissues from each chick at various time points were prepared. The tracheal lumen was flushed with RL buffer repeatedly to lyse the mucosal tissue for RNA extraction, and kidney tissue was homogenized with RL buffer on ice. Total RNA was extracted using a MiniBEST Universal RNA Extraction Kit(TaKaRa, Shiga, Japan), including a gDNA Eraser Spin Column for treating DNA contamination according to the manufacturer's instructions. The concentration of RNA and the A260/A280 ratio were measured using an ultralow volume spectrometer(Biodrop, UK), and agarose gel electrophoresis was used to detect the integrity of the extracted RNA. A PrimeScript RT reagent Kit(TaKaRa)was used in cDNA synthesis for real-time PCR. Five hundred nanograms of total extracted RNA was added to 10 μL of reaction reagent including r and om 6 mers, oligo dT primers, PrimeScript RT Enzyme Mix I, and buffer. The mixture was then incubated at 37 ℃ for 15 min and 85 ℃ for 5 s for preparation of cDNA. cDNA was then stored at –20 ℃.
Absolute and relative quantification analyses were used to determine the absolute copy numbers of IBV RNA and the levels of mRNA expression of target genes, respectively. All primers pairs used for constructing recombinant plasmids and real-time PCR detection are listed in Table 1. Real-time PCR using SYBR Premix Ex Taq Ⅱ mix(TaKaRa)was performed on an iQ5 instrument(Bio-Rad, Hercules, USA)as previously described(Mo et al., 2011). The annealing temperatures for each gene are listed in Table 1. The IBV RNA copy number was calculated according to the st and ard curve using the cycle threshold(Ct). The relative expression ratios of target genes were calculated using Ct values and amplification efficiencies by the Pfaffl method operated in REST software, as previously described(Pfaffl et al., 2002). Several endogenous reference genes were evaluated to determine their suitability in IBV-infected chickens(data not shown), and glyceraldehyde 3-phosphate dehydrogenase(GAPDH) and TATA-box-binding protein(TBP)were used to normalize fold changes in expression.
Gene Sequences (5′-3′) a Amplicon size (bp) Annealing (℃) Accession no. IBV-N F:CAGAAGAAGGGCTCTGCATTAC
200 60 FJ548847.1 TLR3 F:GCAACACTTCATTGAATAGCCTTGAT
92 62.5 EF137861.1 TLR7 F:AAGTCCCGGTATGTTCAGCT
132 58.1 NM_0001011688.2 MyD88 F:CAGCGAGCAATAGCATCCAG
104 61 NM_001030962.1 TRIF F:TTCAGCCATTCTCCGTCCTC
120 60 NM_001081506.1 MDA5 F:CAGCCAGTTGCCCTCGCCTCA
210 60 NM_001193638 LGP2 F:CCAGAATGAGCAGCAGGAC
109 60 XM_004948615 MAVS F:CCTGACTCAAACAAGGGAAG
123 60 NM_001012893 STING F:TGACCGAGAGCTCCAAGAAG
82 60 XM_001232170 TBK1 F:AAGAAGGCACACATCCGAGA
170 58.1 NM_001199558.1 IKKε F:TGGATGGGATGGTGTCTGAAC
117 60 XM_428036.4 TRAF3 F:GGACGCACTTGTCGCTGTTT
107 60 XM_004936348.1 TRAF6 F:GATGGAGACGCAAAACACTCAC
170 60 XM_004941547.1 IKKα F:GAGGGGTGGAGGCTTAGATC
141 60 NM_001012904.1 IKKβ F:TACAGGCAATCCAGACCTTCG
234 60 NM_001031397.1 NF-κB p65 F:CATTGCCAGCATGGCTACTAT
102 60 NM_205129.1 IRF7 F:ACACTCCCACAGACAGTACTGA
134 58.5 NM_205372.1 IFN-α F:GGACATGGCTCCCACACTAC
75 61.3 X92476.1 IFN-β F:TTCTCCTGCAACCATCTTC
82 62.5 AY974089.1 IL-1β F:AACCCGACCAGGTCAACA
101 61.3 AJ245728.1 IL-6 F:ATCCCTCCTCGCCAATCTG
103 61.3 HM179640.1 IL-8 F:CACAGCTCCACAAAACCTCA
118 60 NM_205498 IL-10 F:ATGCTGCGCTTCTACACA
73 61.3 AY647438.1 IL-12 F:TCTGCTAAGACCCACGAGA
82 60 NM_213571 MIP-1β F:TGCTGGTGTTGTGTTCATC
73 63 L34553.1 GAPDH F:GGTGGTGCTAAGCGTGTTA
179 60.5 X01578.1 TBP F:TAGCCCGATGATGCCGTAT
147 62.5 D83127 Note: a Primers for TLR3, IFN-α were previously reported (Karpala et al., 2008), and those for GAPDH and TBP were also previously reported (Li et al., 2005). F indicates forward primer; R indicates reverse primer.
Table . Primers used in this study.
The concentrations of IFN-α, IFN-β, IL-1β, IL-6, IL-8, IL-10, IL-12, and MIP-1β in the trachea and kidney tissues were measured using a commercially available ELISA kit(Chenglin Biotechnology, Beijing, China)according to the manufacturer's instructions. To determine the exact concentration of target protein, st and ards were added to generate a st and ard curve, which was used to calculate the concentration based on the OD values. Each sample was analyzed in triplicate. The IBV-specific IgG antibody in serum was measured according to methods described in our previous report(Li et al., 2012), and the purified IBV N protein was used as antigen for the reaction. The OD values were read using a microplate reader(iMark; Bio-Rad)at 450 nm absorbance.
Data are expressed as the mean ± st and ard error of the mean(SEM). To identify significant differences, mean comparisons were performed using one-way analysis of variance(ANOVA)for viral load and Student's t tests for protein levels in SPSS17.0 software(SPSS Inc., Chicago, IL, USA). The results were considered to be statistically significant when the P value was less than 0.05.