Molecular epidemiology of hydropericardium syndrome outbreak-associated serotype 4 fowl adenovirus isolates in central China

Twelve liver samples of chickens were collected from flocks with HPS outbreaks in 12 different regions of Henan, central China, and frozen at −20 °C. All samples were confirmed to be FAdV-4 positive by polymerase chain reaction (PCR) amplifying a 632-bp fragment (Named fragment I) with primers (Table 1) based on the polymerase gene of FAdV strain MX-SHP95 (GenBank No. KP295475.1). Reactions were performed according to the following protocol: 95 °C for 5 min, followed by 31 cycles of 95 °C for 30 s, 56 °C for 30 s, 72 °C for 50 s, and a final elongation step of 10 min at 72 °C. 24 reference FAdV strains and 12 outbreak-associated FAdV strains used for phylogenetic analyses were listed in Table 2.

The presence of FAdV in each sample was confirmed by virus isolation followed by PCR. Initially, 5 mg of liver was cut into tiny pieces and fully ground in sterilized PBS (weight/volume = 1:3) under aseptic conditions. Following complete grinding, the suspension of liver tissue in PBS was frozen at −20 °C and thawed at 37 °C three times before centrifugation at 8000 rpm at 4 °C for 10 min. The supernatant was then collected, filtered through a 0.22 μm filter, and inoculated onto a confluent monolayer of CEF (chicken embryo fibroblast) cells prepared from 9-day SPF embryonated chicken eggs. After incubation for 1 h at 37 °C, 1.5 ml of DMEM media containing 2% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) were added to the 6-well cell culture plate. 72 h later, supernatants were collected and filtered through a 0.22 μm filter for DNA extraction. Genomic DNA from these field samples was extracted using TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (TaKaRa, Dalian, China), and stored at −20 °C before use. Quantification and quality of DNA extracts were determined with a Nanodrop spectrophotometer (Thermo Scientific, USA).

The hexon gene was divided into two fragments (Named fragment A and B) because of its long length in this study. Primers used for amplifying fragments A and B were designed according to the hexon genes of FAdV-4 strain MX-SHP95 (GenBank No. KP295475.1) and synthesized by Sangon Biotech (Table 1). PCR was performed in a Bio-Rad MyCycler Thermal Cycler. Fragments A and B were amplified in a 25 μl PCR reaction containing 1.5 μl genomic DNA (200–400 ng/μl), 0.25 μl Q₅ High-Fidelity DNA Polymerase, 0.5 μl dNTPs (10 mM), 1.25 μl forward primer (10 pmol/μl), 1.25 μl reverse primer (10 pmol/μl), 10.25 μl sterilized water, 5 μl 5X Q₅ Reaction Buffer, and 5 μl 5X Q5 High GC Enhancer. Two expected size of fragment A (1501 bp) and fragment B (1345 bp) were amplified using the following conditions: initial denaturation at 98 °C for 3 min; 31 cycles of denaturation at 98 °C for 10 s, annealing at 66 °C (for fragment A) and 57 °C (for fragment B) for 30s, extension at 72 °C for 80s; and final extension at 72 °C for 5 min. PCR products for each sample were visualized by electrophoresis in a 1.0% agarose gel containing ethidium bromide, purified according to the manufacturer’s protocol, using Universal DNA Purification Kit (TIANGEN, Beijing), and ligated into pEASY-Blunt Cloning vector using pEASY-Blunt Cloning Kit (TransGen Biotech) according to the manufacturer’s protocol. Upon transformation of each ligation reaction mixture into Trans1-T1 Phage Resistant Chemically Competent cells, a single colony was picked up and identified by PCR and restriction enzyme digestion before sequencing.

The respective bacterial clones harboring recombinant plasmids of fragment A and fragment B were sequenced by Sangon Biotech. Each sample was sequenced twice (one with the primers designed in this study and the other with the sequencing primers of the PEASY-Blunt Cloning Vector). The obtained sequences were edited using DNAStar software, and deposited into GenBank. Homology analyses to identify gene homologs were performed with the BLAST program (NCBI). Phylogenetic trees were constructed using MEGA software by the neighbor-joining analysis with 1000 bootstrap replicates, Maximum Composite Likelihood method. The phylogenetic datasets for analyses included 12 FAdV-4 isolates from this study and 24 reference strains (Table 2).