Virus isolation
Water samples from roosts of wild birds and duck fecal samples were collected in the Izumi plain from November to March of 2018/19, 2019/20, 2020/21, and 2021/22. As shown in Table 1, the total number of water samples was 558; 6, 211, 160, and 181 samples were collected in the 2018/19, 2019/20, 2020/21, and 2021/22 seasons, respectively. Virus isolation was conducted according to the influenza virus isolation procedure from water samples using embryonated chicken eggs [26]. Briefly, water samples were centrifuged at 2,000 × g for 5 min at 4°C to remove debris and a 1/10 volume of 10-fold concentrated phosphate-buffered saline and 100 μL of chicken red blood cells (RBCs) were added and mixed. The mixture was incubated on ice for 1 h to adsorb potential hemagglutinating viruses to RBCs. After incubation, RBCs were collected by centrifugation at 2,000 × g for 5 min at 4°C, and resuspended in 1 mL of viral transport medium containing 0.5% bovine serum albumin, 10,000 U/mL penicillin, 10 mg/mL streptomycin, 0.3 mg/mL gentamicin, and 2.5 μg/mL amphotericin B. The RBC resuspension was directly inoculated into 9- to 11-day-old embryonated chicken eggs.
As shown in Table 1, the total number of fecal samples was 1,021; 96, 475, and 450 samples were collected in the 2018/19, 2019/20, and 2021/22 seasons, respectively. No samples were collected in the 2020/21 season. Virus isolation from fecal samples was conducted using embryonated chicken eggs as previously described [9].
The eggs inoculated with each sample were cultured at 37°C for 3 days. Following cooling of eggs at 4°C for several hours, the allantoic fluid was collected and a hemagglutination (HA) assay was performed. Allantoic fluid with HA activity was tested to confirm the presence of avian influenza virus using ESPLINE® ︎INFLUENZA A&B-N (FUJIREBIO Inc., Tokyo, Japan). When the rapid influenza viral antigen test was negative, the harvested allantoic fluid was stored at -80°C as a viral fluid that may contain APMV-1 for subsequent experiments.
APMV-1 identification using genetic analysis
Nucleic acid extraction from the allantoic fluid containing hemagglutinating virus was performed using the innuPREP Virus DNA/RNA Kit (Analytic Jena, Jena, Germany). To synthesize a DNA strand complementary to the viral genomic RNA, a reverse transcriptase reaction was performed using the PrimeScriptTM II 1st strand cDNA Synthesis Kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer's recommended protocol. Subsequently, the polymerase chain reaction (PCR) was performed using TaKaRa ExTaq® DNA Polymerase (Takara Bio) with the reverse transcription product and APMV-1 genotyping primers (class I: NDV-C1-F and NDV-C1-R, class II: NDV-C2-F and NDV-C2-R) [18]. Following agarose electrophoresis of PCR products, amplified cDNA were purified using the Monarch® DNA Gel Extraction Kit (New England Biolabs, Ipswich, MA). To determine the sequence of the amplified cDNA, sequencing analysis was conducted with Big-dye terminator v3.1 cycle sequencing kit and ABI 3130 sequencer (Applied Biosystems and Fasmac, Kanagawa, Japan). Sequence similarity searches were performed using the Basic Local Alignment Search Tool (BLAST, from the National Center for Biotechnology Information [NCBI]) to identify the hemagglutinating virus isolate as an APMV-1.
Nucleotide sequence accession numbers
The nucleotide sequences determined in this study are available in the DDBJ database under accession numbers LC715129 to LC715143. The strains obtained in this study were named as host or isolation source/Kagoshima/KU-strain number/year. In the text, the strains are referred to by abbreviation listed in Table 2.
Phylogenetic analysis
Based on partial nucleotide sequences (class I: 368 bp and class II: 459 bp) in the Fusion (F) gene of APMV-1 isolates, a molecular phylogenetic tree was constructed using the neighbor-joining method [30] with Molecular Evolutionary Genetics Analysis version 10.1.6. The robustness of the neighbor joining analysis grouping was assessed using resampling with 1,000 bootstraps.
Pathogenicity of APMV-1 isolates in chicken embryos
To evaluate the virulence of APMV-1 isolates against chicken embryos, 100 μL of each virus solution adjusted to 102, 104, and 106 EID50 (50% egg infectious dose) was inoculated into the allantoic cavity of five 9-day-old embryonated chicken eggs, followed by incubation at 37°C. The eggs were observed once every 12 h for 7 days, and the time of embryo death was recorded. The mean death time (MDT) was determined when all embryos inoculated with each dose of the virus were killed. Japanese laws on animal research state that experiments with fertilized eggs performed before hatching do not require animal ethics approval.