From 29 November 2016 to 24 January 2017, sixty-three cases of H5N6 highly pathogenic avian influenza virus (HPAIV) infections were detected in wild birds in Ibaraki Prefecture, Japan. Here, we analyzed the genetic, temporal, and geographic correlations of these 63 HPAIVs to elucidate their dissemination throughout the prefecture. Full-genome sequence analysis of the Ibaraki isolates showed that 7 segments (PB2, PB1, PA, HA, NP, NA, NS) were derived from G1.1.9 strains while the M segment was from G1.1 strains; both groups of strains circulated in south China. Pathological studies revealed severe systemic infection in dead swans (the majority of dead birds and the only species necropsied), thus indicating high susceptibility to H5N6 HPAIVs. Coalescent phylogenetic analysis using the 7 G1.1.9-derived segments enabled detailed analysis of the short-term evolution of these highly homologous HPAIVs. This analysis revealed that the H5N6 HPAIVs isolated from wild birds in Ibaraki Prefecture were divided into 7 groups. Spatial analysis demonstrated that most of the cases concentrated around Senba Lake originated from a single source, and progeny viruses were transmitted to other locations after the infection expanded in mute swans. In contrast, within just a 5-km radius of the area in which cases were concentrated, three different intrusions of H5N6 HPAIVs were evident. Multi-segment analysis of short-term evolution showed that not only was the invading virus spread throughout Ibaraki Prefecture but also that, despite the small size of this region, multiple invasions had occurred during winter 2016-2017.
The European Community’s leukosis key (EC key) is a well-known hematologic method for detecting bovine leukemia virus (BLV) infection in dairy cattle. The key identifies infected cattle with persistent lymphocytosis via a combination of lymphocyte count (LC) and age. Using the EC key to identify BLV-infected Japanese black (JB) cattle is problematic, however, given the inherently lower LCs of JB cattle compared to dairy cattle. We analyzed the LC in BLV-positive and -negative JB cattle and estimated LC cutoff values by age using receiver operating characteristic curve analysis. Among the 716 JB blood samples collected, 452 (63%) JB cattle were confirmed as BLV-positive by an antibody ELISA for ≥1-y-old cattle and by real-time PCR for <1-y-old cattle. The cutoff values for the LC in each age group were calculated as 6.3 × 109/L for <1 y, 5.9 × 109/L for 1 to <2 y, 5.5 × 109/L for 2 to <3 y, 4.5 × 109/L for 3 to <6 y, 4.3 × 109/L for 6 to ≤10 y, and 3.7 × 109/L for >10 y. The sensitivity and specificity of the estimated cutoff values were 0.49 (95% confidence interval: 0.44–0.53) and 0.81 (0.75–0.85), whereas those of the EC key were 0.20 (0.16–0.24) and 0.99 (0.97–1.00). Our LC cutoff values for screening JB cattle for BLV infection appear to be preferable to those of the EC key.
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