Improved method for avian influenza virus isolation from environmental water samples

Abstract

Environmental water-targeted surveillance of migratory aquatic birds at overwintering sites is potentially one of the most effective approaches for understanding the ecology of avian influenza viruses (AIVs). In this study, we improved the method for AIV isolation from environmental water samples by making a minor modification to our previously reported process. We experimentally demonstrated that the AIV recovery efficiency of the modified method was 10-100-fold higher than that of the original method. This improved isolation method allowed us to isolate a considerably larger number of AIV isolates from environmental water samples collected at an overwintering site for tens of thousands of migratory aquatic birds in Japan during the 2018/2019 winter season, compared with those during previous winter seasons.Genetic and phylogenetic analyses revealed that AIVs of the same subtypes with multiple genetic constellations were circulating in a single overwintering site during a single winter season. These findings indicate that our improved isolation method contributes to enhance environmental water-targeted surveillance and to a better understanding of AIV ecology in migratory aquatic bird populations by monitoring ongoing AIV circulation.

Investigating Environmental Matrices for Use in Avian Influenza Virus Surveillance—Surface Water, Sediments, and Avian Fecal Samples

“…Yet, the viral loads are generally too low for virus isolation or further sub- and pathotyping purposes. Specific receptor-binding approaches such as those described in reference 38 have recently been found to be potentially more sensitive, also when smaller water volumes were investigated. Such techniques should be explored further.…”

Newly emerged genotypes of highly pathogenic H5N8 avian influenza viruses in Kagoshima prefecture, Japan during winter 2020/21

“…The AIV surveillance conducted using water samples from the crane roost revealed the temporal changes in the prevalence of circulating AIV strains in the Izumi plain (Figs 2 and 4). Notably, most of the AIVs were isolated at the beginning of November, suggesting that AIV invasion into the Izumi plain during the 2020/21 winter season would have occurred earlier than in the previous winter seasons [14, 18, 21]. However, the timing of migration of wild ducks was not earlier than in previous years (Fig.…”

“…S2c), suggesting that genotype E9 may have emerged via genetic reassortment on the Izumi plain during the 2020/21 winter season. Intriguingly, gene reassortment has been infrequent in the Izumi plain even though we have conducted AIV surveillance every winter season since the 2012/13 winter season [13, 14, 16, 18, 21–23, 29, 30].…”

“…AIVs were isolated from water samples, as was described in a previous study [21]. Briefly, environmental water samples were mixed with ten-fold concentrated phosphate-buffered saline (PBS) for osmotic adjustment and incubated with 100 µl of red blood cells (RBCs) on ice for 1 h. The RBCs were pelleted by centrifuge (3000 r.p.m.…”

“…RNA was extracted from allantoic fluids that showed hemagglutination activity using an InnuPREP virus DNA/RNA Kit (Analytik Jena AG, Jena, Germany) and subjected to real-time reverse transcription (RT)-PCR-based influenza A viral gene detection using the iTaq Universal SYBR Green One-Step Kit (BioRad, Hercules, CA, USA) and primer sets specific for a conserved region of influenza viral M genes [14]. To select samples that were possibly single isolates, M gene-positive samples were subjected to realtime RT-PCR using primer sets specific for the H1, H3, H4, H5, and H6 HA genes (Table 1), which have been frequently detected in the past [14,18,[21][22][23]. RNA samples that reacted with only one HA subtype-specific primer set were reverse transcribed using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) for complementary DNA (cDNA) synthesis and used as templates for conventional PCR-based NA subtyping using Tks Gflex DNA Polymerase (TaKaRa Bio Inc., Otsu, Japan) with a panel of subtype-specific primer sets [24].…”