Collection and transportation of SARS-CoV-2 and influenza A virus diagnostic samples: Optimizing the usage of guanidine-based chaotropic salts for enhanced biosafety and viral genome preservation.

Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.

An Abies Extract Containing Nonvolatile Polyphenols Shows Virucidal Activity against SARS-CoV-2 That Is Enhanced in Increased pH Conditions.

Researching the beneficial health properties of wood byproducts can prevent wastage by turning them into valuable resources. In this study, the virucidal activity of two extracts from Abies sachalinensis byproducts, ASE1, and ASE2, against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was investigated. ASE1 is rich in monoterpenoid volatile compounds, whereas ASE2 contains nonvolatile polyphenols. SARS-CoV-2 solutions were mixed with ASE1 or ASE2, and viral titer reduction was evaluated. At their original acidic pH, ASE2 showed stronger virucidal activity than ASE1. The virucidal activity of ASE2 was also significantly enhanced when pH was increased to neutral or basic, which was not the case for ASE1. At a neutral pH, ASE2 induced statistically significant viral titer reduction in 1 min. HCl and NaOH solutions, which had a pH close to that of acidic and basic ASE2 test mixtures, respectively, exhibited no virucidal activity against SARS-CoV-2. Among the SARS-CoV-2 variants, Omicron showed the highest vulnerability to ASE2. Western blotting, RT-PCR, and electron microscopic analysis revealed that neutral ASE2 interacts with SARS-CoV-2 spike proteins and moderately disrupts the SARS-CoV-2 genome and viral envelope. These findings reveal the virucidal potential of ASE2.

Rapid Virucidal Activity of Japanese Saxifraga Species-Derived Condensed Tannins against SARS-CoV-2, Influenza A Virus, and Human Norovirus Surrogate Viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and norovirus are global threats to human health. The application of effective virucidal agents, which contribute to the inactivation of viruses on hands and environmental surfaces, is important to facilitate robust virus infection control measures. Naturally derived virucidal disinfectants have attracted attention owing to their safety and eco-friendly properties. In this study, we showed that multiple Japanese Saxifraga species-derived fractions demonstrated rapid, potent virucidal activity against the SARS-CoV-2 ancestral strain and multiple variant strains, IAV, and two human norovirus surrogates: feline calicivirus (FCV) and murine norovirus (MNV). Condensed tannins were identified as active chemical constituents that play a central role in the virucidal activities of these fractions. At a concentration of 25 µg/mL, the purified condensed tannin fraction Sst-2R induced significant reductions in the viral titers of the SARS-CoV-2 ancestral strain, IAV, and FCV (reductions of ≥3.13, ≥3.00, and 2.50 log10 50% tissue culture infective doses [TCID50]/mL, respectively) within 10 s of reaction time. Furthermore, at a concentration of 100 µg/mL, Sst-2R induced a reduction of 1.75 log10 TCID50/mL in the viral titers of MNV within 1 min. Western blotting and transmission electron microscopy analyses revealed that Sst-2R produced structural abnormalities in viral structural proteins and envelopes, resulting in the destruction of viral particles. Furthermore, Saxifraga species-derived fraction-containing cream showed virucidal activity against multiple viruses within 10 min. Our findings indicate that Saxifraga species-derived fractions containing condensed tannins can be used as disinfectants against multiple viruses on hands and environmental surfaces. IMPORTANCE SARS-CoV-2, IAV, and norovirus are highly contagious pathogens. The use of naturally derived components as novel virucidal/antiviral agents is currently attracting attention. We showed that fractions from extracts of Saxifraga species, in the form of a solution as well as a cream, exerted potent, rapid virucidal activities against SARS-CoV-2, IAV, and surrogates of human norovirus. Condensed tannins were found to play a central role in this activity. The in vitro cytotoxicity of the purified condensed tannin fraction at a concentration that exhibited some extent of virucidal activity was lower than that of 70% ethanol or 2,000 ppm sodium hypochlorite solution, which are popular virucidal disinfectants. Our study suggests that Saxifraga species-derived fractions containing condensed tannins can be used on hands and environmental surfaces as safe virucidal agents against multiple viruses.

Efficacy Validation of SARS-CoV-2-Inactivation and Viral Genome Stability in Saliva by a Guanidine Hydrochloride and Surfactant-Based Virus Lysis/Transport Buffer.

To enhance biosafety and reliability in SARS-CoV-2 molecular diagnosis, virus lysis/transport buffers should inactivate the virus and preserve viral RNA under various conditions. Herein, we evaluated the SARS-CoV-2-inactivating activity of guanidine hydrochloride (GuHCl)- and surfactant (hexadecyltrimethylammonium chloride (Hexa-DTMC))-based buffer, Prep Buffer A, (Precision System Science Co., Ltd., Matsudo, Japan) and its efficacy in maintaining the stability of viral RNA at different temperatures using the traditional real-time one-step RT-PCR and geneLEAD VIII sample-to-result platform. Although Prep Buffer A successfully inactivated SARS-CoV-2 in solutions with high and low organic substance loading, there was considerable viral genome degradation at 35 °C compared with that at 4 °C. The individual roles of GuHCl and Hexa-DTMC in virus inactivation and virus genome stability at 35 °C were clarified. Hexa-DTMC alone (0.384%), but not 1.5 M GuHCl alone, exhibited considerable virucidal activity, suggesting that it was essential for potently inactivating SARS-CoV-2 using Prep Buffer A. GuHCl and Hexa-DTMC individually reduced the viral copy numbers to the same degree as Prep Buffer A. Although both components inhibited RNase activity, Hexa-DTMC, but not GuHCl, directly destroyed naked viral RNA. Our findings suggest that samples collected in Prep Buffer A should be stored at 4 °C when RT-PCR will not be performed for several days.

Virucidal Activities of Acidic Electrolyzed Water Solutions with Different pH Values against Multiple Strains of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a threat to human health. Acidic electrolyzed water (AEW) has recently been suggested to demonstrate virucidal activity. Many types of AEW with different pH values, generated by the electrolysis of different chemicals, such as sodium chloride, potassium chloride, and hydrochloric acid, are commercially available. In this study, we compared the virucidal activities of these types of AEW against SARS-CoV-2, including the ancestral strain and variant Alpha, Beta, Gamma, Delta, and Omicron strains. Virus solution (viral titer, 6.9 log10 50% tissue culture infective dose [TCID50]/mL) was mixed with AEW (free available chlorine concentration, 34.5 ppm) at mixing ratios of 1:9, 1:19, and 1:49. At mixing ratios of 1:9 and 1:19, AEW with a pH of 2.8 showed stronger virucidal activities than AEW with a pH of 4.1 to 6.5 against the SARS-CoV-2 ancestral strain in 20 s. From the strongest to the weakest virucidal activity, the AEW pH levels were as follows: pH 2.8, pH 4.1 to 5.4, pH 6.4 to 6.5. At a ratio of 1:49, the viral titers of viruses treated with all AEW solutions at pH 2.8 to 6.5 were almost below the detection limit, which was 1.25 log10 TCID50/mL. The virus inactivation efficiency of AEW was reduced in the presence of fetal bovine serum and other substances contained in the virus solution used in this study. AEW with pH values of 2.8 to 6.5 showed virucidal activity against all of the tested SARS-CoV-2 strains, including the ancestral and variant strains. These results provide useful knowledge for the effective application of AEW as a SARS-CoV-2 disinfectant. IMPORTANCE Acidic electrolyzed water (AEW) demonstrates virucidal activity against multiple viruses. Since AEW exhibits low toxicity, is inexpensive, and is environmentally friendly, it can be a useful disinfectant against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the pH values of currently available AEW products vary, the impact of different pH values on SARS-CoV-2 inactivation has not previously been evaluated in detail. In this study, we compared the virucidal activities of multiple AEW solutions with different pH values, under the same experimental conditions. We found that AEW solutions with lower pH values demonstrated more potent virucidal activity. Also, we showed that the extent of virus inactivation by the AEW was based on the balance of the abundance of free available chlorine, virus, and other organic substances in the mixture. AEW exhibited rapid virucidal activity against multiple SARS-CoV-2 strains. This study demonstrated the usefulness of AEW as a disinfectant which can be applied to the inactivation of SARS-CoV-2.

Viral RNA extraction using an automatic nucleic acid extractor with magnetic particles and genetic characterization of bovine viral diarrhea virus in Tokachi Province, Japan, in 2016-2017.

In this study, the viral genome extraction performance of automatic nucleic acid extractors and manual nucleic acid extraction kits was compared. We showed that compared with manual kits, the automatic extractors showed superior genome extraction performance using bovine viral diarrhea virus (BVDV) genome-positive cattle sera and bovine coronavirus/infectious bovine rhinotracheitis virus-spiked cattle nasal swabs. In addition, the subgenotyping of BVDV strains detected in Tokachi Province in Japan during 2016-2017 was performed. Results showed that most of these BVDV strains belonged to subgenotype 1b, while few strains belonged to subgenotypes 1a and 2a. This study showed the high applicability of automatic nucleic acid extractors in extracting multiple viral genomes and the dominant subgenotype of BVDV in Tokachi.

Impact of Theaflavins-Enriched Tea Leaf Extract TY-1 against Surrogate Viruses of Human Norovirus: In Vitro Virucidal Study.

Using an effective natural virucidal substance may be a feasible approach for preventing food-borne viral contamination. Here, the virucidal efficacy of theaflavins (TFs)-enriched tea leaf extract (TY-1) against feline calicivirus (FCV) and murine norovirus (MNV), surrogates of human norovirus (HuNoV), was evaluated. The virus solutions were mixed with various dosages of TY-1 and incubated at 25 °C for different contact times. TY-1 reduced the viral titer of both surrogate viruses in a time- and dosage-dependent manner. A statistically significant reduction in the viral titer of FCV by 5.0 mg/mL TY-1 and MNV by 25.0 mg/mL TY-1 was observed in 10 s and 1 min, respectively. Furthermore, TY-1 reduced the viral titer of FCV and MNV on the dry surface in 10 min. The multiple compounds in TY-1, including TFs and catechins, contributed to its overall virucidal activity. Furthermore, the effect of TY-1 on viral proteins and genome was analyzed using Western blotting, RT-PCR, and transmission electron microscopy. TY-1 was found to promote the profound disruption of virion structures, including the capsid proteins and genome. Our finding demonstrates the potential of using TY-1 as a nature-derived disinfectant in food processing facilities and healthcare settings to reduce viral load and HuNoV transmission.

Application of Copper Iodide Nanoparticle-Doped Film and Fabric To Inactivate SARS-CoV-2 via the Virucidal Activity of Cuprous Ions (Cu+).

As a result of the novel coronavirus disease 2019 pandemic, strengthening control measures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an urgent global issue. In addition to antiviral therapy and vaccination strategies, applying available virucidal substances for SARS-CoV-2 inactivation is also a target of research to prevent the spread of infection. Here, we evaluated the SARS-CoV-2 inactivation activity of a copper iodide (CuI) nanoparticle dispersion, which provides Cu+ ions having high virucidal activity, and its mode of actions. In addition, the utility of CuI-doped film and fabric for SARS-CoV-2 inactivation was evaluated. The CuI dispersion exhibited time-dependent rapid virucidal activity. Analyses of the modes of action of CuI performed by Western blotting and real-time reverse transcription-PCR targeting viral proteins and the genome revealed that CuI treatment induced the destruction of these viral components. In this setting, the indirect action of CuI-derived reactive oxygen species contributed to the destruction of viral protein. Moreover, the CuI-doped film and fabric demonstrated rapid inactivation of the SARS-CoV-2 solution in which the viral titer was high. These findings indicated the utility of the CuI-doped film and fabric as anti-SARS-CoV-2 materials for the protection of high-touch environmental surfaces and surgical masks/protective clothes. Throughout this study, we demonstrated the effectiveness of CuI nanoparticles for inactivating SARS-CoV-2 and revealed a part of its virucidal mechanism of action. IMPORTANCE The COVID-19 pandemic has caused an unprecedented number of infections and deaths. As the spread of the disease is rapid and the risk of infection is severe, hand and environmental hygiene may contribute to suppressing contact transmission of SARS-CoV-2. Here, we evaluated the SARS-CoV-2 inactivation activity of CuI nanoparticles, which provide the Cu+ ion as an antiviral agent, and we provided advanced findings of the virucidal mechanisms of action of Cu+. Our results showed that the CuI dispersion, as well as CuI-doped film and fabric, rapidly inactivated SARS-CoV-2 with a high viral titer. We also demonstrated the CuI's virucidal mechanisms of action, specifically the destruction of viral proteins and the genome by CuI treatment. Protein destruction largely depended on CuI-derived reactive oxygen species. This study provides novel information about the utility and mechanisms of action of promising virucidal material against SARS-CoV-2.

Inactivation Activities of Ozonated Water, Slightly Acidic Electrolyzed Water and Ethanol against SARS-CoV-2.

This study aimed to compare the SARS-CoV-2-inactivation activity and virucidal mechanisms of ozonated water (OW) with those of slightly acidic electrolyzed water (SAEW) and 70% ethanol (EtOH). SARS-CoV-2-inactivation activity was evaluated in a virus solution containing 1%, 20% or 40% fetal bovine serum (FBS) with OW, SAEW or EtOH at a virus-to-test solution ratio of 1:9, 1:19 or 1:99 for a reaction time of 20 s. EtOH showed the strongest virucidal activity, followed by SAEW and OW. Even though EtOH potently inactivated the virus despite the 40% FBS concentration, virus inactivation by OW and SAEW decreased in proportion to the increase in FBS concentration. Nevertheless, OW and SAEW showed potent virucidal activity with 40% FBS at a virus-to-test solution ratio of 1:99. Real-time PCR targeting the viral genome revealed that cycle threshold values in the OW and SAEW groups were significantly higher than those in the control group, suggesting that OW and SAEW disrupted the viral genome. Western blotting analysis targeting the recombinant viral spike protein S1 subunit showed a change in the specific band into a ladder upon treatment with OW and SAEW. OW and SAEW may cause conformational changes in the S1 subunit of the SARS-CoV-2 spike protein.

Isolation and evaluation of virucidal activities of flavanone glycosides and rosmarinic acid derivatives from Dracocephalum spp. against feline calicivirus.

Feline calicivirus is one of the surrogate viruses of human norovirus. This study aimed to identify virucidal compounds, chemical constituents of plants from the genus Dracocephalum, which are rich in flavonoids and phenylpropanoid oligomers. Four undescribed compounds, including a flavanone glucoside, two stilbenoid glycosides, and a phenylpropanoid amide glycoside, as well as 17 known compounds, were isolated from the Mongolian plants Dracocephalum fruticulosum Stephan ex Willd., and D. nutans L. belonging to the family Lamiaceae. The structures of the compounds were determined based on NMR, MS, and electronic CD spectroscopic data. In addition to these 21 compounds, 15 previously reported compounds from D. foetidum Bunge in C.F. von Ledebour were included, and a total of 36 compounds were evaluated for their virucidal activities against feline calicivirus. Some of the flavanone glycosides and phenylpropanoid oligomers showed virucidal activities, and their structural features are discussed. The findings suggest that isosakuranetin glycosides and phenylpropanoid oligomers may have the potential for norovirus inactivation.

Severe Acute Respiratory Syndrome Coronavirus-2 Inactivation Activity of the Polyphenol-Rich Tea Leaf Extract with Concentrated Theaflavins and Other Virucidal Catechins.

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is producing a large number of infections and deaths globally, the development of supportive and auxiliary treatments is attracting increasing attention. Here, we evaluated SARS-CoV-2-inactivation activity of the polyphenol-rich tea leaf extract TY-1 containing concentrated theaflavins and other virucidal catechins. The TY-1 was mixed with SARS-CoV-2 solution, and its virucidal activity was evaluated. To evaluate the inhibition activity of TY-1 in SARS-CoV-2 infection, TY-1 was co-added with SARS-CoV-2 into cell culture media. After 1 h of incubation, the cell culture medium was replaced, and the cells were further incubated in the absence of TY-1. The viral titers were then evaluated. To evaluate the impacts of TY-1 on viral proteins and genome, TY-1-treated SARS-CoV-2 structural proteins and viral RNA were analyzed using western blotting and real-time RT-PCR, respectively. TY-1 showed time- and concentration-dependent virucidal activity. TY-1 inhibited SARS-CoV-2 infection of cells. The results of western blotting and real-time RT-PCR suggested that TY-1 induced structural change in the S2 subunit of the S protein and viral genome destruction, respectively. Our findings provided basic insights in vitro into the possible value of TY-1 as a virucidal agent, which could enhance the current SARS-CoV-2 control measures.

Inactivation of SARS-CoV-2 by Ozonated Glycerol.

We evaluated the SARS-CoV-2-inactivation activity of ozonated glycerol (OG). When a viral solution with 1% fetal bovine serum (FBS) was mixed with test solutions at a ratio of 1:19 and incubated for 20 s, OG with ozone concentrations of over 1000 ppm inactivated ≥ 94.38% of the virus. Extension of the reaction time to 1 h led to the inactivation of ≥ 99.82% of the virus (the viral titer was below the detection limit). Extension to 24 h resulted in concentrations over 200 ppm OG inactivating ≥ 99.87% of the virus (the viral titers were below the detection limit). Next, viral solutions with 1, 20, and 40% FBS were mixed with test solutions at a ratio of 1:19 and incubated for 5 min. Whereas the virucidal activity of 500 ppm OG was very limited in the presence of 1% FBS (79.47% inactivation), it increased in the presence of 20 and 40% FBS (95.13 and 97.95% inactivation, respectively; the viral titers were not below the detection limit). Meanwhile, over 1000 ppm OG inactivated ≥ 99.44% of the virus regardless of the FBS concentration (the viral titers were below the detection limit). Extension of the reaction time to 1 h led to 500 ppm OG inactivating ≥ 99.91 and ≥ 99.95% of the virus with 20 and 40% FBS, respectively (the viral titers were below the detection limit). These results suggested that OG might be useful as a virucidal agent against SARS-CoV-2.

Comparison of the SARS-CoV-2-inactivating activities of the differently manufactured hypochlorous acid water products with various pH.

The use of effective disinfectants is a key method of controlling the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hypochlorous acid water (HAW) has a broad spectrum of virucidal activities. We previously reported that acidic electrolyzed water, one of the HAW products, had potent SARS-CoV-2-inactivating activity and showed promise as a disinfectant. However, different manufacturing methods have produced several HAW products with various pH values. Here, we compared the SARS-CoV-2-inactivating activities of various HAW products. At sufficiently high volume and residual chlorine concentration (RCC), the HAW products inactivated SARS-CoV-2 efficiently regardless of pH or manufacturing method. However, although HAW products at pH 5.0-6.4 maintained high RCC and sustained virucidal activity for 21 days, the RCC rapidly decreased in HAW products at pH ≤ 3.0. Our results may guide in choosing appropriate HAW products for different usage situations.

The SARS-CoV-2-Inactivating Activity of Hydroxytyrosol-Rich Aqueous Olive Pulp Extract (HIDROX®) and Its Use as a Virucidal Cream for Topical Application.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally. Although measures to control SARS-CoV-2, namely, vaccination, medication, and chemical disinfectants are being investigated, there is an increase in the demand for auxiliary antiviral approaches using natural compounds. Here we have focused on hydroxytyrosol (HT)-rich aqueous olive pulp extract (HIDROX®) and evaluated its SARS-CoV-2-inactivating activity in vitro. We showed that the HIDROX solution exhibits time- and concentration-dependent SARS-CoV-2-inactivating activities, and that HIDROX has more potent virucidal activity than pure HT. The evaluation of the mechanism of action suggested that both HIDROX and HT induced structural changes in SARS-CoV-2, which changed the molecular weight of the spike proteins. Even though the spike protein is highly glycosylated, this change was induced regardless of the glycosylation status. In addition, HIDROX or HT treatment disrupted the viral genome. Moreover, the HIDROX-containing cream applied on film showed time- and concentration-dependent SARS-CoV-2-inactivating activities. Thus, the HIDROX-containing cream can be applied topically as an antiviral hand cream. Our findings suggest that HIDROX contributes to improving SARS-CoV-2 control measures.

Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and norovirus (NV) are highly contagious pathogens that threaten human health. Here we focused on the antiviral potential of the medicinal herb, Saxifraga spinulosa (SS). Water-soluble extracts of SS were prepared, and their virus-inactivating activity was evaluated against the human virus pathogens SARS-CoV-2 and IAV; we also examined virucidal activity against feline calicivirus and murine norovirus, which are surrogates for human NV. Among our findings, we found that SS-derived gallocatechin gallate compounds were capable of inactivating all viruses tested. Interestingly, a pyrogallol-enriched fraction (Fr 1C) inactivated all viruses more rapidly and effectively than did any of the component compounds used alone. We found that 25 µg/mL of Fr 1C inactivated >99.6% of SARS-CoV-2 within 10 s (reduction of ≥2.33 log10 TCID50/mL). Fr 1C resulted in the disruption of viral genomes and proteins as determined by gel electrophoresis, electron microscopy, and reverse transcription-PCR. Taken together, our results reveal the potential of Fr 1C for development as a novel antiviral disinfectant.

Crimean-Congo Hemorrhagic Fever Virus, Mongolia, 2013-2014.

During 2013-2014, we collected 1,926 serum samples from humans and 4,583 ticks (Hyalomma asiaticum or Dermacentor nuttalli) in select regions of Mongolia to determine the risk for Crimean-Congo hemorrhagic fever virus (CCHFV) infection among humans in this country. Testing of human serum samples by ELISA demonstrated an overall CCHFV antibody prevalence of 1.4%; Bayankhongor Province had the highest prevalence, 2.63%. We pooled and analyzed tick specimens by real-time reverse transcription PCR; 1 CCHFV-positive H. asiaticum tick pool from Ömnögovi was identified. In phylogenetic analyses, the virus's partial small segment clustered with CCHFV isolates from Central Asia, and the complete medium segment grouped with CCHFV isolates from Africa, Asia, and the Middle East. This study confirms CCHFV endemicity in Mongolia and provides information on risk for CCHFV infection. Further research is needed to better define the risk for CCHFV disease to improve risk mitigation, diagnostics, and surveillance.

Surveillance and characterization of avian influenza viruses from migratory water birds in eastern Hokkaido, the northern part of Japan, 2009-2010.

Avian influenza virus (AIV) surveillance was conducted around a small pond in Obihiro, eastern Hokkaido, Japan. Eleven AIVs were isolated from a total of 1,269 fecal samples of migratory wild birds collected during 2009 and 2010. The sample number covered approximately 60 % of the total number of birds observed during sampling periods. The subtypes of the isolates included H3N8 (4 isolates), H5N2 (3), H6N2 (2), H6N1 (1), and H11N2 (1). The H3N8 subtype was most prevalent as in the previous studies performed in Hokkaido. The three H5N2 isolates genetically characterized as low pathogenic AIV were closely related to the strains previously isolated from aquatic wild birds in Japan and also to the Korean strains isolated from aquatic birds in recent years. In Korea, H5N2 subtype virus has often been isolated from poultry and wild birds, as well as reassortant viruses generated from duck H5N2 viruses and chicken H9N2 virus, and avian-swine-like reassortant H5N2 viruses. Considering the previous chicken outbreaks caused by highly pathogenic H5N2 viruses, which affected many countries, it should be an important priority to continue, monitoring the evolution of H5N2 viruses circulating in the region.