Chemical stability of active ingredients in diluted veterinary disinfectant solutions under simulated storage conditions.

Introduction: The product labels of veterinary disinfectants specify their expiration dates to prevent the use of outdated products, as these may result in disinfection and biosecurity failures during outbreak situations. However, a clear standard for the storage conditions of diluted disinfectant solutions has not yet been established, and the effects of storage conditions have scarcely been investigated. To fill this research gap, our study examined the stability of the active ingredients of diluted veterinary disinfectants based on their change in concentrations when stored at various temperatures for various time periods. Methods: Twenty veterinary disinfectants effective against either foot-and-mouth disease or avian influenza viruses were selected. The disinfectants were diluted to effective concentrations following the manufacturer's instructions. Using selective analytical techniques, the concentrations of the active ingredients of the samples that had been stored for varying intervals at different temperatures (4, 20, 30, and 45°C) were determined. These samples included soaps and detergents, acids, oxidizing agents, aldehydes, and copper compounds. The active ingredient concentrations of two of the samples were determined following freezing/thawing cycle, to establish their stability when exposed to simulated winter conditions. Results: Our results showed that most of the active ingredients had concentrations of 90% or greater of their initial concentrations, indicating ≥90% stability over a 21-day period under the experimental storage conditions. However, there were some exceptions. Glutaraldehyde, formaldehyde, and malic acid are over 90% stable at ≤ 30°C for 21 days, but their concentrations decreased to below 90% of their initial concentrations at 45°C, indicating a decline in stability when stored at 45°C for 21 days. The concentrations of potassium peroxymonosulfate and peracetic acid rapidly declined with increasing time and temperature to less than 90% of their initial concentrations. Discussion: Based on our findings, we propose that diluted disinfectant solutions should preferably be prepared daily. However, if the daily preparation of a diluted disinfectant solution is not feasible, then our results can be used as a reference, providing basic scientific data on the chemical stability of diluted disinfectant solutions commonly used in the veterinary field, thus indicating suitable storage conditions.

Development of a recombinant H9N2 influenza vaccine candidate against the Y280 lineage field virus and its protective efficacy.

Since June 2020, a new H9N2 virus of the Y280 lineage has been epidemic in Korea. Initially, a Korean commercial vaccine against the Y280 and Y439 lineages of H9N2 was evaluated for use in SPF chickens. A single vaccination did not protect chickens against virus of the Y280 lineage, with no significant reduction in virus shedding and a 37.5% inhibition in virus recovery rate in cecal tonsil. rgHS314 was selected as a vaccine candidate, showing immunogenicity in SPF chickens, and was highly productive in eggs. Moreover, rgHS314 protected with high levels of protective immunity and significantly reduced virus shedding, with 100% and 83.3% inhibition of virus recovery in the cecal tonsil against homologous and heterologous challenge viruses, respectively. Taken together, these data suggest that a single vaccination with this recombinant vaccine candidate could elicit cross-reactive immune responses capable of protecting chickens against H9N2 viruses of the Y439 and Y280 lineages.

Pathogenesis and genetic characteristics of a novel reassortant low pathogenic avian influenza A(H7N6) virus isolated in Cambodia in 2019.

The first human case of zoonotic A(H7N4) avian influenza virus (AIV) infection was reported in early 2018 in China. Two months after this case, novel A(H7N4) viruses phylogenetically related to the Jiangsu isolate emerged in ducks from live bird markets in Cambodia. During active surveillance in Cambodia, a novel A(H7N6) reassortant of the zoonotic low pathogenic AIV (LPAIV) A(H7N4) was detected in domestic ducks at a slaughterhouse. Complete genome sequencing and phylogenetic analysis showed that the novel A(H7N6) AIV is a reassortant, in which four gene segments originated from Cambodia A(H7N4) viruses and four gene segments originated from LPAIVs in Eurasia. Animal infection experiments revealed that chickens transmitted the A(H7N6) virus via low-level direct contacts, but ducks did not. Although avian-origin A(H7Nx) LPAIVs do not contain the critical mammalian-adaptive substitution (E627K) in PB2, the lethality and morbidity of the A(H7N6) virus in BALB/c mice were similar to those of A(H7N9) viruses, suggesting potential for interspecies transmission. Our study reports the emergence of a new reassortant of zoonotic A(H7N4) AIVs with novel viral characteristics and emphasizes the need for ongoing surveillance of avian-origin A(H7Nx) viruses.

Single dose of multi-clade virus-like particle vaccine protects chickens against clade 2.3.2.1 and clade 2.3.4.4 highly pathogenic avian influenza viruses.

Virus-like particles (VLPs) are recognized as an alternative vaccine platform that provide effective protection against various highly pathogenic avian influenza viruses (HPAIVs). Here, we developed multi-clade VLPs expressing two HAs (a chimera of clade 2.3.2.1c and clade 2.3.4.4c HA) within a single vector. We then compared its protective efficacy with that of a monovalent VLP and evaluated its potency against each homologous strain. Chickens vaccinated with the multi-clade VLP shed less virus and were better protected against challenge than birds receiving monovalent vaccines. Single vaccination with a multi-clade VLP resulted in 100% survival, with no clinical symptoms and high levels of pre-challenge protective immunity (7.6-8.5 log2). Moreover, the multi-clade VLP showed high productivity (128-256 HAU) both in the laboratory and on a large scale, making it cheaper than whole inactivated vaccines produced in eggs. However, the PD50 (protective dose 50%) of the multi-clade VLP against clades 2.3.2.1c and 2.3.4.4c was < 50 PD50 (28 and 42 PD50, respectively), and effective antibody response was maintained for 2-3 months. This multi-clade VLP protects against both clades of HPAI viruses and can be produced in high amounts at low cost. Thus, the vaccine has potential as a pandemic preparedness vaccine.

Pathogenicity of H9N2 low pathogenic avian influenza viruses of different lineages isolated from live bird markets tested in three animal models: SPF chickens, Korean native chickens, and ducks.

Since its first appearance in 1996, H9N2 avian influenza virus (AIV) of the Y439 lineage persisted in Korean live bird markets (LBMs) until the last documented occurrence in 2018. However, in June 2020, the avian influenza surveillance program detected a novel H9N2 AIV belonging to the Y280 lineage, which has zoonotic potential, in a Korean native chicken (KNC) from a LBM. In this study, we infected KNCs and ducks (the 2 major species held at LBMs), as well as SPF chickens, with Y280-lineage H9N2 AIV LBM261/20 and Y439-equivalent LBM294/18 to compare pathogenicity and transmissibility. In SPF chickens, LBM261/20 replicated mostly in the respiratory tract and spread rapidly among birds. By contrast, LBM294/18 replicated preferentially in the gastrointestinal tract and transmitted more slowly than LBM261/20. LBM261/20 replicated for a longer time in KNCs than in SPF chickens, and only in the respiratory tract; by contrast, LBM294/18 was detected in the oropharynx and cloaca. Ducks did not shed either virus or seroconvert. Taken together, the data suggest that the scheme used to monitor the newly introduced H9N2 AIV of the Y280 lineage needs to be modified to place emphasis on oropharyngeal sampling. Such changes will facilitate better disease control and protect public health.

Multiple Reassortants of H5N8 Clade 2.3.4.4b Highly Pathogenic Avian Influenza Viruses Detected in South Korea during the Winter of 2020-2021.

During October 2020-January 2021, we isolated a total of 67 highly pathogenic avian influenza (HPAI) H5N8 viruses from wild birds and outbreaks in poultry in South Korea. We sequenced the isolates and performed phylogenetic analysis of complete genome sequences to determine the origin, evolution, and spread patterns of these viruses. Phylogenetic analysis of the hemagglutinin (HA) gene showed that all the isolates belong to H5 clade 2.3.4.4 subgroup B (2.3.4.4b) and form two distinct genetic clusters, G1 and G2. The cluster G1 was closely related to the 2.3.4.4b H5N8 HPAI viruses detected in Europe in early 2020, while the cluster G2 had a close genetic relationship with the 2.3.4.4b H5N8 viruses that circulated in Europe in late 2020. A total of seven distinct genotypes were identified, including five novel reassortants carrying internal genes of low pathogenic avian influenza viruses. Our Bayesian discrete trait phylodynamic analysis between host types suggests that the viruses initially disseminated from migratory waterfowl to domestic duck farms in South Korea. Subsequently, domestic duck farms most likely contributed to the transmission of HPAI viruses to chicken and minor poultry farms, highlighting the need for enhanced, high levels of biosecurity measures at domestic duck farms to effectively prevent the introduction and spread of HPAI.

Genetic characterization of H9N2 avian influenza virus previously unrecognized in Korea.

In this study, we describe the isolation and characterization of previously unreported Y280-lineage H9N2 viruses from two live bird markets in Korea in June 2020. Genetic analysis revealed that they were distinct from previous H9N2 viruses circulating in Korea and had highest homology to A/chicken/Shandong/1844/2019(H9N2) viruses. Their genetic constellation showed they belonged to genotype S, which is the predominant genotype in China since 2010, where genotype S viruses have infected humans and acted as internal gene donors to H5 and H7 zoonotic influenza viruses. Active surveillance and control measures need to be enhanced to protect the poultry industry and public health.

Isolation and characterization of low pathogenic H7N7 avian influenza virus from a red-crowned crane in a zoo in South Korea.

BACKGROUND: South Korea conducts annual national surveillance programs to detect avian influenza (AI) in domestic poultry, live bird markets, and wild birds. In March 2017, an AIV was isolated from fecal samples in an outdoor aviary flight cage in a zoo in Korea. RESULTS: Nucleotide sequencing identified the isolate as low pathogenic avian influenza virus (LPAIV) H7N7, and DNA barcoding analysis identified the host species as red-crowned crane. This isolate was designated A/red-crowned crane/Korea/H1026/2017 (H7N7). Genetic analysis and gene constellation analysis revealed that A/red-crowned crane/Korea/H1026/2017 (H7N7) showed high similarity with four H7N7 LPAIVs isolated from wild bird habitats in Seoul and Gyeonggi in early 2017. CONCLUSIONS: Considering the genetic similarity and similar collection dates of the viruses, and the fact that zoo bird cages are vulnerable to AIV, it is likely that fecal contamination from wild birds might have introduced LPAIV H7N7 into the red-crowned crane at the zoo. Therefore, our results emphasize that enhanced biosecurity measures should be employed during the wild bird migration season, and that continued surveillance should be undertaken to prevent potential threats to avian species in zoos and to humans.

Phylogeographic analysis of H5N1 highly pathogenic avian influenza virus isolated in Cambodia from 2018 to 2019.

Since 2004, several outbreaks of highly pathogenic avian influenza (HPAI) have been reported in Cambodia. Until 2013, all H5N1 viruses identified in Cambodia belonged to clade 1 and its subclades. H5N1 HPAI viruses belonging to clade 2.3.2.1c have been dominant since the beginning of 2014, with various genotypes (KH1-KH5) reported. Here, we isolated nine H5N1 HPAI viruses from domestic poultry farms and slaughterhouses in Cambodia during 2018-2019 and performed phylogenetic analysis of whole genome sequences. All isolates were classified as H5 clade 2.3.2.1c viruses and all harbored multi-basic amino acid sequences (PQRERRRKR/GLF) at the haemagglutinin (HA) cleavage site. Phylogenetic analysis revealed that the H5N1 isolates in this study belonged to the KH2 genotype, the dominant genotype in Cambodia in 2015. Phylogenetic analysis of the HA gene showed that the isolates were divided into two groups (A and B). The results of Bayesian discrete phylogeography analysis revealed that the viral migration pathways from Vietnam to Cambodia (Bayes factor value: 734,039.01; posterior probability: 1.00) and from Cambodia to Vietnam (Bayes factor value: 26,199.95; posterior probability: 1.00) were supported by high statistical values. These well-supported viral migrations between Vietnam and Cambodia demonstrate that viral transmission continued in both directions. Several factors may have contributed to this, including the free-grazing duck system and movement of poultry-related products. Thus, the results emphasize the need for an enhanced international surveillance program to better understand transboundary infection and evolution of H5N1 HPAI viruses, along with implementation of more stringent international trade controls on poultry and poultry products.

Genetic characteristics and pathogenesis of H5 low pathogenic avian influenza viruses from wild birds and domestic ducks in South Korea.

H5 and H7 subtypes of low pathogenic avian influenza viruses (LPAIVs) can mutate to highly pathogenic forms and are therefore subject to stringent controls. We characterized H5 LPAIVs isolated from wild-bird habitats and duck farms in South Korea from 2010 to 2017. Through nationwide active surveillance for AIVs, 59 H5 LPAIVs were isolated from wild-bird habitats (a mean annual rate of 5.3% of AIV isolations). In 2015, one LPAI H5N3 strain was isolated on a duck farm. Phylogenetic analysis revealed that the hemagglutinin (HA) gene of H5 isolates belonged to the Eurasian lineage, classified into three subgroups (HA-II, HA-III, and HA-IV). The H5 LPAIVs of the HA-III and HA-IV subgroups appeared in 2015 and 2017 in unusually high proportions (13.1% and 14.4%, respectively). In gene-constellation analysis, H5 LPAIVs isolated from 2015 to 2017 constituted ≥ 35 distinct genotypes, representing high levels of genetic diversity. Representative strains of three HA subgroups replicated restrictively in specific-pathogen-free chickens. Among the 11 isolates that were tested, 10 infected and replicated in mice without prior adaptation. The frequency of recent H5 LPAIV isolates with high genetic diversity indicates the importance of continued surveillance in both wild birds and poultry to monitor genetic and pathobiological changes.

Protection of layers and breeders against homologous or heterologous HPAIv by vaccines from Korean national antigen bank.

Korean government has selected and stocked five type antigens of two clades as Korean national antigen bank having high possibility of introduction to Korea. We aimed to evaluate the efficacy of the clade 2.3.2.1c and 2.3.4.4c H5Nx vaccines from the Korean avian influenza (AI) national antigen bank for emergency preparedness for their potency and protective efficacy against lethal homologous and heterologous viruses in layer and breeder chickens practically. The PD50 (dose of vaccine that protects 50% of chickens from viral challenge) of all vaccinated groups was >50, which was satisfied with minimum antigen requirement of OIE, and the PD50 levels of the two vaccines differed depending on strain and chicken breed. In homologous challenge, all vaccinated groups exhibited 100% survival with no clinical symptoms and high levels of pre-challenge protective immunity (7.2-8.5 log2), although they did not completely prevent virus shedding. On the other hand, against heterologous virus challenge, vaccinated animals exhibited 62.5-80% survival with lower antibody titers (2.3-3.4 log2) and a longer period of virus shedding (14 days post infection [dpi]). Our results suggest that the clade 2.3.2.1c and 2.3.4.4c H5Nx vaccines are good candidates for emergency vaccination of commercial chickens and support the idea that close genetic matching between vaccine and challenge virus provides the best protection.

Sales and immunogenicity of commercial vaccines to H9N2 low pathogenic avian influenza virus in Korea from 2007 to 2017.

The present study was conducted to monitor sales activity and immunogenicity of commercial H9N2 vaccines produced in Korea from 2007 to 2017. Recorded sales of H9N2 vaccine were around 671 million doses, with 10 million doses sold in 2007, rising to a peak of 93 million doses in 2016, with a slight fall in 2017. Multivalent combined vaccines made up around 90% of all vaccine sales, and around 30% of all vaccines were distributed by regional governments for free. The regional vaccination rate was the highest in Gyeonggi and Chungnam, respectively with proportional to the population of layer and breeder chickens. There have been no cases of field infection since 2009. The mean antibody titer was 5.82 log2 across the study period. Our results suggest that continuous genetic monitoring of H9N2 viruses circulating in the field and updating the vaccine seed strain periodically are necessary in order to control H9N2 outbreaks.

The interference effect of maternally-derived antibodies on the serological performance of pigs immunized with a foot-and-mouth disease oil emulsion vaccine.

To control foot-and-mouth disease (FMD) outbreaks that originated in Jincheon County in South Korea between 2014 and 2015, several commercial vaccines were studied for their efficacy and serological performance in the field. In this study, the efficacy of the O SKR 7/10 vaccine was evaluated by challenge with the FMD virus (FMDV) O/Jincheon/SKR/2014 (O Jincheon), which has the same O/SEA/Mya-98 lineage as the O/SKR/7/10 strain that was isolated in 2010 in South Korea, in FMD-seronegative pigs. Full protection against the O Jincheon virus was demonstrated as early as 14 days postvaccination, which was explained by the strong serological relationship (r1 value: ≥ 0.92) between the O Jincheon and O SKR 2010 viruses. However, in the field trial, no satisfactory serological elevations against FMDV were observed, even in the double-vaccinated groups. Therefore, it can be concluded that the O SKR 7/10 vaccine may need to be improved to overcome the interference effects from the high levels of maternally-derived antibodies generated due to the mandatory nationwide vaccination of sows in South Korea.

Genetic and pathogenic characteristics of clade 2.3.2.1c H5N1 highly pathogenic avian influenza viruses isolated from poultry outbreaks in Laos during 2015-2018.

Since 2004, there have been multiple outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses in Laos. Here, we isolated H5N1 HPAI viruses from poultry outbreaks in Laos during 2015-2018 and investigated their genetic characteristics and pathogenicity in chickens. Phylogenetic analysis revealed that the isolates belonged to clade 2.3.2.1c and that they differed from previous Laos viruses with respect to genetic composition. In particular, the isolates were divided into two genotypes, each of which had a different NS segments. The results of possible migration analysis suggested a high likelihood that the Laos isolates were introduced from neighbouring countries, particularly Vietnam. The recent Laos isolate, A/Duck/Laos/NL-1504599/2018, had an intravenous pathogenicity index score of 3.0 and showed a 50% chicken lethal dose of 102.5 EID50 /0.1 ml, indicating high pathogenicity. The isolated viruses exhibited no critical substitution in the markers associated with mammalian adaptation, but possess markers related to neuraminidase inhibitor resistance. These results emphasize the need for ongoing surveillance of circulating influenza virus in South-East Asia, including Laos, to better prepare for and mitigate global spread of H5 HPAI.

Protective efficacy of vaccines of the Korea national antigen bank against the homologous H5Nx clade 2.3.2.1 and clade 2.3.4.4 highly pathogenic avian influenza viruses.

The occurrence of severe outbreaks of highly pathogenic avian influenza in Korea led to establishment of a national antigen bank for emergency preparedness. Here, we developed five vaccines for this bank (clade 2.3.2.1C, clade 2.3.4.4A, B, C, and D) by reverse genetics, inactivated them with formalin, and evaluated the protective efficacy and potency of serial dilutions against lethal homologous challenge in specific-pathogen-free chickens. After vaccination with one dose, each vaccine resulted in 100% survival, with no clinical symptoms, or lack of detectable virus shedding, and high levels of pre-challenge protective immunity (8.4-10.2 log2). After vaccination with one-tenth of the full dose, protection was similar to that with the full dose. After vaccination with one-hundredth of the initial dose, survival was 20-80%, and all vaccines showed virus shedding. Four vaccines (excluding clade 2.3.2.1C) had satisfactory potency. In antibody-persistence tests, all vaccines maintained long-lasting protective immunity. Our results suggest that inactivated reverse-genetics vaccines genetically matched to outbreak viruses provide adequate protection after a single vaccination.

A novel reassortant clade 2.3.4.4 highly pathogenic avian influenza H5N6 virus identified in South Korea in 2018.

Since 2017, clade 2.3.4.4b H5N6 highly pathogenic avian influenza viruses (HPAIVs) have been detected over a broad geographic region, including Eurasia. These viruses have evolved through reassortment with Eurasian low pathogenic avian influenza viruses (LPAIVs), resulting in multiple genotypes. Here, we sequenced the full-length genome of 15 H5N6 HPAIVs collected from wild birds and poultry farms in South Korea from January to March 2018. A comparative phylogenetic analysis was then conducted. Three distinct genotypes were identified in South Korea during 2017/2018, including a novel reassortant genotype, H214. The novel reassortant H5N6 viruses isolated in this study possessed PB2, PA, and NP gene segments of Eurasian LPAIV on a genetic backbone of the H35-like genotype, which was identified in Korea and the Netherlands during 2017. Bayesian molecular clock analysis suggested that the novel reassortant viruses were generated most likely during the fall migration/wintering season of migratory waterfowl in 2017. Considering the continued emergence and spread of clade 2.3.4.4 HPAIV, enhanced surveillance of wild waterfowl is needed for early detection of HPAIV incursions.

Cross-protective efficacy of the O1 Manisa + O 3039 bivalent vaccine and the O 3039 monovalent vaccine against heterologous challenge with FMDV O/Jincheon/SKR/2014 in pig.

After massive foot-and-mouth disease (FMD) outbreaks originated from Jincheon County from Dec. 2014 to Apr. 2015, the effectiveness of the previous FMD vaccine containing only the O1 Manisa as the O antigen, O1 Manisa + A Malaysia 97 + Asia 1 Sharmir trivalent vaccine, was questioned in South Korea, and a change in the O antigen in FMD vaccines was demanded to control the FMD caused by FMDV O/Jincheon/SKR/2014, the O Jincheon strain. Therefore, the efficacies of O1 Manisa + O 3039 bivalent vaccine and O 3039 monovalent vaccine were studied for cross-protection against heterologous challenge with the O Jincheon strain. In this study, the efficacy of the O1 Manisa + O 3039 bivalent vaccine was better than that of the O 3039 monovalent vaccine, even though the serological relationship (r1 value) between O Jincheon and O 3039 was matched according to the OIE Terrestrial Manual. According to serological test results from vaccinated specific pathogen free pigs, virus neutralization test titers against Jincheon were good estimates for predicting protection against challenge. A field trial of the O1 Manisa + O 3039 bivalent vaccine was performed to estimate the possibility of field application in conventional pig farms, especially due to concerns about the effect of maternally derived antibodies (MDA) in field application of the FMD vaccine. According to the result of the field trial, the O1 Manisa + O 3039 bivalent vaccine was considered to overcome MDA. The results of the efficacy and field trials indicated that the O1 Manisa + O3039 vaccine could be suitable to replace previous FMD vaccines to control the FMD field situation caused by O Jincheon FMDV.

Pathogenesis and genetic characteristics of novel reassortant low-pathogenic avian influenza H7 viruses isolated from migratory birds in the Republic of Korea in the winter of 2016-2017.

In this study, we characterized H7 subtype low-pathogenicity (LP) influenza A viruses (IAVs) isolated from wild bird habitats in the Republic of Korea from 2010 to early 2017. Through national surveillance, 104 H7 IAVs were isolated, accounting for an average of 14.9% of annual IAV isolations. In early 2017, H7 subtypes accounted for an unusually high prevalence (43.6%) of IAV detections in wild birds. Phylogenetic analysis revealed that all the viruses isolated in the winter of 2016-2017 fell within cluster II of group C, belonging to the Eurasian lineage of H7 IAVs. Notably, cluster II of group C included the H7 gene from the highly pathogenic H7N7 IAV that was detected in northeastern Italy in April of 2016. Through a gene-constellation analysis, the H7 LPIAVs that we isolated constituted ≥11 distinct genotypes. Because the viruses belonging to the genotypes G2.1 and G1 were observed most frequently, we compared the replication and transmission of representative viruses to these genotypes in specific-pathogen-free chickens. Notably, the representative G2.1 strain was capable of systemic replication and efficient transmission in chickens (as evidenced by virus isolation and histopathological examination) without any clinical signs except mortality (in one infected chicken). The efficient subclinical viral replication and shedding of the G2.1 virus in chickens may facilitate its silent spread among poultry after introduction. Given that wild birds harbor novel strains that could affect poultry, our results highlight the need for enhanced IAV surveillance in both wild birds and poultry in Eurasia.

Genetic evidence for the intercontinental movement of avian influenza viruses possessing North American-origin nonstructural gene allele B into South Korea.

Avian influenza viruses (AIVs) are genetically separated by geographical barriers, resulting in the independent evolution of North American and Eurasian lineages. In the present study, to determine whether AIVs possessing the North American-origin nonstructural (NS) gene were previously introduced into South Korea, we performed a genetic analysis of AIVs isolated from fecal samples of migratory birds. We detected seven viruses possessing the North American-origin NS allele B among 413 AIV-positive samples obtained during AI surveillance between 2012 and 2017. We found evidence for the intercontinental transmission of at least three genetically distinct clusters of the B allele of the North American-origin NS gene into Eurasia at a low frequency. The host species of three viruses were identified as the greater white-fronted goose (Anser albifrons) using a DNA barcoding technique. Moreover, we used GPS-CDMA-based telemetry to determine the migration route of the greater white-fronted goose between the Far East of Russia and South Korea and found that this species may play an important role as an intermediate vector in the intercontinental transmission of AIVs. To improve our understanding of the role of wild birds in the ecology of AIVs, advanced AIV surveillance is required in the Far East of Russia as well as in Alaska region of Beringia accompanied by host identification and wild bird tracking.