Isolation and whole genome sequencing of North American lineage class I avian orthoavulavirus 1 isolated from wild Eurasian teal in South Korea.

We report the first North American origin class I avian orthoavulavirus 1 (AOAV-1) isolated from a faecal dropping of wild Eurasian teal (Anas crecca) in South Korea. Whole genome sequencing and comparative phylogenetic analysis revealed that the AOAV-1/Eurasian teal/South Korea/KU1405-3/2017 virus belongs to the sub-genotype 1.2 of class I AOAV-1. Phylogenetic analysis suggested multiple introductions of the North American sub-genotype 1.2 viruses into Asia and its establishment in the wild bird population in East Asia since May 2011. These results provide information on the epidemiology of AOAV-1, particularly the role of migratory wild birds in exchanging viruses between the Eurasian and North American continents. Enhanced genomic surveillance is required to improve our understanding on the evolution and transmission dynamics of AOAV-1 in wild birds.

Introduction of Avian Influenza A(H6N5) Virus into Asia from North America by Wild Birds.

An avian influenza A(H6N5) virus with all 8 segments of North American origin was isolated from wild bird feces in South Korea. Phylogenetic analysis suggests that this virus may have been introduced into Asia by wild birds, highlighting the role of wild birds in the dispersal of these viruses.

New Reassortant Clade 2.3.4.4b Avian Influenza A(H5N6) Virus in Wild Birds, South Korea, 2017-18.

We isolated new reassortant avian influenza A(H5N6) viruses from feces of wild waterfowl in South Korea during 2017-18. Phylogenetic analysis suggested that reassortment occurred between clade 2.3.4.4b H5N8 and Eurasian low pathogenicity avian influenza viruses circulating in wild birds. Dissemination to South Korea during the 2017 fall migratory season followed.

Reassortant Clade 2.3.4.4 Avian Influenza A(H5N6) Virus in a Wild Mandarin Duck, South Korea, 2016.

A reassortant clade 2.3.4.4 avian influenza A(H5N6) virus was isolated from a fecal sample of a Mandarin duck (Aix galericulata) in South Korea during October 2016. This virus was genetically similar to H5N6 subtype virus isolates from China, Vietnam, Laos, and Hong Kong, including human isolates.

Highly Pathogenic Avian Influenza A(H5N8) Viruses Reintroduced into South Korea by Migratory Waterfowl, 2014-2015.

Highly pathogenic avian influenza A(H5N8) viruses were isolated from migratory waterfowl in South Korea during fall 2014-winter 2015, a recurrence after initial introduction in winter 2014. These reappeared viruses were phylogenetically distinct from isolates circulating in poultry farms in South Korea.

Efficacy of live and inactivated recombinant Newcastle disease virus vaccines expressing clade 2.3.4.4b H5 hemagglutinin against H5N1 highly pathogenic avian influenza in SPF chickens, Broilers, and domestic ducks.

A Newcastle disease virus (NDV)-vectored vaccine expressing clade 2.3.4.4b H5 Hemagglutinin was developed and assessed for efficacy against H5N1 highly pathogenic avian influenza (HPAI) in specific pathogen-free (SPF) chickens, broilers, and domestic ducks. In SPF chickens, the live recombinant NDV-vectored vaccine, rK148/22-H5, achieved complete survival against HPAI and NDV challenges and significantly reduced viral shedding. Notably, the live rK148/22-H5 vaccine conferred good clinical protection in broilers despite the presence of maternally derived antibodies. Good clinical protection was observed in domestic ducks, with decreased viral shedding. It demonstrated complete survival and reduced cloacal viral shedding when used as an inactivated vaccine from SPF chickens. The rK148/22-H5 vaccine is potentially a viable and supportive option for biosecurity measure, effectively protecting in chickens against the deadly clade 2.3.4.4b H5 HPAI and NDV infections. Furthermore, it aligns with the strategy of Differentiating Infected from Vaccinated Animals (DIVA).

Age is a determinant factor in the susceptibility of domestic ducks to H5 clade 2.3.2.1c and 2.3.4.4e high pathogenicity avian influenza viruses.

High pathogenicity avian influenza (HPAI) is a viral disease with devastating consequences for the poultry industry worldwide. Domestic ducks are a major source of HPAI viruses in many Eurasian countries. The infectivity and pathogenicity of HPAI viruses in ducks vary depending on host and viral factors. To assess the factors influencing the infectivity and pathogenicity of HPAI viruses in ducks, we compared the pathobiology of two HPAI viruses (H5N1 clade 2.3.2.1c and H5N6 clade 2.3.4.4e) in 5- and 25-week-old ducks. Both HPAI viruses caused mortality in a dose-dependent manner (104, 106, and 108 EID50) in young ducks. By contrast, adult ducks were infected but exhibited no mortality due to either virus. Viral excretion was higher in young ducks than in adults, regardless of the HPAI strain. These findings demonstrate the age-dependent mortality of clade 2.3.2.1c and clade 2.3.4.4e H5 HPAI viruses in ducks.

Intramuscular administration of recombinant Newcastle disease virus expressing SARS-CoV-2 spike protein protects hACE-2 TG mice against SARS-CoV-2 infection.

Coronavirus disease 2019 (Covid-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) became a pandemic, causing significant burden on public health worldwide. Although the timely development and production of mRNA and adenoviral vector vaccines against SARS-CoV-2 have been successful, issues still exist in vaccine platforms for wide use and production. With the potential for proliferative capability and heat stability, the Newcastle disease virus (NDV)-vectored vaccine is a highly economical and conceivable candidate for treating emerging diseases. In this study, a recombinant NDV-vectored vaccine expressing the spike (S) protein of SARS-CoV-2, rK148/beta-S, was developed and evaluated for its efficacy against SARS-CoV-2 in K18-hACE-2 transgenic mice. Intramuscular vaccination with low dose (106.0 EID50) conferred a survival rate of 76 % after lethal challenge of a SARS-CoV-2 beta (B.1.351) variant. When administered with a high dose (107.0 EID50), vaccinated mice exhibited 100 % survival rate and reduced lung viral load against both beta and delta variants (B.1.617.2). Together with the protective immunity, rK148/beta-S is an accessible and cost-effective SARS-CoV-2 vaccine.

COVID-19 Subunit Vaccine with a Combination of TLR1/2 and TLR3 Agonists Induces Robust and Protective Immunity.

The development of COVID-19 vaccines is critical in controlling global health issues under the COVID-19 pandemic. The subunit vaccines are the safest and most widely used vaccine platform and highly effective against a multitude of infectious diseases. An adjuvant is essential for subunit vaccines to enhance the magnitude and durability of immune responses. In this study, we determined whether a combination of toll-like receptor (TLR)1/2 and TLR3 agonists (L-pampo) can be a potent adjuvant for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine. We measured a neutralizing antibody (nAb) and an angiotensin-converting enzyme 2 (ACE2) receptor-blocking antibody against SARS-CoV-2 receptor-binding domain (RBD). We also detected interferon-gamma (IFN-γ) production by using ELISPOT and ELISA assays. By employing a ferret model, we detected nAbs and IFN-γ producing cells and measured viral load in nasal wash after the challenge of SARS-CoV-2. We found that SARS-CoV-2 antigens with L-pampo stimulated robust humoral and cellular immune responses. The efficacy of L-pampo was higher than the other adjuvants. Furthermore, in the ferret model, SARS-CoV-2 antigens with L-pampo elicited nAb response and antigen-specific cellular immune response against SARS-CoV-2, resulting in substantially decreased viral load in their nasal wash. Our study suggests that SARS-CoV-2 antigens formulated with TLR agonists, L-pampo, can be a potent subunit vaccine to promote sufficient protective immunity against SARS-CoV-2.

Live Recombinant NDV-Vectored H5 Vaccine Protects Chickens and Domestic Ducks From Lethal Infection of the Highly Pathogenic H5N6 Avian Influenza Virus.

The H5 subtype highly pathogenic avian influenza virus (HPAIV) has been introduced to South Korea every 2 or 3 years via wild migratory waterfowls, causing devastating damages to the poultry industry. Although most damages and economic losses by HPAIV are focused on chicken layers, domestic ducks are known to play a major role in the farm-to-farm transmission. However, most HPAIV vaccine studies on poultry have been performed with oil-emulsion inactivated vaccines. In this study, we developed a live recombinant Newcastle disease virus (NDV)-vectored vaccine against H5 HPAIV (rK148/ES2-HA) using a previously established NDV vaccine strain (K148/08) isolated from a wild mallard duck. The efficacy of the vaccine when administered via the oculonasal route or as a spray was evaluated against lethal H5 HPAIV infection in domestic ducks and chickens. Oculonasal inoculation of the rK148/ES2-HA in chickens and ducks elicited antibody titers against HPAIV as early as 1 or 2 week after the single dose of vaccination, whereas spray vaccination in ducks elicited antibodies against HPAIV after the booster vaccination. The chickens and ducks vaccinated with rK148/ES2-HA showed high survival rates and low viral shedding after H5N6 HPAIV challenge. Collectively, vaccination with rK148/ES2-HA prevented lethal infection and decreased viral shedding in both chickens and ducks. The vaccine developed in this study could be useful in suppressing the viral shedding in H5 HPAIV outbreaks, with the ease of vaccine application and fast onset of immunity.

The Microvillar and Solitary Chemosensory Cells as the Novel Targets of Infection of SARS-CoV-2 in Syrian Golden Hamsters.

Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019, suffer from respiratory and non-respiratory symptoms. Among these symptoms, the loss of smell has attracted considerable attention. The objectives of this study were to determine which cells are infected, what happens in the olfactory system after viral infection, and how these pathologic changes contribute to olfactory loss. For this purpose, Syrian golden hamsters were used. First, we verified the olfactory structures in the nasal cavity of Syrian golden hamsters, namely the main olfactory epithelium, the vomeronasal organ, and their cellular components. Second, we found angiotensin-converting enzyme 2 expression, a receptor protein of SARS-CoV-2, in both structures and infections of supporting, microvillar, and solitary chemosensory cells. Third, we observed pathological changes in the infected epithelium, including reduced thickness of the mucus layer, detached epithelia, indistinct layers of epithelia, infiltration of inflammatory cells, and apoptotic cells in the overall layers. We concluded that a structurally and functionally altered microenvironment influences olfactory function. We observed the regeneration of the damaged epithelium, and found multilayers of basal cells, indicating that they were activated and proliferating to reconstitute the injured epithelium.

Subclinical Infection and Transmission of Clade 2.3.4.4 H5N6 Highly Pathogenic Avian Influenza Virus in Mandarin Duck (Aix galericulata) and Domestic Pigeon (Columbia livia domestica).

Since 2014, H5Nx clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIV) have caused outbreaks in wild birds and poultry in multiple continents, including Asia, Europe, Africa, and North America. Wild birds were suspected to be the sources of the local and global spreads of HPAIV. This study evaluated the infectivity, pathogenicity, and transmissibility of clade 2.3.4.4 H5N6 HPAIV in mandarin ducks (Aixgalericulata) and domestic pigeons (Columbia livia domestica). None of the birds used in this study, 20 mandarin ducks or 8 pigeons, showed clinical signs or mortality due to H5N6 HPAI infection. Two genotypes of H5N6 HPAIV showed replication and transmission by direct and indirect contact between mandarin ducks. H5N6 HPAIV replicated and transmitted by direct contact between pigeons, although the viral shedding titer and duration were relatively lower and shorter than those in mandarin ducks. Influenza virus antigen was detected in various internal organs of infected mandarin ducks and pigeons, indicating systemic infection. Therefore, our results indicate mandarin ducks and pigeons can be subclinically infected with clade 2.3.4.4 H5N6 HPAIV and transfer the virus to adjacent birds. The role of mandarin ducks and pigeons in the spread and prevalence of clade 2.3.4.4 H5N6 viruses should be carefully monitored.

Live bird markets as evolutionary epicentres of H9N2 low pathogenicity avian influenza viruses in Korea.

Live bird markets (LBMs) in Korea have been recognized as a reservoir, amplifier, and source of avian influenza viruses (AIVs); however, little was known about the role of LBMs in the epidemiology of AIVs in Korea until recently. Through 10 years of surveillance (2006-2016) we have isolated and sequenced H9N2 viruses in Korean LBMs. To understand how H9N2 evolves and spreads in Korea, a statistical Bayesian phylogenetic model was used. Phylogenetic analysis suggests that three separate introductions of progenitor gene pools, Korean domestic duck-origin and two wild aquatic bird-origin AIVs, contributed to the generation of the five genotypes of H9N2 viruses in Korea. Phylogenetic reconstruction of ecological states infer that the LBMs are where chickens become infected with the virus, with domestic ducks playing a major role in the transmission and evolution of the H9N2 viruses. Three increases in the genetic diversity of H9N2 viruses were observed and coincided with transitions in host species and the locations (domestic farm, LBM, slaughterhouse, and wild aquatic bird habitat) where the viruses were isolated, accompanying genetic reassortment. Following the introduction of a wild aquatic bird-origin AIVs in 2008, six genes of the Korean lineage H9N2 virus were replaced with genes originating from wild aquatic birds, and viruses with this new genotype became predominant in Korean LBMs.

Improvement of PR8-Derived Recombinant Clade 2.3.4.4c H5N6 Vaccine Strains by Optimization of Internal Genes and H103Y Mutation of Hemagglutinin.

Clade 2.3.4.4c H5N6 avian influenza A viruses (AIVs) may have originally adapted to infect chickens and have caused highly pathogenic avian influenza (HPAI) in poultry and human fatalities. Although A/Puerto Rico/8/1934 (H1N1) (PR8)-derived recombinant clade 2.3.4.4c H5N6 vaccine strains have been effective in embryonated chicken eggs-based vaccine production system, they need to be improved in terms of immunogenicity and potential mammalian pathogenicity. We replaced the PB2 gene alone or the PB2 (polymerase basic protein 2), NP (nucleoprotein), M (matrix protein) and NS (non-structural protein) genes together in the PR8 strain with corresponding genes from AIVs with low pathogenicity to remove mammalian pathogenicity and to match CD8+ T cell epitopes with contemporary HPAI viruses, respectively, without loss of viral fitness. Additionally, we tested the effect of the H103Y mutation of hemagglutinin (HA) on antigen productivity, mammalian pathogenicity and heat/acid stability. The replacement of PB2 genes and the H103Y mutation reduced the mammalian pathogenicity but increased the antigen productivity of the recombinant vaccine strains. The H103Y mutation increased heat stability but unexpectedly decreased acid stability, probably resulting in increased activation pH for HA. Interestingly, vaccination with inactivated recombinant virus with replaced NP, M and NS genes halted challenge virus shedding earlier than the recombinant vaccine without internal genes replacement. In conclusion, we successfully generated recombinant clade 2.3.4.4c H5N6 vaccine strains that were less pathogenic to mammals and more productive and heat stable than conventional PR8-derived recombinant strains by optimization of internal genes and the H103Y mutation of HA.

Bioengineering a highly productive vaccine strain in embryonated chicken eggs and mammals from a non-pathogenic clade 2·3·4·4 H5N8 strain.

The clade 2·3·4·4 H5Nx is a highly pathogenic avian influenza (HPAI) virus, which first appeared in China and has spread worldwide since then, including Korea. It is divided into subclades a - d, but the PR8-derived recombinant clade 2·3·4·4 a viruses replicate inefficiently in embryonated chicken eggs (ECEs). High virus titer in ECEs and no mammalian pathogenicity are the most important prerequisites of efficacious and safer vaccine strains against HPAI. In this study, we have synthesized hemagglutinin (HA) and neuraminidase (NA) genes based on the consensus amino acid sequences of the clade 2·3·4·4a and b H5N8 HPAIVs, using the GISAID database. We generated PR8-derived H5N8 recombinant viruses with single point mutations in HA and NA, which are related to efficient replication in ECEs. The H103Y mutation in HA increased mammalian pathogenicity as well as virus titer in ECEs, by 10-fold. We also successfully eradicated mammalian pathogenicity in H103Y-bearing H5N8 recombinant virus by exchanging PB2 genes of PR8 and 01310 (Korean H9N2 vaccine strain). The final optimized H5N8 vaccine strain completely protected against a heterologous clade 2·3·4·4c H5N6 HPAIV in chickens, and induced hemagglutination inhibition (HI) antibody in ducks. However, the antibody titer of ducks showed age-dependent results. Thus, H103Y and 01310PB2 gene have been successfully applied to generate a highly productive, safe, and efficacious clade 2·3·4·4 H5N8 vaccine strain in ECEs.

Different pathogenicity of two strains of clade 2.3.4.4c H5N6 highly pathogenic avian influenza viruses bearing different PA and NS gene in domestic ducks.

H5Nx clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIVs) have been disseminated to wide geographic regions since 2014. In 2016, five distinct genotypes (C-1 to C-5) of clade 2.3.4.4c H5N6 HPAIVs were detected in South Korea. In this study, we evaluated the pathogenicity, susceptibility to infection, and transmissibility of the two strains representing the C-1 and C-4 genotypes of the H5N6 viruses, which have different PA and NS gene, in domestic ducks. Although the susceptibility to infection of domestic ducks to the two strains was similar, the C-4 genotype virus induced higher mortality in ducks than C-1 genotype virus. A higher titer of viral shedding were detected in ducks challenged with the C-4 genotype virus compared with the C-1 genotype virus. These results indicated that the reassortment of HPAIVs with prevailing low pathogenic avian influenza viruses could effect on the pathogenicity in ducks.

Molecular characterization and genetic diversity of avian paramyxovirus type 4 isolated in South Korea from 2013 to 2017.

In recent years, avian paramyxovirus type 4 (APMV-4) frequently isolated from wild and domestic bird populations particularly waterfowls worldwide. However, molecular characteristics and genetic diversity of APMV-4 are uncertain, owing to the limited availability of sequence information. A total of 11 APMV-4 strains from 9850 fecal, swab, and environmental samples were isolated during the surveillance program in wintering seasons of 2013-2017 in South Korea. We performed genetic characterization and phylogenetic analysis to investigate the genetic diversity and relatedness between isolates from the region. We report high APMV-4 genetic diversity (multiple genotypes and sub-genotypes) among wild bird and poultry populations in Korea and that the potential virus exchange occurs between neighboring countries via wild bird migration. Furthermore, our study results suggest the possibility of transcontinental transmission of APMV-4 between Asia and Europe.

Molecular Characterization of Avian Paramyxovirus Types 4 and 8 Isolated from Wild Migratory Waterfowl in Mongolia.

Avian paramyxoviruses (APMVs) constitute some of the most globally prevalent avian viruses and are frequently isolated from wild migratory bird species. Using 1,907 fresh fecal samples collected during the 2012 avian influenza surveillance program, we identified two serotypes of APMV: APMV-4 ( n=10) and APMV-8 ( n=1). Sequences for these isolates phylogenetically clustered with Asian APMV-4 and APMV-8 recently isolated from wild birds in Korea, Japan, China, and Kazakhstan. Analysis by DNA barcoding indicated that the Mongolian APMV-4 and APMV-8 strains were isolated from Anseriformes species including Mallards ( Anas platyrhynchos) and Whooper Swans ( Cygnus cygnus). The close genetic relatedness to Asian isolates, and to similar host species, suggested that wild bird species in the Anatidae family might play an important role as a natural reservoir in the spread of APMV-4 and APMV-8. However, we did not find conclusive evidence to support this hypothesis owing to the limited number of strains that could be isolated. Enhanced surveillance of poultry and wild bird populations in Asia is therefore crucial for the understanding of global AMPV transmission, ecology, evolution, and epidemiology.

Optimization of inactivated H5N9 highly pathogenic avian influenza vaccine and inactivated Salmonella enterica serovar Typhimurium vaccine with antigen dose and prime-boost regimen in domestic ducks.

Owing to the increase in the number of diseases affecting ducks and the demand for food safety by consumers, vaccination has become one of the factors that influence duck meat productivity. The highly pathogenic avian influenza (HPAI) virus is one of the most prevalent and causes one of the most lethal diseases in domestic ducks, and Salmonella enterica serovar Typhimurium is a food-borne pathogen persistent in the domestic duck population. To better understand the optimal usage of HPAI and S. enterica serovar Typhimurium vaccines, we aimed to determine antigen dose, oil and gel adjuvant usage with prime-boost regimen, and vaccination age, inducing the best immune response in ducks, without an effect on body weight gain. In the case of the inactivated H5N9 vaccine, a single dose of vaccine was inadequate to induce proper antibody titer when administered to day-old ducks, which necessitates boost vaccination. Administration of the oil-adjuvanted H5N9 vaccine administration in day-old and 2-week-old ducks resulted in a lower body weight at the time of slaughtering, compared to that of gel-adjuvanted H5N9 vaccine. However, gel-adjuvanted H5N9 vaccine failed to induce proper immune response to an extent recommend by OIE-World Organization for Animal Health. In the case of the Salmonella enterica serovar Typhimurium vaccine, a moderate or low dose of vaccine was appropriate for day-old ducks receiving the gel prime-oil boost vaccination. Single vaccination with oil adjuvants affects the mean body weight of 7-week-old ducks, suggesting that the gel adjuvant is more suitable for meat production. We expect that the use of adjuvants in a prime-boost regimen and at antigen doses set in this study will be helpful to maximize body weight in the case of domestic duck production at the actual farm site.

Efficacy of clade 2.3.2 H5 commercial vaccines in protecting chickens from clade 2.3.4.4 H5N8 highly pathogenic avian influenza infection.

Emerging clade 2.3.4.4 of the highly pathogenic avian influenza (HPAI) virus strain H5N8, which had been detected sporadically in domestic poultry in China, started to affect wild birds and poultry in South Korea in 2014. The virus was spread to Germany, Italy, the Netherlands, United Kingdom, and even United States by migratory birds. Here, we tested currently used commercial clade 2.3.2 H5 vaccines to evaluate mortality, clinical signs, virus shedding, and histological damage after experimental infection of chickens with the clade 2.3.4.4 HPAI H5N8 virus. Although the vaccination protected chickens from death, it failed to prevent chickens from shedding the virus and from tissue damage according to histological examination. These results suggest that the use of appropriate vaccines that match the currently epidemic HPAI virus is recommended, and continuous HPAI surveillance and testing of currently used commercial vaccines should be performed.