Development of a Novel Korean H9-Specific rRT-PCR Assay and Its Application for Avian Influenza Virus Surveillance in Korea.

Since the 2000s, the Y439 lineage of H9N2 avian influenza virus (AIV) has been the predominant strain circulating in poultry in Korea; however, in 2020, the Y280 lineage emerged and spread rapidly nationwide, causing large economic losses. To prevent further spread and circulation of such viruses, rapid detection and diagnosis through active surveillance programs are crucial. Here, we developed a novel H9 rRT-PCR assay that can detect a broad range of H9Nx viruses in situations in which multiple lineages of H9 AIVs are co-circulating. We then evaluated its efficacy using a large number of clinical samples. The assay, named the Uni Kor-H9 assay, showed high sensitivity for Y280 lineage viruses, as well as for the Y439 lineage originating in Korean poultry and wild birds. In addition, the assay showed no cross-reactivity with other subtypes of AIV or other avian pathogens. Furthermore, the Uni Kor-H9 assay was more sensitive, and had higher detection rates, than reference H9 rRT-PCR methods when tested against a panel of domestically isolated H9 AIVs. In conclusion, the novel Uni Kor-H9 assay enables more rapid and efficient diagnosis than the "traditional" method of virus isolation followed by subtyping RT-PCR. Application of the new H9 rRT-PCR assay to AI active surveillance programs will help to control and manage Korean H9 AIVs more efficiently.

Genetic Characterization and Pathogenesis of H5N1 High Pathogenicity Avian Influenza Virus Isolated in South Korea during 2021-2022.

High pathogenicity avian influenza (HPAI) viruses of clade 2.3.4.4 H5Nx have been circulating in poultry and wild birds worldwide since 2014. In South Korea, after the first clade 2.3.4.4b H5N1 HPAI viruses were isolated from wild birds in October 2021, additional HPAIV outbreaks occurred in poultry farms until April 2022. In this study, we genetically characterized clade 2.3.4.4b H5N1 HPAIV isolates in 2021-2022 and examined the pathogenicity and transmissibility of A/mandarin duck/Korea/WA585/2021 (H5N1) (WA585/21) in chickens and ducks. Clade 2.3.4.4b H5N1 HPAI viruses caused 47 outbreaks in poultry farms and were also detected in multiple wild birds. Phylogenetic analysis of HA and NA genes indicated that Korean H5N1 HPAI isolates were closely related to Eurasian viruses isolated in 2021-2022. Four distinct genotypes of H5N1 HPAI viruses were identified in poultry, and the majority were also found in wild birds. WA585/21 inoculated chickens showed virulent pathogenicity with high mortality and transmission. Meanwhile, ducks infected with the virus showed no mortality but exhibited high rates of transmission and longer viral shedding than chickens, suggesting that they may play an important role as silent carriers. In conclusion, consideration of both genetic and pathogenic traits of H5N1 HPAI viruses is required for effective viral control.

Protective efficacy of a bivalent H5 influenza vaccine candidate against both clades 2.3.2.1 and 2.3.4.4 high pathogenic avian influenza viruses in SPF chickens.

Worldwide, high pathogenic avian influenza viruses belonging to clades 2.3.4.4 and 2.3.2.1 have been circulating in both poultry and wild birds. Since 2018, Korea has built a national antigen bank to ensure preparedness in an emergency. In this study, we developed a bivalent vaccine candidate containing antigens derived from two reassortant KA435/2.3.2.1d and H35/2.3.4.4b strains for Korean national antigen bank. We evaluated its immunogenicity and protective efficacy in specific pathogen free chickens. The two vaccine strains, rgKA435-H9N2 PB2/2.3.2.1d and rgH35/2.3.4.4b, both of which were generated successfully by reverse genetics, were highly immunogenic (titres of haemagglutination inhibition: 8.3 and 8.4 log2, respectively) and showed good protective efficacy (100 and 147 50% protective dose, respectively) against lethal challenge with wild-type virus when delivered as a 1:1 mixture. Notably, the vaccine provided complete protection against viral shedding at a full dose (512 HAU) and a 1/10 dose (51.2 HAU), with no clinical signs, after challenge with H35/2.3.4.4b. The bivalent vaccine developed in this study may reduce the cost of vaccine production and could be used as a H5 subtype avian influenza vaccine candidate against two clades simultaneously.

Statistical Analysis of the Performance of Local Veterinary Laboratories in Molecular Detection (rRT-PCR) of Avian Influenza Virus via National Proficiency Testing Performed during 2020-2022.

For the early detection of avian influenza virus (AIV), molecular diagnostic methods such as real-time RT-PCR (rRT-PCR) are the first choice in terms of accuracy and speed in many countries. A laboratory's capability to perform this diagnostic method needs to be measured through external and independent assessment to ensure that the method is validated within the laboratory and in interlaboratory comparison. The Animal and Plant Quarantine Agency of Korea has implemented five rounds of proficiency testing (PT) for rRT-PCR targeting local veterinary service laboratories involved in the AIV national surveillance program from 2020 to 2022. In each round, a portion composed of six or more samples was selected from the entire PT panel consisting of H5, H7, and H9 viruses isolated in Korea and distributed to each participant, and at least one pair of samples was commonly included in each panel for interlaboratory comparison. During the five rounds of PT, a few incorrect and outlying results were detected that required immediate inspection or corrective actions. However, in the quantitative measurement of Ct values, the average standard deviation or coefficient of variation decreased as multiple PT rounds progressed, and a positive correlation between consecutive rounds of PT was observed since 2021. The better consistency or stability in the experimental performance appeared to contribute to the more harmonized results in the latest PTs, and it is assumed that the positive reaction of participants to the challenges of quantitative assessment reports showing their status intuitively might work. We need to continue operating the PT program for local laboratories because they play crucial roles at the front line of the national avian influenza surveillance program, and frequent changes in the human resources or environment for diagnosis in those laboratories are inevitable.

Protection of SPF Chickens by H9N2 Y439 and G1 Lineage Vaccine against Homologous and Heterologous Viruses.

Prior to the identification of low pathogenic avian influenza H9N2 viruses belonging to the Y280 lineage in 2020, Y439 lineage viruses had been circulating in the Republic of Korea since 1996. Here, we developed a whole inactivated vaccine (vac564) by multiple passage of Y439 lineage viruses and then evaluated immunogenicity and protective efficacy in specific-pathogen-free chickens. We found that LBM564 could be produced at high yield in eggs (108.4EID50/0.1 mL; 1024 hemagglutinin units) and was immunogenic (8.0 ± 1.2 log2) in chickens. The vaccine showed 100% inhibition of virus in the cecal tonsil with no viral shedding detected in either oropharyngeal or cloacal swabs after challenge with homologous virus. However, it did not induce effective protection against challenge with heterologous virus. An imported commercial G1 lineage vaccine inhibited viral replication against Y280 and Y439 lineage viruses in major tissues, although viral shedding in oropharyngeal and cloacal swabs was observed up until 5 dpi after exposure to both challenge viruses. These results suggest that a single vaccination with vac564 could elicit immune responses, showing it to be capable of protecting chickens against the Y439 lineage virus. Thus, our results suggest the need to prepare suitable vaccines for use against newly emerging and re-emerging H9N2 viruses.

Current situation and control strategies of H9N2 avian influenza in South Korea.

The H9N2 avian influenza (AI) has become endemic in poultry in many countries since the 1990s, which has caused considerable economic losses in the poultry industry. Considering the long history of the low pathogenicity H9N2 AI in many countries, once H9N2 AI is introduced, it is more difficult to eradicate than high pathogenicity AI. Various preventive measures and strategies, including vaccination and active national surveillance, have been used to control the Y439 lineage of H9N2 AI in South Korea, but it took a long time for the H9N2 virus to disappear from the fields. By contrast, the novel Y280 lineage of H9N2 AI was introduced in June 2020 and has spread nationwide. This study reviews the history, genetic and pathogenic characteristics, and control strategies for Korean H9N2 AI. This review may provide some clues for establishing control strategies for endemic AIV and a newly introduced Y280 lineage of H9N2 AI in South Korea.

Emergence of clade 2.3.4.4b novel reassortant H5N1 high pathogenicity avian influenza virus in South Korea during late 2021.

High pathogenicity H5N1 avian influenza viruses pose a threat to both animal and human health worldwide. In late 2020, outbreaks of H5 high pathogenicity avian influenza viruses belonging to clade 2.3.4.4b emerged in Europe, following on from outbreaks in East Asia in earlier years. However, very recent studies show that clade 2.3.4.4b H5N1, rather than 2.3.4.4b H5N8, has become predominant in wild birds and has infected poultry in several countries. In this study, we describe isolation of a novel H5N1 virus from a captured mandarin duck in South Korea, and another H5N1 virus from a quail farm. We performed genetic analysis of these two viruses to identify their origin and to determine their relationship with the clade 2.3.4.4b H5N1 viruses currently circulating in Europe. Based on our results, it is presumed that the novel H5N1 virus isolated in Korea originated from an unknown reassortant between clade 2.3.4.4b H5N8 viruses circulating from 2020 and other Eurasian viruses, with additional reassortment of genes and point mutations that discriminate them from the recently reported H5N1 virus in Europe.

Genetic Characterization of Novel H7Nx Low Pathogenic Avian Influenza Viruses from Wild Birds in South Korea during the Winter of 2020-2021.

Zoonotic infection with avian influenza viruses (AIVs) of subtype H7, such as H7N9 and H7N4, has raised concerns worldwide. During the winter of 2020-2021, five novel H7 low pathogenic AIVs (LPAIVs) containing different neuraminidase (NA) subtypes, including two H7N3, an H7N8, and two H7N9, were detected in wild bird feces in South Korea. Complete genome sequencing and phylogenetic analysis showed that the novel H7Nx AIVs were reassortants containing two gene segments (hemagglutinin (HA) and matrix) that were related to the zoonotic Jiangsu-Cambodian H7 viruses causing zoonotic infection and six gene segments originating from LPAIVs circulating in migratory birds in Eurasia. A genomic constellation analysis demonstrated that all H7 isolates contained a mix of gene segments from different viruses, indicating that multiple reassortment occurred. The well-known mammalian adaptive substitution (E627K and D701N) in PB2 was not detected in any of these isolates. The detection of multiple reassortant H7Nx AIVs in wild birds highlights the need for intensive surveillance in both wild birds and poultry in Eurasia.

Pathogenicity of H5N8 High Pathogenicity Avian Influenza Virus in Chickens and Ducks from South Korea in 2020-2021.

During the 2020-2021 winter season, an outbreak of clade 2.3.4.4b H5N8 high pathogenicity avian influenza (HPAI) virus occurred in South Korea. Here, we evaluated the pathogenicity and transmissibility of A/mandarin duck/Korea/H242/2020 (H5N8) (H242/20(H5N8)) first isolated from this outbreak in specific pathogen-free (SPF) chickens and commercial ducks in comparison with those of A/duck/Korea/HD1/2017(H5N6) (HD1/17(H5N6)) from a previous HPAI outbreak in 2017-2018. In chickens, the 50% chicken lethal dose and mean death time of H242/20(H5N8) group were 104.5 EID50 and 4.3 days, respectively, which indicate less virulent than those of HD1/17(H5N6) (103.6 EID50 and 2.2 days). Whereas, chickens inoculated with H242/20(H5N8) survived longer and had a higher titer of viral shedding than those inoculated with HD1/17(H5N6), which may increase the risk of viral contamination on farms. All ducks infected with either HPAI virus survived without clinical symptoms. In addition, they exhibited a longer virus shedding period and a higher transmission rate, indicating that ducks may play an important role as a silent carrier of both HPAI viruses. These results suggest that the pathogenic characteristics of HPAI viruses in chickens and ducks need to be considered to effectively control HPAI outbreaks in the field.

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.

Novel reassortants of clade 2.3.4.4 H5N6 highly pathogenic avian influenza viruses possessing genetic heterogeneity in South Korea in late 2017.

Novel H5N6 highly pathogenic avian influenza viruses (HPAIVs) were isolated from duck farms and migratory bird habitats in South Korea in November to December 2017. Genetic analysis demonstrated that at least two genotypes of H5N6 were generated through reassortment between clade 2.3.4.4 H5N8 HPAIVs and Eurasian low pathogenic avian influenza virus in migratory birds in late 2017, suggesting frequent reassortment of clade 2.3.4.4 H5 HPAIVs and highlighting the need for systematic surveillance in Eurasian breeding grounds.

Human telomerase reverse transcriptase-immortalized porcine monomyeloid cell lines for the production of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) shows highly restricted cell tropism and targets subpopulations of differentiated macrophages such as porcine alveolar macrophages (PAM) in the natural host. Although primary PAM cells would be ideal for in vitro virus production, they are not only difficult and expensive for establishment but cannot be frozen reliably for long-term storage and use. Apart from PAM cells, African green monkey kidney derived Marc-145 cells are used commonly for virus propagation. However, concerns have been raised regarding a possible modification of specific epitopes associated with virus neutralization because of distinct virus entry between PAM and Marc-145 cells. In order to overcome these problems, the present study was aimed to generate immortalized porcine monocyte/macrophage cell lines and to evaluate their potential for PRRSV production. Primary PAM cells were transfected stably with the human telomerase reverse transcriptase (hTERT) cDNA by a retrovirus vector so that constitutive expression of the hTERT protein allows cells to proliferate indefinitely. The newly immortalized PAM clones were shown to exert functional telomerase activity, indicating sustained expression of hTERT. In addition, telomerase-immortalization of PAMs did not affect expression levels of the native CD163 receptor on their surface. It was further demonstrated that these continuous PAM cell lines are fully permissive for the efficient growth of both type 1 and 2 PRRSV strains. The findings suggest that the hTERT-immortalized PAM cell lines can enable us to facilitate the continued use of PAMs for virus isolation and production and to provide a promising tool for viral pathogenesis and immune function studies.

Porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-ß production by inhibiting IRF3 activation in immortalized porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection appears to elicit a weak innate immune response suppressing type 1 interferon (IFN) production. Recent studies have revealed that several nonstructural proteins encoded by the PRRSV genome independently antagonize the type 1 IFN system. The present study sought to identify the structural proteins that possess the immune evasion properties in immortalized porcine alveolar macrophages (PAM). Each structural protein gene was stably expressed in a porcine monocyte-derived macrophage cell line, PAM-pCD163, and tested for its potential to inhibit IFN-ß induction. We then focused on the nucleocapsid (N) protein, which has a strong inhibitory effect on dsRNA-induced IFN-ß production. Upon dsRNA stimulation, IFN-ß production was shown to decrease proportionally with increasing levels of N expression. Furthermore, the PRRSV N protein was found to down-regulate IFN-dependent gene production by dsRNA. Taken together, these results indicate the ability of N to modulate the dsRNA-mediated IFN induction pathways. In addition, the N protein significantly interfered with dsRNA-induced phosphorylation and nuclear translocation of IRF3. Our data suggest that the PRRSV N protein is a responsible component, independent of other nonstructural elements, for evading the IFN response by antagonizing IRF3 activation.

Development and characterization of stable cell lines constitutively expressing the porcine reproductive and respiratory syndrome virus nucleocapsid protein.

Despite global efforts to control porcine reproductive and respiratory syndrome virus (PRRSV) infection, the virus continues to cause economic problems in the swine industry worldwide. In this study, we attempted to generate and characterize a panel of stable BHK cell lines that constitutively express the nucleocapsid (N) protein of type 1 or type 2 PRRSV. The established BHK cell lines were found to react well with N-specific antibodies as well as the hyperimmune serum of pigs raised against each genotype of PRRSV. Taken together, the data implicate a potential usefulness for the newly generated stable cell lines as a diagnostic reagent for PRRSV serology.

Differential cellular protein expression in continuous porcine alveolar macrophages regulated by the porcine reproductive and respiratory syndrome virus nucleocapsid protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a leading cause of significant economic losses in the pig industry worldwide. PRRSV infects preferentially porcine alveolar macrophages (PAMs) and subsequently utilizes the host cell biosynthetic machinery for its own replication. To date, a number of studies have been conducted to investigate compensatory changes of cellular gene expression of PAMs upon PRRSV infection. However, very little information exists about differential cellular protein expression of the natural target cells regulated by each viral protein. This study was therefore designed to examine the dynamics of host protein expression of continuous PAM cells by the PRRSV nucleocapsid (N) protein that is the most abundant and multifunctional viral component. We first established sublines of PAM cells to stably express the PRRSV N protein and assessed alterations in cellular protein productions of N-expressing PAM (PAM-pCD163-N) cells at different time courses by the use of proteomic analysis. A total of 23 protein spots were initially found to be differentially expressed in PAM-pCD163-N cells compared with normal PAM cells by high-resolution two-dimensional gel electrophoresis (2DE). Of these spots, 15 protein spots with statistically significant alteration, including 4 up-regulated and 11 down-regulated protein spots, were picked out for subsequent protein identification by peptide mass fingerprinting after matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS). The altered cellular proteins identified in this study were classified into the functions involved in a variety of cellular processes such as cell division, metabolism, inflammation response, stress response, ubiquitin-proteasome pathway, protein folding and synthesis, and transportation. Notably, heat shock 27kDa protein (HSP27) was found to be up-regulated in PAM-pCD163-N cells. The proteomics data will provide insights into the specific cellular response to the N protein during PRRSV infection.