Chang-Seon Song

Intercontinental Spread of Asian-Origin H5N8 to North America through Beringia by Migratory Birds

ABSTRACT Phylogenetic network analysis and understanding of waterfowl migration patterns suggest that the Eurasian H5N8 clade 2.3.4.4 avian influenza virus emerged in late 2013 in China, spread in early 2014 to South Korea and Japan, and reached Siberia and Beringia by summer 2014 via migratory birds. Three genetically distinct subgroups emerged and subsequently spread along different flyways during fall 2014 into Europe, North America, and East Asia, respectively. All three subgroups reappeared in Japan, a wintering site for waterfowl from Eurasia and parts of North America.

Sequence analysis of the hemagglutinin gene of H9N2 Korean avian influenza viruses and assessment of the pathogenic potential of isolate MS96.

Sequence analysis of the hemagglutinin (HA) gene of five Korean H9N2 avian influenza virus (AIV) isolates showed that these viruses were closely related and possibly came from the same source. Phylogenetic analysis of the HA1 subunit of H9 subtype isolates revealed that Korean AIV isolates were different from isolates from the poultry markets in Hong Kong in 1997. None of the Korean AIVs had multiple basic amino acids at the HA cleavage site that confer high pathogenicity to some H5 and H7 AIVs. Phylogenetic analysis of the nucleoprotein and matrix gene demonstrated that Korean isolates cluster with Eurasian origin AIVs. The pathogenic potential of one of the isolates (MS96) was assessed after several passages in 14-day-old embryonated chicken eggs (ECE). Fourteen-day-old ECE derivatives of MS96 showed increased HA titer and embryo mortality in eggs; this was apparent after the third passage in 14-day-old ECE. Sequence analysis of the cleavage site of MS96 after the third and tenth passages in 14-day-old ECE revealed no changes in the amino acid sequence. The pathogenicity of MS96 after the tenth passage in 14-day-old eggs (MS96p10(ECE14)) was tested with 4-wk-old specific-pathogen-free chickens. The 14-day-old derivative, MS96p10(ECE14), showed wider tissue tropism and induced more severe clinical signs than the parent virus. Furthermore, after intranasal inoculation of 86-wk-old broiler breeders and 30-wk-old layers, the MS96p10(ECE14) derivative induced more severe signs of depression than the parent virus as well as a transient drop in egg production.

Intranasal administration of live Lactobacillus species facilitates protection against influenza virus infection in mice.

Influenza virus infections continue to be a significant public health problem. For improved therapies and preventive measures against influenza, there has been an increased tendency in modern medicine involving the use of probiotics. In this study, we compared the protective efficacy of various live and dead Lactobacillus species against challenge with influenza virus in mice according to the administration route and dose. In addition, to understand the underlying mechanism behind this clinical protective effect, we performed immunologic assays including examination of IgA levels and cytokine profiles in the lung. The survival rate of mice receiving intranasal administration of Lactobacillus was higher than after oral administration, and administration of live bacteria was more protective than of dead bacteria. The lung levels of interleukin (IL)-12 and IgA were significantly increased (P<0.05). Conversely, the levels of the pro-inflammatory cytokines tumor necrosis factor-alpha and IL-6 were decreased. Interestingly, there were huge differences in protective effects of various Lactobacillus strains on influenza virus infection. Therefore, for clinical applications, selection of effective strains could be critical and individually optimized application regimens of the selected strains are required.

Analysis of complete genome sequences of swine hepatitis E virus and possible risk factors for transmission of HEV to humans in Korea.

The hepatitis E virus (HEV) is an emerging zoonotic agent, for which pigs are the most important reservoir. Complete genome sequences of two swine HEV strains, designated swKOR‐1 and swKOR‐2, were determined via RT‐PCR and RACE‐PCR. The strains contained genomes composed of 7,222‐ and 7,221‐bp excluding the poly(A) tails, respectively. The swKOR‐1 and swKOR‐2 strains were classified into subtype 3a of genotype 3 via phylogenetic analysis. These strains formed a distinctive cluster in the phylogenetic tree with human and swine HEVs isolated in the USA and human HEVs isolated in Japan. Anti‐HEV antibodies were identified via ELISA in 8 of 99 (8.1%) cats, whereas, among 115 cattle and 213 dogs, no HEV‐specific antibodies were detected. The conserved RNA‐dependent RNA polymerase (RdRp) gene of HEV could be detected via RT‐PCR in 8.7% of raw oysters collected from coastal regions in Korea. The HEV RNAs detected in oysters were identified as belonging to subtype 3a. The HEV RNAs in oysters most closely resembled that of the swKOR‐2 strain. They also showed a close genetic relationship with the swKOR‐1 strain and the swine and human HEVs isolated in the USA. This is the first report describing the detection in oysters of HEV that may have originated from genotype 3 swine HEV in Korea. Pigs and cats infected with HEV, as well as oysters contaminated with HEV, are potential risk factors for HEV transmission to humans. J. Med. Virol. 82:583–591, 2010.

Immune responses and pathogenesis in immunocompromised chickens in response to infection with the H9N2 low pathogenic avian influenza virus

The H9N2 low pathogenic avian influenza (LPAI) viruses have often caused moderate mortality with severe clinical signs in domestic poultry in many Eurasian countries and have occasionally caused clinical respiratory diseases in humans, but the basis for their pathogenesis remains unclear especially in chickens. To better understand the effect of immunosuppression on the risk of H9N2 viral infection, the pathogenesis and host immune responses of the H9N2 LPAI virus in a T-cell-suppressed chicken model were investigated. Cyclosporin A (CsA) treatment led to suppression of cell-mediated immunity such as CD8+ T-cells and reduced expression of IFN-gamma mRNA. T-cell suppression correlated with high viral load in the oropharynx and cloaca of H9N2 LPAI virus-infected specific pathogen free (SPF) chickens. Elevated level of viral RNA in the peripheral blood lymphocytes was found only in immunocompromised chickens. Viral protein and associated cellular apoptosis were observed only in the kidney of the immunocompromised chickens, particularly in those that had died. Our findings suggest that T-cell-mediated responses are important in influenza viral clearance and may help to explain in part the reasons for the increased mortality in chickens infected with H9N2 LPAI viruses in domestic poultry farms.

An emerging recombinant cluster of nephropathogenic strains of avian infectious bronchitis virus in Korea.

Abstract The infectious bronchitis virus (IBV) is continuously evolving through point mutation and recombination of their genome, subsequently the emergence of IBV variants complicates disease control. The objective of this study was to investigate genetic characterization of new IBV variants isolated from commercial chicken flocks in Korea collected between 2005 and 2010. Phylogenetic analysis revealed that all new IBV isolates belonged to Korean group II (K-II), which included the nephropathogenic IBV strains. However, the isolates formed a new gene cluster that was distinguished from the two distinct K-II subgroups (KM91-like and QX-like). Recombination events were identified in the S1 gene, with their putative parental strains being the KM91-like or QX-like subgroup. In addition, two crossover sites were observed in the S1 gene of IBV isolates. These results suggest that natural genetic recombination between heterologous strains classified into different genetic groups has occurred and may have caused the emergence of new IBV strains. This finding provides important information on IBV evolution and is essential for the effective control of IB in Korea.

Prevalence, antibiotic resistance, and molecular characterization of Salmonella serovars in retail meat products.

The prevalence of Salmonella was determined in chicken meat (n = 26), beef (n = 49), and pork (n = 56) collected from wholesale markets, retail stores, and traditional markets in Seoul, South Korea, in 2009. Antibiotic resistance was assessed, and the molecular subtypes of Salmonella isolates were ascertained using an automated repetitive sequence-based PCR (rep-PCR) system (DiversiLab). A total of 18 Salmonella strains were isolated from 17 of 131 samples: 16 strains from each of 16 samples and 2 strains from the same pork sample. The prevalence of Salmonella from the retail meats was 2.0% in beef, 8.9% in pork, and 42.3% in chicken meat. Among 10 different serotypes, Salmonella enterica Panama was recovered from a beef sample, and Salmonella London and Salmonella Montevideo were the predominant serotypes from pork and chicken meat, respectively. The highest antibiotic resistance observed was to erythromycin (100%) followed by streptomycin (22.2%) and tetracycline and chloramphenicol (16.7%). Of the 18 isolates, 5 (27.8%) were resistant to two or more antibiotics, and 1 isolate from chicken meat was resistant to eight antibiotics, including cephalosporins. Differentiation between all of the Salmonella isolates except between Salmonella Montevideo and Salmonella London was successfully performed with the automated rep-PCR system, indicating that it can be added to the toolbox for source tracking of foodborne pathogens associated with outbreaks.

Continuing evolution and interspecies transmission of influenza viruses in live bird markets in Korea.

Abstract Live bird markets (LBMs) provide an ideal environment for the evolution and interspecies transfer of avian influenza viruses (AIVs). In this study, we analyzed AIVs present in LBMs in Korea during the winter seasons of 2006–08. Sixty-five AIVs that belong to four hemagglutination (HA) subtypes of AIV (H3, H4, H6, and H9) were isolated from 644 pooled tissue or swab samples collected in LBMs. Most H9 subtypes of AIVs were isolated from Galliformes (chickens, silky fowls, pheasants, and guinea fowls), and other subtypes were isolated from Anseriformes (Pekin ducks and mallards). In addition, we obtained a single H3N2 virus from nasal swabs of dogs sold in LBMs, and the virus was genetically identical to the canine influenza virus (CIV) isolated from pet dogs in Korea. Phylogenetic analysis suggests that the Korean H9N2 viruses prevalent in chickens have provided their gene segments to AIVs circulating in ducks. These gene transfers facilitated reassortment events among AIVs and likely generated the ancestors of CIV in Korea. An animal challenge study using chickens, quail, mice, and dogs had shown that the H4 and H6 subtypes could replicate in mice and that some H4 and H6 viruses could replicate in chickens without preadaptation. In addition, two H3 subtype viruses (H3N2 and H3N8) induced interstitial pneumonia that accompanied clinical signs and seroconversion in dogs. Our findings indicate that the newly evolved AIVs have been continuously generated by reassortment in ducks, and these reassortments could result in expanding the host range of AIVs.

H9N2 avian influenza virus in Korea: evolution and vaccination

Low pathogenic avian influenza (LPAI) H9N2 viruses have been circulating in the Eurasian poultry industry resulting in great economic losses due to declined egg production and moderate to high mortality. In Korea, H9N2 LPAI was first documented in 1996 and it caused serious economic loss in the Korean poultry industry, including layer and broiler breeder farms. Since then, the H9N2 viruses that belong to the Korea group have been prevalent in chickens and have continuously evolved through reassortment in live bird markets. To control LPAI outbreaks, since 2007, the Korean veterinary authority has permitted the use of the inactivated oil adjuvant H9N2 LPAI vaccine. Although only oil-based inactivated vaccine using the egg-passaged vaccine virus strain (A/chicken/Korea/01310/2001) is permitted and used, several new technology vaccines have been recently suggested for the development of cost-effective and highly immunogenic vaccines. In addition, several different differentiation of infected from vaccinated animals (DIVA) strategies have been suggested using appropriate vaccines and companion serologic tests for discriminating between naturally infected and vaccinated animals. Recent reports demonstrated that the Korean LPAI H9N2 virus underwent antigenic drift and evolved into distinct antigenic groups and thus could escape from vaccine protection. Therefore, improved vaccination strategies including periodic updates of vaccine seed strains are required to achieve efficient control and eradication of LPAI H9N2 in Korea. Further, vaccination should be part of an overall integrated strategy to control the disease, including continued nation-wide surveillance, farm biosecurity, and DIVA strategy.

H9N2 avian influenza virus-like particle vaccine provides protective immunity and a strategy for the differentiation of infected from vaccinated animals

In the present study, virus-like particles (VLPs) were evaluated as a candidate poultry vaccine against avian influenza virus (AIV) subtype H9N2. Specific pathogen-free chickens received a single injection of the VLP vaccine expressing HA and M1 protein of AIV H9N2 (H9 HA VLP) at escalating doses in the presence or absence of ISA70 water-in-oil adjuvant. At 3 weeks post vaccination, we performed hemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) to determine serological immune responses, and challenge studies using SPF chickens. A single dose of H9 HA VLP vaccine induced high levels of HI antibodies and lowered frequencies of virus isolation after the wild-type virus challenge. The addition of ISA70 adjuvant significantly increased the immunogenicity of H9 HA VLP vaccines. Furthermore, it allows differentiation of AIV-infected chickens from vaccinated chickens with an ELISA using nucleocapsid antigen, which offers a promising strategy to differentiate infected from vaccinated animals (DIVA). These results provide support for continued development of the VLP as an animal vaccine against influenza virus.