Haan-Woo Sung

Characterization of Highly Pathogenic H5N1 Avian Influenza A Viruses Isolated from South Korea

ABSTRACT An unprecedented outbreak of H5N1 highly pathogenic avian influenza (HPAI) has been reported for poultry in eight different Asian countries, including South Korea, since December 2003. A phylogenetic analysis of the eight viral genes showed that the H5N1 poultry isolates from South Korea were of avian origin and contained the hemagglutinin and neuraminidase genes of the A/goose/Guangdong/1/96 (Gs/Gd) lineage. The current H5N1 strains in Asia, including the Korean isolates, share a gene constellation similar to that of the Penfold Park, Hong Kong, isolates from late 2002 and contain some molecular markers that seem to have been fixed in the Gs/Gd lineage virus since 2001. However, despite genetic similarities among recent H5N1 isolates, the topology of the phylogenetic tree clearly differentiates the Korean isolates from the Vietnamese and Thai isolates which have been reported to infect humans. A representative Korean isolate was inoculated into mice, with no mortality and no virus being isolated from the brain, although high titers of virus were observed in the lungs. The same isolate, however, caused systemic infections in chickens and quail and killed all of the birds within 2 and 4 days of intranasal inoculation, respectively. This isolate also replicated in multiple organs and tissues of ducks and caused some mortality. However, lower virus titers were observed in all corresponding tissues of ducks than in chicken and quail tissues, and the histological lesions were restricted to the respiratory tract. This study characterizes the molecular and biological properties of the H5N1 HPAI viruses from South Korea and emphasizes the need for comparative analyses of the H5N1 isolates from different countries to help elucidate the risk of a human pandemic from the strains of H5N1 HPAI currently circulating in Asia.

Characterization of a Highly Pathogenic H5N1 Avian Influenza A Virus Isolated from Duck Meat

ABSTRACT Since the 1997 H5N1 influenza virus outbreak in humans and poultry in Hong Kong, the emergence of closely related viruses in poultry has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. In May 2001, an avian H5N1 influenza A virus was isolated from duck meat that had been imported to South Korea from China. Phylogenetic analysis of the hemagglutinin (HA) gene of A/Duck/Anyang/AVL-1/01 showed that the virus clustered with the H5 Goose/Guandong/1/96 lineage and 1997 Hong Kong human isolates and possessed an HA cleavage site sequence identical to these isolates. Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens. The pathogenesis of DK/Anyang/AVL-1/01 virus in Pekin ducks was further characterized and compared with a recent H5N1 isolate, A/Chicken/Hong Kong/317.5/01, and an H5N1 1997 chicken isolate, A/Chicken/Hong Kong/220/97. Although no clinical signs of disease were observed in H5N1 virus-inoculated ducks, infectious virus could be detected in lung tissue, cloacal, and oropharyngeal swabs. The DK/Anyang/AVL-1/01 virus was unique among the H5N1 isolates in that infectious virus and viral antigen could also be detected in muscle and brain tissue of ducks. The pathogenesis of DK/Anyang/AVL-1/01 virus was characterized in BALB/c mice and compared with the other H5N1 isolates. All viruses replicated in mice, but in contrast to the highly lethal CK/HK/220/97 virus, DK/Anyang/AVL-1/01 and CK/HK/317.5/01 viruses remained localized to the respiratory tract. DK/Anyang/AVL-1/01 virus caused weight loss and resulted in 22 to 33% mortality, whereas CK/HK/317.5/01-infected mice exhibited no morbidity or mortality. The isolation of a highly pathogenic H5N1 influenza virus from poultry indicates that such viruses are still circulating in China and may present a risk for transmission of the virus to humans.

Recent Korean isolates of duck hepatitis virus reveal the presence of a new geno- and serotype when compared to duck hepatitis virus type 1 type strains

SummaryDuck hepatitis was first reported in 1985 in Korea. The complete nucleotide sequence of two past Korean isolates, DHV-HS and DHV-HSS, isolated in 1994 and 1995, and four recent Korean isolates, AP-03337, AP-04009, AP-04114 and AP-04203 isolated in 2003 and 2004, were determined. Phylogenetic analysis using the 3D protein sequence confirmed that the previously characterized duck hepatitis virus type 1 strains and the six Korean isolates described here constitute a monophyletic group and form two clades/genotypes in which all except the four recent Korean isolates form one group (A) and the recent Korean isolates of 2003 and 2004 constitute a second group (B). Phylogenetic analysis of the VP1 protein supported the division into two different groups. Antisera raised against viruses of group A showed significant neutralizing cross-reaction against a member of the same genotype but not to a strain of group B and vice versa. These results demonstrated that the two genotypes also could be regarded as two different serotypes.

Epidemiological classification of infectious bronchitis virus isolated in Korea between 1986 and 1997

Forty Korean isolates and four reference strains of infectious bronchitis virus (IBV) were classified by reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism (RFLP) analysis. Each Korean isolate was isolated from different types of commercial chicken flocks between 1986 and 1997. RFLP patterns of an amplified DNA fragment (1722 bp) containing the S1 gene of IBV digested by restriction enzyme HaeIII showed that the 40 Korean isolates were classified into five genotypes, I to V. Six of them belonged to genotype I which had the same HaeIII and XcmI cleavage patterns with Massachusetts type (H120 and M41) but the other four genotypes had a different HaeIII cleavage pattern from the four reference IBV strains used in this study. Genotype III seemed to be the major type as 29 of the 40 isolates belonged to this type which was consistently found in the chicken flocks since 1990. On the other hand, genotypes II, IV and V were found in the field only in 1986, 1995 and 1995, respectively. Five isolates selected from each of the five genotypes were inoculated into 1-day-old specific-pathogen-free chicks to evaluate their pathogenicity. Genotype III induced 50% mortality as well as severe renal urate deposition on the kidneys but the other four genotypes only showed respiratory distress at 1 to 2 days after inoculation. Live H120 vaccine protected chicks against challenge with isolates selected from genotype I, but not genotypes IV to V. A live KM91p120 strain selected from major genotype III did protect chicks against challenge with isolates from genotype III, in addition to other genotypes, including two recent isolates of genotypes IV and V.

Evaluation of a High-Pathogenicity H5N1 Avian Influenza A Virus Isolated from Duck Meat

Abstract The introduction of an influenza A virus possessing a novel hemagglutinin (HA) into an immunologically naive human population has the potential to cause severe disease and death. Such was the case in 1997 in Hong Kong, where H5N1 influenza was transmitted to humans from infected poultry. Because H5N1 viruses are still isolated from domestic poultry in southern China, there needs to be continued surveillance of poultry and characterization of virus subtypes and variants. This study provides molecular characterization and evaluation of pathogenesis of a recent H5N1 virus isolated from duck meat that had been imported to South Korea from China. The HA gene of A/Duck/Anyang/AVL-1/01 (H5N1) isolate was found to be closely related to the Hong Kong/97 H5N1 viruses. This virus also contained multiple basic amino acids adjacent to the cleavage site between HA1 and HA2, characteristic of high-pathogenicity avian influenza viruses (HPAI). The pathogenesis of this virus was characterized in chickens, ducks, and mice. The DK/Anyang/AVL-1/01 isolate replicated well in all species and resulted in 100% and 22% lethality for chickens and mice, respectively. No clinical signs of disease were observed in DK/Anyang/AVL-1/01-inoculated ducks, but high titers of infectious virus could be detected in multiple tissues and oropharyngeal swabs. The presence of an H5N1 influenza virus in ducks bearing a HA gene that is highly similar to those of the pathogenic 1997 human/poultry H5N1 viruses raises the possibility of reintroduction of HPAI to chickens and humans.

Protection of chickens from Newcastle disease with a recombinant baculovirus subunit vaccine expressing the fusion and hemagglutininneuraminidase proteins.

Recombinant baculoviruses containing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein gene of the viscerotropic velogenic (vv) Newcastle disease virus (NDV) isolate, Kr-005/00, and a lentogenic La Sota strain of the NDV were constructed in an attempt to develop an effective subunit vaccine to the recent epizootic vvNDV. The level of protection was determined by evaluating the clinical signs, mortality, and virus shedding from the oropharynx and cloaca of chickens after a challenge with vvNDV Kr-005/00. The recombinant ND F (rND F) and recombinant HN (rND HN) glycoproteins derived from the velogenic strain provided good protection against the clinical signs and mortality, showing a 0.00 PI value and 100% protection after a booster immunization. On the other hand, the combined rND F + HN glycoprotein derived from the velogenic strain induced complete protection (0.00 PI value and 100% protection) and significantly reduced the amount of virus shedding even after a single immunization. The rND F and rND HN glycoproteins derived from the velogenic strain had a slightly, but not significantly, greater protective effect than the lentogenic strain. These results suggest that the combined rND F + HN glycoprotein derived from vvNDV can be an ideal subunit marker vaccine candidate in chickens in a future ND eradication program.

Isolation and characterization of a novel H9N2 influenza virus in Korean native chicken farm.

SUMMARY. An outbreak of avian influenza, caused by an H9N2 low-pathogenic avian influenza virus (AIV), occurred in a chicken farm and caused severe economic losses due to mortality and diarrhea. AIV was isolated and identified in a sample from an affected native Korean chicken. Genetic analysis of the isolate revealed a high sequence similarity to genes of novel reassortant H9N2 viruses isolated from slaughterhouses and live bird markets in Korea in 2008 and 2009. Animal challenge studies demonstrated that the replication kinetics and pathogenicity of the isolate were considerably altered due to adaptation in chickens. Vaccine protection studies indicated that commercial vaccine was not able to prevent virus shedding and clinical disease when chickens were challenged with the isolate. These results suggest that the novel H9N2 virus possesses the capacity to replicate efficiently in the respiratory system against vaccination and to cause severe disease in domestic chickens. The results also highlight the importance of appropriate updating of vaccine strains, based on continuous surveillance data, to prevent the possibility of a new H9N2 epidemic in Korea.

High Virus Titer in Feather Pulp of Chickens Infected with Subgroup J Avian Leukosis Virus

SUMMARY. Subgroup J avian leukosis viruses (ALVs), which are a recombinant virus between exogenous and endogenous ALVs, can spread by either vertical or horizontal transmission. Exogenous and endogenous ALVs can be detected in feather pulp. In this study, virus titers in feather pulp of chickens infected with subgroup J ALV were compared with those of plasma and cloacal swab. All of the broiler chickens inoculated with subgroup J ALV at 1 day old were positive for virus from feather pulp during the experimental period of between 2 wk and 8 wk of age. Virus titers in feather pulp of some broiler chickens infected with subgroup J ALV were very high, ranging from 107 to 108 infective units per 0.2 ml. Virus titers in feather pulp were usually the highest among the samples of plasma, cloacal swab, and feather pulp tested. In another experiment in which layer chickens were inoculated with subgroup J ALV at 1 day old, virus was detected in feather pulp from 2 wk until 18 wk of age, and virus persisted longer in feather pulp than in plasma. Almost all of the layer chickens tested were positive for virus by polymerase chain reaction (PCR) with DNA extracted from feather pulp samples at 2, 4, and 10 wk of age, and the PCR from feather pulp was more sensitive than virus isolation from plasma, cloacal swab, and feather pulp. All above results indicate that samples of feather pulp can be useful for virus isolation and PCR to confirm subgroup J ALV infection.

Molecular Epidemiologic Investigation of Lentogenic Newcastle Disease Virus from Domestic Birds at Live Bird Markets in Korea

SUMMARY. A Newcastle disease surveillance program was conducted at live bird markets in Korea to expand our epidemiologic understanding of the disease in Korea. During the surveillance program, 10 lentogenic Newcastle disease viruses (NDVs) were isolated and identified from apparently healthy chickens and ducks at live bird markets. The lentogenic viruses had sequence motifs of either 112GKQGRL117 (n  =  8) or 112GRQGRL117 (n  =  2) at the F0 cleavage site. Sequencing and phylogenetic analyses of NDV isolates based on the hypervariable region of the F protein revealed two different genotypes: genotypes I (n  =  8) and II (n  =  2). Genotype I viruses were most closely related to the NDV V4 strain (n  =  7) or the NDV Ulster 2C strain (n  =  1). In contrast, genotype II viruses clustered with the NDV vaccine strains (LaSota and VG/GA) that are commonly used as live vaccines in Korea. The epidemiologic importance of NDV at live bird markets in Korea is discussed.

Surveillance of Newcastle Disease Virus in Chicken Slaughterhouses

We conducted a 10-month (March to October 2009) surveillance of Newcastle disease virus (NDV) in 13 slaughter- houses in Korea. NDV was isolated in 13.0%, 13.3%, 16.0%, and 10.8% of chicken farms, transport vehicles, hang rooms, and chilling water, respectively. Of NDV isolates from slaughterhouses, 37% were isolated in July. All NDV isolates were determined to be lentogenic viruses by RT-PCR-based pathotyping, and all NDV isolates had the 112 GKQGR/L 117 motif at the cleavage site of the F protein. Phylogenetic analysis based on the hypervariable region of the F protein gene classified all 25 NDV isolates examined into genotype I within class . Of these, 24 were clustered together with the NDV V4 strain, while the remaining isolate was placed in the cluster belonging to the NDV Ulster 2C strain. Our results indicate that lentogenic NDV was a high-frequency contaminant in the serial process of ranging live birds to slaughtering at slaughterhouses.