Experimental infection of highly pathogenic avian influenza viruses, Clade 2.3.4.4 H5N6 and H5N8, in Mandarin ducks from South Korea.

Outbreaks of highly pathogenic avian influenza (HPAI) have been reported worldwide. Wild waterfowl play a major role in the maintenance and transmission of HPAI. Highly pathogenic avian influenza subtype H5N6 and H5N8 viruses simultaneously emerged in South Korea. In this study, the comparative pathogenicity and infectivity of Clade 2.3.4.4 Group B H5N8 and Group C H5N6 viruses were evaluated in Mandarin duck (Aix galericulata). None of the ducks infected with H5N6 or H5N8 viruses showed clinical signs or mortality. Serological assays revealed that the HA antigenicity of H5N8 and H5N6 viruses was similar to each other. Moreover, both the viruses did not replicate after cross-challenging with H5N8 and H5N6 viruses, respectively, as the second infection. Although both the viruses replicated in most of the internal organs of the ducks, viral replication and shedding through cloaca were higher in H5N8-infected ducks than in H5N6-infected ducks. The findings of this study provide preliminary information to help estimate the risks involved in further evolution and dissemination of Clade 2.3.4.4 HPAI viruses among wild birds.

Experimental Infection of Goats with a Newly Isolated Strain of Akabane Virus that Causes Encephalomyelitis.

In 2010, there was a large-scale outbreak of bovine encephalomyelitis in Korea, and 15 new strains of Akabane virus (AKAV) were isolated. To identify the pathogenicity of one of these strains, we infected adult goats with AKAV-7 via different routes. Twenty-five female goats were used in this study and were divided into five groups: intracerebral (IC) and intrasubarachnoid (IS) viral inoculation (n = 8 each), intravenous (IV) inoculation (n = 4), and vaccinated before IV inoculation (n = 4), in addition to a negative control animal. All animals inoculated with AKAV-7 had AKAV-neutralizing antibodies at 6-8 days post infection (dpi). During the experimental period, infected animals showed no clinical signs. In the IC group, 5/8 goats had non-suppurative encephalomyelitis affecting the cerebrum. Virus S RNA segments were detected in nearly all areas of the brain. In the IS group, 3/8 goats had encephalomyelitis affecting the cerebrum, cerebellum and spinal cord. At 7 and 21 dpi, virus S RNA segments were found mostly in the spinal cord, especially around the area of injection (L5-L6). Antibody titres in the serum of the vaccinated group had an early onset and slightly increased titre compared with the IV group. Histopathologically, there were no obvious lesions in the central nervous tissues in the vaccinated group, while one of four goats in the IV group showed encephalomyelitis in the parietal lobe of the cerebrum. The newly isolated AKAV-7 can cause encephalomyelitis in goats after experimental injection. The attenuated AKAV vaccine currently used in Korea may provide partial protective immunity against AKAV-7 infection, but the real effect of the vaccine requires further investigation in goats.

Prevalence and molecular characterisation of Giardia duodenalis in calves with diarrhoea.

The aim of this study was to investigate the prevalence of Giardia duodenalis in diarrhoeal faeces from calves that were reared in Korea using PCR and ELISA. Diarrhoeal faecal samples were collected from 590 calves (<3 months old) throughout the country from November 2013 through March 2015. Data were analysed according to the region of collection, season, and type of diarrhoea. Of the 590 collected faecal samples, 77 (13.1 per cent) and 78 (13.2 per cent; true prevalence 9.1 per cent) tested positive by PCR and ELISA, respectively. The results from both testing methods were in agreement in 501 (84.9 per cent) samples, with a κ value of 0.34, which suggests fair agreement. The PCR results showed a higher prevalence in summer and in cases of haemorrhagic diarrhoea (P<0.05), while the ELISA results showed a lower prevalence in cases of pasty diarrhoea (P<0.05). Phylogenetic analysis of the ß-giardin gene sequences of G duodenalis showed that the sequences detected in this study belonged to assemblage E, which is specific to cattle and livestock. The higher prevalence of giardiasis in calves with haemorrhagic diarrhoea found in this study differed from previous studies. Therefore, researchers and veterinarians should be aware of the possible involvement of giardiasis in haemorrhagic diarrhoea.

Bovine epizootic encephalomyelitis caused by Akabane virus infection in Korea.

A large-scale epidemic of Akabane virus (AKAV) encephalomyelitis in cattle aged 4-72 months occurred in the southern part of Korea from late summer to late autumn in 2010. Affected cattle exhibited neurological signs including locomotor ataxia, astasia, tremor and hypersensitivity. Samples of brain (n = 116), spinal cord (n = 116) and whole blood (n = 205) were submitted to the National Veterinary Research and Quarantine Service for diagnosis. Microscopical analysis of the brains and spinal cords revealed the presence of non-suppurative encephalomyelitis in 99 of 116 brains and/or spinal cords (85%). The brains and spinal cords were evaluated by reverse transcriptase polymerase chain reaction and AKAV antigens were detected by immunohistochemistry using rabbit antiserum against AKAV strain OBE-1. Fifteen AKAVs were isolated from the brain and spinal cord samples. Antibodies against AKAV in a virus neutralization test were detected in 188 of 205 serum samples (91.7%). This is the first report of a large-scale outbreak of bovine epidemic encephalomyelitis caused by AKAV infection in Korea.

Molecular characterization of chicken infectious anemia viruses detected from breeder and broiler chickens in South Korea.

In South Korea, 32 sequences of chicken infectious anemia virus (CIAV) from various flocks of breeder and commercial chickens were genetically characterized for the first time. Phylogenetic analysis of the viral protein 1 gene, including a hypervariable region of the CIAV genome, indicated that Korean CIAV strains were separated into groups II, IIIa, and IIIb. Strains were commonly identified in great-grandparent and grandparent breeder farms as well as commercial chicken farms. In the field, CIAV strains from breeder farms had no clinical effects, but commercial farm strains were associated with depression, growth retardation, and anemia regardless of the group from which the strain originated. In addition, we identified 7 CIAV genomes that were similar to vaccine strains from vaccinated and unvaccinated breeder flocks. These data suggest that further studies on pathogenicity and vaccine efficacy against the different CIAV group are needed, along with continuous CIAV surveillance and genetic analysis at breeder farms.

Genetic characteristics of virion protein 2 genes of infectious bursal disease viruses isolated from commercial chickens with clinical disease in South Korea.

We examined the molecular identification of 13 infectious bursal disease virus (IBDV) strains isolated in Korea from January 2009 to January 2010. Sequence analysis of the variable virion protein 2 gene suggested that 3 of the isolates were very virulent IBDV, 8 of the isolates were classical virulent IBDV, 1 of the isolates was antigenic variant IBDV, and 1 of the isolates was intermediate plus vaccine strain. However, the clinical effects were evident for these strains regardless of classification because each chick flock was raised in various field situations with a different vaccine program and complications by other viruses or bacteria. Therefore, our observations revealed that IBDV strains of 4 genotypes, including vaccine strain, were recently present in South Korea and that the IBDV strains need to be discriminated using genetic characterization of virion protein 2 gene for efficient diagnosis and disease control.

Pathogenic characteristics of the Korean 2002 isolate of foot-and-mouth disease virus serotype O in pigs and cattle.

Experimental infection of susceptible cattle and pigs showed that the O/SKR/AS/2002 pig strain of foot-and-mouth disease virus (FMDV) causes an infection that is highly virulent and contagious in pigs but very limited in cattle. Pigs directly inoculated with, or exposed to swine infected with, strain O/SKR/AS/2002 showed typical clinical signs, including gross vesicular lesions in mouth and pedal sites. In addition, FMDV was isolated from, and FMDV genomic RNA was detected in, blood, serum, nasal swabs and oesophageal-pharyngeal (OP) fluid early in the course of infection. Antibodies against the non-structural protein (NSP) 3ABC were detected in both directly inoculated and contact pigs, indicating active virus replication. In contrast, the disease in cattle was atypical. After inoculation, lesions were confined to the infection site. A transient viraemia occurred 1 and 2 days after inoculation, and this was followed by the production of antibodies to NSP 3ABC, indicating subclinical infection. No clinical disease was seen, and no antibodies to NSP 3ABC were present in contact cattle. Additionally, no virus or viral nucleic acid was detected in blood, nasal swab and OP fluid samples from contact cattle. Thus, the virus appeared not to be transmitted from infected cattle to contact cattle. In its behaviour in pigs and cattle, strain O/SKR/AS/2002 resembled the porcinophilic FMDV strain of Cathay origin, O/TAW/97. However, the latter, unlike O/SKR/AS/2002, has reduced ability to grow in bovine-derived cells. The porcinophilic character of O/TAW/97 has been attributed to a deletion in the 3A coding region of the viral genome. However, O/SKR/AS/2002 has an intact 3A coding region.

Hepatitis C virus non-structural protein-2 activates CXCL-8 transcription through NF-kappaB.

Hepatitis C is a devastating disease worldwide. Proteins encoded by the etiologic agent, hepatitis C virus (HCV), are believed to play important roles in HCV-associated pathogenesis. However, the biological functions of the non-structural protein-2 (NS2) encoded by HCV are not well characterized. Here, we show that HCV NS2 protein activates CXCL-8 (interleukin-8, IL-8) transcription in HepG2 cells as measured by reverse transcription-polymerase chain reaction and IL-8 promoter-luciferase reporter assays. Furthermore, when the kappaB site on the IL-8 promoter was eliminated by mutagenesis or when intracellular NF-kappaB activity was suppressed by an inhibitor, NS2 did not activate the IL-8 promoter, suggesting a role of NF-kappaB in this process. These results prompted us to hypothesize that HCV NS2 might be able to activate NF-kappaB. This hypothesis was tested by determination of NF-kappaB-driven reporter gene expression and NF-kappaB p65 subunit subcellular localization after HCV NS2 expression. Indeed, NS2 could up-regulate NF-kappaB-driven luciferase activity and was associated with p65 nuclear localization. These results demonstrate that HCV NS2 up-regulates IL-8 transcription through NF-kappaB. This newly identified function increases our understanding of the role of HCV NS2 protein in virus-host interactions.

Vaccination as a control measure during the outbreak of foot-and-mouth disease in 2000 in Korea.

The Republic of Korea had been free from foot-and-mouth disease (FMD) for 66 years until 15 cases were confirmed between 24 March and 15 April in 2000. The FMD virus isolated in Korea was an O Pan Asia type, which was also responsible for the recent outbreaks in Japan and the U.K. Control measures including the stamping-out of infected animals on neighbouring farms, movement restrictions and emergency vaccination were implemented. The decision to vaccinate was made because the cattle affected were showing severe FMD lesions, there was significant possibility that a large amount of virus had already been shed and conditions at the time seemed to favour wind-borne spread. Also, because the spread was limited to cattle, it was assumed that the use of vaccinations would be more effective than if pigs had been affected. All susceptible animals within 10 km radius of the infected farms were vaccinated with inactivated, double-oil emulsion vaccines. Totals of 860,700 and 661,770 animals were vaccinated during the first and second round of booster vaccinations, and were completed within five months of the first outbreak. The government decided to adopt a let-live policy so that the vaccinated animals were not slaughtered. However, they were placed under movement restrictions and had to be identified and registered. Although there were concerns about the vaccinated animals becoming carriers, extensive serological surveillance using NSP ELISA found no evidence of FMD in the remaining vaccinated population. The use of emergency vaccinations in 2000 is regarded as being a major factor in limiting the spread of FMD and containing the outbreak within a month.

Cloning, sequencing, and expression of porcine interleukin-18 in Escherichia coli.

IL-18 is the new name of a novel cytokine that plays an important role in T(H1) response, primarily by its ability to induce IFN-gamma production in T cells and natural killer cells. The porcine IL-18 gene was isolated using RT-PCR from porcine alveolar macrophages. Sequence analysis of the porcine IL-18 gene has demonstrated an open reading frame of 579 base pairs encoding 192 amino acids precursor protein with a predicted molecular mass of 22 kDa. The porcine IL-18 gene shares 84% and 89% similarity to the human and canine equivalents, respectively, at the nucleotide level. The cloned IL-18 was expressed in Escherichia coli and its expression was confirmed by SDS-PAGE and Western blotting.