Construction of an avian hepatitis E virus replicon expressing heterologous genes and evaluation of its potential as an RNA vaccine platform

To evaluate avian hepatitis E virus (aHEV) as an RNA vaccine platform, open reading frame 2 (ORF2) of aHEV was replaced by heterologous genes, such as enhanced green fluorescent protein (eGFP) and hemagglutinin (HA)-tag, in aHEV infectious cDNA clones. eGFP and HA-tag replicons were expressed in leghorn male hepatoma (LMH) cells. To confirm expression of the heterologous protein, ORF2 was replaced with the antigenic S1 gene of infectious bronchitis virus (IBV). The IBVS1 replicon was expressed in LMH cells. To our knowledge, this is the first investigation showing potential as a RNA vaccine platform using an aHEV. In the future, it may be used in the development of RNA vaccines against various pathogens.

Construction of an avian hepatitis E virus replicon expressing heterologous genes and evaluation of its potential as an RNA vaccine platform

To evaluate avian hepatitis E virus (aHEV) as an RNA vaccine platform, open reading frame 2 (ORF2) of aHEV was replaced by heterologous genes, such as enhanced green fluorescent protein (eGFP) and hemagglutinin (HA)-tag, in aHEV infectious cDNA clones. eGFP and HA-tag replicons were expressed in leghorn male hepatoma (LMH) cells. To confirm expression of the heterologous protein, ORF2 was replaced with the antigenic S1 gene of infectious bronchitis virus (IBV). The IBVS1 replicon was expressed in LMH cells. To our knowledge, this is the first investigation showing potential as a RNA vaccine platform using an aHEV. In the future, it may be used in the development of RNA vaccines against various pathogens.

Genomic characteristics of natural recombinant infectious bronchitis viruses isolated in Korea / 대한수의학회지

Two infectious bronchitis virus (IBV) K046-12 and K047-12 strains were isolated and the nearly complete genomes of them were sequenced. Sequence comparisons showed that the K046-12 genome was most similar to Korean IBV strains, and the K047-12 genome was most similar to QX-like IBV strains. Phylogenetic analysis showed that nearly all K046-12 and most K046-12 genes were placed in the same cluster as Korean IBV isolates, but the S1 region was placed in the same cluster as Mass-type IBVs. For K047-12, nearly all K047-12 and most K047-12 genes were located in the same cluster as QX-like IBVs, but the M region was located in the same cluster as Korean IBV isolates with K047-12. Recombination analysis confirmed that K046-12 is a recombinant strain with the primary parental sequence derived from Korean IBVs and minor parental sequence derived from Mass-type IBV, and K047-12 is a recombinant strain with the major parental sequence derived from QX-IBV and minor parental sequence derived from Korean IBVs. This study showed that new IBV recombinants are constantly generated among various IBVs, including those used for vaccination. Therefore, genetic analysis of new virus isolates should be performed for effective infectious bronchitis control and appropriate vaccine development.

Detection and characterization of avian hepatitis E virus from broiler breeders and layers in Korea / 대한수의학회지

The helicase genes and hypervariable regions (HVRs) of three avian hepatitis E viruses (HEVs) detected at three different farms were sequenced and characterized. Two isolates (DW-L and GI-B2) were classified as genotype 2 and one isolate (GR-B) was classified as genotype 1. A phylogenetic tree, based on the helicase gene and HVR nucleotide sequences, revealed the newly detected viruses and other avian HEVs were classified similarly. Unlike previously reported avian HEVs, the DW-L isolate detected in broiler breeders with characteristic lesions of avian HEV had no prolinerich motif in its HVR, suggesting that the proline-rich motif is non-essential for viral replication and infection.

Detection and characterization of avian hepatitis E virus from broiler breeders and layers in Korea

The helicase genes and hypervariable regions (HVRs) of three avian hepatitis E viruses (HEVs) detected at three different farms were sequenced and characterized. Two isolates (DW-L and GI-B2) were classified as genotype 2 and one isolate (GR-B) was classified as genotype 1. A phylogenetic tree, based on the helicase gene and HVR nucleotide sequences, revealed the newly detected viruses and other avian HEVs were classified similarly. Unlike previously reported avian HEVs, the DW-L isolate detected in broiler breeders with characteristic lesions of avian HEV had no prolinerich motif in its HVR, suggesting that the proline-rich motif is non-essential for viral replication and infection.

Patterns of antibiotic resistance in Escherichia coli isolated from fresh and recycled poultry litter

The isolation rate of Escherichia (E.) coli in poultry litter was investigated at 44 broiler farms, 20 that used fresh litter and 24 that used recycled litter. The patterns of resistance to antibiotics of the E. coli isolates were compared. In litter sampled before the rearing period, the isolation rate of E. coli was higher at farms that used fresh litter; E. coli was present in the litter in 94.5% (35 out of 37 flocks tested) of the farms that used fresh litter vs. 51.2% (21 out of 41 flocks) of the farms that used recycled litter. The susceptibility of the 93 isolates of E. coli to 13 antibiotics was studied. Before the rearing period, E. coli isolates from the farms that recycled litter showed higher resistance rates than isolates from farms that replaced litter with fresh litter. Comparing the antibiotic resistance patterns of isolates from litter sampled before and at the end of the rearing period, the antibiotic resistance rates at the end of the rearing period increased dramatically compared with rates before the rearing period.

Reactive oxygen species regulate context-dependent inhibition of NFAT5 target genes

The activation of nuclear factor of activated T cells 5 (NFAT5), a well-known osmoprotective factor, can be induced by isotonic stimuli, such as activated Toll-like receptors (TLRs). It is unclear, however, how NFAT5 discriminates between isotonic and hypertonic stimuli. In this study we identified a novel context-dependent suppression of NFAT5 target gene expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) or a high salt (NaCl) concentration. Although LPS and NaCl both used NFAT5 as a core transcription factor, these stimuli mutually inhibited distinct sets of NFAT5 targets within the cells. Although reactive oxygen species (ROS) are essential for this inhibition, the source of ROS differed depending on the context: mitochondria for high salt and xanthine oxidase for TLRs. Specifically, the high salt-induced suppression of interleukin-6 (IL-6) production was mediated through the ROS-induced inhibition of NFAT5 binding to the IL-6 promoter. The context-dependent inhibition of NFAT5 target gene expression was also confirmed in mouse spleen and kidney tissues that were cotreated with LPS and high salt. Taken together, our data suggest that ROS function as molecular sensors to discriminate between TLR ligation and osmotic stimuli in RAW 264.7 macrophages, directing NFAT5 activity toward proinflammatory or hypertonic responses in a context-dependent manner.

TonEBP and SMIT expression in human placenta / 대한해부학회지

Tonicity-responsive enhancer binding protein (TonEBP) is a signal transcription factor of transporters such as sodium-myo-inositol cotransporter (SMIT), aldose reductase. TonEBP has a variety of functions such as control of intracellular osmolytes and immunomodulating. It is known that TonEBP is abundant in the placenta, but location and function aren't known. The aim of this study is to describe the localization of TonEBP in the placenta. We assayed the immunohistochemistry of TonEBP and performed in situ hybridization of SMIT in normal human full term placenta. In normal human full term placenta, TonEBP was in villous trophoblasts, extravillous trophoblasts and some endothelial cells. The result of the in situ hybridization of SMIT was similar to that of immunohistochemistry of TonEBP. Neither TonEBP nor SMIT was present in TonEBP knockout mouse placenta. This shows TonEBP is a key factor in SMIT transcription. TonEBP may play an important role in transporting of inositol to fetus in placenta.

Inactivation of 2009 Pandemic Human Influenza A Virus H1N1 by Photocatalyst Under UV Irradiation

A large-scale pandemic by human influenza virus H1N1 in 2009 caused severe health, social, and economic impacts. In this study, a photocatalyst technology based on TiO2, was evaluated for inactivation of a human influenza virus H1N1 isolated from a patient. The virus titer was reduced by 103.16-fold within 24 h and more than 104.31-fold inactivation within 48 h and 72 h. These results suggest that the tested photocatalyst technology based on TiO2 can be used for reduction of influenza A virus adherence to other surfaces with Hizen-s inside diverse buildings, enabling effective control of its indirect contact infection. The photocatalyst is expected also to reduce level of the aerosol transmission of the virus.

Sequence analysis of segment A gene of a very virulent infectious bursal disease virus recently isolated in Korea / 대한수의학회지

Infectious bursal disease virus (IBDV) is a member of the Avibirnavirus genus of the Birnaviridae family which genome consists of two segments (A and B) of double stranded RNA. Segment A gene of KNU08010 isolate, which was isolated from a 15-day-old chicken flock in 2008, was sequenced and compared with other IBDV isolates including SH/92 strain, the first Korean very virulent (vv) IBDV isolate. The amino acid sequences of segment A gene showed that KNU08010 had 99.2% homology with SH92 strain. KNU08010 isolate had specific amino acids A222, I242, I256, I294 and S299 which are highly conserved among vvIBDV strains. Phylogenetic analysis based on the nucleotide sequences of variable region of the VP2 gene of 18 IBDV strains revealed that KNU08010 was grouped with vvIBDVs and was closely related to Korean vvIBDVs isolated from wild birds.

Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-gamma

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-gamma were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-gamma (ChIFN-gamma) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-gamma did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV.

Regulation of Urea Transporters by Tonicity-responsive Enhancer Binding Protein

Urea accumulation in the renal inner medulla plays a key role in the maintenance of maximal urinary concentrating ability. Urea transport in the kidney is mediated by transporter proteins that include renal urea transporter (UT-A) and erythrocyte urea transporter (UT-B). UT-A1 and UT-A2 are produced from the same gene. There is an active tonicity-responsive enhancer (TonE) in the promoter of UT-A1, and the UT-A1 promoter is stimulated by hypertonicity via tonicity-responsive enhancer binding protein (TonEBP). The downregulation of UT-A2 raises the possibility that TonEBP also regulates its promoter. There is some evidence that TonEBP regulates expression of UT-A in vivo; (1) during the renal development of the urinary concentrating ability, expression of TonEBP precedes that of UT-A1; (2) in transgenic mice expressing a dominant negative form of TonEBP, expression of UT-A1 and UT-A2 is severely impaired; (3) in treatment with cyclosporine A, TonEBP was significantly downregulated after 28 days. This downregulation involves mRNA levels of UT-A2; (4) in hypokalemic animals, downregulation of TonEBP contributed to the down regulation of UT-A in the inner medulla. These data support that TonEBP directly contributes to the urinary concentration and renal urea recycling by the regulation of urea transporters.

Sequence analysis of the S1 glycoprotein gene of infectious bronchitis viruses: identification of a novel phylogenetic group in Korea

Twelve Korean infectious bronchitis viruses (IBVs) were isolated in the field from chickens suspected of being carriers of infectious bronchitis between 2001 and 2003. The S1 glycoprotein genes of these IBV isolates were amplified by reverse transcriptase-polymerase chain reaction (RTPCR) and analyzed by restriction fragment length polymorphism (RFLP) analysis. These Korean IBV isolates were classified into three groups according to their RFLP patterns obtained using the restriction enzyme HaeIII. Half of the twelve isolates were similar to the KM91 RFLP pattern, which is a common pattern in Korea. Three more isolates were related to the Arkansas strain pattern, but with some unique variations. The other three viruses showed variant RFLP patterns. For a comparison with the published sequences for non-Korean IBV strains, amplified PCR products from the twelve isolates were cloned and sequenced. The Korean IBV field isolates had 71.2-99.7% nucleotide sequence homology with each other and 45.9-80.7% nucleotide sequence homology with non-Korean IBV strains. With respect to the deduced amino acid sequence, the Korean IBV isolates had 71.5-99.3% similarity with each other and 44.9-80.3% similarity with non-Korean IBV strains. Phylogenetic tree analysis revealed that some of the IBV isolates appear to belong to a new group, different from the non-Korean IBV strains or from previously isolated Korean IBV strains. Specifically, the new Korean IBV isolates K10217-03, K3-3 and K1255-03 represented a separate group. These findings suggest that the Korean IBVs appear to be continuously evolving.

Effects of DDA, CpG-ODN, and plasmid-encoded chicken IFN-gamma on protective immunity by a DNA vaccine against IBDV in chickens

This study examined the adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA), CpG oligodeoxynucleotides (CpG-ODN), and chicken interferon-gamma (ChIFN-gamma) on a DNA vaccine (pcDNA-VP243) against the infectious bursal disease virus (IBDV). A plasmid encoding chicken IFN-atilde was constructed. Twice at 2-week intervals, twoweek-old chickens were injected intramuscularly and intraperitoneally with either a DNA vaccine alone or a DNA vaccine together with the respective adjuvants. On week 2 after the second immunization, the chickens were orally challenged with the highly virulent IBDV. The groups that received the DNA vaccines plus either DDA or CpG-ODN showed significantly lower survival rates than the group that received the DNA vaccine alone. However, the survival rates for the DNA vaccine alone and for the DNA vaccine plus ChIFN-gamma were similar. The chickens had no detectable antibodies to the IBDV before the challenge but all the surviving chickens in all groups except for the normal control group showed the induction of antibodies to the IBDV at day 10 after the challenge. As judged by the lymphocyte proliferation assays using the a WST-8 solution performed on the peripheral blood and splenic lymphocytes, the stimulation indices (SI) of the peripheral blood lymphocytes in all groups except for the normal control group were similar immediately before the challenge. At 10 days post-challenge, the SI for DNA vaccine plus either CpG-ODN or ChIFN-gamma was similar to that of the DNA vaccine control group. For splenic lymphocytes, the SI in the DNA vaccine plus CpG-ODN and DNA vaccine plus ChIFN-gamma groups were higher than for the DNA vaccine control. These results suggest that DDA actually compromises the protection against the IBDV by DNA vaccine, and CpG-ODN and IFN-gamma had no significant effect.

Expressions of Matrix Metalloproteinase-9 and Tissue Inhibitor of Metalloproteinase-2 with Changes of Interleukin-6 and Interleukin-18 in Atherosclerotic Lesions of Hypercholesterolemic Rabbits / 대한흉부외과학회지

BACKGROUND: Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by progressive accumulation of lipids,cells,and extracellular matrix.Matrix metalloproteinases(MMPs)and tissue inhibitor of metalloproteinases(TIMPs)contribute to vascular matrix remodeling in atherosclerosis,and some cytokines may play role in the synthesis or activation of MMPs or TIMPs. MATERIALS AND METHOD: We produced experimental atherosclerotic plaques in 9 rabbits by atherogenic hypercholesterol diet for 12 weeks,and 10 other rabbits were used as control group with standard laboratory chow.At that time,19 rabbits were sacrificed and aorta,coronary arteries and blood specimens were prepared.The expressions of MMP-9,TIMP-2 and interleukin(IL)-18,and the bioactivity of IL-6 were investigated with H&E stain,immunohistochemical stain,immunoblotting(Western blot analysis),and bioassay. RESULT: Serum cholesterol in the experimental group increased up to 1258 +/-262 mg/dL(control group:41 +/-7 mg/dL).All experimental group showed well developed atherosclerotic plaques in aorta and coronary artery.The expression of MMP-9 in aorta and coronary artery of the experimental group showed significant increase than that of the control group by immunohistochemistry.Among the experimental group, complicated lesions with intimal rupture or complete luminal occlusion,demonstrated stronger expression of MMP-9.Interestingly,there was no difference in expression of TIMP-2 between the experimental and the control group.These findings were confirmed by Western blot analysis.The bioassay revealed significant up-regulation of serum bioactivity of IL-6 in the experimental group(4819.60 +/-2021.25 IU/ml)compared to that of IL-6 in the control group(27.20 +/-12.19 IU/ml).IL-18 was expressed in all atherosclerotic plaques, whereas little or no expression was detected in the control group. CONCLUSION: The increased MMP-9 expression along with the unchanged TIMP-2 expression seem to be contributory factors in extracellular matrix degradation in atherosclerosis.Focal overexpression of MMP-9 may promote plaque destabilization and cause complications of atherosclerotic plaques such as thrombosis with/without acute coronary syndrome.Elevation of IL-6 and IL-18 may be more than just markers of atherosclerosis but actual participants in lesion development.Identification of critical regulatory pathway is important to improve the understanding of the cellular and molecular basis of atherosclerosis and may open the way for novel therapeutic strategies.

Primary Fibrosarcoma of the Lung with Spontaneous Hemothorax / 대한흉부외과학회지

Primary pulmonary sarcoma is a rare tumor at all ages. It may be present as a solitary nodule or huge tumor of the thoracic cavity and frequently remain asymptomatic until growing large. A 50-year-old woman who presented sudden chest pain was found to have spontaneous hemothorax. The right upper lobectomy with lymph node dissection was performed, the pathologic result was pulmonary fibrosarcoma.

Recurrent Solitary Fibrous Tumor of the Mediastinum / 대한흉부외과학회지

Solitary fibrous tumors of the serosal cavities are rare and usually occur in the visceral or parietal pleura, but they have also been observed in the mediastinum, pericardium, peritoneum, lung parenchyma, orbit, and the meninges. The histological variability of these tumors may contribute to the difficulty in diagnosing these neoplasms, especially when they arise in the mediastinum and extrathoracic sites. The clinical behavior of the tumor is unpredictable. Some tumors that appear histologically benign may behave aggressively. The incidence of aggressive behavior is variously reported as between 13 and 23% of cases in most large series. This report is a case review in a 53-year-old female patient who had re-excision of histologically benign, recurrent solitary fibrous tumor, 1, 6, 11, and 14 years after the initial operation.

A Moderate Case of Hemolytic Disease in a Newborn Caused by Anti-M Antibody / 대한임상병리학회지

Anti-M antibodies are usually assumed to be naturally occurring and to consist of immunoglobulin M reacting at 4degrees C. They are not usually considered to be clinically significant, however, many of them have an immunoglobulin G component reacting at 37degrees C and can be correlated with hemolytic disease of the newborn (HDN). We report a moderate case of HDN by anti-M. A 2-days old baby born from a mother with preeclampsia as a second pregnancy was admitted due to anemia, hyperbilirubinemia and hypoxic encephalopathy. The blood type of mother was AB, ccDEE, NN, and the blood type of baby was A, D+, and MN. Antibody screening and identification identified anti-M antibody which was strong reactive at 37degrees C albumin and antiglobulin phase in both baby's and her mother's serum. The direct antiglobulin test of baby's red blood cells was negative. The infant was transfused with group O red cells which have negative to trace reaction with her mother's serum in antiglobulin phase. Two days later, the hemoglobin level elevated from 6.7 g/dL to 15.9 g/dL falled to below 11 g/dL quite soon. After all, newborn died of cardiac arrest due to her basic disease at age of 49 days; metabolic acidosis and hypernatremia.

Clinical Analysis for Thymic Carcinoma / 대한흉부외과학회지

BACKGROUND: Thymic carcinoma is a very rare disease and treatment modality is not standardized. So, we report our experience of management of thymic carcinoma. MATERIAL AND METHOD: Between 1984 and 1998, eight patients with thymic carcinoma were treated at Keimyung University Dongsan Medical center. RESULT: The median age was 46 years with a range of 23 to 67 years. Chief complaint was a anterior chest pain. Histologic subtypes included two lymphoepithelioma-like carcinoma, two squamous cell carcinoma, one basaloid carcinoma, and three mixed type. Clinical staging was classified to stage I in 2, stage II in 4, stage III in 1, and stage IVA in 1 according to the modified Masaoka staging system. Four patients underwent complete resection and three patients were found to have incomplete resection by histologic evaluation. One patient underwent only biopsy due to pericardial dissemination and invasion of adjacent organ. All patients had adjuvant chemotherapy, radiation therapy was administered to five patients for positive resection margin and above stage III. The median follow up period was 55.3+/-64.6 months, three patiants died and four patients are alive without recurrence. One patient in recurrence had two times re-operations and adjuvant chemoradiotherapy. He is still alive. CONCLUSION: We concluded that completely surgical resection and adequate adjuvant chemoradiotherapy after early diagnosis are useful to management of thymic carcinoma.

Antioxidative Effect of Ginseng Saponin on Cardiac Endothelial Cells in Culture / 대한해부학회지

The cytotoxic effects of oxygen free radicals and the antioxidative effect of ginseng saponin (SPN) on cardiac endothelial cell cultures derived from 3-day old rats were studied. Reactive oxygen species were generated by hypoxanthine (HX) and xanthine oxidase (XO) mixture to the culture medium. Exposure of cardiac endothelial cells to this oxygen-radical-generating system resulted in significant time-dependent decrease of MTT activity and increase of lactate dehydrogenase (LDH) release. These results correlated well with the morphological examination of randomly selected cultured cardiac endothelial cells, which showed large cytoplasmic vacuoles, disordered organelles, pronounced increase of endoplasmic reticular swelling, and decreased maintenance of membrane integrity. The decrease in cell viability and increase of LDH release induced by the oxygen free radicals in cardiac endothelial cell cultures were blocked during the first two hours by antioxidants such as ginseng saponin (SPN), deferoxamine (DFX), and ginseng saponin/deferoxamine mixture (SPN/DFX). These antioxidative effects were significantly greater in the SPN-treated group than in the other antioxidant-treated groups. Especially, the cells of the SPN-treated group showed well developed cytoskeletons, which enabled them to firmly attach to the culture vessel. In conclusion, these results indicate that ginseng saponin has a significant antioxidative effect on cardiac endothelial cells in culture and plays an important role in stimulating the formation of cytoskeleton and maintaining the integrity of cell membrane.