Antiviral activity of adenoviral vector expressing human interferon lambda-4 against influenza virus.

Interferon lambda (IFNλ), classified as a type III IFN, is a representative cytokine that plays an important role in innate immunity along with type I IFN. IFNλ can elicit antiviral states by inducing peculiar sets of IFN-stimulated genes (ISGs). In this study, an adenoviral vector expression system with a tetracycline operator system was used to express human IFNλ4 in cells and mice. The formation of recombinant adenovirus (rAd-huIFNλ4) was confirmed using immunohistochemistry assays and transmission electron microscopy. Its purity was verified by quantifying host cell DNA and host cell proteins, as well as by confirming the absence of the replication-competent adenovirus. The transduction of rAd-huIFNλ4 induced ISGs and inhibited four subtypes of the influenza virus in both mouse-derived (LA-4) and human-derived cells (A549). The antiviral state was confirmed in BALB/c mice following intranasal inoculation with 109 PFU of rAd-huIFNλ4, which led to the inhibition of four subtypes of the influenza virus in mouse lungs, with reduced inflammatory lesions. These results imply that human IFNλ4 could induce antiviral status by modulating ISG expression in mice.

Neurologic Effects of SARS-CoV-2 Transmitted among Dogs.

SARS-CoV-2 induces illness and death in humans by causing systemic infections. Evidence suggests that SARS-CoV-2 can induce brain pathology in humans and other hosts. In this study, we used a canine transmission model to examine histopathologic changes in the brains of dogs infected with SARS-CoV-2. We observed substantial brain pathology in SARS-CoV-2-infected dogs, particularly involving blood-brain barrier damage resembling small vessel disease, including changes in tight junction proteins, reduced laminin levels, and decreased pericyte coverage. Furthermore, we detected phosphorylated tau, a marker of neurodegenerative disease, indicating a potential link between SARS-CoV-2-associated small vessel disease and neurodegeneration. Our findings of degenerative changes in the dog brain during SARS-CoV-2 infection emphasize the potential for transmission to other hosts and induction of similar signs and symptoms. The dynamic brain changes in dogs highlight that even asymptomatic individuals infected with SARS-CoV-2 may develop neuropathologic changes in the brain.

Live recombinant Newcastle disease virus vectored vaccine expressing the haemagglutinin of H9N2 avian influenza virus suppresses viral replication in chickens.

In 2020, the Y280-lineage H9N2 low-pathogenic avian influenza virus (LPAIV) was introduced into South Korea for the first time. Current vaccines are focused on the control of Y439-like viruses; however, there are continuous reports of decrease in egg production and secondary infections caused by Y280-lineage H9N2 LPAI infection in chickens. Therefore, there is an urgent need to develop effective novel vaccines against Y280-lineage H9N2 LPAI. Most commercialized avian influenza vaccines are oil-adjuvanted inactivated vaccines, which are labour-intensive to administer and require higher dosage. In this study, rK148/Y280-HA, a novel recombinant Newcastle disease virus (NDV) vectored vaccine against Y280-lineage H9N2 LPAI, was developed and evaluated using two mass-applicable administration methods, spray vaccination and drinking water vaccination. Regardless of low serum antibody haemagglutination inhibition titres against NDV and Y280-lineage H9N2 LPAI after applying the rK148/Y280-HA vaccine, vaccination with either administration method protected chickens against virulent NDV and Y280-lineage H9N2 LPAIV after the challenge. Taken together, these results indicate that the rK148/Y280 vaccine can be administered using facile mass-application methods to provide protection against the Y280-lineage LPAI.RESEARCH HIGHLIGHTS NDV vectored vaccine harbouring Y280-lineage H9N2 HA protein was successfully generated.NDV vectored vaccine provides protection against NDV.NDV vectored vaccine with H9N2 HA protects against homologous H9N2 LPAIV.

Production of a chimeric porcine reproductive and respiratory syndrome virus (PRRSV)-2 vaccine using a lab-scale packed-bed bioreactor CelCradle.

BACKGROUND: We developed a MARC-145 cell culture and porcine reproductive and respiratory syndrome (PRRS) vaccine production using a novel CelCradle bioreactor. CelCradle is a packed-bed bioreactor capable of both batch and perfusion culture, and the operating parameters are easy to optimize. RESULTS: In this study, CelCradle reached a maximum cell density of 8.94 × 105 cells/mL at 5 days post-seeding when seeded at 8.60 × 104 cells/mL (doubling time = 35.52 h). Inoculation of PRRS vaccine candidate, K418DM1.1, was performed at a multiplicity of infection (MOI) of 0.01 at 5 days post-seeding, which resulted in a high viral titer of 2.04 × 108 TCID50/mL and total viral load of 1.02 × 1011 TCID50/500 mL at 2 days post-infection (dpi). The multilayer cultivation system, BioFactory culture, yielded a higher doubling time (37.14 h) and lower viral titer (i.e., 8.15 × 107 TCID50/mL) compared to the CelCradle culture. Thus, the culture medium productivity of the CelCradle culture was 2-fold higher than that of the BioFactory culture. In the animal experiment, the CelCradle-produced vaccine induced high levels of neutralizing antibodies and effectively protected pigs against homologous challenge, as shown by the significantly lower levels of viremia at 1- and 7-days post-challenge (dpc) compared to the non-vaccinated pigs. CONCLUSIONS: Overall, this study demonstrates that the CelCradle system is an economical platform for PRRS vaccine production.

Gustatory dysfunction perceptions versus objective gustatory dysfunction among older adults.

BACKGROUND: Research on gustatory dysfunction among older adults has been scarce relative to research on olfactory dysfunction, and the relationship between subjective and objective gustatory dysfunction has not been studied in detail. We aimed to evaluate whether subjective recognition of gustatory dysfunction correlates with objective diagnosis of gustatory dysfunction among older adults. METHODS: In this retrospective, cross-sectional study, we reviewed the medical records of 138 patients of ages ≥ 60 years for whom data were available on self-reported and objectively measured gustatory and olfactory function from January 2018 through April 2021 at a specialized smell/taste center of a single institution. We reviewed self-reported and measured outcomes of gustatory and olfactory function using patient data including clinical characteristics, including age, sex, smoking history, and medical history. RESULTS: We found that the subjective recognition of gustatory dysfunction does not correlate with the objective diagnosis of gustatory dysfunction based on the measured results of gustatory function tests. Subjective gustatory dysfunction, however, was correlated with subjective olfactory dysfunction. Among clinical and demographic characteristics, age and sex were significantly associated with measured gustatory function outcomes. CONCLUSION: We suggest that subjective gustatory dysfunction underestimates objective dysfunction and recommend that older men with diminished olfactory function undergo gustatory function testing regardless of their self-reported gustatory function status.

External validation of deep learning-based automated detection algorithm for chest radiograph: practical issues in outpatient clinic.

BACKGROUND: There have been no reports on diagnostic performance of deep learning-based automated detection (DLAD) for thoracic diseases in real-world outpatient clinic. PURPOSE: To validate DLAD for use at an outpatient clinic and analyze the interpretation time for chest radiographs. MATERIAL AND METHODS: This is a retrospective single-center study. From 18 January 2021 to 18 February 2021, 205 chest radiographs with DLAD and paired chest CT from 205 individuals (107 men and 98 women; mean ± SD age: 63 ± 8 years) from an outpatient clinic were analyzed for external validation and observer performance. Two radiologists independently reviewed the chest radiographs by referring to the paired chest CT and made reference standards. Two pulmonologists and two thoracic radiologists participated in observer performance tests, and the total amount of time taken during the test was measured. RESULTS: The performance of DLAD (area under the receiver operating characteristic curve [AUC] = 0.920) was significantly higher than that of pulmonologists (AUC = 0.756) and radiologists (AUC = 0.782) without assistance of DLAD. With help of DLAD, the AUCs were significantly higher for both groups (pulmonologists AUC = 0.853; radiologists AUC = 0.854). A greater than 50% decrease in mean interpretation time was observed in the pulmonologist group with assistance of DLAD compared to mean reading time without aid of DLAD (from 67 s per case to 30 s per case). No significant difference was observed in the radiologist group (from 61 s per case to 61 s per case). CONCLUSION: DLAD demonstrated good performance in interpreting chest radiographs of patients at an outpatient clinic, and was especially helpful for pulmonologists in improving performance.

Th2 cytokines-DUOX2-ROS-HMGB1 translocation axis is important in the pathogenesis of allergic rhinitis.

The function of high-mobility group box 1 (HMGB1) varies according to its location. However, the translocation mechanism behind HMGB1 remains unclear. We hypothesize that type 2 helper T cell (Th2) cytokines are involved in the translocation of HMGB1 in the upper airway epithelium. We investigated the mechanism behind HMGB1 translocation using Th2 cytokine stimulation and examined the clinical significance of HMGB1 translocation in allergic rhinitis (AR). Cytoplasmic and extracellular HMGB1 were increased in AR. Inhibiting HMGB1 translocation with glycyrrhizic acid (GA) decreased the level of antigen-specific immunoglobulin E (IgE), the degree of Periodic Acid-Schiff (PAS), and Sirius Red staining in the murine model. The in vivo reactive oxygen species (ROS) level in the nasal mucosa was higher in the mice with AR than in the controls. Th2 cytokine-induced up-regulation of the ROS and translocation of HMGB1 by Th2 cytokines was dependent on the generated ROS. The ROS level also increased in the murine model. We suggest that the Th2 cytokine-dual oxidase (DUOX)2-ROS-HMGB1 translocation axis is important in AR pathogenesis.

Improvement in the survival rates of extracorporeal membrane oxygenation-supported respiratory failure patients: a multicenter retrospective study in Korean patients.

BACKGROUND: Although the utilization of extracorporeal membrane oxygenation (ECMO) is increasing and its technology is evolving, only a few epidemiologic reports have described the uses and outcomes of ECMO. The aim of this study was to investigate the changes in utilization and survival rate in patients supported with ECMO for severe respiratory failure in Korea. METHODS: This was a multicenter study on consecutive patients who underwent ECMO across 16 hospitals in Korea. The records of all patients who required ECMO for acute respiratory failure between 2012 and 2015 were retrospectively reviewed, and the utilization of ECMO was analyzed over time. RESULTS: During the study period, 5552 patients received ECMO in Korea as a whole, and a total of 2472 patients received ECMO at the participating 16 hospitals. We analyzed 487 (19.7%) patients who received ECMO for respiratory failure. The number of ECMO procedures provided for respiratory failure increased from 104 to 153 during the study period. The in-hospital survival rate increased from 30.8% to 35.9%. The use of prone positioning increased from 6.8% to 49.0% (p < 0.001), and the use of neuromuscular blockers also increased from 28.2% to 58.2% (p < 0.001). Multiple regression analysis showed that old age (OR 1.038 (95% CI 1.022, 1.054)), use of corticosteroid (OR 2.251 (95% CI 1.153, 4.397)), continuous renal replacement therapy (OR 2.196 (95% CI 1.135, 4.247)), driving pressure (OR 1.072 (95% CI 1.031, 1.114)), and prolonged ECMO duration (OR 1.020 (95% CI 1.003, 1.038)) were associated with increased odds of mortality. CONCLUSIONS: Utilization of ECMO and survival rates of patients who received ECMO for respiratory failure increased over time in Korea. The use of pre-ECMO prone positioning and neuromuscular blockers also increased during the same period.

Evidence of hepatitis E virus infection in specific pathogen-free rabbits in Korea.

Rabbits are considered a new natural reservoir of hepatitis E virus (HEV). In this study, HEV infection was verified by the detection of partial genomic sequence of HEV and anti-HEV antibodies in specific pathogen-free (SPF) rabbits. HEV RNA was found in 6.4% serum and 13.5% fecal samples from 126 SPF rabbits. Anti-HEV antibodies were also detected in 4.0% of the SPF rabbits. HEV genetic sequences isolated from the rabbits were clustered into a rabbit HEV clade with other rabbit HEV isolates; they were found to be most closely related with a rabbit HEV sequence previously reported in Korea. Therefore, HEV infection should be diagnosed before conducting experiments involving SPF rabbits.

Evaluation and optimization of a conventional SPCE for FMD post-vaccination monitoring.

BACKGROUND: Foot-and-mouth disease (FMD) can be controlled by either stamping out or vaccination, a choice which depends on both the economic importance of the livestock sector as well as the disease status. In FMD-free countries with vaccination, such as Korea, vaccination programs should guarantee prevention against transmission of FMD. Monitoring of vaccination programs is also essential for ensuring sufficient coverage that will limit the transmission of FMDV. There are several methods to screen FMD virus (FMDV) structural protein (SP) antibodies including SPCE (Solid-phase competitive ELISA), LPBE (Liquid-phase blocking ELISA), and VNT (Virus neutralization test). Among these, SPCE is widely used for serological monitoring since VNT-the gold standard method-has certain practical limitations, such as high costs in terms of time and labor. However, whether SPCE can ensure the vaccination status of individual animals and whole farms is unclear. In this study, SPCE, LPBE and VNT were compared with respect to correlation with each other and sensitivity at commercial pig farms. RESULTS: The positive results obtained by PrioCHECK SPCE differed from those obtained by LPBE and VNT. The sensitivity of SPCE relative to those of the other tests was fairly low. The raw data of SPCE were most highly correlated with those of VNT with XJ strain, while their positivity and negativity were most highly correlated with LPBE. The results of ROC analysis proposed new cut-off for PrioCHECK SPCE higher than the previous 50% inhibition. CONCLUSIONS: The high false positive rate of PrioCHECK SPCE suggested that high seropositivity by SPCE may not guarantee a true vaccination coverage. Adjusting the cut-off percentage (%) inhibition value for SPCE is needed to address this problem, and it is highly recommended that routine FMDV serological monitoring programs using PrioCHECK SPCE should be combined with alternative methods such as LPBE or VNT.

Prevalence and genetic features of rabbit hepatitis E virus in Korea.

Hepatitis caused by hepatitis E virus (HEV) is a public health concern worldwide. HEV strains have been isolated from several animal species, some of which induce zoonosis. Recently, the isolation of HEV from rabbits was reported. Here, the partial capsid gene (320 bp) of HEV was detected in rabbit feces via reverse transcriptase-polymerase chain reaction (RT-PCR). Rabbit HEV was found in two of six rabbit farms and 17 of 264 rabbit fecal samples (6.4%). A phylogenetic analysis of the partial capsid gene classified the 17 HEV isolates into the putative rabbit HEV clade. A full genomic sequence, KOR-Rb-1, was obtained from one rabbit HEV isolate by 5' and 3' rapid amplification of cDNA ends-PCR and RT-PCR, and comprised 7275 bp excluding the 3' poly(A) tail. It shared 77.5-86.8%, 86.6%, and 80.2-84.3% nucleotide identities with rabbit HEV isolates from China, the US, and France, respectively. It also shared 72.3-73.0%, 71.4%, 76.7-78.3%, 72.8-73.3%, and 47.1-47.2% nucleotide identities with representative strains of HEV-1, HEV-2, HEV-3, HEV-4, and avian HEV, respectively. A full-genome phylogenetic analysis classified KOR-Rb-1 into the provisional rabbit HEV clade. This isolate could be used to study the pathogenesis and zoonotic potential of rabbit HEV.

Benign metastasizing leiomyoma presenting as multiple cystic pulmonary nodules: a case report.

BACKGROUND: Benign metastatic leiomyoma (BML) is an extremely rare disease. Although uterine leiomyomas are benign histologically, they can metastasize to distant sites. While the incidence is very low, the lung is the organ most frequently affected by BML. Pulmonary BML usually presents as numerous well-defined nodules of various sizes, while the cavitary or cystic features in the nodules are rarely observed on radiologic images. CASE PRESENTATION: A 52-year-old woman complained of cough and dyspnea for one month. She had been previously diagnosed with uterine leiomyoma and had undergone total hysterectomy about 14 years prior. High-resolution computed tomography (CT) images showed that there were multiple cystic nodules of various sizes in both lungs. Pathologic examination revealed that the pulmonary nodule had complex branching glandular structures lined by a single layer of simple cuboidal to columnar epithelium that was surrounded by abundant spindle cells. Additional immunohistochemistry data suggested that pulmonary nodule diagnosis was BML-associated uterine leiomyoma. CONCLUSION: In this report, we introduce an interesting case of pulmonary BML that presented as a combination of various kinds of nodules including simple round nodules, simple cysts, and cysts with a solid portion, which are very rare radiologic features of BML in lung. In addition, when the patient is a woman of reproductive age, physicians should meticulously review the gynecological history and suspect BML when there are various cystic pulmonary lesions.

A Novel Cysteine Sulfinic Acid Decarboxylase Knock-Out Mouse: Taurine Distribution in Various Tissues With and Without Taurine Supplementation.

Taurine, a sulfur containing amino acid, has various physiological functions including development of the eye and brain, immune function, reproduction, osmo-regulatory function as well as anti-oxidant and anti-inflammatory activities. In order to understand the physiological role, we developed taurine deficient mice deleting a rate-liming enzyme, cysteine sulfinic acid decarboxylase (CSAD) for biosynthesis of taurine. Taurine was measured in various tissues including the liver, brain, lung, spleen, thymus, pancreas, heart, muscle and kidney as well as plasma from CSAD knock-out mice (CSAD KO) with and without treatment of taurine in the drinking water at the age of 2 months (2 M). Taurine was determined using HPLC as a phenylisothiocyanate derivative of taurine at 254 nm. Taurine concentrations in the liver and kidney from homozygotes of CSAD KO (HO), in which CSAD level is high, were 90% and 70% lower than WT, respectively. Taurine concentrations in the brain, spleen and lung, where CSAD level is low, were 21%, 20% and 28% lower than WT, respectively. At 2 M, 1% taurine treatment of HO restored taurine concentrations in all tissues compared to that of WT. To select an appropriate taurine treatment, HO were treated with various concentrations (0.05, 0.2, 1%) of taurine for 4 months (4 M). Restoration of taurine in all tissues except the liver, kidney and lung requires 0.05% taurine to be restored to that of WT. The liver and kidney restore taurine back to WT with 0.2% taurine. To examine which enzymes influence taurine concentrations in various tissues from WT and HO at 2 M, expression of five taurine-related enzymes, two antioxidant enzymes as well as lactoferrin (Lft) and prolactin receptor (Prlr) was determined using RT2 qPCR. The expression of taurine transporter in the liver, brain, muscle and kidney from HO was increased except in the lung. Our data showed expression of glutamate decarboxylase-like 1(Gadl-1) was increased in the brain and muscle in HO, compared to WT, indicating taurine in the brain and muscle from HO was replenished through taurine transporter and increased biosynthesis of taurine by up-regulated Gadl-1. The expression of glutathione peroxidase 3 was increased in the brain and peroxireductase 2 was increased in the liver and lung, suggesting taurine has anti-oxidant activity. In contrast to newborn and 1 month CSAD KO, Ltf and Prlr in the liver from CSAD KO at 2 M were increased more than two times and 52%, respectively, indicating these two proteins may be required for pregnancy of CSAD KO. Ltf in HOT1.0 was restored to WT, while Prlr in HOT1.0 was increased more than HO, explaining improvement of neonatal survival with taurine supplementation.These data are essential for investigating the role of taurine in development of the brain and eye, immune function, reproduction and glucose tolerance.

A Novel Cysteine Sulfinic Acid Decarboxylase Knock-Out Mouse: Immune Function (II).

Taurine deficient mice lacking cysteine sulfinic acid decarboxylase (CSAD KO) were developed for investigating the various physiological roles of taurine including the development of the brain and eye as well as immune function. Due to severe abnormalities of immune function in a taurine deficient cat, the immune function including adoptive and innate immunity in taurine-deficient mice have been studied. Previously we demonstrated that B cell function in CSAD KO was reduced in both females and males. However, T cell function was significantly reduced only in females. In this study, we have examined innate immunity using macrophage activation with LPS or/and IFN-γ and polymorphonuclear leukocytes (PMN) activation with phorbol myristate acetate (PMA). Pro- and anti-inflammatory cytokines including IL-6, TNF-α and IL-10 as well as nitric oxide (NO) were determined using ELISA and Griess reagent, respectively. Peritoneal macrophages were activated with 1 µg/mL of lipopolysaccharide (LPS) and/or 50 U/mL of IFN-γ. In addition, superoxide anion was measured using peritoneal PMN activated with PMA in the presence and absence of superoxide dismutase. Superoxide anion production in activated PMN from CSAD KO homozygotes (HO) was not significantly different from wild-type (WT) with and without 25 mM taurine. IL-10 and TNF-α production in both female and male CSAD KO were not significantly different. IL-6 and NO were significantly lower only in females as previously observed in Con A-activated cellular proliferation of splenocytes. Cytokine production with 10 mM of taurine was not different, indicating the reduction of NO and IL-6 in females may be due to the absence of the CSAD gene, not due to low taurine concentrations.These data indicate that some measures of innate immunity were altered in female CSAD mice.

Experimental evidence of hepatitis A virus infection in pigs.

Hepatitis A virus (HAV) is the leading cause of acute viral hepatitis worldwide, with HAV infection being restricted to humans and nonhuman primates. In this study, HAV infection status was serologically determined in domestic pigs and experimental infections of HAV were attempted to verify HAV infectivity in pigs. Antibodies specific to HAV or HAV-like agents were detected in 3.5% of serum samples collected from pigs in swine farms. When the pigs were infected intravenously with 2 × 10(5) 50% tissue culture infectious dose (TCID50 ) of HAV, shedding of the virus in feces, viremia, and seroconversion were detected. In pigs orally infected with the same quantity of HAV, viral shedding was detected only in feces. HAV genomic RNA was detected in the liver and bile of intravenously infected pigs, but only in the bile of orally infected pigs. In further experiments, pigs were intravenously infected with 6 × 10(5) TCID50 of HAV. Shedding of HAV in feces, along with viremia and seroconversion, were confirmed in infected pigs but not in sentinel pigs. HAV genomic RNA was detected in the liver, bile, spleen, lymph node, and kidney of the infected pigs. HAV antigenomic RNA was detected in the spleen of one HAV-infected pig, suggesting HAV replication in splenic cells. Infiltration of inflammatory cells was observed in the livers of infected pigs but not in controls. This is the first experimental evidence to demonstrate that human HAV strains can infect pigs.

Pre-immune state induced by chicken interferon gamma inhibits the replication of H1N1 human and H9N2 avian influenza viruses in chicken embryo fibroblasts.

BACKGROUND: Interferon gamma (IFN-γ), an immunoregulatory cytokine, is known to control many microbial infections. In a previous study, chicken interferon gamma (chIFN-γ) was found to be up-regulated following avian influenza virus (AIV) infection in specific pathogen-free chickens. We aimed to investigate whether the pre-immune state induced by chIFN-γ could generate an antiviral response against influenza virus. METHODS: We generated a chIFN-γ-expressing plasmid and transfected it into chicken embryo fibroblasts (CEFs) and then infected the cells with human origin H1N1 or avian origin H9N2 influenza viruses. Viral titers of culture medium were evaluated in MDCK cell and the viral RNA and IFN-stimulated genes (ISGs) were then quantified by real-time reverse transcriptase polymerase. To further evaluate the role of the antiviral effect of chIFN-γ by using a backward approach, synthetic small interfering RNAs (siRNA) targeting chIFN-γ were used to suppress chIFN-γ. RESULTS: The chIFN-γ-stimulated CEFs inhibited the replication of viral RNA (vRNA) and showed a mild decrease in the infectious virus load released in the culture medium. Compared to the mock-transfected control, the messenger RNA (mRNA) levels of type I IFNs and IFN-stimulated genes were up-regulated in the cells expressing chIFN-γ. After treatment with the siRNA, we detected a higher expression of viral genes than that observed in the mock-transfected control. CONCLUSIONS: Our results suggest that apart from the important role played by chIFN-γ in the antiviral state generated against influenza virus infection, the pre-immune state induced by chIFN-γ can be helpful in mitigating the propagation of influenza virus.

Genotyping of infectious laryngotracheitis virus using allelic variations from multiple genomic regions.

Live attenuated vaccines are extensively used worldwide to control the outbreak of infectious laryngotracheitis. Virulent field strains showing close genetic relationship with the infectious laryngotracheitis virus (ILTV) vaccines of chicken embryo origin have been detected in the poultry industry. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, a reliable molecular epidemiological method, of multiple genomic regions was performed. The PCR-RFLP is a time-consuming method that requires considerable amount of intact viral genomic DNA to amplify genomic regions greater than 4 kb. In this study, six variable genomic regions were selected and amplified for sequencing. The multi-allelic PCR-sequence genotyping showed better discrimination power than that of previous PCR-sequencing schemes using single or two target regions. The allelic variation patterns yielded 16 strains of ILTV classified into 14 different genotypes. Three Korean field strains, 550/05/Ko, 0010/05/Ko and 40032/08/Ko, were found to have the same genotype as the commercial vaccine strain, Laryngo Vac (Zoetis, Florham Park, NJ, USA). Three other Korean field strains, 40798/10/Ko, 12/07/Ko, and 30678/14/Ko, showed recombined allelic patterns. The multi-allelic PCR-sequencing method was proved to be an efficient and practical procedure to classify the different strains of ILTV. The method could serve as an alternate diagnostic and differentiating tool for the classification of ILTV, and contribute to understanding of the epidemiology of the disease at a global level.

Pathogenicity of the Korean H5N8 highly pathogenic avian influenza virus in commercial domestic poultry species.

In 2014, the highly pathogenic avian influenza (HPAI) virus H5N8 triggered outbreaks in wild birds and poultry farms in South Korea. In the present study, we investigated the pathogenicity of the H5N8 HPAI virus, belonging to the clade 2.3.4.4, in different species of poultry. For this, we examined clinical signs and viral shedding levels following intranasal inoculation of the virus in 3-week-old commercial layer chickens and quails, 10-week-old Korean native chickens, and 8-week-old Muscovy ducks. Intranasal inoculation with 10(6.0) viruses at 50% egg-infective dose resulted in 100% mortality in the layer chickens (8/8) and quails (4/4), but 60% and 0% deaths in the Korean native chickens (3/5) and Muscovy ducks (0/4), respectively. In addition, transmission of the inoculated virus to contact-exposed birds was evident in all the species used in this study. Based on our results, we conclude that the H5N8 HPAI virus has lower pathogenicity and transmissibility in poultry species compared with previously reported H5N1 HPAI viruses.

Analysis of cytokine production in a newly developed canine tracheal epithelial cell line infected with H3N2 canine influenza virus.

The Madin-Darby canine kidney (MDCK) cell line is typically used to analyze pathological features after canine influenza virus (CIV) infection. However, MDCK cells are not the ideal cell type, because they are kidney epithelial cells. Therefore, we generated an immortalized canine tracheal epithelial cell line, KU-CBE, to more reliably study immune responses to CIV infection in the respiratory tract. KU-CBE cells expressed the influenza virus receptor, α-2,3-sialic acid (SA), but not α-2,6-SA. KU-CBE and MDCK cells infected with H3N2 CIV demonstrated comparable virus growth kinetics. Gene expression levels of interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-ß were estimated in both KU-CBE and MDCK cells infected with CIV by real-time reverse transcription polymerase chain reaction (qRT-PCR). Of these cytokines, IL-4, IL-10, TNF-α, and IFN-ß mRNAs were detected in both cell lines. Gene expression of IL-4, IL-10, and TNF-α was not significantly different in the two cell lines. However, MDCK cells exhibited a significantly higher level of IFN-ß mRNA than KU-CBE cells at 18 h post infection. Additionally, the protein concentrations of these four cytokines were determined by enzyme-linked immunosorbent assay (ELISA) using cell culture supernatants obtained from the two CIV-infected cell lines. MDCK cells produced significantly higher amounts of IL-4 and IFN-ß than KU-CBE cells. However, KU-CBE cells produced a significantly higher amount of TNF-α than MDCK cells. These data indicated that the newly developed canine tracheal epithelial cells exhibited different cytokine production patterns compared to MDCK cells when infected with CIV. Inflammation of the respiratory tract of dogs induced by CIV infection may be attributed to the elevated expression level of TNF-α in canine tracheal epithelial cells.