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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.

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BACKGROUND: Hydrogen sulfide is a highly toxic, flammable, and colorless gas. Hydrogen sulfide has been identified as a potential terrorist chemical threat agent in mass-casualty events. Our previous studies showed that cobinamide, a vitamin B12 analog, effectively reverses the toxicity from hydrogen sulfide poisoning. In this study, we investigate the effectiveness of intratracheally administered cobinamide in treating a lethal dose hydrogen sulfide gas inhalation and compare its performance to saline control administration. METHODS: A total of 53 pathogen-free New Zealand White rabbits were used for this study. Four groups were compared: (i) received no saline solution or drug intratracheally (n = 15), (ii) slow drip saline intratracheally (n = 15), (iii) fast drip saline intratracheally (n = 15), and (iv) slow drip cobinamide intratracheally (n = 8). Blood pressure was continuously monitored, and deoxy- and oxyhemoglobin concentration changes were monitored in real-time in vivo using continuous wave near-infrared spectroscopy. RESULTS: The mean (± standard deviation) weight for all animals (n = 53) was 3.87 ± 0.10 kg. The survival rates of the slow cobinamide and the fast saline groups were 75 percent and 60 percent, respectively, while the survival rates in the slow saline and control groups were 26.7 percent and 20 percent, respectively. A log-rank (Mantel-Cox) test showed that survival in fast saline and slow cobinamide groups were significantly greater than those of no saline control and slow saline groups (P < 0.05). The slow and no saline control groups were not significantly different (P = 0.59). The slow cobinamide group did significantly better than the slow saline group (P = 0.021). DISCUSSION: The ability to use intratracheal cobinamide as an antidote to hydrogen sulfide poisoning is a novel approach to mass-casualty care. The major limitations of this study are that it was conducted in a single species at a single inhaled hydrogen sulfide concentration. Repeated investigations in other species and at varying levels of hydrogen sulfide exposure will be needed before any definitive recommendations can be made. CONCLUSIONS: We demonstrated that intratracheal cobinamide and fast saline drip improved survival for hydrogen sulfide gas inhalation in rabbit models. Although further study is required, our results suggest that intratracheal administration of cobinamide and fast saline may be useful in hydrogen sulfide mass-casualty events.

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Many denture frameworks are currently manufactured using metal 3-dimensional (3D) printing technology. However, this advanced technology is still limited to conventional denture designs. A metal 3D printing-based denture design concept was developed and is presented based on a nonwear occlusal surface, a nonfracture denture base, and a novel connection between metal and resin.

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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.

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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.

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Rottlerin (R) is a natural extract from Mallotus philippensis with antiviral properties. Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV) that is characterized by systemic granulomatous inflammation and high mortality. We investigated the antiviral effect of liposome-loaded R, i.e., rottlerin-liposome (RL), against FCoV. We demonstrated that RL inhibited FCoV replication in a dose-dependent manner, not only in the early endocytosis stage but also in the late stage of replication. RL resolved the low solubility issue of rottlerin and improved its inhibition efficacy at the cellular level. Based on these findings, we suggest that RL is worth further investigation as a potential treatment for FCoV.

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Salmonella Enteritidis is a common foodborne pathogen transmitted through poultry products, which are its main carriers. Poultry are vaccinated against Salmonella Enteritidis in many countries, despite the absence of clinical symptoms, using commercially available live-attenuated vaccines. We previously constructed a highly attenuated temperature-sensitive (ts) Salmonella Enteritidis mutant, 2S-G10. In the present study, we describe the construction and attenuation-associated characteristics of 2S-G10. We infected 1-day-old chicks with 2S-G10 and the parental strains to evaluate the attenuation. One week after infection, 2S-G10 was not detected in the liver, cecum, or cecal tonsil tissues of the orally inoculated chicks, contrary to the parental strain. This indicates that 2S-G10 was highly attenuated when compared to the parental stain. In vitro experiments revealed the inability of 2S-G10 to grow at the normal body temperature of chickens and invade chicken liver epithelial cells. Moreover, single nucleotide polymorphism (SNP) analysis between the complete genome sequence of 2S-G10 and its parental strain revealed SNPs in bcsE, recG, rfaF, and pepD_1 genes, which are involved in epithelial cell invasion and persistence in host systems, growth, lipopolysaccharide core biosynthesis, and cellular survival under heat stress, respectively. These potential characteristics are consistent with the findings of in vitro experiments. Conclusively, chemical treatment-induced random genetic mutations highly attenuated 2S-G10, implying its potential to be developed as a novel live-attenuated vaccine against Salmonella Enteritidis.

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N-linked glycans covering GP5 neutralizing epitopes of porcine reproductive and respiratory syndrome virus (PRRSV) have been proposed to act as a sheath blocking the production of neutralizing antibodies. Herein, we genetically engineered PRRSV with serine (S) substitution on the 44th asparagine (N) on the GP5 ectodomain of PRRSV-2 lineage-1. To evaluate the recombinant PRRSV, in vivo experiments were performed in piglets. The recombinant virus group showed no viremia until 42 days post-inoculation (dpi), and the rectal temperature and average daily weight gain were in the normal range at the same time point as the negative control group. On the 42 dpi, both groups were challenged with the wild-type virus. The recombinant PRRSV group showed lower rectal temperature, viremia, and the lung lesions than that of the negative control group for 19 days post-challenge (dpc). Additionally, the recombinant virus induced 4.50 ± 3.00 (log2) and 8.25 ± 0.96 (log2) of neutralizing antibody before and after challenge, respectively. Taken together, this study confirmed that N44S substitution can create an infectious PRRSV that strongly induces neutralizing antibodies. In addition, the vCSL1-GP5-N44S mutant that we produced was confirmed to have potential as a vaccine candidate, showing good safety and protective effects in pigs.

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Background: Subjects with diabetes are at higher risk of serious influenza-related complications. We aimed to investigate the yearly trend of influenza vaccination and factors associated with being unvaccinated for influenza in subjects with diabetes using a nationwide observational study performed within the recent decade. Methods: Among 105,732 subjects from the Korea National Health and Nutrition Examination Survey between 2007 and 2019, 8,632 with diabetes were included. We investigated the yearly trend of influenza vaccination and factors associated with being unvaccinated for influenza. Results: During the study period, the prevalence of influenza vaccination in subjects with diabetes showed a tendency to increase every year, reaching almost 60% in 2019, which was higher than the rate in subjects without diabetes. Younger age (adjusted hazard ratio (aHR) [95% CI] 11.29 [8.63-14.75] for < 50 years; 6.16 [5.21-7.29] for 50-65 years), male (aHR 1.67 [1.52-1.87]), current smoker (aHR 1.31 [1.00-1.72], lower-income status (aHR 1.46 [1.17, 1.84]), and high education level (aHR 1.30 [1.01-1.67]) were associated with being unvaccinated. Also, a poorer glycemic control with HbA1c ≥ 9% was found to be correlated with unvaccinated status (aHR 1.48 [1.15-1.90]). Conclusion: The influenza vaccination rate is still unsatisfactory in subjects with diabetes. Young age, males, low-income level, high education level, and poor glycemic control were associated with unvaccinated status. Considering the risk-benefits of influenza vaccination in patients with diabetes, physicians should make an effort to increase vaccination rates, especially in low vaccination rate groups.

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The emergence of multidrug-resistant (MDR) bacteria and the spread of antimicrobial resistance among various bacteria are major threats to the global community. Due to the increased failure of classical antibiotic treatments against MDR bacterial infections, probiotics and their antimicrobial compounds have been suggested as promising alternatives to deal with MDR bacteria. Various strains of lactic acid bacteria have been reported to produce antagonistic molecules against pathogens. A new strain of Ligilactobacillus animalis, L. animalis SWLA-1, isolated from the feces of healthy dogs, shows strong antimicrobial activity against not only common pathogens but also MDR bacteria. In this study, we compared the antimicrobial activity of L. animalis SWLA-1 with that of other lactobacilli and antibiotics using an agar spot assay. Additionally, a novel spot inhibition index was developed and validated to quantitively evaluate the inhibitory activities of lactobacilli and antibiotics. A competitive coculture assay of L. animalis SWLA-1 with MDR bacteria further demonstrated its antibacterial activity. Furthermore, we evaluated the antimicrobial activity of the cell-free supernatant (CFS) of L. animalis SWLA-1 and its stability under various conditions in vitro. We found that L. animalis SWLA-1 and its CFS are potential alternatives to classic antimicrobial agents.

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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.

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Torque teno canis virus (TTCaV) is an approximately 2.8 kb circular single-stranded DNA virus known to cause infections in dogs. However, its incidence in Republic of Korea remains unknown. In this study, 135 dog fecal samples were collected to determine TTCaV infection status in Republic of Korea. Based on polymerase chain reaction (PCR) analysis, 13 of 135 (9.6%) dogs tested positive for TTCaV. Three full-length genome sequences (GenBank IDs: MZ503910, MZ503911, and MZ503912) were obtained from the positive specimens. Phylogenetic tree construction and sequence identity, similarity plot, and recombination analyses were performed using these three full-length genomic sequences. Among the three full-length genomes, MZ503912 was determined to be a recombinant virus based on analysis with the reference TTCaV strains. This novel virus strain might have been generated by recombination between TTCaV strain KX827768 discovered in China and MZ503910 discovered in Republic of Korea. This is the first report to determine the incidence, genetic variation, and recombination of TTCaV in dogs in Republic of Korea. Further studies are needed to elucidate TTCaV pathogenesis in dogs.

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The number of companion animals living with humans has continually increased over the last few decades, and so has the interest of owners and stakeholders in the animal food and probiotics industry. Currently, the probiotic bacteria added to the feed of companion animals predominantly originate from the lactic acid bacteria (LAB) used for humans; however, there are differences between the microbiota of humans and that of their companion animals. This study aimed to determine the dominant LAB in dog feces and investigate their functional properties. Ligilactobacillus animalis (formerly called Lactobacillus animalis) was identified as the dominant lactic acid bacterium in dog feces. It displayed various inhibitory effects against pathogenic and enteropathogenic bacteria. This finding suggests that Ligilactobacillus animalis can potentially be used in novel probiotics or as a food additive for dogs.

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Equine parvovirus-hepatitis (EqPV-H) is one of the etiological agents of Theiler's disease, causing fulminant hepatitis; however, its transmission route and pathogenesis remain unclear. In the present study, we aimed to determine EqPV-H shedding in oral/nasal/vaginal swabs or semen samples from horses living in Korea using nested polymerase chain reaction. We then used the data obtained to investigate various risk factors associated with EqPV-H including viral shedding, hepatopathological changes, and genetic diversity. Our data revealed occurrence of EqPV-H shedding in these animals (oral: 3/102 [2.9%]; nasal: 3/102 [2.9%]; semen: 1/9 [11.1%]) and identified that both age and country of foaling were significantly associated with EqPV-H shedding (p < .05). In addition, we noted that one of the newly isolated strains clustered separately from the other strains in the phylogenetic tree, revealing unique nucleotide and amino acid substitutions. This is a field surveillance study providing evidence of natural and venereal shedding of EqPV-H and describing its presence in both oral/nasal fluids and semen. This epidemiological and clinical analysis may help specify the clinicopathological features of EqPV-H and facilitate the development of novel disease prevention strategies.

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An ideal vaccine for controlling Salmonella infection in chicken flocks should be safe, inducing both humoral and cellular immunity. Live attenuated vaccines against Salmonella Enteritidis (S. Enteritidis) have been used as a potential control method of Salmonella infection in the poultry industry. However, live attenuated vaccines can persistently infect poultry for long periods and can become virulent revertant strains. In this study, we assessed the immune responses and protective efficacy of a temperature-sensitive attenuated S. Enteritidis mutant as a potential vaccine candidate. In addition, we evaluated the combined vaccine administration methods to maximize both humoral and cellular immune responses in chickens induced by the vaccine candidate. Immune responses and protective efficacy were compared between the Oral/IM group, vaccinated using one oral dose at four weeks old and a booster intramuscular dose at seven weeks old, and the IM/Oral group, vaccinated using one intramuscular dose at four weeks old and a booster oral dose at seven weeks old. The Oral/IM group showed stronger immune responses than those of the IM/Oral group. Spleens from the Oral/IM group showed a promising tendency of reduction of challenged Salmonella compared with those of other groups. Overall, the results indicated that the S. Enteritidis mutant strain is a promising live attenuated vaccine candidate with good efficacy.

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Canine coronavirus (CCoV), canine parvovirus (CPV), and canine distemper virus (CDV) are highly contagious canine pathogens; dogs with these diseases are difficult to treat. In a previous study, we developed a recombinant adenovirus expressing canine interferon lambda 3 (Ad-caIFNλ3) in canine epithelial cells. In this study, we aimed to investigate the antiviral activity of Ad-caIFNλ3 against CCoV, CPV, and CDV in two canine cell lines, A72 and MDCK. Ad-caIFNλ3 transduction suppressed replication of these viruses without cytotoxicity. Our results suggest that Ad-caIFNλ3 may be a therapeutic candidate for canine viral diseases.

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We investigated the cross-species transmission of rabbit hepatitis E virus (rb HEV) to pigs and evaluated the cross-protection of a swine (sw) HEV-3 virus-like particle (VLP) vaccine against rb HEV infection in pigs. Twelve 4-week-old conventional pigs were divided into negative control (n = 3), positive control (rb HEV-infected, n = 4), and vaccinated (vaccinated and rb HEV-challenged, n = 5) groups. The vaccine was administered at weeks 0 and 2, and viral challenge was conducted at week 4. Serum HEV RNA, anti-HEV antibody, cytokine, and liver enzyme levels were determined. Histopathological lesions were examined in abdominal organs. Viral RNA was detected and increased anti-HEV antibody and alanine aminotransferase (ALT) levels were observed in positive control pigs; liver fibrosis, inflammatory cell infiltration in the lamina propria of the small intestine and shortened small intestine villi were also observed. In vaccinated pigs, anti-HEV antibody and Th1 cytokine level elevations were observed after the second vaccination; viral RNA was not detected, and ALT level elevations were not observed. The results verified the cross-species transmission of rb HEV to pigs and cross-protection of the sw HEV-3 VLP vaccine against rb HEV infection in pigs. This vaccine may be used for cross-protection against HEV infection in other species.

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Here, rabbits were immunized with a virus-like particle (VLP) vaccine prepared by expressing 239 amino acids of the swine hepatitis E virus (HEV)-3 capsid protein using a baculovirus system. Thirty specific-pathogen-free rabbits were divided into five groups (negative and positive control and 10, 50, and 100 µg VLP-vaccinated). Positive control group rabbits showed viremia and fecal viral shedding, whereas rabbits vaccinated with 10 µg VLP showed transient fecal viral shedding, and rabbits vaccinated with 50 and 100 µg VLP did not show viremia or fecal viral shedding. Serum anti-HEV antibody titers increased in a dose-dependent manner. Anti-HEV antibody titers were significantly higher (p < 0.05) in 100 µg VLP-vaccinated rabbits than in the negative control rabbits at week 4. Anti-HEV antibody titers were significantly higher in 50 and 10 µg VLP-vaccinated rabbits than in the negative control rabbits at weeks 8 and 11, respectively. Serum IFN-γ and IL-12 levels were significantly higher (p < 0.01) in rabbits vaccinated with 50 and 100 µg VLP than in the negative control rabbits at weeks 4 and 6. Liver tissues of 50 and 100 µg VLP-vaccinated rabbits displayed significantly less (p < 0.05) fibrosis than those of the positive control rabbits. The prepared VLP vaccine demonstrated dose-dependent immunogenicity sufficient for inducing anti-HEV antibody production, thus protecting rabbits against swine HEV-3.

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Hepatitis E virus (HEV) is an emerging zoonotic pathogen causing hepatitis worldwide. Despite the prevalent evidence of interspecies HEV infection in various animal species, the role of horses in HEV epidemiology remains unclear. In this study, we investigated the prevalence of HEV infection in 283 blood and 114 fecal samples from 397 horses using sandwich enzyme-linked immunosorbent assay and nested reverse transcription-polymerase chain reaction. Among the 283 serum samples, 35 were positive for anti-HEV antibodies (12.4%; 95% confidence interval: 8.8-16.8), and four of the five sampling regions (80%) had these seropositive individuals. Analyses of the potential risk factors for HEV infection revealed that racing horses had a significantly higher risk of infection (P = 0.01). However, HEV RNA was not detected in any of the tested serum and fecal samples. To the best of our knowledge, this is the first epidemiological HEV study on horses in Republic of Korea, thereby providing evidence of HEV exposure in the horse population in Korea and specifying the risk factors for HEV infection.

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