Cross-species transmission and histopathological variation in specific-pathogen-free minipigs infected with different hepatitis E virus strains.

Hepatitis E virus (HEV) is a major cause of viral hepatitis worldwide. Pigs are the natural host of HEV genotype 3 and the main reservoir of HEV. As the host range of HEV genotype 3 expands, the possibility that HEV from various species can be transmitted to humans via pigs is increasing. We investigated the potential cross-species transmission of HEV by infecting minipigs with swine HEV (swHEV), rabbit HEV (rbHEV), and human HEV (huHEV) and examining their histopathological characteristics and distribution in various organs. Fifteen specific-pathogen-free Yucatan minipigs were infected with swHEV, rbHEV, huHEV, or a mock control. In the present study, we analysed faecal shedding, viremia, and serological parameters over a seven-week period. Our results indicated that swHEV exhibited more robust shedding and viremia than non-swHEVs. Only swHEV affected the serological parameters, suggesting strain-specific differences. Histopathological examination revealed distinct patterns in the liver, pancreas, intestine, and lymphoid tissues after infection with each HEV strain. Notably, all three HEVs induced histopathological changes in the pancreas, supporting the association of HEVs with acute pancreatitis. Our results also identified skeletal muscle as a site of HEV antigen presence, suggesting a potential link to myositis. In conclusion, this study provides valuable insights into the infection dynamics of different HEV strains in minipigs, emphasizing the strain-specific variations in virological, serological, and histological parameters. The observed differences in infection kinetics and tissue tropism will contribute to our understanding of HEV pathogenesis and the potential for cross-species transmission.

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.

Assessment of nematicidal and plant growth-promoting effects of Burkholderia sp. JB-2 in root-knot nematode-infested soil.

Root-knot nematodes (RKN), Meloidogyne spp., are plant-parasitic nematodes that are responsible for considerable economic losses worldwide, because of the damage they cause to numerous plant species and the inadequate biological agents available to combat them. Therefore, developing novel and eco-friendly nematicides is necessary. In the present study, Burkholderia sp. JB-2, isolated from RKN-infested rhizosphere soil in South Korea, was evaluated to determine its nematicidal and plant growth-promoting effects under in vitro and in vivo conditions. Cell-free filtrates of the JB-2 strain showed high levels of nematicidal activity against second-stage juveniles (J2) of M. incognita, with 87.5% mortality following two days of treatment. In addition, the assessment of the activity against other six plant parasitic nematodes (M. javanica, M. hapla, M. arenaria, Ditylenchus destructor, Aphelenchoides subtenuis, and Heterodera trifolii) showed that the cell-free filtrates have a broad nematicidal spectrum. The three defense-responsive (MiMIF-2, MiDaf16-like1, and MiSkn1-like1) genes were activated, while Mi-cm-3 was downregulated when treated with cell-free filtrates of JB-2 cultures on J2. The greenhouse experiments suggested that the cell-free filtrates of the JB-2 strain efficiently controlled the nematode population in soil and egg mass formations of M. incognita in tomato (Solanum lycopersicum L., cv. Rutgers). An improvement in the host plant growth was observed, in which the shoot length and fresh weights of shoots and roots increased. The treatment with 10% of JB-2 cell-free filtrates significantly upregulated the expression levels of plant defenses (SlPR1, SlPR5, and SlPAL) and growth-promoting (ACO1, Exp18, and SlIAA1) genes compared with the corresponding parameters of the control group. Therefore, JB-2 could be a promising candidate for the sustainable management of RKN.

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.

Rottlerin-Liposome Inhibits the Endocytosis of Feline Coronavirus Infection.

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.

Characteristics of a Temperature-Sensitive Mutant Strain of Salmonella Enteritidis and Its Potential as a Live Vaccine Candidate.

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.

Porcine Reproductive and Respiratory Syndrome Virus Engineered by Serine Substitution on the 44th Amino Acid of GP5 Resulted in a Potential Vaccine Candidate with the Ability to Produce High Levels of Neutralizing Antibody.

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.

Antimicrobial Activity of Ligilactobacillus animalis SWLA-1 and Its Cell-Free Supernatant against Multidrug-Resistant Bacteria and Its Potential Use as an Alternative to Antimicrobial Agents.

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.

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.

Stability and inactivation of SARS-CoV-2 on food contact surfaces.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 269 million people and killed more than 5.3 million people worldwide. Although fomite transmission of SARS-CoV-2 has been continuously reported, few studies have been conducted on food contact surfaces. Therefore, this study aimed to investigate the viability of coronaviruses on food contact surfaces and to remove SARS-CoV-2 contaminated on food contact surfaces with disinfectants. At 20 °C, SARS-CoV-2 was inactivated within 48 h on all food contact surfaces. At 4 °C, it was inactivated at 48 h on kraft paper and 96 h on parchment paper, but it was viable up to 5 days in low-density polyethylene (LDPE). At -20 °C, SARS-CoV-2 did not decrease by even 1 log on all food contact surfaces until 5 days. Treatment with 70% ethanol or 1000 ppm sodium hypochlorite for 5 min was sufficient to completely remove SARS-CoV-2 from 6 food contact surfaces. Similarly, UV-C irradiation at 60 mJ/cm2 eliminated SARS-CoV-2 contaminated on food contact surfaces. Also, the wiping test showed that even wiping an area contaminated with SARS-CoV-2 with a cloth moistened with 70% ethanol or 1000 ppm sodium hypochlorite, it took 5 min to inactivate the virus. Our findings suggested that SARS-CoV-2 contaminated on food contact surfaces in local retail may be viable enough to be transported home. However, if the type and method of use of the disinfectant suggested in this study are followed, it is possible to sufficiently control the fomite transmission of SARS-CoV-2 through food contact surfaces at home.

Genetic Analysis of Torque Teno Canis Virus Identified in Republic of Korea.

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.

Antimicrobial activity of dominant Ligilactobacillus animalis strains in healthy canine feces and their probiotic potential.

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.

Molecular surveillance of equine parvovirus-hepatitis from oral, nasal, vaginal, and semen specimens collected from horses living in Korea.

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.

Antiviral activity of canine interferon lambda 3 expressed using a recombinant adenovirus against canine coronavirus, canine parvovirus, and canine distemper virus.

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.

Evaluation of Immune Responses and Protective Efficacy of a Novel Live Attenuated Salmonella Enteritidis Vaccine Candidate in Chickens.

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.

Characterization of Heterodera sojae Virulence Phenotypes in Korea.

The white soybean cyst nematode Heterodera sojae, isolated from the roots of soybean in Korea, is widespread in most provinces of the country and has the potential to be as harmful to soybean as H. glycines. Determining the virulence phenotypes of H. sojae is essential to devising management strategies that use resistant cultivars. Consequently, virulence phenotypes of 15 H. sojae populations from Korea were determined on seven soybean lines and one susceptible check variety. Two different HS types were found to be present in Korea; the more common HS type 2.5.7, comprising 73.3% of the H. sojae populations and the less common HS type 0, constituting only 26.7% of the tested populations. Considering the high frequency of H. sojae adaptation to soybean indicator lines, the PI 88788 group may not be a possible source of resistance while PI 548402, PI 90763, PI 437654, and PI 89772 can be used as resistance sources for soybean breeding programs aimed at developing H. sojae-resistant soybean cultivars in Korea.

Cross-Species Transmission of Rabbit Hepatitis E Virus to Pigs and Evaluation of the Protection of a Virus-like Particle Vaccine against Rabbit Hepatitis E Virus Infection in Pigs.

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.

Immunization with Virus-Like Particle Vaccine Protects Rabbits against Hepatitis E-3 Virus Infection.

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.