Anticoronavirus activity of rhizome of Dryopteris crassirhizoma via multistage targeting of virus entry and viral proteases, Mpro and PLpro.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Dryopteris crassirhizoma Nakai (Dryopteridaceae, RDC), a traditional East Asian herbal medicine, possesses a broad spectrum of medicinal properties, including anti-inflammatory, anticancer, antibacterial, and antiviral activities. AIM OF THE STUDY: This study investigates the 30% ethanolic extract of RDC's antiviral potential against human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and its variants infections. MATERIALS AND METHODS: A 30% ethanolic extract of RDC or its components, filixic acid ABA (PubChem CID: 15081408) and dryocrassin ABBA (PubChem CID: 3082025) were treated with Human Coronavirus infection (HCoV-OC43, SARS-CoV-2 and its variants). The base peak chromatogram of RDC was evaluated using UPLC-Q/TOF Mass to identify the RDC, and the quantitative analysis of RDC compounds was performed using LC-MS/MS. A cytopathic effect (CPE) reduction assay, Western blot, immunofluorescence staining of viral protein expression, and qRT-PCR were performed to quantify the viral RNA copy numbers and determine the antiviral activity. The time-of-addition assay, the virus attachment, penetration, and virucidal assays, and SARS-CoV-2 Mpro and PLpro activity assay were used to elucidate the mode of action. RESULTS: RDC exhibited dose-dependent inhibition of HCoV-OC43-induced cytopathic effects, reducing viral RNA copy numbers and viral protein levels. Time-of-addition assays indicated that RDC targets the early stages of the HCoV-OC43 life cycle, inhibiting virion attachment and penetration with virucidal activity. Notably, filixic acid ABA and dryocrassin ABBA, constituents of RDC, reduced HCoV-OC43 viral RNA loads. Furthermore, RDC effectively blocked viral entry in pseudotyped lentivirus assays, involving spike proteins of SARS-CoV-2 Delta plus and South Africa variants, as well as control lentiviral particles expressing vesicular stomatitis virus glycoprotein G. Additionally, RDC demonstrated inhibition of SARS-CoV-2 infection and its variants by targeting viral proteases, namely main protease (Mpro) and papain-like protease (PLpro). CONCLUSIONS: These findings underscore RDC's multistage approach to targeting viral infections by impeding virus entry and inhibiting viral protease activity. Therefore, RDC holds promise as a potent, broad-spectrum anticoronaviral therapeutic agent.

Intradermal Inoculation of Inactivated Foot-and-Mouth Disease Vaccine Induced Effective Immune Responses Comparable to Conventional Intramuscular Injection in Pigs.

All pigs in the Republic of Korea are given the foot-and-mouth disease virus (FMDV) vaccine intramuscularly (IM) as part of the country's vaccination policy. However, the IM administration of the FMDV vaccine to pig results in residual vaccine components in the muscle and undesirable changes in muscle and soft tissues, causing economic losses in swine production. In this study, we evaluated whether intradermal (ID) vaccination could be proposed as an alternative to IM administration. ID vaccination (0.2 mL on each side of the neck muscle) and IM vaccination (2 mL on each side of the neck muscle) were performed twice, separated by 14 days, using a commercial FMD vaccine in specific-pathogen-free pigs. We observed growth performance, gross and microscopic lesions at the inoculation site, FMDV-specific antibodies, and neutralizing antibodies for 35 days after vaccination. Side effects on the skin grossly appeared following ID administration, but most were reduced within two weeks. All ID-vaccinated pigs showed inflammatory lesions limited to the dermis, but IM-vaccinated pigs had abnormal undesirable changes and pus in the muscle. ID-vaccinated pigs performed comparably to IM-vaccinated pigs in terms of growth, FMD virus-specific antibodies, protection capability against FMDV, and T-cell induction. This study demonstrated that the ID inoculation of the inactivated FMD vaccine induced immune responses comparable to an IM injection at 1/10 of the inoculation dose and that the inoculation lesion was limited to the dermis, effectively protecting against the formation of abnormal undesirable changes in muscle and soft tissues.

Genetic trends in parrot Bornavirus: a clinical analysis.

Parrot Bornavirus (PaBV) has been reported to cause indigestion and other wasting symptoms such as weight loss and lethargy. The pathogenesis of PaBV has yet to be fully elucidated. This study reports PaBV infections in South Korea and suggests a trend in the genetic information gathered from clinical cases. A total of 487 birds with or without clinical symptoms were tested for bornavirus. Twelve of 361 asymptomatic birds tested positive for bornavirus, while 15 of 126 birds with various symptoms tested positive. A segment of approximately 1,540 bps including the N, X, P and M proteins were obtained from 23 of the positive strains and analyzed with other strains found on GenBank that had clinical information. PaBV was type 2 and 4 in South Korea, and certain amino acid sequences showed a difference between symptom presenting animals and asymptomatic animals in the X protein and P protein. When considering that some asymptomatic cases may have been latent infections at the time of examination, it is plausible these trends may grow stronger with time. Majority of PaBV was type 4 in South Korea. If these trends are confirmed, diagnosis of potentially pathogenic PaBVs in a clinical manner will be possible during the early stages of infection.

Intranasal immunization with the recombinant measles virus encoding the spike protein of SARS-CoV-2 confers protective immunity against COVID-19 in hamsters.

BACKGROUND: As the nasal mucosa is the initial site of infection for COVID-19, intranasal vaccines are more favorable than conventional vaccines. In recent clinical studies, intranasal immunization has been shown to generate higher neutralizing antibodies; however, there is a lack of evidence on sterilizing immunity in the upper airway. Previously, we developed a recombinant measles virus encoding the spike protein of SARS-CoV-2 (rMeV-S), eliciting humoral and cellular immune responses against SARS-CoV-2. OBJECTIVES: In this study, we aim to provide an experiment on nasal vaccines focusing on a measles virus platform as well as injection routes. STUDY DESIGN: Recombinant measles viruses expressing rMeV-S were prepared, and 5 × 105 PFUs of rMeV-S were administered to Syrian golden hamsters via intramuscular or intranasal injection. Subsequently, the hamsters were challenged with inoculations of 1 × 105 PFUs of SARS-CoV-2 and euthanized 4 days post-infection. Neutralizing antibodies and RBD-specific IgG in the serum and RBD-specific IgA in the bronchoalveolar lavage fluid (BALF) were measured, and SARS-CoV-2 clearance capacity was determined via quantitative reverse-transcription PCR (qRT-PCR) analysis and viral titer measurement in the upper respiratory tract and lungs. Immunohistochemistry and histopathological examinations of lung samples from experimental hamsters were conducted. RESULTS: The intranasal immunization of rMeV-S elicits protective immune responses and alleviates virus-induced pathophysiology, such as body weight reduction and lung weight increase in hamsters. Furthermore, lung immunohistochemistry demonstrated that intranasal rMeV-S immunization induces effective SARS-CoV-2 clearance that correlates with viral RNA content, as determined by qRT-PCR, in the lung and nasal wash samples, SARS-CoV-2 viral titers in lung, nasal wash, BALF samples, serum RBD-specific IgG concentration, and RBD-specific IgA concentration in the BALF. CONCLUSION: An intranasal vaccine based on the measles virus platform is a promising strategy owing to the typical route of infection of the virus, the ease of administration of the vaccine, and the strong immune response it elicits.

Outbreak of severe diarrhea due to zoonotic Cryptosporidium parvum and C. xiaoi in goat kids in Chungcheongbuk-do, Korea.

Severe diarrhea was reported in goat kids in Chungcheongbuk-do, Korea, from 2021 to 2023, and Cryptosporidium infection was suspected. To confirm the cause of this outbreak, fecal samples were collected from goat farms where diarrhea had been reported and analyzed for Cryptosporidium infection using a molecular assay. A total of 65 fecal samples, including 37 from goats with diarrhea and 28 from goats without diarrhea, were collected from six goat farms. Forty-eight of the goats were kids (<2 months) and 17 were adults (>1 year). Cryptosporidium was identified in 53.8% (35/65) of total samples. Overall, 86.5% (32/37) of the diarrheic fecal samples tested positive; however, Cryptosporidium was not detected in any fecal sample from non-diarrheic adult goats. Therefore, cryptosporidiosis was significantly associated with diarrhea in goat kids, and adult goats were not responsible for transmission of Cryptosporidium to them. Phylogenetic analysis and molecular characterization revealed two Cryptosporidium species, namely, C. parvum (n = 28) and C. xiaoi (n = 7). In the C. parvum-positive samples, gp60 gene analysis revealed three zoonotic subtypes-IIaA18G3R1, IIdA15G1, and IIdA16G1. To the best of our knowledge, this study is the first to identify C. parvum IIaA18G3R1 and IIdA16G1 in goats, as well as the first to identify C. xiaoi in goats in Korea. These results suggest that goat kids play an important role as reservoir hosts for different Cryptosporidium species and that continuous monitoring with biosecurity measures is necessary to control cryptosporidiosis outbreaks.

Morphological and Molecular Characterization of Diplogasteroides sp., a Cryptic Population of the Haslacheri Group (Diplogastridae), and Parasitorhabditis terebranus (Rhabditidae) from Korea.

Diplogasteroides sp., a cryptic population of D. haslacheri, and Parasitorhabditis terebranus were reported from the frass of Monochamus alternatus galleries in dead Pinus thunbergii for the first time in Korea. Females and males are morphologically characterized and their linked DNA barcodes (18S-rRNA, 28S-rRNA, ITS-rRNA and COI) supplied. Females and males of the two species from Korea conform to the original species descriptions from Europe and the USA, with variations in a few details in morphometrics. Specifically, Diplogasteroides sp. is morphologically very similar to D. haslacheri. However, it cannot be designated as D. haslacheri due to the existence of cryptic species complex within the haslacheri group (D. haslacheri, D. asiaticus, D. nix, D. andrassyi, and D. carinthiacus), a condition requiring hybridization studies to test species identity within the group. Based on analysis of COI sequences, differences among these cryptic species are evident. Thus, in addition to hybridization tests, the COI might be a powerful DNA barcoding marker for the precise identification of these cryptic species within the genus. Additionally, this is the first molecular characterization of P. terebranus, and the species is herein recorded for the first time outside its type locality.

Heterologous vaccination utilizing viral vector and protein platforms confers complete protection against SFTSV.

Severe fever with thrombocytopenia syndrome virus was first discovered in 2009 as the causative agent of severe fever with thrombocytopenia syndrome. Despite its potential threat to public health, no prophylactic vaccine is yet available. This study developed a heterologous prime-boost strategy comprising priming with recombinant replication-deficient human adenovirus type 5 (rAd5) expressing the surface glycoprotein, Gn, and boosting with Gn protein. This vaccination regimen induced balanced Th1/Th2 immune responses and resulted in potent humoral and T cell-mediated responses in mice. It elicited high neutralizing antibody titers in both mice and non-human primates. Transcriptome analysis revealed that rAd5 and Gn proteins induced adaptive and innate immune pathways, respectively. This study provides immunological and mechanistic insight into this heterologous regimen and paves the way for future strategies against emerging infectious diseases.

Change in intestinal alkaline phosphatase activity is a hallmark of antibiotic-induced intestinal dysbiosis.

OBJECTIVE: Intestinal alkaline phosphatase (IAP) maintains intestinal homeostasis by detoxifying bacterial endotoxins and regulating gut microbiota, and lipid absorption. Antibiotics administered to animals can cause gut dysbiosis and barrier disruption affecting animal health. Therefore, the present study sought to investigate the role of IAP in the intestinal environment in dysbiosis. METHODS: Young male mice aged 9 weeks were administered a high dose of antibiotics to induce dysbiosis. They were then sacrificed after 4 weeks to collect the serum and intestinal organs. The IAP activity in the ileum and the level of cytokines in the serum samples were measured. Quantitative real-time polymerase chain reaction analysis of RNA from the intestinal samples was performed using primers for tight junction proteins (TJPs) and proinflammatory cytokines. The relative intensity of IAP and toll-like receptor 4 (TLR4) in intestinal samples was evaluated by western blotting. RESULTS: The IAP activity was significantly lower in the ileum samples of the dysbiosisinduced group compared to the control. The interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha concentrations were significantly higher in the ileum samples of the dysbiosis-induced group. The RNA expression levels of TJP2, claudin-3, and claudin-11 showed significantly lower values in the intestinal samples from the dysbiosis-induced mice. Results from western blotting revealed that the intensity of IAP expression was significantly lower in the ileum samples of the dysbiosis-induced group, while the intensity of TLR4 expression was significantly higher compared to that of the control group without dysbiosis. CONCLUSION: The IAP activity and relative mRNA expression of the TJPs decreased, while the levels of proinflammatory cytokines increased, which can affect intestinal integrity and the function of the intestinal epithelial cells. This suggests that IAP is involved in mediating the intestinal environment in dysbiosis induced by antibiotics and is an enzyme that can potentially be used to maintain the intestinal environment in animal health care.

A standardized method to study immune responses using porcine whole blood.

BACKGROUND: Peripheral blood mononuclear cells (PBMCs) are commonly used to assess in vitro immune responses. However, PBMC isolation is a time-consuming procedure, introduces technical variability, and requires a relatively large volume of blood. By contrast, whole blood assay (WBA) is faster, cheaper, maintains more physiological conditions, and requires less sample volume, laboratory training, and equipment. OBJECTIVES: Herein, this study aimed to develop a porcine WBA for in vitro evaluation of immune responses. METHODS: Heparinized whole blood (WB) was diluted (non-diluted, 1/2, 1/8, and 1/16) in RPMI-1640 media, followed by phorbol myristate acetate and ionomycin. After 24 h, cells were stained for interferon (IFN)-γ secreting T-cells followed by flow cytometry, and the supernatant was analyzed for tumor necrosis factor (TNF)-α. In addition, diluted WB was stimulated by lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), reference strain KCTC3557 (RS), field isolate (FI), of heat-killed (HK) Streptococcus suis, and porcine reproductive and respiratory syndrome virus (PRRSV). RESULTS: The frequency of IFN-γ+CD3+ T-cells and concentration of TNF-α in the supernatant of WB increased with increasing dilution factor and were optimal at 1/8. WB TNF-α and interleukin (IL)-10 cytokine levels increased significantly following stimulation with LPS or poly I:C. Further, FI and RS induced IL-10 production in WB. Additionally, PRRSV strains increased the frequency of IFN-γ+CD4-CD8+ cells, and IFN-γ was non-significantly induced in the supernatant of re-stimulated samples. CONCLUSIONS: We propose that the WBA is a rapid, reliable, and simple method to evaluate immune responses and WB should be diluted to trigger immune cells.

Recombinant measles virus encoding the spike protein of SARS-CoV-2 efficiently induces Th1 responses and neutralizing antibodies that block SARS-CoV-2 variants.

Owing to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, the development of effective and safe vaccines has become a priority. The measles virus (MeV) vaccine is an attractive vaccine platform as it has been administered to children for more than 40 years in over 100 countries. In this study, we developed a recombinant MeV expressing the full-length SARS-CoV-2 spike protein (rMeV-S) and tested its efficacy using mouse and hamster models. In hCD46Tg mice, two-dose rMeV-S vaccination induced higher Th1 secretion and humoral responses than one-dose vaccination. Interestingly, neutralizing antibodies induced by one-dose and two-dose rMeV-S immunization effectively blocked the entry of the α, ß, γ, and δ variants of SARS-CoV-2. Furthermore, two-dose rMeV-S immunization provided complete protection against SARS-CoV-2 in the hamster model. These results suggest the potential of rMeV-S as a vaccine candidate for targeting SARS-CoV-2 and its variants.

Inactivation of mammalian spermatozoa on the exposure of TiO2 nanorods deposited with noble metals.

Titanium dioxide (TiO2) nanorods (NRs) are well-known semiconducting and catalytic material that has been widely applied, but their toxicities have also attracted recent interest. In this study, we investigated and compared the toxic effects of TiO2 NRs and TiO2 NRs loaded with Ag or Au NPs on boar spermatozoa. As a result, sperm incubated with Ag-TiO2 NRs showed lower motility than sperm incubated with controls (with or without TiO2 NRs) or Au-TiO2 NRs. In addition, sperm viability and acrosomal integrity were defective in the presence of Ag-TiO2 NRs, and the generation of intracellular reactive oxygen species (ROS) increased significantly when spermatozoa were incubated with 20 µg/ml Ag-TiO2 NRs. We discussed in depth the charge transfer mechanism between enzymatic NADPH and Ag-TiO2 NRs in the context of ROS generation in spermatozoa. The effects we observed reflected the fertilization competence of sperm incubated with Ag-TiO2 NRs; specifically sperm penetration and embryonic development rates by in vitro fertilization were reduced by Ag-TiO2 NRs. To summarize, our findings indicate that exposure to Ag-TiO2 NRs could affect male fertilization fecundity and caution that care be exercised when using these NRs.

Distribution of Gastrointestinal Parasitic Infection in Domestic Pigs in the Republic of Korea: Nationwide Survey from 2020-2021.

This study aimed to examine the distribution of gastrointestinal parasitic infections in domestic pigs in the Republic of Korea. From May 2020 to October 2021, 364 pig fecal samples were collected from 75 farms in 7 Provinces and microscopically examined. A total of 170 (46.7%) pigs were infected with at least one of the following parasites: Balantioides coli, strongyles, Ascaris suum, Trichuris suis, and coccidia. By parasite species, B. coli, strongyles, A. suum, T. suis, and coccidia oocysts or eggs were detected in 144 (39.6%), 24 (6.6%), 14 (3.8%), 4 (1.1%), and 1 (0.3%) samples, respectively. One hundred fifty-four, 15, and 1 cases showed single, double, and triple infections, respectively. Of the swine fecal samples from 75 farms, 69 specimens (92.0%) were infected with 1 or more parasites. All surveyed farms across the country exhibited a positive rate of over 30%, among which the highest positive rate was 65.0% in Chungcheongnam-do, and Jeollabuk-do was followed by 61.9%. Winter showed a statistically lower prevalence than other seasons. This study showed that gastrointestinal parasites are prevalent in pigs in Korea, although the diversity of parasites is low.

Non-invasive administration of AAV to target lung parenchymal cells and develop SARS-CoV-2-susceptible mice.

Adeno-associated virus (AAV)-mediated gene delivery holds great promise for gene therapy. However, the non-invasive delivery of AAV for lung tissues has not been adequately established. Here, we revealed that the intratracheal administration of an appropriate amount of AAV2/8 predominantly targets lung tissue. AAV-mediated gene delivery that we used in this study induced the expression of the desired protein in lung parenchymal cells, including alveolar type II cells. We harnessed the technique to develop severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-susceptible mice. Three kinds of immune function-relevant gene knockout (KO) mice were transduced with AAV encoding human angiotensin-converting enzyme 2 (hACE2) and then injected with SARS-CoV-2. Among these mice, type I interferon receptor (IFNAR) KO mice showed increased viral titer in the lungs compared to that in the other KO mice. Moreover, nucleocapsid protein of SARS-CoV-2 and multiple lesions in the trachea and lung were observed in AAV-hACE2-transduced, SARS-CoV-2-infected IFNAR KO mice, indicating the involvement of type I interferon signaling in the protection of SARS-CoV-2. In this study, we demonstrate the ease and rapidness of the intratracheal administration of AAV for targeting lung tissue in mice, and this can be used to study diverse pulmonary diseases.

Evaluation of Photobiogoverning Role of Blue Light Irradiation on Viral Replication.

Most recently, severe acute respiratory syndrome coronavirus-2 has triggered a global pandemic without successful therapeutics. The goal of the present study was to define the antiviral effect and therapeutic action of blue light irradiation in SARS-CoV-2-infected cells. Vero cells were infected with SARS-CoV-2 (NCCP43326) or mock inoculum at 50 pfu/well. After blue light irradiation, the inhibitory effect was assessed by qPCR and plaque reduction assay. When Vero cells were irradiated to blue light ranging from 1.6 to 10 J cm-2 , SARS-CoV-2 replication was inhibited by up to 80%. The antiviral effect of blue light irradiation was associated with translation suppression via the phosphorylation of eIF2α by prolonging endoplasmic reticulum (ER) stress. The levels of LC3A/B and Beclin-1, which are key markers of autophagy, and the levels of PERK and PDI for ER stress were highly increased, whereas caspase-3 cleavage was inhibited after blue light irradiation in the later stage of infection. Our data revealed that blue light irradiation exerted antiviral and photo-biogoverning activities by prolonging ER stress and stimulating autophagy progression during viral infection. The findings increase our understanding of how photo-energy acts on viral progression and have implications for use in therapeutic strategies against COVID-19.

Sirtuin 6 is a negative regulator of FcεRI signaling and anaphylactic responses.

BACKGROUND: Binding IgE to a cognate allergen causes aggregation of Fcε receptor I (FcεRI) in mast cells, resulting in activation of receptor-associated Src family tyrosine kinases, including Lyn and Syk. Protein tyrosine phosphatase, receptor type C (PTPRC), also known as CD45, has emerged as a positive regulator of FcεRI signaling by dephosphorylation of the inhibitory tyrosine of Lyn. OBJECTIVE: Sirtuin 6 (Sirt6), a NAD+-dependent deacetylase, exhibits an anti-inflammatory property. It remains to be determined, however, whether Sirt6 attenuates mast cell-associated diseases, including anaphylaxis. METHODS: FcεRI signaling and mast cell degranulation were measured after IgE cross-linking in murine bone marrow-derived mast cells (BMMCs) and human cord blood-derived mast cells. To investigate the function of Sirt6 in mast cell activation in vivo, we used mast cell-dependent animal models of passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA). RESULTS: Sirt6-deficient BMMCs augmented IgE-FcεRI-mediated signaling and degranulation compared to wild-type BMMCs. Reconstitution of mast cell-deficient KitW-sh/W-sh mice with BMMCs received from Sirt6 knockout mice developed more severe PSA and PCA compared to mice engrafted with wild-type BMMCs. Similarly, genetic overexpression or pharmacologic activation of Sirt6 suppressed mast cell degranulation and blunted responses to PCA. Mechanistically, Sirt6 deficiency increased PTPRC transcription via acetylating histone H3, leading to enhanced aggregation of FcεRI in BMMCs. Finally, we recapitulated the Sirt6 regulation of PTPRC and FcεRI signaling in human mast cells. CONCLUSIONS: Sirt6 acts as a negative regulator of FcεRI signaling cascade in mast cells by suppressing PTPRC transcription. Activation of Sirt6 may therefore represent a promising and novel therapeutic strategy for anaphylaxis.

Peptides Derived From S and N Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 Induce T Cell Responses: A Proof of Concept for T Cell Vaccines.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that escape vaccine-induced neutralizing antibodies has indicated the importance of T cell responses against this virus. In this study, we highlight the SARS-CoV-2 epitopes that induce potent T cell responses and discuss whether T cell responses alone are adequate to confer protection against SARS-CoV-2 and describe the administration of 20 peptides with an RNA adjuvant in mice. The peptides have been synthesized based on SARS-CoV-2 spike and nucleocapsid protein sequences. Our study demonstrates that immunization with these peptides significantly increases the proportion of effector memory T cell population and interferon-γ (IFN-γ)-, interleukin-4 (IL-4)-, tumor necrosis factor-α (TNF-α)-, and granzyme B-producing T cells. Of these 20 peptides, four induce the generation of IFN-γ-producing T cells, elicit CD8+ T cell (CTL) responses in a dose-dependent manner, and induce cytotoxic T lymphocytes that eliminate peptide-pulsed target cells in vivo. Although it is not statistically significant, these peptide vaccines reduce viral titers in infected hamsters and alleviate pulmonary pathology in SARS-CoV-2-infected human ACE2 transgenic mice. These findings may aid the design of effective SARS-CoV-2 peptide vaccines, while providing insights into the role of T cells in SARS-CoV-2 infection.

Ultra- and micro-structural changes of respiratory tracts in SARS-CoV-2 infected Syrian hamsters.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a global crisis. It is still unresolved. Although many therapies and vaccines are being studied, they are still in their infancy. As this pandemic continues, rapid and accurate research for the development of therapies and vaccines is needed. Therefore, it is necessary to understand characteristics of diseases caused by SARS-CoV-2 through animal models. Syrian hamsters are known to be susceptible to SARS-CoV-2. They were intranasally inoculated with SARS-CoV-2. At 2, 4, 8, 12, and 16 days post-infection (dpi), these hamsters were euthanized, and tissues were collected for ultrastructural and microstructural examinations. Microscopic lesions were prominent in the upper and lower respiratory tracts from 2 and 4 dpi groups, respectively. The respiratory epithelium in the trachea, bronchiole, and alveolar showed pathological changes. Inflammatory cells including neutrophils, lymphocytes, macrophages, and eosinophils were infiltrated in/around tracheal lamina propria, pulmonary vessels, alveoli, and bronchiole. In pulmonary lesions, alveolar wall was thickened with infiltrated inflammatory cells, mainly neutrophils and macrophages. In the trachea, epithelial damages started from 2 dpi and recovered from 8 dpi, consistent with microscopic results, High levels of SARS-CoV-2 nucleoprotein were detected at 2 dpi and 4 dpi. In the lung, lesions were most severe at 8 dpi. Meanwhile, high levels of SARS-CoV-2 were detected at 4 dpi. Electron microscopic examinations revealed cellular changes in the trachea epithelium and alveolar epithelium such as vacuolation, sparse micro-organelle, and poor cellular margin. In the trachea epithelium, the number of cytoplasmic organelles was diminished, and small vesicles were prominent from 2 dpi. Some of these electron-lucent vesicles were filled with virion particles. From 8 dpi, the trachea epithelium started to recover. Because of shrunken nucleus and swollen cytoplasm, the N/C ratio of type 2 pneumocyte decreased at 8 and 12 dpi. From 8 dpi, lamellar bodies on type 2 pneumocyte cytoplasm were increasingly observed. Their number then decreased from 16 dpi. However, there was no significant change in type 1 pneumocyte. Viral vesicles were only observed in the cytoplasm of type 2 pneumocyte. In conclusion, ultra- and micro-structural changes presented in this study may provide useful information for SARS-CoV-2 studies in various fields.

Protective immunity induced by concurrent intradermal injection of porcine circovirus type 2 and Mycoplasma hyopneumoniae inactivated vaccines in pigs.

Vaccines against porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhp) are routinely used by intramuscular injection. However, since intramuscular vaccination causes stress and increases the risk of cross-contamination among pigs, research on intradermal vaccination is currently being actively conducted. This study was designed to evaluate the efficacy of intradermally administered inactivated vaccines against PCV2 and Mhp in pigs. Three-week-old specific pathogen-free pigs were divided into three groups (5 pigs per group). Pigs in the two groups were intradermally vaccinated with the PCV2 or Mhp vaccine using a needle-free injector. Pigs in the third group were kept as nonvaccinated controls. At 21 days post-vaccination, pigs in one of these vaccinated groups and the nonvaccinated group were intranasally challenged with PCV2b and Mhp, while the other vaccinated group pigs were maintained as vaccine controls. Vaccine efficacy was evaluated by observing weight gain, pathogen load, pathological changes, and humoral or cellular immune responses. As a result, vaccinated pigs revealed significantly higher body weight gain, with lower clinical scores. Vaccinated pigs also showed higher antibody responses but lower PCV2b or Mhp loads in sera, nasal swabs, or lungs than nonvaccinated pigs. Intriguingly, vaccinated pigs upregulated cytotoxic T cells (CTLs), helper T type 1 cells (Th1 cells), and helper T type 17 cells (Th17 cells) after immunization and showed significantly higher levels of CTLs, Th1 and Th17 cells at 14 days post-challenge than nonvaccinated and challenged pigs. This study demonstrated that protective immune responses against PCV2 and Mhp could be efficiently induced in pigs using a relatively small volume of intradermal vaccines, probably due to effective antigen delivery to antigen-presenting cells in the dermis.

Potential Therapeutic Effect of Micrornas in Extracellular Vesicles from Mesenchymal Stem Cells against SARS-CoV-2.

Extracellular vesicles (EVs) are cell-released, nanometer-scaled, membrane-bound materials and contain diverse contents including proteins, small peptides, and nucleic acids. Once released, EVs can alter the microenvironment and regulate a myriad of cellular physiology components, including cell-cell communication, proliferation, differentiation, and immune responses against viral infection. Among the cargoes in the vesicles, small non-coding micro-RNAs (miRNAs) have received attention in that they can regulate the expression of a variety of human genes as well as external viral genes via binding to the complementary mRNAs. In this study, we tested the potential of EVs as therapeutic agents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. First, we found that the mesenchymal stem-cell-derived EVs (MSC-EVs) enabled the rescue of the cytopathic effect of SARS-CoV-2 virus and the suppression of proinflammatory responses in the infected cells by inhibiting the viral replication. We found that these anti-viral responses were mediated by 17 miRNAs matching the rarely mutated, conserved 3'-untranslated regions (UTR) of the viral genome. The top five miRNAs highly expressed in the MSC-EVs, miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p, and miR-181a-5p, were tested. They were bound to the complemented sequence which led to the recovery of the cytopathic effects. These findings suggest that the MSC-EVs are a potential candidate for multiple variants of anti-SARS-CoV-2.