Inflammatory & Apoptotic Factor Fluctuations Associated with Japanese Encephalitis Virus Infection in Transgenic IFNAR1-/- Mice.

Japanese encephalitis virus (JEV) is an orthoflavivirus that causes Japanese encephalitis, a mosquito-borne viral infection that primarily affects humans and animals. JEV is a major cause of encephalitis in many parts of Asia, particularly in rural and agricultural areas. In this study, we used the IFNAR1-/- mice model to investigate alterations in cytokine and apoptotic factor levels in IFNAR1-/- mice upon JEV infection. A 5-week-adult female C57BL/6 IFN-α/ß receptor knockout (IFNAR1-/-) transgenic mice were intramuscularly inoculated with several viral titers and monitored within 10 dpi. The weight changes and survival rates were evaluated during the study period. Gene expression analysis was performed using RT-qPCR, targeting genes related to specific cytokines and apoptotic factors, to identify the inflammatory factors fluctuations associated with JEV strain KBPV-VR-27 infection in IFNAR1-/- mice. The expression of cytokine genes was enhanced in IFNAR1-/- mice infected with JEV KBPV-VR-27. Notably, a significant induction of cytokines, such as IL-13, IL-17α, IFN-ß, and IFN-γ, was observed in the brain, while upregulation of IL-6, IFN-ß, and IFN-γ was exhibited in the lung. In addition, among the targeted apoptotic factors, only significant induction of Bak was observed in the brain. We also found that the spleen exhibited a higher viral load compared to the brain and lungs. In conclusion, the findings of this study shed light on the varying viral loads across targeted organs, with the brain exhibiting a lower viral load but pronounced expression of targeted pro-inflammatory cytokines in IFNAR1-/- mice.

Bivalent Hemagglutinin Cleavage-Site Peptide Vaccines Protect Chickens from Lethal Infections with Highly Pathogenic H5N1 and H5N6 Avian Influenza Viruses.

BACKGROUND: Outbreaks of highly pathogenic avian influenza viruses cause huge economic losses to the poultry industry worldwide. Vaccines that can protect chickens from infections caused by various variants of highly pathogenic H5Nx avian influenza viruses are needed owing to the continuous emergence of new variants. We previously showed that vaccines containing the H5 cleavage-site peptide from clade 2.3.4.4. H5N6 avian influenza virus protects chickens from infection with homologous clade 2.3.4.4. H5N6 avian influenza virus, but not from infection with the heterologous clade 1 H5N1 avian influenza virus. Therefore, we developed bivalent peptide vaccines containing H5 cleavage sites of viruses from both clades to protect chickens from both H5N1 and H5N6 avian influenza viruses. METHODS: Chickens were vaccinated with two doses of a combined peptide vaccine containing cleavage-site peptides from clade 1 and clade 2.3.4.4. highly pathogenic H5N1 and H5N6 avian influenza viruses and then challenged with both viruses. The infected chickens were monitored for survival and their tracheae and cloacae were sampled to check for viral shedding based on the median tissue culture infectious dose of 50 (log10TCID50/mL) in Madin-Darby canine kidney cells. RESULTS: Antibody production was induced at similar levels in the sera of chickens immunized with two doses of the combined peptide vaccines containing cleavage-site peptides from highly pathogenic H5N1 and H5N6 avian influenza viruses. The immunized chickens were protected from infection with both H5N1 and H5N6 avian influenza viruses without viral shedding in the tracheae and cloacae. CONCLUSIONS: Dual-peptide vaccines containing cleavage-site peptides of both clades can protect chickens from highly pathogenic avian influenza virus infections.

Differential Protection of Chickens against Highly Pathogenic H5 Avian Influenza Virus Using Polybasic Amino Acids with H5 Cleavage Peptide.

BACKGROUND: Highly pathogenic H5Nx viruses cause avian influenza, a zoonotic disease that can infect humans. The vaccine can facilitate the prevention of human infections from infected poultry. Our previous study showed that an H5 cleavage-site peptide vaccine containing the polybasic amino acid RRRK could protect chickens from lethal infections of the highly pathogenic H5N6 avian influenza virus. METHODS: Chickens immunized with the various polybasic amino combinations (RRRK, RRR, RR, R, RK, and K) of H5 cleavage-site peptides were challenged with highly pathogenic H5N6 avian influenza viruses. The challenged chickens were monitored for survival rate, and viral titers in swabs and tissue samples were measured in Madin-Darby canine kidney (MDCK) cells using the median tissue culture infectious dose 50 (log10 TCID50/mL). RESULTS: Most H5 cleavage-site vaccines containing various combinations of polybasic amino acids protected chickens from lethal infection. Chickens immunized with the RK-containing peptide combination of the H5 cleavage site were not protected. CONCLUSIONS: The polybasic amino acids (RRRK) of H5 cleavage cleavage-site peptide vaccines are important for protecting chickens against HP H5N6 avian influenza virus. The H5 cleavage cleavage-site peptide containing RK did not protect chickens against the virus.

Comparison of the Pathogenicity of SARS-CoV-2 Delta and Omicron Variants by Analyzing the Expression Patterns of Immune Response Genes in K18-hACE2 Transgenic Mice.

BACKGROUND: The recently emerged variants of the severe acute respiratory coronavirus 2 (SARS-CoV-2) pose a threat to public health. Understanding the pathogenicity of these variants is a salient factor in the development of effective SARS-CoV-2 therapeutics. This study aimed to compare the expression patterns of genes involved in immune responses in K18-hACE2 mice infected with the wild-type, Delta, and Omicron SARS-CoV-2 variants. METHODS: K18-hACE2 mice were intranasally infected with either wild-type (B.1), Delta (B.1.617.2), or Omicron (B.1.1.529) variants. On day 6 post-infection, lung, brain, and kidney tissues were collected from each variant-infected group. The mRNA expression levels of 39 immune response genes in all three groups were compared by RT-qPCR. Viral titers were measured using the median tissue culture infectious dose (TCID50) assay and expressed as Log10 TCID50/0.1 g. The statistical significance of the differences in gene expression was determined by one-way analysis of variance (ANOVA) (alpha = 0.05). RESULTS: The expression of toll-like receptors (TLRs) was upregulated in the lung and brain tissues of the wild-type- and Delta-infected groups but not in those of the Omicron-infected group. The highest expression of cytokines, including interleukin (IL)-1α, IL-1ß, IL-17α, interferon, and tumor necrosis factors, was observed in the lungs of mice infected with the wild-type variant. Additionally, CCL4, CCL11, CXCL9, and CXCL10 were upregulated (>3-fold) in wild-type-infected mice, with markedly higher expressions in the brain than in the lungs. Most of the apoptotic factors were mainly expressed in the brain tissues of Omicron-infected mice (caspase 8, caspase 9, p53, Bax, Bak, BCL-2, and Bcl-XL), whereas neither the lung nor kidney showed more than 3-fold upregulation of these apoptotic factors. CONCLUSIONS: Collectively, our findings revealed that the wild-type SARS-CoV-2 variant exhibited the highest pathogenicity, followed by the Delta variant, then the Omicron variant.

Evaluation of Efficacy of Oil Adjuvanted H5N6 Inactivated Vaccine against Highly Pathogenic H5N6 and H5N1 Influenza Viruses Infected Chickens.

BACKGROUND: Over the last 20 years, circulating highly pathogenic (HP) Asian H5 subtype avian influenza viruses have caused global pandemics in poultry and sporadic infections in humans. Vaccines are a desirable solution to prevent viral infections in poultry and reduce transmission to humans. Herein, we investigated the efficacy of an oil-adjuvanted inactivated H5N6 vaccine against highly pathogenic H5N6 and H5N1 influenza virus infections in chickens. METHODS: The polybasic amino acid cleavage site depleted HA gene and NA gene of A/Waterfowl/Korea/S57/2016 (clade 2.3.4.4) (H5N6) was assembled with the rest of the A/PR/8/34 (H1N1) genes to construct the vaccine virus. The vaccine virus was propagated in fertilized eggs, partially purified using a tangential flow filtration (TFF) system, and inactivated using formalin. The chickens were intramuscularly immunized with 384 HA, 192HA, and 96HA units of oil-adjuvanted inactivated H5N6 vaccine. Antibody titer, survival rate, and lung pathology were evaluated against the homologous H5N6: A/waterfowl/Korea/S57/2016 (clade 2.3.4.4) and heterologous H5N1: A/Hong Kong/213/2003 (clade 1) viruses 12 and 4 weeks post-vaccination (p.v.), respectively. Data were statistically analyzed using the Mann-Whitney U test. RESULTS: The 384HA (n = 10) and 192HA (n = 5) antigen-immunized chickens showed 100% survival after lethal infections with homologous H5N6, and no virus shedding was observed from tracheal and cloacal routes. All chickens that received the 384HA vaccine survived the challenge of heterologous H5N1 after 4 weeks of immunization. The chickens that received the 384HA vaccine showed mean HI titers of 60 and 240 after 12 and 4 weeks of vaccination, respectively, against HP H5N6, whereas a mean HI titer of 80 was observed in sera collected 4 weeks after vaccination against HP H5N1. CONCLUSIONS: Our findings indicate that one dose of 384HA oil-adjuvanted inactivated H5N6 vaccine can induce a long-lasting immune response against both homologous H5N6 and heterologous H5N1 infections in chickens.

Ginseng Protects ACE2-Transgenic Mice from SARS-CoV-2 Infection.

BACKGROUND: The pandemic caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is ongoing, and despite massive vaccination campaigns, individuals continue to be infected with new SARS-CoV-2 variants. We studied the effects of ginseng, an immune-enhancing agent, on conferring immunity against SARS-CoV-2 in transgenic mice expressing the SARS-CoV-2 human angiotensin-converting enzyme 2 (ACE2) receptor. METHODS: Human ACE2-transgenic (ACE2-tg) mice were fed ginseng extract for 180 days before they were intranasally infected with SARS-CoV-2. The mortality and morbidity were monitored for 10 days. The amount of antiviral interferon in the lung tissues was measured using enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: Thirty percent of the mice fed ginseng extract prior to infection survived, whereas all those that were not fed ginseng extract prior to infection died. Viral titers in the lungs were significantly lower in mice fed ginseng extract than in those not fed ginseng extract. The induction of antiviral interferon-gamma (IFN-γ) was significantly higher in the lungs of mice fed ginseng extract than in those that were not. CONCLUSIONS: Our data indicate that a ginseng-containing diet may enhance immunity against SARS-CoV-2 in a mouse model.

H5 cleavage-site peptide vaccine protects chickens from lethal infection by highly pathogenic H5 avian influenza viruses.

Highly pathogenic H5Nx avian influenza viruses constantly threaten the poultry industry and humans and have pandemic potential. These viruses continuously evolve, requiring a universal vaccine to protect chickens from members of diverse clades. The purpose of this study was to develop an H5 cleavage-site peptide vaccine containing polybasic amino acids (RRRK) to completely protect chickens from H5N6, H5N8, and H5N1 avian influenza viruses. Chickens were immunized with various doses of a keyhole limpet hemocyanin (KLH)-conjugated H5 cleavage-site peptide vaccine containing RRRK. The effect of RRRK was evaluated by comparing the survival rates of chickens immunized with vaccines either containing or lacking RRRK. The ability of the RRRK-containing vaccine to confer long-term protective immunity was also assessed. We found that protection was dependent on the number of antigens in the vaccine containing RRRK. Chickens immunized intramuscularly with two doses of 5 µg of the vaccine containing RRRK were completely protected, but those immunized with fewer than two doses of 3 or 1 µg were not protected. Chickens immunized with the vaccine lacking RRRK were not protected, suggesting the importance of the polybasic amino acids in conferring immunity. Our results suggest that conserved H5 cleavage-site peptides with polybasic amino acids may be a potential universal vaccine to protect chickens from various emerging clades of H5Nx avian influenza viruses.

Development and evaluation of inhalable composite niclosamide-lysozyme particles: A broad-spectrum, patient-adaptable treatment for coronavirus infections and sequalae.

Niclosamide (NIC) has demonstrated promising in vitro antiviral efficacy against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Though NIC is already FDA-approved, administration of the currently available oral formulation results in systemic drug levels that are too low for the inhibition of SARS-CoV-2. We hypothesized that the co-formulation of NIC with an endogenous protein, human lysozyme (hLYS), could enable the direct aerosol delivery of the drug to the respiratory tract as an alternative to oral delivery, thereby effectively treating COVID-19 by targeting the primary site of SARS-CoV-2 acquisition and spread. To test this hypothesis, we engineered and optimized composite particles containing NIC and hLYS suitable for delivery to the upper and lower airways via dry powder inhaler, nebulizer, and nasal spray. The novel formulation demonstrates potent in vitro and in vivo activity against two coronavirus strains, MERS-CoV and SARS-CoV-2, and may offer protection against methicillin-resistance staphylococcus aureus pneumonia and inflammatory lung damage occurring secondary to SARS-CoV-2 infections. The suitability of the formulation for all stages of the disease and low-cost development approach will ensure rapid clinical development and wide-spread utilization.

Cold-Adapted Live Attenuated SARS-Cov-2 Vaccine Completely Protects Human ACE2 Transgenic Mice from SARS-Cov-2 Infection.

A safe and effective vaccine that can provide herd immunity against severe acute respiratory syndrome coronavirus (SARS-CoV-2) is urgently needed to stop the spread of this virus among humans. Many human viral vaccines are live, attenuated forms of viruses that elicit humoral and cellular immunity. Here, we describe a cold-adapted live-attenuated vaccine (SARS-CoV-2/human/Korea/CNUHV03-CA22 °C/2020) developed by gradually adapting the growth of SARS-CoV-2 from 37 °C to 22 °C in Vero cells. This vaccine can be potentially administered to humans as a nasal spray. Its single dose strongly induced neutralising antibodies (titre > 640), cellular immunity, and mucosal IgA antibodies in intranasally immunised K18-hACE2 mice, which are very susceptible to SARS-CoV-2 and SARS-CoV infections. The one-dose vaccinated mice were completely protected from SARS-CoV-2 infection and did not show body weight loss, death, or the presence of virus in tissues, such as the nasal turbinates, brain, lungs, and kidneys. These results demonstrate that the cold-adapted live attenuated SARS-CoV-2 vaccine we have developed may be a candidate SARS-CoV-2 vaccine for humans.

Gene expression pattern differences in primary human pulmonary epithelial cells infected with MERS-CoV or SARS-CoV-2.

Coronaviruses such as MERS-CoV and SARS-CoV-2 infect the human respiratory tract and can cause severe pneumonia. Disease severity and outcomes are different for these two infections: the human mortality rate for MERS-CoV and SARS-CoV-2 is over 30% and less than 10%, respectively. Here, using microarray assay, we analyzed the global alterations in gene expression induced by MERS-CoV or SARS-CoV-2 infections in primary human pulmonary epithelial cells. Overall, the number of differentially expressed genes was higher in human lung cells infected with MERS-CoV than in cells with SARS-CoV-2. Out of 44,556 genes analyzed, 127 and 50 were differentially expressed in cells infected with MERS-CoV and SARS-CoV-2, respectively (> 2-fold increase, compared to uninfected cells). Of these, only eight genes, including the one coding for CXCL8, were similarly modulated (upregulated or downregulated) by the two coronaviruses. Importantly, these results were virus-specific and not conditioned by differences in viral load, and viral growth curves were similar in human lung cells infected with both viruses. Our results suggest that these distinct gene expression profiles, detected early after infection by these two coronaviruses, may help us understand the differences in clinical outcomes of MERS-CoV and SARS-CoV-2 infections.

Age-Dependent Lethality in Ducks Caused by Highly Pathogenic H5N6 Avian Influenza Virus.

Ducks show notably higher resistance to highly pathogenic avian influenza viruses as compared to chickens. Here, we studied the age-dependent susceptibility in ducks to the infections caused by highly pathogenic avian influenza viruses. We intranasally infected ducks aged 1, 2, 4, and 8 weeks with highly pathogenic H5N6 avian influenza viruses isolated in South Korea in 2016. All the 1-and 2-week-old ducks died after infection, 20% of 3-week-old ducks died, and from the ducks aged 4 and 8 weeks, all of them survived. We performed microarray analysis and quantitative real-time PCR using total RNA isolated from the lungs of infected 2- and 4-week-old ducks to determine the mechanism underlying the age-dependent susceptibility to highly pathogenic avian influenza virus. Limited genes were found to be differentially expressed between the lungs of 2- and 4-week-old ducks. Cell damage-related genes, such as CIDEA and ND2, and the immune response-related gene NR4A3 were notably induced in the lungs of infected 2-week-old ducks compared to those in the lungs of infected 4-week-old ducks.

Higher virulence of swine H1N2 influenza viruses containing avian-origin HA and 2009 pandemic PA and NP in pigs and mice.

Pigs are capable of harbouring influenza A viruses of human and avian origin in their respiratory tracts and thus act as an important intermediary host to generate novel influenza viruses with pandemic potential by genetic reassortment between the two viruses. Here, we show that two distinct H1N2 swine influenza viruses contain avian-like or classical swine-like hemagglutinins with polymerase acidic (PA) and nucleoprotein (NP) genes from 2009 pandemic H1N1 influenza viruses that were found to be circulating in Korean pigs in 2018. Swine H1N2 influenza virus containing an avian-like hemagglutinin gene had enhanced pathogenicity, causing severe interstitial pneumonia in infected pigs and mice. The mortality rate of mice infected with swine H1N2 influenza virus containing an avian-like hemagglutinin gene was higher by 100% when compared to that of mice infected with swine H1N2 influenza virus harbouring classical swine-like hemagglutinin. Further, chemokines attracting inflammatory cells were strongly induced in lung tissues of pigs and mice infected by swine H1N2 influenza virus containing an avian-like hemagglutinin gene. In conclusion, it is necessary for the well-being of humans and pigs to closely monitor swine influenza viruses containing avian-like hemagglutinin with PA and NP genes from 2009 pandemic H1N1 influenza viruses.

Histamine contributes to severe pneumonia in pigs infected with 2009 pandemic H1N1 influenza virus.

Histamine is a biogenic amine that influences many immune cells. In this study, we investigated the effect of histamine on the pathogenesis of 2009 pandemic H1N1 influenza virus in pigs. Histamine was not detected in the tracheal tissues of infected pigs, and no difference was found in the pathological damage found in infected pigs with and without treatment with a histamine antagonist. Lung tissues from untreated infected pigs showed severe interstitial pneumonia with accumulation of histamine, in contrast to those from infected pigs that were treated with the histamine antagonist. The expression of inflammatory cytokines was much higher in the lungs of untreated infected pigs than in infected pigs treated with the histamine antagonist. These data suggest that histamine necessary for the development of the severe pneumonia in infected pigs.

Inactivated H5 Antigens of H5N8 Protect Chickens from Lethal Infections by the Highly Pathogenic H5N8 and H5N6 Avian Influenza Viruses.

INTRODUCTION: Highly pathogenic Asian H5-subtype avian influenza viruses have been found in poultry and wild birds worldwide since they were first detected in southern China in 1996. Extensive control efforts have not eradicated them. Vaccination prevents such viruses infecting poultry and reduces the number lost to compulsory slaughter. The study showed the efficacy of inactivated H5 vaccine from the H5N8 virus against highly pathogenic H5N8 and H5N6 avian influenza viruses in chickens. MATERIAL AND METHODS: Reverse genetics constructed an H5 vaccine virus using the HA gene of the 2014 H5N8 avian influenza virus and the rest of the genes from A/PR/8/34 (H1N1). The vaccine viruses were grown in fertilised eggs, partially purified through a sucrose gradient, and inactivated with formalin. Chickens were immunised i.m. with 1 µg of oil-adjuvanted inactivated H5 antigens. RESULTS: Single dose H5 vaccine recipients were completely protected from lethal infections by homologous H5N8 avian influenza virus and shed no virus from the respiratory or intestinal tracts but were not protected from lethal infections by heterologous H5N6. When chickens were immunised with two doses and challenged with homologous H5N8 or heterologous H5N6, all survived and shed no virus. CONCLUSION: Our results indicate that two-dose immunisations of chickens with H5 antigens with oil adjuvant are needed to provide broad protection against different highly pathogenic H5 avian influenza viruses.

Genetic and pathogenic analysis of a novel reassortant H5N6 influenza virus isolated from waterfowl in South Korea in 2016.

A novel reassortant highly pathogenic H5N6 influenza virus was isolated from waterfowl in South Korea in 2016. Seven genes of this virus originated from an H5N6 virus from China, whereas the remaining gene, PB1, was from an unknown virus. This virus productively infected pigs, which showed viral shedding through their noses and developed severe interstitial pneumonia.

Genetic analysis of a novel reassortant H11N9 Isolated from waterfowl in South Korea in 2016.

Aquatic birds are known to harbor all the known influenza A viruses. In the winter of January 2016, we surveyed influenza A virus in the feces of migratory birds in South Korea. The novel re-assorted H11N9 avian influenza virus, which contains genes from avian influenza viruses of poultry and wild birds, was isolated. The polymerase basic 2 (PB2), polymerase basic 1 (PB1), hemagglutinin (HA), and nucleoprotein (NP) genes were most closely related to those of domestic duck-origin avian influenza viruses, while the non-structural (NS) gene was closely related to that of domestic goose-origin avian influenza virus. The polymerase acidic (PA), neuraminidase (NA), and matrix (M) genes were most similar to those of wild bird-origin avian influenza viruses. Our results suggested that the interaction between wild birds and domestic poultry could possibly create novel re-assorted avian influenza viruses circulating in wild birds.

Porcine mast cells infected with H1N1 influenza virus release histamine and inflammatory cytokines and chemokines.

Mast cells reside in many tissues, including the lungs, and might play a role in enhancing influenza virus infections in animals. In this study, we cultured porcine mast cells from porcine bone marrow cells with IL-3 and stem cell factor to study the infectivity and activation of the 2009 pandemic H1N1 influenza virus of swine origin. Porcine mast cells were infected with H1N1 influenza virus, without the subsequent production of infectious viruses but were activated, as indicated by the release of histamines. Inflammatory cytokine- and chemokine-encoding genes, including IL-1α, IL-6, CXCL9, CXCL10, and CXCL11, were upregulated in the infected porcine mast cells. Our results suggest that mast cells could be involved in enhancing influenza-virus-mediated disease in infected animals.

Inactivated Antigen of the H7N9 Influenza Virus Protects Mice from Its Lethal Infection.

The H7N9 influenza virus emerged in February 2013 in China, and underlies over 20% of human mortality in the country. Many efforts are being made to develop an effective vaccine against this highly pathogenic virus. We made H7N9 vaccine virus with six internal genes of A/PR/8/34 (H1N1) and two surface genes of hemagglutinin and neuraminidase from A/Anhui/1/2013 (H7N9) by reverse genetics, and the H7N9 vaccine antigens were produced in eggs. Protective antibodies were induced in mice immunized with a single dose (7.5 µg) of the H7N9 antigen. These mice survived lethal infection by the H7N9 virus, although few viruses were found in their lung tissues. However, mice administered with two doses of the H7N9 antigen survived without any viral antigen being detected in their lung tissues. Furthermore, the IgG antibody subtypes were also pronounced in lung tissues of the immunized mice. Therefore, our results suggest that the inactivated whole antigen of the H7N9 influenza virus might protect animals and humans from its lethal infection.

Isolation of a novel H3N2 influenza virus containing a gene of H9N2 avian influenza in a dog in South Korea in 2015.

We isolated a serotype H3N2 influenza virus from a dog with severe respiratory distress in an animal clinic in South Korea in 2015 and characterized the sequences of its eight genes. The following seven genes were derived from canine influenza virus: PB2, PB1, HA, NP, NA, M, and NS. However, the PA gene was derived from avian H9N2 influenza virus that is circulating in poultry in Korea. These findings suggest that the continued surveillance of the influenza virus in dogs is warranted because humans have close contact with dogs, which may promote viral transmission.