Search Results (35)

Background: Respiratory viruses spread through airborne droplets and aerosols, causing highly contagious acute respiratory syndromes in humans. This study evaluated the antiviral potential of vapours of catmint-oil-based formulations against respiratory viruses. Methods: The antiviral activity of formulations with or without catmint oil (CO) in solution or in aerosolised form was determined against influenza virus H1N1 ATCC VR-1469 and mouse hepatitis virus (MHV-1) ATCC/VR261. In solution, both viruses were exposed to CO formulations for 2–3 h. In aerosolised form, H1N1 was exposed to formulations for 2 min in a closed cylinder and MHV-1 for 10 min in a booth. The antiviral effect of the formulations was evaluated by growing H1N1 in a Madin–Darby canine kidney (MDCK; ATCC-CRL-2936) and MHV-1 in A9 ATCC/CCL 1.4 cells using TCID50 and a plaque assay, respectively. Transmission electron microscopy (TEM) was conducted to investigate the mode of action of the formulations. Results: In solution, the formulation containing hydrogenated CO (HCO), bromelain, N-acetylcysteine and Tween 20 (Formulation (1)) reduced the viability of H1N1 by 2.6 ± 0.07 log₁₀ (p = 0.025) and MHV-1 by 4.5 ± 0.14 log₁₀ (p = 0.014) within 2–3 h. In vapourised form, Formulation (1) produced similar antiviral effects against H1N1, reducing it by 3.00 ± 0.07 log₁₀ (p = 0.002) within 2 min, and Formulation (1) produced a 3.00 ± 0.07 log₁₀ reduction of MHV-1 (p < 0.001) within 10 min (the minimum time needed to detect infective viral particles in the experimental set-ups). Formulation (3) (without bromelain) reduced H1N1 by 1.57 ± 0.14 log₁₀ (p = 0.008) after 2 min and MHV-1 by 1.3 ± 0.04 log₁₀ (p = 0.057) after 10 min. In the absence of catmint oil (Formulation (4)) or in the absence of catmint oil and bromelain (Formulation (5)), there were only slight reductions in the viability of aerosolised H1N1 (1.00 ± 0.14 log₁₀, p = 0.046; <1 log₁₀, p = 0.966, respectively) and MHV-1 (1.07 ± 0.02 log₁₀, p = 0.013; 0.16 ± 0.03 log₁₀, p = 0.910, respectively). The TEM analysis showed that the formulation disrupted the H1N1 envelopes and caused a reduction in size of the viral particles. Conclusions: The catmint-oil-based formulations reduced the H1N1 and MHV-1 by disrupting the vial envelopes. Full article

Madin–Darby Canine Kidney (MDCK) cells are a key cell line for influenza vaccine production, due to their high viral yield and low mutation resistance. In our laboratory, we established a tertiary cell bank (called M60) using a standard MDCK cell line imported from American Type Culture Collection (ATCC) in the USA. Due to their controversial tumourigenicity, we domesticated non-tumourigenic MDCK cells (named CL23) for influenza vaccine production via monoclonal screening in the early stage of this study, and the screened CL23 cells were characterised based on their low proliferative capacity, which had certain limitations in terms of expanding their production during cell resuscitation. It was thus our objective to enhance the proliferation efficiency of MDCK cells for influenza vaccine production following cell resuscitation, with a view to improving the production of non-tumourigenic MDCK cells for vaccines and enhancing the production of influenza virus lysate vaccines from MDCK cells through genetic intervention. We concentrated on the protein thrombosponin-1 (THBS1), which was markedly differentiated in the proteomics data of the two MDCK cells. By integrating this finding with related studies, we were able to ascertain that THBS1 exerts a significant influence on the level of cell proliferation and apoptosis. Consequently, our objective was to investigate the impact of THBS1 expression on MDCK cell apoptosis by verifying the differences in THBS1 expression between the two MDCK cells and by interfering with THBS1 expression in the MDCK cells. We found that the knockdown of THBS1 significantly increased the proliferation and apoptosis of CL23 cells without causing significant changes in cell migration and invasion, and its overexpression significantly decreased the proliferation of M60 cells and increased cell migration, invasion, and apoptosis. In addition, the TGF-β/Smad pathway target genes transforming growth factor-β1 (TGF-β1), mothers against decapentaplegic homolog 2 (Smad2), and mothers against decapentaplegic homolog 3 (Smad3), were significantly down-regulated in CL23 cells after THBS1 knockdown and up-regulated in M60 cells after overexpression, with consistent expression identified at both the mRNA and protein levels. The treatment of cells with TGF-β activators and inhibitors further demonstrated that THBS1 regulated MDCK cell proliferation and apoptosis through the TGF-β/Smad signalling pathway. Finally, we found that THBS1 also regulated H1N1 influenza virus replication. These findings enable a comprehensive understanding of the regulatory mechanisms of THBS1 regarding MDCK cell proliferation and apoptosis functions and the effects of influenza virus replication. Full article

Canine influenza virus (CIV) H3N2 causes a highly contagious respiratory disease in dogs and has been the source of outbreaks across North America since 2015. An injectable RNA Particle (RP)-CIV H3N2 vaccine has been developed to protect dogs against this disease. To demonstrate efficacy, dogs were randomized into two treatment groups, then vaccinated subcutaneously twice, 21 days apart, with a placebo vaccine (n = 20) or an RP-CIV H3N2 vaccine (n = 20). Three weeks later, dogs were challenged intranasally with virulent CIV H3N2 and observed daily for 10 days for clinical signs of disease. Nasal swabs were also collected daily to evaluate the shedding of the challenge virus. Ten days post-challenge, the dogs were euthanized, and the lungs were examined for consolidation. RP-CIV H3N2 vaccination demonstrated a significant reduction in the duration of clinical signs, duration and amount of virus shed, lung consolidation, and the incidence of suppurative pneumonia. To evaluate safety, dogs from multiple geographic regions were vaccinated subcutaneously, 3–4 weeks apart, with an RP-CIV H3N2 vaccine and observed for adverse events for 14 days after each administration. The RP-CIV H3N2 vaccine was deemed safe, with lethargy being the most reported adverse event at a rate of 1.6%. Full article

The influenza A virus poses a serious threat to human health and is an important global public health issue. The drugs currently used for treatment are becoming increasingly ineffective against influenza A viruses and require the development of new antiviral drugs. Angelica tenuissima Nakai (ATN), a traditional herbal medicine belonging to the Umbelliferae family, exhibits a broad range of pharmacological activities, including inflammation, headache, and cold symptoms. In the present study, based on target protein identification, functional enrichment analysis, and gene set comparisons, we first suggested that ATN has potential therapeutic effects against influenza A virus infection. Next, methylthiazol tetrazolium (MTT) and sulforhodamine B colorimetric (SRB) assay results revealed that ATN exhibited low cytotoxicity in Madin–Darby canine kidney (MDCK) cells. The antiviral properties of ATN were observed against H1N1 and H3N2 virus strains. Microscopy confirmed the increased survival rate of the host cells. Further time-of-addition experiments revealed that the addition of ATN before virus adsorption showed similar results to the whole period of treatment. The pre- and co-treated groups showed lower levels of viral RNA (M1 protein). The results of this study suggest that ATN exhibits antiviral properties against the influenza A virus. These therapeutic properties of ATN can serve as a theoretical basis for further research on the applicability of ATN in the development of antiviral agents. Full article

Influenza vaccines, which are recommended by the World Health Organization (WHO), are the most effective preventive measure against influenza virus infection. Madin–Darby canine kidney (MDCK) cell culture is an emerging technology used to produce influenza vaccines. One challenge when purifying influenza vaccines using this cell culture system is to efficiently remove impurities, especially host cell double-stranded DNA (dsDNA) and host cell proteins (HCPs), for safety assurance. In this study, we optimized ion-exchange chromatography methods to harvest influenza viruses from an MDCK cell culture broth, the first step in influenza vaccine purification. Bind/elute was chosen as the mode of operation for simplicity. The anion-exchange Q chromatography method was able to efficiently remove dsDNA and HCPs, but the recovery rate for influenza viruses was low. However, the cation-exchange SP process was able to simultaneously achieve high dsDNA and HCP removal and high influenza virus recovery. For the SP process to work, the clarified cell culture broth needed to be diluted to reduce its ionic strength, and the optimal dilution rate was determined to be 1:2 with purified water. The SP process yielded a virus recovery rate exceeding 90%, as measured using a hemagglutination units (HAUs) assay, with removal efficiencies over 97% for HCPs and over 99% for dsDNA. Furthermore, the general applicability of the SP chromatography method was demonstrated with seven strains of influenza viruses recommended for seasonal influenza vaccine production, including H1N1, H3N2, B (Victoria), and B (Yamagata) strains, indicating that the SP process could be utilized as a platform process. The SP process developed in this study showed four advantages: (1) simple operation, (2) a high recovery rate for influenza viruses, (3) a high removal rate for major impurities, and (4) general applicability. Full article

Influenza is a potentially fatal acute respiratory viral disease caused by the influenza virus. Influenza viruses vary in antigenicity and spread rapidly, resulting in seasonal epidemics. Vaccination is the most effective strategy for lowering the incidence and fatality rates of influenza-related disorders, and it is also an important method for reducing seasonal influenza infections. Mammalian Madin–Darby canine kidney (MDCK) cell lines are recommended for influenza virus growth, and such cell lines have been utilized in several commercial influenza vaccine productions. The limit dilution approach was used to screen ATCC-MDCK cell line subcellular strains that are especially sensitive to H1N1, H3N2, BV, and BY influenza viruses to increase virus production, and research on influenza virus culture media was performed to support influenza virus vaccine development. We also used RNA sequencing to identify differentially expressed genes and a GSEA analysis to determine the biological mechanisms underlying the various levels of susceptibility of cells to influenza viruses. MDCK cell subline 2B6 can be cultured to increase titer and the production of the H1N1, H3N2, BV, and BY influenza viruses. MDCK-2B6 has a significantly enriched and activated in ECM receptor interaction, JAK-STAT signaling, and cytokine receptor interaction signaling pathways, which may result in increased cellular susceptibility and cell proliferation activity to influenza viruses, promote viral adsorption and replication, and elevate viral production, ultimately. The study revealed that MDCK-2B6 can increase the influenza virus titer and yield in vaccine production by increasing cell sensitivity and enhancing proliferative activity. Full article

Influenza antiviral drugs are important tools in our fight against both annual influenza epidemics and pandemics. Polyphenols are a group of compounds found in plants, some of which have demonstrated promising antiviral activity. Previous in vitro and mouse studies have outlined the anti-influenza virus effectiveness of the polyphenol epigallocatechin-3-gallate (EGCG); however, no study has utilised the ferret model, which is considered the gold-standard for influenza antiviral studies. This study aimed to explore the antiviral efficacy of EGCG in vitro and in ferrets. We first performed studies in Madin-Darby Canine Kidney (MDCK) and human lung carcinoma (Calu-3) cells, which demonstrated antiviral activity. In MDCK cells, we observed a selective index (SI, CC50/IC50) of 77 (290 µM/3.8 µM) and 96 (290 µM/3.0 µM) against A/California/07/2009 and A/Victoria/2570/2019 (H1N1)pdm09 influenza virus, respectively. Calu-3 cells demonstrated a SI of 16 (420 µM/26 µM) and 18 (420 µM/24 µM). Ferrets infected with A/California/07/2009 influenza virus and treated with EGCG (500 mg/kg/day for 4 days) had no change in respiratory tissue viral titres, in contrast to oseltamivir treatment, which significantly reduced viral load in the lungs of treated animals. Therefore, we demonstrated that although EGCG showed antiviral activity in vitro against influenza viruses, the drug failed to impair viral replication in the respiratory tract of ferrets. Full article

A series of 2,4-disubstituted pyrrolo[2,1-f][1,2,4]triazines containing both aryl and thienyl substituents were synthesized by exploiting the 1,3-cycloaddition reaction of N(1)-ethyl-1,2,4-triazinium tetrafluoroborates with dimethyl acetylenedicarboxylate. The antiviral activity of the synthesized compounds against influenza virus strain A/Puerto Rico/8/34 (H1N1) was studied in experiments on Madin-Darby canine kidney (MDCK) cell culture. Among the pyrrolo[2,1-f][1,2,4]triazine derivatives, compounds with low toxicity and high antiviral activity were identified. Dimethyl 4-(4-methoxyphenyl)-7-methyl-2-p-tolylpyrrolo[2,1-f][1,2,4]triazine-5,6-dicarboxylate was found to demonstrate the best antiviral activity (IC₅₀ 4 µg/mL and selectivity index 188). Based on the results of in vitro tests and molecular docking studies performed, a plausible mechanism of action for these compounds was suggested to involve inhibition of neuraminidase. Full article

Influenza is a highly contagious respiratory illness that commonly causes outbreaks among human communities. Details about the exact nature of the droplets produced by human respiratory activities such as breathing, and their potential to carry and transmit influenza A and B viruses is still not fully understood. The objective of our study was to characterize and quantify influenza viral shedding in exhaled aerosols from natural patient breath, and to determine their viral infectivity among participants in a university cohort in tropical Singapore. Using the Gesundheit-II exhaled breath sampling apparatus, samples of exhaled breath of two aerosol size fractions (“coarse” > 5 µm and “fine” ≤ 5 µm) were collected and analyzed from 31 study participants, i.e., 24 with influenza A (including H1N1 and H3N2 subtypes) and 7 with influenza B (including Victoria and Yamagata lineages). Influenza viral copy number was quantified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Infectivity of influenza virus in the fine particle fraction was determined by culturing in Madin–Darby canine kidney cells. Exhaled influenza virus RNA generation rates ranged from 9 to 1.67 × 10⁵ and 10 to 1.24 × 10⁴ influenza virus RNA copies per minute for the fine and coarse aerosol fractions, respectively. Compared to the coarse aerosol fractions, influenza A and B viruses were detected more frequently in the fine aerosol fractions that harbored 12-fold higher viral loads. Culturable virus was recovered from the fine aerosol fractions from 9 of the 31 subjects (29%). These findings constitute additional evidence to reiterate the important role of fine aerosols in influenza transmission and provide a baseline range of influenza virus RNA generation rates. Full article

Canine infectious respiratory disease complex (CIRDC) is the primary cause of respiratory disease in the canine population and is caused by a wide array of viruses and bacterial pathogens with coinfections being common. Since its recognition in late 2019, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been reported to cause respiratory disease in dogs. Therefore, the rapid detection and differentiation of SARS-CoV-2 from other common viral and bacterial agents is critical from a public health standpoint. Here, we developed and validated a panel of four one-step multiplex qPCR/RT-qPCR assays for the detection and identification of twelve pathogens associated with CIRDC (canine adenovirus-2, canine distemper virus, canine herpesvirus-1, canine influenza A virus, canine parainfluenza virus, canine pneumovirus, canine respiratory coronavirus, SARS-CoV-2, Bordetella bronchiseptica, Streptococcus equi subsp. zooepidemicus, Mycoplasma cynos, and M. canis), as well as the identification of three main CIV subtypes (i.e., H3N2, H3N8, and H1N1). All developed assays demonstrated high specificity and analytical sensitivity. This panel was used to test clinical specimens (n = 76) from CIRDC-suspected dogs. M. canis, M. cynos, and CRCoV were the most frequently identified pathogens (30.3%, 25.0%, and 19.7% of samples, respectively). The newly emerging pathogens CPnV and SARS-CoV-2 were detected in 5.3% of samples and coinfections were identified in 30.3%. This new multiplex qPCR/RT-qPCR panel is the most comprehensive panel developed thus far for identifying CIRDC pathogens, along with SARS-CoV-2. Full article

Chitosan, poly[β-(1–4)-linked-2-amino-2-deoxy-d-glucose], is a polysaccharide with broad applications in various biomaterial and biomedical fields, primarily because of its nontoxicity. Because the biodegradable biopolymer chitosan can be catalyzed by the alkaline partial deacetylation of chitin in shrimp and crab shells, its degree of deacetylation is crucial for proper application. However, the antiviral properties of deacetylated chitosan in combination with other compounds remain unclear. We used a plaque assay to examine the antiviral activity of deacetylated chitosan against human influenza A virus (H1N1) and parainfluenza virus type 3 (HPIV3) when supplemented with grapefruit seed extract (GSE). We demonstrated that deacetylated chitosan reduced H1N1- and HPIV3-induced plaque formation in Madin–Darby canine kidney (MDCK) cells. Chitosan also decreased viral RNA expression in H1N1- and HPIV3-infected A549 cells. Furthermore, antiviral activity analysis in human A549 cells revealed that deacetylated chitosan prevented virus-induced p38 MAPK activation and mitochondrial fragmentation. These findings indicate that deacetylated chitosan with GSE may be a promising antiviral biomaterial against influenza and parainfluenza viruses. Full article

The innate immune pathway serves as the first line of defense against viral infections and plays a crucial role in the host’s immune response in clearing viruses. Prior research has indicated that the influenza A virus has developed various strategies to avoid host immune responses. Nevertheless, the role of the NS1 protein of the canine influenza virus (CIV) in the innate immune pathway remains unclear. In this study, eukaryotic plasmids of NS1, NP, PA, PB1, and PB2 were constructed, and it was found that these proteins interact with melanoma differentiation-associated gene 5 (MDA5) and antagonize the activation of IFN-β promoters by MDA5. We selected the NS1 protein for further study and found that NS1 does not affect the interaction between the viral ribonucleoprotein (RNP) subunit and MDA5, but that it downregulates the expression of the laboratory of genetics and physiology 2 (LGP2) and retinoic acid-inducible gene-I (RIG-I) receptors in the RIG-I pathway. Additionally, NS1 was found to inhibit the expression of several antiviral proteins and cytokines, including MX dynamin like GTPase 1 (MX1), 2′-5′oligoadenylate synthetase (OAS), Signal Transducers and Activators of Transcription (STAT1), tripartite motif 25 (TRIM25), interleukin-2 (IL-2), IFN, IL-8, and IL-1β. To further investigate the role of NS1, a recombinant H3N2 virus strain (rH3N2) and an NS1-null virus (rH3N2ΔNS1) were rescued using reverse-genetic technology. The rH3N2ΔNS1 virus exhibited lower viral titers compared to rH3N2, but had a stronger activation effect on the receptors LGP2 and RIG-I. Furthermore, when compared to rH3N2, rH3N2ΔNS1 exhibited a more pronounced activation of antiviral proteins such as MX1, OAS, STAT1, and TRIM25, as well as antiviral cytokines such as IL-6, IFN-β, and IL-1β. These findings suggest a new mechanism by which NS1, a nonstructural protein of CIV, facilitates innate immune signaling and provides new avenues for the development of antiviral strategies. Full article

Canine influenza virus (CIV) significantly threatens the canine population and public health. Tetherin, an innate immune factor, plays an important role in the defense against pathogen invasion and has been discovered to restrict the release of various enveloped viruses. Two isoforms of canine tetherin (tetherin-X1 and tetherin-X2) were identified in peripheral blood leukocytes of mixed-breed dogs using reverse transcription polymerase chain reaction (RT–PCR). Amino acid alignment revealed that relative to full-length tetherin (tetherin-X1) and truncated canine tetherin (tetherin-X2) exhibited deletion of 34 amino acids. The deletion occurred at the C-terminus of the coiled-coiled ectodomain and the N-terminus of the glycosylphosphatidylinositol (GPI)-anchor domain. Tetherin-X2 was localized subcellularly at the cell membrane, which was consistent with the localization of tetherin-X1. In addition, canine tetherin-X1 and tetherin-X2 restricted the release of H3N2 CIV. However, canine tetherin-X1 had higher antiviral activity than canine tetherin-X2, indicating that the C-terminus of the coiled-coiled ectodomain and the N-terminus of the GPI-anchor domain of canine tetherin (containing the amino acids deleted in tetherin-X2) are critical for its ability to restrict H3N2 CIV release. This study provides insights for understanding the key functional domains of tetherin that restrict CIV release. Full article

Human infections with avian-origin H7N9 influenza A viruses were first reported in China, and an approximately 38% human mortality rate was described across six waves from February 2013 to September 2018. Vaccination is one of the most cost-effective ways to reduce morbidity and mortality during influenza epidemics and pandemics. Egg-based platforms for the production of influenza vaccines are labor-intensive and unable to meet the surging demand during pandemics. Therefore, cell culture-based technology is becoming the alternative strategy for producing influenza vaccines. The current influenza H7N9 vaccine virus (NIBRG-268), a reassortant virus from A/Anhui/1/2013 (H7N9) and egg-adapted A/PR/8/34 (H1N1) viruses, could grow efficiently in embryonated eggs but not mammalian cells. Moreover, a freezing-dry formulation of influenza H7N9 vaccines with long-term stability will be desirable for pandemic preparedness, as the occurrence of influenza H7N9 pandemics is not predictable. In this study, we adapted a serum-free anchorage-independent suspension Madin-Darby Canine Kidney (MDCK) cell line for producing influenza H7N9 vaccines and compared the biochemical characteristics and immunogenicity of three influenza H7N9 vaccine antigens produced using the suspension MDCK cell-based platform without freeze-drying (S-WO-H7N9), the suspension MDCK cell-based platform with freeze-drying (S-W-H7N9) or the egg-based platform with freeze-drying (E-W-H7N9). We demonstrated these three vaccine antigens have comparable biochemical characteristics. In addition, these three vaccine antigens induced robust and comparable neutralizing antibody (NT; geometric mean between 1016 and 4064) and hemagglutinin-inhibition antibody (HI; geometric mean between 640 and 1613) titers in mice. In conclusion, the serum-free suspension MDCK cell-derived freeze-dried influenza H7N9 vaccine is highly immunogenic in mice, and clinical development is warranted. Full article

Betulinic acid (BA) and its derivatives exhibit a variety of biological activities, especially their anti-HIV-1 activity, but generally have only modest inhibitory potency against influenza virus. The entry of influenza virus into host cells can be competitively inhibited by multivalent derivatives targeting hemagglutinin. In this study, a series of hexa-, hepta- and octavalent BA derivatives based on α-, β- and γ-cyclodextrin scaffolds, respectively, with varying lengths of flexible oligo(ethylene glycol) linkers was designed and synthesized using a microwave-assisted copper-catalyzed 1,3-dipolar cycloaddition reaction. The generated BA-cyclodextrin conjugates were tested for their in vitro activity against influenza A/WSN/33 (H1N1) virus and cytotoxicity. Among the tested compounds, 58, 80 and 82 showed slight cytotoxicity to Madin-Darby canine kidney cells with viabilities ranging from 64 to 68% at a high concentration of 100 μM. Four conjugates 51 and 69–71 showed significant inhibitory effects on influenza infection with half maximal inhibitory concentration values of 5.20, 9.82, 7.48 and 7.59 μM, respectively. The structure-activity relationships of multivalent BA-cyclodextrin conjugates were discussed, highlighting that multivalent BA derivatives may be potential antiviral agents against influenza infection. Full article