Coronavirus M Protein Trafficking in Epithelial Cells Utilizes a Myosin Vb Splice Variant and Rab10.

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two-hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV-CoV) M protein with Myosin Vb (MYO5B), specifically with the alternative splice variant of cellular MYO5B including exon D (MYO5B+D), which mediates interaction with Rab10. When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with the MHV-CoV M protein, as well as with the M proteins from Porcine Epidemic Diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2). Co-expressed M proteins and MYO5B+D co-localized with endogenous Rab10 and Rab11a. We identified point mutations in MHV-CoV M that blocked the interaction with MYO5B+D in yeast 2-hybrid assays. One of these point mutations (E121K) was previously shown to block MHV-CoV virion assembly and its interaction with MYO5B+D. The E to K mutation at homologous positions in PEDV-CoV, MERS-CoV and SARS-CoV-2 M proteins also blocked colocalization with MYO5B+D. The knockdown of Rab10 blocked the co-localization of M proteins with MYO5B+D and was rescued by re-expression of CFP-Rab10. Our results suggest that CoV M proteins traffic through Rab10-containing systems, in association with MYO5B+D.

Cell Bank Origin of MDCK Parental Cells Shapes Adaptation to Serum-Free Suspension Culture and Canine Adenoviral Vector Production.

Phenotypic variation in cultured mammalian cell lines is known to be induced by passaging and culture conditions. Yet, the effect these variations have on the production of viral vectors has been overlooked. In this work we evaluated the impact of using Madin-Darby canine kidney (MDCK) parental cells from American Type Culture Collection (ATCC) or European Collection of Authenticated Cell Cultures (ECACC) cell bank repositories in both adherent and suspension cultures for the production of canine adenoviral vectors type 2 (CAV-2). To further explore the differences between cells, we conducted whole-genome transcriptome analysis. ECACC's MDCK showed to be a less heterogeneous population, more difficult to adapt to suspension and serum-free culture conditions, but more permissive to CAV-2 replication progression, enabling higher yields. Transcriptome data indicated that this increased permissiveness is due to a general down-regulation of biological networks of innate immunity in ECACC cells, including apoptosis and death receptor signaling, Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling, toll-like receptors signaling and the canonical pathway of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. These results show the impact of MDCK source on the outcome of viral-based production processes further elucidating transcriptome signatures underlying enhanced adenoviral replication. Following functional validation, the genes and networks identified herein can be targeted in future engineering approaches aiming at improving the production of CAV-2 gene therapy vectors.

Establishment of a Simple and Efficient Reverse Genetics System for Canine Adenoviruses Using Bacterial Artificial Chromosomes.

Canine adenoviruses (CAdVs) are divided into pathotypes CAdV1 and CAdV2, which cause infectious hepatitis and laryngotracheitis in canid animals, respectively. They can be the backbones of viral vectors that could be applied in recombinant vaccines or for gene transfer in dogs and in serologically naïve humans. Although conventional plasmid-based reverse genetics systems can be used to construct CAdV vectors, their large genome size creates technical difficulties in gene cloning and manipulation. In this study, we established an improved reverse genetics system for CAdVs using bacterial artificial chromosomes (BACs), in which genetic modifications can be efficiently and simply made through BAC recombineering. To validate the utility of this system, we used it to generate CAdV2 with the early region 1 gene deleted. This mutant was robustly generated and attenuated in cell culture. The results suggest that our established BAC-based reverse genetics system for CAdVs would be a useful and powerful tool for basic and advanced practical studies with these viruses.

3D-quantitative structure-activity relationship and antiviral effects of curcumin derivatives as potent inhibitors of influenza H1N1 neuraminidase.

Curcumin derivatives have been shown to inhibit replication of human influenza A viruses (IAVs). However, it is not clear whether curcumin and its derivatives can inhibit neuraminidase (NA) of influenza virus. In this study, a meaningful 3D quantitative structure-activity relationship model (comparative molecular field analysis R2 = 0.997, q2 = 0.527, s = 0.064, F = 282.663) was built to understand the chemical-biological interactions between their activities and neuraminidase. Molecular docking was used to predict binding models between curcumin derivatives and neuraminidase. Real-time polymerase chain reactions showed that the five active curcumin derivatives might have direct effects on viral particle infectivity in H1N1-infected lung epithelial (MDCK) cells. Neuraminidase activation assay showed that five active curcumin derivatives decreased H1N1-induced neuraminidase activation in MDCK cells. Indirect immunofluorescence assay indicated that two active curcumin derivatives (tetramethylcurcumin and curcumin) down-regulated the nucleoprotein expression. Curcumin inhibited IAV in vivo. The therapeutic mechanism of curcumin in the treatment of influenza viral pneumonia is related to improving the immune function of infected mice and regulating secretion of tumor necrosis-α, interleukin-6, and interferon-γ. These results indicate that curcumin derivatives inhibit IAV by blocking neuraminidase in the cellular model and curcumin also has anti-IAV activity in the animal model.

Isocorilagin, isolated from Canarium album (Lour.) Raeusch, as a potent neuraminidase inhibitor against influenza A virus.

Canarium album (Lour.) Raeusch (C. album) as a normally medicinal and edible plant has been used widely in Asian countries and is considered a source of phytochemicals that are beneficial to human health. Here, we showed at the first time isocorilagin, a polyphenolic compound isolated from C. album, displayed antiviral activity against diverse strains of influenza A virus (IAV), including A/Puerto Rico/8/34 (H1N1), A/Aichi/2/68 (H3N2) and NA-H274Y (H1N1) with IC50 value of 9.19 ±â€¯1.99, 23.72 ±â€¯2.51 and 4.64 ±â€¯3.01 µM, respectively. Further mechanistic studies revealed that it clearly inhibited neuraminidase activity of IAV and directly influenced the virus release. The molecular docking studies presented isocorilagin could bind to the highly conserved residues in the active sites of NA, implying that isocorilagin may be effective against various influenza strains and not susceptible to produce drug resistance. Taken together, the results strongly suggest that isocorilagin has potential to be an effective, safe and affordable neuraminidase inhibitor against a diverse panel of IAV strains. More importantly, our work expands the biological activities of C. album extracts and provide a new option for the development of anti-influenza drug.

Lanostane Triterpenes from Gloeophyllum odoratum and Their Anti-Influenza Effects.

In an in vitro screening for anti-influenza agents from European polypores, the fruit body extract of Gloeophyllum odoratum dose-dependently inhibited the cytopathic effect of the H3N2 influenza virus A/Hong Kong/68 (HK/68) in Madin Darby canine kidney cells with a 50% inhibitory concentration (IC50) of 15 µg/mL, a noncytotoxic concentration. After a chromatographic work-up, eight lanostane triterpenes (1: -8: ) were isolated and their structures were elucidated based on high-resolution electrospray ionization mass spectrometry analyses, and one- and two-dimensional nuclear magnetic resonance experiments. Constituents 1: (gloeophyllin K) and 2: (gloeophyllin L) are reported here for the first time, and compounds 5: , 7: , and 8: have not been described for the investigated fungal material so far. The highest activity was determined for trametenolic acid B (3: ) against HK/68 and the 2009 pandemic H1N1 strain A/Jena/8178/09 with IC50 values of 14 and 11 µM, respectively. In a plaque reduction assay, this compound was able to bind to cell-free viruses and to neutralize their infectivity.

Synthesis of a Novel Class of 1,3-oxathiolane Nucleoside Derivatives of T- 705 and Evaluation of Their Anti-influenza A Virus and Anti-HIV Activity.

BACKGROUND: T-705 (Favipiravir) is a broad spectrum antiviral agent approved for stockpiling in Japan and currently in Phase 3 testing in the United States. Against influenza, it acts as a prodrug, converted intracellularly to selectively inhibit viral RNA-dependent RNA polymerase or similar enzymes. This is regarded as a novel antiviral mechanism of action, reducing crossresistance to other existing anti-influenza drugs. OBJECTIVE: To develop new analogs, a class of 1,3-oxathiolane nucleoside derivatives of T-705 was designed and synthesized in this work. RESULTS: Anti-influenza activity and Anti-HIV activity of these compounds were evaluated. Compound 1a displayed activity against A H1N1 with an IC50 of 40.4 µmol/L. Compound 1b showed weak activity against HIV with a viral suppression rate of 70-80% at 30 µmol/L. CONCLUSION: A class of 1,3-oxathiolane nucleoside derivatives of T-705 was designed and synthesized, and one of them was identified as a novel scaffold against viral infection.

Silver Nanoparticles Impair Retinoic Acid-Inducible Gene I-Mediated Mitochondrial Antiviral Immunity by Blocking the Autophagic Flux in Lung Epithelial Cells.

Silver nanoparticles (AgNPs) are microbicidal agents which could be potentially used as an alternative to antivirals to treat human infectious diseases, especially influenza virus infections where antivirals have generally proven unsuccessful. However, concerns about the use of AgNPs on humans arise from their potential toxicity, although mechanisms are not well-understood. We show here, in the context of an influenza virus infection of lung epithelial cells, that AgNPs down-regulated influenza induced CCL-5 and -IFN-ß release (two cytokines important in antiviral immunity) through RIG-I inhibition, while enhancing IL-8 production, a cytokine important for mobilizing host antibacterial responses. AgNPs activity was independent of coating and was not observed with gold nanoparticles. Down-stream analysis indicated that AgNPs disorganized the mitochondrial network and prevented the antiviral IRF-7 transcription factor influx into the nucleus. Importantly, we showed that the modulation of RIG-I-IRF-7 pathway was concomitant with inhibition of either classical or alternative autophagy (ATG-5- and Rab-9 dependent, respectively), depending on the epithelial cell type used. Altogether, this demonstration of a AgNPs-mediated functional dichotomy (down-regulation of IFN-dependent antiviral responses and up-regulation of IL-8-dependent antibacterial responses) may have practical implications for their use in the clinic.

Facile functionalization at the C4 position of pyrimidine nucleosides via amide group activation with (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and biological evaluations of the products.

Reactions of O-t-butyldimethylsilyl-protected thymidine, 2'-deoxyuridine, and 3'-azidothymidine (AZT) with (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) leads to activation of the C4 amide carbonyl by formation of putative O4-(benzotriazol-1-yl) derivatives. Subsequent substitution with alkyl and aryl amines, thiols, and alcohols leads to facile functionalization at this position. Reactions with amines and thiols were conducted either as a two-step, one-pot transformation, or as a one-step conversion. Reactions with alcohols were conducted as two-step, one-pot transformations. In the course of these investigations, the formation of 1-(4-pyrimidinyl)-1H-benzotriazole-3-oxide derivatives from the pyrimidine nucleosides was identified. However, these too underwent conversion to the desired products. Products obtained from AZT were converted to the 3'-amino derivatives by catalytic reduction. All products were assayed for their abilities to inhibit cancer cell proliferation and for antiviral activities. Many were seen to be active against HIV-1 and HIV-2, and one was active against herpes simplex virus-1 (HSV-1).

Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production.

Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-(13)C]glucose and [U-(13)C]glutamine, we apply for the first time (13)C-Metabolic flux analysis ((13)C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and (13)C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. (13)C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production.

Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity.

Phosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non-structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post-translational events yet. Here, we show that the newly identified phospho-site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double-stranded RNA and TRIM25 as well as complex formation with RIG-I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon-antagonistic activity are needed.

Analysis of cytokine production in a newly developed canine tracheal epithelial cell line infected with H3N2 canine influenza virus.

The Madin-Darby canine kidney (MDCK) cell line is typically used to analyze pathological features after canine influenza virus (CIV) infection. However, MDCK cells are not the ideal cell type, because they are kidney epithelial cells. Therefore, we generated an immortalized canine tracheal epithelial cell line, KU-CBE, to more reliably study immune responses to CIV infection in the respiratory tract. KU-CBE cells expressed the influenza virus receptor, α-2,3-sialic acid (SA), but not α-2,6-SA. KU-CBE and MDCK cells infected with H3N2 CIV demonstrated comparable virus growth kinetics. Gene expression levels of interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-ß were estimated in both KU-CBE and MDCK cells infected with CIV by real-time reverse transcription polymerase chain reaction (qRT-PCR). Of these cytokines, IL-4, IL-10, TNF-α, and IFN-ß mRNAs were detected in both cell lines. Gene expression of IL-4, IL-10, and TNF-α was not significantly different in the two cell lines. However, MDCK cells exhibited a significantly higher level of IFN-ß mRNA than KU-CBE cells at 18 h post infection. Additionally, the protein concentrations of these four cytokines were determined by enzyme-linked immunosorbent assay (ELISA) using cell culture supernatants obtained from the two CIV-infected cell lines. MDCK cells produced significantly higher amounts of IL-4 and IFN-ß than KU-CBE cells. However, KU-CBE cells produced a significantly higher amount of TNF-α than MDCK cells. These data indicated that the newly developed canine tracheal epithelial cells exhibited different cytokine production patterns compared to MDCK cells when infected with CIV. Inflammation of the respiratory tract of dogs induced by CIV infection may be attributed to the elevated expression level of TNF-α in canine tracheal epithelial cells.

Comparison of influenza virus yields and apoptosis-induction in an adherent and a suspension MDCK cell line.

Cell culture-based manufacturing of influenza vaccines is ideally based on easily scalable platforms using suspension cells that grow in chemically defined media. Consequently, different adherent cell lines selected for high virus yields were adapted to grow in suspension culture. This includes the MDCK suspension cell line MDCK.SUS2, which was shown to be a suitable substrate for influenza virus propagation in previous studies. In this study, we investigated options for further improvement of influenza A/PR/8 (H1N1) virus titres and cell-specific virus yields. Best results were achieved by performing a 1:2 dilution with fresh medium at time of infection. In shake flask cultivations, even for multiplicities of infection as low as 10⁻5, all cells were infected at 36 h post infection as determined by flow cytometry. In addition, these cells showed a better viability than cells infected without previous washing steps, which was reflected by a reduced level of apoptotic cells, and virus yields exceeding 3 log10 HAU/100 µL. Comparison of bioreactor infections of MDCK.SUS2 cells to the parental adherent MDCK cells showed similar HA titres of 2.94 and 3.15 log10 HAU/100 µL and TCID50 of 1 × 109 and 2.37 × 109 infectious virions/mL. Surprisingly, virus-induced apoptosis differed between the two cell lines, with the MDCK.SUS2 cells showing a much stronger apoptosis induction than the adherent MDCK cells. Obviously, despite their resistance to anoikis, the suspension cells were more susceptible to virus-induced apoptosis. Whether this is related to the adaptation process itself and/or to changes in cell survival pathways influenced by adhesion molecules or influenza virus proteins needs to be clarified in additional studies.

High yield production of influenza virus in Madin Darby canine kidney (MDCK) cells with stable knockdown of IRF7.

Influenza is a serious public health problem that causes a contagious respiratory disease. Vaccination is the most effective strategy to reduce transmission and prevent influenza. In recent years, cell-based vaccines have been developed with continuous cell lines such as Madin-Darby canine kidney (MDCK) and Vero. However, wild-type influenza and egg-based vaccine seed viruses will not grow efficiently in these cell lines. Therefore, improvement of virus growth is strongly required for development of vaccine seed viruses and cell-based influenza vaccine production. The aim of our research is to develop novel MDCK cells supporting highly efficient propagation of influenza virus in order to expand the capacity of vaccine production. In this study, we screened a human siRNA library that involves 78 target molecules relating to three major type I interferon (IFN) pathways to identify genes that when knocked down by siRNA lead to enhanced production of influenza virus A/Puerto Rico/8/1934 in A549 cells. The siRNAs targeting 23 candidate genes were selected to undergo a second screening pass in MDCK cells. We examined the effects of knockdown of target genes on the viral production using newly designed siRNAs based on sequence analyses. Knockdown of the expression of a canine gene corresponding to human IRF7 by siRNA increased the efficiency of viral production in MDCK cells through an unknown process that includes the mechanisms other than inhibition of IFN-α/ß induction. Furthermore, the viral yield greatly increased in MDCK cells stably transduced with the lentiviral vector for expression of short hairpin RNA against IRF7 compared with that in control MDCK cells. Therefore, we propose that modified MDCK cells with lower expression level of IRF7 could be useful not only for increasing the capacity of vaccine production but also facilitating the process of seed virus isolation from clinical specimens for manufacturing of vaccines.

Relationship between polyphenol content and anti-influenza viral effects of berries.

BACKGROUND: Berries are known to have many kinds of biological activities. We focused on their antiviral effect, which has not yet been well evaluated. RESULTS: We compared the anti-influenza viral effects of berries belonging to the genus Vaccinium - 35 species of blueberry (Vaccinium cyanococcus), the Natsuhaze (Vaccinium oldhamii), bilberry (Vaccinium myrtillus) and cranberry (Vaccinium oxycoccos)- with those belonging to the genus Ribes, i.e. blackcurrant (Ribes nigrum). Only Elliott and Legacy among Northern Highbush varieties but many Rabbiteye varieties such as Austin, Baldwin, Brightblue, Festival, T-100 and Tifblue showed anti-influenza viral activity. Natsuhaze, bilberry, cranberry and blackcurrant had high antiviral effects. A relationship was observed between the antiviral effect and total polyphenol content. CONCLUSIONS: Antiviral effects were found to differ markedly between berry species. Rabbiteye varieties tended to have higher antiviral effects than Northern, Southern and Half Highbush blueberry varieties. We also found that Natsuhaze, which has recently been harvested in Japan as a potential functional food, had an antiviral effect comparable to that of bilberry, cranberry and blackcurrant. There was a positive relationship between antiviral activity and polyphenol content, indicating the possibility that polyphenol is one of the key factors in the antiviral effects of berries.