Preoperative recovery sleep ameliorates postoperative cognitive dysfunction aggravated by sleep fragmentation in aged mice by enhancing EEG delta-wave activity and LFP theta oscillation in hippocampal CA1.

Sleep fragmentation (SF) is a common sleep problem experienced during the perioperative period by older adults, and is associated with postoperative cognitive dysfunction (POCD). Increasing evidence indicates that delta-wave activity during non-rapid eye movement (NREM) sleep is involved in sleep-dependent memory consolidation and that hippocampal theta oscillations are related to spatial exploratory memory. Recovery sleep (RS), a self-regulated state of sleep homeostasis, enhances delta-wave power and memory performance in sleep-deprived older mice. However, it remains unclear whether RS therapy has a positive effect on cognitive changes following SF in older mouse models. Therefore, this study aimed to explore whether preoperative RS can alleviate cognitive deficits in aged mice with SF. A model of preoperative 24-h SF combined with exploratory laparotomy-induced POCD was established in 18-month-old mice. Aged mice were treated with preoperative 6-h RS following SF and postoperative 6-h RS following surgery, respectively. The changes in hippocampus-dependent cognitive function were investigated using behavioral tests, electroencephalography (EEG), local field potential (LFP), magnetic resonance imaging, and neuromorphology. Mice that underwent 24-h SF combined with surgery exhibited severe spatial memory impairment; impaired cognitive performance could be alleviated by preoperative RS treatment. In addition, preoperative RS increased NREM sleep; enhanced EEG delta-wave activity and LFP theta oscillation in the hippocampal CA1; and improved hippocampal perfusion, microstructural integrity, and neuronal damage. Taken together, these results provide evidence that preoperative RS may ameliorate the severity of POCD aggravated by SF by enhancing delta slow-wave activity and hippocampal theta oscillation, and by ameliorating the reduction in regional cerebral blood flow and white matter microstructure integrity in the hippocampus.

COVID-19 instigates adipose browning and atrophy through VEGF in small mammals.

Patients with COVID-19 frequently manifest adipose atrophy, weight loss and cachexia, which significantly contribute to poor quality of life and mortality1,2. Browning of white adipose tissue and activation of brown adipose tissue are effective processes for energy expenditure3-7; however, mechanistic and functional links between SARS-CoV-2 infection and adipose thermogenesis have not been studied. In this study, we provide experimental evidence that SARS-CoV-2 infection augments adipose browning and non-shivering thermogenesis (NST), which contributes to adipose atrophy and body weight loss. In mouse and hamster models, SARS-CoV-2 infection activates brown adipose tissue and instigates a browning or beige phenotype of white adipose tissues, including augmented NST. This browning phenotype was also observed in post-mortem adipose tissue of four patients who died of COVID-19. Mechanistically, high levels of vascular endothelial growth factor (VEGF) in the adipose tissue induces adipose browning through vasculature-adipocyte interaction. Inhibition of VEGF blocks COVID-19-induced adipose tissue browning and NST and partially prevents infection-induced body weight loss. Our data suggest that the browning of adipose tissues induced by COVID-19 can contribute to adipose tissue atrophy and weight loss observed during infection. Inhibition of VEGF signaling may represent an effective approach for preventing and treating COVID-19-associated weight loss.

Multi-target direct-acting SARS-CoV-2 antivirals against the nucleotide-binding pockets of virus-specific proteins.

The nucleotide-binding pockets (NBPs) in virus-specific proteins have proven to be the most successful antiviral targets for several viral diseases. Functionally important NBPs are found in various structural and non-structural proteins of SARS-CoV-2. In this study, the first successful multi-targeting attempt to identify effective antivirals has been made against NBPs in nsp12, nsp13, nsp14, nsp15, nsp16, and nucleocapsid (N) proteins of SARS-CoV-2. A structure-based drug repurposing in silico screening approach with ADME analysis identified small molecules targeting NBPs in SARS-CoV-2 proteins. Further, isothermal titration calorimetry (ITC) experiments validated the binding of top hit molecules to the purified N-protein. Importantly, cell-based antiviral assays revealed antiviral potency for INCB28060, darglitazone, and columbianadin with EC50 values 15.71 µM, 5.36 µM, and 22.52 µM, respectively. These effective antivirals targeting multiple proteins are envisioned to direct the development of antiviral therapy against SARS-CoV-2 and its emerging variants.

Colloidal dispersion of poly(ionic liquid)/Cu composite particles for protective surface coating against SAR-CoV-2.

Herein, we report a waterproof anti-SARS-CoV-2 protective film prepared by spray-coating of an aqueous colloidal dispersion of poly(ionic liquid)/copper (PIL/Cu) composite nanoparticles onto a substrate. The PIL dispersion was prepared by suspension polymerization of 3-dodecyl-1-vinylimdiazolium bromide in water at 70°C. The copper acetate salt was added into the PIL nanoparticle dispersion and in situ reduced into copper nanoparticles anchoring onto the PIL nanoparticles. Despite being waterborne, the PIL in bulk is intrinsically insoluble in water and the formed coating is stable in water. The formed surface coating by PIL/copper composite nanoparticles was able to deactivate SARS-CoV-2 virions by 90.0% in 30 minutes and thus may effectively prevent the spread of SARS-CoV-2 through surface contact. This method may provide waterborne dispersions for a broad range of antivirus protective surface coatings for both outdoor and indoor applications.

Porcine reproductive and respiratory syndrome virus nsp4 positively regulates cellular cholesterol to inhibit type I interferon production.

Cellular cholesterol plays an important role in the life cycles of enveloped viruses. Previous studies by our group and other groups have demonstrated that the depletion of cellular cholesterol by methyl-ß-cyclodextrin (MßCD) reduces the proliferation of porcine reproductive and respiratory syndrome virus (PRRSV), a porcine Arterivirus that has been devastating the swine industry worldwide for over two decades. However, how PRRSV infection regulates cholesterol synthesis is not fully understood. In this study, we showed that PRRSV infection upregulated the activity of protein phosphatase 2 (PP2A), which subsequently activated 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme in the cholesterol synthesis pathway, to increase the levels of cellular cholesterol. By screening the PRRSV-encoded proteins, we showed that nsp4 dominated the upregulation of cellular cholesterol, independently of the 3C-like protease activity of nsp4. A mutation analysis showed that domain I (amino acids 1-80) of PRRSV nsp4 interacted with PR65 alpha (PR65α), the structural subunit, and PP2Ac, the catalytic subunit, of PP2A. Importantly, domain I of nsp4 inhibited Sendai virus-induced interferon ß production, and this inhibitory effect was eliminated by Lovastatin, an HMGCR inhibitor, indicating that the upregulation of cellular cholesterol by nsp4 is a strategy used by PRRSV to suppress the antiviral innate immunity of its host. Collectively, we here demonstrated the mechanism by which PRRSV regulates cellular cholesterol synthesis and reported a novel strategy by which PRRSV evades its host's antiviral innate immune response.

Antiviral Activity of Silver, Copper Oxide and Zinc Oxide Nanoparticle Coatings against SARS-CoV-2.

SARS-CoV-2 is responsible for several million deaths to date globally, and both fomite transmission from surfaces as well as airborne transmission from aerosols may be largely responsible for the spread of the virus. Here, nanoparticle coatings of three antimicrobial materials (Ag, CuO and ZnO) are deposited on both solid flat surfaces as well as porous filter media, and their activity against SARS-CoV-2 viability is compared with a viral plaque assay. These nanocoatings are manufactured by aerosol nanoparticle self-assembly during their flame synthesis. Nanosilver particles as a coating exhibit the strongest antiviral activity of the three studied nanomaterials, while copper oxide exhibits moderate activity, and zinc oxide does not appear to significantly reduce the virus infectivity. Thus, nanosilver and copper oxide show potential as antiviral coatings on solid surfaces and on filter media to minimize transmission and super-spreading events while also providing critical information for the current and any future pandemic mitigation efforts.

Porcine Deltacoronavirus nsp5 Cleaves DCP1A To Decrease Its Antiviral Activity.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus. The nonstructural protein nsp5, also called 3C-like protease, is responsible for processing viral polyprotein precursors in coronavirus (CoV) replication. Previous studies have shown that PDCoV nsp5 cleaves the NF-κB essential modulator and the signal transducer and activator of transcription 2 to disrupt interferon (IFN) production and signaling, respectively. Whether PDCoV nsp5 also cleaves IFN-stimulated genes (ISGs), IFN-induced antiviral effector molecules, remains unclear. In this study, we screened 14 classical ISGs and found that PDCoV nsp5 cleaved the porcine mRNA-decapping enzyme 1a (pDCP1A) through its protease activity. Similar cleavage of endogenous pDCP1A was also observed in PDCoV-infected cells. PDCoV nsp5 cleaved pDCP1A at glutamine 343 (Q343), and the cleaved pDCP1A fragments, pDCP1A1-343 and pDCP1A344-580, were unable to inhibit PDCoV infection. Mutant pDCP1A-Q343A, which resists nsp5-mediated cleavage, exhibited a stronger ability to inhibit PDCoV infection than wild-type pDCP1A. Interestingly, the Q343 cleavage site is highly conserved in DCP1A homologs from other mammalian species. Further analyses demonstrated that nsp5 encoded by seven tested CoVs that can infect human or pig also cleaved pDCP1A and human DCP1A, suggesting that DCP1A may be the common target for cleavage by nsp5 of mammalian CoVs.IMPORTANCE Interferon (IFN)-stimulated gene (ISG) induction through IFN signaling is important to create an antiviral state and usually directly inhibits virus infection. The present study first demonstrated that PDCoV nsp5 can cleave mRNA-decapping enzyme 1a (DCP1A) to attenuate its antiviral activity. Furthermore, cleaving DCP1A is a common characteristic of nsp5 proteins from different coronaviruses (CoVs), which represents a common immune evasion mechanism of CoVs. Previous evidence showed that CoV nsp5 cleaves the NF-κB essential modulator and signal transducer and activator of transcription 2. Taken together, CoV nsp5 is a potent IFN antagonist because it can simultaneously target different aspects of the host IFN system, including IFN production and signaling and effector molecules.

Fatty Acids Regulate Porcine Reproductive and Respiratory Syndrome Virus Infection via the AMPK-ACC1 Signaling Pathway.

Lipids play a crucial role in the replication of porcine reproductive and respiratory syndrome virus (PRRSV), a porcine virus that is endemic throughout the world. However, little is known about the effect of fatty acids (FAs), a type of vital lipid, on PRRSV infection. In this study, we found that treatment with a FA biosynthetic inhibitor significantly inhibited PRRSV propagation, indicating the necessity of FAs for optimal replication of PRRSV. Further study revealed that 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), a key kinase antagonizing FA biosynthesis, was strongly activated by PRRSV and the pharmacological activator of AMPK exhibited anti-PRRSV activity. Additionally, we found that acetyl-CoA carboxylase 1 (ACC1), the first rate-limiting enzyme in the FA biosynthesis pathway, was phosphorylated (inactive form) by PRRSV-activated AMPK, and active ACC1 was required for PRRSV proliferation, suggesting that the PRRSV infection induced the activation of the AMPK-ACC1 pathway, which was not conducive to PRRSV replication. This work provides new evidence about the mechanisms involved in host lipid metabolism during PRRSV infection and identifies novel potential antiviral targets for PRRSV.

Japanese Encephalitis Virus infection induces inflammation of swine testis through RIG-I-NF-ĸB signaling pathway.

Japanese Encephalitis Virus (JEV) is an important zoonotic flavivirus transmitted by mosquitos. JEV infection in sows primarily manifests as a reproductive disease such as abortion and transient infertility while in infected boars, it can cause orchitis. Previous studies mainly focused on the pathogenesis of human encephalitis caused by JEV infection, while few concentrations have been made to unveil the potential mechanism of reproductive dysfunction in JEV-infected pigs. In this study, histopathological analysis and immunohistochemistry staining was performed on testis of JEV-infected boars, indicating that JEV could infect testicular cells and cause inflammatory changes in testis. In vitro assays reveal that primary swine testicular cells and swine testis (ST) cells are highly permissive to JEV and significant inflammatory response was shown during JEV infection. Mechanically, we found that JEV infection increases the expression of retinoic acid-inducible gene I (RIG-I) and activates transcription factor NF-κB. Production of pro-inflammatory cytokines was greatly reduced in JEV infected testicular cells after knockout of RIG-I or treatment with the NF-κB specific inhibitor. In addition, activation of NF-κB was also significantly suppressed upon RIG-I knockout. Taken together, our results reveal that JEV could infect boar testicles, and RIG-I-NF-κB signaling pathway is involved in JEV-induced inflammation in swine testicular cells.

Cholesterol 25-Hydroxylase Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication through Enzyme Activity-Dependent and -Independent Mechanisms.

Cholesterol 25-hydroxylase (CH25H) has recently been identified as a host restriction factor that exerts antiviral effects by catalyzing the production of 25-hydroxycholesterol (25HC). CH25H can be rapidly induced upon infection with some viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, has ranked among the most important swine pathogens since it was discovered in the late 1980s. In this study, we found that PRRSV infection significantly downregulated the expression of CH25H in cells by a so-far unknown mechanism, suggesting that CH25H exerts antiviral activity against PRRSV. Indeed, overexpression of CH25H inhibited PRRSV replication, whereas knockdown of CH25H by short interfering RNA (siRNA) promoted PRRSV infection. The anti-PRRSV effect of 25HC operates via inhibition of viral penetration. Interestingly, a CH25H mutant (CH25H-M) lacking hydroxylase activity still inhibited PRRSV infection. Screening using a yeast two-hybrid system followed by coimmunoprecipitation and immunofluorescence colocalization analyses confirmed that both CH25H and CH25H-M interact with the nonstructural protein 1 alpha (nsp1α) of PRRSV. Unexpectedly, the expression of nsp1α decreased following coexpression with CH25H or CH25H-M. Detailed analyses demonstrated that CH25H/CH25H-M could degrade nsp1α through the ubiquitin-proteasome pathway and that site K169 in the nsp1α protein is the key site of ubiquitination. Taken together, our findings demonstrate that CH25H restricts PRRSV replication by targeting viral penetration as well as degrading nsp1α, revealing a novel antiviral mechanism used by CH25H.IMPORTANCE PRRSV has been a continuous threat to the global swine industry, and current vaccines are insufficient to provide sustainable control. CH25H has been found to exert a broad antiviral effect; thus, it is an attractive target for the development of anti-PRRSV drugs. Here, we demonstrate that CH25H is an interferon-stimulated gene that is highly expressed in porcine alveolar macrophages. CH25H exerts its anti-PRRSV effect not only via the production of 25HC to inhibit viral penetration but also by degrading viral protein through the ubiquitin-proteasome pathway, suggesting that CH25H is a candidate for the development of antiviral therapeutics. However, PRRSV infection appears to actively decrease CH25H expression to promote viral replication, highlighting the complex game between PRRSV and its host.

The nucleocapsid proteins of mouse hepatitis virus and severe acute respiratory syndrome coronavirus share the same IFN-ß antagonizing mechanism: attenuation of PACT-mediated RIG-I/ MDA5 activation.

Coronaviruses (CoVs) are a huge threat to both humans and animals and have evolved elaborate mechanisms to antagonize interferons (IFNs). Nucleocapsid (N) protein is the most abundant viral protein in CoV-infected cells, and has been identified as an innate immunity antagonist in several CoVs, including mouse hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV. However, the underlying molecular mechanism(s) remain unclear. In this study, we found that MHV N protein inhibited Sendai virus and poly(I:C)-induced IFN-ß production by targeting a molecule upstream of retinoic acid-induced gene I (RIG-I) and melanoma differentiation gene 5 (MDA5). Further studies showed that both MHV and SARS-CoV N proteins directly interacted with protein activator of protein kinase R (PACT), a cellular dsRNA-binding protein that can bind to RIG-I and MDA5 to activate IFN production. The N-PACT interaction sequestered the association of PACT and RIG-I/MDA5, which in turn inhibited IFN-ß production. However, the N proteins from porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV), which are also classified in the order Nidovirales, did not interact and counteract with PACT. Taken together, our present study confirms that both MHV and SARS-CoV N proteins can perturb the function of cellular PACT to circumvent the innate antiviral response. However, this strategy does not appear to be used by all CoVs N proteins.