The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Recombinant 3CL(pro) was expressed in Pichia pastoris GS115 as a 42 kDa protein that displayed a K ( m ) of 15 ± 2 μM with Dabcyl-KTSAVLQSGFRKME-Edans as substrate. Purified 3CL(pro) was used for inhibition and kinetic assays with seven flavonoid compounds. The IC(50) of six flavonoidcompounds were 47-381 μM. Quercetin, epigallocatechin gallate and gallocatechin gallate (GCG) displayed good inhibition toward 3CL(pro) with IC(50) values of 73, 73 and 47 μM, respectively. GCG showed a competitive inhibition pattern with K ( i) value of 25 ± 1.7 μM. In molecular docking experiments, GCG displayed a binding energy of -14 kcal mol(-1) to the active site of 3CL(pro) and the galloyl moiety at 3-OH position was required for 3CL(pro) inhibition activity.

The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Recombinant 3CL(pro) was expressed in Pichia pastoris GS115 as a 42 kDa protein that displayed a K ( m ) of 15 ± 2 μM with Dabcyl-KTSAVLQSGFRKME-Edans as substrate. Purified 3CL(pro) was used for inhibition and kinetic assays with seven flavonoid compounds. The IC(50) of six flavonoidcompounds were 47-381 μM. Quercetin, epigallocatechin gallate and gallocatechin gallate (GCG) displayed good inhibition toward 3CL(pro) with IC(50) values of 73, 73 and 47 μM, respectively. GCG showed a competitive inhibition pattern with K ( i) value of 25 ± 1.7 μM. In molecular docking experiments, GCG displayed a binding energy of -14 kcal mol(-1) to the active site of 3CL(pro) and the galloyl moiety at 3-OH position was required for 3CL(pro) inhibition activity.

Forsythoside A is a polyphenolic constituent of the fruits of Forsythia suspensa Vahl. which is widely used as an antiinflammatory agent in traditional Chinese medicine. In the present study, the effects of forsythoside A on cell infection by avian infectious bronchitis virus were assessed. A real-time fluorescence quantitative PCR assay was used to determine mRNA content of IBV N gene. The pretreatment of cells with forsythoside A, adding forsythoside A post infection of cells, and treatment of virus with forsythoside A were analysed. The inhibitory effect of forsythoside A was confirmed by infecting primary chicken embryo kidney cells. Infected cells were inhibited by forsythoside A treatment. The data indicated that forsythoside A has the potential to prevent IBV infection in vitro. Copyright© 2010 John Wiley&Sons, Ltd.

Forsythoside A is a polyphenolic constituent of the fruits of Forsythia suspensa Vahl. which is widely used as an antiinflammatory agent in traditional Chinese medicine. In the present study, the effects of forsythoside A on cell infection by avian infectious bronchitis virus were assessed. A real-time fluorescence quantitative PCR assay was used to determine mRNA content of IBV N gene. The pretreatment of cells with forsythoside A, adding forsythoside A post infection of cells, and treatment of virus with forsythoside A were analysed. The inhibitory effect of forsythoside A was confirmed by infecting primary chicken embryo kidney cells. Infected cells were inhibited by forsythoside A treatment. The data indicated that forsythoside A has the potential to prevent IBV infection in vitro. Copyright© 2010 John Wiley&Sons, Ltd.

Northern Italy, especially Lombardia and Veneto, has been affected by the COVID-19 coronavirus epidemic for weeks. Trying to isolate the carriers of the disease in connection with China is not an effective strategy to contain the disease, since the virus is transmitted like the flu virus and we can expect to have patients who have had no contact with possible carriers. According to Coronavirus genetic map, there are 3 isolated viruses in Italy. After that of Spallanzani (Rome) Covid 1 (Chinese), that of S. Raffaele Covid 2 (mutation of Chinese already known from the references) called Milanese, here is that of Sacco Hospital which is only a relative of the first two. It seems to be the same as that isolated in Finland, Germany, South America and it is hypothesized to have arrived in Europe long before the Chinese epidemic (November 2019 - March 2020). In Italy, however, the number of swabs and the procedures for carrying them out is essentially left to the arbitration of the Regions; a chaos that prevents any reference model from being determined. With this situation, it is not clear why among the various strategies that could have been adopted, after the home isolation of the population not engaged in certain work activities, there is even a quarantine for all people. Measure that it is not clear how long it can last.The real number of people infected in Italy is at least 4 or five times higher than that declared by the recovered patiens. Lombardia is the region in the first place for using tampons; this is the reason combined with the casual praxis to present as "coronavirus deaths" patients who, on the other hand, could have deaths associated with coronavirus (ie suffering from previous pathologies that caused death) that could explain the "mortality peak for COVID19" of Lombardia. It would be appropriate before identifying the primary cause of death in COVID19, carrying out the necessary pathological investigations and, above all, defining a standard to be applied throughout the national territory.

Northern Italy, especially Lombardia and Veneto, has been affected by the COVID-19 coronavirus epidemic for weeks. Trying to isolate the carriers of the disease in connection with China is not an effective strategy to contain the disease, since the virus is transmitted like the flu virus and we can expect to have patients who have had no contact with possible carriers. According to Coronavirus genetic map, there are 3 isolated viruses in Italy. After that of Spallanzani (Rome) Covid 1 (Chinese), that of S. Raffaele Covid 2 (mutation of Chinese already known from the references) called Milanese, here is that of Sacco Hospital which is only a relative of the first two. It seems to be the same as that isolated in Finland, Germany, South America and it is hypothesized to have arrived in Europe long before the Chinese epidemic (November 2019 - March 2020). In Italy, however, the number of swabs and the procedures for carrying them out is essentially left to the arbitration of the Regions; a chaos that prevents any reference model from being determined. With this situation, it is not clear why among the various strategies that could have been adopted, after the home isolation of the population not engaged in certain work activities, there is even a quarantine for all people. Measure that it is not clear how long it can last.The real number of people infected in Italy is at least 4 or five times higher than that declared by the recovered patiens. Lombardia is the region in the first place for using tampons; this is the reason combined with the casual praxis to present as "coronavirus deaths" patients who, on the other hand, could have deaths associated with coronavirus (ie suffering from previous pathologies that caused death) that could explain the "mortality peak for COVID19" of Lombardia. It would be appropriate before identifying the primary cause of death in COVID19, carrying out the necessary pathological investigations and, above all, defining a standard to be applied throughout the national territory.

Northern Italy, especially Lombardia and Veneto, has been affected by the COVID-19 coronavirus epidemic for weeks. Trying to isolate the carriers of the disease in connection with China is not an effective strategy to contain the disease, since the virus is transmitted like the flu virus and we can expect to have patients who have had no contact with possible carriers. According to Coronavirus genetic map, there are 3 isolated viruses in Italy. After that of Spallanzani (Rome) Covid 1 (Chinese), that of S. Raffaele Covid 2 (mutation of Chinese already known from the references) called Milanese, here is that of Sacco Hospital which is only a relative of the first two. It seems to be the same as that isolated in Finland, Germany, South America and it is hypothesized to have arrived in Europe long before the Chinese epidemic (November 2019 - March 2020). In Italy, however, the number of swabs and the procedures for carrying them out is essentially left to the arbitration of the Regions; a chaos that prevents any reference model from being determined. With this situation, it is not clear why among the various strategies that could have been adopted, after the home isolation of the population not engaged in certain work activities, there is even a quarantine for all people. Measure that it is not clear how long it can last.The real number of people infected in Italy is at least 4 or five times higher than that declared by the recovered patiens. Lombardia is the region in the first place for using tampons; this is the reason combined with the casual praxis to present as "coronavirus deaths" patients who, on the other hand, could have deaths associated with coronavirus (ie suffering from previous pathologies that caused death) that could explain the "mortality peak for COVID19" of Lombardia. It would be appropriate before identifying the primary cause of death in COVID19, carrying out the necessary pathological investigations and, above all, defining a standard to be applied throughout the national territory.

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV) infection, leads to significant economic losses in the swine industry worldwide. In our studies, we found that glycyrrhizin, the major component of licorice root extracts, could moderately inhibit PEDV infection in Vero cells, when analyzed by western blot, qRT-PCR and a plaque formation assay. We also revealed that glycyrrhizin inhibited the entry and replication of PEDV. In addition, we demonstrated that glycyrrhizin decreased the mRNA levels of proinflammatory cytokines. Since glycyrrhizin is a competitive inhibitor of high mobility group box-1 (HMGB1), we confirmed that TLR4 and RAGE (£ associated with PEDV pathogenesis during the infection in Vero cells. In summary, our studies provide a molecular basis for developing novel therapeutic methods to control PEDV infection, based on glycyrrhizin and its derivatives.

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV) infection, leads to significant economic losses in the swine industry worldwide. In our studies, we found that glycyrrhizin, the major component of licorice root extracts, could moderately inhibit PEDV infection in Vero cells, when analyzed by western blot, qRT-PCR and a plaque formation assay. We also revealed that glycyrrhizin inhibited the entry and replication of PEDV. In addition, we demonstrated that glycyrrhizin decreased the mRNA levels of proinflammatory cytokines. Since glycyrrhizin is a competitive inhibitor of high mobility group box-1 (HMGB1), we confirmed that TLR4 and RAGE (£ associated with PEDV pathogenesis during the infection in Vero cells. In summary, our studies provide a molecular basis for developing novel therapeutic methods to control PEDV infection, based on glycyrrhizin and its derivatives.

The outbreak of SARS warrants the search for antiviral compounds to treat the disease. At present, no specific treatment has been identified for SARS-associated coronavirus infection. We assessed the antiviral potential of ribavirin, 6-azauridine, pyrazofurin, mycophenolic acid, and glycyrrhizin against two clinical isolates of coronavirus (FFM-1 and FFM-2) from patients with SARS admitted to the clinical centre of Frankfurt University, Germany. Of all the compounds, glycyrrhizin was the most active in inhibiting replication of the SARS-associated virus. Our findings suggest that glycyrrhizin should be assessed for treatment of SARS.

The outbreak of SARS warrants the search for antiviral compounds to treat the disease. At present, no specific treatment has been identified for SARS-associated coronavirus infection. We assessed the antiviral potential of ribavirin, 6-azauridine, pyrazofurin, mycophenolic acid, and glycyrrhizin against two clinical isolates of coronavirus (FFM-1 and FFM-2) from patients with SARS admitted to the clinical centre of Frankfurt University, Germany. Of all the compounds, glycyrrhizin was the most active in inhibiting replication of the SARS-associated virus. Our findings suggest that glycyrrhizin should be assessed for treatment of SARS.

BACKGROUND:Zinc (Zn) supplementation has been shown to reduce the incidence of diarrhea and to protect animals from intestinal diseases, but the mechanisms of this protective effect against virus infection in vivo have not yet been elucidated. Transmissible gastroenteritis virus (TGEV) causes diarrhea in piglets with an age-dependent decrease of severity.

RESULTS:We used 60 weaned piglets that were divided into three groups to evaluate the effect of different Zn levels added to a conventional diet (50 mg Zn/kg diet, Znlow, control group). The other groups received the diet supplemented with ZnO at final concentrations of 150 mg Zn/kg diet (Znmed), or 2,500 mg/kg diet (Znhigh). Oral challenge infection with TGEV was performed when the pigs had been fed for 1 week with the respective diet. Half of the piglets of each group were sacrificed at day 1 and 18 after challenge infection. Fecal consistency was improved and body weights increased in the Znhigh group when compared to the other groups, but no direct effect of Zn concentrations in the diet on fecal TGEV shedding and mucosal immune responses was detectable. However, in the Znhigh group, we found a prevention of villus atrophy and decreased caspase-3-mediated apoptosis of jejunal epithelium. Furthermore, pigs receiving high Zn diet showed a down-regulation of interferon (IFN)-α, oligoadenylate synthetase (OAS), Zn transporter SLC39A4 (ZIP4), but up-regulation of metallothionein-1 (MT1), as well as the Zn transporters SLC30A1 (ZnT1) and SLC30A5 (ZnT5). In addition, forskolin-induced chloride secretion and epithelial resistance were controlled at a physiological level inthe Znhigh but not the other groups. Finally, in the Znhigh group, we documented an earlier and higher systemic TGEV-specific serum antibody response.

CONCLUSIONS:These results suggest that high dietary Zn could provide enhanced protection in the intestinal tract and stimulate the systemic humoral immune response against TGEV infection.

BACKGROUND:Zinc (Zn) supplementation has been shown to reduce the incidence of diarrhea and to protect animals from intestinal diseases, but the mechanisms of this protective effect against virus infection in vivo have not yet been elucidated. Transmissible gastroenteritis virus (TGEV) causes diarrhea in piglets with an age-dependent decrease of severity.

RESULTS:We used 60 weaned piglets that were divided into three groups to evaluate the effect of different Zn levels added to a conventional diet (50 mg Zn/kg diet, Znlow, control group). The other groups received the diet supplemented with ZnO at final concentrations of 150 mg Zn/kg diet (Znmed), or 2,500 mg/kg diet (Znhigh). Oral challenge infection with TGEV was performed when the pigs had been fed for 1 week with the respective diet. Half of the piglets of each group were sacrificed at day 1 and 18 after challenge infection. Fecal consistency was improved and body weights increased in the Znhigh group when compared to the other groups, but no direct effect of Zn concentrations in the diet on fecal TGEV shedding and mucosal immune responses was detectable. However, in the Znhigh group, we found a prevention of villus atrophy and decreased caspase-3-mediated apoptosis of jejunal epithelium. Furthermore, pigs receiving high Zn diet showed a down-regulation of interferon (IFN)-α, oligoadenylate synthetase (OAS), Zn transporter SLC39A4 (ZIP4), but up-regulation of metallothionein-1 (MT1), as well as the Zn transporters SLC30A1 (ZnT1) and SLC30A5 (ZnT5). In addition, forskolin-induced chloride secretion and epithelial resistance were controlled at a physiological level inthe Znhigh but not the other groups. Finally, in the Znhigh group, we documented an earlier and higher systemic TGEV-specific serum antibody response.

CONCLUSIONS:These results suggest that high dietary Zn could provide enhanced protection in the intestinal tract and stimulate the systemic humoral immune response against TGEV infection.

Coronaviruses (CoVs) act as cross-species viruses and have the potential to spread rapidly into new host species and cause epidemic diseases. Despite the severe public health threat of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome CoV (MERS-CoV), there are currently no drugs available for their treatment; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are urgently needed. To search for effective inhibitory agents, we performed high-throughput screening (HTS) of a 2,000-compound library of approved drugs and pharmacologically active compounds using the established genetically engineered human CoV OC43 (HCoV-OC43) strain expressingluciferase (rOC43-ns2Del-Rluc) and validated the inhibitors using multiple genetically distinct CoVsWe screened 56 hits from the HTS data and validated 36 compoundsusing wild-type HCoV-OC43. Furthermore, we identified seven compounds (lycorine, emetine, monensin sodium, mycophenolate mofetil, mycophenolic acid, phenazopyridine, and pyrvinium pamoate) as broad-spectrum inhibitors according to their strong inhibition of replication by four CoVsat low-micromolar concentrations. Additionally, we found that emetine blocked MERS-CoV entry according to pseudovirus entry assays and that lycorine protected BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This represents the first demonstration ofreal-time bioluminescence imaging to monitor the effect of lycorine on the spread and distribution of HCoV-OC43 in a mouse model. These results offer critical information supporting the development of an effective therapeutic strategy against CoV infection.Currently, there is no approved therapy to treat coronavirus infection; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are needed. Based on our high-throughput screening assay using a compound library, we identified seven compounds with broad-spectrum efficacy against the replication of four CoVsAdditionally, one compound (lycorine) was found to protect BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This inhibitor might offer promising therapeutic possibilities for combatting novel CoV infections in the future.

Coronaviruses (CoVs) act as cross-species viruses and have the potential to spread rapidly into new host species and cause epidemic diseases. Despite the severe public health threat of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome CoV (MERS-CoV), there are currently no drugs available for their treatment; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are urgently needed. To search for effective inhibitory agents, we performed high-throughput screening (HTS) of a 2,000-compound library of approved drugs and pharmacologically active compounds using the established genetically engineered human CoV OC43 (HCoV-OC43) strain expressingluciferase (rOC43-ns2Del-Rluc) and validated the inhibitors using multiple genetically distinct CoVsWe screened 56 hits from the HTS data and validated 36 compoundsusing wild-type HCoV-OC43. Furthermore, we identified seven compounds (lycorine, emetine, monensin sodium, mycophenolate mofetil, mycophenolic acid, phenazopyridine, and pyrvinium pamoate) as broad-spectrum inhibitors according to their strong inhibition of replication by four CoVsat low-micromolar concentrations. Additionally, we found that emetine blocked MERS-CoV entry according to pseudovirus entry assays and that lycorine protected BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This represents the first demonstration ofreal-time bioluminescence imaging to monitor the effect of lycorine on the spread and distribution of HCoV-OC43 in a mouse model. These results offer critical information supporting the development of an effective therapeutic strategy against CoV infection.Currently, there is no approved therapy to treat coronavirus infection; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are needed. Based on our high-throughput screening assay using a compound library, we identified seven compounds with broad-spectrum efficacy against the replication of four CoVsAdditionally, one compound (lycorine) was found to protect BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This inhibitor might offer promising therapeutic possibilities for combatting novel CoV infections in the future.

The development of new therapeutic agents against the coronavirus causing Middle East Respiratory Syndrome (MERS) is a continuing imperative. The initial MERS-CoV epidemic was contained entirely through public health measures, but episodic cases continue, as there are currently no therapeutic agents effective in the treatment of MERS-CoV, although multiple strategies have been proposed. In this study, we screened 30,000 compounds from three different compound libraries against one of the essential proteases, the papain-like protease (PL), using a fluorescence-based enzymatic assay followed by surface plasmon resonance (SPR) direct binding analysis for hit confirmation. Mode of inhibition assays and competition SPR studies revealed two compounds to be competitive inhibitors. To improve upon the inhibitory activity of the best hit compounds, a small fragment library consisting of 352 fragments was screened in the presence of each hit compound, resulting in one fragment that enhanced the ICvalue of the best hit compound by 3-fold. Molecular docking and MM/PBSA binding energy calculations were used to predict potential binding sites, providing insight for design and synthesis of next-generation compounds.

The development of new therapeutic agents against the coronavirus causing Middle East Respiratory Syndrome (MERS) is a continuing imperative. The initial MERS-CoV epidemic was contained entirely through public health measures, but episodic cases continue, as there are currently no therapeutic agents effective in the treatment of MERS-CoV, although multiple strategies have been proposed. In this study, we screened 30,000 compounds from three different compound libraries against one of the essential proteases, the papain-like protease (PL), using a fluorescence-based enzymatic assay followed by surface plasmon resonance (SPR) direct binding analysis for hit confirmation. Mode of inhibition assays and competition SPR studies revealed two compounds to be competitive inhibitors. To improve upon the inhibitory activity of the best hit compounds, a small fragment library consisting of 352 fragments was screened in the presence of each hit compound, resulting in one fragment that enhanced the ICvalue of the best hit compound by 3-fold. Molecular docking and MM/PBSA binding energy calculations were used to predict potential binding sites, providing insight for design and synthesis of next-generation compounds.

More than 200 Chinese medicinal herb extracts were screened for antiviral activities against Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV) using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay for virus-induced cytopathic effect (CPE). Four of these extracts showed moderate to potent antiviral activities against SARS-CoV with 50% effective concentration (EC50) ranging from 2.4 +/- 0.2 to 88.2 +/- 7.7 microg/ml. Out of the four, Lycoris radiata was most potent. To identify the active component, L. radiata extract was subjected to further fractionation, purification, and CPE/MTS assays. This process led to the identification of a single substance lycorine as an anti-SARS-CoV component with an EC50 value of 15.7 +/- 1.2 nM. This compound has a CC50 value of 14980.0 +/- 912.0 nM in cytotoxicity assay and a selective index (SI) greater than 900. The results suggested that four herbal extracts and the compound lycorine are candidates for the development of new anti-SARS-CoV drugs in the treatment of SARS.

More than 200 Chinese medicinal herb extracts were screened for antiviral activities against Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV) using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay for virus-induced cytopathic effect (CPE). Four of these extracts showed moderate to potent antiviral activities against SARS-CoV with 50% effective concentration (EC50) ranging from 2.4 +/- 0.2 to 88.2 +/- 7.7 microg/ml. Out of the four, Lycoris radiata was most potent. To identify the active component, L. radiata extract was subjected to further fractionation, purification, and CPE/MTS assays. This process led to the identification of a single substance lycorine as an anti-SARS-CoV component with an EC50 value of 15.7 +/- 1.2 nM. This compound has a CC50 value of 14980.0 +/- 912.0 nM in cytotoxicity assay and a selective index (SI) greater than 900. The results suggested that four herbal extracts and the compound lycorine are candidates for the development of new anti-SARS-CoV drugs in the treatment of SARS.