In this study, 221 phytocompounds were evaluated for activity against anti-severe acute respiratory syndrome associated SARS-CoV-induced cytopathogenic effect on Vero E6 cells. Ten diterpenoids (1-10), two sesquiterpenoids (11 and 12), two triterpenoids (13 and 14), five lignoids (15-19), curcumin (20), and reference controls niclosamide (21) and valinomycin (22) were potent inhibitors at concentrations between 3.3 and 10 microM. The concentrations of the 22 compounds to inhibit 50% of Vero E6 cell proliferation (CC50) and viral replication (EC50) were measured. The selective index values (SI = CC50/EC50) of the most potent compounds 1, 5, 6, 8, 14, and 16 were 58, >510, 111, 193, 180, and >667, respectively. Betulinic acid (13) and savinin (16) were competitive inhibitors of SARS-CoV 3CL protease with Ki values = 8.2 +/- 0.7 and 9.1 +/- 2.4 microM, respectively. Our findings suggest that specific abietane-type diterpenoids and lignoids exhibit strong anti-SARS-CoV effects.

OBJECTIVE:The causal agent for SARS is considered as a novel coronavirus that has never been described both in human and animals previously. The stability of SARS coronavirus in human specimens and in environments was studied.

METHODS:Using a SARS coronavirus strain CoV-P9, which was isolated from pharyngeal swab of a probable SARS case in Beijing, its stability in mimic human specimens and in mimic environment including surfaces of commonly used materials or in household conditions, as well as its resistance to temperature and UV irradiation were analyzed. A total of 10(6) TCID50 viruses were placed in each tested condition, and changes of the viral infectivity in samples after treatments were measured by evaluating cytopathic effect (CPE) in cell line Vero-E6 at 48 h after infection.

RESULTS:The results showed that SARS coronavirus in the testing condition could survive in serum, 1:20 diluted sputum and feces for at least 96 h, whereas it could remain alive in urine for at least 72 h with a low level of infectivity. The survival abilities on the surfaces of eight different materials and in water were quite comparable, revealing reduction of infectivity after 72 to 96 h exposure. Viruses stayed stable at 4 degrees C, at room temperature (20 degrees C) and at 37 degrees C for at least 2 h without remarkable change in the infectious ability in cells, but were converted to be non-infectious after 90-, 60- and 30-min exposure at 56 degrees C, at 67 degrees C and at 75 degrees C, respectively. Irradiation of UV for 60 min on the virus in culture medium resulted in the destruction of viral infectivity at an undetectable level.

CONCLUSION:The survival ability of SARS coronavirus in human specimens and in environments seems to be relatively strong. Heating and UV irradiation can efficiently eliminate the viral infectivity.

Severe acute respiratory syndrome (SARS) is an infectious disease and some of its symptoms were clinically indistinguishable of those from similar diseases. This study aimed to find the symptom combinations associated with adverse outcome and the therapeutic effects in a cohort of patients with probable SARS retrospectively. In 2003, 123 SARS cases in Beijing were subjected to a strictly western medicine (WM) treatment, or a combined treatment (WM plus Herba houttuyniae injection, addition of individualized herbal treatments when necessary), of which 115 were followed till death or discharge; 8 were transferred and lost to follow-up. In both treatment groups, clinical manifestations were evaluated daily; development of signs and symptoms, and their possible relationship with outcome, were assessed. The relationships between these sign/symptom complexes and outcome under two treatment protocols were evaluated and differences were noted. Dynamic symptom combinations, dividing into the early, the medium-term and the durational symptom clusters, were identified as likely being related to the adverse outcomes of SARS (p<0.05, p<0.01). Compared with a strictly WM treatment, the combined treatment resulted in a longer hospital stay (p = 0.028), a non-statistically significant mortality rate decrease (combined treatment: 9.6% versus WM: 11.1%), and a significant improvement of arthralgia and myalgia (p<0.05) in the early symptom cluster. Additionally, the combined protocol improved arterial oxyhemoglobin saturation significantly at day 22 (p<0.05). In conclusion, the progress and outcome of SARS may be associated with specific temporal patterns of development in combination of several non-specific signs and symptom complexes, which are also helpful for evaluating the therapeutic effects on SARS patients.

Severe acute respiratory syndrome (SARS) is an infectious disease and some of its symptoms were clinically indistinguishable of those from similar diseases. This study aimed to find the symptom combinations associated with adverse outcome and the therapeutic effects in a cohort of patients with probable SARS retrospectively. In 2003, 123 SARS cases in Beijing were subjected to a strictly western medicine (WM) treatment, or a combined treatment (WM plus Herba houttuyniae injection, addition of individualized herbal treatments when necessary), of which 115 were followed till death or discharge; 8 were transferred and lost to follow-up. In both treatment groups, clinical manifestations were evaluated daily; development of signs and symptoms, and their possible relationship with outcome, were assessed. The relationships between these sign/symptom complexes and outcome under two treatment protocols were evaluated and differences were noted. Dynamic symptom combinations, dividing into the early, the medium-term and the durational symptom clusters, were identified as likely being related to the adverse outcomes of SARS (p<0.05, p<0.01). Compared with a strictly WM treatment, the combined treatment resulted in a longer hospital stay (p = 0.028), a non-statistically significant mortality rate decrease (combined treatment: 9.6% versus WM: 11.1%), and a significant improvement of arthralgia and myalgia (p<0.05) in the early symptom cluster. Additionally, the combined protocol improved arterial oxyhemoglobin saturation significantly at day 22 (p<0.05). In conclusion, the progress and outcome of SARS may be associated with specific temporal patterns of development in combination of several non-specific signs and symptom complexes, which are also helpful for evaluating the therapeutic effects on SARS patients.

The aim of our experiment was to study how synbiotics are able to deal with the problems of post-weaning piglets. Lactobacillus plantarum - Biocenol(TM) LP96 (CCM 7512), Lactobacillus fermentum - Biocenol(TM) LF99 (CCM 7514) and flaxseed (rich in n-3 polyunsaturated fatty acids) were administered to 36 conventional piglets from a problematic breed with confirmed presence of enterotoxigenic Escherichia coli and Coronavirus. The experimental piglets were supplied with probiotic cheeses and crushed flax-seed in the period starting 10 days before weaning and lasting up to 14 days post-weaning. Piglets in the control group were supplied only control cheese. The impact of such additives on the release of lactate dehydrogenase (LDH; spectroscopic and electrophoretic assay), alteration of immunity (index of metabolic activity), jejunum histology (light microscopy), and health of conventional piglets from a problematic breed (monitoring of hematology, consistency and moisture of feces and body temperature) were examined. We found significant decrease in LDH leakage in the blood serum and tissue extracts, indicating better cell membrane integrity in the individual organs of animals. Probiotics and flaxseed applied together seem to be a good source of nutrients to improve the immune status and the integrity of jejunum mucosa during infection.© 2015 Japanese Society of Animal Science.

The aim of our experiment was to study how synbiotics are able to deal with the problems of post-weaning piglets. Lactobacillus plantarum - Biocenol(TM) LP96 (CCM 7512), Lactobacillus fermentum - Biocenol(TM) LF99 (CCM 7514) and flaxseed (rich in n-3 polyunsaturated fatty acids) were administered to 36 conventional piglets from a problematic breed with confirmed presence of enterotoxigenic Escherichia coli and Coronavirus. The experimental piglets were supplied with probiotic cheeses and crushed flax-seed in the period starting 10 days before weaning and lasting up to 14 days post-weaning. Piglets in the control group were supplied only control cheese. The impact of such additives on the release of lactate dehydrogenase (LDH; spectroscopic and electrophoretic assay), alteration of immunity (index of metabolic activity), jejunum histology (light microscopy), and health of conventional piglets from a problematic breed (monitoring of hematology, consistency and moisture of feces and body temperature) were examined. We found significant decrease in LDH leakage in the blood serum and tissue extracts, indicating better cell membrane integrity in the individual organs of animals. Probiotics and flaxseed applied together seem to be a good source of nutrients to improve the immune status and the integrity of jejunum mucosa during infection.© 2015 Japanese Society of Animal Science.

Aryl diketoacid (ADK) is well known for antiviral activity which can be enhanced by introduction of an aromatic arylmethyl substituent. A natural flavonoid quercetin has a 3,5-dihydroxychromone pharmacophore which is in bioisosteric relationship with the 1,3-diketoacid moiety of the ADK. Thus, it was of our interest to test the antiviral activity of the quercetin derivatives with an arylmethyl group attached. In this study, we prepared a series of the 7-O-arylmethylquercetin derivatives with various aromatic substituents and evaluated their antiviral activity against the SARS-associated coronavirus (SARS-CoV, SCV) as well as hepatitis C virus (HCV). Single difference in the aromatic substituent fine-tuned the biological activity of the 7-O-arylmethylquercetin derivatives to result in two different classes of derivatives selectively active against SCV and HCV.

Aryl diketoacid (ADK) is well known for antiviral activity which can be enhanced by introduction of an aromatic arylmethyl substituent. A natural flavonoid quercetin has a 3,5-dihydroxychromone pharmacophore which is in bioisosteric relationship with the 1,3-diketoacid moiety of the ADK. Thus, it was of our interest to test the antiviral activity of the quercetin derivatives with an arylmethyl group attached. In this study, we prepared a series of the 7-O-arylmethylquercetin derivatives with various aromatic substituents and evaluated their antiviral activity against the SARS-associated coronavirus (SARS-CoV, SCV) as well as hepatitis C virus (HCV). Single difference in the aromatic substituent fine-tuned the biological activity of the 7-O-arylmethylquercetin derivatives to result in two different classes of derivatives selectively active against SCV and HCV.

In the search for anti-SARS-CoV, tanshinones derived from Salvia miltiorrhiza were found to be specific and selective inhibitors for the SARS-CoV 3CL(pro) and PL(pro), viral cysteine proteases. A literature search for studies involving the seven isolated tanshinone hits showed that at present, none have been identified as coronaviral protease inhibitors. We have identified that all of the isolated tanshinones are good inhibitors of both cysteine proteases. However, their activity was slightly affected by subtle changes in structure and targeting enzymes. All isolated compounds (1-7) act as time dependent inhibitors of PL(pro), but no improved inhibition was observed following preincubation with the 3CL(pro). In a detail kinetic mechanism study, all of the tanshinones except rosmariquinone (7) were identified as noncompetitive enzyme isomerization inhibitors. However, rosmariquinone (7) showed a different kinetic mechanism through mixed-type simple reversible slow-binding inhibition. Furthermore, tanshinone I (5) exhibited the most potent nanomolar level inhibitory activity toward deubiquitinating (IC(50)=0.7μM). Additionally, the inhibition is selective because these compounds do not exert significant inhibitory effects against other proteases including chymotrysin, papain, and HIV protease. These findings provide potential inhibitors for SARS-CoV viral infection and replication.

In the search for anti-SARS-CoV, tanshinones derived from Salvia miltiorrhiza were found to be specific and selective inhibitors for the SARS-CoV 3CL(pro) and PL(pro), viral cysteine proteases. A literature search for studies involving the seven isolated tanshinone hits showed that at present, none have been identified as coronaviral protease inhibitors. We have identified that all of the isolated tanshinones are good inhibitors of both cysteine proteases. However, their activity was slightly affected by subtle changes in structure and targeting enzymes. All isolated compounds (1-7) act as time dependent inhibitors of PL(pro), but no improved inhibition was observed following preincubation with the 3CL(pro). In a detail kinetic mechanism study, all of the tanshinones except rosmariquinone (7) were identified as noncompetitive enzyme isomerization inhibitors. However, rosmariquinone (7) showed a different kinetic mechanism through mixed-type simple reversible slow-binding inhibition. Furthermore, tanshinone I (5) exhibited the most potent nanomolar level inhibitory activity toward deubiquitinating (IC(50)=0.7μM). Additionally, the inhibition is selective because these compounds do not exert significant inhibitory effects against other proteases including chymotrysin, papain, and HIV protease. These findings provide potential inhibitors for SARS-CoV viral infection and replication.

Extracts of Anthemis hyalina (Ah), Nigella sativa (Ns) and peels of Citrus sinensis (Cs) have been used as folk medicine to fight antimicrobial diseases. To evaluate the effect of extracts of Ah, Ns and Cs on the replication of coronavirus (CoV) and on the expression of TRP genes during coronavirus infection, HeLa-CEACAM1a (HeLa-epithelial carcinoembryonic antigen-related cell adhesion molecule 1a) cells were inoculated with MHV-A59 (mouse hepatitis virus-A59) at moi of 30. 1/50 dilution of the extracts was found to be the safe active dose. ELISA kits were used to detect the human IL-8 levels. Total RNA was isolated from the infected cells and cDNA was synthesized. Fluidigm Dynamic Array nanofluidic chip 96.96 was used to analyze the mRNA expression of 21 TRP genes and two control genes. Data was analyzed using the BioMark digital array software. Determinations of relative gene expression values were carried out by using the 2(-∆∆Ct) method (normalized threshold cycle (Ct) value of sample minus normalized Ct value of control). TCID50/ml (tissue culture infectious dose that will produce cytopathic effect in 50% of the inoculated tissue culture cells) was found for treatments to determine the viral loads. The inflammatory cytokine IL-8 level was found to increase for both 24 and 48 h time points following Ns extract treatment. TRPA1, TRPC4, TRPM6, TRPM7, TRPM8 and TRPV4 were the genes which expression levels changed significantly after Ah, Ns or Cs extract treatments. The virus load decreased when any of the Ah, Nsor Cs extracts was added to the CoV infected cells with Ah extract treatment leading to undetectable virus load for both 6 and 8 hpi. Although all the extract treatments had an effect on IL-8 secretion, TRP gene expression and virus load after CoV infection, it was the Ah extract treatment that showed the biggest difference in virus load. Therefore Ah extract is the best candidate in our hands that contains potential treatment molecule(s).

Extracts of Anthemis hyalina (Ah), Nigella sativa (Ns) and peels of Citrus sinensis (Cs) have been used as folk medicine to fight antimicrobial diseases. To evaluate the effect of extracts of Ah, Ns and Cs on the replication of coronavirus (CoV) and on the expression of TRP genes during coronavirus infection, HeLa-CEACAM1a (HeLa-epithelial carcinoembryonic antigen-related cell adhesion molecule 1a) cells were inoculated with MHV-A59 (mouse hepatitis virus-A59) at moi of 30. 1/50 dilution of the extracts was found to be the safe active dose. ELISA kits were used to detect the human IL-8 levels. Total RNA was isolated from the infected cells and cDNA was synthesized. Fluidigm Dynamic Array nanofluidic chip 96.96 was used to analyze the mRNA expression of 21 TRP genes and two control genes. Data was analyzed using the BioMark digital array software. Determinations of relative gene expression values were carried out by using the 2(-∆∆Ct) method (normalized threshold cycle (Ct) value of sample minus normalized Ct value of control). TCID50/ml (tissue culture infectious dose that will produce cytopathic effect in 50% of the inoculated tissue culture cells) was found for treatments to determine the viral loads. The inflammatory cytokine IL-8 level was found to increase for both 24 and 48 h time points following Ns extract treatment. TRPA1, TRPC4, TRPM6, TRPM7, TRPM8 and TRPV4 were the genes which expression levels changed significantly after Ah, Ns or Cs extract treatments. The virus load decreased when any of the Ah, Nsor Cs extracts was added to the CoV infected cells with Ah extract treatment leading to undetectable virus load for both 6 and 8 hpi. Although all the extract treatments had an effect on IL-8 secretion, TRP gene expression and virus load after CoV infection, it was the Ah extract treatment that showed the biggest difference in virus load. Therefore Ah extract is the best candidate in our hands that contains potential treatment molecule(s).

Chinese medicine (CM) has been used to control infectious diseases for thousands of years. In 2003 outbreaks of severe acute respiratory syndrome (SARS) occurred in China, Hong Kong and Taiwan. In view of the possible beneficial effect of CM on SARS, we conducted this study to examine whether CM is of any benefit as a supplementary treatment of SARS. Four severe laboratory-confirmed SARS patients received routine western-medicine treatment plus different supplementary treatment: CM A, CM B and CM C (placebo control). We reported the course of the cases in terms of changes in chest radiographic scores. Case 1 treated as a placebo control passed away on the 9th day after onset of disease. The other three cases treated with CM A or CM B survived. The initial findings seemed to indicate a favorable effect of CM on management of SARS. The findings need to be verified with a larger sample. Using CM as a supplementary treatment of severe SARS seems to indicate that natural herbal medicine can be used against avian influenza. Hence, such related experience or clinical trials should be taken into consideration when facing the possible outbreak of avian influenza in the future.

Chinese medicine (CM) has been used to control infectious diseases for thousands of years. In 2003 outbreaks of severe acute respiratory syndrome (SARS) occurred in China, Hong Kong and Taiwan. In view of the possible beneficial effect of CM on SARS, we conducted this study to examine whether CM is of any benefit as a supplementary treatment of SARS. Four severe laboratory-confirmed SARS patients received routine western-medicine treatment plus different supplementary treatment: CM A, CM B and CM C (placebo control). We reported the course of the cases in terms of changes in chest radiographic scores. Case 1 treated as a placebo control passed away on the 9th day after onset of disease. The other three cases treated with CM A or CM B survived. The initial findings seemed to indicate a favorable effect of CM on management of SARS. The findings need to be verified with a larger sample. Using CM as a supplementary treatment of severe SARS seems to indicate that natural herbal medicine can be used against avian influenza. Hence, such related experience or clinical trials should be taken into consideration when facing the possible outbreak of avian influenza in the future.