Potential Antiviral Effect of Korean Forest Wild Mushrooms against Feline Coronavirus (FCoV).

Coronaviruses (CoV) are among the major viruses that cause common cold in humans. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a high-risk human pathogen that derived from bat coronaviruses, although several other animals serve as CoV hosts, contributing to human infection. As the human activity area expanded, viruses previously prevalent only in animals mutated and became threats to humans as well, leading to worldwide epidemics. Therefore, controlling CoV infections in animals is essential to prevent CoV-related human infections. Feline coronavirus (FCoV) could be reportedly used as an alternative model for SARS-CoV-2. Traditionally, mushrooms are not only foods but are also consumed to prevent diseases. Importantly, certain edible and medicinal mushrooms display antibacterial and antiviral effects against respiratory pathogens; therefore, they could be tested as potential coronavirus treatment agents. In this study, we investigated if wild forest mushrooms with various reported physiological activities could exhibit an antiviral activity against CoV, using FCoV as a SARS-CoV-2 model infecting Crandell Rees feline kidney cells. We measured the antiviral activity of 11 wild mushrooms overall and our results demonstrated that Pleurotus ostreatus and Phallus luteus displayed the highest antiviral efficacy of 55.33%, followed by Tricholoma bakamatsutake at 43.77%. Grifola frondosa, Morchella esculenta, and Sarcodon imbricatus exhibited mild efficacy of 29.21%. We also tested Amanita caesareoides, Marasmius siccus, Pachyma hoelen, Phallus rubrovolvata, and Sparassis latifolia but could not detect any antiviral activity in their case. Our study confirms that wild forest mushrooms could be used as potential functional foods or pharmacological materials against coronavirus.

Comparison of the Performance of Bioresonance, Electrophoresis and RT-PCR in the Diagnosis of Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) continues to be one of the most researched infectious diseases of cats. The diagnosis of FIP is challenging, and diverse techniques have been developed for its accurate diagnosis. However, they have some limitations. The present study was conducted to investigate the efficacy of specific modulation frequency (SMF), compared to other routine diagnostic methods for detecting feline coronavirus. Blood samples were collected from 30 diseased cats suspected of having FIP based on clinical signs. Electrophoresis, polymerase chain reaction (PCR), and SMF tests were performed for each sample. The sensitivity and specificity of each test, as well as the agreement between the tests and the gold standard (the combination of PCR, electrophoresis, and bioresonance results), were calculated using the Kappa coefficient method. The sensitivity and specificity of electrophoresis, PCR, and SMF for the diagnosis of FIP were 70.6%, 70.6%, 100%, and 100%, 72.7%, 81.8%, respectively. According to the findings of the present study, SMF is effective and safe in FIP diagnosis, which is a challenge in veterinary medicine diagnosis.

Detection of anti-feline infectious peritonitis virus activity of a Chinese herb extract using geneLEAD VIII, a fully automated nucleic acid extraction/quantitative PCR testing system.

The geneLEAD VIII is a fully-automated nucleic acid extraction/quantitative PCR equipment developed by Precision System Science Co., Ltd., (PSS). To take advantage of its capability, we developed a quantitative assay system to measure growth of animal viruses. The system was used to assay one of the Chinese herbal extracts whose anti-malarial activities were previously reported and demonstrated its dose-dependent anti-viral activity against feline infectious peritonitis virus (FIPV), a feline coronavirus causing the fatal diseases in cats, and relatively low cell toxicity. The assay developed in this study is useful to screen antiviral drugs and the anti-FIPV activity of the herbal extract identified have a potential to lead to development of new drugs against FIPV and other coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

In vitro efficacy of Artemisia extracts against SARS-CoV-2.

BACKGROUND: Traditional medicines based on herbal extracts have been proposed as affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Teas and drinks containing extracts of Artemisia annua and Artemisia afra have been widely used in Africa in efforts to prevent SARS-CoV-2 infection and fight COVID-19. METHODS: The plant extracts and Covid-Organics drink produced in Madagascar were tested for plaque reduction using both feline coronavirus and SARS-CoV-2 in vitro. Their cytotoxicities were also investigated. RESULTS: Several extracts as well as Covid-Organics inhibited SARS-CoV-2 and FCoV infection at concentrations that did not affect cell viability. CONCLUSIONS: Some plant extracts show inhibitory activity against FCoV and SARS-CoV-2. However, it remains unclear whether peak plasma concentrations in humans can reach levels needed to inhibit viral infection following consumption of teas or Covid-Organics. Clinical studies are required to evaluate the utility of these drinks for COVID-19 prevention or treatment of patients.

Virucidal and antiviral effects of Thymus vulgaris essential oil on feline coronavirus.

Feline infectious peritonitis (FIP) is a fatal systemic disease of felids caused by a Coronavirus (CoV) (FIPV). In spite of its clinical relevance and impact on feline health, currently the therapeutic possibilities for treatment of FIP in cats are limited. The emergence of the pandemic Severe Respiratory Syndrome (SARS) coronavirus (CoV) type 2 (SARS-CoV-2), etiological agent of the 2019 Coronavirus Disease (COVID-19), able to infect a broad spectrum of animal species including cats, triggered the interest for the development of novel molecules with antiviral activity for treatment of CoV infections in humans and animals. Essential oils (EOs) have raised significant attention for their antiviral properties integrating and, in some cases, replacing conventional drugs. Thymus vulgaris EO (TEO) has been previously shown to be effective against several RNA viruses including CoVs. In the present study the antiviral efficacy of TEO against FIPV was evaluated in vitro. TEO at 27 µg/ml was able to inhibit virus replication with a significant reduction of 2 log10 TCID50/50 µl. Moreover, virucidal activity was tested using TEO at 27 and 270 µg/ml, over the cytotoxic threshold, determining a reduction of viral titre as high as 3.25 log10 TCID50/50 µl up to 1 h of time contact. These results open several perspectives in terms of future applications and therapeutic possibilities for coronaviruses considering that FIPV infection in cats could be a potential model for the study of antivirals against CoVs.

Structural-based virtual screening and in vitro assays for small molecules inhibiting the feline coronavirus 3CL protease as a surrogate platform for coronaviruses.

Feline infectious peritonitis (FIP) which is caused by feline infectious peritonitis virus (FIPV), a variant of feline coronavirus (FCoV), is a member of family Coronaviridae, together with severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. So far, neither effective vaccines nor approved antiviral therapeutics are currently available for the treatment of FIPV infection. Both human and animal CoVs shares similar functional proteins, particularly the 3CL protease (3CLpro), which plays the pivotal role on viral replication. We investigated the potential drug-liked compounds and their inhibitory interaction on the 3CLpro active sites of CoVs by the structural-bases virtual screening. Fluorescence resonance energy transfer (FRET) assay revealed that three out of twenty-eight compounds could hamper FIPV 3CLpro activities with IC50 of 3.57 ± 0.36 µM to 25.90 ± 1.40 µM, and Ki values of 2.04 ± 0.08 to 15.21 ± 1.76 µM, respectively. Evaluation of antiviral activity using cell-based assay showed that NSC629301 and NSC71097 could strongly inhibit the cytopathic effect and also reduced replication of FIPV in CRFK cells in all examined conditions with the low range of EC50 (6.11 ± 1.90 to 7.75 ± 0.48 µM and 1.99 ± 0.30 to 4.03 ± 0.60 µM, respectively), less than those of ribavirin and lopinavir. Analysis of FIPV 3CLpro-ligand interaction demonstrated that the selected compounds reacted to the crucial residues (His41 and Cys144) of catalytic dyad. Our investigations provide a fundamental knowledge for the further development of antiviral agents and increase the number of anti-CoV agent pools for feline coronavirus and other related CoVs.

Natural product-derived phytochemicals as potential agents against coronaviruses: A review.

Coronaviruses are responsible for a growing economic, social and mortality burden, as the causative agent of diseases such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), avian infectious bronchitis virus (IBV) and COVID-19. However, there is a lack of effective antiviral agents for many coronavirus strains. Naturally existing compounds provide a wealth of chemical diversity, including antiviral activity, and thus may have utility as therapeutic agents against coronaviral infections. The PubMed database was searched for papers including the keywords coronavirus, SARS or MERS, as well as traditional medicine, herbal, remedy or plants, with 55 primary research articles identified. The overwhelming majority of publications focussed on polar compounds. Compounds that show promise for the inhibition of coronavirus in humans include scutellarein, silvestrol, tryptanthrin, saikosaponin B2, quercetin, myricetin, caffeic acid, psoralidin, isobavachalcone, and lectins such as griffithsin. Other compounds such as lycorine may be suitable if a therapeutic level of antiviral activity can be achieved without exceeding toxic plasma concentrations. It was noted that the most promising small molecules identified as coronavirus inhibitors contained a conjugated fused ring structure with the majority being classified as being polyphenols.

In silico and in vitro analysis of small molecules and natural compounds targeting the 3CL protease of feline infectious peritonitis virus.

The computational search of chemical libraries has been used as a powerful tool for the rapid discovery of candidate compounds. To find small molecules with anti-feline infectious peritonitis virus (FIPV) properties, we utilized a virtual screening technique to identify the active site on the viral protease for the binding of the available natural compounds. The protease 3CL (3CLpro) plays an important role in the replication cycle of FIPV and other viruses within the family Coronaviridae. The 15 best-ranked candidate consensus compounds, based on three docking tools, were evaluated for further assays. The protease inhibitor assay on recombinant FIPV 3CLpro was performed to screen the inhibitory effect of the candidate compounds with IC50 ranging from 6.36 ± 2.15 to 78.40 ± 2.60 µM. As determined by the cell-based assay, the compounds NSC345647, NSC87511, and NSC343256 showed better EC50 values than the broad-spectrum antiviral drug ribavirin and the protease inhibitor lopinavir, under all the test conditions including pre-viral entry, post-viral entry, and prophylactic activity. The NSC87511 particularly yielded the best selective index (>4; range of SI = 13.80-22.90). These results indicated that the natural small-molecular compounds specifically targeted the 3CLpro of FIPV and inhibited its replication. Structural modification of these compounds may generate a higher anti-viral potency for the further development of a novel therapy against FIP.

Crystal Structure of Feline Infectious Peritonitis Virus Main Protease in Complex with Synergetic Dual Inhibitors.

UNLABELLED: Coronaviruses (CoVs) can cause highly prevalent diseases in humans and animals. Feline infectious peritonitis virus (FIPV) belongs to the genus Alphacoronavirus, resulting in a lethal systemic granulomatous disease called feline infectious peritonitis (FIP), which is one of the most important fatal infectious diseases of cats worldwide. No specific vaccines or drugs have been approved to treat FIP. CoV main proteases (M(pro)s) play a pivotal role in viral transcription and replication, making them an ideal target for drug development. Here, we report the crystal structure of FIPV M(pro) in complex with dual inhibitors, a zinc ion and a Michael acceptor. The complex structure elaborates a unique mechanism of two distinct inhibitors synergizing to inactivate the protease, providing a structural basis to design novel antivirals and suggesting the potential to take advantage of zinc as an adjunct therapy against CoV-associated diseases. IMPORTANCE: Coronaviruses (CoVs) have the largest genome size among all RNA viruses. CoV infection causes various diseases in humans and animals, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). No approved specific drugs or vaccinations are available to treat their infections. Here, we report a novel dual inhibition mechanism targeting CoV main protease (M(pro)) from feline infectious peritonitis virus (FIPV), which leads to lethal systemic granulomatous disease in cats. M(pro), conserved across all CoV genomes, is essential for viral replication and transcription. We demonstrated that zinc ion and a Michael acceptor-based peptidomimetic inhibitor synergistically inactivate FIPV M(pro). We also solved the structure of FIPV M(pro) complexed with two inhibitors, delineating the structural view of a dual inhibition mechanism. Our study provides new insight into the pharmaceutical strategy against CoV M(pro) through using zinc as an adjuvant therapy to enhance the efficacy of an irreversible peptidomimetic inhibitor.

Peptides corresponding to the predicted heptad repeat 2 domain of the feline coronavirus spike protein are potent inhibitors of viral infection.

BACKGROUND: Feline infectious peritonitis (FIP) is a lethal immune-mediated disease caused by feline coronavirus (FCoV). Currently, no therapy with proven efficacy is available. In searching for agents that may prove clinically effective against FCoV infection, five analogous overlapping peptides were designed and synthesized based on the putative heptad repeat 2 (HR2) sequence of the spike protein of FCoV, and the antiviral efficacy was evaluated. METHODS: Plaque reduction assay and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay were performed in this study. Peptides were selected using a plaque reduction assay to inhibit Feline coronavirus infection. RESULTS: The results demonstrated that peptide (FP5) at concentrations below 20 µM inhibited viral replication by up to 97%. The peptide (FP5) exhibiting the most effective antiviral effect was further combined with a known anti-viral agent, human interferon-α (IFN-α), and a significant synergistic antiviral effect was observed. CONCLUSION: Our data suggest that the synthetic peptide FP5 could serve as a valuable addition to the current FIP prevention methods.

Synergistic antiviral effect of Galanthus nivalis agglutinin and nelfinavir against feline coronavirus.

Feline infectious peritonitis (FIP) is a fatal disease in domestic and nondomestic felids caused by feline coronavirus (FCoV). Currently, no effective vaccine is available for the prevention of this disease. In searching for agents that may prove clinically effective against FCoV infection, 16 compounds were screened for their antiviral activity against a local FCoV strain in Felis catus whole fetus-4 cells. The results showed that Galanthus nivalis agglutinin (GNA) and nelfinavir effectively inhibited FCoV replication. When the amount of virus preinoculated into the test cells was increased to mimic the high viral load present in the target cells of FIP cats, GNA and nelfinavir by themselves lost their inhibitory effect. However, when the two agents were added together to FCoV-infected cells, a synergistic antiviral effect defined by complete blockage of viral replication was observed. These results suggest that the combined use of GNA and nelfinavir has therapeutic potential in the prophylaxis and treatment of cats with early-diagnosed FIP.

Type IV hypersensitivity in the pathogenesis of FIPV-induced lesions.

In focal lesions of feline infectious peritonitis (FIP), the cells involved in the delayed-type hypersensitivity were identified in formalin-fixed paraffin-embedded and frozen samples taken from 35 affected cats. The clinical diagnosis of FIP was confirmed by necropsy, histology and direct immunofluorescence against the coronaviruses on cryostatic sections. The immune cells were detected immunohistochemically by the Avidin-Biotin-Complex (ABC) method using either polyclonal antibodies against lymphoid antigens (CD3) or monoclonal antibodies against lymphoid (PAN-T, CD4, CD8) and myeloid antigens (MAC387). Better identification of T cells and macrophages was found on formalin-fixed paraffin-embedded sections than on cryostatic ones, while T lymphocyte subpopulations could be differentiated only in cryostatic sections. Type IV hypersensitivity was detected in focal feline infectious peritonitis virus (FIPV)-induced lesions from progressive activation of T lymphocytes, mainly CD4+, and the presence of granulocytes and macrophages. The FIPV-induced lesions could be studied as examples of granulomas caused by unconventional antigens, such as viruses or immune complexes.

Detection of feline infectious peritonitis virus infection in cell cultures and peripheral blood mononuclear leukocytes of experimentally infected cats using a biotinylated cDNA probe.

A dot blot hybridization assay, using a biotinylated cDNA probe, was able to detect feline infectious peritonitis virus (FIPV) RNA in Felis catus whole fetus (fcwf-4) cells infected with the FIPV isolates DF2, 79-1146, UCD1, and UCD2. The probe cross-hybridized in the dot blot assay with nucleic acid of a closely related feline coronavirus, feline enteric coronavirus (FEVC)-79-1683. To construct the probe, a 2.5 kilobase cDNA, prepared from FIPV-DF2 genomic RNA, was molecularly cloned. The recombinant cDNA clone was digested with the restriction endonuclease Rsa I, and an 870 basepair Rsa I fragment was isolated from vector DNA by agarose electrophoresis and glass-milk purification. This fragment was complementary to the 3' three fourths of the nucleocapsid gene. The hybridization probe was prepared by random primed labeling in the presence of biotin-11-dUTP. Using an avidin-alkaline phosphatase conjugate and chemiluminescent substrate detection system, virus could be detected in as few as 3000 infected cells. In an in vivo study, the probe was used to detect FIPV RNA in peripheral blood mononuclear leukocytes (PBML) isolated at various post-infection days (PID) from cats experimentally infected with the FIP-producing coronavirus isolate FIPV-79-1146 or FIPV-DF2. Viral RNA could be detected in as few as 12,000 PBML isolated from cats at PID 7 and in 50,000 PBML at PID 22. There was no consistent pattern, however, between hybridization results and prognosis or severity of disease at the time of sampling. Despite some cross-hybridization with FECV RNA, this probe should be useful for diagnosis of FIP, because cats infected with FECV most likely do not become viremic.