The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (M^pro) and papain like protease (PL^pro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851M^pro inhibitors and 205 PL^pro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both M^pro and PL^pro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of M^pro inhibitors and over 20% of PL^pro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 M^pro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.
Humans ; Antiviral Agents/chemistry* ; COVID-19 ; COVID-19 Drug Treatment ; High-Throughput Screening Assays ; Molecular Docking Simulation ; Protease Inhibitors/chemistry* ; SARS-CoV-2/enzymology* ; Viral Nonstructural Proteins

OBJECTIVE: To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene. METHODS: A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay. RESULTS: The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses. CONCLUSION A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.
African Swine Fever/virology* ; African Swine Fever Virus/isolation & purification* ; Animals ; Nucleic Acid Amplification Techniques/methods* ; Recombinases/chemistry* ; Sensitivity and Specificity ; Swine ; Viral Proteins/genetics*

Hepatitis B virus core protein (HBc) has become a hot spot in drug carrier protein research due to its natural particle self-assembly ability and ease of modification. The truncation of the C-terminal polyarginine domain (CTD, aa 151-183) of HBc does not affect the self-assembly of the particles. However, it does affect the internal and external charges of the particles, which may subsequently affect drug encapsulation. Thus, the truncated C-terminal polyarginine domain (CTD) of HBc and the inserted RGD peptide were selected to construct and express three HBc variants (RH) encapsulated with ICG (RH/ICG) with different C-terminal lengths to compare the stability and drug activity of their nanoformulations. RH160/ICG was found to have a great advantages in encapsulation efficiency and biological imaging. Compared with other HBc variants, RH160/ICG significantly improved encapsulation efficiency, up to 32.77%±1.23%. Cytotoxicity and hemolysis assays further demonstrated the good biocompatibility of RH160/ICG. Cell uptake and in vivo imaging experiments in mice showed that RH160/ICG could efficiently deliver ICG in tumor cells and tumor sites with good imaging effect. This research provides a new direction for further expanding the diagnosis and treatment application of ICG and development of HBc-based nanoparticle drug carrier platform.
Animals ; Hepatitis B/drug therapy* ; Hepatitis B Core Antigens ; Indocyanine Green/chemistry* ; Mice ; Nanoparticles/chemistry* ; Viral Core Proteins

ORF3 protein, the single accessory protein encoded by porcine epidemic diarrhea virus (PEDV), is related to viral pathogenicity. In order to determine the cytoplasmic location signal of PEDV ORF3, we constructed a series of recombinant plasmids carrying full-length or truncated segments of PEDV DR13 ORF3 protein. When the acquired plasmids were transfected into Vero cells, expression and distribution of the EGFP-fused full-length ORF3 protein and its truncated forms in the cells were observed by laser confocal microscopy. The results showed that ORF3 protein or their truncated forms containing 40-91 aa segment including two transmembrane domains were localized in the cytoplasm, whereas ORF3 truncated peptides without the 40-91 aa segment were distributed in the whole cell (in both cytoplasm and nucleus). This suggests that the 40-91 aa is the key structural domain determining cytoplasmic location of PEDV ORF3 protein. The discovery provides reference for further clarifying intracellular transport and biological function of PEDV ORF3 protein.
Amino Acid Sequence ; Animals ; Chlorocebus aethiops ; Coronavirus Infections ; virology ; Cytoplasm ; virology ; Porcine epidemic diarrhea virus ; genetics ; Protein Domains ; Swine ; Vero Cells ; Viral Proteins ; chemistry ; metabolism

OBJECTIVE: Lung-toxin Dispelling Formula No. 1, referred to as Respiratory Detox Shot (RDS), was developed based on a classical prescription of traditional Chinese medicine (TCM) and the theoretical understanding of herbal properties within TCM. Therapeutic benefits of using RDS for both disease control and prevention, in the effort to contain the coronavirus disease 2019 (COVID-19), have been shown. However, the biochemically active constituents of RDS and their mechanisms of action are still unclear. The goal of the present study is to clarify the material foundation and action mechanism of RDS. METHODS: To conduct an analysis of RDS, an integrative analytical platform was constructed, including target prediction, protein-protein interaction (PPI) network, and cluster analysis; further, the hub genes involved in the disease-related pathways were identified, and the their corresponding compounds were used for in vitro validation of molecular docking predictions. The presence of these validated compounds was also measured in samples of the RDS formula to quantify the abundance of the biochemically active constituents. In our network pharmacological study, a total of 26 bioinformatic programs and databases were used, and six networks, covering the entire Zang-fu viscera, were constructed to comprehensively analyze the intricate connections among the compounds-targets-disease pathways-meridians of RDS. RESULTS: For all 1071 known chemical constituents of the nine ingredients in RDS, identified from established TCM databases, 157 passed drug-likeness screening and led to 339 predicted targets in the constituent-target network. Forty-two hub genes with core regulatory effects were extracted from the PPI network, and 134 compounds and 29 crucial disease pathways were implicated in the target-constituent-disease network. Twelve disease pathways attributed to the Lung-Large Intestine meridians, with six and five attributed to the Kidney-Urinary Bladder and Stomach-Spleen meridians, respectively. One-hundred and eighteen candidate constituents showed a high binding affinity with SARS-coronavirus-2 3-chymotrypsin-like protease (3CL), as indicated by molecular docking using computational pattern recognition. The in vitro activity of 22 chemical constituents of RDS was validated using the 3CL inhibition assay. Finally, using liquid chromatography mass spectrometry in data-independent analysis mode, the presence of seven out of these 22 constituents was confirmed and validated in an aqueous decoction of RDS, using reference standards in both non-targeted and targeted approaches. CONCLUSION RDS acts primarily in the Lung-Large Intestine, Kidney-Urinary Bladder and Stomach-Spleen meridians, with other Zang-fu viscera strategically covered by all nine ingredients. In the context of TCM meridian theory, the multiple components and targets of RDS contribute to RDS's dual effects of health-strengthening and pathogen-eliminating. This results in general therapeutic effects for early COVID-19 control and prevention.
Antiviral Agents ; chemistry ; therapeutic use ; Betacoronavirus ; chemistry ; enzymology ; Coronavirus Infections ; drug therapy ; prevention & control ; virology ; Cysteine Endopeptidases ; chemistry ; Drugs, Chinese Herbal ; chemistry ; therapeutic use ; Humans ; Mass Spectrometry ; Medicine, Chinese Traditional ; Molecular Docking Simulation ; Pandemics ; prevention & control ; Pneumonia, Viral ; drug therapy ; prevention & control ; virology ; Protein Interaction Maps ; Viral Nonstructural Proteins ; chemistry

BACKGROUND: The incidence and etiology of hepatocellular carcinoma (HCC) vary widely according to race and geographic regions. The insertional mutagenesis of adeno-associated virus 2 (AAV2) has recently been considered a new viral etiology of HCC. The aim of this study was to investigate the frequency and clinical characteristics of AAV2 in Korean patients with HCC. METHODS: A total of 289 unrelated Korean patients with HCC, including 159 Hepatitis-B-related cases, 16 Hepatitis-C-related cases, and 114 viral serology-negative cases, who underwent surgery at the Samsung Medical Center in Korea from 2009 to 2014 were enrolled in this study. The presence of AAV2 in fresh-frozen tumor tissues was investigated by DNA PCR and Sanger sequencing. The clinical and pathological characteristics of AAV2-associated HCC in these patients were compared with previous findings in French patients. RESULTS: The AAV2 detection rate in Korean patients (2/289) was very low compared with that in French patients (11/193). Similar to the French patients, the Korean patients with AAV2-related HCC showed no signs of liver cirrhosis. The Korean patients were younger than the French patients with the same AAV2-associated HCC; the ages at diagnosis of the two Korean patients were 47 and 39 yr, while the median age of the 11 French patients was 55 yr (range 43-90 yr). CONCLUSIONS: AAV2-associated HCC was very rare in Korean patients with HCC. Despite a limited number of cases, this study is the first to report the clinical characteristics of Korean patients with AAV2-associated HCC. These findings suggest epidemiologic differences in viral hepatocarcinogenesis between Korean and European patients.
Adult ; Asian Continental Ancestry Group ; Capsid Proteins/genetics ; Carcinoma, Hepatocellular/etiology/*pathology/virology ; DNA, Viral/chemistry/genetics/metabolism ; DNA-Binding Proteins/genetics ; Dependovirus/*genetics/isolation & purification/pathogenicity ; Female ; Humans ; Incidence ; Inverted Repeat Sequences/genetics ; Liver Neoplasms/etiology/*pathology/virology ; Male ; Middle Aged ; Parvoviridae Infections/complications/epidemiology ; Polymerase Chain Reaction ; Republic of Korea ; Sequence Analysis, DNA ; Viral Proteins/genetics

To analyze the genetic characterization of epidemic mumps virus strains in Liaoning Province and provide the basis for mumps control. A total of 32 mumps viruses strains were isolated during 2008-2104. The fragment of SH genes and HN genes were amplified by RT-PCR, the PCR products were sequenced and analyzed. Basing on the 316 nucleotides of SH gene, The phylogenetic analyses were processed with the data of WHO mumps reference strains downloaded from GenBank and 32 mumps viruses strains. It showed that the 31 mumps virus strains belong to F genotype except MuVi/Liaoning. CHN/16.11 which was G genotype . Comparing to the A reference strains (Jeryl-Lynn and S-79), F genotype MuV were mutated on 12 amino acids sites and 27 amino acids siteson on HN gene. F genotype MuV added one N-glycosylation site in 464th-466th amino acids. The antigenic sites on HN were mutated on 121th, 123th, 279th, 287th, 336th, 356th and 442th. Maybe, it will influence the MuV antigenic.
Base Sequence ; China ; Genotype ; HN Protein ; chemistry ; genetics ; Humans ; Molecular Sequence Data ; Mumps ; virology ; Mumps virus ; chemistry ; classification ; genetics ; isolation & purification ; Phylogeny ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics

We wished to sequence the full-length genomes of the DHL10M110 strain of the Akabane virus (AKV) isolated from mosquitoes in Yunnan Province, China, in 2010. We also wished to analyze the characteristics of these complete nucleotide sequences. The complete genomic sequence of the DHL10M110 strain from Yunnan Province was obtained by reverse transcription-polymerase chain reaction and direct sequencing. We found that the length of the L, M and S gene nucleotide sequences of the DHL10M110 strain were 6 869-bp, 4 309-bp and 856-bp, respectively, including the open reading frame (ORF) nucleotide sequences of 6 756-bp (L), 4 206-bp (M) and 702-bp (S), encoding 2252, 1402 and 234 amino-acid polyproteins, respectively. Phylogenetic analyses based on L-fragment ORF showed that the DHL10M110 strain had a close relationship with the OBE-1 strain of the AKV from Japan and AKVS-7/SKR/2010 strain of the AKV from South Korea. Phylogenetic analyses based on M- and S-fragment ORF showed that the DHL10M110 strain had a close relationship with the epidemic strains of the AKV from Japan, South Korea and Taiwan, but that the DHL10M110 strain had a lone evolutionary branch. In terms of nucleotide (amino acid) homology, the similarity of L-, M- and S-fragment ORFs of the DHL10M110 strain to the OBE-1 strain from Japan was 92.6% (98%), 88.5% (94%) and 96.4% (99.1%), respectively. When comparing the DHL10M110 strain with the OBE-1 strain, we noted 45, 84, and 2 different sites in the amino acids of L, M and S fragments, respectively. Homology and phylogenetic analyses also suggested that the DHL10M110 strain had a distant relationship with the epidemic strains of the AKV from Kenya and Australia. Also, we confirmed by complete genomic sequence analyses that the DHL10M110 strain was clade-Asia of the AKV. However, differences between the DHL10M110 strain compared with strains from Japan and South Korea were also noted. These results suggest that the DHL10M110 strain harbored relatively stable genetic characteristics and distinct regional features. This is the first time that full-length genomic sequences of the DHL10M110 strain of the AKV in mainland China have been obtained.
Amino Acid Sequence ; Animals ; Base Sequence ; Bunyaviridae Infections ; transmission ; virology ; China ; Culicidae ; virology ; Female ; Genome, Viral ; Humans ; Insect Vectors ; virology ; Male ; Molecular Sequence Data ; Open Reading Frames ; Orthobunyavirus ; classification ; genetics ; isolation & purification ; Phylogeny ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics

Avian leukosis virus subgroup J (ALV-J) is an avian retrovirus that can induce myelocytomas. A high-frequency mutation in gene envelope endows ALV-J with the potential for cross-species transmission. We wished to ascertain if the ALV-J can spread across species under selection pressure in susceptible and resistant hosts. First, we inoculated (in turn) two susceptible host birds (specific pathogen-free (SPF) chickens and turkeys). Then, we inoculated three resistant hosts (pheasants, quails and ducks) to detect the viral shedding, pathologic changes, and genetic evolution of different isolates. We found that pheasants and quails were infected under the selective pressure that accumulates stepwise in different hosts, and that ducks were not infected. Infection rates for SPF chickens and turkeys were 100% (16/16), whereas those for pheasants and quails were 37.5% (6/16) and 11.1% (3/27). Infected hosts showed immune tolerance, and inflammation and tissue damage could be seen in the liver, spleen, kidneys and cardiovascular system. Non-synonymous mutation and synonymous ratio (NS/S) analyses revealed the NS/S in hypervariable region (hr) 2 of pheasants and quails was 2.5. That finding suggested that mutation of isolates in pheasants and quails was induced by selective pressure from the resistant host, and that the hr2 region is a critical domain in cross-species transmission of ALV-J. Sequencing showed that ALV-J isolates from turkeys, pheasants and quails had moved away from the original virus, and were closer to the ALV-J prototype strain HPRS-103. However, the HPRS-103 strain cannot infect pheasants and quails, so further studies are needed.
Amino Acid Sequence ; Animals ; Avian Leukosis ; transmission ; virology ; Avian Leukosis Virus ; classification ; genetics ; physiology ; Chickens ; Ducks ; virology ; Galliformes ; virology ; Host Specificity ; Molecular Sequence Data ; Poultry Diseases ; transmission ; virology ; Quail ; virology ; Sequence Alignment ; Turkeys ; virology ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

Faldaprevir analogue molecule (FAM) has been reported to effectively inhibit the catalytic activity of HCV NS3/4A protease, making it a potential lead compound against HCV. A series of HCV NS3/4A protease crystal structures were analyzed by bioinformatics methods, and the FAM-HCV NS3/4A protease crystal structure was chosen for this study. A 20.4 ns molecular dynamics simulation of the complex consists of HCV NS3/4A protease and FAM was conducted. The key amino acid residues for interaction and the binding driving force for the molecular recognition between the protease and FAM were identified from the hydrogen bonds and binding free energy analyses. With the driving force of hydrogen bonds and van der Waals, FAM specifically bind to the active pocket of HCV NS3/4A protease, including V130-S137, F152-D166, D77-D79 and V55, which agreed with the experimental data. The effect of R155K, D168E/V and V170T site-directed mutagenesis on FAM molecular recognition was analyzed for their effect on drug resistance, which provided the possible molecular explanation of FAM resistance. Finally, the system conformational change was explored by using free energy landscape and conformational cluster. The result showed four kinds of dominant conformation, which provides theoretical basis for subsequent design of Faldaprevir analogue inhibitors based on the structure of HCV NS3/4A protease.
Antiviral Agents ; chemistry ; Carrier Proteins ; chemistry ; Drug Resistance, Viral ; Endopeptidases ; Hepacivirus ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; Oligopeptides ; chemistry ; Protease Inhibitors ; chemistry ; Serine Proteases ; Thiazoles ; chemistry ; Viral Nonstructural Proteins ; chemistry