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SARS-CoV-2 related coronaviruses (SARS-CoV-2r) from Guangdong and Guangxi pangolins have been implicated in the emergence of SARS-CoV-2 and future pandemics. We previously reported the culture of a SARS-CoV-2r GX_P2V from Guangxi pangolins. Here we report the GX_P2V isolate rapidly adapted to Vero cells by acquiring two genomic mutations: an alanine to valine substitution in the nucleoprotein and a 104-nucleotide deletion in the hypervariable region (HVR) of the 3-terminus untranslated region (3-UTR). We further report the characterization of the GX_P2V variant in in vitro and in vivo infection models. In cultured Vero and BGM cells, the GX_P2V variant produced minimal cell damage and small plaques. The GX_P2V variant infected golden hamsters and BALB/c mice but was highly attenuated. Golden hamsters infected intranasally had a short duration of productive infection. These productive infections induced neutralizing antibodies against pseudoviruses of GX_P2V and SARS-CoV-2. Collectively, our data show that the GX_P2V variant is highly attenuated in in vitro and in vivo infection models. Attenuation of the variant is likely due to the 104-nt deletion in the HVR in the 3-UTR. This study furthers our understanding of pangolin coronaviruses pathogenesis and provides novel insights for the design of live attenuated vaccines against SARS-CoV-2.
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As a member of the dibenzyl isoquinoline alkaloid family, cepharathine is an alkaloid from the traditional Chinese medicine cepharathine, which is mainly used for treatment of leukopenia and other diseases. Recent studies of the inhibitory effect of cepharathine against SARS-CoV-2 have attracted widespread attention and aroused heated discussion. As the original discoverer of the anti-SARS-CoV-2 activity of cepharanthine, here we briefly summarize the discovery of cepharanthine and review important progress in relevant studies concerning the discovery and validation of anti-SARS-CoV-2 activity of cepharathine, its antiviral mechanisms and clinical trials of its applications in COVID-19 therapy.
Subject(s)
Humans , Antiviral Agents/therapeutic use , Benzylisoquinolines/therapeutic use , COVID-19 , SARS-CoV-2ABSTRACT
Since the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in several somatic cells, little is known about the infection of SASRS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that SARS-CoV-2 pseudovirus and GX_P2V could infect lung progenitor and even anterior foregut endoderm cells causing these cells death, which differentiated from human embryonic stem cells (hESCs). The infection and replication of SARS-CoV-2 and GX_P2V were inhibited when treated with whey protein of breastmilk and Remdesivir, confirming that these two viruses could infect lung progenitor and even anterior foregut endoderm. Moreover, we found that SARS-CoV-2 pseudovirus could infect endoderm and ectoderm. We found that whey protein blocked SARS-CoV-2 infecting these cells. In line with the SARS-CoV-2 results, GX_P2V could also infected endoderm and ectoderm, and also was inhibited by Remdesivir treatment. Although expressing coronavirus related receptor such as ACE2 and TMPRSS2, mesoderm cells are not permissive for SARS-CoV-2 and GX_P2V infection, which needed further to study the mechanisms. Interestingly, we also found that hESCs, which also express ACE2 and TMPRSS2 markers, are permissive for GX_P2V but not SARS-CoV-2 pseudovirus infection and replication, indicating the widespread cell types for GX_P2V infection. Heparin treatment blocked efficiently viral infection. These results provided insight that these stem cells maybe provided a stable repository of coronavirus function or genome. The potential consequence of SARS-CoV-2 and animal coronavirus such as GX_P2V infection in hESCs, germ layer and induced progenitors should be closely monitored.
ABSTRACT
Since the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human breastmilk, little is known about the antiviral property of human breastmilk to SARS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that whey protein from human breastmilk effectively inhibited both SARS-CoV-2 and GX_P2V by blocking viral attachment, entry and even post-entry viral replication. Moreover, human whey protein inhibited infectious virus production proved by the plaque assay. We found that whey protein from different species such as cow and goat also showed anti-coronavirus properties. And commercial bovine milk also showed similar activity. Interestingly, the main antimicrobial components of breastmilk, such as Lactoferrin and IgA antibody, showed limited anti-coronavirus activity, indicating that other factors of breastmilk may play the important anti-coronavirus role. Taken together, we reported that whey protein inhibits SARS-CoV-2 and its related virus of GX_P2V. These results rule out whey protein as a direct-acting inhibitor of SARS-CoV-2 and GX_P2V infection and replication and further investigation of its molecular mechanism of action in the context of COVID-19.
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The ongoing coronavirus disease 2019 (COVID-19) pandemic is a serious threat to global public health, and imposes severe burdens on the entire human society. The severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) can cause severe respiratory illness and death. Currently, there are no specific antiviral drugs that can treat COVID-19. Several vaccines against SARS-CoV-2 are being actively developed by research groups around the world. The surface S (spike) protein and the highly expressed internal N (nucleocapsid) protein of SARS-CoV-2 are widely considered as promising candidates for vaccines. In order to guide the design of an effective vaccine, we need experimental data on these potential epitope candidates. In this study, we mapped the immunodominant (ID) sites of S protein using sera samples collected from recently discharged COVID-19 patients. The SARS-CoV-2 S protein-specific antibody levels in the sera of recovered COVID-19 patients were strongly correlated with the neutralising antibody titres. We used epitope mapping to determine the landscape of ID sites of S protein, which identified nine linearized B cell ID sites. Four out of the nine ID sites were found in the receptor-binding domain (RBD). Further analysis showed that these ID sites are potential high-affinity SARS-CoV-2 antibody binding sites. Peptides containing two out of the nine sites were tested as vaccine candidates against SARS-CoV-2 in a mouse model. We detected epitope-specific antibodies and SARS-CoV-2-neutralising activity in the immunised mice. This study for the first time provides human serological data for the design of vaccines against COVID-19.
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The ongoing outbreak of viral pneumonia in China and beyond is associated with a novel coronavirus, provisionally termed 2019-nCoV. This outbreak has been tentatively associated with a seafood market in Wuhan, China, where the sale of wild animals may be the source of zoonotic infection. Although bats are likely reservoir hosts for 2019-nCoV, the identity of any intermediate host facilitating transfer to humans is unknown. Here, we report the identification of 2019-nCoV related coronaviruses in pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identified pangolin associated CoVs that belong to two sub-lineages of 2019-nCoV related coronaviruses, including one very closely related to 2019-nCoV in the receptor-binding domain. The discovery of multiple lineages of pangolin coronavirus and their similarity to 2019-nCoV suggests that pangolins should be considered as possible intermediate hosts for this novel human virus and should be removed from wet markets to prevent zoonotic transmission.
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Background@#Medicines for the treatment of 2019-novel coronavirus (2019-nCoV) infections are urgently needed. However, drug screening using live 2019-nCoV requires high-level biosafety facilities, which imposes an obstacle for those without such facilities or 2019-novel coronavirus (2019-nCoV). This study aims to repurpose the clinically approved drugs for the treatment of coronavirus disease 2019 (COVID-19) in a 2019-nCoV related coronavirus model.@*Methods@#A 2019-nCoV related pangolin coronavirus GX_P2V/pangolin/2017/ Guangxi was described. Whether GX_P2X uses angiotensin-converting enzyme 2 (ACE2) as the cell receptor was investigated by using small interfering RNA (siRNA) -mediated silencing of ACE2. The pangolin coronavirus model was used to identify drug candidates for treating 2019-nCoV infection. Two libraries of 2406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on Vero E6 cells by GX_P2X infection. The antiviral activities and antiviral mechanisms of potential drugs were further investigated. Viral yields of RNAs and infectious particles were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and plaque assay, respectively.@*Results@#The spike protein of coronavirus GX_P2V shares 92.2% amino acid identity with that of 2019-nCoV isolate Wuhan-hu-1, and uses ACE2 as the receptor for infection just like 2019-nCoV. Three drugs-cepharanthine (CEP), selamectin and mefloquine hydrochloride exhibited complete inhibition of cytopathic effects in cell culture at 10 μmol/L. CEP demonstrated the most potent inhibition of GX_P2V infection, with a concentration for 50% of maximal effect [EC50] of 0.98 μmol/L. The viral RNA yield in cells treated with 10 μmol/L CEP was 15,393-fold lower than in cells without CEP treatment ([6.48±0.02]×10-4 vs. 1.00±0.12, t=150.38, P<0.001) at 72 h post-infection (p.i.). Plaque assays found no production of live viruses in media containing 10 μmol/L CEP at 48 h p.i. Furthermore, we found CEP has potent antiviral activities against both viral entry (1.00±0.37 vs. 0.46±0.12, t=2.42, P<0.05) and viral replication (1.00±0.43 vs. [6.18±0.95]×10-4, t=3.98, P<0.05).@*Conclusions@#Our pangolin coronavirus GX_P2V is a workable model for 2019-nCoV research. CEP, selamectin and mefloquine hydrochloride are potential drugs for treating 2019-nCoV infection. Our results strongly suggest that CEP is a wide-spectrum inhibitor of pan-betacoronavirus, and clinical trial of CEP for treatment of 2019-nCoV infection is warranted.
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BACKGROUND@#Medicines for the treatment of 2019-novel coronavirus (2019-nCoV) infections are urgently needed. However, drug screening using live 2019-nCoV requires high-level biosafety facilities, which imposes an obstacle for those institutions without such facilities or 2019-nCoV. This study aims to repurpose the clinically approved drugs for the treatment of coronavirus disease 2019 (COVID-19) in a 2019-nCoV-related coronavirus model.@*METHODS@#A 2019-nCoV-related pangolin coronavirus GX_P2V/pangolin/2017/Guangxi was described. Whether GX_P2V uses angiotensin-converting enzyme 2 (ACE2) as the cell receptor was investigated by using small interfering RNA (siRNA)-mediated silencing of ACE2. The pangolin coronavirus model was used to identify drug candidates for treating 2019-nCoV infection. Two libraries of 2406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on Vero E6 cells by GX_P2V infection. The anti-viral activities and anti-viral mechanisms of potential drugs were further investigated. Viral yields of RNAs and infectious particles were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and plaque assay, respectively.@*RESULTS@#The spike protein of coronavirus GX_P2V shares 92.2% amino acid identity with that of 2019-nCoV isolate Wuhan-hu-1, and uses ACE2 as the receptor for infection just like 2019-nCoV. Three drugs, including cepharanthine (CEP), selamectin, and mefloquine hydrochloride, exhibited complete inhibition of cytopathic effects in cell culture at 10 μmol/L. CEP demonstrated the most potent inhibition of GX_P2V infection, with a concentration for 50% of maximal effect [EC50] of 0.98 μmol/L. The viral RNA yield in cells treated with 10 μmol/L CEP was 15,393-fold lower than in cells without CEP treatment ([6.48 ± 0.02] × 10vs. 1.00 ± 0.12, t = 150.38, P < 0.001) at 72 h post-infection (p.i.). Plaque assays found no production of live viruses in media containing 10 μmol/L CEP at 48 h p.i. Furthermore, we found CEP had potent anti-viral activities against both viral entry (0.46 ± 0.12, vs.1.00 ± 0.37, t = 2.42, P < 0.05) and viral replication ([6.18 ± 0.95] × 10vs. 1.00 ± 0.43, t = 3.98, P < 0.05).@*CONCLUSIONS@#Our pangolin coronavirus GX_P2V is a workable model for 2019-nCoV research. CEP, selamectin, and mefloquine hydrochloride are potential drugs for treating 2019-nCoV infection. Our results strongly suggest that CEP is a wide-spectrum inhibitor of pan-betacoronavirus, and further study of CEP for treatment of 2019-nCoV infection is warranted.
Subject(s)
Humans , Betacoronavirus , Genetics , Cell Line , Clinical Laboratory Techniques , Coronavirus Infections , Diagnosis , Drug Therapy , Drug Approval , Pandemics , Pneumonia, Viral , Diagnosis , Drug Therapy , RNA, Small Interfering , Genetics , Real-Time Polymerase Chain Reaction , Viral LoadABSTRACT
The highly pathogenic avian influenza (HPAI) H5N1 virus has caused several outbreaks in domestic poultry. Despite great efforts to control the spread of this virus, it continues to evolve and poses a substantial threat to public health because of a high mortality rate. In this study, we sequenced whole genomes of eight H5N1 avian influenza viruses isolated from domestic poultry in eastern China and compared them with those of typical influenza virus strains. Phylogenetic analyses showed that all eight genomes belonged to clade 2.3.2.1 and clade 7.2, the two main circulating clades in China. Viruses that clustered in clade 2.3.2.1 shared a high degree of homology with H5N1 isolates located in eastern Asian. Isolates that clustered in clade 7.2 were found to circulate throughout China, with an east-to-west density gradient. Pathogenicity studies in mice showed that these isolates replicate in the lungs, and clade 2.3.2.1 viruses exhibit a notably higher degree of virulence compared to clade 7.2 viruses. Our results contribute to the elucidation of the biological characterization and pathogenicity of HPAI H5N1 viruses.
Subject(s)
Animals , China , Influenza A Virus, H5N1 Subtype , Genetics , Virulence , Influenza in Birds , Virology , Mice, Inbred BALB C , Phylogeny , PoultryABSTRACT
To confirm the hypothesis that the high frequency sequences of high throughput sequencing are the terminal sequences of the bacteriophage genome. An adaptor of specific sequence was linked to the end of the bacteriophage T3 genomic DNA, which was then subject to high throughput sequencing; as a control, the same T3 genomic DNA without adaptor was also analyzed by high throughput sequencing. The sequencing results were examined with bioinformatics software. Similar high throughput sequencing technique was applied to analyze the genomic sequence of N4-like bacteriophage IME11. Bioinformatics study showed that the sequences tagged with adaptors were consistent with the high frequency sequences without adaptor labeling. Our analysis also indicated that the end of the T4-like phage genome had specific sequences instead of random sequences, disagreeing with the previous assertion. Evidences were provided that N4-like bacteriophage had a particular terminal sequence: the left end of the genome was unique while the right end was permuted. The high throughput sequencing technique was convenient and practical to be used to simultaneously detect the terminal sequence and the complete sequence of bacteriophage genome.
Subject(s)
Caudovirales , Genetics , Computational Biology , Genome, Viral , High-Throughput Nucleotide Sequencing , MethodsABSTRACT
<p><b>OBJECTIVES</b>To construct a stable HCV-producing cell model for anti-HCV drug research.</p><p><b>METHODS</b>The HCV-ribozyme recombinant plasmid pJFH1-Rbz was constructed to generate the exact 5' and 3' ends of HCV genomic RNA by placing two self-cleaving ribozymes at both ends of the HCV JFH-1 cDNA. The plasmid was then transfected into HepG2 cells and the resultant clones were screened with G418. Subsequently, immunofluorescence and Western blot were performed to detect the expression of HCV core protein, HCV RNA level was quantitated by TaqMan real-time PCR method and HCV particles was detected by electron microscopy.</p><p><b>RESULTS</b>HCV core protein was detected in the screened cell clone, and the level of HCV RNA was up to 1000,0000 copies/ml in the culture medium. Electron microscopy showed the viral particles in the culture suspension were approximately 55 nm in diameter. IFN-treating experiment demonstrated that the HCV RNA level decreased with the increasing concentration of IFN alpha.</p><p><b>CONCLUSION</b>We constructed a stable HCV-producing cell model which can be used for anti-HCV drug research.</p>
Subject(s)
Humans , DNA, Complementary , Genome, Viral , Hep G2 Cells , Hepacivirus , Genetics , Plasmids , RNA, Catalytic , Genetics , Transfection , Viral Core Proteins , Genetics , Virion , Virus ReplicationABSTRACT
Objective To study the serological characterization of indeterminate Western blot(WB)results of HIV antibody and to find a new way to verify the HIV antibody indeterminate results and provide references for editing"National Guideline for Detection of HIV/AIDS".Methods All of the 42 subjects who were confirmed as indeterminate HIV antibody in People'Libaretion Amry HIV Confirmation Laboratory from 2005 to 2006,were collected.Line immunoassay.HIV viral load test and HIV-1 p24 were tested and followed up for 3-6 months'to compare the changes of WB bands patterns.Results (1)For the 42 individuals with indeterminate HIV antibody.a total of 8 different patterns of bands were found in WB test including 45.2% of them were p24 monoband,30.9% were gp160 monoband,11.9% were gp160 with p24,2.4%(only one case)were gp160gp120 ±,gp41p24,p24p17,gp41 or gp120respectively.It was noticed that the most patterns of common bands with indeterminate results were p24 monoband.gpl60 monoband and gpl60 with p24.which composed 88.0% of the whole indeterminate WB band patterns.(2)Twenty three cases had been followed up for more than 3 months with 22 giving no WB band image change and were confirmed as HIV sero-negative.The other one with case gp160 and p24 had developed to more bands in the period of 77 days follow-up with more bands,including gpl60,gp120,p66,p31,p24 and p17,showed up and was confirmed as HIV primary infection.(3)Line immunoassay was applied to all of those 23 cases who had been followed up and the results showed that only one serological change was found and the case was confirmed to be HIV-positive.Among the other 22 cases without serological changes.16 cases were proved to be HIV-negative,6 cases were still indeterminate.The specificitv was 72.7%.P24 antigen test showed negative in all the 23 cases,including the case which later was confirmed as HIV-positive.Of all the 23 originally indeterminate cases,viral loads were tested in 7 eases.Positive result was found in the case which was proved later to be HIV-positive.No viral loads were detected in the other 6 cases(＜LDL).Conclusion The most common band patterns of indeterminate HIV antibody were mainly p24 monoband,gp160 monoband or with p24.Most of them (95.6%)were not infected by HIV,the bands showed up in WB test and demonstrated as non-specific reactions.Line immunoassay could determine about 70% of the indeterminate reactions.Results from viral load test also suggested that it was an efficient method to discriminate indeterminate results.With these two techniques,HIV serology could be diagnosed without 3 months'follow-up in primary infection which gave indeterminate WB results.
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At the time of phage’s discovery, phage therapy was regarded as a possible treatment method against bacterial infection. Although phage therapy was used to treat and prevent bacterial infection in the former Soviet Union and Eastern Europe, it was abandoned by the West in the 1940s with the arrival of the antibiotic era. However, the ongoing evolution of bacterial multidrug-resistance has recently motivated the Western scientific community to reevaluate phage therapy for bacterial infections that are incurable by conventional chemotherapy. With the indepth study of phages, it’s increasingly acknowledged that phages, as the medicine to cure bacterial infection, are convenient, safe and efficient therapeutics. This paper summarizes the recent years’ advanced researches in this area.
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<p><b>OBJECTIVES</b>To construct an eucaryotic expression plasmid carrying the BMP7 gene and express in MSCs.</p><p><b>METHODS</b>The BMP7 gene was cloned into the eucaryotic expression vector pcDNA3.1. At the same time, mesenchymal stem cells (MSCs) were isolated and cultured in vitro. The plasmid carrying the BMP7 gene was transfected into MSCs.</p><p><b>RESULTS</b>PCR and digesting demonstrated that the eucaryotic expression plasmid -pcDNA-BMP7 was obtained. RT-PCR and immunohistochemical methods showed that the BMP7 gene was expressed in MSCs.</p><p><b>CONCLUSION</b>Construction of an eucaryotic expression plasmid carrying BMP7 gene and expression in MSCs provide a sound basis for gene therapy using the BMP7 gene and the ideal seeds for tissue engineering.</p>
