Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a...Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.展开更多
Dear Editor,Zika virus（ZIKV）used to be an unknown mosquito-borne flavivirus,and maintained its limited sylvatic circulation in a few African and Asian countries（Enfissi et al.,2016）.Based on available clinical dat...Dear Editor,Zika virus（ZIKV）used to be an unknown mosquito-borne flavivirus,and maintained its limited sylvatic circulation in a few African and Asian countries（Enfissi et al.,2016）.Based on available clinical data,the symptoms in human infections with ZIKV are supposed to be similar to other arbovirus infections such as dengue,and characterized by fever,skin rashes,conjunctivitis,muscle and joint pain,malaise,and headache（Duffy et al.,2009）.展开更多
Guaico Culex virus(GCXV)is a newly identified segmented Jingmenvirus from Culex spp.mosquitoes in Central and South America.The genome of GCXV is composed of four or five single-stranded positive RNA segments.However,...Guaico Culex virus(GCXV)is a newly identified segmented Jingmenvirus from Culex spp.mosquitoes in Central and South America.The genome of GCXV is composed of four or five single-stranded positive RNA segments.However,the infection kinetics and transmission capability of GCXV in mosquitoes remain unknown.In this study,we used reverse genetics to rescue two GCXVs(4S and 5S)that contained four and five RNA segments,respectively,in C6/36 cells.Further in vitro characterization revealed that the two GCXVs exhibited comparable replication kinetics,protein expression and viral titers.Importantly,GCXV RNAs were detected in the bodies,salivary glands,midguts and ovaries of Culex quinquefasciatus at 4–10 days after oral infection.In addition,two GCXVs can colonize Cx.quinquefasciatus eggs,resulting in positive rates of 15%–35%for the second gonotrophic cycle.In conclusion,our results demonstrated that GCXVs with four or five RNA segments can be detected in Cx.quinquefasciatus eggs during the first and second gonotrophic cycles after oral infection.展开更多
Cap-dependent endonuclease(CEN)in the polymerase acidic protein(PA)of influenza A virus(IAV)represents a promising drug target due to its critical role in viral gene transcription.The CEN inhibitor,baloxavir marboxil(...Cap-dependent endonuclease(CEN)in the polymerase acidic protein(PA)of influenza A virus(IAV)represents a promising drug target due to its critical role in viral gene transcription.The CEN inhibitor,baloxavir marboxil(BXM),was approved in Japan and the US in 2018 and several other countries subsequently.Along with the clinical use of BXM,the emergence and spread of IAV variants with reduced susceptibility to BXM have aroused serious concern.Herein,we comprehensively characterized the in vitro and in vivo antiviral activities of ZX-7101A,an analogue of BXM.The active form of prodrug ZX-7101 showed broad-spectrum antiviral potency against various IAV subtypes,including pH1N1,H3N2,H7N9 and H9N2,in MDCK cells,and the 50%effective concentration(EC_(50))was calculated to nanomole level and comparable to that of baloxavir acid(BXA),the active form of BXM.Furthermore,in vivo assays showed that administration of ZX-7101A conferred significant protection against lethal pH1N1 challenge in mice,with reduced viral RNA loads and alleviated pulmonary damage.Importantly,serial passaging of H1N1 virus in MDCK cells under selection pressure of ZX-7101 led to a resistant variant at the 15th passage.Reverse genetic and sequencing analysis demonstrated that a single E18G substitution in the PA subunit contributed to the reduced susceptibility to both ZX-7101 and BXA.Taken together,our results not only characterized a new CEN inhibitor of IAV but also identified a novel amino acid substitution responsible for CEN inhibitor resistance,which provides critical clues for future drug development and drug resistance surveillance.展开更多
Coxsackievirus A16(CA16) is one of the major causes of hand, foot, and mouth disease(HFMD) worldwide, which is a common illness that affects children. The frequent occurrence of HFMD outbreaks has become a serious pub...Coxsackievirus A16(CA16) is one of the major causes of hand, foot, and mouth disease(HFMD) worldwide, which is a common illness that affects children. The frequent occurrence of HFMD outbreaks has become a serious public health problem in Asia. Therefore, it is important to understand the pathogenesis and replication of CA16. In this study, a stable infectious c DNA clone of an epidemic strain of Coxsackievirus A16(CA16) was assembled, and subsequently a reporter virus(e GFP-CA16) was constructed by inserting the e GFP gene between the 5'-UTR and the N-terminus of VP4, with the addition of a 2A protease cleavage site(ITTLG) at its C-terminus. This was transfected into Vero cells to generate infectious recombinant viruses. The growth characteristics and plaque morphology, in vitro, in mammalian cells were found to be indistinguishable between the parental and recombinant viruses. Although the e GFP-CA16 showed smaller plaque size as compared to recombinant CA16, both were found to exhibit similar growth trends and EC50 of NITD008. In summary, this stable infectious c DNA clone should provide a valuable experimental system to study CA16 infection and host response. The e GFP-CA16 is expected to provide a powerful tool to monitor e GFP expression in infected cells and to evaluate the antiviral activity of potential antiviral agents in the treatment of CA16 infections.展开更多
The Orthopoxvirus genus,especially variola virus(VARV),monkeypox virus(MPXV),remains a significant public health threat worldwide.The development of therapeutic antibodies against orthopoxviruses is largely hampered b...The Orthopoxvirus genus,especially variola virus(VARV),monkeypox virus(MPXV),remains a significant public health threat worldwide.The development of therapeutic antibodies against orthopoxviruses is largely hampered by the high cost of antibody engineering and manufacturing processes.mRNA-encoded antibodies have emerged as a powerful and universal platform for rapid antibody production.Herein,by using the established lipid nanoparticle(LNP)-encapsulated mRNA platform,we constructed four mRNA combinations that encode monoclonal antibodies with broad neutralization activities against orthopoxviruses.In vivo characterization demonstrated that a single intravenous injection of each LNP-encapsulated mRNA antibody in mice resulted in the rapid production of neutralizing antibodies.More importantly,mRNA antibody treatments showed significant protection from weight loss and mortality in the vaccinia virus(VACV)lethal challenge mouse model,and a unique mRNA antibody cocktail,Mix2a,exhibited superior in vivo protection by targeting both intracellular mature virus(IMV)-form and extracellular enveloped virus(EEV)-form viruses.In summary,our results demonstrate the proof-of-concept production of orthopoxvirus antibodies via the LNP-mRNA platform,highlighting the great potential of tailored mRNA antibody combinations as a universal strategy to combat orthopoxvirus as well as otheremerging viruses.展开更多
AIM： To identify clonality and genetic alterations in focal nodular hyperplasia （FNH） and the nodules derived from it. METHODS： Twelve FNH lesions were examined. Twelve hepatocellular adenomas （HCAs） and 22 hepa...AIM： To identify clonality and genetic alterations in focal nodular hyperplasia （FNH） and the nodules derived from it. METHODS： Twelve FNH lesions were examined. Twelve hepatocellular adenomas （HCAs） and 22 hepatocellular carcinomas （HCCs） were used as references. Nodules of different types were identified and isolated from FNH by microdissection. An X-chromosome inactivation assay was employed to describe their clonality status. Loss of heterozygosity （LOH） was detected, using 57 markers, for genetic alterations.RESULTS： Nodules of altered hepatocytes （NAH）, the putative precursors of HCA and HCC, were found in all the FNH lesions. Polyclonality was revealed in 10 FNH lesions from female patients, and LOH was not detected in any of the six FNH lesions examined, the results apparently showing their polyclonal nature. In contrast, monoclonality was demonstrated in all the eight HCAs and in four of the HCCs from females, and allelic imbalances were found in the HCAs （9/9） and HCCs （15/18）, with chromosomal arms 11p, 13q and 17p affected in the former, and 6q, 8p, 11p, 16q and 17p affected in the latter lesions in high frequencies （≥ 30%）. Monodonality was revealed in 21 （40%） of the 52 microdissected NAH, but was not found in any of the five ordinary nodules. LOH was found in all of the 13 NAH tested, being highly frequent at six loci on 8p, 11p, 13q and 17p. CONCLUSION： FNH, as a whole, is polyclonal, but some of the NAH lesions derived from it are already neoplastic and harbor similar allelic imbalances as HCAs.展开更多
Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprise...Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.展开更多
Seasonal influenza is a highly contagious, acute respiratory illness that affects people of all ages. The major pathogens, influenza A viruses, are classified into serologically defined antigenic subtypes of the hemag...Seasonal influenza is a highly contagious, acute respiratory illness that affects people of all ages. The major pathogens, influenza A viruses, are classified into serologically defined antigenic subtypes of the hemagglutinin （HA） and neuraminidase （NA）. Of 16 identified HA and 9 NA subtypes, only H1N1 and H3N2 subtypes are now circulating among humans. Influenza vaccines have been available for over 60 years and well proven to be an effective public health intervention to control seasonal influenza epidemics. Seasonal influenza vaccines presently available, inactivated or split, contain the circulating strains of influenza A virus H3N2, H1N1 and influenza B virus. The composition of the vaccine is renewed semi-annually, as necessary, based on surveillance data.展开更多
Recently,many SARS-CoV-2 variants including 501Y.V1,501Y.V2 and 501Y.V3 were detected in different regions(Table S1)and drew great attention from all over the world.The 501Y.V1 was firstly isolated in the United Kingd...Recently,many SARS-CoV-2 variants including 501Y.V1,501Y.V2 and 501Y.V3 were detected in different regions(Table S1)and drew great attention from all over the world.The 501Y.V1 was firstly isolated in the United Kingdom(UK)(Davies et al.,2020)and featured with 7 substitutions including N501Y as well as 3 deletions in S protein.This variant was identified to increase the viral transmissibility by 56%in comparison with the preexisting strains.Days after this report,another SARS-CoV-2 variant(501Y.V2)featured with N501Y,K417N and E484K substitutions in S protein was supposed to rapidly outcompete the preexisting strains(Tegally et al.,2020)in South Africa.Besides,the 501Y.V3 variant was initially detected in Brazil and has caused rapidly increased infections with SNPs N501Y,K417T and E484K.Of them,N501Y,K417N/T and E484K are of particular interest because the N501Y was shared in all three variants and the K417N/T and E484K were detected simultaneous appeared with N501Y in 501Y.V2 and 501Y.V3.展开更多
Background： Epilepsy is one of the most common serious neurological disorders. The present study aimed to investigate the influence of occupational status on the quality of life of Chinese adult patients with epileps...Background： Epilepsy is one of the most common serious neurological disorders. The present study aimed to investigate the influence of occupational status on the quality of life of Chinese adult patients with epilepsy. Methods： This study surveyed 819 subjects clinically diagnosed with epilepsy for more than 1 year in 11 hospitals in Beijing; 586 were employed （71.55%）. All subjects completed the case report form with inquiries on demographic data, social factors, and illness. The patients＇ quality of life was assessed using the quality of life in patients with epilepsy-31 items （QOLIE-31） questionnaire. Results： The QOLIE-31 score in the employed group was significantly higher than that in the unemployed group. Furthermore, the scores in all the sections （overall quality of life, energy/fatigue, emotional well-being, seizure worry, cognition, social function, and medication effects） of the employed group were higher than those of the unemployed group. Both the employed and unemployed groups achieved the highest difference in social function. The QOLIE-31 score of students was higher than those of farmers and workers. Both the students and workers scored higher in the quality of life compared with the adult peasants living with epilepsy. The students and farmers showed significant differences in QOL1E-31 score, cognition, emotional well-being, overall quality of life, energy/fatigue, and social function. In contrast, no significant difference was noted in seizure worry and medication effects across the three different kinds of occupation. Conclusion： Occupational status might affect the quality of life of Chinese adult patients with epilepsy, and social function is the most important contributing factor.展开更多
Background Previous serological studies of human bocavirus(HBoV)1 could not exclude cross-reactivity with the other three HBoVs,particularly HBoV2.Methods To search for genotype-specific antibodies against HBoV1 and H...Background Previous serological studies of human bocavirus(HBoV)1 could not exclude cross-reactivity with the other three HBoVs,particularly HBoV2.Methods To search for genotype-specific antibodies against HBoV1 and HBoV2,the divergent regions(DRs)located on the major capsid protein VP3 were defined through viral amino acid alignment and structure prediction.DR-deduced peptides were used as antigens to harvest corresponding anti-DR rabbit sera.To determine their genotype specificities for HBoV1 and HBoV2,these sera samples were used as antibodies against the antigens VP3 of HBoV1 and HBoV2(expressed in Escherichia coli)in western blotting(WB),enzyme-linked immunosorbent assay(ELISA),and bio-layer interferometry(BLI)assays.Subsequently,the antibodies were evaluated with clinical specimens from pediatric patients with acute respiratory tract infection by indirect immunofluorescence assay(IFA).Results There were four DRs(DR1–4)located on VP3 with different secondary and tertiary structures between HBoV1 and HBoV2.Regarding the reactivity with VP3 of HBoV1 or HBoV2 in WB and ELISA,high intra-genotype cross-reactivity of anti-HBoV1 or HBoV2 DR1,DR3,and DR4,but not anti-DR2,was observed.Genotype-specific binding capacity of anti-DR2 sera was confirmed by BLI and IFA,in which only anti-HBoV1 DR2 antibody reacted with HBoV1-positive respiratory specimens.Conclusion Antibodies against DR2,located on VP3 of HBoV1 or HBoV2,were genotype specific for HBoV1 and HBoV2,respectively.展开更多
The constantly mutating severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)poses great risk of efficacy loss to the present neutralizing therapeutics.Thus,it is urgently needed to develop versatile strategies ...The constantly mutating severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)poses great risk of efficacy loss to the present neutralizing therapeutics.Thus,it is urgently needed to develop versatile strategies that enable rapid design and engineering of potent neutralizing therapeutics for newly emerging variants.Herein,we present an unprecedented DNA nanocrown that can topologically match and multivalently bind the S-trimer of SARS-CoV-2 and thereby inhibit its infection to host cells.A neutralizing aptamer binding the N-terminal domain(NTD)supersite of the S protein was first screened and identified.It was further elaborately engineered onto the best fitting tetrahedral DNA nanostructure to form a spike protein-capping nanocrown,which can effectively block not only wild-type(WT)SARS-CoV-2 pseudovirus,but also several important mutants including D614G,N501Y,andΔ69–70.Significantly,it can evidently diminish the RNA copies of authentic WT SARS-CoV-2 in host cells by 4.6 orders of magnitude.Therefore,utilizing the aptamer selection method and the dedicated engineering route,our topology-matching DNA framework provides a versatile platform for SARS-CoV-2 inhibition and has the potential to be facilely expanded to newly emerging variants and other fatal coronaviruses.展开更多
Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome repli...Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstmctural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV（Mammalian orthoreovimses）, which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region （335-742） was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28℃. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80（335-742） protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody （mouse） and NS80〈335.742） specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.展开更多
基金National Natural Science Foundation ofChina (Grant Nos 30470074, 30671615)Innovation Projectof the Chinese Academy of Sciences (KSCX2- YW-N- 021)Science and technology foundation of Zhejiang Province(2007C22052)
文摘Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.
基金supported by the State Key Laboratory of Pathogen and Biosecurity, ChinaCheng-Feng Qin was supported by the Excellent Young Scientist Program from the National Natural Science Foundation of China (81522025)the Newton Advanced Fellowship from the UK Academy of Medical Sciences and the National Natural Science Foundation of China (8151101191)
文摘Dear Editor,Zika virus（ZIKV）used to be an unknown mosquito-borne flavivirus,and maintained its limited sylvatic circulation in a few African and Asian countries（Enfissi et al.,2016）.Based on available clinical data,the symptoms in human infections with ZIKV are supposed to be similar to other arbovirus infections such as dengue,and characterized by fever,skin rashes,conjunctivitis,muscle and joint pain,malaise,and headache（Duffy et al.,2009）.
基金supported by the National Key Research and Development Project(2023YFC2305901)J.J.G.was supported by the China Postdoctoral Science Fund(No.2022M713868)。
文摘Guaico Culex virus(GCXV)is a newly identified segmented Jingmenvirus from Culex spp.mosquitoes in Central and South America.The genome of GCXV is composed of four or five single-stranded positive RNA segments.However,the infection kinetics and transmission capability of GCXV in mosquitoes remain unknown.In this study,we used reverse genetics to rescue two GCXVs(4S and 5S)that contained four and five RNA segments,respectively,in C6/36 cells.Further in vitro characterization revealed that the two GCXVs exhibited comparable replication kinetics,protein expression and viral titers.Importantly,GCXV RNAs were detected in the bodies,salivary glands,midguts and ovaries of Culex quinquefasciatus at 4–10 days after oral infection.In addition,two GCXVs can colonize Cx.quinquefasciatus eggs,resulting in positive rates of 15%–35%for the second gonotrophic cycle.In conclusion,our results demonstrated that GCXVs with four or five RNA segments can be detected in Cx.quinquefasciatus eggs during the first and second gonotrophic cycles after oral infection.
基金supported by the National Science and Technology Major Project (2018ZX097110003-005-002)the Key-Area Research and Development Program of Guangdong Province (2022B1111020002)+3 种基金the National Natural Science Foundation of China (NSFC) (32170159,and 82174055)supported by the National Science Fund for Distinguished Young Scholars (81925025)the Innovative Research Group (81621005)of the NSFCthe Innovation Fund for Medical Sciences (2019-I2M-5-049)of the Chinese Academy of Medical Sciences.
文摘Cap-dependent endonuclease(CEN)in the polymerase acidic protein(PA)of influenza A virus(IAV)represents a promising drug target due to its critical role in viral gene transcription.The CEN inhibitor,baloxavir marboxil(BXM),was approved in Japan and the US in 2018 and several other countries subsequently.Along with the clinical use of BXM,the emergence and spread of IAV variants with reduced susceptibility to BXM have aroused serious concern.Herein,we comprehensively characterized the in vitro and in vivo antiviral activities of ZX-7101A,an analogue of BXM.The active form of prodrug ZX-7101 showed broad-spectrum antiviral potency against various IAV subtypes,including pH1N1,H3N2,H7N9 and H9N2,in MDCK cells,and the 50%effective concentration(EC_(50))was calculated to nanomole level and comparable to that of baloxavir acid(BXA),the active form of BXM.Furthermore,in vivo assays showed that administration of ZX-7101A conferred significant protection against lethal pH1N1 challenge in mice,with reduced viral RNA loads and alleviated pulmonary damage.Importantly,serial passaging of H1N1 virus in MDCK cells under selection pressure of ZX-7101 led to a resistant variant at the 15th passage.Reverse genetic and sequencing analysis demonstrated that a single E18G substitution in the PA subunit contributed to the reduced susceptibility to both ZX-7101 and BXA.Taken together,our results not only characterized a new CEN inhibitor of IAV but also identified a novel amino acid substitution responsible for CEN inhibitor resistance,which provides critical clues for future drug development and drug resistance surveillance.
基金supported by the Science and Technology Plan Projects of Wuhan (grant No. 2013060501010157)
文摘Coxsackievirus A16(CA16) is one of the major causes of hand, foot, and mouth disease(HFMD) worldwide, which is a common illness that affects children. The frequent occurrence of HFMD outbreaks has become a serious public health problem in Asia. Therefore, it is important to understand the pathogenesis and replication of CA16. In this study, a stable infectious c DNA clone of an epidemic strain of Coxsackievirus A16(CA16) was assembled, and subsequently a reporter virus(e GFP-CA16) was constructed by inserting the e GFP gene between the 5'-UTR and the N-terminus of VP4, with the addition of a 2A protease cleavage site(ITTLG) at its C-terminus. This was transfected into Vero cells to generate infectious recombinant viruses. The growth characteristics and plaque morphology, in vitro, in mammalian cells were found to be indistinguishable between the parental and recombinant viruses. Although the e GFP-CA16 showed smaller plaque size as compared to recombinant CA16, both were found to exhibit similar growth trends and EC50 of NITD008. In summary, this stable infectious c DNA clone should provide a valuable experimental system to study CA16 infection and host response. The e GFP-CA16 is expected to provide a powerful tool to monitor e GFP expression in infected cells and to evaluate the antiviral activity of potential antiviral agents in the treatment of CA16 infections.
基金supported by the National Natural Science Foundation of China (No.82241069,and 82350801)to C.F.Q.the National Key Research and Development Project of China (2021YFC2302400)to C.F.Q.+2 种基金the Key-Area Research and Development Program of Guangdong Province (2022B1111020002).C.F.Q.supported by the National Science Fund for Distinguished Young Scholars (81925025)the Innovation Fund for Medical Sciences (2019-12M-5-049)from the Chinese Academy of Medical Sciences.
文摘The Orthopoxvirus genus,especially variola virus(VARV),monkeypox virus(MPXV),remains a significant public health threat worldwide.The development of therapeutic antibodies against orthopoxviruses is largely hampered by the high cost of antibody engineering and manufacturing processes.mRNA-encoded antibodies have emerged as a powerful and universal platform for rapid antibody production.Herein,by using the established lipid nanoparticle(LNP)-encapsulated mRNA platform,we constructed four mRNA combinations that encode monoclonal antibodies with broad neutralization activities against orthopoxviruses.In vivo characterization demonstrated that a single intravenous injection of each LNP-encapsulated mRNA antibody in mice resulted in the rapid production of neutralizing antibodies.More importantly,mRNA antibody treatments showed significant protection from weight loss and mortality in the vaccinia virus(VACV)lethal challenge mouse model,and a unique mRNA antibody cocktail,Mix2a,exhibited superior in vivo protection by targeting both intracellular mature virus(IMV)-form and extracellular enveloped virus(EEV)-form viruses.In summary,our results demonstrate the proof-of-concept production of orthopoxvirus antibodies via the LNP-mRNA platform,highlighting the great potential of tailored mRNA antibody combinations as a universal strategy to combat orthopoxvirus as well as otheremerging viruses.
基金Supported by The National Natural Science Foundation of China (NSFC), Grants 30171052, 30572125 and 30772508the CAMS Cancer Hospital Clinical Research Project LC2007A21
文摘AIM： To identify clonality and genetic alterations in focal nodular hyperplasia （FNH） and the nodules derived from it. METHODS： Twelve FNH lesions were examined. Twelve hepatocellular adenomas （HCAs） and 22 hepatocellular carcinomas （HCCs） were used as references. Nodules of different types were identified and isolated from FNH by microdissection. An X-chromosome inactivation assay was employed to describe their clonality status. Loss of heterozygosity （LOH） was detected, using 57 markers, for genetic alterations.RESULTS： Nodules of altered hepatocytes （NAH）, the putative precursors of HCA and HCC, were found in all the FNH lesions. Polyclonality was revealed in 10 FNH lesions from female patients, and LOH was not detected in any of the six FNH lesions examined, the results apparently showing their polyclonal nature. In contrast, monoclonality was demonstrated in all the eight HCAs and in four of the HCCs from females, and allelic imbalances were found in the HCAs （9/9） and HCCs （15/18）, with chromosomal arms 11p, 13q and 17p affected in the former, and 6q, 8p, 11p, 16q and 17p affected in the latter lesions in high frequencies （≥ 30%）. Monodonality was revealed in 21 （40%） of the 52 microdissected NAH, but was not found in any of the five ordinary nodules. LOH was found in all of the 13 NAH tested, being highly frequent at six loci on 8p, 11p, 13q and 17p. CONCLUSION： FNH, as a whole, is polyclonal, but some of the NAH lesions derived from it are already neoplastic and harbor similar allelic imbalances as HCAs.
基金National Basic Research Program ofChina (973 Program) (2009CB118701)National NaturalScientific Foundation of China (30671615, 30871940)+1 种基金Innovation Project of the Chinese Academy of Sciences(KSCX2-YW-N-021)Science and Technology Foundation of Zhejiang Province (2007C22052)
文摘Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.
文摘Seasonal influenza is a highly contagious, acute respiratory illness that affects people of all ages. The major pathogens, influenza A viruses, are classified into serologically defined antigenic subtypes of the hemagglutinin （HA） and neuraminidase （NA）. Of 16 identified HA and 9 NA subtypes, only H1N1 and H3N2 subtypes are now circulating among humans. Influenza vaccines have been available for over 60 years and well proven to be an effective public health intervention to control seasonal influenza epidemics. Seasonal influenza vaccines presently available, inactivated or split, contain the circulating strains of influenza A virus H3N2, H1N1 and influenza B virus. The composition of the vaccine is renewed semi-annually, as necessary, based on surveillance data.
基金The National Key Plan for Scientific Research and Development of China(2016YFD0500301)CAMS Initiative for Innovative Medicine(CAMS-I2M,2016-I2M-1-005)+4 种基金Hang-Yu Zhou was supported by China postdoctoral science foundation grant(2019M660548,2020T130007ZX)Youthful Teacher Project of Peking Union Medical College(3332019114)Cheng-Feng Qin was supported by the National Science Fund for Distinguished Young Scholar(No.81925025)the Innovative Research Group(No.81621005)from the NSFCthe Innovation Fund for Medical Sciences(No.2019RU040)from the Chinese Academy of Medical Sciences(CAMS).
文摘Recently,many SARS-CoV-2 variants including 501Y.V1,501Y.V2 and 501Y.V3 were detected in different regions(Table S1)and drew great attention from all over the world.The 501Y.V1 was firstly isolated in the United Kingdom(UK)(Davies et al.,2020)and featured with 7 substitutions including N501Y as well as 3 deletions in S protein.This variant was identified to increase the viral transmissibility by 56%in comparison with the preexisting strains.Days after this report,another SARS-CoV-2 variant(501Y.V2)featured with N501Y,K417N and E484K substitutions in S protein was supposed to rapidly outcompete the preexisting strains(Tegally et al.,2020)in South Africa.Besides,the 501Y.V3 variant was initially detected in Brazil and has caused rapidly increased infections with SNPs N501Y,K417T and E484K.Of them,N501Y,K417N/T and E484K are of particular interest because the N501Y was shared in all three variants and the K417N/T and E484K were detected simultaneous appeared with N501Y in 501Y.V2 and 501Y.V3.
文摘Background： Epilepsy is one of the most common serious neurological disorders. The present study aimed to investigate the influence of occupational status on the quality of life of Chinese adult patients with epilepsy. Methods： This study surveyed 819 subjects clinically diagnosed with epilepsy for more than 1 year in 11 hospitals in Beijing; 586 were employed （71.55%）. All subjects completed the case report form with inquiries on demographic data, social factors, and illness. The patients＇ quality of life was assessed using the quality of life in patients with epilepsy-31 items （QOLIE-31） questionnaire. Results： The QOLIE-31 score in the employed group was significantly higher than that in the unemployed group. Furthermore, the scores in all the sections （overall quality of life, energy/fatigue, emotional well-being, seizure worry, cognition, social function, and medication effects） of the employed group were higher than those of the unemployed group. Both the employed and unemployed groups achieved the highest difference in social function. The QOLIE-31 score of students was higher than those of farmers and workers. Both the students and workers scored higher in the quality of life compared with the adult peasants living with epilepsy. The students and farmers showed significant differences in QOL1E-31 score, cognition, emotional well-being, overall quality of life, energy/fatigue, and social function. In contrast, no significant difference was noted in seizure worry and medication effects across the three different kinds of occupation. Conclusion： Occupational status might affect the quality of life of Chinese adult patients with epilepsy, and social function is the most important contributing factor.
基金supported by the Beijing Natural Science Foundation(7192029)the National Natural Science Foundation of China(82172277)the Beijing Municipal Commission of Health(2060399 PXM2017_026268_00005_00254486).
文摘Background Previous serological studies of human bocavirus(HBoV)1 could not exclude cross-reactivity with the other three HBoVs,particularly HBoV2.Methods To search for genotype-specific antibodies against HBoV1 and HBoV2,the divergent regions(DRs)located on the major capsid protein VP3 were defined through viral amino acid alignment and structure prediction.DR-deduced peptides were used as antigens to harvest corresponding anti-DR rabbit sera.To determine their genotype specificities for HBoV1 and HBoV2,these sera samples were used as antibodies against the antigens VP3 of HBoV1 and HBoV2(expressed in Escherichia coli)in western blotting(WB),enzyme-linked immunosorbent assay(ELISA),and bio-layer interferometry(BLI)assays.Subsequently,the antibodies were evaluated with clinical specimens from pediatric patients with acute respiratory tract infection by indirect immunofluorescence assay(IFA).Results There were four DRs(DR1–4)located on VP3 with different secondary and tertiary structures between HBoV1 and HBoV2.Regarding the reactivity with VP3 of HBoV1 or HBoV2 in WB and ELISA,high intra-genotype cross-reactivity of anti-HBoV1 or HBoV2 DR1,DR3,and DR4,but not anti-DR2,was observed.Genotype-specific binding capacity of anti-DR2 sera was confirmed by BLI and IFA,in which only anti-HBoV1 DR2 antibody reacted with HBoV1-positive respiratory specimens.Conclusion Antibodies against DR2,located on VP3 of HBoV1 or HBoV2,were genotype specific for HBoV1 and HBoV2,respectively.
基金This work was supported by the Key Grant(grant no.21834003)from the National Natural Science Foundation of Chinathe National Key R&D Program of China(grant no.2018YFC0910301)from the Ministry of Science and Technology of Chinathe Excellent Research Program of Nanjing University(grant no.ZYJH004)to Z.L.
文摘The constantly mutating severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)poses great risk of efficacy loss to the present neutralizing therapeutics.Thus,it is urgently needed to develop versatile strategies that enable rapid design and engineering of potent neutralizing therapeutics for newly emerging variants.Herein,we present an unprecedented DNA nanocrown that can topologically match and multivalently bind the S-trimer of SARS-CoV-2 and thereby inhibit its infection to host cells.A neutralizing aptamer binding the N-terminal domain(NTD)supersite of the S protein was first screened and identified.It was further elaborately engineered onto the best fitting tetrahedral DNA nanostructure to form a spike protein-capping nanocrown,which can effectively block not only wild-type(WT)SARS-CoV-2 pseudovirus,but also several important mutants including D614G,N501Y,andΔ69–70.Significantly,it can evidently diminish the RNA copies of authentic WT SARS-CoV-2 in host cells by 4.6 orders of magnitude.Therefore,utilizing the aptamer selection method and the dedicated engineering route,our topology-matching DNA framework provides a versatile platform for SARS-CoV-2 inhibition and has the potential to be facilely expanded to newly emerging variants and other fatal coronaviruses.
基金National Basic Research Program of China (973 Program, Grant No. 2009CB118701)National Natural Scientific Foundation of China (Grant Nos. 30671615, 30871940)Innovation project of the Chinese Academy of Sciences (Grant No.KSCX2-YW-N-021)
文摘Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstmctural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV（Mammalian orthoreovimses）, which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region （335-742） was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28℃. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80（335-742） protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody （mouse） and NS80〈335.742） specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.
