AIMTo investigate the role of non-structural open reading frame 1 “Y-domain” sequences in the hepatitis E virus (HEV) life cycle.METHODSSequences of human HEV Y-domain (amino acid sequences 216...AIMTo investigate the role of non-structural open reading frame 1 “Y-domain” sequences in the hepatitis E virus (HEV) life cycle.METHODSSequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzed in silico. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. In vitro transcribed mRNA (pSK-GFP) constructs were transfected into S10-3 cells and viral RNA replicating GFP-positive cells were scored by flow cytometry. Mutant virions’ infectivity was assayed on naïve HepG2/C3A cells.RESULTSIn silico analysis identified a potential palmitoylation-site (C<sub>336</sub>C<sub>337</sub>) and an α-helix segment (L<sub>410</sub>Y<sub>411</sub>S<sub>412</sub>W<sub>413</sub>L<sub>414</sub>F<sub>415</sub>E<sub>416</sub>) in the HEV Y-domain. Molecular characterization of C<sub>336</sub>A, C<sub>337</sub>A and W<sub>413</sub>A mutants of the three universally conserved residues showed non-viability. Further, of the 10 consecutive saturation mutants covering the entire Y-domain nucleotide sequences (nts 650-1339), three constructs (nts 788-994) severely affected virus replication. This revealed the indispensability of the internal sequences but not of the up- or downstream sequences at the transcriptional level. Interestingly, the three mutated residues corresponded to the downstream codons that tolerated saturation mutation, indicating their post-translational functional/structural essentiality. In addition, RNA secondary structure prediction revealed formation of stable hairpins (nts 788-994) where saturation mutation drastically inhibited virion infectivity.CONCLUSIONThis is the first demonstration of the critical role of Y-domain sequences in HEV life cycle, which may involve gene regulation and/or membrane binding in intracellular replication complexes.展开更多
Hepatitis C virus(HCV)is a major cause of chronic liver diseases,including steatosis,cirrhosis and hepatocellular carcinoma,and its infection is also associated with insulin resistance and type 2 diabetes mellitus.HCV...Hepatitis C virus(HCV)is a major cause of chronic liver diseases,including steatosis,cirrhosis and hepatocellular carcinoma,and its infection is also associated with insulin resistance and type 2 diabetes mellitus.HCV,belonging to the Flaviviridae family,is a small enveloped virus whose positive-stranded RNA genome encoding a polyprotein.The HCV core protein is cleaved first at residue 191 by the host signal peptidase and further cleaved by the host signal peptide peptidase at about residue 177 to generate the mature core protein(a.a.1-177)and the cleaved peptide(a.a.178-191).Core protein could induce insulin resistance,steatosis and even hepatocellular carcinoma through various mechanisms.The peptide(a.a.178-191)may play a role in the immune response.The polymorphism of this peptide is associated with the cellular lipid drop accumulation,contributing to steatosis development.In addition to the conventional open reading frame(ORF),in the+1 frame,an ORF overlaps with the core proteincoding sequence and encodes the alternative reading frame proteins(ARFP or core+1).ARFP/core+1/F protein could enhance hepatocyte growth and may regulate iron metabolism.In this review,we briefly summarized the current knowledge regarding the production of different core gene products and their roles in viral pathogenesis.展开更多
The hepatitis E virus(HEV),a member of the Hepeviridae family,is a small,non-enveloped icosahedral virus divided into eight distinct genotypes(HEV-1 to HEV-8).Only genotypes 1 to 4 are known to cause diseases in human...The hepatitis E virus(HEV),a member of the Hepeviridae family,is a small,non-enveloped icosahedral virus divided into eight distinct genotypes(HEV-1 to HEV-8).Only genotypes 1 to 4 are known to cause diseases in humans.Genotypes 1 and 2 commonly spread via fecal-oral transmission,often through the consum-ption of contaminated water.Genotypes 3 and 4 are known to infect pigs,deer,and wild boars,often transferring to humans through inadequately cooked meat.Acute hepatitis caused by HEV in healthy individuals is mostly asymptomatic or associated with minor symptoms,such as jaundice.However,in immunosup-pressed individuals,the disease can progress to chronic hepatitis and even escalate to cirrhosis.For pregnant women,an HEV infection can cause fulminant liver failure,with a potential mortality rate of 25%.Mortality rates also rise amongst cirrhotic patients when they contract an acute HEV infection,which can even trigger acute-on-chronic liver failure if layered onto pre-existing chronic liver disease.As the prevalence of HEV infection continues to rise worldwide,highlighting the particular risks associated with severe HEV infection is of major medical interest.This text offers a brief summary of the characteristics of hepatitis developed by patient groups at an elevated risk of severe HEV infection.展开更多
Reading in Word Groups is one of the techniques at early stages of reading. Some tried techniques are recommended in this paper to help develop the students’ability to read in word groups. They are 1)Reading flashcar...Reading in Word Groups is one of the techniques at early stages of reading. Some tried techniques are recommended in this paper to help develop the students’ability to read in word groups. They are 1)Reading flashcards 2)Read and look up 3)Eye-span exercises. I hope these tech-niques will give you a hand to increase the speed and accuracy of your reading, both required reading and leisure reading.展开更多
The outbreak of coronavirus disease 2019(COVID-19)in Wuhan,China,was caused by a novel coronavirus(CoV),named severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).The rapid detection of viral nucleic acids is c...The outbreak of coronavirus disease 2019(COVID-19)in Wuhan,China,was caused by a novel coronavirus(CoV),named severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).The rapid detection of viral nucleic acids is critical for the early identification of infected cases.We have developed two TaqMan real-time reverse transcription-PCR assays to detect SARS-CoV-2.The designed primers target the nucleocapsid(N)and open reading frame(ORF)1b gene regions,where the probes discriminate SARS-CoV-2 from other human and animal CoVs.The sensitivities are one genomic copy per reaction for theN gene assay and ten copies for the ORF 1b gene assay.The overall linear detection ranges are 1–10^(6)and 10–10^(6)copies per reaction for the N gene assay and the ORF 1b gene assay,respectively.Surveillance of 23 suspected COVID-19 patients demonstrated that SARS-CoV-2 could be detected from 100%(23/23)and 62.5%(16/23)of clinical specimens by the N gene assay and the ORF 1b gene assay,respectively.All of the samples not detected by the ORF 1b gene assay were throat swabs,indicating a lower viral load in the upper respiratory tract and the relatively lower sensitivity of the ORF 1b gene assay.The assays developed in the present study offer alternative diagnostic tests for COVID-19.展开更多
Background DNA analysis has shown a lack of significant compatibility between couples affected by unexplained recurrent spontaneous abortion (URSA) compared with normal fertile couples, 8 although one study that ma...Background DNA analysis has shown a lack of significant compatibility between couples affected by unexplained recurrent spontaneous abortion (URSA) compared with normal fertile couples, 8 although one study that made use of a PCR-sequence-specific oligonucleotide (SSO) method did observe evidence of significant compatibility in the HLA-DQA1 and DQB1 alleles between patients and aborted fetuses. 9 This study was designed to investigate whether URSA were associated with particular DQ alleles or promoter alleles.Methods Thirty-two patients with URSA and 54 women who had had at least one successful pregnancy were included in this study. HLA-DQ genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The HLA-DQB1 promoter was detected by the SSO and sequence-specific primer (SSP) methods. The DQA1, DQB1, and DQB1 promoter (QBP) gene frequencies in the patients were compared with the gene frequencies in normal controls. The data were analyzed statistically with the χ 2 and Fisher’s exact tests.Results The results showed that the frequency of DQB1 *0604/0605 was significantly higher and the frequency of DQB1 *0501/0502 was significantly lower in the patient group as compared with the normal controls. In addition, the frequencies of the DQA1 *01-DQB1 *0604/0605 and QBP6.2-DQB1 *0604/0605 haplotypes were overrepresented in the patients relative to the controls. Our results did not show any differences between URSA patients and the controls with regard to DQA1 and QBP allele frequencies. Conclusions Our data suggest that URSA is associated with the HLA-DQB1 coding region, and is not associated with its upstream regulatory region. The DQB1 *0604/0605, DQA1 *01-DQB1 *0604/0605, and QBP6.2-DQB1 *0604/0605 haplotypes may confer susceptibility to URSA, while the DQB1 *0501/0502 allele may protect women from URSA.展开更多
基金Supported by the Deanship of Scientific Research at King Saud University,Riyadh,No.RG-1435-053
文摘AIMTo investigate the role of non-structural open reading frame 1 “Y-domain” sequences in the hepatitis E virus (HEV) life cycle.METHODSSequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzed in silico. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. In vitro transcribed mRNA (pSK-GFP) constructs were transfected into S10-3 cells and viral RNA replicating GFP-positive cells were scored by flow cytometry. Mutant virions’ infectivity was assayed on naïve HepG2/C3A cells.RESULTSIn silico analysis identified a potential palmitoylation-site (C<sub>336</sub>C<sub>337</sub>) and an α-helix segment (L<sub>410</sub>Y<sub>411</sub>S<sub>412</sub>W<sub>413</sub>L<sub>414</sub>F<sub>415</sub>E<sub>416</sub>) in the HEV Y-domain. Molecular characterization of C<sub>336</sub>A, C<sub>337</sub>A and W<sub>413</sub>A mutants of the three universally conserved residues showed non-viability. Further, of the 10 consecutive saturation mutants covering the entire Y-domain nucleotide sequences (nts 650-1339), three constructs (nts 788-994) severely affected virus replication. This revealed the indispensability of the internal sequences but not of the up- or downstream sequences at the transcriptional level. Interestingly, the three mutated residues corresponded to the downstream codons that tolerated saturation mutation, indicating their post-translational functional/structural essentiality. In addition, RNA secondary structure prediction revealed formation of stable hairpins (nts 788-994) where saturation mutation drastically inhibited virion infectivity.CONCLUSIONThis is the first demonstration of the critical role of Y-domain sequences in HEV life cycle, which may involve gene regulation and/or membrane binding in intracellular replication complexes.
基金Supported by Grants from the National Science Council of Taiwan,NSC 101-2320-B-320-011 to Lo SYfrom the Tzu Chi University,TCMRC-P-101015 and TCRPP101017 to Li HC and Lo SY
文摘Hepatitis C virus(HCV)is a major cause of chronic liver diseases,including steatosis,cirrhosis and hepatocellular carcinoma,and its infection is also associated with insulin resistance and type 2 diabetes mellitus.HCV,belonging to the Flaviviridae family,is a small enveloped virus whose positive-stranded RNA genome encoding a polyprotein.The HCV core protein is cleaved first at residue 191 by the host signal peptidase and further cleaved by the host signal peptide peptidase at about residue 177 to generate the mature core protein(a.a.1-177)and the cleaved peptide(a.a.178-191).Core protein could induce insulin resistance,steatosis and even hepatocellular carcinoma through various mechanisms.The peptide(a.a.178-191)may play a role in the immune response.The polymorphism of this peptide is associated with the cellular lipid drop accumulation,contributing to steatosis development.In addition to the conventional open reading frame(ORF),in the+1 frame,an ORF overlaps with the core proteincoding sequence and encodes the alternative reading frame proteins(ARFP or core+1).ARFP/core+1/F protein could enhance hepatocyte growth and may regulate iron metabolism.In this review,we briefly summarized the current knowledge regarding the production of different core gene products and their roles in viral pathogenesis.
基金Supported by Interreg V-A Romania-Hungary Programme,No.ROHU339.
文摘The hepatitis E virus(HEV),a member of the Hepeviridae family,is a small,non-enveloped icosahedral virus divided into eight distinct genotypes(HEV-1 to HEV-8).Only genotypes 1 to 4 are known to cause diseases in humans.Genotypes 1 and 2 commonly spread via fecal-oral transmission,often through the consum-ption of contaminated water.Genotypes 3 and 4 are known to infect pigs,deer,and wild boars,often transferring to humans through inadequately cooked meat.Acute hepatitis caused by HEV in healthy individuals is mostly asymptomatic or associated with minor symptoms,such as jaundice.However,in immunosup-pressed individuals,the disease can progress to chronic hepatitis and even escalate to cirrhosis.For pregnant women,an HEV infection can cause fulminant liver failure,with a potential mortality rate of 25%.Mortality rates also rise amongst cirrhotic patients when they contract an acute HEV infection,which can even trigger acute-on-chronic liver failure if layered onto pre-existing chronic liver disease.As the prevalence of HEV infection continues to rise worldwide,highlighting the particular risks associated with severe HEV infection is of major medical interest.This text offers a brief summary of the characteristics of hepatitis developed by patient groups at an elevated risk of severe HEV infection.
文摘Reading in Word Groups is one of the techniques at early stages of reading. Some tried techniques are recommended in this paper to help develop the students’ability to read in word groups. They are 1)Reading flashcards 2)Read and look up 3)Eye-span exercises. I hope these tech-niques will give you a hand to increase the speed and accuracy of your reading, both required reading and leisure reading.
基金funded in part by the Project from the Ministry of Science and Technology in China(2020YFC0841200)the National Major Science&Technology Project for Control and Prevention of Major Infectious Diseases in China(2017ZX10103004)+2 种基金the Chinese Academy of Medical Sciences(CAMS)Innovation Fund for Medical Sciences(2020HY320001)the non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences(2019PT310029)the Chinese Academy of Medical Sciences(CAMS)Innovation Fund for Medical Sciences(2016-I2M-1-014).
文摘The outbreak of coronavirus disease 2019(COVID-19)in Wuhan,China,was caused by a novel coronavirus(CoV),named severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).The rapid detection of viral nucleic acids is critical for the early identification of infected cases.We have developed two TaqMan real-time reverse transcription-PCR assays to detect SARS-CoV-2.The designed primers target the nucleocapsid(N)and open reading frame(ORF)1b gene regions,where the probes discriminate SARS-CoV-2 from other human and animal CoVs.The sensitivities are one genomic copy per reaction for theN gene assay and ten copies for the ORF 1b gene assay.The overall linear detection ranges are 1–10^(6)and 10–10^(6)copies per reaction for the N gene assay and the ORF 1b gene assay,respectively.Surveillance of 23 suspected COVID-19 patients demonstrated that SARS-CoV-2 could be detected from 100%(23/23)and 62.5%(16/23)of clinical specimens by the N gene assay and the ORF 1b gene assay,respectively.All of the samples not detected by the ORF 1b gene assay were throat swabs,indicating a lower viral load in the upper respiratory tract and the relatively lower sensitivity of the ORF 1b gene assay.The assays developed in the present study offer alternative diagnostic tests for COVID-19.
基金ThisstudywassupportedbytheNationalNaturalScienceFoundationofChina (No 3 9870 775 )
文摘Background DNA analysis has shown a lack of significant compatibility between couples affected by unexplained recurrent spontaneous abortion (URSA) compared with normal fertile couples, 8 although one study that made use of a PCR-sequence-specific oligonucleotide (SSO) method did observe evidence of significant compatibility in the HLA-DQA1 and DQB1 alleles between patients and aborted fetuses. 9 This study was designed to investigate whether URSA were associated with particular DQ alleles or promoter alleles.Methods Thirty-two patients with URSA and 54 women who had had at least one successful pregnancy were included in this study. HLA-DQ genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The HLA-DQB1 promoter was detected by the SSO and sequence-specific primer (SSP) methods. The DQA1, DQB1, and DQB1 promoter (QBP) gene frequencies in the patients were compared with the gene frequencies in normal controls. The data were analyzed statistically with the χ 2 and Fisher’s exact tests.Results The results showed that the frequency of DQB1 *0604/0605 was significantly higher and the frequency of DQB1 *0501/0502 was significantly lower in the patient group as compared with the normal controls. In addition, the frequencies of the DQA1 *01-DQB1 *0604/0605 and QBP6.2-DQB1 *0604/0605 haplotypes were overrepresented in the patients relative to the controls. Our results did not show any differences between URSA patients and the controls with regard to DQA1 and QBP allele frequencies. Conclusions Our data suggest that URSA is associated with the HLA-DQB1 coding region, and is not associated with its upstream regulatory region. The DQB1 *0604/0605, DQA1 *01-DQB1 *0604/0605, and QBP6.2-DQB1 *0604/0605 haplotypes may confer susceptibility to URSA, while the DQB1 *0501/0502 allele may protect women from URSA.
