Concentration of hepatitis A virus from mimicked water samples by membrane filtration method / 中华实验和临床病毒学杂志

Objective@#To optimize the membrane filtration method for hepatitis A virus concentration from mimicked water samples.@*Methods@#Mimicked water samples containing HAV particles were prepared and concentrated by positively charged membrane and negatively charged membrane respectively. Then different method including direct lysis, shaker, vortex and ultrasonication were used to elute HAV followed by the quantification of HAV by Taqman Real-time RT-PCR. The data were analyzed by professional statistical software.@*Results@#In the present study, when mimicked water samples contained 300 TCID50 of HAV, there was no significant difference between the concentration effects by negatively charged membrane and positively charged membrane (P=0.825>0.05). However, when HAV in mimic water samples was up to 1500 TCID50 and 7500 TCID50, the recycle efficiency by negatively charged membrane was higher than that by positively charged membrane (P<0.01). Additionally, this study found that HAV recycle ratio could be up to (68.17±16.79)% when direct lysis was used for viral elution, therefore direct lysis was proved much better than shaker, vortex and ultrasonication. They were significantly different (P<0.0001).@*Conclusions@#Elution process played the key role in HAV concentration when membrane filtration method was used. Direct lysis was proved much better than other method and it was the most efficient way in HAV recycle from mimicked water samples.

Sero-epidemiological characteristics of hepatitis E in population aged 1-29 years in China, 2014 / 中华流行病学杂志

Objective@#To analyze the epidemiological characteristics of hepatitis E Virus antibody (anti-HEV) in people aged 1-29 years in China in 2014.@*Methods@#Based on database of the national serologic survey of hepatitis B in people aged 1-29 years in China, in 2014, the sample size was estimated. The serum samples of the people surveyed were randomly selected to detect anti-HEV IgG by using enzyme- linked immunosorbent assay (ELISA). Statistical software SAS 9.1.3 was used to calculate the positive rate of anti-HEV and 95% confidence interval (CI) in different age, gender groups, urban and rural areas and geographic areas by using the Taylor series linear method with sampling weight. The difference was determined by comparing 95%CI.@*Results@#A total of 14 106 serum samples were detected from people aged 1-29 years, including 6 996 males (49.60%), 7 013 urban residents (49.72%). The positive rate of anti-HEV was 8.12%(95%CI: 7.19-9.15) in people aged 1-29 years. There was no statistical difference between the men and women, between urban area and rural area. The positive rates of anti-HEV in western area(11.36%, 95%CI: 9.45-13.62) was higher than those in eastern and central areas. The positive rates of anti-HEV were 2.46%, 2.24%, 4.50%, 7.58%, 11.89% and 17.27% in people aged 1-, 5-, 10-, 15-, 20- and 25-29 years, respectively. As the age increased, the positive rate of anti-HEV gradually increased. In different ethnic groups, the positive rate of anti-HEV was higher in Tibetan (18.32%, 95%CI: 12.02-26.90), Zhuang (9.54%, 95%CI: 4.33-19.73) ethnic groups.@*Conclusion@#The positive rate of anti-HEV declined slightly in China in 2014. It is still necessary to pay close attention to the HEV infection, morbidity of hepatitis E and risk factors in people aged 1-29 years.

Preparation of peptide mimotope-based diagnostic antigen of Epstein-Barr virus infection / 中华实验和临床病毒学杂志

Objective@#To prepare peptide minotope-based recombinant diagnostic antigen of Epstein-Barr virus (EBV) infection and evaluate its antigenicity preliminarily.@*Methods@#With Trx at the N-terminal and His tag at the C-terminal, the peptide minotope of EBV (GP125, F1, A2, A3C2) was expressed in Escherichia coli and purified by affinity and anion exchange chromatography (designated 'H58’); based on antigenicity of H58 identified by Western blotting (WB), we constructed and evaluated a novel early diagnostic ELISA for EBV infection.@*Results@#The soluble H58 protein with high concentration (2.8 mg/ml) and purity (99.01) was obtained; WB analysis found that there was an obvious band (28 ×103) on the NC membrane, using H58 anti-Trx monoclonal antibody or acute-phase sera of EBV infection as the first antibody. With the novel ELISA, 50 positive sera of EBV infection and 50 negative sera were detected, displaying that the grouping of OD value of positive serum (95%CI: 1.233-1.489) and negative serum (95%CI: 0.113-0.159) was different (P<0.05) with the sensitivity 98.0%, specificity 96.0% and kappa value 0.940.@*Conclusions@#By E. coli expression and affinity and ion exchange chromatography purification, the peptide minotope-based recombinant diagnostic antigen of EBV infection was obtained with excellent antigenicity, which could be applied for serological detection of EBV infection.

Expression and purification of the chimeric virus-like particles displaying enterovirus 71 epitopes / 中华实验和临床病毒学杂志

Objective@#To study the expression and purification of the chimeric virus-like particles displaying epitopes of EV71 as a candidate of enterovirus 71 gene recombined vaccinet.@*Methods@#The fusion protein hepatitis B core (HBc)-SP70 was constructed by inserting SP70 into the main immunogenic region of truncated hepatitis B core antigen (HBcAg) sequence, expressed in E. Coli, and purified through sonication, ion exchange chromatography, CsCl cushion centrifugation and density gradient centrifugation. Then its antigenicity was detected by ELISA and Western blot assay.@*Results@#Recombinant plasmid pHBc-SP70 was successfully constructed. And the soluble fusion protein was efficiently expressed induced by IPTG. The purity of the chimeric virus-like particles (VLPs) was up to 90% after the purification process described in method . The purified fusion protein HBc-SP70 could be spontaneously folded and assembled into empty virus-like particles and react with the monoclonal antibodies against HBc and SP70.@*Conclusions@#The chimeric VLPs displaying epitopes of EV71 were efficiently expressed and purified in E. Coli. with excellent antigenicity, which laid a foundation for evaluation of the immune effect evoked by this EV71 gene recombined vaccine.

Whole-genome and quasispecies sequences analysis of three hepatitis A virus isolates in China / 中华实验和临床病毒学杂志

Objective@#To analyze the genetic characteristics of whole-genome and quasispecies sequences from three hepatitis A virus (HAV) strains in China.@*Methods@#Serum samples from acute hepatitis A patients were collected and viral RNA extraction, transcription, nested PCR, sequencing and assembling were performed to gain near full-length sequences; cloning-based sequencing of the full-length VP1-2 A region was also performed.@*Results@#Genotyping showed that the nucleotide and amino acid identities among three strains on VP1-2 A junction region were both 100% and all belonged to subgenotype IA; the nucleotide and amino acid identities on whole-genome region were 99.9%-100% and 100% respectively, and shared the highest identities with AH2 strain from GenBank of 98.5% in nucleotide and 99.7% in amino acid level; no amino acid variation was found among published neutralizing antigenic sites. Within cloning sequences from each strain, the nucleotide and amino acid identities were 99.0%-100% and 98.1%-100%, while among all cloning sequences were 99.0%-100% and 97.2%-100%. The variation rate of nucleotide and amino acid in VP1-2 A junction region were both higher than that of partial VP1 region.@*Conclusions@#Sequences among three strains in VP1-2 A region were identical, the nucleotide and amino acid identities in both whole-genome region and among quasispecies sequences were relatively high to deduce that they were from the same outbreak. This study provides new insight for identification of HAV transmissions and tracing investigations.

Comparative analyze on hepatitis B seroepidemiological surveys among population aged 1-29 years in different epidemic regions of China in 1992 and 2014 / 中华预防医学杂志

Objective@#To evaluate the effect of hepatitis B prevention and control by comparative analysis on the results of HBsAg, anti-HBs and anti-HBc prevalence from national hepatitis B seroepidemiological surveys in 1992 and 2014 in different epidemic regions of China.@*Methods@#Data was from the national seroepidemiological surveys of hepatitis B conducted in 1992 and 2014. The survey in 1992 was conducted in 145 disease surveillance points of 30 provinces (excluding Hong Kong, Macao Special Administrative Region and Taiwan province) in China. The survey in 2016 was conducted in 160 disease surveillance points of 31 provinces (excluding Hong Kong, Macao Special Administrative Region and Taiwan province) in China. In the two surveys, face-to-face interviews with the subject by door to door or on the investigation site were conducted by trained staff using standard questionnaires to obtain basic information including birth date, gender, ethnicity, resident place and so on. And then 5 ml venous blood was collected to test the sero-markers of HBsAg, anti-HBs and anti-HBc. We analyzed unweighted point prevalence and 95% CI of HBsAg, anti-HBs and anti-HBc in 1992 which had no design weighting, and analyzed weighted point prevalence and 95%CI of HBsAg, anti-HBs and anti-HBc in 2014 which had design weighting.@*Results@#34 291 and 31 713 people aged 1-29 years were involved in 1992 and 2014 national serosurveys of China, respectively. For the people aged 1-29 years, HBsAg prevalence was 2.64% (95%CI: 2.28%-3.06%) in 2014 and decreased by 73.92% as compared with the rate 10.13% (95% CI: 9.81%-10.45%) in 1992. Anti-HBc prevalence was 13.01% (95%CI: 12.09%-14.00%) in 2014 and decreased by 71.61% as compared with the rate 45.84% (95% CI: 45.31%-46.37%) in 1992. Anti-HBs prevalence was 57.79% (95%CI: 56.33%-59.25%) in 2014 and ascended by 127.41% as compared with the rate 25.41% (95% CI: 24.95%-25.87%) in 1992. In high, medium and low epidemic region, for the people who born during 1992-2001 when hepatitis B vaccine was introduced in routine immunization management, HBsAg prevalence was 4.74% (95%CI: 3.79%-5.69%), 1.59% (95%CI: 1.09%-2.10%) and 2.53% (95%CI: 1.66%-3.39%), respectively, and anti-HBs prevalence was 64.25% (95% CI: 62.11%-66.39%), 56.34% (95% CI: 54.50%-58.57%), 54.49% (95%CI: 51.75%-57.23%), respectively, and anti-HBc prevalence was 15.16% (95%CI: 13.56%-16.76%), 11.07% (95%CI: 9.80%-12.33%), 7.61% (95%CI: 6.15%-9.07%), respectively. In high, medium and low epidemic region, for the people who born during 2002-2013 the duration which hepatitis B vaccine was integrated in expanded immunization program born during when HBsAg prevalence was 0.88% (95%CI: 0.66%-1.11%), 0.37% (95%CI: 0.24%-0.49%)and 0.71% (95%CI: 0.48%-0.94%), respectively, and anti-HBs prevalence was 60.74% (95%CI: 59.57%-61.90%), 59.46% (95%CI: 58.44%-60.49%), 52.56% (95% CI: 51.20%-53.92%), respectively, and anti-HBc prevalence was 3.30% (95% CI: 2.87%-3.72%), 1.91% (95%CI: 1.63%-2.20%), 2.25% (95%CI: 1.85%-2.66%), respectively.@*Conclusion@#China had made great achievement in hepatitis B prevention and control. HBsAg prevalence among people aged 1-29 years old in 2014 decreased dramatically as compared with that in 1992. Since hepatitis B vaccine was integrated into expanded immunization program, China reduced HBsAg prevalence to less than 1% among people aged 1-12 years in 2014 in different epidemic region.

The influence of pVR-IL15 gene adjuvant on immunization with recombinant viruses expressing HBsAg / 中华实验和临床病毒学杂志

Objective@#To explore the influence of pVR-IL15 gene adjuvant on the immune responses of different immunization strategies.@*Methods@#The sequential immunization strategies were used in BALB/c mice with a DNA vaccine, recombinant modified vaccinia virus Ankara and recombinant adenovirus expressing HBsAg respectively and combined with a gene adjuvant pVR-IL15. Cellular and humoral immune responses were evaluated by IFN-γ enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA), respectively.@*Results@#The levels of humoral and cellular immune responses in the immune groups combined with pVR-IL15 were significantly higher than those of the non-combined with pVR-IL15.@*Conclusions@#The pVR-IL15 gene adjuvant can enhance the immune responses induced by the recombinant viruses expressing HBsAg.

Comparisons for kits HAVAb2.0 and HAV-IgG / 中华实验和临床病毒学杂志

Objective@#Two kits for detecting anti-HAV antibody produced by Abbott Laboratories are evaluated for their performance in order to use in the anti-HAV antibodies detections.@*Methods@#Serially diluted standard reference serum were detected 4 times with HAVAb2.0 kit and HAV-IgG kit , then fit standard curves and calculated Interclass correlation coefficient (ICC). 120 serum that have different levels of anti-HAV antibodies were chosen to be detected by two kits and calculated sensitivity and specificity, receive operation characteristic (ROC) curve and area under curve (AUC) while the results of HAV-IgG were used as golden standard.@*Results@#R2 and ICC for HAV-IgG were 0.9977 and 0.999, respectively, higher than 0.9893 and 0.995(P>0.05) for HAVAb2.0. Sensitivity and specificity for HAVAb2.0 were 93.15% and 100% and AUC was 0.994 when kit HAV-IgG was chosen as golden standard.@*Conclusion@#There is little difference in performance between HAV-IgG and HAVAb2.0, both of them can be used in anti-HAV antibodies detection.

Parallel test of anti-HCV and HCV-RNA can enhance the ability to detect HCV infection / 中华实验和临床病毒学杂志

Objective@#To explore the method of enhancing the ability to detect HCV infection and to provide the advice for practical work.@*Methods@#A total of 487 samples from a village in Hebei Province were selected by cluster sampling. Serum samples were detected by anti-HCV and HCV-RNA assays. The results were compared between the two tests. The correlation of HCV-RNA quantification and its anti-HCV s/n value was analyzed.@*Results@#The positive values of anti-HCV and HCV-RNA were 22.1% and 14.2%, respectively, which were lower than that of the parallel test (χ2=4.0000, P=0.0455, χ2=42.0000, P <0.0001). The HCV-RNA quantification of the samples was positively correlated with the anti-HCV s/n value, which means anti-HCV s/n value increased with HCV-RNA quantification.@*Conclusion@#The parallel test of anti-HCV and HCV-RNA can enhance the ability to detect HCV infection.

Sequence analysis of HBV in primary hepatomas patients infected with HBV / 中华实验和临床病毒学杂志

Objective@#To study the relationship between the development of hepatocellular carcinoma(HCC) and HBV gene characteristics among the HCC patients with hepatitis B virus (HBV) infection.@*Methods@#Some acute and chronic hepatitis B patients were collected as control group and HBV associated HCC patients as HCC group. Serum samples of subjects were tested for HBV serological markers. HBV DNA of those samples had been extracted and nested PCR was used to amplify the sequence of HBV DNA. Furthermore, MEGA 6.0 and Bioedit softwares were used to made phylogenetic trees and analyze the gene mutations.@*Results@#The sequences of S region and BCP/Precore region of HBV were amplified from 86 samples in study group and 39 samples in control group. The prevalence of PreS deletion, A1762T and A1762T/G1764A in HCC group were 39.53%, 74.42% and 72.09% respectively, and in control group were 20.51%, 53.85% and 53.85% respectively. The statistical differences of them were significant. The prevalence of A1762T and A1762T/G1764A in ≥ 50 years group were higher than that of < 50 years group. The prevalence of A1762T, G1764A and A1762T/G1764A of subjects who infected genotype C were higher than those infected genotype B. On the contrary, the prevalence of G1896A of subjects who infected genotype C were lower than that of genotype B. It was found that ≥ 50 years, genotype C and G1896A mutation were independently associated with HCC. The risk for suffer from HCC of ≥50 years group, genotype C group and G1896A group were 9.349, 28.875 and 7.648 times compared with < 50 years group genotype B group and without G1896A mutation group, respectively.@*Conclusions@#The population of ≥50 years or genotype C had a higher prevalence of A1762T, A1762T/G1764A, ≥50years、genotype C、G1896A were independently associated with HCC, as compared with the subjects of the control group.

Recent advances in molecular epidemiological methods for analyzing hepatitis C virus transmission / 中华微生物学和免疫学杂志

Hepatitis C virus ( HCV) infection is one of the main reasons causing liver cirrhosis and hepatocellular carcinoma. The quasispecies composition and the evolution of HCV are very complicated. An in-depth analysis of the quasispecies composition of HCV is critical for elucidating the mechanisms of HCV transmission. The regions encoding the envelope glycoproteins (E1 and E2) and the nonstructural protein 2 (NS2) are hyper variable regions (HVR). Analyzing the quasispecies of HCV HVR with advanced sequen-cing and bio-information technologies would be beneficial for understanding the sources of HCV infection, the routes of transmission and the evolution of HCV. Currently, three generations of sequencing methods and some bio-information soft-wares are available for analyzing HCV HVR quasispecies. When an outbreak of HCV infection happens, using the new generation of sequencing and bio-information methods to seek the first case and the routes of transmission would provide scientific basis for the prevention and treatment of HCV in-fection.

Whole-sequence Analyses for 12 HBV C/D Recombinants from a Population in Tibet (China) / 病毒学报

We wished to undertake molecular genetic typing and evaluate recombinants of the hepatitis-B virus (HBV) in Tibet (China). Multistage random sampling was used to collect HBsAg-positive samples. Nested polymerase chain reactions were used to amplify the whole sequence of the HBV. DNAstar, MEGA6 and SimPlot were used to assemble sequences, create phylogenetic trees, and undertake recombination analyses. Twelve whole sequences of the HBV of a Tibetan population were collected using these methods. Results showed that all 12 strains were C/D recombinants. Nine of the recombinations were at nt750, and the other three at nt1526. Therefore, the 12 strains could be divided into two types of recombinants: C/Da and C/Db. Analyses of the sequence of the whole genome revealed that the 12 strains belonged to genotype C, and that the nucleotide distance was > 4% between the 12 strains and sub-genotypes C1 to C15 in Genbank. The most likely sub-genotype was C1. Individuals with C/Da were from central and northern Tibet (e.g., Lasa, Linzhi, Ali) and those with C/Db recombinants were from Shannan in southern Tibet. These data suggest that the two types of recombinants had a good distribution in Tibet. Also, they can provide important information for studies on HBV recombination, gene features, virus evolution, as well as the control and prevention of HBV infection in Tibet.

Cloning and expression and preliminary antigenicity identification for the diagnostic antigen of hepatitis E virus / 中华流行病学杂志

Objective To clone,express and purify thioredoxin (named as N5),a specific diagnostic antigen of hepatitis E virus (HEV),and to initially evaluate its antigenicity and serological test performance.Methods Based on the gene sequences of HEV-ORF2 and carboxyl terminal ORF3 on GenBank,the codon was optimized by the Escherichia coli codon preference,inserted it into prokaryotic expression vector M48 following total gene synthesization,and expressed in Escherichia coli fusion protein N5 recombined with Thioredoxin (TRX).Fusion protein was purified in affinity chromatography,evaluating its antigenicity with Western blot technology,then evaluating its serological test performance using the negative and positive serum samples confirmed of HEV infection with laboratory and clinical tests.Results The recombinant plasmid expressing N5 diagnostic antigen was successfully established; high-level expression and purification to obtain soluble diagnostic antigens; Western blot results indicating fusion protein N5 can be bound specifically with the serum of HEV IgM antibody positive,showing satisfactory antigencity; using fusion protein N5 as the capture antigen to build indirect ELISA,testing 40 serum samples of HEV cases confirmed by pathogen detection and clinical diagnosis and 40 serum samples of healthy people,with the sensitivity and specificity of 95％ (38/40) and 90％ (36/40) respectively.Conclusion Recombinant plasmid expressing the HEV diagnostic antigen recombined with thioredoxin was successfully established,and soluble fusion protein N5 was obtained with high expression and strong antigenicity,promising in its future applications.

Analysis on genotype distribution and mutation of major hydroponic region of hepatitis B virus in Liaoning / 中华流行病学杂志

<p><b>OBJECTIVE</b>To understand the genotype and the mutation of amino acid (aa) in the major hydrophilic region (MHR) of hepatitis B virus (HBV) among the hepatitis B cases under surveillance in Liaoning province.</p><p><b>METHODS</b>The serum samples were collected from hepatitis B cases under surveillance in Liaoning. The complete S gene of HBV were amplified and sequenced. The aa sequences were analyzed with bioinformatics software.</p><p><b>RESULTS</b>A total of 81 sequences of S gene of HBV were ontained, including 7 sequences of genotype B (8.64%), 70 sequences of genotype C (86.42%) and 4 sequences of genotype D (4.94%). The mutation rate of aa of MHR of S gene was 4.87%. The mutation rate of T126I was highest (8.64%). The overall prevalence of mutant strain of MHR was 49.38% (40/81), and it was 42.86% for genotype B (3/7), 47.14% for genotype C (33/70) and 100% for genotype D (4/4). Statistical analysis revealed that there was no age, sex, genotypes and anti-HBc IgM (±) specific significant differences in aa mutant strains prevalence, while the difference between ALT abnormal group (ALT ≥ 43 IU/L) and ALT normal group (ALT<43 IU/L) was significant (P < 0.05).</p><p><b>CONCLUSION</b>Among the hepatitis B cases under surveillance in Liaoning, HBV genotype C predominant, followed by genotype B and genotype D. The mutation of aa in MHR of HBV detected in this study was consistent with previous research results. It is necessary to strengthen the surveillance for HBV mutation to provide accurate information for the development of hepatitis B prevention and control measures.</p>

Association of T cell receptor diversity of CD4+ T lymphocytes with viral load in individuals with HIV-1 infection / 中华微生物学和免疫学杂志

Objective To assess the impact of the virus on the complementary determining region 3 (CDR3) length diversity of T cell receptor(TCR) Vβ repertoires of CD4+ T lymphocytes and to explore its association with viral load in individuals with HIV-1 infection. Methods The TCR repertoire was examined using spectratyping of CDR3 length diversity within CD4+ T cells in HIV infected and healthy adults. Separation of CD4+ T cells from peripheral blood mononuclear cells ( PBMCs) was carried out by using immunomagnetic beads coated with anti-CD4 antibody. Total RNAs from the purified CD4 + T lymphocytes were isolated and used to perform nested-PCR amplifications in CDR3 of 22 TCR gene families. CDR3 diversity and its association with viral load in individuals with HIV-1 infection were analyzed. Results An average diversity for all CDR3 profiles in CD4+ T cells from 25 HIV-infected individuals was significantly different as compared to 10 age-matched healthy donors (P＜0.05) with the HIV-infected individuals losing diversity in the CDR3 profiles. There was positive correlation between changes in TCR CDR3 diversity and viral load (r = 0. 494, P ＜ 0. 05). The changes in CDR3 length diversity of Vβ families in HIV-infected individuals, particular in Vβ8, Vβ22, Vβ23 were statistically different from the healthy controls. Conclusion HIV-1 infection might induce the loss of TCR Vp repertoire diversity and disrupt the CDR3 Gaussian distributions within CD4 + T cells. There should be positive correlation between changes in TCR CDR3 diversity and the viral load in HIV-1 infected patients.

Enhancement of the immune response of a novel DNA vaccine encoding conserved NS3 and Core fusion gene of HCV injected by intradermal electrotransfer in mice / 中华微生物学和免疫学杂志

Objective To characterize the immunogenicity in gene immunization of the conserved regions of hepatitis C virus(HCV) based on different delivery strategies. Methods We first constructed a novel DNA vaccine encoding a fusion gene(from partial NS3 and Core) of HCV. Then we compared different protocols based on naked DNA injection twice or DNA injection with gene electrotransfer(GET) in BALB/c mice. The immune response was measured by antibody ELISA and by IFN-gamma ELISPOT. Results Our data showed that a protocol based on intradermally injection of DNA with optimal GET induced the strongest humoral and cellular immunity, and DNA with GET induced a substantially higher anti-NS3/Core T cell re-spoase than naked DNA injection. Conclusion Our data suggest that DNA vaccines encoding NS3/Core fu-sion protein of HCV immunized by the present strategy could merit further study in the context of future prophylactic and therapeutic HCV T cell based vaccines.

The structural characterization and antigenicity of the S protein of SARS-CoV / 基因组蛋白质组与生物信息学报·英文版

The corona-like spikes or peplomers on the surface of the virion under electronic microscope are the most striking features of coronaviruses. The S (spike) protein is the largest structural protein, with 1,255 amino acids, in the viral genome. Its structure can be divided into three regions: a long N-terminal region in the exterior, a characteristic transmembrane (TM) region, and a short C-terminus in the interior of a virion. We detected fifteen substitutions of nucleotides by comparisons with the seventeen published SARS-CoV genome sequences, eight (53.3%) of which are non-synonymous mutations leading to amino acid alternations with predicted physiochemical changes. The possible antigenic determinants of the S protein are predicted, and the result is confirmed by ELISA (enzyme-linked immunosorbent assay) with synthesized peptides. Another profound finding is that three disulfide bonds are defined at the C-terminus with the N-terminus of the E (envelope) protein, based on the typical sequence and positions, thus establishing the structural connection with these two important structural proteins, if confirmed. Phylogenetic analysis reveals several conserved regions that might be potent drug targets.

The E protein is a multifunctional membrane protein of SARS-CoV / 基因组蛋白质组与生物信息学报·英文版

The E (envelope) protein is the smallest structural protein in all coronaviruses and is the only viral structural protein in which no variation has been detected. We conducted genome sequencing and phylogenetic analyses of SARS-CoV. Based on genome sequencing, we predicted the E protein is a transmembrane (TM) protein characterized by a TM region with strong hydrophobicity and alpha-helix conformation. We identified a segment (NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH) in the carboxyl-terminal region of the E protein that appears to form three disulfide bonds with another segment of corresponding cysteines in the carboxyl-terminus of the S (spike) protein. These bonds point to a possible structural association between the E and S proteins. Our phylogenetic analyses of the E protein sequences in all published coronaviruses place SARS-CoV in an independent group in Coronaviridae and suggest a non-human animal origin.

The structure analysis and antigenicity study of the N protein of SARS-CoV / 基因组蛋白质组与生物信息学报·英文版

The Coronaviridae family is characterized by a nucleocapsid that is composed of the genome RNA molecule in combination with the nucleoprotein (N protein) within a virion. The most striking physiochemical feature of the N protein of SARS-CoV is that it is a typical basic protein with a high predicted pI and high hydrophilicity, which is consistent with its function of binding to the ribophosphate backbone of the RNA molecule. The predicted high extent of phosphorylation of the N protein on multiple candidate phosphorylation sites demonstrates that it would be related to important functions, such as RNA-binding and localization to the nucleolus of host cells. Subsequent study shows that there is an SR-rich region in the N protein and this region might be involved in the protein-protein interaction. The abundant antigenic sites predicted in the N protein, as well as experimental evidence with synthesized polypeptides, indicate that the N protein is one of the major antigens of the SARS-CoV. Compared with other viral structural proteins, the low variation rate of the N protein with regards to its size suggests its importance to the survival of the virus.

The R protein of SARS-CoV: analyses of structure and function based on four complete genome sequences of isolates BJ01-BJ04 / 基因组蛋白质组与生物信息学报·英文版

The R (replicase) protein is the uniquely defined non-structural protein (NSP) responsible for RNA replication, mutation rate or fidelity, regulation of transcription in coronaviruses and many other ssRNA viruses. Based on our complete genome sequences of four isolates (BJ01-BJ04) of SARS-CoV from Beijing, China, we analyzed the structure and predicted functions of the R protein in comparison with 13 other isolates of SARS-CoV and 6 other coronaviruses. The entire ORF (open-reading frame) encodes for two major enzyme activities, RNA-dependent RNA polymerase (RdRp) and proteinase activities. The R polyprotein undergoes a complex proteolytic process to produce 15 function-related peptides. A hydrophobic domain (HOD) and a hydrophilic domain (HID) are newly identified within NSP1. The substitution rate of the R protein is close to the average of the SARS-CoV genome. The functional domains in all NSPs of the R protein give different phylogenetic results that suggest their different mutation rate under selective pressure. Eleven highly conserved regions in RdRp and twelve cleavage sites by 3CLP (chymotrypsin-like protein) have been identified as potential drug targets. Findings suggest that it is possible to obtain information about the phylogeny of SARS-CoV, as well as potential tools for drug design, genotyping and diagnostics of SARS.