Abstract Objective:To define the epidemiology and clinical manifestations of hepatitis D virus infection in an institutionalized population. Design:A case-control study of hepatitis B carriers with...

Hepatitis E is recognized as a zoonosis, and swine are known reservoirs, but how broadly enzootic its causative agent, hepatitis E virus (HEV), is remains controversial. To determine the prevalence of HEV infection in animals, a serological assay with capability to detect anti-HEV-antibody across a wide variety of animal species was devised. Recombinant antigens comprising truncated capsid proteins generated from HEV-subgenomic constructs that represent all four viral genotypes were used to capture anti-HEV in the test sample and as an analyte reporter. To facilitate development and validation of the assay, serum samples were assembled from blood donors (n = 372), acute hepatitis E patients (n = 94), five laboratory animals (rhesus monkey, pig, New Zealand rabbit, Wistar rat, and BALB/c mouse) immunized with HEV antigens, and four pigs experimentally infected with HEV. The assay was then applied to 4,936 sera collected from 35 genera of animals that were wild, feral, domesticated, or otherwise held captive in the United States. Test positivity was determined in 457 samples (9.3%). These originated from: bison (3/65, 4.6%), cattle (174/1,156, 15%), dogs (2/212, 0.9%), Norway rats (2/318, 0.6%), farmed swine (267/648, 41.2%), and feral swine (9/306, 2.9%). Only the porcine samples yielded the highest reactivities. HEV RNA was amplified from one farmed pig and two feral pigs and characterized by nucleotide sequencing to belong to genotype 3. HEV infected farmed swine primarily, and the role of other animals as reservoirs of its zoonotic spread appears to be limited.

ABSTRACT Currently available serological assays for detection of antibodies to hepatitis C virus (HCV) cannot reliably discriminate acute from chronic HCV infection. We developed a multiplexed, flow-cytometric microsphere immunoassay to measure anti-HCV-IgG reactivities to the core, NS3, NS4, and NS5 HCV recombinant proteins and applied it to 99 serum samples from 24 anti-HCV seroconverters and 141 anti-HCV-IgG and HCV RNA-positive plasma specimens from chronically infected people. Differences in the geometric means or means of signal/cutoff ratios between the two sample sets were statistically significant for all the antigens tested. A multivariate logistic regression model correctly classified the samples in two groups, with a cross-validation accuracy of 90.8% for the acute group and 97.2% for the chronic group. The immunoassay described has the potential to distinguish acute from chronic HCV infection.

The new highly sensitive test system "DS-EIA-HBsAg-0.01" (Priority Certificate No. 2006129019 of August 10, 2006) in detecting hepatitis B surface antigen (HBsAg) was assessed. The sensitivity of the test was estimated using the federal standards sample HBsAg 42-28-311-06, panels' samples Boston Biomedica Inc. (West Bridgewater, Mass, USA) and ZeptoMetrix Corp. (Buffalo, NY, USA). The findings have indicated that "DS-EIA-HBsAg-0.01" is equally effective in detecting different subtypes of HBsAg during a seroconversion period earlier than alternative assays. Along with its high analytical and diagnostic sensitivity, the system shows a high diagnostic specificity.

An artificial antigen composed of 12 small antigenic regions derived from the ORF2 and ORF3 HEV proteins was designed. The gene encoding for this artificial antigen was assembled from synthetic oligonucleotides by a new method called Restriction Enzyme-Assisted Ligation (REAL). The diagnostic relevance of this second generation HEV mosaic protein (HEV MA-II) was demonstrated by testing this antigen against a panel of 142 well defined anti-HEV positive and anti-HEV negative serum samples. The data obtained in this study support the substantial diagnostic potential of this HEV mosaic antigen.

ABSTRACT An assay to measure avidity index (AI) was developed to diagnose incident hepatitis C virus (HCV) infections. The assay demonstrated an AI value statistically significantly lower in primary HCV infections than in chronic infections. When the assay was applied to past resolved infections, the difference in AI values was not as significant as the difference between incident and chronic infections. Lower AI values obtained in past resolved infections may be directly related to lower levels of immunoglobulin G anti-HCV in past resolved infections than in either new infections or chronic infections.

BACKGROUND: This study was conducted by the International Consortium for Blood Safety (ICBS) and its Collaborating Center, the Paul Ehrlich Institute, to identify high‐quality, affordable assays for the detection of hepatitis C virus (HCV) antibodies and make available information on their performance for the benefit of developing countries.STUDY DESIGN AND METHODS: Forty‐four assays were evaluated for their sensitivity and specificity. The assays’ sensitivity was evaluated on a characterized panel of 200 anti‐HCV‐positive samples comprising major HCV genotypes 1 through 6. Three seroconversion panels were used to estimate sensitivity in the early infectious phase. Specificity was evaluated with a characterized ICBS‐negative panel of 181 verified negative samples.RESULTS: Sensitivity was 100 percent for 15 assays, 99.5 percent for 11 assays, 99.0 percent for 6 assays, and less than 99.0 percent for 12 assays. The false‐negative results found were not linked to the genotype. Anti‐HCV detection in the early infectious phase was, on average, 16.7 days later than for tests licensed in the European Union. Specificity in 25 tests was 100 percent, whereas 11 assays showed 1 false‐positive result (99.45%) and the other assays were nonspecific in 2 or more samples. Two assays were not supplied in sufficient quantity to test for specificity.CONCLUSIONS: On applying criteria for highest sensitivity (100%) and high specificity (≥99.5%), 11 tests met the criteria. An additional 19 tests reached a performance comparable to WHO’s criteria for human immunodeficiency virus antibody assays. The genotype diversity of HCV was found not to influence sensitivity of the assays.

Objective: We have reexamined hepatitis B virus subtypes to determine the role of specific HBsAg amino acids in serologic reactivity because of problematic genotype/subtype associations seen in a set of geographically diverse serum specimens. Methods: We obtained DNA sequences for 491 HBsAg-positive specimens from geographically distinct locations, determined their genotypes through phylogenetic analysis, and subtyped the specimens using an algorithm derived from published data on the molecular basis of HBsAg subtype reactivity. Problematic samples were subtyped serologically to resolve conflicts based on the amino acid sequence alone. Results: Three isolates were found to have unusual genotype/subtype associations. Examination of the isolates’ amino acid sequences suggested amino acid positions 122, 127, 140, 159 and 160 can be used to determine subtype reactivity from HBsAg amino acid sequences, while position 134, previously thought to play a role, is no longer important. Conclusions: This re-examination of hepatitis B virus subtypes shows the involvement of amino acid positions 122, 127, 140, 159 and 160 in HBsAg reactivity. While d, y, and r reactivities are controlled by single amino acid changes, w reactivity is determined by positions 122, 127, 140, and 159.

We developed a conceptually new subtraction strategy for the detection and isolation of target DNA and/or RNA from complex nucleic acid mixtures, called Primer Extension Enrichment Reaction (PEER). PEER uses adapters and class IIS restriction enzymes to generate tagged oligonucleotides from dsDNA fragments derived from specimens containing an unknown target ('tester'). Subtraction is achieved by selectively disabling these oligonucleotides by extension reaction using ddNTPs and a double stranded DNA template generated from a pool of normal specimens ('driver'). Primers that do not acquire ddNTP are used to capture and amplify the unique target DNA from the original tester dsDNA. We successfully applied PEER to specimens containing known infectious agents (Hepatitis B Virus and Walrus Calicivirus) and demonstrated that it has higher efficiency than the best comparable technique. The strategy used for PEER is versatile and can be adapted for the identification of known and unknown pathogens and mutations, differential expression studies and other applications that allow the use of subtractive strategies.

OBJECTIVETo identify the location of major immunodominant antigenic region and study the relationship between the gene heterogeneity and immunoreactivity via detecting antigenic reactivity of synthetic peptides deriving from immunodominant region in different genotypes of hepatitis C virus (HCV) NS5a gene.METHODSIn total, 305 non-identical 30-mer long and overlapping by 15 aa peptides derived from HCV NS5a region from codon 2,182 to 2,343 among 45 unique published HCV sequences in GenBank corresponding to different genotype were designed and synthesized. The amino acid sequences of all peptides were compared with DNA Star software. The antigenic reactivity of those peptides was detected with indirect ELISA with both anti-HCV and anti-NS5 positive serum.RESULTSThe sequences showed highly conserved among HCV genotype in regions 2,272-2,301 and 2,302-2,331 as compared to regions 2,212-2,241 and 2,257-2,286. The peptides basing on amino acid residues among 2,212-2,241, 2,272-2,301 and 2,302-2,331 showed stronger immunoreactivity than any other peptides. Eighteen peptides derived from this region showed a broad immunoreactivity, 3 of them could react with 96% of anti-HCV positive sera. Whereas the immunoreactivity of the peptides derived from the region showing highly variable among HCV genotype was found to react more strongly with homologous-genotype sera.CONCLUSIONThe major linear antigenic region was located at amino acid residues among 2,212-2,241, 2,272-2,301 and 2,302-2,331; the short synthetic peptide derived from NS5a region at position 2,212-2,241, 2,272-2,301 and 2,302-2,331 can be used for efficient detection of HCV antibody; some peptides showed genotype specific immuunoreactivity.

The identification and characterization of the hepatitis C virus (HCV) was accomplished by the application of molecular methods by Choo et al. in 1989 using high titered acute phase sera obtained from an experimentally infected chimpanzee.1 Over the ensuing years, important aspects of the epidemiology, molecular virology, pathogenesis, and natural history of HCV have been elucidated. In addition, specific and sensitive assays for the detection of important markers of infection have been developed. These tests have been used in various settings including the clinical diagnostic laboratory to diagnose infection, the blood transfusion services to virtually eliminate the occurrence of post-transfusion HCV and, in the therapeutic setting, to monitor the effect of various treatment modalities.

BACKGROUND AND AIM:The purpose of the present study was to develop enzyme immunoassay (EIA) for the detection of IgG anti-hepatitis E virus (HEV) activity using two new recombinant proteins as antigenic targets, and to evaluate these EIA with the aid of statistical methods.METHODS:Two proteins, a mosaic protein and pB166 containing region 452-617 aa of the ORF2 of the HEV Burma strain, were used to develop the new HEV EIA. This EIA was evaluated using several panels of serum specimens obtained from: (i) acutely HEV-infected patients; (ii) patients with non-A, non-C hepatitis; (iii) normal blood donors (NBD) from non-endemic countries; and (iv) experimentally infected chimpanzees.RESULTS:A new HEV EIA was developed using two new recombinant proteins. This assay was able to detect anti-HEV activity in all specimens from acutely HEV-infected patients. When NBD were tested, more than 15% of specimens were found to be IgG anti-HEV positive. All NBD anti-HEV-positive specimens were tested with overlapping synthetic peptides spanning the entire HEV ORF2-encoded protein. More than 90% of the anti-HEV-positive NBD specimens immunoreacted with an average of 15 synthetic peptides derived from different regions of the HEV ORF2 protein. These data suggest that the HEV EIA is at least 90% specific in detecting remote HEV infections.CONCLUSION:The new HEV EIA developed in the present study is a highly specific diagnostic assay for the detection of anti-HEV activity in serum specimens obtained from different epidemiologic settings.

目的:探讨不同基因型和亚型戊型肝炎病毒(HEV)重组蛋白氨基酸序列的变化对HEV抗原性的影响,筛选出最佳的HEV抗原用于戊型肝炎的免疫诊断.方法:用已知序列的HEV基因工程表达蛋白作为包被抗原,对已知血清标本进行酶联免疫吸附试验测定.结果:6种不同抗原对30份正常献血者血清无抗原性,对17份急性戊型肝炎病人血清和5份实验感染动物血清均呈阳性反应,但血清抗体滴度的高低与所用抗原的基因型有关,尤其是受到该抗原第76位氨基酸变化的影响.结论:多基因型HEV抗原混合物是HEV免疫诊断的最佳抗原.

OBJECTIVE:To study the effect of sequence variability between different genotypes of HCV on the antigenic properties of the NS5a protein of strong antigenic region at position 2212-2313 by using recombinant proteins.METHODS:Thirteen representative sequences from HCV genotypes 1 to 6 were selected to design synthetic genes encoding for this antigenic region. These genes were assembled by PCR from synthetic olionucleotides and expressed in E.coli as hybrid proteins with glutathione S-transferase. All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n=61) obtained from patients infected with HCV genotypes 1 through 6.RESULTS:A comparison of sequences derived from different HCV genotypes showed that the primary structure of this strong antigenic region is highly variable. Percent homology between different genotype sequences varied from 40.4% to 72.5%. All but one protein immunoreacted with 62% to 93% of serum samples. Although a variable degree of genotype specific antigenic reactivity was detected, only one protein demonstrated a noticeable preference to immunoreact with antibodies against the homologous HCV genotype. On the other hand, closely related proteins derived from the same subtype or genotype immunoreacted with significantly different efficiency with HCV antibodies.CONCLUSIONS:Different genotype HCV genes were successfully cloned, expressed and purified. Sequence variability has a profound effect on the antigenic properties of the HCV NS5a immunodominant region. This finding should be considered in the development of diagnostic tests for the efficient detection of anti-HCV activity in serum specimens.

第3代诊断丙型肝炎病毒(HCV)感染免疫酶分析法(EIA)诊断试剂盒中增加了来自HCV非结构蛋白NS5抗原,但其敏感性和特异性均较低.