ABSTRACT
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.
Subject(s)
Endemic Diseases , Hepatitis E virus/isolation & purification , Hepatitis E/veterinary , Animals , Antigens, Viral , Genotype , Hepatitis Antibodies/blood , Hepatitis E/epidemiology , Hepatitis E virus/classification , Hepatitis E virus/genetics , Humans , Molecular Sequence Data , RNA, Viral/genetics , Sequence Analysis, DNA , Seroepidemiologic Studies , United States/epidemiologyABSTRACT
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.
Subject(s)
Antigens, Viral , Clinical Laboratory Techniques/methods , Hepatitis C Antibodies/blood , Hepatitis C/diagnosis , Hepatitis C/immunology , Viral Nonstructural Proteins , Viral Structural Proteins , Flow Cytometry , Humans , MicrospheresABSTRACT
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.
Subject(s)
Antibody Affinity , Hepatitis C Antibodies/immunology , Hepatitis C, Chronic/diagnosis , Hepatitis C/diagnosis , Immunoglobulin G/immunology , Acute Disease , Diagnosis, Differential , Enzyme-Linked Immunosorbent Assay , Humans , RNA, Viral/blood , Time Factors , Urea/pharmacologyABSTRACT
The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenic properties of the NS5 protein was studied by using recombinant proteins. A strong antigenic region was identified within the HCV NS5A protein at amino acids 2212 to 2313. Forty-five unique sequences encompassing this region were selected from GenBank and were compared to each other. The results of this analysis 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%. Thirteen representative sequences from all six HCV genotypes were selected to design synthetic genes coding for this antigenic region. These genes were assembled by PCR from synthetic oligonucleotides and expressed in Escherichia 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 = 91) obtained from patients infected with HCV genotypes 1 through 6. All but two proteins immunoreacted with 62 to 93% of HCV anti-NS5-positive 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. Thus, sequence variability has a profound effect on the antigenic properties of the NS5A immunodominant regions. This observation should be taken into consideration in the development of diagnostic tests for the efficient detection of anti-HCV activity in serum specimens.