We made recombinant antigen GE containing fragment of VZV glycoprotein E (Gly48 - Glu135) fused to E. coli beta-galactosidase and confirmed its antigen specificity by Western blotting and competitive-inhibition enzyme immunoassay (EIA) in comparison with commercial analogues and natural viral antigens. We showed interaction of recombinant GE protein with IgG antibodies from rabbits immunized by vaccine viral strain. GE protein also specifically reacted in ELISA with 66% of sera from zoster patients and 35% of sera from control groups including sera containing antibodies to other herpes viruses, sera from healthy donors, and sera from patients with different forms of intestinal disorders. Consequently, we demonstrated possibility of application of our recombinant GE VZV as antigen for diagnostics and research use.

Introduction. The diagnostic efficacy of methods for hepatitis E serodiagnostic varies over a wide range; therefore, the combined use of tests of various formats is recommended. The aim of the research was to develop a test system for the detection of IgG antibodies to hepatitis E virus (HEV) in human serum by linear immunoassay (LIA). Material and methods. Serum samples from patients with hepatitis and healthy individuals were tested using commercial enzyme-linked immunosorbent assay systems for the presence of IgG antibodies to viral agents causing hepatitis and other infections associated with liver pathology. Recombinant antigens ORF2 and ORF3 of HEV genotypes 1 and 3 were used. The “RecomLine HEV IgG/IgM” reagent kit (Mikrogen GmbH, Germany) was used as a comparison test system. Results. The first Russian diagnostic kit “Blot-HEV”, designed to detect IgG antibodies to individual HEV proteins in human serum using LIA, was developed. The antigenic base is represented by strips of a nitrocellulose membrane with immobilized recombinant antigens ORF2 (aa 406–660) and ORF3 (aa 1–113) of HEV genotypes 1 and 3, and control antigens in the form of discrete lines. The conjugate was mouse monoclonal antibodies to human class G immunoglobulins labeled with horseradish peroxidase. The chromogen solution contained the 3,3’,5,5’-tetramethylbenzidine. A visual and digital recording of results was provided. The analytical sensitivity of the test kit was 0.625 IU/ml for ORF2 antigens and 2.5 IU/ml for ORF3 antigens. The absence of the influence of endogenous interfering substances on the results of the analysis and the absence of cross-reactions with antibodies to hepatitis pathogens of the other etiologies had been shown. The sensitivity of the test system compared to the “RecomLine HEV IgG/IgM” kit was 92%, specificity 97%. Shelf life in condition of storage was determined to be 12 months. Conclusions. The developed test can be used to confirm the results of ELISA in laboratory diagnosis of hepatitis E.

No abstract

Hepatitis E is an acute viral infectious disease transmitted by fecal-oral route mainly through fecally contaminated drinking water, with cyclic outbreaks and frequent development of acute hepatic encephalopathy in pregnant women. Hepatitis E epidemic outbreaks occur in Central Asia, Africa and Latin America, whereasChina,India,Turkmenistan,Kazakhstan,Tajikistan,Uzbekistan,Kyrgyzstan,Bolivia,Mexico, andTaiwanrepresent endemic geographic regions. Hepatitis E in the structure of acute viral hepatitis morbidity during outbreaks ranges from 64.7% to 80%, whereas sporadic morbidity may be up to 10 to 18.8%. In contrast, percentage of hepatitis E in acute viral hepatitis varies from 0.5% to 12.6% in European countries and some territories of theRussian Federation. The latent active virus circulation was confirmed in various regions of theRussian Federation. All introduced cases were related to recent traveling to the regions with high incidence of hepatitis E, which course clinically did not differ from standard hepatitis E infection, but no cases of infection were recorded after exposure. Lack of contact transmission in this case was associated with low virus survival in environment. Patients with any clinical form including anicteric serve as a source of infection. An increased risk of hepatitis E infection is typical for livestock workers dealing with pigs, employe es of meat processing plants engaged in primary meat carcass processing and working at slaughterhouse. According to the World Health Organization, 20 million cases of hepatitis E virus infection are recorded annually, among which 3 million cases account for acute hepatitis E and related 70 000 lethal outcomes. Chronic liver disorders comprising up to 70% followed by death of pregnant women (40%) as well as acute liver and kidney failure reaching as low as 4% result in lethal outcome in hepatitis E patients. Creating a mathematical model for development of hepatitis E infection could allow to predict changes in its morbidity rate at controlled area. Here, for the first time we propose a mathematical model for developing hepatitis E in human population based on disease course, which may potentially predict an incidence rate for the most dangerous icteric hepatitis E as well as assess amount of individuals susceptible to it at morbidity rise in the geographic region.

Aim. The development of the hepatitis E virus (HEV) genotype 1 recombinant capsid protein. Materials and methods. Escherichia coli strains, plasmid vectors, serological and clinical samples, ELISA reagent kits, molecular biological, bioinformatic, biotechnological, biochemical and serological methods. Results. Using HEV genotype 1 DNA copy of subgenomic virus RNA we made E.coli strains producing recombinabt capsid protein, containing C-terminal fragment of ORF2 protein fused to E.coli beta-galactosidase. Recombinant protein ORF2 had been isolated from the inclusion bodies of the E.coli biomass and purified by size exclusion chromatography. By Western blotting it had been shown specific interaction of the recombinant polypeptide with anti-HEV IgG from pool of positive sera. Antigenic specificity of the recombinant polypeptide had been confirmed by enzyme-linked immunosorbent assay with sera of hepatitis E patients and reference groups: healthy donors, patients with hepatitis А, В, C, infectious mononucleosis and cytomegalovirus infection, HIV-infected patients. Conclusion. HEV genotype 1 ORF2 recombinant antigen had been developed, and its possible use in diagnostic tests had been experimentally shown.