[Line immunoassay for detection of IgG antibodies to hepatitis E virus (Hepeviridae, Orthohepevirus, Orthohepevirus A)].

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.

[The estimation of the hepatitis E proportion in the etiological structure of acute viral hepatitis in certain regions of of Kyrgyzstan.]

Despite the fact that the Kyrgyz Republic (KR) belongs to the highly endemic regions of the world for hepatitis E, the true extent of the spread of this infection in the country remains poorly understood. It was estimated the prevalence of serological markers of hepatitis E virus (HEV) infection among patients with acute viral hepatitis (AVH) from the regions of the Kyrgyz Republic with a high level of seroprevalence previously established by us. Blood sera samples of hepatitis patients who were admitted to hospitals of Kyrgyzstan in the period 2018-2019 were examined by the enzyme immunoassay method using the kits «DS-ELISA-Anti-HEVIgG¼ and «DS-ELISA-ANTI-HEV-IgM¼ (RPC Diagnostic Systems, Russia). IgG and IgM antibodies to HEV were detected in 103 of 344 studied samples (29.9%). Most often, seropositive specimens were detected among people of age groups under 20 and over 40 years old. Hepatitis with the fecal-oral mode of transmission was dominated in the structure of AVH: the specific gravity of hepatitis E was 47.9%, hepatitis A - 35.32%. Markers of mixed infections with other hepatitis viruses have been detected in 40.4% IgM-positive individuals. Thus, high prevalence of serological markers of HEV infection in the territory of Kyrgyzstan during the interepidemic period had been shown. The necessity of including the determination of serological markers of hepatitis E into the algorithm for the comprehensive diagnosis of AVH in patients of all age groups with liver pathology had been confirmed.

[Prevalence of antibodies to hepatitis E virus among residents of different climate and geographic zones of Russia Federation]. / Chastota vstrechaemosti antitel k virusu gepatita E u naseleniia razlichnykh klimatogeograficheskikh zon Rossiiskoi Federatsii.

The detection frequency of antibodies to Hepatitis E Virus (HEV) was studied in residents of the South and of the Middle European Part of the Russia Federation as well as of Siberia. Antibodies to HEV were most often found both in patients with hepatic pathologies and in subjects with diseases unrelated with a primary hepatic lesion, in particular, in patients with skin and venereal diseases and with HEV. A higher concentration of antibodies to HEV was noted also in blood donors, medical personnel and isolated communities, like prisons or psychiatric clinics. A correlation was established between the rate antibodies to HEV are registered and such risk factor as contacting with blood or a gross violation of the hygienic rules.

[Propagation of hepatitis E virus in an endemic and nonendemic regions]. / Osobennosti rasprostraneniia virusa gepatita E v éndemichnom i neéndemichnom regionakh.

The role of hepatitis E (HE) in sporadic morbidity at an endemic territory (Southern Uzbekistan) and the incidence of anti-HEV in different populations of a nonendemic region (Russia) were evaluated. Antibodies to HEV were detected in 22.1% of patients with acute HE, including mixed infections (+ HA or HB), in the Dekhkanabad district of Kashkadarya region in 1993. The estimated incidence of acute HE was 51.7 per 100,000 population. Analysis of monthly incidence of acute HE demonstrated a seasonal pattern of the morbidity: more than 80% of total recorded cases occurred in August-September. These data indicate the presence of group HEV infections and an important role of this infection in the structure of acute viral hepatitis at the endemic territory. Anti-HEV were found in some population groups at a nonendemic territory: in free-of-charge blood donors in Surgut (4%), in patients with HIV infection (1.6%), and in medical workers in Moscow (1.1%).

[Antigenic specificity of recombinant polypeptides, containing fragments of hepatitis E virus proteins]. / Antigennaia spetsifichnost' rekombinantnykh polipeptidov, soderzhashchikh fragmenty belkov virusa gepatita E.

Antigenic specificity of recombinant polypeptides HE40 and HE60 containing fragments of gene ORF2 and ORF3 protein products of hepatitis E, strain Burma, produced in E. coli cells, is analyzed. Blood sera from patients with acute hepatitis from an endemic region in Uzbekistan were tested for IgG to recombinant antigens by solid-phase enzyme immunoassay with a protein fragment coded by PRF3 gene, a synthetic peptide previously characterized in a commercial test system, as the positive control. 93% sera reacting with recombinant polypeptide HE60 and 32% reacting with HE40 reacted with the synthetic peptide. No antibodies to the studied polypeptides were detected in the sera of Moscow patients with hepatitis A, B, or C confirmed by laboratory findings. Antigenic specificity of recombinant polypeptide HE60 was confirmed by competitive enzyme immunoassay with the same peptide as the competitive antigen. Test system based on recombinant polypeptides HE40 and HE60 was used for deciphering the etiological structure of acute viral hepatitis which occurred in a hepatitis E endemic region of Uzbekistan in 1993.

[Preparation of recombinant polypeptides containing antigenic determinants of hepatitis E virus]. / Poluchenie rekombinantnykh polipeptidov, soderzhashchikh antigennye determinanty virusa gepatita E.

DNA fragments complementary to hepatitis E Burma strain ORF2 and ORF3 obtained by oligonucleotide synthesis were cloned in expressing bacterial system. Recombinant polypeptides isolated from E. coli producer strains, immobilized on solid phase (polystyrene plates and nitrocellulose membranes), are studied in enzyme immunoassay to detect their ability to react with sera of patients with acute viral hepatitis from an Uzbekistan region endemic for hepatitis E. Two polypeptides reacting with the greatest number of sera, containing hepatitis E virus ORF2 and ORF3 gene fragments, were selected for further study of antigenic specificity.