Search

Rationale. Hepatitis E (HE) is a zooanthroponosis. Domestic pigs are the main reservoir for hepatitis E virus (HEV) in the Republic of Belarus (RB). Considering the well-developed pig farming, there is a high risk of infection spread among the population; however, the scale of virus circulation and patterns of HE epidemiology in the above region are still insufficiently explored.The aim of the study is to assess HEV seroprevalence specific for the HE epidemic process in RB.Materials and methods. Serum samples (n = 2,784) collected from patients of infectious disease departments at hospitals (n = 1,669) and relatively healthy people (n = 1,114) from different RB regions were used to measure the activity of alanine aminotransferase (ALT) by a kinetic rate method as well as IgG antibodies to HEV by the enzyme-linked immunosorbent assay (ELISA).Results. In the group of healthy people, anti-HEV IgG were detected in 7.3% (95% CI, 5.8–9.0). In the group of patients with liver disorders, the detection frequency was significantly higher, reaching 11.2% (95% CI, 9.6–12.9). In the groups of healthy people and patients with elevated ALT levels, the HEV seroprevalence did not depend on their gender or the region of residence. The anti-HEV IgG detection frequency gradually increased among olderage patients and reached the peak levels (15.9% on average) in the over-64 age group.Conclusions. RB is characterized by intensive HEV circulation, while the HE epidemic process is characterized by a latent nature. The actual prevalence of HЕ seromarkers among the RB population exceeds the frequency of diagnosed cases, suggesting insufficient vigilance of healthcare workers towards HE and subclinical forms of infection in most of the patients.

Introduction. The relevance of studying the circulation of human respiratory viruses among laboratory primates is associated with the need to test vaccines and antiviral drugs against these infections on monkeys. The aim of this work was to study the prevalence of serological and molecular markers of human respiratory viral infections in laboratory primates born at the Adler Primate Center and in imported monkeys. Material and methods. Blood serum samples (n = 1971) and lung autopsy material (n = 26) were obtained from different monkey species. These samples were tested for the presence of serological markers of measles, parainfluenza (PI) types 1, 2, 3, influenza A and B, respiratory syncytial (RS) and adenovirus infections using enzyme immunoassay (ELISA). Detection of RS virus, metapneumovirus, PI virus types 1–4, rhinovirus, coronavirus, and adenoviruses B, C, E and bocavirus nucleic acids in this material was performed by reverse transcription polymerase chain reaction (RT-PCR). Results and discussion. The overall prevalence of antibodies (Abs) among all monkeys was low and amounted 11.3% (95% CI: 9.2–13.7%, n = 811) for measles virus, 8.9% (95% CI: 6.2–12.2%, n = 381) for PI type 3 virus, 2.5% (95% CI: 0.8–5.6%, n = 204) for PI type 1 virus, and 7.7% (95% CI: 3.8–13.7%, n = 130) for adenoviruses. When testing 26 autopsy lung samples from monkeys of different species that died from pneumonia, 2 samples from Anubis baboons (Papio аnubis) were positive for of parainfluenza virus type 3 RNA. Conclusion. Our data suggest the importance of the strict adherence to the terms of quarantine and mandatory testing of monkey sera for the presence of IgM antibodies to the measles virus that indicate the recent infection. The role of PI virus type 3 in the pathology of the respiratory tract in Anubis baboons has been established.

Hepatitis D (delta, δ) virus (HDV) was discovered more than 40 years ago, but the understanding of its origin and evolution is poor. This is mainly due to the lack, until recently, of data on the existence of any viruses similar to HDV. The discovery in recent years of sequences of new delta-like agents in a wide range of vertebrate (Vertebrata) and invertebrate (Invertebrata) species has facilitated a revision of views on the origin of HDV and contributed to understanding the place of this unique virus among other animals’ viral agents. The purpose of this review is to analyze the latest published data on new delta-like agents and their biological characteristics.

Introduction. Viral hepatitis E is a zooanthroponotic disease that occurs in humans and various animals, including monkeys. It is caused by hepatitis E virus (HEV) (Hepeviridae, Orthohepevirus: Orthohepevirus A), for which 8 genotypes have been described to date. Among them, strains of genotypes 1 and 2 have been isolated from humans, strains of genotypes 3 and 4 from humans and animals, and strains of genotypes 5–8 from animals only. The main threat of the disease is associated with the documented zoonotic transmission of HEV genotypes 3, 4, 7, and 8, to humans through infected meat, blood and milk. Thus, monkeys could be involved in the transmission of HEV. The aim of this work was to study serological and molecular genetic markers of HEV infection in strepsirrhines (Old World monkeys, Cercopithecoidea), imported to the Adler Primate Center from various regions of the world (Tanzania, Vietnam, Mauritius). Material and methods. Fecal (n = 224) and blood serum samples (n = 395) from cynomolgus (Macaca fascicularis) and vervet monkeys (Chlorocebus pygerythrus) were examined by the enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Results and discussion. The data obtained show the high detection rate (51.8%) of IgG antibodies to HEV among 5 groups of cynomolgus monkeys imported from Vietnam, with a predominance of highly reactive sera (84%). High detection rate of IgM antibodies in these animals (10.4%) was observed, with the large number of IgM-reactive sera in one particular group of animals (36.8%). The fact of detection of HEV RNA in two groups of cynomolgus monkeys (11.9% and 5.7%) is of particular importance. All HEV sequences of isolated from monkeys belonged to genotype 4. Conclusion. Our data indicate that monkeys (in particular, cynomolgus monkeys) can serve as a natural reservoir of HEV genotype 4 for humans. This requires an appropriate set of anti-epidemic measures in a number of situations.

The problem of choosing a strategy to control hepatitis A in the Russian Federation remains relevant for the health care of our country. By strategy we mean a scientifically based program of action that defines the priorities and resources needed to effectively control hepatitis A.The aim of the work is to present a strategy to control hepatitis A in the Russian Federation, taking into account the realities of today. Based on the analysis of Russian and international studies, we present answers to the questions that should be addressed before the choosing a strategy to control hepatitis A in the Russian Federation. The low incidence of hepatitis A creates a false image of the favorable situation with this infection. The rationale is given to include vaccination against hepatitis A in the National Vaccination Schedule in the section of mandatory vaccinations. Routine vaccination of children aged 24 and 36 months will lay the foundation for future protection against this infection. We consider the creating a National Hepatitis A Vaccination Program as an important aim. The implementation of such a program will allow full control of hepatitis A in the Russian Federation.

Introduction. Hepatitis D (delta, 5) is caused by an RNA virus (hepatitis D virus, HDV) from genus Deltavirus, and is the most severe and difficult to treat disease among both viral hepatitis and infectious diseases in general. The development of HDV infection in the host organism is possible only in the presence of hepatitis B virus (HBV). Coinfection with HBV and HDV is associated with a more rapid progression of chronic viral hepatitis (CVH) to liver cirrhosis (LC) and an unfavorable outcome in comparison with HBV monoinfection. Data on the influence of clinical, biochemical and virological factors on the infectious process in patients with hepatitis D are limited due to the insufficient amount of research on this theme. The study aimed to determine demographic, clinical, biochemical, and virological factors influencing the course and progression of CVH D in patients followed during 10 years, residing in the territory of the Tuva Republic, one of the endemic regions of the Russian Federation. Material and methods. Changes in clinical and laboratory parameters were analyzed in dynamics in 121 HDV infected patients with a different course of the disease, who were under observation from 2009 to 2019. Three groups of patients were identified: group 1 - 61 patients with disease progression of chronic hepatitis to LC (Child-Pugh class B-C), group 2 - 49 patients with non-progressive chronic hepatitis, and group 3 - 11 patients with slowly progressive LC (class A). Demographic data, the presence of detectable HBV DNA, indicators of the functional state of the liver: alanine aminotransferase (ALT/GPT), aspartate aminotransferase (AST/GOT), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), and total bilirubin content were analyzed. The severity of hepatic encephalopathy was assessed by the duration of the numbers connection test (NCT). Results. All patients belonged to the same ethnic group (Tuvinians), were infected with HDV genotype 1 and were positive for HDV RNA throughout the entire follow-up period. There were no significant differences in sex ratio and mean age at the time of inclusion in the study between the groups. In group 1, the average number of years from inclusion in the study to the formation of LC was 3.65 ± 2.3 years, years to the lethal outcome: 4.5 ± 3 years. Significantly higher levels of AST/GOT, ALP, GGT, total bilirubin (TB) and NCT grade were found in group 1 compared to group 2. ALT/GPT levels did not differ significantly in these groups. When comparing groups with disease progression and slowly progressive LC (groups 1 and 3), no significant differences were found in any of the clinical and biochemical parameters. ALT/GPT, GGT, TB and NCT values were significantly higher in patients with slowly progressive LC (group 3) compared to group 2. No differences in AST/GOT and ALP levels were found between these groups. Detectable HBV DNA was significantly more frequent in patients with progressive disease and with chronic viral hepatitis than in patients with slowly progressive LC. There were no significant differences in the frequency of HBV DNA detection in patients from groups 1 and 2. Conclusion. The results obtained on a relatively homogeneous cohort demonstrated that age and gender are not the factors influencing the progression of chronic viral hepatitis D to cirrhosis. The lack of detectable HBV DNA is associated with the slow progression of LC. The revealed differences in clinical and biochemical parameters reflect the degree of functional liver damage in chronic viral hepatitis D and HDV-associated cirrhosis.

Introduction. Hepatitis E (HE) is an important public health problem worldwide and is especially significant for pregnant women, among whom the associated mortality rate reaches 25%. The distribution of HE serological markers in this cohort in the endemic regions of Central Asia is poorly understood. The aim of the study was to assess the seroprevalence of HEV among pregnant women in the region of Kyrgyzstan where an increased incidence of HE is reported. Material and methods. Blood serum of pregnant women, obtained from medical institutions in Bishkek, city of Osh and Osh region in the period from September 2016 to October 2019, and the control group of clinically healthy women were tested using the test systems “DS-IFA-ANTI-HEV-G” and “DS-IFA-ANTI-HEV-M” (NPO “Diagnostic Systems”, Russia). Results. IgG antibodies to HEV were detected in 87 (5.9%) of 1472 examined pregnant women, IgM antibodies -in 64 (4.8%) of 1378, while 5 (0.34%) samples were simultaneously positive for IgG and IgM antibodies. The rates of detection of antibodies to HEV in women of three age categories from 17 to 36 years old in the studied and control groups were similar. The analysis of the seasonal dynamics of HEV seroprevalence in pregnant women in the period from February to September 2019 showed a tendency towards an increase in the values of the positivity coefficient of specific IgM antibodies by the beginning of the autumn. Antibodies to HEV were detected with highest frequency in women from Osh region. Conclusions. A high risk of HEV infection for pregnant women in the surveyed region had been shown.

Introduction. Belgorod region is the territory with the highest incidence of hepatitis E in the Russian Federation. The aim of the study was to comprehensively characterize the circulation of hepatitis E virus (HEV) in the Belgorod region, including the study of population immunity to the virus, determining the prevalence of infection among the pig population and analysis of the genetic diversity of HEV from patients and animals. Material and methods. Serum samples of a conditionally healthy population (n = 2027) of all age groups were tested for anti-HEV IgG and IgM by ELISA with commercial assays. HEV RNA was determined in fecal samples from pigs aged 2–4 months (n = 526), in sewage samples from pig farms (n = 10), as well as in stool samples from patients with hepatitis E (n = 6) using reverse transcription polymerase chain reaction (RT-PCR). Phylogenetic analysis was performed for an amplified 300 nt fragment corresponding to HEV open reading frame 2. Results and discussion. The prevalence of anti-HEV IgG in general population averaged 16.4% (95% CI: 14.8–18.1; 332/2027). The proportion of individuals who had both anti-HEV IgM and IgG averaged 2.8% (95% CI: 2.2–3.6; 57/2027). The incidence rate of anti-HEV IgG increased with age, from 2.8% (95% CI: 1.3-5.8) in children aged 1–14 years to 40.1% (95% CI: 34.9-45.6) in people 70 years or older. The detection rate of HEV RNA in pigs was 20% (95% CI: 16.8-23.6; 105/526). HEV RNA was detected in 2 out of 10 sewage samples. The HEV sequences isolated from patients with hepatitis E, pigs, and sewage samples in Belgorod region belonged to the HEV genotype 3, had a 95-100% homology, and formed common clusters on a phylogenetic tree. Conclusions. The high prevalence of HEV in pigs population has led to the formation of an endemic territory in the Belgorod region, which is the center of pig breeding. Measures aimed at reducing the circulation of HEV among pig population and decontamination of sewage from pig farms are necessary to control HEV infection.

Hepatitis A is a widespread viral infection. The HAV strains of “human” and “monkey” origin are similar in their morphological and antigenic properties, but differ genotypically. The aim of this research was a comparative study of serological and molecular-genetic markers of HAV infection in monkeys born at the Adler Primate Center and in those imported from different countries. Material and methods. Fecal samples (n = 313) and serum (n = 266) from various species of monkey using ELISA and RT-PCR were studied. Results and discussion. The frequency of anti-HAV-IgG was high (78.9%) in imported animals (vervet monkeys from Tanzania and cynomolgus monkeys from Vietnam) and as well as in various species of monkeys (rhesus monkeys, cynomolgus monkeys, green monkeys and papio hamadryas) of the Center (88.6%). At the same time, in the imported monkeys, the markers of “fresh” HAV infection (IgM-27.2%, Ag-HAV-16.7%, RNA-22.0%) were detected significantly more often (p 0.05) than in monkeys kept at the Colony (IgM-7.5%, HAV-Ag – 5.2%, RNA – 3.6%). In general, anti-IgG reactivity ranged from 1.064 to 2.073 OD450, anti-IgM ranged from 0.546 to 1.059 OD450. The number of HAV-Ag was 0.496 – 1.995 OD450. RNA HAV only in rhesus monkeys and cynomolgys monkeys born at the Colony, as well as in imported vervet monkeys was detected. Conclusions. The data obtained indicate a wide circulation of HAV among monkeys born in the Adler Primate Center and among the imported animals. Markers of “fresh” HAV infection varied depending on the species of monkeys and their origin.

This article presents the results of a study of the epidemiology of hepatitis E in the Russian Federation. Obtained data explaining the phenomenon, known as the Balayan paradox — the wide distribution of anamnestic antibodies to the hepatitis E virus in the absence of a registered incidence. It was shown that imported cases of infection are not able to support the epidemiological process of hepatitis E in Russia. The most cases of HEV infection are autochthonous in nature and are associated with zoonotic transmission of genotype 3 virus from pigs.

Aim. The development of the hepatitis E virus (HEV) genotype 3 recombinant capsid protein. Materials and methods. E.coli strains, plasmid vectors, serological and clinical samples, ELISA reagent kits, molecular biological, bioinformatic, biotechnological, biochemical and serological methods. Results. Using viruscontaining material from pigs of Belgorod region (Russian Federation) we made E.coli strains producing recombinant capsid protein, containing C-terminal of viral ORF2 protein fragment fused to E.coli β-galactosidase. Recombinant protein ORF2 had been isolated from the bacterial inclusion bodies and purified by size exclusion chromatography. Antigenic specificity of the recombinant polypeptide was confirmed by ELISA and Western blotting with sera of hepatitis E patients and reference groups (healthy donors, patients with hepatitis A, B, C, infectious mononucleosis, cytomegalovirus infection and HIV-infected patients). Conclusion. HEV genotype 3 ORF2 recombinant antigen had been developed, and the possibility to use it in diagnostic tests had been experimentally shown.

Aim. Design аис1 construction of the hepatitis E virus (HEV) genotype 3 full-size ORF3 recombimnt polypeptide. Materials and methods. Escherichia coli strains, ptasmid vectors, serologiral and biological amples, molecular biological, bioinformatic, biotechnological, biochemical and serological methods.Results. RNA was isolated from pig fecal extracts collected on Belgorod farms and was used in RT-PCR to obtain the fragment of the orf3 gene of the hepatitis E virus genotype 3. Using A/T-cloning a recombinant plasmid was obtained with insertion of a DNA fragment (230 bp) encoding the N-terminal region of the ORF3 protein. The primary structure of the missing C-terminal region of the ORF3 VGE of the genotype 3 was calculated by bioinformatics methods. Codon optimization of the sequence for biosynthesis in E.coli cells was performed. For constructing the recombinant plasmid a chemically synthesized DNA fragment encoding the fulllength ORF3 protein had been used. E.coli strain producing full-size recombinant protein ORF3 fused to E.coli beta-galactosidase was developed. Recombinant protein ORF3 had been isolated from the inclusion bodies of the E.coli biomass and purified by size exclusion chromatography. Antigenic specificity of recombinant polypeptide had been confirmed in immunochemical reactions (ELISA, Western blot) with sera from patients with hepatitis E and control groups of patients. Conclusion. HEV genotype 3 ORF3 recombinant antigen had been designed, and itfs applicability in diagnostic tests had been experimentally confirmed.

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.

Aim. The development of the hepatitis E virus (HEV) genotype 1 full-size ORF3 recombinant polypeptide. Materials and methods. Escherichia coli strains, plasmid vectors, serological and clinical samples, ELISA reagent kits, molecular biological, bioinformatic, biotechnological, biochemical and serological methods. Results. HEV genotype 1 RNA had been isolated from clinical samples collected in Kyrgyzstan. DNA copy of subgenomic virus RNA had been cloned and used for further development of E.coli strains producing full-size recombinant protein ORF3 fused to E.coli beta-galactosidase. Codons optimization method was used in aim to increase expression level of recombinant protein. Recombinant protein ORF3 had been isolated from the inclusion bodies of the E.coli biomass and purified by size exclusion chromatography. Antigenic specificity of recombinant polypeptide had been confirmed by enzyme-linked immunosorbent assay and Western blotting with the specific sera. Conclusion. HEVgenotype 1 ORF3 recombinant antigen had been designed, and it’s applicability in diagnostic tests had been experimentally confirmed.

Aim. Experimental reproduction of HEV-infection in mini-pigs and comparative analysis of virological and immunological characteristics of experimental infection. Materials and methods. Wiesenauer minipigs (2 females and 4 males, age 50 - 60 days, weight 5-10 kg) were infected by HEV genotype 3 strain isolated from swine feces in Belgorod region in 2013. The virus was administered as a 10% clarified feces extract (800 pi). Blood (weekly) and feces (daily) were sampled from the animals for 49 days. Anti-HEV IgG were determined in sera samples using DS- ElA-ANTI-HEV-G (Diagnostic Systems) system, HEV RNA in samples of feces extracts and blood sera - RT-PCR. Results. Wiesenauer minipigs were sensitive to HEV genotype 3 infection, infection developed in all the animals after administration of the virus, that was accompanied by the presence of HEV RNA in feces for 2 weeks and seroconversion by anti-HEV. Comparative analysis of virological characteristics of experimental H EV-infection in mini- and standard pigs has shown, that the duration of the infection in mini-pigs is shorter, and seroconversion by anti-HEV occurs earlier. Conclusion. Sensitivity to HEV genotype 3 and lower weight make mini-pigs a comfortable alternative to standard swine and primates for modelling HEV infection in vivo.

Serum from humans (n = 646) and monkeys (n = 1867) collected during the period 1999-2013 was tested by enzyme immunoassay. Anti-HEV IgG was detected significantly more frequently (P ≥ 0.001) in rhesus macaques (Macaca mulatta) - 45.1 ± 1.6% (n = 1001) than in cynomolgus macaques (M. fascicularis) 16.2 ± 1.8% (n = 426). Single seropositive individuals were found among M. nemestrina - 4.0±2.8% (n = 50). Anti-HEV was not detected in the sera of green monkeys (Chlorocebus aethiops) - n = 162, Papio hamadryas (n = 124), and Papio anubis - n = 104. The presence of the anti-HEV IgM indicating the cases of fresh infection in Macaca mulatta - 2.1 ± 0.5% (n = 717) and M. fascicularis - 3.5 ± 1.3% (n = 266) is of great significance. The overall frequency of detection of the anti-HEV IgG among the staff of the Adler Primate Center - 6.8 ± 2.3% (n = 118) was significantly lower (P ≤ 0.001) than among the population of the Greater Sochi - 15.9% ± 1.6 (n = 528). It is important that only in patients of medical institutions (clinic, hospital, cancer center), anti-HEV IgM were detected (2.7-11.8%) along with anti-HEV IgG (15-23.5%), thereby indicating the presence of acute cases of HEV infection among this population. HEV RNA was not detected in the serum of anti-HEV IgM-positive people and monkeys. Seroepidemiological data do not confirm the assumption on the ability of seropositive monkeys of Macaca genus to be a natural reservoir of HEV infection for humans.

Aim. Determine spread of hepatitis C vims genotypes, circulating in Moscow Region over the last decade. Materials and methods. The presence of HCV RNA, genotype and subtype of the vims were determined in blood sera samples obtained in 2006 - 2014 from 2847 individuals with the presence of HCV infection, who had or did not have injectable dmg administration in anamnesis. Results, lb and 3a remain the main subtypes, circulating in the population over the last decade. A notable reduction of la subtype circulation took place among injectable dmg users (IDU). Recombinant form RFl_2k/lb of hepatitis vims is present only among this risk group and constitutes 2% of the overall amount of subtypes in both 2007 and 2014. Genotype 4d was detected in 2014, that is not typical for Russian Federation. Genotype 3a is dominant in IDU age groups of 20 - 29 and older than 40, and in the 30 - 39 group the main - subtype lb. Phylogenetic analysis has shown the lack of certain genetic variants of subtypes lb and 3a, characteristic for IDU, that gives evidence on a wide circulation of the main subtypes of the vims in the whole population of individuals, infected by HCV. Conclusion. Spread of main genotypes/subtypes of HCV in the overall population and among IDU in Moscow remains stable over the last decade.

Aim. Description of the first documented case of imported hepatitis E, associated with genotype 4 of HEV and introduced from southern France. Materials and methods. Clinical, epidemiologic and laboratory analysis of the imported case of disease of hepatitis E was carried out. Phylogenetic analysis of nucleotide sequences of HEV isolate, taken from the patient, was carried out. Results. Epidemiologic analysis allowed to assume imported character of the detected case of HEV-infection. Comparative analysis of nucleotide sequences of regions of the open reading frame 2 (300 nt) and open reading frame 1 (721 nt) of HEV genome, isolated from the patient, showed identity of this isolate with variants of genotype 4 HEV, isolated in France in 2009 - 2011 from patients with autochthonous hepatitis E. Conclusion. The results obtained confirm the case of import into Russia of genotype 4 HEV from south-eastern France (Corsica), where spread of this virus genotype is observed in recent years.

Aim. Frequency of detection determination for past and current hepatitis E virus (HEV) infection markers in children with immune suppression, as well as children with normal immune status. Materials and methods. The presence of HEV markers (anti-HEV IgG and IgM, HEV RNA) was studied in 609 sera samples of children with neurologic pathologies, 87 samples - from children with immune deficiencies, as well as 3122 samples from conditionally healthy children of 6 regions of Russia. The children were divided into 5 age groups. Anti-HEV IgG and IgM determination was carried out in EIA, HEV RNA - by RT-PCR. Results. The frequency of detection of anamnestic anti-HEV IgG turned out to be significantly higher among immune-compromised children compared with healthy children (5.7% against 1.4%, p