[Animal delta-like viruses (Kolmioviridae: Deltavirus) and the origin of the human hepatitis D virus (HDV)].

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.

[Clinical course and outcomes of chronic viral hepatitis D in patients from Republic of Tuva as endemic region].

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.

[Detection of markers of hepatitis B and D virus infection in biological media and dried blood spots.]

The aim of this study was to assess the rates of detection of the major markers of infection with hepatitis B and Delta (D) viruses in serum, saliva and dry blood dots (DBS) as a possible option for serological studies among the population of the endemic region in conditions of limited laboratory resources. For this purpose, paired samples of blood serum and DBS, blood serum and saliva from patients with chronic hepatitis B with Delta agent living in the Republic of Tyva, which is endemic for this disease. HBsAg was detected in 289 (100%) serum samples, in 88/92 (95.7%) saliva samples, in 60/80 (75%) DBS samples, stored three years at room temperature, and in 111/117 (94.9%) DBS stored one year at the same conditions. Anti-HBcore was detected in 209 (100%) serum samples, while in saliva and DBS samples this marker was detected in only 13.04% (12/92) and 19.7% (23/117), respectively. Anti-HDV antibodies in serum were detected in 209 (100%) samples collected from patients in 2017-2018. In saliva and DBS anti-HDV were not detected in any sample. This difference in the detection rates of anti-HBcore and anti-HDV might be accounted for the fact that the HBV core protein is a very strong immunogen, indusing the production of anti-HBcore in high concentrations. Probably, the concentration of anti-HDV is much lower, which explains its absence in saliva and DBS in patients with hepatitis B+D. Samples of biological media (saliva), as well as DBS can serve as an alternative material for the detection of HBsAg in screening and research prevalence studies. Meanwhile, the definition of anti-HDV in such media is not possible due to the false negative results. Due to the high probability of superinfection with HDV in patients with HBV in endemic areas, the detection of HBsAg in alternative media (saliva or DBS) should be followed by testing for anti-HDV in serum samples.

[Belgorod region - the territory endemic for hepatitis E.]

INTRODUCTION: Belgorod region is the territory with the highest incidence of hepatitis E in the Russian Federation. OBJECTIVES: 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.

Frequency of Interferon-Resistance Conferring Substitutions in Amino Acid Positions 70 and 91 of Core Protein of the Russian HCV 1b Isolates Analyzed in the T-Cell Epitopic Context.

Amino acid substitutions R70Q/H and L91M in HCV subtype 1b core protein can affect the response to interferon and are associated with the development of hepatocellular carcinoma. We found that the rate of R70Q/H in HCV 1b from Russia was 31.2%, similar to that in HCV strains from Asia (34.0%), higher than that in the European (18.0%, p = 0.0010), but lower than that in the US HCV 1b strains (62.8%, p < 0.0001). Substitution L91M was found in 80.4% of the Russian HCV 1b isolates, higher than in Asian isolates (43.8%, p < 0.0001). Thus, a significant proportion of Russian HCV 1b isolates carry the unfavorable R70Q/H and/or L91M substitution. In silico analysis of the epitopic structure of the regions of substitutions revealed that both harbor clusters of T-cell epitopes. Peptides encompassing these regions were predicted to bind to a panel of HLA class I molecules, with substitutions impairing peptide recognition by HLA I molecules of the alleles prevalent in Russia. This indicates that HCV 1b with R70Q/H and L91M substitutions may have evolved as the immune escape variants. Impairment of T-cell recognition may play a part in the negative effect of these substitutions on the response to IFN treatment.

[The duration of preservation of anamnestic antibodies to hepatitis E virus.]

The purpose of the study was to determine the duration of antibody response against hepatitis E virus (anti-HEV). Veterans of the war in Afghanistan who were in this endemic region in the late 1970s and early 1980s were tested for anti-HEV. On average, 20 years after the end of military service in Afghanistan, the rate of seropositivity was 30.0% (95/317), which was significantly higher compared to positivity rates in males who were at military service in the territory of Russia during the same period (3.9%, 8/208). At an average of 29.5 years after the visit to Afghanistan anti-HEV prevalence in veterans dropped to 20.0% (21/105), but still significantly exceeded the seroprevalence in general population (3.8%). Serum samples from elderly individuals (>60 years) without known risk factors of infection were also tested for anti-HEV IgG and IgM (n = 896). Anti-HEV IgG detection rates in the elderly from two regions of Russia exceeded those observed of primary blood donors (18.0-27.8% vs. 4.5-10.0%, p<0.01). The detection of anti-HEV IgM in individuals above 60 years (2.7-6.9%) indicates a current or recent infection. Thus, anti-HEV IgG can persist several decades after the infection. This might account for the wide anti-HEV prevalence among the elderly. At the same time, a significant proportion of individuals exposed to HEV lose detectable anti-HEV IgG within 20-30 years. The detection of anti-HEV IgM among the elderly indicates the viral circulation in older age groups, suggesting the need for testing for hepatitis E markers in elderly patients with liver diseases.

Simulation of Viral Hepatitis E in Marmosets.

We developed a model of hepatitis E virus infection in common marmosets (Callithrix jacchus) and determined optimal route of infection, duration, clinical and virological characteristics of infection in laboratory animals. Using this model, we demonstrated that replication of hepatitis E virus primarily occurs in the liver, while virus replication presumed to take place in the intestine was not confirmed in this experiment.

[A CASE OF IMPORT OF GENOTYPE 4 HEPATITIS E VIRUS INTO RUSSIA].

AIM: Description of the first documented case of imported hepatitis E, associated with geno- type 4 of HEV and introduced from southern France. MATERIALS AND METHODS: Clinical, epidemio- logic 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 ofnucleotide sequences of regions ofthe 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.

[The standardization of diagnostic of hepatitis E.]

The study was carried out to develop approaches to standardization of laboratory diagnostic of hepatitis E. The three stages of standardization are establishment of analytical sensitivity of molecular test for detection of RNA of virus of hepatitis E; establishment of analytical sensitivity in International Units of enzyme-linked immunosorbent assay testing widely applied in Russia for detection of anti-virus of hepatitis E; And development of national reference material - standard anti-virus of hepatitis E IgG validated relatively to International standard. The results of study permitted to develop tools for standardizing of laboratory diagnostic of hepatitis E and epidemiological control of the given function - molecular test for detecting RNA of virus of hepatitis E with sensitivity within range of 1250250 IU/ml, data concerning analytical sensitivity of commercial enzyme-linked immunosorbent assay testing for detecting anti-virus of hepatitis E (0.25 IU/ml) and national standard of anti-virus of hepatitis E with concentration of 5 IU/ml.

[GENUS ANELLOVIRIDAE VIRUSES IN CHRONIC LIVER DISEASE].

AIM: Viruses from genus Anelloviridae (TTV, TTMDV and TTMV) are small DNA viruses that are widespread in human popu- lation. Data on tissue tropism, cell localization and morphometry of anelloviruses are scarce. The purpose of this study was to determine the prevalence of TTV, TTMDV and TTMV in persons with liver disease and in healthy individuals, as well as electron-microscopic verification of Anelloviridae species. METHODS: Detection of anelloviral DNA was performed in serum samples from 203 patients with liver diseases of various etiology and 115 voluntary blood donors using PCR with primers allowing to differentiate TTV, TTMDV TTMV based on the length of amplified fragment. Histopathological and electron microscopic studies were performed for liver biopsy specimens from 203 patients with liver disease. RESULTS: High prevalence (70-90%) of all three anelloviruses in healthy individuals and patients with liver disease was demonstrated, with high frequency of triple TTV, TTMDV and TTMV infection (52.2-55.7%). Electron-microscopic study of liver biopsy specimens from TTMDV monoinfected patients gave a submicroscopic image of TTMDV virions with diameter 35.86 ± 2.04 nm. Electron microscopic studies confirmed the nature of liver damage in TTMDV monoinfection: accumulation of virus in the hepatocytes, significant cyropathy with enlightenment matrix of the cytoplasm and reduction of intracellula organelles involved in protein synthesis, portal and perivascular perisinusoidal fibrosis. TTV, TTMDV and TTMV virions were dentified in hepatocytes, confirming these viruses to be hepatotropic. CONCLUSION: Our results demonstrate that anelloviruses are lymphotropic viruses, individual genotypes of those might be hepatotropic and pathogenic to liver.

The 2010 outbreak of poliomyelitis in Tajikistan: epidemiology and lessons learnt.

A large outbreak of poliomyelitis, with 463 laboratory-confirmed and 47 polio-compatible cases, took place in 2010 in Tajikistan. Phylogenetic analysis of the viral VP1 gene suggested a single importation of wild poliovirus type 1 from India in late 2009, its further circulation in Tajikistan and expansion into neighbouring countries, namely Kazakhstan, Russia, Turkmenistan and Uzbekistan. Whole-genome sequencing of 14 isolates revealed recombination events with enterovirus C with cross-overs within the P2 region. Viruses with one class of recombinant genomes co-circulated with the parental virus, and representatives of both caused paralytic poliomyelitis. Serological analysis of 327 sera from acute flaccid paralysis cases as well as from patients with other diagnoses and from healthy people demonstrated inadequate immunity against polio in the years preceding the outbreak. Evidence was obtained suggesting that vaccination against poliomyelitis, in rare cases, may not prevent the disease. Factors contributing to the peculiarities of this outbreak are discussed. The outbreak emphasises the necessity of continued vaccination against polio and the need, at least in risk areas, of quality control of this vaccination through well planned serological surveillance.

[Methodological features of DNA isolation for nested PCR for the differential diagnosis of malaria].

Current PCR assays for the differential diagnosis of malaria need to be further developed and certified in the Russian Federation. PCR may serve as a control diagnostic method. These investigations have demonstrated that an erythrocytic clot may be used as an adjunct to microscopy and the described procedure can be employed to isolate DNA for PRC in an inpatient setting.

[The panels of serums, containing various subtypes and mutant forms of HBsAg, to evaluate the diagnostics sensitivity of kits oa reagents detecting HBsAg].

The detection of HBsAg in blood serum using immune-enzyme analysis techniques decisively matters both for diagnostics of acute and chronic hepatitis B and screening of donor's blood and its components, controls of persons from risk groups of hepatitis B injection. The making of panels containing wide specter of samples of blood serums with sero-variants and mutant forms of surface antigen of hepatitis B virus widespread on the territory of the Russian Federation is necessary to control analytic and diagnostic sensitivity of test systems for detecting HBsAg. The testing of reagents kits to detect HBsAg using twi panels containing recombinant and native variants of HBsAg, demonstrated that these kits enable to detect various sero-vatriants of HBsAg (ayw2, adw2, ayw3varA, ayw3varB, adrq-) in concentration 0.1-0.01 IU/l and the so called elusive mutant forms of HBsAg of recombinant and native origin (G145R, Q129R, Q129H, Q129L, T143K, T126N, T126S, D144A, M133L, K141E and P142S). At that, the sensitivity can differ during the detection of native and recombinant mutant forms. The results testify the importance of using the panels both with recombinant and native samples containing the mutant forms of HBsAg in evaluation of sensitivity of reagents kits.

[Epidemiological and molecular biological analysis of causes of rise of hepatitis A incidence in Republic of Tuva in 2008].

AIM: Analysis of hepatitis A (HA) incidence in Tyva Republic in 2008 exploiting traditional epidemiological and molecular methods. MATERIALS AND METHODS: Epidemiological analysis of HA cases and contact persons in Erzinsky and Kyzyl regions of Tyva Republic was performed. Class M and G antibodies to HAV were determined in serum samples (n = 28), and HAV RNA--in stool samples (n = 16). Phylogenetic analysis of HAV RNA sequence was performed for VP1/2A region of the HAV genome with length 394 nucleotides. RESULTS: Cases of HA were registered during 3.5 months. Water supply sources did not have deviations from established standards. According to results of interviews, common food factor, which was able to cause the rise of HA incidence, was not determined. Signs of fecal contamination were revealed on environmental objects in preschool institutions and schools that demonstrate the low level of hygienic behaviour. It was shown that all cases of HA are related with different variants of the virus belonged to genotype IA that points to the absence of common source of infection. CONCLUSION: Results of epidemiological and genetic analysis of HAV demonstrate that observed rise in HA incidence in Tyva Republic are connected with phase of seasonal rise of HA incidence, which is characteristic for its perennial dynamics with active realization of contact route of virus transmission.

[GBV-C infection in HIV-infected patients in the Russian Federation].

The spread and genotypical variability of GBV-C virus were determined among the HIV-positive patients in the Russian Federation. More than a fourth (26.2%) of the HIV-infected patients were shown to have GBV-C coinfection; all virus isolates belonged to genotype 2 with a predominance of subtype 2a. Analysis of the impact of GBV-C coinfection on HIV burden and CD4 lymphocyte levels showed no significant impact on these basic characteristics of HIV infection. However, coinfection with GBV-C and HIV was associated with the higher frequency of undetectably low ( < 400 copies/ml) of HIV burden, which enables GBV-C infection to be regarded as a potentially favorable factor in HIV infection.

[Endophytic yeast fungi in plant storage tissues].

It was found that plant storage tissues (fleshy sugar-containing fruits, subsurface metamorphically altered plant organs (storage roots, tubers, etc.), and starch-containing seed lobes) nearly always contain yeasts that are able to actively reproduce in these tissues causing no visible damage. Within storage tissues, yeast cells were detected both in the intercellular space and inside plant cells. In the tissues of fleshy fruits, endophytic yeasts are represented by the same species as epiphytic ones; cryptococci of the order Filobasidiales and ascomycetes belonging to the genera Hanseniaspora and Metschnikowia are predominant. In subsurface plant organs, red pigmented basidiomycetous yeasts of the genus Rhodotorula prevail. Selective growth of representatives of one species, Candida railenensis, is typical of starch-containing storage tissues of seeds. The results obtained change the established notion of the distributional patterns of yeast fungi in natural habitats and suggest that internal storage tissues of plants can be considered as a new interesting model for studies ofcoevolving plant-microbial associations.

[Quality assessment of HCV RNA detection by PCR method in Federal System of External Quality Assessment in 2005 - 2007].

Part of the Federal System of External Quality Assessment of Clinical Laboratory Tests named "PCR-identification of hepatitis C virus (HCV)" and aimed at detection of HCV RNA by polymerase chain reaction (PCR) assay is functioning from 2000. Ninety, 98, and 112 laboratories from more than 50 regions of Russian Federation participated in it in 2000, 2006, and 2007 respectively. Analysis of results of control samples tests, which were performed by participated laboratories, showed increasing proportion of method of amplification in the presence of fluorescent-marked probes both in real-time and in end-point regimens. In these circumstances, significant increase of specificity and sensitivity of tests was observed. In 2007, proportion of correct results obtained by the participants for tests with negative control samples was 96 - 97%, whereas during detection of HCV RNA in concentration 10(3) IU/ml the proportion of correct results was 80 - 83%. Significant proportion of laboratories, which did not detect HCV RNA in concentration 10(3) IU/ml, points on the necessity to improve sensitivity of this important method of laboratory tests.