[New approaches to the treatment of the flavivirus infections].

Since spontaneous mutagenesis and quasi-species rearrangements of the RNA-containing viruses, as well as an absence of both viral and cellular RNA reparation systems, causes resistance to originally effective antiviral drugs, combination therapy with nucleoside and non-nucleoside inhibitors of the viral enzymes in combination with immunomodulators is recommended. The use of specific immunoglobulins does not result in complete elimination of the flaviviruses but rather in possible antibody-dependent enhancement of the flavivirus infection by means of increased penetration of complexes of virions with specific antibodies into cells with receptors for Fc-fragments of immunoglobulins.

[Human bocavirus].

Human bocavirus (HBoV) is a newly identified parvovirus associated with acute respiratory infections in young children in different parts of the world. It is not inconceivable that this virus is also capable of causing acute gastroenteritis and asymptomatically persisting in infected children. HBoV is the third widespread human respiratory virus after respiratory syncytial virus and rhinovirus. Polymerase chain reaction remains the most reliable of HBoV detection in clinical samples. Phylogenetic analysis shows the presence of at least 2 circulating variants (genotypes) of HBoV.

[Human metapneumovirus as a common cause of respiratory tract disease].

Human metapneumovirus (HMPV), the newly identified paramyxovirus, causes respiratory infections in children, immunosuppressed patients, and the elderly in different countries of the world. The epidemiology and clinical manifestations of HMPV infection are similar to those in human respiratory syncytial virus infection. The diagnosis of HMPV infection is based on the polymerase chain reaction detection of viral RNA or the recording of rising serum antibody titers. There are at least two genotypes and several subtypes of HMPV in the human population. The use of cell cultures and laboratory animals have provided new evidence for the pathogenesis of HMPV infection, the specific features of antiviral immunity and enabled recombinant HMPV vaccine candidates to be designed.

[Infection of mice with TT virus]. / Infitsirovanie myshei virusom TT.

The sera pool of the human blood containing TTV DNA were administered to mice intravenously and then every 10 days examinations were made of blood serum and different organs of inoculated mice by polymerase chain reaction for presence of the viral nucleotide sequences. The viral DNA was revealed in the blood serum, the liver, kidneys and other organs 20 days after infection and persisted in most of the animals during 80 days. The data obtained show the possibility of TTV-infection of mice.

Performance characteristics of a transcription-mediated nucleic acid amplification assay for qualitative detection of hepatitis C virus RNA.

The detection of hepatitis C virus (HCV) RNA by nucleic acid amplification techniques is the method of choice to differentiate between ongoing and past infection, and can be used to monitor the course of HCV infection. In this study, we evaluated the performance characteristics of a newly developed transcription-mediated amplification (TMA)-based assay, the VERSANT HCV RNA qualitative assay, which was designed to qualitatively detect HCV RNA. Samples tested by the TMA assay included 100 HCV antibody negative sera; serial dilutions of an HCV genotype 1a panel; the WHO HCV RNA standard 76/790; an HCV genotyping panel; and 150 clinical specimens, including sera from patients who had received alpha interferon (IFN) treatment or liver transplants. TMA test results were compared with the Cobas Amplicor HCV polymerase chain reaction (PCR) assay. The analytical specificity of the HCV TMA assay was > 98%. No carry-over contaminations were observed. The assay demonstrated an analytical sensitivity of 100% at 41 HCV RNA copies/mL (genotype 1a panel) and 5 IU/mL (WHO standard), respectively. HCV genotypes and subtypes did not affect the results. Qualitative RNA detection by diagnostic Amplicor PCR and TMA was in agreement in > 97% of all 150 clinical samples tested. In our study, the TMA-based assay proved to be a specific and sensitive method for qualitative HCV RNA detection. The test may turn out to be an attractive alternative to already established techniques for HCV RNA amplification in routine clinical laboratories.

Genotyping of hepatitis C virus isolates using CLIP sequencing.

Determination of hepatitis C virus (HCV) genotypes and subtypes has become increasingly important for the clinical management and prognosis of HCV infections. The aim of the present study was to assess the specificity and reliability of a newly developed, commercially available HCV genotyping kit (TRUGENE HCV 5'NC genotyping kit). This technique utilizes PCR fragments previously generated by the diagnostic Roche AMPLICOR HCV test, which are subsequently subjected to simultaneous PCR amplification and direct sequencing (CLIP sequencing) of the 5' noncoding region (5'NCR). HCV isolates from 100 randomly chosen patients were genotyped by both the TRUGENE HCV 5'NC genotyping kit and DNA enzyme immunoassay (DEIA). Typing results obtained by both methods were in complete concordance in 91% of the cases. HCV RNA from the samples with discordant genotype assignment in both assays was additionally amplified with primers from the HCV core and NS5B regions. Phylogenetic analysis of the obtained sequences supported the results obtained from DEIA in six cases and CLIP sequencing in two cases. In the former six cases, the TRUGENE HCV 5'NC genotyping kit could not correctly differentiate between subtypes of genotypes 1 and 2 due to the high conservation of the 5'NCR. However, since there was not any misclassification between HCV genotypes 1 and non-1 types, the results obtained with this system are, in general, reliable and can be used in clinical practice. The TRUGENE HCV 5'NC genotyping kit in our hands proved to be a fast and convenient technique that might be an attractive option for HCV genotyping in laboratories already using the Roche AMPLICOR HCV test for diagnostic reverse transcription-PCR.

Sequence heterogeneity of TT virus and closely related viruses.

TT virus (TTV) is a recently discovered infectious agent originally obtained from transfusion-related hepatitis. However, the causative link between the TTV infection and liver disease remains uncertain. Recent studies demonstrated that genome sequences of different TTV strains are significantly divergent. To assess genetic heterogeneity of the TTV genome in more detail, a sequence analysis of PCR fragments (271 bp) amplified from open reading frame 1 (ORF1) was performed. PCR fragments were amplified from 5 to 40% of serum specimens obtained from patients with different forms of hepatitis who reside in different countries (e.g., China, Egypt, Vietnam, and the United States) and from normal human specimens obtained from U.S. residents. A total of 170 PCR fragments were sequenced and compared to sequences derived from the corresponding TTV genome region deposited in GenBank. Genotypes 2 and 3 were found to be significantly more genetically related than any other TTV genotype. Moreover, three sequences were shown to be almost equally related to both genotypes 2 and 3. These observations suggest a merger of genotypes 2 and 3 into one genotype, 2/3. Additionally, five new groups of TTV sequences were identified. One group represents a new genotype, whereas the other four groups were shown to be more evolutionary distant from all known TTV sequences. The evolutionary distances between these four groups were also shown to be greater than between TTV genotypes. The phylogenetic analysis suggested that these four new genetic groups represent closely related yet different viral species. Thus, TTV exists as a "swarm" of at least five closely related but different viruses. These observations suggest a high degree of genetic complexity within the TTV population. The finding of the additional TTV-related species should be taken into consideration when the association between TTV infections and human diseases of unknown etiology is studied.

[Detection of TT DNA virus (TTV) in blood donors and patients with hepatitis of unclear etiology]. / Vyiavlenie DNk virusa TT (TTV) u donorov krovi i bol'nykh gepatitom neiasnoi étiologii.

A new human infective agent: TT virus (TTV) has been recently identified. The polymerase chain reaction detected TTV DNA in the sera of 5 (31.3%) out of 16 children with acute hepatitis, 5 (17.2%) out of 29 children and 3 (14.3%) out of 21 adults with liver diseases of unknown etiology, and 18 (13.2%) out of 136 free-of-charge blood donors. These results indicate a high prevalence of TTV infection in Russia and absence of an obvious correlation between this infection and nonA, nonB, nonC hepatitis in examined patients. Phylogenetic analysis of amplified fragments of viral DNA from 3 patients selected at random showed that the isolated strains belong to subtype 1a, most prevalent in the world.

[Persistence of hepatitis C virus in newborn mice brain cell culture]. / Persistentsiia virusa gepatita C v kul'turakh kletok golovnogo mozga novorozhdennykh myshei.

A model of chronic infection of primary cultures of suckling mouse brain (SMB) cells actively producing hepatitis C virus (HCV) is developed. Destruction and repopulation of cells was observed for at least 6 months; this phenomenon was paralleled by virus release in culture medium. Persistent HCV contained in SMB cultures induced a cytopathogenic effect in PS, BHK-21, Vero, HAK, and click embryo cell cultures, its infective titers being 10.0-12.0 lg TCD50/0.2 ml. Persistent HCV formed heterogeneous plaques under agar in chick embryo cells. The polymerase chain reaction (PCR) regularly detected the HCV RNA at the stage of cell destruction in the culture fluid of HCV-infected cell cultures. The cytopathogenic activity of persistent HCV was neutralized by anti-HCV positive patients' sera with the neutralization index of 8.0-9.0 lg. The results of persistent HCV neutralization were confirmed by PCR. Immunofluorescence detected virus-specific HCV antigens in 15-40% of infected cells. Hence, the SMB-HCV system realized the cytopathogenic potential of HCV circulating in the blood of patients with hepatitis C. This system is promising for the study of the pathogenesis of HCV infection at a cellular level, for screening for specific and nonspecific antiviral agents, and for preparing native virus-specific proteins and RNA.

[Lethal infection of newborn mice, caused by hepatitis C virus]. / Letal'naia infektsiia novorozhdennykh myshei, vyzvannaia virusom gepatita C.

Intracerebral infection of mice aged 2-3 days with hepatitis C virus (HCV) from chronically infected suckling mouse brain cultures and porcine embryo cells resulted in the death of animals after 12-14 days. Repeated passages of HCV in animals decreased the incubation period to just 3-4 days. Blood serum of surviving mice collected during the 4th week postinfection contained antiHCV antibodies detected by enzyme immunoassay and hemagglutination inhibition test. The results are proof of creation of a new in vivo model of experimental HCV infection.

[Chronic infection of swine embryonal kidney cells (PS), caused by hepatitis C virus]. / Khronicheskaia infektsiia kul'tur kletok pochki émbriona svin'i (PS), vyzvannaia virusom gepatita C.

Hepatitis C virus (HCV) circulating in patient's blood acquired cytopathogenic properties after infection of suckling mouse brain cells. HCV infection of PS cells was studied for 11 months. Three stages of infection were distinguished; noncytocidal infection, poorly manifest destruction of PS cells, and intensive cytodestruction and cell repopulation. Persistent HCV was steadily released in PS culture and caused destruction of BHK-21, Vero, PS, HAK, and chick embryo cells. Infective titers of HCV in culture fluid collected from these cultures were 10.0 = 11.0 lg TCD50/0.2 ml. Cytopathogenic activity of HCV regularly confirmed by the polymerase chain reaction was neutralized by anti-HCV-positive sera of patients with hepatitis C. Persistent HCV formed heterogeneous plaques in chick embryo fibroblast cultures and agglutinated goose erythrocytes. Indirect immunofluorescence demonstrated that 25 to 45% of infected cells contained virus-specific antigens. The PS-HCV system holds the best promise for theoretical and practical studies of hepatitis C.

[Genotype of hepatitis C virus in hemophiliacs]. / Genotipy virusa gepatita C u bol'nykh gemofiliei.

Testing of blood sera of 63 hemophiliacs revealed markers of hepatitides B and C in 97 and 98% cases, respectively. Polymerase chain reaction confirmed the presence of hepatitis C virus (HCV) RNA in 31 (94%) out of 33 sera tested which contained antibodies to HCV. Gene typing of the isolated strains revealed the presence of three types of HCV: 1b (94%), 2a (3%), and 3a (3%). Similar distribution of HCV genotypes was revealed in the blood sera of 39 patients with chronic hepatitis C.

[Determination of the nucleotide sequence of the Russian variant of the hepatitis C virus]. / Opredelenie posledovatel'nostei nukleotidov rossiiskogo varianta virusa gepatita C.

From a blood serum of patients with chronic posttransfusional non-A, non-B hepatitis the genomic RNA of hepatitis C virus (HCV) was isolated. Using RT-PCR (reverse transcription-polymerase chain reaction) there were synthesized and cloned cDNA fragments, representing 3 regions of the genome of a new virus isolate (HCV-R): 5'-nontranslating region, a core gene and a part of the nonstructural region NS3/NS4. Analysis of the nucleotide and of the amino acid sequences of a core and NS3/NS4 regions revealed significant difference between isolates from Russia (HCV-R) and from Japan (HCV-J). Nucleotide sequence homology between them was 90.0-90.87%, while homology between Russian and American isolates (USA-PT) complised 95.27-97.32%. No essential variations were found in the nucleotide sequences of 5'-nontranslating region of all three HCV isolates.

[The incidence of detecting influenza virus antigens in the peripheral blood lymphocytes of donors in relation to the epidemic activity of influenza viruses A and B]. / Chastota vyiavleniia antigenov virusov grippa v limfotsitakh perifericheskoi krovi donorov v zavisimosti ot épidemicheskoi aktivnosti virusov grippa A i B.

Antigens of influenza A and B viruses in the peripheral blood lymphocytes of normal human subjects are found regularly both in epidemic and interepidemic periods. The level of detection of viral proteins in lymphocytes varies widely and correlates with the epidemic activity of the viruses. Influenza virus antigens were found several months before a rise in the incidence of the disease, the per cent ratio of the identified antigens correlating with the pattern of antigen detection in nasopharyngeal washings during an epidemic outbreak. Most frequently, the antigen found in lymphocytes was that of the main etiological agent of a definite epidemic; less frequently the hemagglutinin of the virus accompanying the one dominant in a given epidemic was found.

[Chronic hepatitis C in children]. / Khronicheskii gepatiti C u detei.

The clinico-laboratory manifestations and outcomes of chronic hepatitis C have been studied in 49 children. The proportion of chronic hepatitis C in the structure of chronic viral hepatitides in children is 20.5%. Among chronic hepatitis C patients, in 18.4% chronic persisting hepatitis, in 20.4% chronic active hepatitis and in 61.4% chronic active hepatitis with transition to cirrhosis of the liver have been diagnosed. In patients with chronic hepatitis C manifested as chronic persisting hepatitis or chronic active hepatitis the course of the disease is characterized by intermittent periods of prolonged exacerbations and short remissions. In cases of chronic active hepatitis with cirrhosis of the liver the signs of the active process can be constantly detected in the course of prolonged observations. In some patients with chronic active hepatitis the lethal outcome is possible as the consequence of progressing liver insufficiency. The data obtained in this investigation indicate that autoimmune chronic hepatitis in children, extensively described in Russian and foreign literature, may be etiologically linked with hepatitis C virus.

[The importance of the blood transfusion factor in the epidemic process in hepatitis C in the patients of chronic hemodialysis units studied using a computer simulation model]. / Issledovanie znachimosti faktora gemotransfuzii v épidemicheskom protsesse pri gepatitie C u patsientov otdelenii khronicheskogo gemodializa s pomoshch'iu imitatsionnoi komp'iuternoi modeli.

Examinations for the presence of antibody to hepatitis C virus (HCV) were carried out in 144 patients of chronic hemodialysis wards and 83 blood donors. The anti-HCV were found in 26.4% of the patients and only in 1.2% of the blood donors. A definite increase in the incidence of HCV infection in the patients of hemodialysis wards was established in relation to the duration of the treatment, namely from 17.4% in the patients treated for up to 1 year to 37.5% in those treated for 6 years or more. Significant differences were observed in the rate of anti-HCV findings in the patients with kidney transplantation and in those who had not experience this operation. The results obtained by an imitation computer model indicated that the hemotransfusion factor is not the only one determining the high rate of HCV infection in patients of chronic hemodialysis wards, however its influence on the intensity of the epidemic process in hepatitis C in these wards was sufficiently high.

[Localization of an immunodominant site in the hepatitis delta viral antigen using synthetic peptides]. / Lokalizatsiia immunodominantnogo saita v sostave antigena virusa gepatita del'ta s pomoshch'iu sinteticheskikh peptidov.

A set of 8 peptides from the immunodominant region (65-80 aa) of delta-antigen was prepared by solid-phase synthesis. Peptide 71-80 was synthesized in two variants--with different amino acid residues in positions 73, 74 and 76. Free peptides and their conjugates with bovine serum albumin were tested for antigenicity in ELISA. The correlation between the peptide chain's length and its antigenic activity was noted. Peptides 65-80 and 69-80 displayed a positive reaction with all individual sera and pools of sera from HDV chronic patients. Both variants of the peptide 71-80 reacted with 100% of sera pools but only with 83% of individual sera. Smaller peptides from the same 65-80 region (73-80, 69-78, 71-78, 71-76) did not bind to any anti-delta positive serum. All synthesized peptides reacted strongly with rabbit antisera raised to the conjugate of peptide 65-80 with bovine albumin. These findings suggest that delta-antigen contains multiple highly immunogenic epitopes associated with the single immunodominant site between 69 and 80 amino acid residues.

[Synthesis and antigenic activity of peptides from core- and NS3-proteins of hepatitis C virus]. / Sintez i antigennaia aktivnost' peptidov iz sostava core- i NS3- belkov virusa gepatita C.

We synthesized the 20-34 and 22-34 fragments of the core and 1269-1282 fragment of the NS3 protein of the hepatitis C virus. The peptides were prepared by the solid-phase synthesis using activated esters and symmetrical anhydrides of Boc-amino acids. Peptide 20-34 demonstrated positive reaction in ELISA with all individual sera from HCV chronic patients. Peptide 1269-1282 reacted with 33% patients sera.