1 NEW LOOK AT THE MULTIVIRUS INFECTION IN CHRONIC LIVER DISEASESThe XXII Annual International Congress "Hepatology Today" on March , 2017 NEW LOOK AT THE MULTIVIRUS INFECTION IN CHRONIC LIVER DISEASES Morozov Igor A., Ilchenko Ludmila Yu. Chumakov Institute of Poliomyelitis and Viral Encephalitides, Moscow

2 WITH PARENTERAL TRANSFERHEPATOTROPIC VIRUSES WITH PARENTERAL TRANSFER HBV HDV HCV HGV/GBV-C SEN (A,B, C, D,E, F,G,H) NF-V EBV CMV Herpes simple TTV TTmidiV TTminiV

3 INTRASTRUCTURE OF HDV VIRIONSHBV virion 42 nm

4 HCV HBV

5 HGV HBV

6 HCV HGV

7 HISTORY of Anelloviridae DISCOVEREDDivision of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi-Ken, Japan Department of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan 1997 – Alphatorquevirus (TTV) Nishizawa T, Okamoto H, Konishi K, Yoshizava H, Miyakawa Y, Mayumi M A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology. Biochem Biophys Res Commun (1997) 241:92–97 2000 – Betatorquevirus (TTMV) Takahashi K., Iwasa Y., Hijikata M., Mishiro S. Identification of a new human DNA virus (TTV-like mini virus, TLMV) intermediately related to TT virus and chicken anemia virus. Arch Virol (2000) 145, 979–993. 2007 – Gammatorquevirus (TTMDV) Masashi Ninomiya, Tsutomu Nishizawa, Masaharu Takahashi, Felipe R. Lorenzo, Tooru Shimosegawa, Hiroaki Okamoto (2007) Identification and genomic characterization of a novel human torque teno virus of 3.2 kb Journal of General Virology , 88: 1939–1944 o

8 WORLD OF VIRAL HEPATITIS, 2006 №2. C2-8Based on 18 biopsies of patients with TTV monoinfection WORLD OF VIRAL HEPATITIS, 2006 №2. C2-8

9 Based on 18 biopsies of patients with TTV monoinfection

10 PREVALENCE OF ANELLOVIRIDAE FAMILY VIRUSES IN HEALTHY PEOPLE OF VARIOUS AGE Ninomiya M, Takahashi M, Nishizawa T, et al (2008). J Clin Microbiol 46: Age (years) and sample type No. of samples tested No. of samples (%) with: TTV DNA TTMDV DNA TTMV DNA <1 Serum 44 35 (79.5) 22 (50.0) 33 (75.0) 1 28 28 (100) 2-4. 29 29 (100) 28 (96.6) 5-9. 42 41 (97.6) 35 (85.7) 10-19. 62 61 (98.4) 48 (77.4) 55 (88.7) 20-49 32 32 (100) 22 (68.8) 24 (75.0) 50-81 68 68 (100) 53 (77.9) 58 (85.3) Subtotal 261 259 (99.2) 215 (82.4) 234 (89.7) Total 315 294 (93.3) 237 (75.2) 267 (84.8)

11 From 2007 to 2015, out of every 100 patients with complaints of pain various character in the liver and symptoms of its defeat, which produced a percutaneous thin needle biopsy of the liver the following clinical diagnoses were made: Chronic hepatitis В % Chronic hepatitis С % Chronic hepatitis В + С % Autoimmune lesions of the liver % Medicinal lesions % Primary biliary cirrhosis % Chronic hepatitis of alcoholic etiology % Non-alcoholic steatohepatitis % Chronic hepatitis of unknown etiology % Other diseases %

12 The frequency of detection of anelovirus (TTV, TTMDV, TTMV) at chronic liver pathology and primary blood donors Primary Clinical diagnosis Quan- tity The viruses, detected in PCR, IFA and morphological study The number of positive individuals (%) HBV anti-HBcore + HCV TTV TTMDV TTMV Alcoholic liver disease 80 35(43,8) 77(96,3) 47(57,5) 62(72,5) Chronic hepatitis of unknown etiology 57 29(50,9) 54(94,7) 33(57,9) 45(78,9) Nonalcoholic steatohepatitis 14 5(35,7) 13(92,9) 9(56,3) 11(78,6) Chronic hepatitis C 30 1 15(50,0) 29(96,7) 25(83,3) 26(86,7) Chronic hepatitis B 22 - 20(90,9) 15(68,2) 19(86,4) “Isolated” anti-HBcore 84 84(100) 78(92,9) 52(61,9) 70(83,3) Primary blood donors 115 111(96,5) 84(73,0) 103(89,6)

13 Anti-HBcore positive patient with clinical diagnosis"Chronic hepatitis of unknown etiology"

14 TT-like mini virus LYMPHOCYT IN SINUSOIDE HBV

15 Harald zur Hausen and Ethel-Michele de VilliersVirus target cell conditioning model to explain some epidemiologic characteristics of childhood leukemias and lymphomas Int. J. Cancer: 115, 1–5 (2005) Persistent infections with TT virus-like agents increase the risk for specific chromosomal translocations. The emerging phenotype of specific leukemias and lymphomas may be determined by different genotypes of TTV-like infections. Virus-like particles in the nuclei of cells of the Epstein-Barr virus-negative L1236 line of Hodgkin’s lymphoma origin. The size of the particles is in the range of 25–30 nm

16 Mori M. , Dempo K. , Abe M. , Onoe TMori M., Dempo K., Abe M., Onoe T. Electron microscopic study of intranuclear glycogen. J. Electron Microscopy, 1970;19(2):163-9.

17 Ttlike TT like mini virus nM HBV

18 x 20-22 nm

19 EM immunocytochemistry of TTMV in the hepatocyte nucleus with serum (1: 100) of the patient with hepatitis C and monoinfection with TTMV and Protein A - Colloidal Gold 10 nm 50 nm

20 Frequency of detection of mono- and multiple anelloviral infectionwith chronic liver pathology and primary blood donors Group Quantity Monoinfection Triple infection TTV, TTMDV and TTMV Without  infection TTV, TTMDV and TTMV TTV TTMDV TTMV The number of positive individuals (%) Patients with chronic hepatitis B, C 52 4(7,7) 1(1,9) 36(69,2) Patients with hepatitis of non-viral origin 151 16(10,6) 2(1,3) ! 75(49,7) 1(0,66) Primary blood donors 115 4(3,5) 1(0,9) 60(52,2) 3(2,6)

21 Results of the sequencing of two patients with TTMDV monoinfectionPatient M. 39 years old Donor - source of anti-TTMDV for the immunogold method Patient P. 65 years old

22 TTMDV monoinfection

23 Intracellular cholestasis Regeneration

24 TTMDV monoinfection

25 TTMDV monoinfection

26 EM immunocytochemistry with serum (1:50) of the blood donorwith a high concentration of TTMDV and Protein A - Colloidal Gold 10 nm № 76 № 540

27

28 Primary Clinical diagnosis n 80 35 16 24 39 57 30 9 19 29 14 5 4 10The viruses, detected in PCR, IFA and morphological study Anelloviridae (AV), n Confirmation of a clinical diagnosis anti-HBcore + Without AV 1 virus 2 viruses 3 viruses Alcoholic disease of the liver 80 35 16 24 39 Chronic hepatitis of unknown etiology 57 30 9 19 29 Nonalcoholic steatohepatitis 14 5 4 10 Chronic hepatitis C 2 6 22 Chronic hepatitis B -

29 Order of Rospotrebnadzor of 27.06.2011 № 621 "On the improvement of surveillance and measures to prevent viral hepatitis" Heads of departments of Rospotrebnadzor for the subjects of the Russian Federation: 1.4. Take the necessary measures to improve the organizational and methodological foundations of laboratory diagnostics of viral hepatitis, including the introduction and improvement of molecular genetic methods, including to determine the tactics and effectiveness of treatment of patients. Ensure the necessary volume of diagnostic studies of the whole spectrum of viral hepatitis. 1.6. To organize work on creating registers of patients with chronic viral hepatitis. To strengthen control over the organization and conduct of dispensary follow-up of patients who underwent hepatitis B and C.

30 At present, our knowledge Hidden under water - we do not know!About multiviral chronic hepatitis are nothing more than a "above-water part of the iceberg". Hidden under water - we do not know! The time has come when it became necessary to determine the entire spectrum of known hepatotropic viruses in patients with chronic hepatitis for establishing an etiologically justified diagnosis and prescribing adequate treatment.

31 Karen K. Kyuregyan, Anastasia A. Karlsen, Olga V. Isaeva,Many thanks to the participants of the work! Karen K. Kyuregyan, Anastasia A. Karlsen, Olga V. Isaeva, Il`ya G. Fedorov, Tatyana V. Kozhanova, Il`ya V. Gordeychuk, Natalya V. Petrenko, Mikhail I. Mikhailov