THE FIRST TWO CASES OF CHRONIC HEPATITIS ASSOCIATED WITH TORQUE TENO MIDI...

Section 1. Clinical Medicine Morozov Igor Aleksandrovich, Head of the Department pathomorphology, Institute of Poliomyelitis and Viral Encephalitides FSBSI “Chumakov FSC R&D IBP RAS”, E-mail: [email protected] Ilchenko Liudmila Yur’evna, Institute of Poliomyelitis and Viral Encephalitides FSBSI “Chumakov FSC R&D IBP RAS”, researcher Pirogov Russian National Research Medical University Professor of the Department of Hospital Therapy E-mail: [email protected] Kyuregyan Karen Karenovich, Mechnikov Research Institute of Vaccines and Sera, Head of the Department of Viral Hepatitis Research Center at Russian Academy of Postgraduate Education E-mail: [email protected] Fediukina Elena Sergeevna, researcher, Institute of Poliomyelitis and Viral Encephalitides FSBSI “Chumakov FSC R&D IBP RAS”, E-mail: [email protected] Kozhanova Tatyana Viktorovna, researcher, Institute of Poliomyelitis and Viral Encephalitides FSBSI “Chumakov FSC R&D IBP RAS” E-mail: [email protected] Soboleva Nataliya Vasil’evna, researcher, Institute of Poliomyelitis and Viral Encephalitides FSBSI “Chumakov FSC R&D IBP RAS” E-mail: [email protected] THE FIRST TWO CASES OF CHRONIC HEPATITIS ASSOCIATED WITH TORQUE TENO MIDI (TTMDV), GENUS GAMMATORQUEVIRUS Abstract: The prevalence ofAnelloviridae within general population is high in many countries. The hepatotropic and hepatopathogenic features were confirmed for some genotypes of

3 Section 1. Clinical Medicine this family. Two case studies of chronic hepatitis associated with Torque teno midi virus (TTMDV, genus Gammatorquevirus) in patients negative for other hepatotropic viruses (HBV, HCV, GBV–C, CMV, TTV, TTMV) were described using biomolecular, histopathological, immunohistochemical and electron microscopy methods. Both patients were diagnosed with chronic cholestatic hepatitis with marked intracellular cholestasis, I–II degree portal and pericellular fibrosis, moderate portal and lobular lymphocytic infiltration, and necrobiosis of biliary epithelium. The structural hepatic changes were similar to those observed in cases of TTV infection. Keywords: Torque teno midi virus, hepatotropic, hepatopathogenic, chronic hepatitis. Within a decade starting in 1997 Japanese virolo- As for TTMDV, its role in human pathology is gists (Okamoto H., Nishizawa T., Ninomiya M. et al.) currently not defined at all, its virion size is unknown, have discovered three new human viruses with sin- and its morphology is not described. gle-stranded circular DNA genome: Torque teno vi- In retrospective studies of the prevalence of rus (TTV, genus Alphatorquevirus) with 3.7–3.8 kb infection among patients with viral genome [1], Torque teno mini virus (TTMV, genus or non-viral chronic liver diseases and healthy vol- ) with 2.8–2.9 kb genome [2] and untary blood donors, we detected three cases of Torque teno midi virus (TTMDV), genus Gamma- TTMDV monoinfection [8, 9]: two patients with torquevirus) with 3.2 kb genome [3]. These viruses chronic alcoholic hepatitis and chronic unspecified have been combined into the phylogenetically un- hepatitis, respectively, and one voluntary blood do- classified family Anelloviridae, which includes simi- nor. All three cases were negative for markers of lar viruses harbored by a number of mammals [4]. other hepatotropic viruses (HBV, HCV, GBV–C, Within 2–3 years after the discovery of TTV, the CMV, TTV, TTMV). In patients with chronic hepa- extremely high prevalence of these viruses has been titis the virus was identified in hepatocytes using demonstrated among the population of many coun- electron microscopy, its size was determined, and tries worldwide. Not only hepatotropic nature, but the nature of chronic TTMDV-induced hepatic pa- also the hepatopathogenicity of some viral genotypes thology was described [8, 9]. As this was the first have been confirmed. The nature of chronic TTV- description of pathology possibly induced by TT- induced hepatic pathology has been described, elec- MDV, we decided to provide a complete descrip- tron-microscopic images of this virus isolated from tion of these case studies. serum, feces [5] and cytoplasm of hepatocytes [6] Materials and Methods have been obtained. Nevertheless, some researchers Blood sera and appropriately prepared liver are still convinced that TTV is not pathogenic which biopsy samples were used for the in-depth study is a result of a long-lasting (centuries-long) coevolu- using pathohistological, electron microscopic and tion of the virus with its host. immunohistochemical test methods, as well as us- Numerous epidemiological and clinical studies ing biomolecular methods (ELISA, PCR) in the of TTMV provide evidence of its association with a laboratories of the Chumakov Institute of Poliomy- number of pathological conditions of the immune elitis and Viral Encephalitides. Within 3 minutes and respiratory system, blood, and also with cancer from the sample collection, the blood serum and a and lymphomas. At the same time, these studies do part of the liver biopsy sample (2 mm) were frozen not give an accurate and complete picture of the at –20 °C for up to 1 week, followed by their trans- role of TTMV in human pathology and, in particu- portation (in compliance with the cold chain) to lar, in liver pathology [7]. the laboratory for analysis.

4 THE FIRST TWO CASES OF CHRONIC HEPATITIS ASSOCIATED WITH TORQUE TENO MIDI...

Electron microscopy. Liver biopsy samples were manufactured by Diagnostic Systems LTD (Russia) placed in a fixative consisting of 2% paraformalde- according to the manufacturer’s protocols. Serum hyde solution and 2.5% glutaraldehyde in Hank’s buf- samples were also tested for GBV–C RNA (a virus fer, and put into the Epon-Araldite resin mixture after previously known as hepatitis G virus) by PCR, fixation and dehydration. Subsequently, the prepared combined with reverse transcription according to ultrathin sections after contrasting with uranyl acetate the protocol described by J. Hattori et al. [11]. and lead citrate were studied in the JEM‑100C elec- The isolation of nucleic acids from serum sam- tron microscope (Jeol, Japan). The morphometric ples was performed using a “Plasma or Serum DNA analysis of the virions was performed using BioVision / RNA Isolation Kit on Magnetic Beads MP@SiO2” program (Austria) with × 30 000 magnification in the (manufactured by ZAO “Sileks”) according to the reverse image of 400% of the original size. manufacturer’s protocol. Immunoelectron microscopy with Protein A-Colloi- To detect the anelloviral DNA, serum samples dal Gold. Ultrathin sections were mounted on nickel were analyzed in PCR with nested primers suggested grids (200 mesh), coated with formwar and spray- by M. Ninomiya and colleagues [12], that allows for coated with carbon (Agar Scientific Ltd), and, after differentiation of TTV, TTMDV and TTMV on the osmium plating with Na-metaperiodate, were used basis of the size of the amplicon. for immunocytochemical verification of TTMDV To confirm the specificity of TTV, TTMDV, and virions. The sample handling procedure for immu- TTMV detection in PCR, the nucleotide sequence noelectron microscopy using Protein A-Colloidal of the obtained viral genome fragments was deter- Gold (10 nm, EMS) was performed according to the mined by direct sequencing of amplicons. To confirm method detailed by M. Bendayan [10]. Due to the the amplification specificity, a search was performed absence of commercial anti-virus antibodies stud- using the BLAST function in the NCBI (National ied, we used blood serum (1:50 dilution) from the Center for Biotechnology Information) database. donor, who also had a monoinfection with only one Results and Discussion anellovirus TTMDV, and had no biopsy material, Case study 1. Patient P., female, 39 years. Patient which excluded the morphological study. was admitted to the clinic on 28.01.2009 with com- The omission of antibody incubation step, bi- plaints of general weakness, an abdominal expansion. opsy sample incubation with 1:50 diluted serum of Patient considered herself ill after having drunk a healthy donor, who had no viruses of the family moderate doses of alcoholic drinks during the New Anelloviridae, was used as the negative control. Year holidays, after which she had noticed a temper- ELISA & PCR methods. To exclude the occult form ature rise to 38 °C and abdominal expansion. She of chronic hepatitis B in patients an immunohisto- denied the systematic intake of alcoholic drinks in chemical detection of HBsAg was performed on par- past medical history. affin sections from archival blocks from the pathology A moderate hyperchromic anemia and minor department of Buyanova City Clinical Hospital using leukocytosis were diagnosed on an ambulatory monoclonal anti-HBsAg antibodies (anti-HBs, Cell basis. Blood biochemistry: total bilirubin – 50 Marque S1–210 USA) according to the manufacturer’s μmol/L, direct bilirubin – 28.5 μmol/L, aspartic standard procedure. Semithin sections for histological aminotransferase (AST) – 321 IU/L, alanine ami- examination were stained with toluidine blue. notransferase (ALT) – 49 IU/L. No HBsAg and Serological markers of HBV and HCV (HBsAg, HCV were detected. She was referred for the City anti-HBc, anti-HCV) were determined in serum Clinical Hospital named V. M. Buyanova for exami- samples using enzyme immunoassay test systems nation and treatment.

5 Section 1. Clinical Medicine

Condition on admission: moderate. The body received. Since September 2013, pain syndrome, mass index – 21 kg/m2. Skin and sclera were bile dyspeptic disorders have increased; elevated ALT, stained. No peripheral edema. Psoriatic plaques on GGTP levels. Given the past medical history data, the skin of the legs. Respiratory, circulatory and uri- the clinical findings were considered as a presenta- nary systems: no abnormalities detected. The liver tion of chronic hepatitis. is enlarged, indurated, even surface, edge is slightly According to medical history, Sjogren’s syn- pointed. The spleen was non-palpable. drome was diagnosed in 1973, anti-human herpes Complete blood count was without pathological virus‑3, 4, 5 IgG, IgM antibodies are detected since findings. Blood chemistry dd. 17.02.2009: AST‑223 November 2011. During the follow-up period, the IU/L, ALT‑48 IU/L, gamma-glutamyltranspepti- antibody titer has returned to normal. dase 1681IU/L, alkaline phosphatase 372IU/L, to- Complete blood count dd. 10.10.2013 was with- tal bilirubin 24.3 μmol, direct bilirubin – 9.6 μmol. out pathological findings. Blood chemistry: AST – The ultrasound of the hepatobiliary system –34 IU/L, ALT – 52 IU/L, ALP – 235 IU/L, total showed enlarged liver, increased echogenicity, dif- bilirubin – 10.9 μmol, direct bilirubin – 4.1 μmol. No fusely heterogeneous structure, not dilated hepatic HBsAg and anti-HCV antibodies detected. ducts and the hepatic veins, normal vascular pattern. The ultrasound of the hepatobiliary system To assess the fibrosis activity and its stage, a liver bi- showed that liver is not enlarged; diffuse changes opsy was performed. in the parenchyma were detected. To diagnose During the pathohistological study of the bi- pathological processes differentially, determine the opsy sample, an enlargement of portal tracts due histological activity index, a puncture biopsy was to sclerosis with single portal-portal septa was de- performed. tected, with a tendency to annular displacement Pathogistological analysis of the liver biopsy of lobules and fibrosis around the central veins. sample demonstrated that portal tracts were not Vascularization of sinusoids, moderate inflamma- enlarged, minor lymphoid infiltration, release of tory infiltration of lymphocytes in portal tracts and individual lymphoid elements beyond the bor- lobules were detected. Hepatocytes with common der plate. Sinusoids were not dilated, lymphoid small and large lipid droplets of fatty infiltrate with elements were in the lumen. The proliferation of the effacement of the beam structure, with intra- reticuloendotheliocytes, presence of small intra- cellular cholestasis, with necrosis of individual lobular lymphoid collections were observed. There hepatocyte. Morphological conclusion: moderate was the deposition of bile pigment in the cytoplasm steatohepatitis (A2), II degree fibrosis. of individual hepatocytes. Hepatocytes with granu- Case study 2. Patient M., female, 65 years. Pa- lar dystrophy; groups of hepatocytes with hydropic tient was admitted to the hospital on 09.10.2013 dystrophy were found near the perivenular zone. with complaints of persistent aching pain in the Some hepatocytes had nuclear dysplasia, two-nu- upper abdomen; expressed nausea, not associated clear hepatocytes and apoptotic bodies were ob- with food intake and time of day; bringing relief served. Van Gieson’s staining demonstrated fibrosis episodic cholemesis,; weight loss of 7 kg during the of the portal tracts, collagen fibers in the walls of year; temperature rise to a febrile level (mainly in the central veins, minor focal perisinusoidal fibro- the morning and evening) with chills; pain in tibia; sis. Sudan III staining revealed no fatty infiltration dyspnea at rest; faint. of hepatocytes. Morphological conclusion: chron- Patient considered herself ill since March 2013, ic hepatitis with minor histological activity (A1), when the above complaints arose; no treatment I degree fibrosis.

6 THE FIRST TWO CASES OF CHRONIC HEPATITIS ASSOCIATED WITH TORQUE TENO MIDI...

At the beginning of the in-depth study of the nome sequences available at GeneBank. All TTVDV material obtained from these two patients, first of sequences from our patients, including the donors all, we determined the nucleotide sequence of de- whose blood serum was the source of anti-TTMDV tected fragments of the viral genomes using direct antiserum, harbored nucleotide polymorphisms that sequencing of amplicons in order to compare them are absent among 23 comparison genomes even on with the corresponding fragments of 23 TTMDV ge- such a short fragment of the genome (Fig. 1).

Figure 1. Heterogenicity of TTMDV genome fragments from patients examined (No. 24, No. 26) and the donor (No. 25), the source of anti-TTMDV antiserum Through carefully performed light-optical ex- miophilic mass were found. Given the EM results amination, it was established that the signs of HG in of such cells, it has been determined that their cy- both patients were similar. There was a minor lym- toplasm contains a huge amount of lipofuscin and phoid infiltration of the portal tracts beyond the bor- small drops of triglycerides, which are likely to be der plate, small intralobular clusters of lymphocytes, the product of lysosomal hydrolysis of bile elements, significant clarification of the cytoplasm (cytopathy) intracellular elements and possibly virions (Fig. 3a). of the hepatocytes. In both cases, the intracellular The degeneration and necrobiosis of the biliary epi- cholestasis (Fig. 2a, b, 3b), as well as degeneration thelium mentioned above (Fig. 4a), most likely hin- and necrobiosis of the epithelium of bile ducts (Fig. der the intra-cellular transport of bile and are the 4a) were the most characteristic signs of pathology. main pathogenic cause of intracellular cholestasis. The I–II degree moderate fibrosis of the portal tracts, Its indirect cause includes fibrosis, which has devel- as well as fibrosis around the central veins and in the oped in patients not only in the portal tract, but also perisinusoidal space (Fig. 4a, b) was observed. in the space of Disse, as well as in the structures of Besides hepatocytes in state of cytopathy, cells the hepatic lobes between the hepatocytes, i. e., peri- with a very dark cytoplasm (Fig. 2a), filled with os- cellularly (Fig. 5a).

7 Section 1. Clinical Medicine

a) b) Figure 2. Pronounced intracellular cholestasis: a – case 1 × 400; b – case 2 × 1200

a) b) Figure 3 a. – Ultrastructure of the hepatocyte with a dark cytoplasm (refer to text); b – Binuclear hepatocytes (arrows) as a sign of regeneration × 1200

8 THE FIRST TWO CASES OF CHRONIC HEPATITIS ASSOCIATED WITH TORQUE TENO MIDI...

a) b) Figure 4. а – Fibrosis of the portal tract; necrobiotic changes in the biliary epithelium × 400; b – Collagen fibers (arrow) in the space of Disse

a) b) Figure 5. а – Collagen fibers between hepatocytes (arrow); b –TTMDV virions in the cytoplasm of the hepatocyte with cleared cytoplasm

9 Section 1. Clinical Medicine

Chronic hepatitis with the presence of minimal molecular and immune staining methods, was fur- portal cholangitis, moderate II degree portal and ther confirmed by electron microscopy of liver bi- perisinusoidal fibrosis, associated with TTMDV opsy samples. Large amounts of TTMDV virions was detected. The first observation also takes the have been even detected in all hepatocytes at low possible role of the alcohol factor in liver damage magnification (Fig. 4b, 5a). At high magnification, into account. However, a certain similarity of the TTMDV was represented by rounded electron- morphological pattern with liver tissue changes in dense particles, although their density was some- the patient with unspecified hepatitis raises doubts what lower than that of calcium inclusions in the that only a toxic factor played the leading role in the matrix of mitochondria (Fig. 5b). development of chronic hepatitis. Virion verification using immune staining and The association of chronic hepatitis in these pa- electron microscopy with Protein A-Colloidal Gold tients with anelloviral infection, wich was initially is a direct evidence that detected virions are exactly detected in serum and liver biopsy samples using TT midi virus fromAnelloviridae family (Fig. 6a, b).

a) b) Figure 6. Verification of TTMDV virions by IEM using Protein A –Colloidal Gold. а – Patient М.; b – Patient P. Slices are not contrasted It can be seen quite clearly that the staining is Conclusion observed in the virion location area only, with direct In the last 15 years, an issue about the role of re- contact with virions, and is absent on the mitochon- cently discovered hepatitis G, TT and SEN viruses in drial profiles. The absence of a nonspecific reaction in human pathology, and in particular in hepatopathy, negative controls confirms that TTMDV monoinfec- has been repeatedly raised in the research literature, tion detected using molecular methods in serum and but it is still a pending one. This is also true for infec- liver tissue, is the cause of chronic hepatitis in patients. tion caused by TTMDV, accidentally discovered by

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Japanese researches during the retrospective study of in the liver of patients with TTMDV monoinfec- the collection of patient blood serum samples with tion, except for our two case studies [8, 9, 13]. TTV less than 10 years ago. Partly this lack of morphological data is due to a Many clinicians ignore the notion of “therapeu- recent decrease in indications for a fine-needle liver tic infection”, when almost all population has bac- biopsy, which cannot be completely replaced by fi- terial or viral infection, but only 10–15% of them broelastometry. We hope that this report, which have clinical sings of the associated disease. At the provides evidence of the association of chronic same time, no liver biopsy studies are done, which cholestatic hepatitis with TTMDV monoinfection, would make it possible to determine not only the will draw hepatologists and pathologists’ attention hepatotrophic character of the infection, but also to this issue. its hepatopathogenicity. So, for example, we have The authors declare that they have no conflicts found no published data on morphological changes of interest. References: 1. Nishizawa T., Okamoto H., Konishi K. et al. A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology // Biochem. Biophys. Res. Commun. – 1997. – Vol. 241. – P. 92–97. 2. 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. – Vol. 145. – P. 979–993. 3. Ninomiya M., Nishizawa T., Takahashi M. et al. Identification and genomic characterization of a novel human torque teno virus of 3.2 kb // J. General Virology. – 2007. – Vol. 88. – P. 1939–1944. 4. Okamoto H. History of discoveries and pathogenicity of TT Viruses // TT Viruses: the still elusive human pathogens / E. – M. de Villiers, H. zur Hausen (eds.), Springer Verlag Berlin Heidelberg, – 2009. – P. 2–15. 5. Itoh Y., Takahashi M., Fukuda M. et al. Visualization of TT virus particles recoveredfrom the sera and feces of Infected humans // Bioch. Biophys. Res. Commun. – 2000. – Vol. 279. – P. 718–724. 6. Khomeriki S.G., Ilchenko L.Y., Morozov I.А., Karlovich T.I. Clinico–morphological features of liver disease in patients infected with hepatitis TT // V Mire Virusn Gepatitov. – 2006;(2):2–8. (In Russian). 7. Hausen H., de Villiers E–M. Virus target cell conditioning model to explain some epidemiologic characteristics of childhood leukemias and lymphomas // Int. J. Cancer. – 2005. – Vol. 115. – Р. 1–5. 8. Morozov I., Zverkova E., Kyuregyan K. et al. The viruses of Anelloviridae family in chronic liver disease // Experimental and Сlinical Gastroenterol. – 2015. – No. 7. – P. 4–11 (In Russian). 9. Morozov I. A., Kyuregyan K. K., Karlsen A. A., Ilchenko L. Yu., Fedorov I. G. Viruses of the Anelloviridae family in cases of chronic liver pathology and in primary blood donors // European Journal of Biomedical and Life Sciences, “East West” Association for Advanced Studies and Higher Education GmbH. – Vienna. – No. 1. – 2017. – P. 27–35. 10. Bendayan M. Protein A. – Gold and Protein G Postembedding Immynoelectron Microscopy // In: Colloidal G old. Principles, Methods and Applications // Ed. M. A. Hayat, Academic Press Inc. (London). – 1989. – P. 33–94. 11. Hattori J., Okumura N., Yamazaki Y. et al. Beneficial effect of GB virus C co–infection in human immunodeficiency virus type 1–infected individuals // Microbiol. Immunol. – 2007. – Vol. 51. – Р. 193–200.

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12. Ninomiya M., Takahashi M., Nishizawa T. et al. Development of PCR assays with nested primers specific for differential detection of three human anelloviruses and early acquisition of dual or triple infection during infancy // J. Clin Microbiol. – 2008. – Vol. 46. – P. 507–514. 13. Morozov I., Ilchenko L. TT midi virus (Genus Gammatorquevirus) – commensal or pathogen? In: XXII Int. Congress “Hepatology Today 2016” // Russian Journal of Gastroenterology Hepatology Coloproctology. – 2016. – No. 1. – Suppl 47. – 15 p. (In Russian).

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