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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: moroz38@gmail.com 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: Ilchenko-med@yandex.ru 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: karen-kyuregyan@yandex.ru Fediukina Elena Sergeevna, researcher, Institute of Poliomyelitis and Viral Encephalitides FSBSI"Chumakov FSC R&D IBP RAS", E-mail: elena_fedyukina@mail.ru Kozhanova Tatyana Viktorovna, researcher, Institute of Poliomyelitis and Viral Encephalitides FSBSI"Chumakov FSC R&D IBP RAS" E-mail: vkozhanov@bk.ru Soboleva Nataliya Vasil'evna, researcher, Institute of Poliomyelitis and Viral Encephalitides FSBSI"Chumakov FSC R&D IBP RAS" E-mail: soboleva.natalia@inbox.ru
THE FIRST TWO CASES OF CHRONIC HEPATITIS ASSOCIATED WITH TORQUE TENO MIDI VIRUS (TTMDV), GENUS GAMMATORQUEVIRUS
Abstract: The prevalence of Anelloviridae viruses within general population is high in many countries. The hepatotropic and hepatopathogenic features were confirmed for some genotypes of
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 1997Japanese virologists (Okamoto H., Nishizawa T., Ninomiya M. et al.) have discovered three new human viruses with single-stranded circular DNA genome: Torque teno virus (TTV genus Alphatorquevirus) with 3.7-3.8 kb genome [1], Torque teno mini virus (TTMV, genus Betatorquevirus) with 2.8-2.9 kb genome [2] and Torque teno midi virus (TTMDV), genus Gammatorquevirus) with 3.2 kb genome [3]. These viruses have been combined into the phylogenetically unclassified family Anelloviridae, which includes similar viruses harbored by a number of mammals [4].
Within 2-3 years after the discovery of TTV, the extremely high prevalence of these viruses has been demonstrated among the population of many countries worldwide. Not only hepatotropic nature, but also the hepatopathogenicity of some viral genotypes have been confirmed. The nature of chronic TTV-induced hepatic pathology has been described, electron-microscopic images of this virus isolated from serum, feces [5] and cytoplasm of hepatocytes [6] have been obtained. Nevertheless, some researchers are still convinced that TTV is not pathogenic which is a result of a long-lasting (centuries-long) coevolu-tion of the virus with its host.
Numerous epidemiological and clinical studies of TTMV provide evidence of its association with a number of pathological conditions of the immune and respiratory system, blood, and also with cancer and lymphomas. At the same time, these studies do not give an accurate and complete picture of the role of TTMV in human pathology and, in particular, in liver pathology [7].
As for TTMDV its role in human pathology is currently not defined at all, its virion size is unknown, and its morphology is not described.
In retrospective studies of the prevalence of Anelloviridae infection among patients with viral or non-viral chronic liver diseases and healthy voluntary blood donors, we detected three cases of TTMDV monoinfection [8, 9]: two patients with chronic alcoholic hepatitis and chronic unspecified hepatitis, respectively, and one voluntary blood donor. All three cases were negative for markers of other hepatotropic viruses (HBV HCV GBV-C, CMV, TTV, TTMV). In patients with chronic hepatitis the virus was identified in hepatocytes using electron microscopy, its size was determined, and the nature of chronic TTMDV-induced hepatic pathology was described [8, 9]. As this was the first description of pathology possibly induced by TT-MDV, we decided to provide a complete description of these case studies.
Materials and Methods
Blood sera and appropriately prepared liver biopsy samples were used for the in-depth study using pathohistological, electron microscopic and immunohistochemical test methods, as well as using biomolecular methods (ELISA, PCR) in the laboratories of the Chumakov Institute of Poliomyelitis and Viral Encephalitides. Within 3 minutes from the sample collection, the blood serum and a part of the liver biopsy sample (2 mm) were frozen at -20 °C for up to 1 week, followed by their transportation (in compliance with the cold chain) to the laboratory for analysis.
Electron microscopy. Liver biopsy samples were placed in a fixative consisting of 2% paraformaldehyde solution and 2.5% glutaraldehyde in Hank's buffer, and put into the Epon-Araldite resin mixture after fixation and dehydration. Subsequently, the prepared ultrathin sections after contrasting with uranyl acetate and lead citrate were studied in the JEM-100C electron microscope (Jeol, Japan). The morphometric analysis of the virions was performed using BioVision program (Austria) with x 30000 magnification in the reverse image of400% of the original size.
Immunoelectron microscopy with Protein A-Colloi-dal Gold. Ultrathin sections were mounted on nickel grids (200 mesh), coated with formwar and spray-coated with carbon (Agar Scientific Ltd), and, after osmium plating with Na-metaperiodate, were used for immunocytochemical verification of TTMDV virions. The sample handling procedure for immunoelectron microscopy using Protein A-Colloidal Gold (10 nm, EMS) was performed according to the method detailed by M. Bendayan [10]. Due to the absence of commercial anti-virus antibodies studied, we used blood serum (1:50 dilution) from the donor, who also had a monoinfection with only one anellovirus TTMDV and had no biopsy material, which excluded the morphological study.
The omission of antibody incubation step, biopsy sample incubation with 1:50 diluted serum of a healthy donor, who had no viruses of the family Anelloviridae, was used as the negative control.
ELISA & PCR methods. To exclude the occult form of chronic hepatitis B in patients an immunohisto-chemical detection of HBsAg was performed on paraffin sections from archival blocks from the pathology department of Buyanova City Clinical Hospital using monoclonal anti-HBsAg antibodies (anti-HBs, Cell Marque S1-210 USA) according to the manufacturer's standard procedure. Semithin sections for histological examination were stained with toluidine blue.
Serological markers of HBV and HCV (HBsAg, anti-HBc, anti-HCV) were determined in serum samples using enzyme immunoassay test systems
manufactured by Diagnostic Systems LTD (Russia) according to the manufacturer's protocols. Serum samples were also tested for GBV-C RNA (a virus previously known as hepatitis G virus) by PCR, combined with reverse transcription according to the protocol described by J. Hattori et al. [11].
The isolation of nucleic acids from serum samples was performed using a "Plasma or Serum DNA / RNA Isolation Kit on Magnetic Beads MP@SiO2" (manufactured by ZAO "Sileks") according to the manufacturer's protocol.
To detect the anelloviral DNA, serum samples were analyzed in PCR with nested primers suggested by M. Ninomiya and colleagues [12], that allows for differentiation of TTV, TTMDV and TTMV on the basis of the size of the amplicon.
To confirm the specificity of TTV, TTMDV, and TTMV detection in PCR, the nucleotide sequence of the obtained viral genome fragments was determined by direct sequencing of amplicons. To confirm the amplification specificity, a search was performed using the BLAST function in the NCBI (National Center for Biotechnology Information) database.
Results and Discussion
Case study 1. Patient P., female, 39 years. Patient was admitted to the clinic on 28.01.2009 with complaints of general weakness, an abdominal expansion.
Patient considered herself ill after having drunk moderate doses of alcoholic drinks during the New Year holidays, after which she had noticed a temperature rise to 38 °C and abdominal expansion. She denied the systematic intake of alcoholic drinks in past medical history.
A moderate hyperchromic anemia and minor leukocytosis were diagnosed on an ambulatory basis. Blood biochemistry: total bilirubin - 50 ^mol/L, direct bilirubin - 28.5 ^mol/L, aspartic aminotransferase (AST) - 321 IU/L, alanine ami-notransferase (ALT) - 49 IU/L. No HBsAg and HCV were detected. She was referred for the City Clinical Hospital named V. M. Buyanova for examination and treatment.
Condition on admission: moderate. The body mass index - 21 kg/m2. Skin and sclera were bile stained. No peripheral edema. Psoriatic plaques on the skin of the legs. Respiratory, circulatory and urinary systems: no abnormalities detected. The liver is enlarged, indurated, even surface, edge is slightly pointed. The spleen was non-palpable.
Complete blood count was without pathological findings. Blood chemistry dd. 17.02.2009: AST-223 IU/L, ALT-48 IU/L, gamma-glutamyltranspepti-dase 1681IU/L, alkaline phosphatase 372IU/L, total bilirubin 24.3 ^mol, direct bilirubin - 9.6 ^mol.
The ultrasound of the hepatobiliary system showed enlarged liver, increased echogenicity, diffusely heterogeneous structure, not dilated hepatic ducts and the hepatic veins, normal vascular pattern. To assess the fibrosis activity and its stage, a liver biopsy was performed.
During the pathohistological study of the biopsy sample, an enlargement of portal tracts due to sclerosis with single portal-portal septa was detected, with a tendency to annular displacement of lobules and fibrosis around the central veins. Vascularization of sinusoids, moderate inflammatory infiltration of lymphocytes in portal tracts and lobules were detected. Hepatocytes with common small and large lipid droplets of fatty infiltrate with the effacement of the beam structure, with intracellular cholestasis, with necrosis of individual hepatocyte. Morphological conclusion: moderate steatohepatitis (A2), II degree fibrosis.
Case study 2. Patient M., female, 65 years. Patient was admitted to the hospital on 09.10.2013 with complaints of persistent aching pain in the upper abdomen; expressed nausea, not associated with food intake and time of day; bringing relief episodic cholemesis,; weight loss of 7 kg during the year; temperature rise to a febrile level (mainly in the morning and evening) with chills; pain in tibia; dyspnea at rest; faint.
Patient considered herself ill since March 2013, when the above complaints arose; no treatment
received. Since September 2013, pain syndrome, dyspeptic disorders have increased; elevated ALT, GGTP levels. Given the past medical history data, the clinical findings were considered as a presentation of chronic hepatitis.
According to medical history, Sjogren's syndrome was diagnosed in 1973, anti-human herpes virus-3, 4, 5 IgG, IgM antibodies are detected since November 2011. During the follow-up period, the antibody titer has returned to normal.
Complete blood count dd. 10.10.2013 was without pathological findings. Blood chemistry: AST --34 IU/L, ALT - 52 IU/L, ALP - 235 IU/L, total bilirubin - 10.9 ^mol, direct bilirubin - 4.1 ^mol. No HBsAg and anti-HCV antibodies detected.
The ultrasound of the hepatobiliary system showed that liver is not enlarged; diffuse changes in the parenchyma were detected. To diagnose pathological processes differentially, determine the histological activity index, a puncture biopsy was performed.
Pathogistological analysis of the liver biopsy sample demonstrated that portal tracts were not enlarged, minor lymphoid infiltration, release of individual lymphoid elements beyond the border plate. Sinusoids were not dilated, lymphoid elements were in the lumen. The proliferation of reticuloendotheliocytes, presence of small intra-lobular lymphoid collections were observed. There was the deposition of bile pigment in the cytoplasm of individual hepatocytes. Hepatocytes with granular dystrophy; groups of hepatocytes with hydropic dystrophy were found near the perivenular zone. Some hepatocytes had nuclear dysplasia, two-nuclear hepatocytes and apoptotic bodies were observed. Van Gieson's staining demonstrated fibrosis of the portal tracts, collagen fibers in the walls of the central veins, minor focal perisinusoidal fibrosis. Sudan III staining revealed no fatty infiltration of hepatocytes. Morphological conclusion: chronic hepatitis with minor histological activity (A1), I degree fibrosis.
At the beginning of the in-depth study of the material obtained from these two patients, first of all, we determined the nucleotide sequence of detected fragments of the viral genomes using direct sequencing of amplicons in order to compare them with the corresponding fragments of 23 TTMDV ge-
nome sequences available at GeneBank. All TTVDV sequences from our patients, including the donors whose blood serum was the source of anti-TTMDV antiserum, harbored nucleotide polymorphisms that are absent among 23 comparison genomes even on such a short fragment of the genome (Fig. 1).
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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 examination, it was established that the signs of HG in both patients were similar. There was a minor lymphoid infiltration of the portal tracts beyond the border plate, small intralobular clusters oflymphocytes, significant clarification of the cytoplasm (cytopathy) of the hepatocytes. In both cases, the intracellular cholestasis (Fig. 2a, b, 3b), as well as degeneration and necrobiosis of the epithelium of bile ducts (Fig. 4a) were the most characteristic signs of pathology. The I-II degree moderate fibrosis of the portal tracts, as well as fibrosis around the central veins and in the perisinusoidal space (Fig. 4a, b) was observed.
Besides hepatocytes in state of cytopathy, cells with a very dark cytoplasm (Fig. 2a), filled with os-
miophilic mass were found. Given the EM results of such cells, it has been determined that their cytoplasm contains a huge amount of lipofuscin and small drops of triglycerides, which are likely to be the product of lysosomal hydrolysis of bile elements, intracellular elements and possibly virions (Fig. 3a). The degeneration and necrobiosis of the biliary epithelium mentioned above (Fig. 4a), most likely hinder the intra-cellular transport of bile and are the main pathogenic cause of intracellular cholestasis. Its indirect cause includes fibrosis, which has developed in patients not only in the portal tract, but also in the space of Disse, as well as in the structures of the hepatic lobes between the hepatocytes, i.e., peri-cellularly (Fig. 5a).
a) b)
Figure 2. Pronounced intracellular cholestasis: a - case 1 x 400; b - case 2 x 1200
Figure 3 a. - Ultrastructure of the hepatocyte with a dark cytoplasm (refer to text); b - Binuclear hepatocytes (arrows) as a sign of regeneration x 1200
Figure 4. a - Fibrosis of the portal tract; necrobiotic changes in the biliary epithelium x 400; b - Collagen fibers (arrow) in the space of Disse
a) b)
Figure 5. a - Collagen fibers between hepatocytes (arrow); b -TTMDV virions in the cytoplasm of the hepatocyte with cleared cytoplasm
Chronic hepatitis with the presence of minimal portal cholangitis, moderate II degree portal and perisinusoidal fibrosis, associated with TTMDV was detected. The first observation also takes the possible role of the alcohol factor in liver damage into account. However, a certain similarity of the morphological pattern with liver tissue changes in the patient with unspecified hepatitis raises doubts that only a toxic factor played the leading role in the development of chronic hepatitis.
The association of chronic hepatitis in these patients with anelloviral infection, wich was initially detected in serum and liver biopsy samples using
molecular and immune staining methods, was further confirmed by electron microscopy of liver biopsy samples. Large amounts of TTMDV virions have been even detected in all hepatocytes at low magnification (Fig. 4b, 5a). At high magnification, TTMDV was represented by rounded electron-dense particles, although their density was somewhat lower than that of calcium inclusions in the matrix of mitochondria (Fig. 5b).
Virion verification using immune staining and electron microscopy with Protein A-Colloidal Gold is a direct evidence that detected virions are exactly TT midi virus from Anelloviridae family (Fig. 6a, b).
a) b)
Figure 6. Verification of TTMDV virions by IEM using Protein A -Colloidal Gold. a - Patient M.; b - Patient P. Slices are not contrasted
It can be seen quite clearly that the staining is observed in the virion location area only, with direct contact with virions, and is absent on the mitochon-drial profiles. The absence of a nonspecific reaction in negative controls confirms that TTMDV monoinfection detected using molecular methods in serum and liver tissue, is the cause ofchronic hepatitis in patients.
Conclusion
In the last 15 years, an issue about the role of recently discovered hepatitis G, TT and SEN viruses in human pathology, and in particular in hepatopathy, has been repeatedly raised in the research literature, but it is still a pending one. This is also true for infection caused by TTMDV, accidentally discovered by
Japanese researches during the retrospective study of the collection of patient blood serum samples with TT V less than 10 years ago.
Many clinicians ignore the notion of "therapeutic infection", when almost all population has bacterial or viral infection, but only 10-15% of them have clinical sings of the associated disease. At the same time, no liver biopsy studies are done, which would make it possible to determine not only the hepatotrophic character of the infection, but also its hepatopathogenicity. So, for example, we have found no published data on morphological changes
in the liver of patients with TTMDV monoinfection, except for our two case studies [8, 9, 13]. Partly this lack of morphological data is due to a recent decrease in indications for a fine-needle liver biopsy, which cannot be completely replaced by fi-broelastometry. We hope that this report, which provides evidence of the association of chronic cholestatic hepatitis with TTMDV monoinfection, will draw hepatologists and pathologists' attention to this issue.
The authors declare that they have no conflicts of interest.
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