Hepatitis G virus (HGV) infection & its pathogenic significance in patients of cirrhosis
In the present study the hepatitis G virus (HGV) infection and its pathogenic significance in patients of cirrhosis were assessed using reverse transcription plus nested polymerase chain reaction (RT-PCR). Serum samples were collected from a total of 50 patients of histologically proven non-alcoholic cirrhosis and from a control group consisting of 50 healthy voluntary blood donors. HGV RNA was detected by RT-PCR using primer sequences located in the conserved NS3 helicase region of HGV genome. Serological evaluation for markers of chronic infection with HBV (HBsAg, IgG anti-HBc, HBeAg) and HCV (anti-HCV) was carried out using commercially available kits. HBV DNA and HCV RNA were also tested by PCR in those samples that were found to be non-B, non-C by serological assays. Serological evidence of exposure to HBV was found in 31 (62 %) and to HCV in 15 (30%) patients. HGV RNA was detected in 6 (12%) cirrhosis patients and in 2 (4%) healthy blood donors but the difference between the two groups was not statistically significant. Of the 6 HGV positive patients, 2 were coinfected with HBV, 1 with HCV, while the remaining 3 belonged to non-B, non-C category. No significant difference was observed in the clinical and biochemical profiles of HGV-positive and HGV-negative patients except that a history of blood transfusion was significantly (P
Key words Cirrhosis – HBV – HCV – hepatitis G virus – reverse transcription PCR – voluntary blood donors
Hepatic cirrhosis is an important public health problem; the major burden being caused by infection with primary hepatotropic viruses viz. 7 hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus (HDV). Many individuals who have clinical evidence of virus-induced chronic liver disease fall in the non-B, non-C category, suggesting the existence of one or more yet unidentified additional viral agent(s). HGV, a novel human flavivirus is considered to be a putative hepatotropic agent. This virus is widely distributed throughout the world and its presence has been reported from different geographical areas’-10. However, so far there has been no comprehensive study on the occurrence of HGV in India. In view of this paucity of information, the present study was designed to study the HGV infection and also to evaluate the pathogenic significance of this virus in patients of cirrhosis and in healthy voluntary blood donors by using reverse transcription plus nested polymerase chain-reaction (RTPCR).
Material & Methods
Subjects : The present study was conducted during the period from April 1997 to March 1998 and included a total of 100 subjects comprising 50 consecutive patients of postnecrotic cirrhosis and 50 healthy controls. The diagnosis of cirrhosis was made when a patient presented to the medical out-patient department of Lok Nayak Hospital, New Delhi, with features of chronic liver disease associated with portal hypertension, and biochemical evidence of hepatic failure and barium swallow or endoscopy showed presence of oesophageal varices. Non-viral causes of cirrhosis were carefully excluded on the basis of a detailed history, clinical features, liver function tests, liver biopsy and specific investigations, if considered necessary. The control subjects were healthy voluntary blood donors without any history or symptomatology suggestive of liver disease or high risk behaviour.
Sample collection and clinical and biochemical evaluation : Drawing of blood and separation of serum was done under complete aseptic conditions and serum was stored at -70 deg C until use. After initial clinical assessment, a complete biochemical evaluation [haemogram, blood sugar, blood urea, serum bilirubin and its fractions, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, prothrombin time, albumin and globulin ratio] of the patients was carried out. All patients were subjected to oesophagogastroscopy for detection of oesophageal varices and abdominal sonography for assessment of liver size and echotexture. A percutaneous liver biopsy was also performed in all the cases for histopathological confirmation of the clinical diagnosis.
Serology: The serological tests were performed for detection of any evidence of infection with HBV and HCV in all the study samples using commercially available third generation ELISA kits. HBsAg was detected using Eliscan micro ELISA strips (Ranbaxy Diagnostics, England), IgG anti-HBc antibodies by Melotest antiHBc kit (Melotec, Spain), HBeAg by Melotest HBeAg/ anti HBe kit (Melotec, Spain) and anti-HCV antibodies using Innotest HCV Ab III (Innogenetics NV, Ghent, Belgium).
Reverse transcription-polymerase chain reaction (RTPCR) for HGV RNA : RT-PCR was performed for detection of HGV RNA in all study samples. Those samples which were found to be negative for HBV and HCV related serological markers were further subjected to PCR for detection of HBV DNA and HCV RNA so as to confirm their non-B, non-C status.
RNA extraction : RNA was extracted from serum by standard acid guanidinium-phenol-chloroform method as described by Chomczynski and Sacchi11 with slight modifications12. Briefly, 100(mu)l of patient’s serum was used to which 500(mu)l of lysis buffer containing 4 M guanidine isothiocyanate (GITC), 0.7 M sodium citrate (pH 7.0), 2 per cent sarcosyl and 0.1 M (final concentration) beta-mercaptoethanol was added. To this, 50 (mu)l of 3 M sodium acetate (pH 5.0) was added, extraction carried out once with phenol and chloroform: isoamyl alcohol (49:1) and left on ice. After centrifugation at 12,000 rpm for 20 min at 4degC, the pellet was resuspended in 150 (mu)l lysis buffer (without P-mercaptoethanol), precipitated in chilled isopropanol, kept at -70 deg C for 90 min, centrifuged and washed once with 70 per cent ethanol. Finally, the pellet was dissolved in 25 (mu)l of diethyl pyrocarbonate (DEPC water and stored at -70 deg C until use.
RT-PCR for HGV-RNA : Reverse transcription for : preparation of complementary DNA (cDNA) with AMV reverse transcriptase (Promega, USA) and the PCR were performed in a single reaction tube using DNA Thermal Cycler (Perkin-Elmer Cetus, Roche, USA). The outer and inner primer sequences for nested PCR were selected from NS3 helicase region of HGV genome and were as follows:
Sense S^sub 1^: 5′-GGC ACC TCG TGT TCT GCC A-3′ Antisense A^sub 1^ : 5′-AGG TCT CCG TCT TTG ATG at-3′
Sense S^sub 2^: 5′-CAT TC (A/C) AAG GCG GAG TGC GAG-3′ Antisense A^sub 2^ : 5′-(A/G) TC (T/C) TT GAT GGA ACT GTC -3′
For reverse transcription and first round of PCR, 5pl of extracted template RNA was added to 25mul of PCR master mix containing 1OmM Tris-HCI, (pH 8.4), 50 mM KCl, 1.5 mM MgCl ^sub 2^, 250 muM dNTPs (Promega, USA), 20 picomoles each of HGV outer primers (S^sub 1^ and a^sub 1^) and Taq DNA polymerase (0.5 units; Promega, USA) and reverse transcriptase (10 units) were added and incubated at 42degC for 1 h. Subsequently, PCR amplification was carried out after inactivating reverse transcriptase at 94 deg C for 5 min and 35 cycles of PCR with 30 sec each of denaturation at 94 deg C, annealing at 50 deg C and extension at 72 deg C. Finally, in the last cycle, the extension was prolonged for 8 min. The second round of PCR was performed by using 5(mu)l of first PCR product as a template and amplified with the second PCR mix containing HGV inner primers (S^sub 2^ and A^sub 2^) All compositions and temperature profile were same as that of the first PCR. Appropriate positive and negative controls were run in parallel to the test samples and all safety precautions applicable to RT-PCR procedure were strictly followed.
Detection of PCR products: 15pl of amplified products obtained after the second round of PCR were run on a gel mini using 3 per cent NuSieve agarose (FMC BioProducts, USA) containing ethidium bromide. An expected PCR amplimer of 101 bp was identified under UV transilluminator by using HaeIII-digested OX 174 DNA molecular weight marker (Promega, USA) (Fig.).
Serum RT-PCR for HCV RNA : The HCV RNA PCR was performed in serum samples found to be non-B, non-C by serology on lines similar to the procedure of HGV PCR. The sequences of HCV primers used were as follows:
#46 (+): 5′-CTG TGA GGA ACT ACT GCT T-3′
#317(-): 5′-GTG CTC ATG GTG CAC GGT CTA CGA GAC CTC CGG-3′
#64(+): 5′-TrC ACG CAG AAA GCG TCT AG-3′
#287(-): 5′-CAC TCG CAA GCA CCC TAT CAG GCA TGC A-3′
The final amplified PCR product was detected as a 251 bp fragment on 3 per cent NuSieve agarose gel electrophoresis,
Serum PCR for HBV DNA : The HBV DNA was extracted from serum samples by conventional phenolchloroform method13,14 . For this, an aliquot of 125(mu)l of serum was incubated at 70 deg C for 2 h in presence of 400 mg proteinase k/ml, 2.5 mM disodium EDTA, 25mM sodium acetate and 1 per cent sodium dodecyl sulphate. The suspension was subsequently extracted with phenol and then chloroform: isoamyl alcohol. The DNA was precipitated with 0.3M (final concentration) sodium acetate (pH 5.0) and 2.5 volume of absolute ethanol, washed with 70 per cent ethanol, vacuum dried, and resuspended in 50mul of TE buffer (10 mMTris-HCI+ 1 mM EDTA: pH 8.0). The primer sequences used were as follows
2269(+) : 5′ -GGA GTG TGG ATT CGC ACT-3′
2415(-) : 5′ -TGA GAT CTT CTG CGA CGC -3′
The final amplified PCR product was identified as a 147 bp fragment on an agarose gel electrophoresis.
Statistical analysis The data were analyzed for statistical significance using Chi-square test and Ficher’s exact test.
Results & Discussion
The mean age of cirrhosis patients was 44.34 12.04 yr and the male: female ratio was 4.6: 1. The HGV RNA was detected by RT-PCR in 6 of the 50 (12%) cirrhosis patients. This seems to be considerably lower than that reported by Panda et al15 the other Indian study on HGV, in which only four patients of chronic liver disease were evaluated and one of them (25%) was found to harbour HGV RNA. The HGV positivity of 12 per cent observed in the present study is in good agreement with the reports from other parts of the world in patients of cirrhosis9,16 and hepatocellular carcinoma9,10,17. HGV infection was frequently detected in patients coinfected with other hepatotropic viruses (3/6; 50%) and the most common coinfecting agent was HBV (2/3) followed by HCV (1/3). This is consistent with the widely accepted view that HGV is frequently associated with HBV and/or HCV infection(s). Furthermore, HGV was detected-in three out of thirteen (23%) non-B, non-C patients. This observation is also comparable with the reported presence of HGV genome in 10-20 per cent of cryptogenic (non-B, non-C) liver disease patients3,16,18,19
On serological testing HBsAg was detected in 20 (40%) patients, IgG anti-HBc was present in 29 (58%) and HBeAg in 13 (26%) patients. The overall percentage of cirrhotic subjects with one or more serological markers suggestive of exposure to HBV was 62 per cent. In contrast, anti-HCV antibodies were present in 15 (30%) patients only. No serological markers could be found in 13 (26%) patients, but evidence of infection with both HBV and HCV was seen in 9 (18%) patients. These findings are in good agreement with the earlier reports from India 21-22 and indicate that HBV is the principal hepatotropic virus responsible for majority of chronic liver diseases in India. The occurrence of HGV coinfection among patients of HBV related cirrhosis was 6.5 per cent (2/31). Similarly, HGV coinfection in anti-HCV positive patients was 6.7 per cent (1/15). This correlates well with the findings reported from other parts of the world9,18,23,24.
The various biological attributes of HGV-positive and HGV-negative patients are presented in the Table. On statistical analysis, no significant difference was observed in the age and sex distribution, symptomatology, physical signs, biochemical parameters or liver function profile of the two groups. Three risk factors, viz., blood transfusion, surgery and tattooing appeared to be more commonly associated with HGV infection, but the difference was statistically significant only with respect to blood transfusion (P
The prevalence of HGV in the normal population of India has not been reported so far. We found detectable hepatitis G viraemia in two of the 50 (4%) healthy blood donors studied. The prevalence of HGV in voluntary blood donors is known to vary considerably in different geographical regions, ranging from 0.33 per cent in China2 to 10.9 per cent in Uzbekistan26. Many factors such as ethnic, geographic or demographic characteristics of the population or the sequence of the primers selected for amplification may be responsible for the wide variations observed among different studies.
Though the presence of HGV RNA in patients of cirrhosis was apparently higher than that of the control population of healthy blood donors, the difference was statistically not significant. This is indicative of the fact that-the presence of HGV RNA in cirrhosis patients may be a mere reflection of high level of prevalence of the virus in the general population of India. This observation is significant as it may imply that HGV is unlikely to have any pathogenic significance. Essentially similar results have been reported from other geographical regions of the world in patients of chronic liver disease 27 and hepatocellular carcinoma 10.
It is therefore, concluded that the hepatitis G virus is commonly observed in the patients with cirrhosis. Its frequent association with the transfusion of blood and / or blood products is suggestive of a parenteral route of transmission. This study also suggests that the etiologic role of HGV in the causation of hepatic cirrhosis is probably insignificant but this observation needs to be confirmed in larger studies involving a longer followup period.
The authors thank Dr Gert Frosner of Max von Pettenkofer– Institute, Germany for giving information on HGV primer sequences and Dr Kendo Kiyosawa, Japan for the gift of HGV positive control. This work was partly supported by a financial grant from the Indian Council of Medical Research, New Delhi.
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Anil Jain, P. Kar, V Gopalkrishna% R Gangwal, S. Katiyar* & B.C. Das*
Department of Medicine, Maulana Azad Medical College & *Division of Molecular Oncology Institute of Cytology & Preventive Oncology (ICMR), New Delhi
Reprint requests: Dr P. Kar, Professor of Medicine, Maulana Azad Medical College& Associate L.N. Hospital Bahadur Shah Zafar Marg, New Delhi 110002
Copyright Indian Council of Medical Research Aug 1999
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