J Med Virol

Prospective study of mother-to-infant transmission of hepatitis C virus (HCV) infection. Study Group for Vertical Transmission.

Mazza C. Ravaggi A. Rodella A. Padula D. Duse M. Lomini M. Puoti M. Rossini A. Cariani E.
III Laboratory of Clinical Chemistry, Hospital of Brescia, Italy.
Seventy-five women with anti-hepatitis C virus (HCV) antibody were enrolled prospectively during pregnancy or at delivery for study of mother-to-child transmission of HCV. Twenty-three women were coinfected with the human immunodeficiency virus (HIV). Seventy babies were monitored for at least 6 months. HCV infection was diagnosed in six infants (8.6%), four of whom were born to anti-HIV-positive mothers. HCV RNA was first detected between 2 and 6 months, and the genotypes of infected babies matched those of their mothers (type 1: n = 4; type 3: n = 2). Identical master sequences of the hypervariable region (HVR1) were detected in a mother-infant pair. In three babies coinfected with HCV and HIV, anti-HCV disappeared between 2 and 7 months, being persistently negative in two cases monitored for 11 and 26 months. Transmitting mothers did not differ significantly from those who did not transmit the infection with anti-HIV, HCV genotypes, and viral load at delivery, but had lower rate of reactivity to C100 by the recombinant immunoblot assay (RIBA) (P < .01). This prospective study confirms transmission of HCV from anti-HIV-negative mothers (4.4% in this series). Absence of anti-C100 antibodies at delivery is apparently related to increased risk of vertical transmission. Seronegative HCV infection can be observed in children coinfected with HIV.

Characteristics of hepatitis C virus before and after interferon treatment in patients with ongoing viraemia but sustained biochemical response.

Donadel E. Pontisso P. Ruvoletto MG. Gerotto M. De Salvo G. Chemello L. Casarin C. Alberti A.
Dipartimento di Medicina Clinica e Sperimentale, Universita di Padova, Italy.
In hepatitis C virus (HCV) infection, persistent viraemia can occur after successful biochemical response to interferon treatment. To assess whether this unusual profile might be due to trivial amounts of remaining virus or to the emergence of less pathogenic HCV strains, pre- and posttreatment sera from 27 patients who remained with HCV-RNA, despite sustained transaminase normalisation after interferon therapy, were investigated. All but one had infection by genotype 2 (P < 0.0001), and levels of HCV-RNA were not decreased after therapy. Sequence comparison of the 5' untranslated region revealed mixed viral populations and "not compensatory" nucleotide transitions localised at the stem level of the secondary structure of this region in samples taken before and after treatment. Neither quantitative nor qualitative viral changes, at least for the 5' untranslated region, are responsible for interferon-induced biochemical remission in these patients typically infected by genotype 2.

Prevalence of antibodies to hepatitis E virus among hemodialysis patients in Sweden.

Sylvan SP. Jacobson SH. Christenson B.
Department of Communicable Disease Control and Prevention, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden.
In order to study the prevalence of antibody to hepatitis E virus (HEV) among hemodialysis patients and to evaluate whether chronic hemodialysis is associated with an increased risk of exposure to HEV in developed countries, the IgG anti-HEV was determined in serum samples obtained from 182 patients on chronic hemodialysis and 349 statistically selected, healthy Swedish control subjects. Serum specimens from 11 of the 182 (6.0%) hemodialysis patients and from 18 of the 349 (5.2%) control subjects were repeatedly positive for HEV antibodies (the difference was not significant: P = .67). Analysis of serial serum samples obtained at the initiation of hemodialysis and consecutively during follow-up periods of several years demonstrated no IgG anti-HEV seroconversion during chronic hemodialysis. The seroprevalence of anti-HEV antibody in the adult Swedish population was associated significantly with age. In persons younger than 40 years, the percentage of seropositive individuals was 2.5%, whereas the seroprevalence rate of anti-HEV was 7.4% in subjects older than 40 years (P < .05). This study indicates that nosocomial transmission of HEV to patients on maintenance hemodialysis was non-existent in three dialysis centers in Sweden (a developed country) and that chronic hemodialysis is not associated with an increased risk of exposure to HEV infection in this region.

GB virus C (GBV-C) infection in patients with chronic hepatitis C. Influence on liver disease and on hepatitis virus behaviour: effect of interferon alfa therapy.

Pawlotsky JM. Roudot-Thoraval F. Muerhoff AS. Pellerin M. Germanidis G. Desai SM. Bastie A. Darthuy F. Remire J. Zafrani ES. Soussy CJ. Mushahwar IK. Dhumeaux D.
Department of Bacteriology and Virology, Hopital Henri Mondor, Universite Paris XII, Creteil, France. pawlotsky@univ-paris12.fr
The aim of this study was to evaluate, in patients with chronic hepatitis C, 1) the prevalence and the epidemiological characteristics of GB virus C (GBV-C) infection, 2) the influence of GBV-C on hepatitis C virus (HCV) infection, 3) the pathogenicity of GBV-C in the absence of treatment and under interferon therapy, and 4) the effect of interferon alfa on GBV-C and HCV replications. One hundred fifteen patients with chronic hepatitis C were studied. Before treatment, they were tested for GBV-C RNA by PCR and GBV-C genotype was determined for positive samples. Pretreatment information was collected, including age, gender, source of HCV, estimated duration of HCV infection, alanine aminotransferase and gamma-glutamyl transpeptidase activities, cirrhosis and Knodell's score on liver biopsy, HCV genotype, HCV viral burden and anti-HCV core IgM antibodies. The genetic complexity of the hypervariable region 1 (HVR1) of HCV was studied by PCR-Single Strand Conformation Polymorphism. All patients were treated with 3 to 9 mega units of interferon alfa-2a three times per week for 3 to 6 months. The influence of GBV-C on the evolution of ALT and HCV replication during and after treatment was studied, and GBV-C and HCV RNA were monitored monthly by PCR during this period. Eighteen patients (16%) were GBV-C RNA-positive. Among 11 samples studied, GBV-C genotype 2a was present in 9 cases, 2b in one case and type 3 in one case. GBV-C RNA-positive patients were significantly younger than GBV-C RNA-negative ones (38.4 +/- 11.5 vs. 47.4 +/- 14.0, P = 0.012), a result independent of the route of transmission and the disease duration. No difference between GBV-C RNA-positive and -negative patients was found for other epidemiological parameters (e.g. gender, risk factor for parenteral viral infections, disease duration and HCV genotypes), or for the characteristics of HCV infection and related liver disease (e.g. HCV RNA level, genetic complexity of the HVR1, anti-HCV core IgM, alanine aminotransferase and gamma-glutamyl transpeptidase activities, cirrhosis and Knodell's score). GBV-C did not influence the rates of ALT normalization at months 3, 6 and 12 and of sustained hepatitis C virological response at month 12 of treatment follow-up. During treatment, GBV-C viremia became undetectable in 12 patients (67%) but relapse occurred after treatment withdrawal in all the nine patients with sufficient follow-up. In the remaining six patients (33%), GBV-C resisted interferon. Whatever the effect of interferon on GBV-C replication, the ALT levels correlated with the presence of HCV RNA. In conclusion, GBV-C infection is frequent in patients with chronic hepatitis C, who are mainly, but not exclusively, infected by GBV-C genotype 2a. GBV-C positive patients are significantly younger than GBV-C negative ones. GBV-C does not seem to affect HCV replication, liver disease and responses of HCV infection and liver disease to interferon therapy. GBV-C is sensitive to 3 mega units of interferon alfa administered three times per week in two-thirds of the patients, but relapse is constant with this dosage after treatment withdrawal.

High-grade dysplasia in genital warts from two patients infected with the human immunodeficiency virus.

Bryan JT. Stoler MH. Tyring SK. McClowry T. Fife KH. Brown DR.
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-5124, USA.
Cancer-associated human papillomavirus (HPV) types are detected in genital warts removed from immunosuppressed individuals more commonly than from those occurring in otherwise healthy individuals. The prognosis of genital warts containing cancer-associated HPV types is not known. Because it is assumed that genital warts are benign lesions, they are usually treated by destructive therapies without prior knowledge of histopathology. The aim of the present study was to determine whether genital warts from individuals with or without human immunodeficiency virus (HIV) contain high-risk HPV types or areas of dysplasia. The study design was a nonrandomized analysis of genital warts removed by excision biopsy from 15 HIV-infected patients and 15 HIV-negative patients. The tissue was analyzed for HPV DNA by hybrid capture, and microscopic sections of each biopsy were examined for areas of dysplasia. Genital warts from HIV-infected patients contained cancer-associated ("high risk") HPV types in 9 of 15 cases, including 1 that contained only a high-risk type. High-grade dysplastic abnormalities were present in 2 of the 15 lesions from this group, both of which contained high-risk HPV types. Four genital warts removed from HIV-negative patients contained high-risk HPV types, but none contained dysplastic abnormalities. It is concluded that genital warts from HIV-infected patients often contain high-risk HPV types. Such lesions may exhibit dysplastic changes. The frequency of dysplastic changes in genital warts from HIV-infected patients is not known. Biopsy of genital warts may be indicated prior to additional therapy in HIV-infected patients, and surgical removal should be considered as a preferred treatment option in these patients.

GBV-C/HGV infection in hepatitis C virus-infected deferred Swedish blood donors.

Year 1998
Shev S. Bjorkman P. Norkrans G. Foberg U. Fryden A. Lindh G. Lindholm A. Weiland O. Widell A.
Department of Infectious Diseases, Sahlgrenska University Hospital/Ostra, Goteborg, Sweden. steven.shev@medfak.gu.se
Sera from 62 hepatitis C virus (HCV)-infected Swedish blood donors were tested by a nested polymerase chain reaction using primers targeting the 5'-noncoding region of the GB virus-C/hepatitis G (GBV-C/HGV) genome and an enzyme-linked immunosorbent assay that detects antibodies to the envelope protein E2 of GBV-C/HGV (anti-E2). Fourteen (22%) and 21 (34%) of the 62 blood donors were found to be GBV-C/HGV RNA and anti-E2 positive, respectively. None of the blood donors was positive for both GBV-C/HGV RNA and anti-E2. Thus, 35 of 62 (56%) HCV-infected donors had been exposed to GBV-C/HGV infection. At sequencing of the 14 GBV-C/HGV isolates, 12 were identified as subtype 2a and 2 as subtype 2b. One of 7 (14%) donors with mild liver disease such as steatosis and nonspecific reactive hepatitis had been exposed to GBV-C/HGV vs. 34 of 55 (62%) with chronic hepatitis with or without cirrhosis (P = 0.04). All other differences in histology were small between HCV and dual HCV GBV-C/HGV-infected donors. In conclusion, more than half of HCV-infected Swedish blood donors in this study were positive for either GBV-C/HGV RNA or anti-E2. GBV-C/HGV viremia and seropositivity were mutually exclusive.

Quasispecies nature of hepatitis C virus (HCV) in patients with chronic hepatitis C with mixed HCV subtypes.

Year 1998
Toyoda H. Fukuda Y. Nakano I. Katano Y. Takayama T. Kumada T. Nakano S. Takamatsu J. Saito H. Hayakawa T.
Second Department of Internal Medicine, Nagoya University School of Medicine, Japan.
The quasispecies nature of hepatitis C virus (HCV) in patients with mixed HCV subtype infection was compared with that in patients with single HCV subtype infection. The number of HCV quasispecies was compared between 35 patients with mixed HCV subtype infection and 83 patients with single subtype infection. Subtype was determined by primers deduced from the core region and by line probe assay respectively. The number of quasispecies was evaluated by polymerase chain reaction amplification of hypervariable region 1 and by fluorescence single-strand conformation polymorphism analysis. There was no difference in clinical background between patients with mixed subtype infection and patients with single subtype infection. The number of quasispecies in patients with multiple subtype HCV infection was larger than in patients with single subtype HCV infection. The immunologic environments which allow the coexistence of more HCV quasispecies in patients with multiple HCV subtype infection differs from that in patients with single HCV subtype infection.

Baseline level and early suppression of serum HCV RNA for predicting sustained complete response to alpha-interferon therapy.

Year 1998
Izopet J. Payen JL. Alric L. Sandres K. Charlet JP. Vinel JP. Duffaut M. Pascal JP. Puel J.
Laboratoire de Virologie, CHU purpan, Toulouse, France. izopet@cict.fr
The relationship between serum hepatitis C virus (HCV) RNA and the outcome of alpha-interferon (alpha-IFN) therapy in patients with chronic hepatitis C has important implications for therapeutic research and clinical care. Serum HCV RNA was tested for HCV genotype and quantified by a standardized reverse transcriptase-polymerase chain reaction assay as a measure of viral load in a cohort of 130 patients with chronic hepatitis C treated with alpha-IFN at a standard dose of 3 million units three times a week scheduled for 6 (n = 50) or 12 months (n = 76). Twenty-one of 126 evaluable patients (16.7%) developed a sustained complete response to alpha-IFN according to biochemical and virological criteria. The 3 pretreatment independent factors associated with a sustained complete response were a low baseline serum HCV RNA concentration, non-1 HCV genotype, and female sex. A multivariate logistic regression model, with pretreatment and month 1 variables, showed that a lower baseline serum HCV RNA concentration, female sex, and a greater suppression of RNA were the significant predictors of sustained complete response. The lowest baseline serum HCV RNA concentration was observed in patients with genotype 2 infection and the greatest decrease in HCV RNA from baseline to month 1 in those with genotype 3. The findings suggest that measuring HCV RNA in serum before and soon after beginning treatment can be helpful for selecting patients who are most likely to have a sustained complete response to standard schedule of alpha-IFN and for identifying patients in whom alternative strategies should be examined.

Distinct prevalence of antibodies to the E2 protein of GB virus C/hepatitis G virus in different parts of the world.

Year 1998
Ross RS. Viazov S. Schmitt U. Schmolke S. Tacke M. Ofenloch-Haehnle B. Holtmann M. Muller N. Da Villa G. Yoshida CF. Oliveira JM. Szabo A. Paladi N. Kruppenbacher JP. Philipp T. Roggendorf M.
Institute of Virology, University of Essen, Germany.
Since the identification of the new human virus, GB virus C (GBV-C)/hepatitis G-virus (HGV), in 1995/1996, reverse transcription polymerase chain reaction remained the sole available diagnostic tool for GBV-C/HGV infection. Recently, a serologic test based on the detection of antibodies to the putative envelope protein 2 (anti-E2) has been introduced. We used this assay for a seroepidemiological survey including 3,314 healthy individuals from different parts of the world, 123 patients from Germany who were suspected to have an increased risk of acquiring GBV-C/HGV infection, 128 multiple organ donors, and 90 GBV-C/HGV RNA positive persons. In European countries, anti-E2 seropositivity ranged from 10.9% (Germany) to 15.3% (Austria). In South Africa (20.3%) and Brazil (19.5%), even higher anti-E2 prevalence rates were recorded. In Asian countries like Bhutan (3.9%), Malaysia (6.3%), and the Philippines (2.7%), anti-E2 positivity was significantly lower. GBV-C/HGV anti-E2 prevalence in potential "risk groups," i.e., patients on hemodialysis and renal transplant recipients, did not vary significantly from anti-E2 seroprevalence in German blood donors. Anti-E2 and GBV-C/HGV RNA were found to be mutually exclusive, confirming the notion that anti-E2 has to be considered as a marker of past infection.

Multicenter trial on mother-to-infant transmission of GBV-C virus. The Lombardy Study Group on Vertical/Perinatal Hepatitis Viruses Transmission.

Year 1998
Zanetti AR. Tanzi E. Romano L. Principi N. Zuin G. Minola E. Zapparoli B. Palmieri M. Marini A. Ghisotti D. Friedman P. Hunt J. Laffler T.
Institute of Virology, University of Milan, Italy. zanetti@imiucca.csi.unimi.it
Evidence indicates that the GBV-C or hepatitis G virus can cause persistent infection in humans, but little is known on the importance of vertical transmission. To assess the risk of mother-to-infant transmission and the clinical outcome of infected babies, we investigated 175 anti-HCV positive mothers and followed-up their children for 3-33 months. GBV-C RNA was detected by RT-PCR and anti-E2 antibody was assayed by EIA. Thirty-four (19.4%) women were GBV-C RNA positive and transmission occurred to 21 (61.8%) babies; 20 (95.2%) acquired GBV-C alone, and one (4.8%) GBV-C and HCV. Maternal factors such as intravenous drug use, HIV coinfection, HCV-RNA positivity, and type of feeding were not correlated with GBV-C transmission. GBV-C RNA remained persistently positive in all infected babies but one baby who seroconverted to anti-E2. Seven (35%) babies with GBV-C alone developed marginally elevated ALT; the baby with HCV and GBV-C co-infection had the highest ALT peak value (664 IU/l). Seven of the 141 (5%) babies born to the GBV-C RNA negative mothers acquired HCV and six (85.7%) had abnormal ALT. The mean ALT peak value was significantly higher (P < 0.05) for babies with HCV than for those with GBV-C. None of the children with GBV-C or with HCV became icteric. GBV-C is frequently present in anti-HCV positive women. The infection is transmitted efficiently from mother to baby and rate of transmission is much higher than that for HCV. GBV-C can cause persistent infection in babies but usually without clear evidence of liver disease.

Prevalence of antibodies to the Hawaii strain of human calicivirus as measured by a recombinant protein based immunoassay.

Year 1998
Cubitt WD. Green KY. Payment P.
Department of Virology, Camelia Botnar Laboratories, Hospital for Children, London, United Kingdom. d.cubitt/ich.ucl.ac.uk
The evaluation of an enzyme immunoassay using recombinant Hawaii virus-like particles (rHV-LPs) with a panel of sera which had been screened previously for antibodies to Norwalk virus (NV) and Mexico virus (MxV) is described. The assay was also applied to study the epidemiology of Hawaii virus. Adult volunteers challenged with the prototype (genogroup II, human calicivirus) HV developed significant IgG responses (16-32 fold rises) following challenge whereas adults challenged or naturally infected with NV (genogroup I) did not. Lesser antibody responses (4-8 fold rises) were demonstrated in volunteers challenged with Snow Mountain agent (SMA) and patients infected by SRSV 'UK3' and 'UK4' strains, indicating a degree of antigenic relatedness among viruses within genogroup II. Comparison of the seroprevalence of IgG antibodies to rHV, rMxV and rNV in 338 children in London showed that infections with genogroup II viruses are prevalent and occur earlier in life than NV. Many young children had antibodies to MxV but not HV indicating that genogroup II viruses have both conserved and antigenically distinct epitopes. A serological study on 566 Canadians aged between 9 and 79 years showed that the prevalence of antibodies to rHV rose with age from 65-100% and from 53-100% for NV. Measurement of antibody response in a heart transplant patient infected with an MxV-like virus showed significant responses to both rMxV and rHV. Continuous monitoring of the patient over two years showed that antibody levels declined rapidly to prechallenge levels after a year.

Tumor necrosis factor alpha promoter polymorphism at position -238 is associated with chronic active hepatitis C infection.

Year 1998
Hohler T. Kruger A. Gerken G. Schneider PM. Meyer zum Buschenfelde KH. Rittner C.
I. Department of Internal Medicine, Johannes Gutenberg University, Mainz, Germany. hoehler@goofy.zdv.uni-mainz.de
Tumor necrosis factor alpha (TNF-alpha) is involved in the pathogenesis of chronic hepatitis C virus infection. The gene for TNF-alpha is encoded in the major histocompatibility locus (MHC). Two polymorphisms at positions -308 and -238 in the TNF-alpha promoter region might influence TNF-alpha expression. These promoter polymorphisms have been linked previously to a number of infectious diseases. TNF-alpha promoter polymorphisms at positions -238 and -308 were studied by DNA sequencing and sequence-specific oligonucleotide hybridization in 82 individuals with chronic hepatitis C and 99 control subjects. Subjects had been HLA class I and class II typed in a previous study. The frequency of the TNF238.2 promoter allele was significantly higher in the hepatitis C group (18.7%) compared to the controls (3.5%; P < 0.0001; pcorr < 0.009). No significant differences in the frequency of the TNF308.2 allele were observed between patients and controls. The increased frequency of the TNF238.2 allele could not be explained by linkage disequilibrium to HLA-B or -DR genes. These findings show an association between the TNF238.2 promoter variant and chronic active hepatitis C. They suggest that this polymorphism or a linked gene may be a host factor contributing to the development of chronic active hepatitis C.

Association of hepatitis E virus with an outbreak of hepatitis at a military training camp in Nepal.

Year 1998
Clayson ET. Vaughn DW. Innis BL. Shrestha MP. Pandey R. Malla DB.
Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.
From 29 January 1995 to 15 March 1995, an outbreak of hepatitis occurred among 692 soldiers at an isolated training camp 25 km east of Kathmandu. Thirty-two cases occurred approximately 8 weeks after arrival of soldiers at the camp. To determine the etiology of the outbreak, patient sera were examined for evidence of infection with hepatitis A, B, C, and E viruses using commercially available enzyme-linked immunosorbent assay (ELISA) kits. The polymerase chain reaction (PCR) was used to detect hepatitis E virus (HEV) RNA. Evidence of recent infection (IgM to HEV and/or HEV RNA) was found in all but two patients, whereas none had evidence of recent infection with hepatitis A, B, or C viruses. Therefore, the outbreak was attributed to HEV. Fecally contaminated drinking water was suspected as the source of the outbreak. To determine the extent of HEV infections among those without clinical hepatitis, sera from the remaining soldiers were examined for markers of HEV infection. Evidence of past infection (IgG to HEV in the absence of IgM or HEV RNA) was found among 204 soldiers (prevalence = 30%), leaving 488 individuals susceptible to infection at the onset of the outbreak. Evidence of recent infection was found among another 83 individuals. We conclude that most exposed, susceptible soldiers sustained HEV infection without experiencing overt hepatitis. If the levels of virus inoculum and prior immunity in this population were typical, inapparent infection may be the usual adult response to virus exposure in an endemic area.

Epidemiological study and genetic analysis of GB virus C infection in general population from an area endemic for hepatitis C.

Year 1998
Zhang X. Shinzawa H. Shao L. Ishibashi M. Jiang Q. Saito K. Misawa H. Togashi H. Takahashi T.
Second Department of Internal Medicine, Yamagata University School of Medicine, Japan.
The aim of this work was to study the prevalence, potential risk factors, clinical and laboratory features of GB virus C (GBV-C) infection in general population from an area endemic for hepatitis C. A reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of GBV-C RNA was used to examine the prevalence of GBV-C RNA in both hepatitis C virus (HCV) endemic (R town) and nonendemic areas (M town) in Yamagata prefecture, Japan. In R town, GBV-C RNA was detected in 23 (2.9%) out of the 800 residents, whereas anti-HCV and HCV-RNA were found in 226 (28.3%) and 163 (20.4%), respectively. The prevalence of GBV-C RNA in R town (2.9%) was higher than that in M town (1.0%), although the difference was not statistically significant. The individuals with anti-HCV had significantly higher frequency of active GBV-C-infection than those without anti-HCV in both towns. No evidence indicating that GBV-C infection affected the severity of hepatitis C was obtained. The multivariate analysis revealed that the young anti-HCV positive individuals with a history of blood transfusion had higher incidence of active GBV-C infection. The phylogenetic analysis showed that the GBV-C isolates from both R and M towns were divided into two separate branch groups designated HG and Asia GB groups.

Prevalence of hepatitis B, hepatitis C, and GB virus C/hepatitis G virus infections in liver disease patients and inhabitants in Ho Chi Minh, Vietnam.

Year 1998
Kakumu S. Sato K. Morishita T. Trinh KA. Nguyen HB. Banh VD. Do HC. Nguyen HP. Nguyen VT. Le TT. Yamamoto N. Nakao H. Isomura S.
First Department of Internal Medicine, Aichi Medical University, Japan.
The prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), and GB virus C or hepatitis G virus (GBV-C/HGV) infections was determined in 289 patients with liver disease in Ho Chi Minh City and 890 healthy inhabitants of its rural area, Dalat City, Vietnam, respectively. Serum HCV RNA and GBV-C/HGV RNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). HBsAg, HCV antibodies, and GBV-C/HGV RNA were detected in 139 (47%), 69 (23%), and ten (3%) subjects, respectively, often accompanied by elevated serum levels of alanine aminotransferase. HBsAg and HCV antibodies or HCV antibodies and GBV-C/HGV RNA were detectable simultaneously in 8% and 2% of the patients, respectively. In the inhabitants, HBsAg, HCV antibodies, and GBV-C/HGV RNA were found in 51 (5.7%), nine (1.0%), and 11 (1.2%) subjects, respectively. Thus, the prevalence of HBsAg, HCV antibodies, and GBV-C/HGV RNA was significantly higher in liver disease patients than those in the general population. In the samples from 69 patients and nine inhabitants who were seropositive for HCV antibodies, HCV RNA was detectable in 42 (61%) and 4 (44%), respectively. In patients with liver disease, ten belonged to HCV genotype 1a, ten to HCV 1b, three to HCV 2a, four to HCV 2b, and two to HCV 3a by PCR with genotype-specific primers. Nine patients had mixed genotypes, and the remaining four were not classified. Of the GBV-C/HGV RNA-positive individuals, two patients and two inhabitants were positive for HBsAg, while none of the residents had HCV antibodies, although six HCV antibodies (60%) and four HCV RNA (40%) were found in patients. When a phylogenetic tree of GBV-C/HGV was constructed based on the nucleotide sequences, the 21 isolates were classified into at least two genotypes; four isolates belonged to G2, and 17 to G3. The results indicate that in Ho Chi Minh HCV infection prevails with broad distribution of genotypes together with HBV infection among patients with liver disease. This study suggests that GBV-C/HGV infection occurs independently in the two different districts in association with HCV infection.

Hepatitis B virus DNA is frequently found in liver biopsy samples from hepatitis C virus-infected chronic hepatitis patients.

Year 1998
Koike K. Kobayashi M. Gondo M. Hayashi I. Osuga T. Takada S.
Department of Gene Research, Cancer Institute (JFCR), Tokyo, Japan.
Human hepatitis B virus (HBV) and hepatitis C virus (HCV) are two major etiologic agents of chronic hepatitis, which is closely related to the development of hepatocellular carcinoma (HCC). A possible involvement of HBV co-infection was investigated in ongoing HCV-related liver diseases in HCV-infected patients. A prevalence of anti-HBc in anti-HCV-positive/HBsAg-negative chronic hepatitis patients and a low copy number of HBV DNA were found in most of the liver biopsy samples of anti-HCV-positive/HBsAg-negative patients. The present data suggest that HBV co-infects frequently with HCV and may play an important role in the development of HCC in the anti-HCV-positive/HBsAg-negative patients with chronic hepatitis.

Genetic complexity of the hypervariable region 1 (HVR1) of hepatitis C virus (HCV): influence on the characteristics of the infection and responses to interferon alfa therapy in patients with chronic hepatitis C.

Year 1998
Pawlotsky JM. Pellerin M. Bouvier M. Roudot-Thoraval F. Germanidis G. Bastie A. Darthuy F. Remire J. Soussy CJ. Dhumeaux D.
Department of Bacteriology and Virology, Hopital Henri Mondor, Universite Paris XII, Creteil, France.
HCV exists within its host as pools of related genetic variants referred to as quasispecies. The hypervariable region 1 (HVR1) of the E2 envelope gene is subjected to strong selective pressure from neutralizing antibodies. The genetic complexity of this region is defined as the total number of genetic variants within the quasispecies population. The genetic complexity of the HVR1 region was examined in patients with chronic hepatitis C and its relationship with the epidemiology of HCV infection, and its influence on liver disease and the response to interferon treatment were determined in 114 patients with chronic hepatitis C. The genetic complexity of the HVR1 major variants was measured before treatment by using a polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) technique, and was compared with epidemiological, clinical, virological and histological features. The patients were treated with 3 megaunits of interferon (IFN) alfa for 3 to 6 months and the response to treatment was assessed at 3, 6 and 12 months. The HVR1 could be studied in 101 of the 114 patients (89%). Genetic complexity was significantly higher in patients infected through blood transfusion than intravenous drug use (mean complexity index: 5.7 +/- 2.3 vs. 4.7 +/- 1.5, respectively; P = 0.04). This relationship was independent of age and the estimated time since infection. No significant relationship was found with other parameters of infection or liver disease. In univariate analysis, the genetic complexity of HVR1 major variants did not affect the rates of ALT normalization at months 3 and 6 of IFN treatment. HVR1 genetic complexity was lower in patients with a sustained virological response than in non-responders (4.0 +/- 1.7 vs. 5.4 +/- 2.0, respectively; P = 0.07). In multivariate analysis of pretreatment parameters associated with a sustained virological response to treatment, three parameters appeared to be independent predictors of such a response: a low viral load (P < 0.04), a low anti-HCV core IgM titer (P = 0.03) and a low genetic complexity of HVR1 major variants (P < 0.04). In conclusion, the HVR1 of HCV has a quasispecies distribution in infected individuals. Its genetic complexity is significantly higher in transfusion recipients than in intravenous drug users, suggesting that the size of the initial inoculum affects the later emergence and development of viral quasispecies. The genetic complexity of HVR1, together with viral load and the anti-HCV IgM titer, are independent predictors of a sustained virological response to IFN alfa in patients with chronic hepatitis.

Predominance of HCV type 2a in saliva from intravenous drug users.

Year 1998
Roy KM. Bagg J. McCarron B. Good T. Cameron S. Pithie A.
University of Glasgow Dental School, Scotland.
Paired serum and saliva samples were collected simultaneously from 50 intravenous drug users with serologically proven hepatitis C virus infection. The oral health of the volunteers was also assessed. Hepatitis C virus RNA was detected by nested PCR, employing primers from the 5' noncoding region. Positive PCR products were sequenced using the Sequenase PCR Product Sequencing Kit (Amersham Life Sciences). HCV RNA was detected in 33 (66%) of the 50 serum samples. HCV RNA was detected in 19 (57.6%) of the corresponding 33 saliva samples. There was no correlation between oral health status or HIV seropositivity and the detection of HCV in saliva. However, subjects with HCV in their saliva were significantly more likely to complain of xerostomia (P < 0.05). Isolate genotypes were identified in paired serum and saliva of 15 intravenous drug users. HCV genotypes 1, 2, 3 and 6 were detected in both specimens. In seven cases, a differing HCV genotype was found in serum compared to the paired saliva specimen. The distributions of genotypes in serum and saliva were very different, with genotype 2a more common in saliva than serum (P < 0.005). These data suggest that in some cases the source of salivary HCV may not be serum transudation along the periodontal membrane or across damaged mucosa, and that an alternative local source, possibly the salivary glands themselves, should be considered.

Homotypic and heterotypic IgG and IgM antibody responses in adults infected with small round structured viruses.

Year 1998
Hale AD. Lewis DC. Jiang X. Brown DW.
Enteric and Respiratory Virus Laboratory, Central Public Health Laboratory, London, England.
Antibody responses to recombinant Norwalk (rNV) and Mexico (rMXV) viral capsid proteins were studied in 39 adults involved in outbreaks of gastroenteritis associated with genogroup 2 small round structured viruses (SRSVs). Nineteen individuals were involved in outbreaks associated with MXV-like strains and 20 in outbreaks associated with four other genogroup 2 SRSVs. IgG antibodies were measured in acute and convalescent sera using indirect enzyme-linked immunosorbent assay (ELISA), and IgM was measured by indirect and capture ELISAs. Nineteen (49%) patients demonstrated a significant rise in IgG to rMXV with four (10%) patients also showing anamnestic responses to rNV. Fourteen patients were positive in the rMXV IgM-capture ELISA, representing 74% of patients demonstrating IgG rises. IgG and IgM responses to rMXV were observed in both groups, although higher levels of responses were seen in adults infected with MXV-like strains than those infected with non-MXV genogroup 2 viruses. No significant IgM responses were observed to rNV. These results indicate that, following SRSV infection, adults show a rise in antibody which is broadly reactive to viruses within but not between genogroups, although greater homotypic than heterotypic responses are produced. These findings have implications for interpretation of seroepidemiological studies and serodiagnosis of SRSV infections using recombinant capsids.

Production of cytokines in patients infected by hepatitis C virus.

Year 1998
Cribier B. Schmitt C. Rey D. Lang JM. Kirn A. Stoll-Keller F.
INSERM U 74, Strasbourg, France.
T helper type 1 (Th1) cytokines play an important role in antiviral defence. The purpose of this study was to quantify by ELISA IL2, soluble receptor of IL2 (IL2Rs), IFNgamma TNFbeta, IL4, IL6 and IL10 levels in the sera of 134 HCV-positive patients, 69 of whom were coinfected with HIV, and in 54 HIV-HCV-negative patients. The mean IL2Rs and IFN serum levels were much higher in patients with anti-HCV than in the control group, whereas the mean IL4 and IL6 levels were lower in patients infected with HCV. There were no significant differences in cytokine levels between patients with and without HIV. There were significantly less patients with HCV than controls with IFNgamma levels under cut-off, and significantly more patients with HCV with IL4 levels under cut-off. Although serum level of cytokines must be interpreted with caution, the results suggest that Th1 response is enhanced in HCV infection.

Evidence for high genetic diversity and long-term endemicity of hepatitis C virus genotypes 1 and 2 in West Africa.

Year 1998
Jeannel D. Fretz C. Traore Y. Kohdjo N. Bigot A. Pe Gamy E. Jourdan G. Kourouma K. Maertens G. Fumoux F. Fournel JJ. Stuyver L.
ICRF Cancer Epidemiology Unit, University of Oxford, The Radcliffe Infirmary, England.
During 1994 and 1995, the prevalence of hepatitis C virus (HCV) and its genotypes were studied in several rural and urban populations in three West African countries: Guinea, Burkina Faso, and Benin. The following groups were screened for antibodies to HCV (anti-HCV): 459 villagers in the forest region of Guinea; 965 individuals in urban, suburban, and rural populations of the Bobo Dioulasso area, Burkina Faso; and 582 blood donors in Cotonou, Benin. In Benin, 60 patients with sickle cell anemia (30 with and 30 without history of multiple transfusion) and 13 hospital patients with liver disease were also tested. RT-PCR detection of HCV-RNA was carried out on all anti-HCV positive samples, followed by genotyping and sequencing of unrecognized subtypes. The prevalence rates of anti-HCV were 1.1% in the Guinean population group, 1.4% among blood donors in Benin, and 4.9% in residents of Burkina Faso. In patients with sickle cell anemia, five of the 30 polytranfused patients (17%) had anti-HCV, whereas none of the patients without a history of blood transfusion had anti-HCV (P < 0.05). Among the 13 patients with liver disease, five had anti-HCV, of whom four had history of blood transfusion. HCV-RNA was detected in 41 anti-HCV positive sera. All belonged to genotypes 1 or 2, with a high genomic diversity; 18 different subtypes were identified, including 2c, 2d, and 16 new subtypes. Such genetic diversity poses a challenge for vaccine development and also implies that HCV infection is long-established in these West African regions.

GBV-C/HGV infection in chronic hepatitis C patients: its effect on clinical features and interferon therapy.

Year 1998
Oshita M. Hayashi N. Mita E. Iio S. Hiramatsu N. Hijioka T. Kato M. Masuzawa M. Sasaki Y. Kasahara A. Hori M.
First Department of Medicine, Osaka University School of Medicine, Suita, Japan.
A novel virus (GBV-C/HGV) may be associated with some liver diseases including fulminant hepatitis and acute and chronic hepatitis. On the other hand, many investigations showed that this infection does not contribute to liver disease. GBV-C/HGV has been found to occur in association with infection with other hepatitis viruses. We investigated the effect of GBV-C/HGV infection on the clinical features and interferon treatment in patients with chronic hepatitis C. A total of 262 hepatitis C virus (HCV) RNA positive patients with chronic hepatitis were examined in this study. The detection of serum GBV-C/HGV RNA was done by RT-PCR using specific primers from the NS5 regions. Interferon-alpha was given at a dose of 6 MU/day for 16 or 24 weeks. A responder was defined as a patient with ALT normalization and HCV RNA disappearance after treatment. GBV-C/HGV RNA was detected in 28 (11%) patients. No significant difference was detected in clinical features (age, sex, liver-related biochemical tests, and histological examination) between the 28 GBV-C/HGV-positive patients and the GBV-C/HGV-negative patients. Using interferon therapy for hepatitis C, the responder rates of GBV-C/HGV-positive and -negative patients were 14% and 20%, respectively. Of the 28 patients with GBV-C/HGV RNA, GBV-C/HGV RNA was tested after interferon therapy in 16 and of these GBV-C/HGV RNA was not detected in nine patients after therapy. These findings suggest that GBV-C/HGV infection dose not affect the clinical features in patients with HCV and the efficacy of interferon therapy for chronic hepatitis C.

GB virus C prevalence in blood donors and high risk groups for parenterally transmitted agents from Gauteng, South Africa.

Year 1998
Casteling A. Song E. Sim J. Blaauw D. Heyns A. Schweizer R. Margolius L. Kuun E. Field S. Schoub B. Vardas E.
National Institute for Virology, University of the Witwatersrand, Department of Virology, Sandringham, South Africa.
The prevalence of GBV-C infection in voluntary blood donors and in groups at high risk for parenteral exposure to infectious agents was studied. The high risk groups included chronic renal failure patients on haemodialysis, renal transplant patients and haemophiliacs from Gauteng. The presence of GBV-C RNA in these populations was determined using reverse transcription polymerase chain reaction (RT-PCR) in the 5' non-coding region (NCR) of the virus. Of the blood donors, 11.1% (95% CI 7.6, 15.8) were positive, whereas 23.8% (95% CI 12.6, 40.2) of haemodialysis patients and 23.5% (95% CI 15.9, 33.3) of the haemophiliacs were infected with GBV-C. The highest proportion of infection was in the renal transplant patients, where 41.2% (95% CI 35.1, 47.7) were found to have circulating GBV-C RNA. Serological markers for hepatitis B (HBV) and hepatitis C viruses (HCV) were also measured as indicators of other hepatitis viruses with important parenteral transmission routes. Of the GBV-C positive blood donors, 3.6% were also HBsAg positive and none were positive for HCV. The GBV-C positive patients on haemodialysis were not positive for either HBsAg or antibodies to HCV, but had evidence of past infection with HBV since 40% were anti-HBc positive. The greatest proportion of HCV positives was in the haemophiliac group, 91.3%, none of these were HBsAg positive but 39.1% had anti-HBc. In the GBV-C positive renal transplant patients, 4% had HBsAg, 13.3% had anti-HBc and 2.1% had antibodies to HCV. This is the first report describing the prevalence of GBV-C in South African populations.

Heterogeneity in E2 region of GBV-C/hepatitis G virus and hepatitis C virus.

Year 1998
Kato T. Mizokami M. Nakano T. Orito E. Ohba K. Kondo Y. Tanaka Y. Ueda R. Mukaide M. Fujita K. Yasuda K. Iino S.
Second Department of Medicine, Nagoya City University Medical School, Mizuho, Nagoya, Japan.
GB virus C/hepatitis G virus (GBV-C/HGV) is related distantly to hepatitis C virus (HCV). HCV has a hypervariable region (HVR), and exists as quasispecies in vivo. Although GBV-C/HGV also has replaceable amino acids in the presumed antigenic region, the existence and fluctuation of population of heterogeneous virus have not been evaluated. In this study, the heterogeneity of GBV-C/HGV and HCV was investigated by the single-strand conformation polymorphism (SSCP) analysis in six concomitantly infected patients. Two patients were observed for 4 years without any treatment, and four were treated with interferon-alpha (IFN). By SSCP analysis, amplicons of GBV-C/HGV RNA were separated into 1-5 bands on gels for each patient. The amplicons had different nucleotide but the same amino acid sequences in the presumed antigenic region. The amplicons of HCV RNA, separated into 1-4 bands, had different nucleotide and amino acid sequences in the HVR. In the two patients without treatment, the predominant strain of GBV-C/HGV was unchanged for the 4 years. In the four patients administered IFN, some strains of GBV-C/HGV disappeared after IFN therapy, whereas other strains persisted. The mean genetic distance among GBV-C/HGV strains represented by SSCP analysis was significantly lower than that of HCV (P < 0.05). The data indicate that: 1) GBV-C/HGV can be devoid of antigenic drift unlike HCV; 2) GBV-C/HGV has no HVR as seen in HCV in the presumed antigenic region; and 3) the sensitivity to IFN differs among GBV-C/HGV strains in the same hosts, as with HCV.

Molecular analysis of GB virus C isolates in Belgian hemodialysis patients.

Year 1998
Liu HF. Cornu C. Jadoul M. Dahan K. Loute G. Goubau P.
Unit of Virology, Cliniques Universitaires St-Luc, Universite Catholique de Louvain, Brussels, Belgium.
GB virus C (GBV-C) has been detected in Belgian hemodialysis patients. To study their genomic diversity and phylogenetic relationship, a 592 nucleotide fragment extending from the 5' non-coding region to part of the E1 gene of the GBV-C genome was amplified and sequenced from 12 Belgian hemodialysis patients in two different centers. Together with strains from different geographical origins, these sequences were analyzed phylogenetically using three different methods. A consistent tree topology was obtained with all methods. Three GBV-C genotypes were observed with two subtypes in type 2 and a questionable subtyping in type 1. Except for one isolate falling into type 1 cluster which mainly consists of African strains, all the other Belgian strains clustered within the type 2a branch. Two GBV-C isolates in two patients from the same hemodialysis center clustered together closely, suggesting a nosocomial transmission. In view of their long branch length, it seems unlikely that the other Belgian strains evolved recently from a common ancestor. Our results indicate that the major type circulating among Belgian hemodialysis patients seems to be 2a, which is usual for Europe and North America, but that the African type 1 also exists to a minor extent. Although patient to patient transmission of GBV-C in Belgian hemodialysis centers did occur, it may not account for the majority of infections.

Infection with GB virus C, hepatitis C and B viruses in 1,044 cases autopsied at the Medical Examiners Office in Tokyo.

Year 1998
Takamatsu J. Tsuda F. Okudaira M.
Medical Examiner's Office, Tokyo Metropolitan Government, Japan.
Markers of GB virus C (GBV-C), hepatitis C virus (HCV) and hepatitis B virus (HBV) were sought in sera from 1,044 cases autopsied at the Medical Examiner's Office, Tokyo Metropolitan Government. GBV-C RNA was detected in 35 (3%) cases at a frequency significantly higher (P < 0.05) than in blood donors in Tokyo (4 of 448 or 1%). Three genotypes of GBV-C provisionally designated G1, G2 and G3 were determined by selective amplification with type-specific primers, and G3 (Asian type) was detected in 31 (89%), G2 (European/American type represented by the prototype hepatitis G virus) in three (9%) and G1 (West African type represented by the prototype GBV-C) in one (3%). Antibody to HCV (anti-HCV) was detected in 116 (11%) cases and accompanied by HCV RNA in 88. HCV genotypes were I/1a in one (1%), II/1b in 55 (63%), III/2a in 17 (19%) and IV/2b in 13 (15%). Antibodies to hepatitis B virus (HBV) was detected in 335 (32%) cases and hepatitis B surface antigen in 14 (1%). Subtypes were determined in 12 of them, adw was found in seven (58%), adr in four (33%) and adyr in one (8%). GBV-C RNA was detected significantly more frequently (P< 0.01) in the cases with liver disease (9 of 70 or 13%) than in those with the other causes of death (26 of 974 or 3%). Anti-HCV was more frequent in the cases with GBV-C RNA than in those without it (15 of 35 or 43% vs. 101 of 1,009 or 10%, P< 0.001). These results indicate that infection with GBV-C as well as HCV was common, while infection with HBV was not common in the Medical Examiner's autopsy cases in Tokyo.

Lack of anti-GOR antibody among subjects with GB virus C/hepatitis G virus RNA.

Year 1998
Nakano T. Mizokami M. Cao K. Noguchi S. Sata M. Park YM. Kim BS. Oyunsuren T. Pereira LB. Ruzibakiev R. Gurtsevitch V. Hayami M.
Second Department of Medicine, Nagoya City University Medical School, Mizuho, Nagoya, Japan.
Homologies were sought between the putative amino acid sequences of GB virus C/hepatitis G virus (GBV-C/HGV) and the GOR epitope or the liver/kidney microsome-1 (LKM-1) epitope, which share partial sequence identity with the hepatitis C virus (HCV) polyprotein. Anti-GOR antibody (anti-GOR) was assayed among 100 subjects with GBV-C/HGV RNA. Twenty-one and 25 subjects were coinfected with hepatitis B virus (HBV) or HCV, respectively. Homologies were found between the NS5 or E2 polyproteins of GBV-C/HGV and the GOR epitope or the LKM-1 epitope, respectively. These segments of GBV-C/HGV polyproteins sharing identity with the GOR or the LKM-1 epitope were well conserved among three genotypes of GBV-C/HGV. However, only 1 of 55 subjects (1.8%) with GBV-C/HGV RNA, but not with HBV or HCV, was positive for anti-GOR. The positivity for anti-GOR among the group with GBV-C/HGV RNA alone was significantly lower than that among the groups with HCV RNA (P < 0.01 and P < 0.05, respectively). Only 2 of 55 subjects (3.6%) with GBV-C/HGV RNA alone exhibited elevation of alanine aminotransferase. The incidence of liver dysfunction among the group with GBV-C/HGV RNA alone was significantly lower than the incidence among the groups with GBV-C/HGV RNA and hepatitis B surface antigen (HBsAg) or HCV RNA (P< 0.01 and P< 0.01, respectively). These data indicate that 1) there is no association between GBV-C/HGV infection and the presence of anti-GOR, and 2) GBV-C/HGV infection is not related to chronic liver dysfunction.

Comparison of tests for antibody to hepatitis E virus.

Year 1998
Ghabrah TM. Tsarev S. Yarbough PO. Emerson SU. Strickland GT. Purcell RH.
Department of Community Medicine and Primary Health Care, College of Medicine and Allied Sciences, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
The results of serologic tests for hepatitis E virus have varied widely from laboratory to laboratory, making interpretation of seroepidemiologic studies difficult. The present study compares serologic results with different antigens and tests developed in two laboratories for their ability to diagnose hepatitis E and measure antibody prevalence in a high risk population in Saudi Arabia. The results confirm that tests based upon open reading frame (ORF) 3 of HEV are of limited value for seroepidemiologic studies, whereas ORF2-based antigens have broad utility and yield data that are reproducible in more than one laboratory.

Serological and genomic characterization of human rotaviruses detected in China.

Year 1998
Wu H. Taniguchi K. Urasawa T. Urasawa S.
Department of Hygiene, Sapporo Medical University School of Medicine, Japan.
A total of 1,385 stool specimens were collected from children with diarrhea at two hospitals in Wuhan, Hubei Province, China, in 1994 and 1995, and screened for rotavirus by polyacrylamide gel electrophoresis of viral RNA. Group A rotavirus was detected with high frequency; 56.5% (87/154) and 40.8% (502/1,231) of the specimens collected in 1994 and 1995, respectively, were positive for rotavirus. Assignment of G serotype and P type (VP4 genotype) of group A rotavirus by ELISA with monoclonal antibodies and/or PCR, respectively, showed that strains of G2-P[4] and G1-P[8] specificity were predominant in 1994 and in 1995, respectively. In contrast, a single strain was found to have a P[9] type specificity, and no G4 strain was detected. Unusual combinations of RNA pattern-subgroup-G serotype-P type, such as long pattern-subgroup I-G1-P[8], short pattern-subgroup II-G3-P[4] and short pattern-subgroup I-G1-P[4], were detected in four specimens. Nucleotide sequences of the VP8* and/or NSP5 genes from two Chinese P[8] strains 470 and 582 and one Chinese P[9] strain 512 as well as five Japanese P[9] strains (K8, AU1, M318, 0264, and 0265) were determined and compared with the published sequences of the corresponding gene. In the phylogenetic tree of VP8* sequences of P[9] strains, which formed two clusters each having strain K8 or AU-1 as the representative strain, the Chinese P[9] strain was found in the cluster represented by AU-1, although it was most distantly related to other strains. While NSP5 sequences of human strains with P[9] specificity were related to simian and bovine strains, that of Chinese P[8] strains was most closely related to those of porcine strains. A single group C rotavirus (No. 208) was detected. Nucleotide sequences of its VP4, VP6, VP7, and NSP4 genes were very similar to those of group C human rotaviruses detected worldwide.

Mutation of nucleotide 1,762 in the core promoter region during hepatitis B e seroconversion and its relation to liver damage in hepatitis B e antigen carriers.

Year 1998
Lindh M. Gustavson C. Mardberg K. Norkrans G. Dhillon AP. Horal P.
Department of Clinical Virology, Goteborg University, Sweden. magnus.lindh@microbio.gu.se
In chronic hepatitis B virus (HBV) infection, mutations develop frequently at nucleotides 1,762/1,764 in the X protein open reading frame, where the core promoter is also located. By using a modified allele-specific polymerase chain reaction method, the longitudinal emergence of the A-->T mutation at nucleotide 1,762 was studied in relation to precore mutations, genotype, and liver damage. First, samples from 38 carriers that were drawn before and after hepatitis B e (HBe) seroconversion were tested. T-1,762 mutant strains increased during HBe seroconversion (P = 0.004). In the HBe antigen-negative (HBeAg-) phase, T-1,762 mutants were found in 71% (12 of 17) of patients without compared with 33% (6 of 18) of patients with a concomitant precore mutation that prevents HBeAg synthesis (P = 0.08). Second, in 55 HBeAg+ patients, the T-1,762 mutant was found to be associated with more liver inflammation (P = 0.04) and fibrosis (P = 0.02), as measured by histology activity index (HAI) scores. The results show that the nucleotide (nt) 1,762 A-->T mutation often develops during HBe seroconversion, particularly in strains without precore mutations that prevent HBeAg production. For unknown reasons, the T-1,762 mutant was rare in genotype B strains. The presence of a T-1,762 mutant in the HBeAg+ phase may be useful for identifying immunoactivation in previously immunotolerant carriers, which could be valuable for selecting patients for interferon therapy.

Effect of genotypes on the quantification of hepatitis C virus (HCV) RNA in clinical samples using the Amplicor HCV Monitor Test and the Quantiplex HCV RNA 2.0 assay (bDNA).

Year 1998
Tong CY. Hollingsworth RC. Williams H. Irving WL. Gilmore IT.
Department of Medical Microbiology, University of Liverpool, United Kingdom. cywtong@liv.ac.uk
The Amplicor HCV Monitor test and the Quantiplex HCV RNA 2.0 (bDNA) assay are two commercially available assays for the quantification of hepatitis C virus (HCV) RNA in clinical samples. A direct comparison of the two assays was carried out using sera frozen previously from patients known to be chronically infected with HCV. Overall, 61 samples from 51 patients were tested simultaneously by the two methods: 67% (28/42) of the patients were infected by HCV genotype/serotype 1, 10% (4/42) with type 2, and 24% (10/42) with type 3. When the absolute value from each assay was examined, the Quantiplex assay gave a consistently higher reading and the mean logarithmic difference between the two assays was 1.4 (1.0 in type 1, 2.0 in type 2, and 2.2 in type 3). When analyzed according to genotype, strong correlation was observed between the two assays for type 1 (r = 0.83, 95% CI 0.63-0.93, P < 0.01), but not for nontype 1 samples. Despite the difference in absolute level reported by the two assays, there was a consistent trend of change in HCV RNA concentration by both assays in patients whose consecutive samples were analyzed and the differences between the two assays in consecutive samples were within 0.4 log of each other. The results suggested that with samples containing genotype 1, the Amplicor assay was more sensitive than the Quantiplex assay by about one log. However, the sensitivities of the two assays with nontype 1 samples were much closer probably due to the failure of the Amplicor assay to quantify nontype 1 genotypes effectively.

Experimental African HEV infection in cynomolgus macaques (Macaca fascicularis).

Year 1998
van Cuyck-Gandre H. Cockman-Thomas R. Caudill JD. Asher LS. Armstrong KL. Hauroeder B. Clements NJ. Binn LN. Longer CF.
Department of Virus Diseases, Walter Reed Army Institute of Research, Washington, D.C., USA.
Experimental infection with hepatitis E virus (HEV) from Africa has not been investigated. Our purpose was to study hepatitis E produced by HEV from Chad (North Africa) and to analyze the genetic sequence of the HEV obtained after animal passage. An HEV-containing fecal sample from Chad was intravenously inoculated in four cynomolgus macaques. When serum Alanine Amino Transferase (ALT) levels rose, open liver biopsy and bile aspiration were performed. In all the monkeys, an ALT rise occurred 25 to 32 days after inoculation and new anti-HEV was detected by Enzyme Immuno Assay (EIA). Hepatic histopathology was consistent with acute viral hepatitis. HEV was detected by polymerase chain reaction (PCR) in bile (3/4 animals) and feces (2/4 animals) and by imunoelectron microscopy (IEM) in the inoculum and one bile specimen. A genetic variant HEV was identified in one monkey. The Chad HEV produced hepatitis E with pathophysiologic and histopathologic findings similar to those observed with HEV from other geographic origins. A genomic variant HEV population was produced after one passage in a macaque.

Sexual transmission of GB virus C/hepatitis G virus.

Year 1998
Scallan MF. Clutterbuck D. Jarvis LM. Scott GR. Simmonds P.
Department of Medical Microbiology, University of Edinburgh, Scotland.
Although it is established that infection with GB virus C (GBV-C) or hepatitis G virus (HGV) can be transmitted parenterally, the prevalence of GBV-C/HGV viremia in the general population (2-5%) is relatively high compared with other parenterally borne viruses such as hepatitis C virus. To investigate the possibility of sexual transmission of GBV-C/HGV, we determined the frequency of viremia by the polymerase chain reaction and serological reactivity to the E2 protein by ELISA in samples collected from individuals at risk for sexually transmitted diseases attending a city genitourinary medicine clinic. GBV-C/HGV viremia was detected in 27 of 87 male homosexuals (31%) and 9 of 50 prostitutes (18%), frequencies significantly greater than those in matched controls (2/63) and local blood donors (2.3%). Among nonviremic individuals, a high frequency of serological reactivity to the E2 protein of GBV-C/HGV was also observed in the risk groups (male homosexuals: 14/60; prostitutes: 11/41), although these figures are likely to be underestimates of the frequency of past infection as detectable anti-E2 reactivity may attenuate rapidly over time following resolution of infection. Infection with GBV-C/HGV was more frequent among those coinfected with human immunodeficiency virus type 1. Among male homosexuals from whom retrospective samples were available, evidence for de novo infection was found in 9 of 22 individuals over a mean sampling time of 2.9 years, predicting an annualized incidence of GBV-C/HGV infection of approximately 11% in this group. The high prevalence and incidence of GBV-C/HGV infection in these individuals and prostitutes provides strong evidence for its spread by sexual contact. Further studies are required to investigate the mechanism of its transmission and the clinical significance of acute and persistent infection in these risk groups.

Absence of detectable measles virus genome sequence in inflammatory bowel disease tissues and peripheral blood lymphocytes.

Year 1998
Afzal MA. Armitage E. Begley J. Bentley ML. Minor PD. Ghosh S. Ferguson A.
Division of Virology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Herts, England.
A highly sensitive measles-specific RT-PCR-nested PCR system was established, which consistently amplified measles virus genome sequence from control samples containing as little as 5.5 x 10(-3) pfu per reaction. This method failed to detect the presence of measles virus in 93 colonoscopic biopsies and 31 peripheral blood lymphocyte preparations, examined and obtained from patients with inflammatory bowel disease (IBD) and noninflammatory controls. All patients had detectable levels of serum neutralization antibody against measles virus. Each biopsy was estimated to have about one million cells, based on the amplification of the beta actin gene. The assay was calibrated by use of a known number of lymphocytes. The method applied was able to amplify measles virus RNA from a nucleic acid mixture equivalent to 18 cells derived from subacute sclerosing panencephalitis (SSPE) brain material. The level of measles RNA present, if any, in the biopsies is therefore at least 50,000-fold less than in SSPE.

Double-blind, randomized controlled trial of interleukin-2 treatment of chronic hepatitis B.

Year 1998
Artillo S. Pastore G. Alberti A. Milella M. Santantonio T. Fattovich G. Giustina G. Ryff JC. Chaneac M. Bartolome J. Carreno V.
Department of Hepatology, Fundacion Jimenez Diaz and Fundacion Estudio Hepatitis Virales, Madrid, Spain.
Pilot studies have demonstrated that recombinant interleukin 2 (rIL-2) has an indirect antiviral activity against hepatitis B virus, but the minimal dose of rIL-2 for induction of this effect was not defined. The aim of the study was to ascertain the most efficient dose of rIL-2 for induction of the loss of detectable serum HBV-DNA or a 50% or greater decrease in its level. Thirty-one patients with chronic hepatitis B, hepatitis B e antigen and serum HBV-DNA positive were enrolled in this double-blind randomized controlled trial. Patients were divided: Group I (n = 8) placebo; Group II (n = 7) treated with 0.9 MU of rIL-2 subcutaneously administered daily for 8 weeks; Group III (n = 8) treated with 1.8 MU of rIL-2 under the same schedule; Group IV (n = 8) which received 3.6 MU of rIL-2 under the same conditions. At the end of treatment 25% of the patients in the placebo group, and 13% and 25% in rIL-2 groups III and IV, respectively, had a decrease in HBV-DNA higher than 50% of the basal value. None of the patients lost serum HBV-DNA. Only three patients (one from group II and two from group IV) normalized the ALT levels. Overall, during treatment, ALT levels decreased in the treated groups. This decrease occurred simultaneously with an increase in serum HBV-DNA concentration. Since the response rate in the treated groups was similar to that of the placebo group, rIL-2 is not useful as monotherapy for the treatment of chronic hepatitis B at the doses and schedules used in this study.

Quantitation of hepatitis C virus in liver and peripheral blood mononuclear cells from patients with chronic hepatitis C virus infection.

Year 1998
Martin J. Navas S. Quiroga JA. Colucci G. Pardo M. Carreno V.
Department of Hepatology, Fundacion Jimenez Diaz, Madrid, Spain.
Since the natural history of hepatitis C virus-associated liver disease and the therapeutic responsiveness might vary according to liver and blood mononuclear cells viral levels, it may be important to quantitate viral RNA in liver, blood mononuclear cells and serum, and to compare these data with genotype, biochemical and histologic data. A polymerase chain reaction-based assay available for serum hepatitis C virus RNA quantitation has been optimized to quantitate viral genomes in liver and peripheral blood mononuclear cells from 47 chronic hepatitis C patients. The procedure permitted hepatitis C virus RNA quantitation in freshly isolated mononuclear cells and in total RNA extracted from frozen mononuclear cells and liver tissue. The intrahepatic viral amount (median: 2.6 x 10(3) copies/microgram RNA; range: 0 to 3.6 x 10(4) copies/microgram RNA) correlated significantly with the hepatitis C virus RNA concentration in serum (r = 0.76, P < .001), but not in mononuclear cells. Viral RNA concentrations in liver (P < .001), serum (P < 0.01) and PBMC (P < 0.05) were significantly higher in hepatitis C virus genotype 1 patients (essentially type 1b) than in non-1 type cases, but were unrelated to biochemical or histologic indexes of disease activity. In conclusion, the optimized assay permit HCV RNA quantitation in liver and peripheral blood mononuclear cells, suggesting that serum viral level is an accurate measurement of intrahepatic viral burden.

Clinical significance of hepatic HCV RNA in patients with chronic hepatitis C demonstrating long-term sustained response to interferon-alpha therapy.

Year 1998
Larghi A. Tagger A. Crosignani A. Ribero ML. Bruno S. Portera G. Battezzati PM. Maggioni M. Fasola M. Zuin M. Podda M.
Department of Internal Medicine, Ospedale San Paolo, Milan, Italy.
Whether sustained biochemical response and absence of serum HCV RNA in the 6-12 months following suspension of interferon-alpha (IFN-alpha) therapy reflect definitive viral clearance in patients with chronic hepatitis C virus (HCV) infection is controversial. To obtain more information on this topic, HCV RNA was sought in both liver and serum samples of 25 long-term responders who were followed for a median period of 39 months (range 21-79) after discontinuation of IFN-alpha. Liver biopsy was undertaken before and 6 to 12 months after IFN-alpha withdrawal. Liver and serum HCV RNA were tested by a nested polymerase chain reaction. Twenty-two patients (88%) tested negative for both liver and serum HCV RNA, two patients had detectable HCV RNA in both liver and serum, and one patient showed persistent HCV RNA only in the liver. Post-treatment liver histology improved markedly in all patients, including those with viral persistence. During further follow-up, biochemical remission was maintained in all patients except one in whom both serum and liver specimens remained HCV RNA positive. The data indicate that the large majority of long-term responders test negative for HCV RNA in the liver, which suggests definitive eradication of HCV RNA infection.

Floating density of hepatitis C virus particles and response to interferon treatment.

Year 1998
Sakai A. Kaneko S. Matsushita E. Kobayashi K.
First Department of Internal Medicine, Kanazawa University School of Medicine, Ishikawa, Japan.
Hepatitis C virus (HCV) particles can be classified into two major fractions according to their floating density in serum. However, the genomic heterogeneity of each fraction and the relationship between this viral characteristic and interferon (IFN) response in patients with chronic hepatitis are not known. In this study, floating density and single strand conformation polymorphism (SSCP) of the hypervariable region 1 (HVR1) of HCV were examined in 16 patients with chronic hepatitis prior to IFN treatment. The ratio of HCV RNA titers in the top (T) and bottom (B) fractions, or T:B ratio, was 10:1 in 4 patients, 1:1 in 7, and 1:10 in 5. Three of the 4 patients with a 10:1 ratio showed a sustained response to IFN, while none of the 5 patients with a 1:10 ratio demonstrated a sustained response (P < 0.05). All 4 patients with a 10:1 ratio had 1 or 2 SSCP bands, and 4 of the 5 patients with a 1:10 ratio had 4 or 5 bands (P < 0.01). Furthermore, the number of SSCP bands in the top fraction from 6 sustained responders (1.8 +/- 0.3) was significantly smaller than from 10 non sustained responders (4.1 +/- 0.8) (P < 0.05). Thus, patients with a high T:B ratio and low heterogeneity in HVR1 demonstrated sustained responses to IFN, while those with low T:B ratios and high heterogeneity did not.

Large-scale analysis of hepatitis C virus serological typing assay: effectiveness and limits.

Year 1998
Leruez-Ville M. Nguyen QT. Cohen P. Cocco S. Nouyou M. Ferriere F. Deny P.
Laboratorie de Bacteriologie-Virologie, Hopital Avicenne, UFR Bobigny Universite Paris-Nord, France.
The HCV (hepatitis C virus) Serotyping 1-6 Assay (Murex Laboratories) was evaluated on 303 French HCV-infected patients. Serological typing results were compared to the genotypes obtained from sequence analyses of the 5' noncoding regions of the virus genome from 46 HCV-infected patients, and assay specificity was found to be high (97.6%). The serological typing assay, run in 257 consecutive HCV-infected patients, yielded an assay sensitivity lower (70.6%) than that previously reported. This finding was attributed mainly to nonreactive sera from human immunodeficiency virus (HIV)-positive patients (P < 0.001) and perhaps reflected cryoglobulin positivity in others. No anti-type 6 reactivity was detected, and the overall serological type distribution values for types 1 to 5 were 67.3, 7.9, 16.4, 6.6, and 0.9%, respectively. A higher prevalence of type 4 was noted among HIV-infected patients (P < 0.001). In addition, serotype 2 was significantly more frequent in cryoglobulinemia positive than in cryoglobulinemia-negative patients (P < 0.05). Although an initial high level (7%) of mixed serological typing reactivities was found, after predilution of serum only two mixed infections could be confirmed (0.9%). It is suggested, therefore, that mixed reactivities have to be interpreted carefully and retested with prediluted serum, particularly when the optical density of the reactivity is > 2.5 or remains > 0.4 after competition with all type-specific peptides. The high specificity and relatively good sensitivity even in immunocompromised patients obtained with this assay indicate that it can be used routinely. Because response to treatment is linked to HCV type, this assay could be used to identify HCV serotype to guide therapeutic decisions.

HCV antibodies in saliva and urine.

Year 1998
Elsana S. Sikuler E. Yaari A. Shemer-Avni Y. Abu-Shakra M. Buskila D. Katzman P. Naggan L. Margalith M.
Department of Virology, Soroka Medical Centre, Beer-Sheva, Israel.
Infection with hepatitis C virus (HCV) is usually established by detection of serum antibodies (anti-HCV). This study was conducted in order to evaluate whether saliva and urine may substitute serum for anti-HCV detection. Serum, saliva, and urine were obtained simultaneously from 141 patients with a variety of liver diseases and from 52 patients with autoimmune diseases (systemic lupus erythematosus n = 27 and rheumatoid arthritis n = 25). The cell free fraction of saliva and urine samples was tested for anti-HCV using a modification of a serum anti-HCV kit. Western blot analysis was used as a confirmation method. Of the patients with liver diseases, 73 were anti-HCV-seropositive. Salivary and urinary anti-HCV could be detected in 66 (90%) and 36 (49%) of the anti-HCV-seropositive patients, respectively. The presence of anti-HCV in saliva or urine was not related to the severity of liver disease. All the anti-HCV-seronegative liver patients were negative for salivary anti-HCV and 22 (32%) had urinary anti-HCV. The patients with autoimmune diseases were all anti-HCV-seronegative. None had detectable salivary anti-HCV while 33 (63%) were positive for urinary anti-HCV. Western Blot analysis confirmed the presence of anti-HCV in all serum and saliva samples tested but only in 2/12 urine samples. The results suggest that saliva, but not urine, may serve as a substitute for serum for the determination of anti-HCV positivity.

Clinical and molecular virological differences between fulminant hepatic failures following acute and chronic infection with hepatitis B virus.

Year 1998
Inoue K. Yoshiba M. Sekiyama K. Okamoto H. Mayumi M.
Division of Gastroenterology, Showa University Fujigaoka Hospital, Yokohama, Japan.
Clinical and molecular biological characteristics were compared between patients who presented with fulminant hepatic failure following acute infection with hepatitis B virus (HBV) and those who developed hepatic failure during they carried HBV. The 11 patients with acute HBV infection had higher levels of alanine aminotransferase (mean +/- SD: 4943 +/- 2867 vs 1157 +/- 678 IU/L, P < 0.01), more often with a single peak (91% vs. 0%, P < 0.001), and lower total bilirubin level (15.3 +/- 4.4 vs 28.1 +/- 14.3 mg/1000 ml, P < 0.01) than the 13 patients with chronic HBV infection. Hepatitis B surface antigen was detected less often (55% vs. 100%, P < 0.05) and viral DNA polymerase less frequently (0% vs. 46%, P < 0.05) in the patients with acute than chronic HBV infection. Hepatitis B e antigen was detected in one (9%) patient with acute infection, less frequently than in six (46%) patients with chronic infection (P < 0.05). Mutations in the precore region was detected in HBV DNA clones from ten (91%) patients with acute infection and only in those from eight (62%) patients with chronic infection. All HBV DNA clones from the five (38%) patients with chronic infection that did not have precore mutations, however, possessed mutations in the core promoter. These results indicate that HBV mutants incapable of translating hepatitis B e antigen would play a major role in fulminant hepatic failure occurring after acute HBV infection. In contrast, HBV variants with core promoter mutations for reducing the transcription of hepatitis B e antigen would play an additional role in fulminant hepatic failure developing during chronic infection.