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SUMMARYHistorical enquiry into diseases with morbidity or mortality predilections for particular demographic groups can permit clarification of their emergence, endemicity, and epidemicity. During community-wide outbreaks of hepatitis A in the pre-vaccine era, clinical attack rates were higher among juveniles rather than adults. In community-wide hepatitis E outbreaks, past and present, mortality rates have been most pronounced among pregnant women. Examination for these characteristic predilections in reports of jaundice outbreaks in the USA traces the emergence of hepatitis A and also of hepatitis E to the closing three decades of the 19th century. Thereafter, outbreaks of hepatitis A burgeoned, whereas those of hepatitis E abated. There were, in addition, community-wide outbreaks that bore features of neither hepatitis A nor E; they occurred before the 1870s. The American Civil War antedated that period. If hepatitis A had yet to establish endemicity, then it would not underlie the jaundice epidemic that was widespread during the war. Such an assessment may be revised, however, with the discovery of more extant outbreak reports.

HCV strains belonging to genotype 4 may be gaining endemicity across Continental Europe but the extent of their spread in the United Kingdom is unknown. Sera referred from patients attending hospitals in England between 2004 through 2008 were characterised. A total of 243 sera carried HCV genotype 4. The most common subtypes were 4a (33%) and 4d (35%). Compared to patients infected by 4d, those infected by 4a were 20 times more likely to be Middle Eastern than English, and those infected by non-4a/4d were older, tended to be female, and 50 or 12 times more likely to be Middle Eastern or South Asian, respectively, than English. Persons infected by 4d tended to be English rather than Middle Eastern or South Asian. One group of 4d strains clustered with strains reported from persons in Europe engaged in male homosexual activity. Surveillance of trends in the importation and subsequent spread of HCV genotype 4 and its subtypes is advocated.

Background A large outbreak of hepatitis B virus (HBV) infection in the UK occurred between 2001 and 2005 in Bristol, UK. Objectives To identify HBV strains circulating amongst risk groups in the HBV outbreak cohort. Study design Cross-sectional study of acute HBV outbreak cases in Bristol. Results HBV sequences from sera of 95 of the 237 cases (40%) were characterised. The majority of cases (77%) were found to carry an HBV variant belonging to genotype D, designated HBVBV. Eighty-eight percent (36/41) of sequences from injection drug users were HBVBV as were 70% (19/27) from those with heterosexual intercourse as the primary identified risk factor. Of 15 sequences characterised from cases of pre-outbreak acute or chronic hepatitis B residing in Bristol, 40% also carried HBVBV; the earliest was from a case identified in 1994. Conclusion The findings from this study link the spread of HBVBV from injecting drug users to the general population through heterosexual intercourse during the outbreak. The molecular sequencing of specimens from this outbreak reports the emergence of HBVBV, a HBV strain circulating in Bristol and South West England, as the cause of one of the largest outbreaks of acute hepatitis B in the UK.

Introduction: Studies were conducted to investigate changes in the extent of human herpesvirus 8 (HHV-8) shedding and diversity of HHV-8 strains in the mouth of a renal allograft recipient who developed cutaneous post-transplantation Kaposi’s sarcoma. Methods: Matched oral and blood samples were obtained from a Saudi Arabian renal allograft recipient from 3 days before to 38 weeks after transplantation, and from his kidney donor. Polymerase chain reaction (PCR) protocols to amplify selected HHV-8 sub-genomic regions were applied to detect and quantify HHV-8 DNA. Sequence diversity was determined by cloning the PCR products and subjecting them to denaturing gradient gel electrophoresis and to nucleotide sequencing. Results: Before transplantation, the recipient was seropositive for anti-HHV-8 immunoglobulin G, but the donor was seronegative; HHV-8 DNA could be detected in the recipient’s blood, whole-mouth saliva (WMS) and buccal exfoliates, and the salivary viral load was estimated as 2.6 million genome-copies/ml. Post-transplantation, the recipient’s salivary viral load initially increased to 4.1 million genome-copies/ml, and thereafter declined precipitously, coinciding with an increase in the dosage of valaciclovir given; HHV-8 DNA was detected most often in WMS compared with parotid saliva, and buccal and palatal exfoliates. Carriage of multiple HHV-8 strains was evident in blood and oral samples; whereas before transplantation strains belonging to genotypes A1 and A5 were observed, after transplantation genotype A5 strains became dominant and A2 strains emerged. Conclusion: Immunosuppression and antiviral prophylaxis may interact to influence the spectrum of oral HHV-8 strains and the extent of post-transplantation HHV-8 shedding into the mouth.

Background: Increasing data shows the relevance of HBV genotypes in the outcome of infection. Most studies investigating the relationship between the genotypic characteristics of hepatitis B virus (HBV) and the clinical or epidemiological aspects of HBV infection originate from studies of patients with chronic rather than acute hepatitis B. Objectives: To study a convenience sample representing ca. 5% of reported acute hepatitis B in England between 1997 and 2001 to investigate the distribution of HBV genotypes and specific HBV variants with epidemiological risk factors, thereby providing baseline data for ongoing surveillance. Study design: From 160 serum samples, PCR was carried out to amplify the first 600 bases of the HBV S gene. Amplicons were sequenced and subjected to phylogenetic analysis and risk factor analysis. Results: Fifty-seven percent of the study samples carried HBV belonging to subtype A2, 13% to subtype D2, and the rest to genotype E (8%) and subtypes C2 and D3 (each 6%), D1 and D4 (each 3%) and B4 (1%). One particular A2 isolate was dominant, accounting for 23% of the total sample set. Drug use and homosexual transmission were equally implicated as risks within genotype A2. No mutations associated with vaccine escape or resistance to antiviral therapy were identified. Conclusion: Immigration and travel likely shape the observed genotype distribution and consequent prevalence of genotypes other than A2 or D in this population. Data suggests no genetic separation of parenteral and sexually transmitted virus. These data demonstrate the value in pursuing more extensive and recent surveillance. © 2008 Elsevier B.V. All rights reserved.

(Accepted 7 November 2008; first published online 23 December 2008)SUMMARYSurveillance reports and prevalence studies have indicated that injecting drug users (IDUs)contribute more to the hepatitis C epidemic in the United Kingdom than any other risk group.Information on both the prevalence and incidence of hepatitis C in IDUs is therefore essential tounderstanding the epidemiology of this infection. The prevalence of hepatitis C in specimens fromthe Unlinked Anonymous Prevalence Monitoring Programme collected in 1995, 1996, 1998, 1999,2000, and 2001 was determined using residual syphilis serology specimens from IDUs attending15 genitourinary medicine (GUM) clinics in and outside London. These specimens were tested forantibodies to hepatitis C virus (anti-HCV). Using this cross-sectional design, anti-HCV-negativespecimens were tested for HCV RNA to identify incident infections during the ‘window’ period ofinfection, and thus to estimate HCV incidence. Results of the multivariable analysis showed thatthere was marked variation in prevalence by clinic (P

Inspired by the Ancient Greeks, past students of in fectious diseases have often personified in that many headed monster, Hydra, the terrifying effects of scourges such as cholera, scarlet fever, diphtheria, yellow fever and malaria [1-4]. Hercules-like, these pioneers laboured to prod and probe the beast, in tending to identify its vulnerabilities and deliver the final, mortal blows [5]. They have largely been suc cessful, although cholera and malaria remain as some of the few heads to slay.

Background The hepatitis B virus (HBV) pol gene overlaps the S gene encoding surface antigen (HBsAg). It has been reported previously that drug-induced changes in HBsAg alter its binding to sera from humans immunized against HBV. We investigate here the changes to specific epitopes in the a determinant (the major target of neutralizing antibody) caused by a number of drug-resistant mutations. Methods Recombinant HBsAgs, produced by transfection of Chinese hamster ovary cells with S gene plasmids into which lamivudine, adefovir and entecavir resistance and common antibody-escape mutations had been introduced, were probed with monoclonal antibodies to epitopes in the first and second loops of the a determinant. Results The mutations rtF166L/sF158Y (lamivudine-associated, compensatory) and rtI169T/sF161L (entecavir-associated, primary) acting alone, and the mutations rtV173L/sE164D (lamivudine-associated, compensatory) and rtSilent/sD144E (antibody escape-associated) each when combined with rtM204V/sI195M (lamivudine-associated, primary) led to decreases in antibody reactivity to epitopes in the first or second loop, or in both loops. The rtM204V/ sI195M+rtV173L/sE164D mutations yielded an epitope–antibody profile similar to the rtR153Q/sG145R vaccine escape mutant. The rtM204V/sI195M mutation combined with the rtF166L/sF158Y or rtR153Q/sG145R mutation restored reactivity to second-loop epitopes previously abrogated by single mutations. Conclusions Mutations associated with resistance to nucleos(t)ide analogue therapy, singly or in combination with each other or antibody escape-associated mutations, alter HBsAg immunoreactivity through concomitant amino acid substitutions at codons within and downstream of the a determinant. The findings have implications for understanding the native structure of HBsAg, optimizing treatment of chronic hepatitis B and evaluating the success of immunization programmes.

An HIV-1/hepatitis C virus (HCV) co-infected patient with haemophilia received a 48-week course of pegylated interferon-α-2b and ribavirin therapy for genotype 5a HCV infection. Virological response was achieved at week 24. At the end of treatment, HCV RNA in serum was detected and identified to belong to genotype 2b, rather than genotype 5a. A sensitive method for identifying minority HCV genotypes in pre-treatment serum showed genotype 2b HCV carriage prior to treatment. Sequencing the interferon sensitivity-determining region of the HCV NS5A gene obtained from pre-, intra- and post-treatment sera revealed emergence of quasispecies bearing R → K and M → A/T mutations at codons 2222 and 2223, respectively. Occult presence of minority HCV subpopulations and their acquisition of mutations following therapy can result in poor treatment outcome.

Summary. Whether differences in the natural history of hepatitis C virus (HCV) can be explained by differences in the infecting HCV type is unknown. The aim of this study was to investigate whether the HCV type might influence the clinical outcome of infection. Study serum samples were assembled from 749 individuals enrolled into the UK HCV National Register from which data on clinical outcomes were extracted. HCV-RNA-positive specimens were genotyped and HCV-RNA-negative specimens serotyped. Logistic regression analysis was used to investigate the independent effect of HCV type on viral clearance by comparing patients who were HCV RNA negative (n = 86) with those who were HCV RNA positive (n = 508). The same method was used to investigate whether HCV type was associated with histological stage of liver disease. The prevalence of HCV type 1 among those who cleared infection was 69% and among those who remained HCV RNA positive was 51%: Type 1 infections were more likely to be HCV RNA negative than non-1 types (OR 0.47, 95% CI 0.29–0.78, P = 0.003). Type 1 infections were also more likely to be associated with histological stage scores above the median when compared with non-1 types (OR 2.03, 95% CI 1.07–3.83, P = 0.03). In conclusion, HCV type 1 infection was more often HCV RNA negative, suggesting that spontaneous clearance may occur more commonly with this type. Among the RNA-positive infections, type 1 infection may be more aggressive than types 2/3.

Renal allograft recipients in the Middle East are at high risk of developing Kaposi's sarcoma. This report describes the extent of oral human herpesvirus 8 shedding and the genomic diversity of the virus in five Saudi Arabian kidney transplantation patients in whom Kaposi's sarcoma had developed. PCR protocols were applied to amplify three fragments of the viral genome from whole-mouth saliva, parotid saliva, buccal and palatal exfoliates, plasma, peripheral blood leukocytes and biopsy of the Kaposi's sarcoma lesion, and to quantify the viral load in whole-mouth saliva. Viral DNA was detected in all plasma and biopsy samples, 80% of whole-mouth saliva, 20% of each of the other oral samples, and none of the leukocyte samples. The viral load in the cell-free fraction of whole-mouth saliva ranged between approximately 1.2 x 10(3) and 2.2 x 10(6) genome-copies/ml. Genotypically distinct viral strains were evident: intra-lesionally in 1 patient; intra-orally in one patient; between an oral sample and biopsy in two patients; and in four patients, between an oral sample and plasma, and between plasma and biopsy. Thus, in the patients studied, salivary shedding of human herpesvirus 8 was frequent and could be extensive, and they were prone to multiple infections. Measures to curtail salivary viral transmission to pre- and post-transplantation patients might reduce the incidence of post-transplantation Kaposi's sarcoma.

The prevalence of concurrent multitypic hepatitis C virus (HCV) infection is uncertain. A sensitive and specific approach to identifying minority HCV genotypes in blood is presented. Following serum extraction and reverse transcription PCR to amplify cDNA originating from the viral 5′ noncoding region, the amplified product mixture was treated with genotype-specific restriction endonuclease to digest the dominant genotype. Residual amplicons were subjected to PCR cloning and then to real-time DNA sequencing using a Pyrosequencer to identify the remaining genotypes. Dilution experiments showed that minority genotypes may be detected when they represent 1:10,000 of the total population and in serum specimens with viral loads as low as 1,000 IU/ml. Of 37 patients with bleeding disorders and 44 injecting drug users, infection by more than one HCV genotype was found in 7 (19%) and 4 (9%) patients, respectively. The low rate of detection in people at high risk of repeated HCV infection suggests that multitypic HCV carriage is uncommon.

Sequence polymorphisms in the gN and gO genes of cytomegalovirus (CMV) amplified from mouth rinse and urine samples of 19 Malawian patients with Kaposi's sarcoma (KS) and 58 of their first-degree relatives were investigated. CMV-DNA was amplified from 41 people (53%) from either the gN or gO region in at least one sample, from 14 people (18%) in both domains in at least one sample, and from 13 (17%) in either domain in both samples. Twenty-one (51%) were seropositive for human immunodeficiency virus-1 (HIV). Identical gN sequences were recovered from eight families and non-identical sequences in six, while identical gO sequences were found in three families and non-identical sequences in five. Five people, four of whom were children, each carried multitypic gN sequences or gO sequences. The findings are consistent with CMV spread along intra- and extra-household routes, and with multiple intra-host CMV infection.

Objective: To determine the prevalence and genetic diversity of hepatitis C virus in genitourinary medicine clinic attenders and to assess the extent of sexual transmission of the virus. Methods: A cross sectional, unlinked, anonymous survey in 14 genitourinary medicine clinics situated in England, Wales, and Northern Ireland. Serum specimens from genitourinary medicine clinic attenders, retained as part of the Unlinked Anonymous Prevalence Monitoring Programme (UAPMP) serum archive, were tested in small pools, for the presence of antibody to hepatitis C virus (anti-HCV). The main outcome measures were prevalence of antibodies to hepatitis C virus and identification of hepatitis C virus genotypes. Results: Testing of 17 586 specimens from 1995 showed an adjusted prevalence of anti-HCV in genitourinary medicine clinic attenders of 1.03% (95% CI: 0.89 to 1.16) overall and 0.65% (95% CI: 0.51 to 0.78) among those who did not report injecting drug use. Prevalence in injecting drug users attending genitourinary medicine clinics was 36.9% in both 1995 and 1996. Heterosexual injecting drug users had a higher prevalence of anti-HCV than homosexual/bisexual injectors. The most common hepatitis C genotypes were types 3a and 1a. There was a high degree of concordance between genotype and serotype. Conclusions: The low prevalence of anti-HCV in genitourinary medicine clinic attenders who deny injecting drugs suggests that the majority of hepatitis C infections have been acquired in adult life, mostly by injecting drug use, and that the hepatitis C virus is rarely transmitted sexually. The use of needle exchanges may explain the relatively low prevalence observed in the injecting drug users.

Denaturing gradient gel electrophoresis (DGGE) was used to study the diversity of hepatitis C virus (HCV) quasispecies. Optimized DGGE running conditions were applied to screen for variations in sequences cloned from amplicons originating from the nonstructural 5b (NS5b) gene of HCV in blood of hemophilia patients, intravenous drug users, and blood donors (five specimens from each study group, ca. 40 clones studied per specimen). Clones identified by DGGE as unique were sequenced. NS5b sequence entropy and mean genetic distance in hemophiliacs did not differ significantly from those in the other groups, pointing to a lack of correlation between HCV diversity and the multiplicity of past HCV exposures. DGGE was also applied to investigate variation in the HCV envelope 2/hypervariable region 1 (E2/HVR-1) in serum samples serially taken from two patients during the seroconversion phase of HCV infection. E2/HVR-1 sequence entropy changes were small and not correlated with rising anti-HCV antibody levels, reflecting mutational changes not mediated by antibody selection.

The first nucleotide sequences of hepatitis E virus (HEV) acquired in the United Kingdom are described. The sequences are novel and are related most closely to HEV isolated from Greece (Greece 2 strain), consistent with their having been derived from an indigenous European virus. HEV was assumed until recently to be rare in the United Kingdom and other industrialised countries and, consequently, hepatitis E may be under-diagnosed in industrialised countries.

A novel TT virus (TTV)-like DNA sequence was detected in the serum of a patient (PM) with acute non-A–E hepatitis. The full-length genome sequence, referred to here as PM virus (PMV), was obtained and its relationship to other full or near full-length TTV sequences examined. Although it shares a common genomic arrangement and short conserved regions, the majority of the genome is extremely divergent, displaying an average genetic distance of 0·60 from all other TTV sequences. By comparing PMV with TTV genomes representing the most divergent types so far described, six major groups can be distinguished. The level of genetic diversity seen between these genomes is higher than would be expected within a single virus species. Indeed, PMV could be considered the prototype of an independent taxonomic group within the Circoviridae family. A genoprevalence study of sera from blood donors and patients with acute hepatitis suggests that PMV is rare.

Unregulated skin-piercing procedures potentially facilitate the transmission of bloodborne pathogens. In February, 1998, a patient who had recently received autohaemotherapy at an alternative medicine clinic in the UK was diagnosed with acute hepatitis B. The autohaemotherapy procedure involved the drawing of 1 mL of the patient's blood, mixing with saline, and reinjection of the autologous blood mixture. We investigated the extent of hepatitis B virus (HBV) infection in patients and staff of the clinic.Patients who had attended the clinic between January, 1997, and February, 1998, were tested for serological markers of HBV, and for HBV DNA by PCR. HBV DNA was sequenced to assess the relatedness of the virus identified in the cases. We analysed the number and dates of visits with regard to HBV status.Serum samples were received from 352 patients and four staff members. Serological evidence of exposure to HBV was found in 57 (16%). Of the 33 patients and staff who were positive for hepatitis B surface antigen, 30 (91%) showed complete nucleotide identity in the DNA segments derived from the surface and core genes. Five patients with linked infection had markers of chronic hepatitis B, and one of these was regarded as the likely source of the outbreak. The attack rate was associated with the number of visits (p0.0001) and the week of visit (p=0.011). Contaminated saline in a repeatedly used bottle was the probable vehicle of transmission.We have described a large community-based outbreak of hepatitis B due to transmission by a single HBV variant. Our findings emphasise the continuing risk of transmission of bloodborne viruses in all health-care settings where skin-piercing procedures are used.

The effect of human immunodeficiency virus-1 protease inhibitors on the frequency of human herpesvirus 8 DNA detection from peripheral blood of human immunodeficiency virus-positive persons was evaluated. Thirty-three human immunodeficiency virus-seropositive male patients were studied longitudinally. DNA from open reading frame 26 of the human herpesvirus 8 genome was amplified by the polymerase chain reaction from the CD45+ fraction of peripheral blood before and after the introduction of protease inhibitor therapy. Human herpesvirus 8 IgG status, CD4+ cell counts, and human immunodeficiency virus-1 plasma viral load were also assessed before and after therapy. When both reverse transcriptase inhibitor and protease inhibitor treatment were introduced at the same time, there was an increase in CD4+ T cell counts (P=0.0041), a decrease in human immunodeficiency virus plasma load (P=0.0584), and a decrease in the detection rate of human herpesvirus 8 DNA (P=0.0077). Introducing protease inhibitor to patients already receiving reverse transcriptase inhibitor treatment was associated with an increase in CD4+ T cell counts (P=0.0003), a decrease in human immunodeficiency virus plasma viral load (P=0.0911), and a decrease in the human herpesvirus 8 detection rate (P=0.0412). No significant changes in the titters of anti-human herpesvirus 8 IgG were observed. Treatment with human immunodeficiency virus-1 protease inhibitors is therefore associated with the clearance of human herpesvirus 8 DNA from peripheral blood of human immunodeficiency virus-infected patients. The concomitant decrease in the human immunodeficiency virus plasma load and increase in the peripheral CD4+ cell count suggest that an amelioration in the immune defect following reduction in the burden of human immunodeficiency virus-1 infection is responsible for the clearance of human herpesvirus 8 by protease inhibitors.

The existence of HBV as quasispecies is thought to be favoured by the infidelity of HBV RT, which would account for the emergence of the many natural mutants with point substitutions. RT infidelity may also underlie the hypermutation phenomenon. Indeed, the oft-reported point mutation in the preC gene that leads to failure of HBeAg synthesis may be driven by a hypermutation-related mechanism. The presence of mutants with deletions and insertions involving single nucleotides and oligonucleotides at specific positions in the genome, and of mutants with deletions of even longer stretches particularly in the C gene, suggests that other mutagenic mechanisms operate. Candidates include slippage during mispairing between template and progeny DNA strand, the action of cellular topoisomerase I, and gene splicing using alternative donor and acceptor sites. Natural substitutions, deletions or insertions involving the Cp/ENII locus in the X gene can significantly alter the extent of viral replicative activity. Similar mutations occurring at other locations of Cp/ENII, and at B-cell epitope sites of the S gene are associated with failure to detect serological markers of HBV infection. HBV variation can also arise from recombination between coinfecting strains. S gene mutations that become evident following HBIG administration and HBV vaccination are all point substitutions, as are mutations in functional RT domains of the P gene after treatment with viral RT-inhibitory drugs. Widespread and long-term use of prophylactic and therapeutic agents may potentially generate serologically occult HBV variants that might become difficult to eradicate.

The cause of sarcoidosis is unknown, although mycobacteria have been implicated. We examined sarcoid tissues for human herpesvirus 8 (HHV-8) in addition to mycobacterial genomic sequences.Biopsy samples from 17 patients with sarcoidosis were studied (eight transbronchial, 27 lymph node, two skin, and two oral mucosa). We used tissues (n = 137) from 96 patients without sarcoidosis as negative controls. A nested PCR was applied to amplify a segment of open reading frame (ORF) 26 of the HHV-8 genome, and a heminested PCR was to amplify a segment of ORF 25 of HHV-8 and of the 16 S rRNA gene of mycobacteria. Differences in base sequences of the amplified fragments were resolved with single-strand conformation polymorphism and dideoxy sequencing.HHV-8 ORF 26 DNA was detected in significantly higher proportions of sarcoid than of non-sarcoid tissue samples from lung (8/8 vs 0/54; p0.0001), lymph nodes (26/27 vs 6/29; p0.0001), skin (2/2 vs 0/17; p = 0.006), and oral tissues (2/2 vs 1/13; p = 0.029). 31 (82%) of the 38 ORF 26 DNA-positive sarcoid specimens were also positive for ORF 25 DNA. For mycobacteria-like 16 S rRNA DNA, the proportion positive was significantly higher in sarcoid than non-sarcoid tissues for lymph node samples (11/27 vs 2/29; p = 0.003) but not for other tissues (lung 3/8 vs 22/54; skin 2/2 vs 15/17; and oral tissues 1/2 vs 0/13). Overall, the prevalence of HHV-8 ORF 26 sequences was higher in sarcoid tissues than in non-sarcoid tissues (p0.0001). When patients whose tissues were included in a masked phase of the study were treated as units of analysis, eight of eight sarcoidosis patients were positive for HHV-8 ORF 26 DNA, compared with three of 56 control patients (p0.0001); for mycobacteria-like sequences, three of eight sarcoidosis patients were positive, compared with four of 56 controls (p = 0.0464). The HHV-8 ORF 26 sequences, ten of which were unique, could be segregated into four groups according to peptide motifs. In seven of nine patients from whom biopsy samples were taken from various sites, different sequences were recovered. The mycobacterial sequences amplified from sarcoid tissues were also varied, but none was homologous to those of known species.Variant HHV-8 DNA sequences are found in a wide range of sarcoid but not non-sarcoid tissues. Mycobacteria-like 16 S rRNA sequences are more frequently present in sarcoid lymph nodes and not in other tissue types, but do not indicate infection by a particular mycobacterial species.

A 210-bp DNA segment specific to the human herpesvirus 8 (HHV-8) genome was amplified by nested polymerase chain reaction from 10 of 14 archived oral biopsy samples of HIV-positive patients in London who had no evidence of oral Kaposi's sarcoma (KS). Various oral sites were represented. Oral tissues from 20 general dental patients not known to be HIV-infected were negative. When DNA sequences of these products were compared with sequences derived from 5 oral KS tissues of AIDS patients in London and 10 skin biopsies of Italian patients with Mediterranean KS (total number of positive tissues = 25), 11 were found to be unique. DNA and predicted peptide motifs of these sequences were also different from those in 28 of 36 HHV-8-positive lesions previously reported from American and African patients. HHV-8 is tropic for the oral mucosa of HIV-infected persons, and HHV-8 variants, though diverse, may be geographically restricted.

IgG and IgA antibodies with specificities to a latent and a lytic antigen of human herpes virus 8 (HHV-8) were detectable in the saliva and serum of eight patients whose Kaposi's sarcoma had regressed, seven of whom were HIV-1 infected. The measurement of antibody-specific activity and secretion rate, and the detection of secretory IgA all indicate anti-HHV-8 antibody activity in saliva. The specific humoral responses possibly influence mucosal replication of HHV-8, and in turn, that of HIV.

The detection of compensatory mutations that abrogate negative fitness effects of drug-resistance and vaccine-escape mutations indicates the important role of epistatic connectivity in evolution of viruses, especially under the strong selection pressures. Mapping of epistatic connectivity in the form of coordinated substitutions should help to characterize molecular mechanisms shaping viral evolution and provides a tool for the development of novel anti-viral drugs and vaccines. We analyzed coordinated variation among amino acid sites in 370 the hepatitis B virus (HBV) polymerase sequences using Bayesian networks. Among the HBV polymerase domains the spacer domain separating terminal protein from the reverse-transcriptase domain, showed the highest network centrality. Coordinated substitutions preserve the hydrophobicity and charge of Spacer. Maximum likelihood estimates of codon selection showed that Spacer contains the highest number of positively selected sites. Identification of 67% of the domain lacking an ordered structure suggests that Spacer belongs to the class of intrinsically disordered domains and proteins whose crucial functional role in the regulation of transcription, translation and cellular signal transduction has only recently been recognized. Spacer plays a central role in the epistatic network associating substitutions across the HBV genome, including those conferring viral virulence, drug resistance and vaccine escape. The data suggest that Spacer is extensively involved in coordination of HBV evolution.

SUMMARYThe aim of this study was to estimate the amount of childhood hepatitis B virus transmission in children born in the UK, a very low-prevalence country, that is preventable only by universal hepatitis B immunization of infants. Oral fluid specimens were collected from schoolchildren aged 7–11 years in four inner city multi-ethnic areas and tested for the presence of antibody to hepatitis B core antigen (anti-HBc). Those found positive or indeterminate were followed up with testing on serum to confirm their hepatitis B status. The overall prevalence of anti-HBc in children was low [0·26%, 95% confidence interval (CI) 0·14–0·44]. The estimated average annual incidence of hepatitis B was estimated to be 29·26/100 000 children (95% CI 16·00–49·08). The total incidence that is preventable only by a universal infant immunization programme in the UK was estimated to be between 5·00 and 12·49/100 000. The study demonstrates that the extent of horizontal childhood hepatitis B virus transmission is low in children born in the UK and suggests that schools in the UK are an uncommon setting for the transmission of the virus. Targeted hepatitis B testing and immunization of migrants from intermediate- and high-prevalence countries is likely to be a more effective measure to reduce childhood transmission than a universal infant immunization programme.

A new IgG antibody avidity test for hepatitis C virus (HCV) has been developed and was validated using sera from 12 renal dialysis patients infected with HCV. In primary HCV infection low avidity antibody (mean avidity index 24%) was detected within 50 days of seroconversion whereas in long-term infection (at least 300 days after seroconversion), the mean avidity index was high (88%); in five patients, the avidity index was shown to increase rapidly as time elapsed after primary infection, whereas immunosuppressive therapy was found to delay maturation of the immune response in two further patients. The assay was then employed to confirm that a spurious outbreak of primary HCV infection in eight bone marrow transplant patients was explicable by passive acquisition of high avidity anti-HCV after intravenous immunoglobulin therapy. It is concluded that this avidity test will have an important role in the investigation of HCV infection in patients.

Hepatitis B virus (HBV) DNA from regions coding for surface and core antigens were amplified and radiolabeled from hepatitis B surface antigen (HBsAg)-positive serum samples by polymerase chain reaction, heat-denatured, and analyzed for conformation-dependent polymorphisms by gel electrophoresis under nondenaturation conditions. Analysis of serum samples representative of diverse and identical HBsAg subtypes showed a wide range of autoradiographic banding patterns, each unique to the specimen. Serial samples of a long-term carrier showed relative stability of banding patterns over time. Epidemiologic analyses using this procedure showed banding patterns of case subjects to be identical to those of persons implicated as the source. This facile and discriminatory approach to the differentiation of viral strains should be useful in the study of HBV transmission.

Differentiation between acute and chronic HCV infections is clinically important given that early treatment of infected patients leads to high rates of sustained virological response. Analysis of HVR1 sequences (n=2179) from samples obtained from patients with acute (n=49) and chronic (n=102) HCV infections showed that intra-host HVR1 diversity was 1.8 times higher in patients with chronic than acute infection. Analysis of molecular variance showed significant differences between sequences from acute and chronic patients. We found statistically significant differences in polarity, volume and hydrophobicity of amino acids at 10 HVR1 positions. A classification model constructed using the 10 positions in HVR1 distinguished between acute and chronic cases with accuracy of 88% in cross-validation experiments. The results indicate that progression from acute to chronic stage of HCV infection is accompanied by characteristic changes in amino acid composition of HVR1, suggesting a substantial regularity of the intra-host HVR1 evolution.

AIM: To determine if a scheme for validating enzyme immunoassay (EIA) results could be devised that did not require costly and methodically elaborate supplemental assays. METHODS: Samples (n = 525) from patients with haemophilia A, leukaemia, and chronic liver disease and at increased risk of hepatitis C virus infection were tested by EIA-1 (Ortho Diagnostics), an assay which uses recombinant HCV fusion proteins as antigens, and by EIA-2 (United Biomedical), an assay based on synthetic HCV oligopeptide antigens. RESULTS: Samples (n = 193) were repeatedly reactive in both EIAs. Of these, 190 (98%) yielded reactivities in both of two supplemental assays used, one an immunoblot assay (RIBA) using recombinant HCV polypeptides similar to EIA-1 antigens, and the other a neutralisation EIA (EIA-2N) based on antigenic competition with HCV peptides similar to EIA-2 antigens. The three samples not reactive in supplemental tests exhibited low EIA optical density (OD) values (signal/cutoff ratios of less than 3). Hence, all specimens reactive and yielding high OD values in both EIAs were also reactive in supplemental assays. Twenty four samples were reactive in EIA-1 only and nine (38%) of these were reactive in RIBA. Fourteen of the 15 (93%) specimens reactive in EIA-1 but not RIBA were derived from patients with chronic liver dysfunction. Two samples were reactive in EIA-2 only, of which one was reactive in EIA-2N and none in RIBA. CONCLUSIONS: Compared with EIA-2, EIA-1 yielded more validated reactive samples and resulted in more non-validated reactivities. It is therefore suggested that for clinical diagnosis: (i) EIA-1 be used for anti-HCV testing and EIA-2 for validation of EIA-1 reactivities; (ii) samples concordantly reactive in EIA-1 and EIA-2 and displaying high OD readings be considered HCV antibody positive without supplemental testing; (iii) supplemental testing by RIBA be limited to samples reactive in EIA-1 but equivocal or unreactive in EIA-2 and those concordantly reactive but exhibiting low absorbance readings.

The presence and distribution of Epstein-Barr Virus receptors (EBVR's) on a range of normal (n = 18), dysplastic (n = 10) and malignant (n = 20) oral mucosa were studied by immunocytochemical methods using the monoclonal antibodies (MAb's) HB5 and B2. EBVR's were demonstrated as membrane staining of the spinous layers of normal non- and parakeratinized epithelium, indicating that EBVR's are differentiation-linked. This distribution was retained in dysplastic epithelium. Tissue from oral squamous cell carcinomas (SCC's) showed variable reactivity of only a few cells scattered randomly within the samples. Furthermore, a sensitive in situ hybridization (ISH) technique was used to determine if Epstein-Barr virus (EBV) was present in normal (n = 15) and oral squamous cell carcinoma tissue (n = 20). No EBV DNA was demonstrated within either normal or malignant epithelium, suggesting that the virus does not persist in normal oral stratified squamous epithelium nor is there any evidence for a role in oral carcinogenesis.

Although autochthonous hepatitis E has been reported in developed countries, its extent and nature in the United Kingdom are unclear. The aim of the present study was to report the natural history, lifestyle risk factors and molecular epidemiology of autochthonous hepatitis E infection in southwest England. Three hundred and thirty-three patients with unexplained hepatitis were tested for markers of hepatitis E virus (HEV) infection over a 7-year period. HEV RNA isolated from the cases was amplified and characterized. Of the 333 patients, 21 had autochthonous hepatitis E. Patients were middle-aged or elderly and males were more commonly affected. Clinical manifestations ranged from asymptomatic infection to severe hepatitis. Of the 21 patients, 20 recovered within 6 weeks. None of the cases had travelled to an area endemic for HEV. None of the patients were vegetarian and all ate pork. Of the 21 cases, 20 occurred in the spring, summer and autumn months. All polymerase-chain-reaction-confirmed cases carried HEV genotype 3, which bore close sequence homology to HEV circulating in UK pigs. In the United Kingdom, autochthonous hepatitis E may be more common than previously recognized. Although the mode of transmission remains to be determined, it may be a zoonosis with pigs as a reservoir. Hepatitis E should be considered a public health issue in the United Kingdom.

Polymorphisms along the hepatitis B virus (HBV) genome have an impact on disease outcome, sensitivity to antiviral treatment, escape from vaccination, and laboratory diagnosis. We have designed a diagnostic tool based on duplex amplification of the whole HBV genome and a high-density DNA chip designed to detect 245 mutations, 20 deletions, and 2 insertions at 151 positions and to determine the genotype of the virus in serum. Assay performances were evaluated with 170 samples, characterized by determination of viral load and sequencing of the Pol, S, and precore genes and the basal core promoter. One hundred fifty-three samples (90%) could be amplified and analyzed by the chip. Only two samples with more than 103genome copies/ml could not be analyzed. Genotype had no impact on analytical sensitivity. Reproducibility studies showed no difference between repeats for codon and genotype determination. Genotype determination by sequencing and the chip were concordant in 148 of 151 samples. Twelve thousand one hundred sixty-one codons were analyzed by both techniques. Only 89.4% could be determined by sequencing, and among the remaining 11,335 codons, 92.8% were identical by sequencing and the chip. Failures to identify an amino acid by the chip were mainly due to reduced hybridization efficiency attributed to unexpected polymorphisms. Optimization of the chip-based reagent for the analysis of the HBV genome is ongoing. This first evaluation showed that DNA chip technology can provide important information in relation to the clinical management of chronic hepatitis B.

A 44-year-old lady was diagnosed with acute hepatitis C virus (HCV) infection 8 weeks after hysterectomy at which the attending anesthetist was known to be hepatitis C seropositive. Comparative nucleotide sequence analysis and phylogenetic comparison proved that transmission had occurred from the anesthetist to the patient. The patient had received general anesthesia with endotracheal intubation and peripheral intravenous cannulation. No exposure-prone anesthetic procedures had been performed. This is the first case described in UK involving transmission from an anesthetist to a patient during anesthesia where no exposure prone procedures were carried out. It is the first example in which the anesthetist was known to be seropositive for hepatitis C prior to the operation.

Human herpesvirus 8 (HHV-8) open reading frame K1 sequences amplified from the urine of 5 of 78 (6.4%) infected people in Malawi were monotypic. In two people, urinary and oral sequences were genotypically different. Comprehensive evaluation of HHV-8 transmission may require characterization of HHV-8 shed both in urine and orally.

Background/Aims Lamivudine is an antiviral drug that is used to treat hepatitis B virus (HBV) infection. Long-term therapy does not completely suppress viral replication, and resistant mutants emerge. Resistance is mediated by changes in the tyrosine-methionine-apartate-aspartate (YMDD) motif in the catalytic site of the HBV polymerase gene. We describe a method to detect and quantify mutant viral populations using amplification refractory mutation system (ARMS) PCR. Methods We developed a real-time ARMS-PCR to detect point mutations in the polymerase gene. Using real-time PCR (LightCycler) with a ResonSense probe, PCRs were performed using clones of the HBV polymerase gene containing the different YMDD mutations. Dilution series of the templates were made and tested against each of the primer pairs. This method was applied to quantify mutant virus in patient serum samples. Results As little as 0.01% mutant DNA in 10 5 –10 9 copies wild-type DNA were detected. The method is more sensitive than amplicon sequencing, which is the current method of mutant determination in the YMDD motif. Conclusions This study demonstrates a rapid, highly sensitive and reproducible method of quantifying mutant HBV virus in lamivudine treated patients. It can be used to monitor patients before and during lamivudine therapy.

AIM--To evaluate the performance of three newly introduced enzyme immunoassays (EIAs) for hepatitis C virus (HCV) antibodies, based on synthetic oligopeptides as antigens. METHODS--Referred serum samples (n = 173) from people representing groups at high risk of HCV infection were studied. An EIA based on second generation recombinant polypeptide antigens was used for comparison. EIA reactivities were validated by testing repeatedly reactive samples in two recombinant antigen based immunoblot assays. RESULTS--In samples from patients with liver dysfunction and those with bleeding disorders sensitivity of the three peptide based EIAs, manufactured by Innogenetics NV, Biokit SA, and United Biomedical Inc., were all 93%; specificity and efficiency were all greater than 95%. In samples from blood donors (previously tested as positive by the Ortho and Abbott Second Generation EIA) specificity, sensitivity, and efficiency were 95% or greater in all three peptide assays. Sensitivity, specificity, and efficiency of the recombinant antigen based Ortho Second Generation EIA were 100%, 89%, and 93%, respectively, in sera of patients with liver disease and those with bleeding disorders; and 100%, 43%, and 83%, respectively, in prescreened samples from blood donors. CONCLUSION--The peptide EIAs are more specific but less sensitive than the Ortho EIA. Peptide based EIAs should be useful in validating the specificity of Ortho EIA reactivities.

Oral hairy leukoplakia (OHL) and Kaposi's sarcoma (KS) are commonly encountered in the HIV-infected patient. A unique feature of OHL is non-cytolytic high level of replication of Epstein-Barr virus (EBV) in the glossal epithelium. The expression of viral-encoded anti-apoptotic proteins concomitant to replicative proteins probably underlies this phenomenon. The question of whether OHL arises from activation of EBV latent in the tongue, or from superinfection by endogenous EBV shed via nonglossal sites or by exogenous EBV remains unresolved. Human herpesvirus 8 (HHV8) is now seen as necessary but not sufficient cause of KS. Expression of HHV8-encoded oncogenic proteins in endothelial cells probably explains the aberrant proliferation of these cells in KS lesions. Studies into why KS is so commonly observed at the palate in HIV-infected patients may provide important clues to its pathogenesis.

The effect of human immunodeficiency virus-1 protease inhibitors on the frequency of human herpesvirus 8 DNA detection from peripheral blood of human immunodeficiency virus-positive persons was evaluated. Thirty-three human immunodeficiency virus-seropositive male patients were studied longitudinally. DNA from open reading frame 26 of the human herpesvirus 8 genome was amplified by the polymerase chain reaction from the CD45+ fraction of peripheral blood before and after the introduction of protease inhibitor therapy. Human herpesvirus 8 IgG status, CD4+ cell counts, and human immunodeficiency virus-1 plasma viral load were also assessed before and after therapy. When both reverse transcriptase inhibitor and protease inhibitor treatment were introduced at the same time, there was an increase in CD4+ T cell counts (P=0.0041), a decrease in human immunodeficiency virus plasma load (P=0.0584), and a decrease in the detection rate of human herpesvirus 8 DNA (P=0.0077). Introducing protease inhibitor to patients already receiving reverse transcriptase inhibitor treatment was associated with an increase in CD4+ T cell counts (P=0.0003), a decrease in human immunodeficiency virus plasma viral load (P=0.0911), and a decrease in the human herpesvirus 8 detection rate (P=0.0412). No significant changes in the titters of anti-human herpesvirus 8 IgG were observed. Treatment with human immunodeficiency virus-1 protease inhibitors is therefore associated with the clearance of human herpesvirus 8 DNA from peripheral blood of human immunodeficiency virus-infected patients. The concomitant decrease in the human immunodeficiency virus plasma load and increase in the peripheral CD4+ cell count suggest that an amelioration in the immune defect following reduction in the burden of human immunodeficiency virus-1 infection is responsible for the clearance of human herpesvirus 8 by protease inhibitors.

The existence of HBV as quasispecies is thought to be favoured by the infidelity of HBV RT, which would account for the emergence of the many natural mutants with point substitutions. RT infidelity may also underlie the hypermutation phenomenon. Indeed, the oft-reported point mutation in the preC gene that leads to failure of HBeAg synthesis may be driven by a hypermutation-related mechanism. The presence of mutants with deletions and insertions involving single nucleotides and oligonucleotides at specific positions in the genome, and of mutants with deletions of even longer stretches particularly in the C gene, suggests that other mutagenic mechanisms operate. Candidates include slippage during mispairing between template and progeny DNA strand, the action of cellular topoisomerase I, and gene splicing using alternative donor and acceptor sites. Natural substitutions, deletions or insertions involving the Cp/ENII locus in the X gene can significantly alter the extent of viral replicative activity. Similar mutations occurring at other locations of Cp/ENII, and at B-cell epitope sites of the S gene are associated with failure to detect serological markers of HBV infection. HBV variation can also arise from recombination between coinfecting strains. S gene mutations that become evident following HBIG administration and HBV vaccination are all point substitutions, as are mutations in functional RT domains of the P gene after treatment with viral RT-inhibitory drugs. Widespread and long-term use of prophylactic and therapeutic agents may potentially generate serologically occult HBV variants that might become difficult to eradicate.

Hepatitis C virus (HCV) genotypes were assigned to 567 individuals by restriction fragment length polymorphism analysis of the 5' noncoding region of the HCV genome following reverse transcription-polymerase chain reaction. The groups of individuals in this study included hemophilia patients, injecting drug users (IDUs), blood donors, antenatal patients, those attending genitourinary medicine (GUM) clinics, and patients with chronic liver disease, all from England and Wales. The majority of HCV infections were types 1a (32%), 1b (15%), or 3a (37%). The genotype distribution in individual groups was similar to the overall genotype distribution except for hemophilia patients, in whom the frequencies were 1a (39%), 1b (23%), and 3a (21%). With the exception of hemophilia patients, subpopulations in England and Wales appear to share common modes of HCV transmission. There is a need for continued surveillance to monitor the spread of possibly more virulent or drug-resistant HCV genotypes.

Investigation of the death of a 77 year old woman from acute hepatitis B infection revealed that she had undergone orthopaedic surgery two and a half months earlier. The surgeon was found to be a hepatitis B surface antigen carrier, hepatitis B e antigen (HBeAg)negative, but with antibodies to HBeAg. Viruses from the surgeon and the patient were identical, apart from a single nucleotide substitution. Both had a precore mutation, which prevents expression of e antigen. A look back exercise was undertaken on the patients operated on by the surgeon during the previous year. The surgeon had performed exposure prone procedures on 253 patients, 188 of whom provided blood specimens. No HBsAg carriers were detected, and no serological markers of recent transmission were found.

A retrospective case-control study was conducted to determine why some infants born full-term without obstetric intervention to hepatitis B e antigen (HBeAg)-seropositive mothers become infected by hepatitis B virus (HBV) despite having received passive-active immunoprophylaxis. Cases and controls comprised 12 hepatitis B surface antigen (HBsAg)-seropositive infants and 22 HBsAg-seronegative infants, respectively. Infants infected by putative vaccine-escape mutants were excluded. Risk factors, after adjustment for the level of maternal viremia, were the following allelic base changes in maternal HBV:C158, A328, G365, and A479 (P = .017, .005, .003, and .005, respectively). High-level maternal viremia (i.e., > or = 10(8) genome equivalents/mL) was a significant factor only after adjustment for G365 (P = .027). HBV DNA sequences recovered from one of the cases, the case's mother, and three infected contacts all had the high-risk mutations. Specific allelic mutations in maternal HBV and level of maternal viremia are potential predictors of vertical breakthrough infection.

Hepatitis B virus (HBV) infection occurred despite full passive-active immunoprophylaxis in 20 of 321 infants born to mothers seropositive for hepatitis B e antigen. In 2 (12%) of 17 infected infants, mother-infant DNA sequence mismatches were found in a segment of the HBV S gene coding for antigenic determinants of the HBV surface antigen (HBsAg) amplified from sera by polymerase chain reaction (PCR). Point substitutions occurred in codons 120, 134, and 144 of the HBsAg polypeptide in the variant sequence of 1 infant and in codon 126 in the other; all were missense mutations. Mutant sequences could not be recovered from maternal sera by PCR cloning but were selectively generated using an amplification refractory mutation system. The frequency of potential vaccine escape mutants is therefore low, and these preexist maternally as minor variants.

Nucleotide sequence variability studies were conducted on a 263-base pair fragment of the core-coding genomic region of hepatitis B virus (HBV), amplified by the polymerase chain reaction (PCR) from three surgeons with varying circulating levels of HBV, all of whom were thought to have transmitted HBV to their patients post-surgically. DNA sequencing was applied to amplicons obtained directly from serum and those cloned into plasmid vectors, and from single HBV molecules in serum separated by a limiting dilution procedure. In one surgeon, who had a titre of approximately 3 x 10(5) genome equivalents ml-1, the direct sequence was identical to none of 29 other sequences and differed by one base substitution from the sequence amplified from the single patient he infected. In another surgeon, who had a titre of approximately 2 x 10(6) genome equivalents ml-1, the direct sequence was identical to 17 of 36 (47%) sequences; however, the sequence common to all three infected patients was identical to a unique sequence in the surgeon that differed by three base substitutions from the direct sequence. By contrast, the direct sequence in the third surgeon, who had a titre of approximately 4 x 10(7) genome equivalents ml-1, was identical to 25 of 38 (66%) sequences, and to the sequence common to all 11 infected patients. Assessment of HBV DNA sequences directly amplified from clinical specimens may not be appropriate to studies of transmission in which the source of infection harbours a relatively dilute, heterogeneous mix of viral variants.