No evidence of transfusion transmission of Adenovirus and Epstein-Barr virus infections in paediatric recipients post-bone marrow transplant.

Adenovirus and Epstein-Barr virus can cause significant morbidity and mortality in paediatric patients post-bone marrow transplant. The source of infection is thought to be either reactivation of latent viruses or primary infection. We have investigated whether transfusion of blood components from viraemic donors could provide a route of primary infection in these patients and sought the prevalence of viraemia in the blood donor population from England. In 32 linked donor/recipient samples and 300 unselected blood donors, we found no evidence to suggest that these infections in paediatric bone marrow transplant recipients had been acquired from transfused blood components.

Therapy-induced clearance of HCV core antigen from plasma predicts an end of treatment viral response.

During viral assembly, viral proteins are released into plasma and can be used to infer viral load. The Architect hepatitis C virus (HCV) core antigen (Ag) assay is a potential alternative to HCV RNA quantification for measuring response to therapy and predicting an end of treatment viral response (EOTR). The HCVp22Ag assay was used to infer viral load in 68 window RNA-containing samples and in 284 samples from baseline to week 14 of ribavirin/interferon treatment in 23 patients with EOTR including three who relapsed, 20 not achieving EOTR and 11 controls. HCV Ag and RNA correlated well (r = 0.86) with linear dose responses on dilution. In patients on therapy and control patients, plasma HCV antigen was detected in 51 of 54 with an interpolated LOD cut off between 10(3) and 10(4) RNA IU/mL. Plasma HCV antigenaemia and plasma RNA levels were significantly different in EOTR from non-EOTR patients at 3 days after treatment start and all times thereafter. Positive and negative EOTR predictive values for HCV RNA >2 log drop and HCV Ag loss at 12 weeks were 70% and 74%, 85% and 93% respectively. HCV Ag reactivity has a linear dose response independent of genotype and correlates well with HCV RNA. The failure to clear HCV Ag is as accurate as the failure to clear HCV RNA at twelve weeks into therapy in predicting the likelihood of failure to achieve EOTR. HCV Ag potentially offers a convenient alternative to RNA measurement for defining a futility flag in HCV therapy.

Can plasma HHV8 viral load be used to differentiate multicentric Castleman disease from Kaposi sarcoma?

We measured plasma human herpesvirus 8 (HHV8) DNA load in consecutive patients presenting with HIV-associated multicentric Castleman disease (MCD) and in contemporaneous patients who had Kaposi sarcoma (KS), lymphoma or other diagnoses. All 11 patients with MCD had detectable plasma HHV8 DNA compared with 18 (72%) of 25 patients with KS, none with lymphoma and one of 38 patients with other diagnoses. Detectable plasma HHV8 DNA levels were higher among MCD patients, median (interquartile range [IQR]) = 43,500 (5200-150,000) copies/mL, when compared with those with KS, median (IQR) = 320 (167-822) copies/mL and those with lymphoma and other diagnoses (one-way analysis of variance; P = 0.0303). Using receiver operating characteristic analysis, a cut-off of >1000 copies HHV8 DNA/mL of plasma helped to discriminate between MCD and other diagnoses, with a specificity of 94.7% and a negative predictive value of 97.3%. The level of HHV8 viraemia, while not diagnostic, may aid discrimination between patients with MCD and those with KS and other systemic illnesses.

Distribution and quantification of human herpesvirus 6 in multiple sclerosis and control brains.

Multiple sclerosis (MS) is thought to be precipitated by environmental factors, potentially including viruses, in genetically susceptible individuals and recently human herpesvirus 6 (HHV-6) has been associated with the disease. We have analysed post mortem brain for the presence, variant type and quantity of HHV-6 by PCR. A total of 124 samples from seven anatomically defined regions of brain were tested from MS cases and controls. HHV-6 DNA was detected in 41% and 44% of MS case and control samples. The median viral loads were 11 and 9 genome copies/microg DNA in cases and controls respectively and the viral load was not increased in lesions. Except in one instance, the HHV-6 DNA detected was variant B. There was no apparent difference in the distribution of HHV-6 DNA in the brains of MS cases versus controls, nor between normal appearing and lesional tissue in MS cases. Periventricular regions of the brain were more frequently positive for HHV-6 DNA in both MS cases and controls, although this difference was not statistically significant. These studies confirm the neurotropism of HHV-6 but do not demonstrate differences in the distribution, variant type or quantity of HHV-6 in brains from patients with MS versus controls.

Infrequency of detection of particle-associated MSRV/HERV-W RNA in the synovial fluid of patients with rheumatoid arthritis.

OBJECTIVES: To determine whether the recently identified multiple sclerosis-associated retrovirus, MSRV, is detectable in the serum and synovial fluid of patients with rheumatoid arthritis (RA). METHODS: A reverse transcription-polymerase chain reaction (RT-PCR) assay was used to seek evidence of particle-associated MSRV/HERV-W RNA in the plasma and synovial fluid of patients with RA and controls. Stringent precautions were taken to avoid detection of contaminating human genomic DNA and cellular RNA sequences. RESULTS: Thirty-seven plasma samples were tested (20 from RA patients and 17 from controls) but none had detectable MSRV/HERV-W RNA. Synovial fluid samples were available from nine patients with RA and 10 controls. Particle-associated MSRV/HERV-W RNA was reproducibly detected in two of nine synovial fluid samples from RA patients and in one control sample. The identity of RT-PCR products was confirmed by sequencing. CONCLUSION: MSRV/HERV-W RNA sequences are detectable in the synovial fluid of a small proportion of RA patients, but this phenomenon may not be specific to RA.

Detection of reverse transcriptase activity in the serum of patients with motor neurone disease.

The recognition that both human and murine retroviruses can cause motor neurone disease-like syndromes has raised the possibility that a retrovirus may be involved in the aetiology of motor neurone disease. This possibility was explored by looking for evidence of reverse transcriptase in the serum of motor neurone disease patients. Sera from 56 patients with motor neurone disease and 58 controls were tested by the product-enhanced reverse transcriptase assay, a technique that is approximately a million fold more sensitive than conventional reverse transcriptase assays and capable of detecting very low numbers of retroviral particles. Cell-free reverse transcriptase activity was detected in the serum of 33 of the 56 motor neurone disease patients (59%) but in only 3 of the controls (P < 0.00001). The reverse transcriptase activity was detectable in the presence of a large excess of an effective inhibitor of human cellular DNA polymerases and was therefore tentatively considered to be compatible with a retroviral origin. The reverse transcriptase activity, however, was not found to be due to the presence of known human exogenous retroviruses including HIV-1, HIV-2, HTLV-I, HTLV-II, HRV-5 or human foamy virus, as assessed by PCR-based assays. Further investigations will be required to determine the source of the reverse transcriptase activity observed in these motor neurone disease patient sera.

Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis. The Collaborative Research Group on Multiple Sclerosis.

The partial molecular characterization of multiple sclerosis (MS)-associated retrovirus (MSRV), a novel retrovirus previously called LM7, is reported. MSRV has been isolated repeatedly from leptomeningeal, choroid plexus and from Epstein-Barr virus-immortalized B cells of MS patients. A strategy based on reverse transcriptase PCR with RNA-purified extracellular virions yielded an initial pol fragment from which other regions of the retroviral genome were subsequently obtained by sequence extension. MSRV-specific PCR primers amplified a pol region from RNA present at the peak of reverse transcriptase activity, coinciding with extracellular viral particles in sucrose density gradients. The same sequence was detected in noncellular RNA from MS patient plasma and in cerebrospinal fluid from untreated MS patients. MSRV is related to, but distinct from, the endogenous retroviral sequence ERV9. Whether MSRV represents an exogenous retrovirus with closely related endogenous elements or a replication-competent, virion-producing, endogenous provirus is as yet unknown. Further molecular epidemiological studies are required to determine precisely the apparent association of virions containing MSRV RNA with MS.

Development of a pan-retrovirus detection system for multiple sclerosis studies.

INTRODUCTION: Although recent claims implicating HTLV-1 in multiple sclerosis (MS) have been refuted, several reports suggest that another, hitherto uncharacterised, retrovirus may be involved. We have developed and applied a novel PCR-based strategy to explore this possibility. METHODS: Degenerate oligonucleotides were used in a semi-nested format to amplify, from reverse-transcribed RNA, a region of the pol gene which is well conserved amongst all known retroviruses. RESULTS: The 'pan-retrovirus' detection system was shown to be capable of detecting diverse retroviruses including human lentivirus, human oncovirus, simian D-type virus and murine oncovirus. The 'pan-retrovirus' technique identified a novel retroviral sequence, designated MSRV-cpol, in the serum of an MS patient and also in purified virions from MS patient-derived tissue cultures. Sequence comparisons suggest that in the pol gene MSRV is related (approximately 75% homology) to the endogenous retroviral element ERV9. CONCLUSION: These findings lend further support to the concept of retroviral involvement in MS.

Cell cultures and associated retroviruses in multiple sclerosis. Collaborative Research Group on MS.

Retroviral particles associated with reverse transcriptase (RT) activity in cell-cultures from MS patients have been reported by different groups. Cell-cultures have been used for the study and characterization of the corresponding retroviral genome which we have shown is related to ERV9 in the pol region. Previously unpublished details of a study with monocyte cultures are presented together with observations on leptomeningeal and choroid-plexus cultures. The generation of self-transformed cultures after inhibition of interferon, followed by the loss of retroviral expression and recurrent apoptosis, is analyzed. Retroviral particles with RT-activity are produced in monocyte cultures with an apparent correlation with MS disease activity. However, though leptomeningeal and choroid plexus cells from MS can be passaged for a limited period, their evolution in vitro is not compatible with stable retroviral expression. These culture limitations greatly hampered progress on the elucidation of the retroviral genome sequence.

Characterisation of a panel of monoclonal antibodies raised against recombinant HCV core protein.

Hepatitis C virus (HCV) has as yet no practical culture system so any antigen or antibody studies must be carried out using recombinant antigens. In this study, HCV core sequence was amplified by PCR, inserted into pRSET, and expressed in E. coli. The resultant core protein was purified using nickel affinity chromatography which bound the six histidine tag attached to the N-terminus of the protein. After elution in imidazole buffer, the core protein was used to immunise Balb/c mice and monoclonal antibodies against HCV core were raised. Six monoclonals were examined in a variety of assays. All of them recognised the p27 kDa protein which they were raised against and 2D2 and 3D7 recognised the core component of an HCV Recombinant Immunoblot Assay (RIBA). None of the antibodies recognised the linear peptides in an Innolia HCV assay. 2D2 showed cytoplasmic granular staining in 1-5% of cells in frozen section of HCV infected livers. Cross-competition assays between themselves and human anti-HCV core positive sera divided the antibodies into two main groups (I and II), with a sub-division of group I into a and b. Group I antibodies were unable to be inhibited by human anti-HCV sera whereas group II antibodies were inhibited by these sera (up to 62%). Epitopes recognised by all the monoclonals were probably conformational with the group I epitope being located within the first 105 amino acids of the core sequence and the group II epitope between amino acids 105 and 160.

Prevalence of antibodies to human T cell leukaemia/lymphoma virus in blood donors in north London.

OBJECTIVES: To determine the prevalence of antibodies to the human T cell leukaemia/lymphoma viruses (HTLV-I and HTLV-II) in blood donors in north London in order to assess the economic impact and the logistic effects that routine screening would have on the blood supply. DESIGN: All donations collected by the north London blood transfusion centre between January 1991 and June 1991 were screened for antibodies to HTLV-I and HTLV-II by modified, improved Fujirebio gel particle agglutination test. Positive samples were titrated and retested as necessary. SUBJECTS: 96,720 unpaid volunteers, who gave 105,730 consecutive donations of blood and plasma. SETTING: North London blood transfusion centre. MAIN OUTCOME MEASURE: Observed numbers of donors confirmed to be seropositive for HTLV by reference laboratories. RESULTS: Of 2622 (2.5%) initially reactive samples, 414 (0.4% of all samples) gave a titre of > or = 1 in 16 on the modified agglutination test. Thirty five of the 414 serum samples yielded positive results on one of two enzyme linked immunosorbent assays (ELISA (Cambridge Biotech and Abbot)), and none of these results were confirmed by either reference laboratory. Five samples yielded positive results on both ELISAs and all five of these were confirmed to contain antibodies to HTLV. One of the five contained antibodies to HTLV-II and the others antibodies to HTLV-I. Four seropositive donors were white women whose only risk factor for infection was sexual contact. The fifth (positive for antibodies to HTLV-II) was an Anglo-Caribbean man who admitted to previous misuse of intravenous drugs. CONCLUSION: The prevalence of antibodies to HTLV in blood donors in north London was one in 19,344 (0.005%). Up to 100 donors a year might be identified in the United Kingdom as being infected with HTLV, although prevalence in different regions may vary considerably.

Hepatitis C viral RNA in clotting factor concentrates and the development of hepatitis in recipients.

The polymerase chain reaction (PCR) was used to detect hepatitis C (HCV) viral sequences (HCV-RNA) in clotting factor concentrates that had been stored at 4 degrees C for 1 to 16 years. A total of 43 concentrates were tested, comprising 31 batches of factor VIII, 6 of factor IX, 2 of antithrombin III, 3 of FEIBA, and 1 of factor VII. HCV-RNA was detected in 13 of the 43 batches (30.2%). Concentrates that had not undergone viral inactivation during manufacture were significantly more likely to contain detectable HCV-RNA than concentrates that had been virally inactivated (56.3% v 14.5%, P = .006). HCV sequences were more commonly detected in concentrates made from paid donor plasma than in those made from volunteer donor plasma (44% v 11%, P = .041), and more commonly in virally inactivated concentrates with pre-1989 than with post-1989 expiration dates (50% v 0%, P = .004). Of the four batches of heat-treated products that were HCV-RNA positive, at least three transmitted non-A, non-B hepatitis (NANBH). An association between the presence of HCV-RNA in concentrates and the development of NANBH was demonstrated in nine previously untreated patients on prospective follow-up. HCV-RNA was detected in the concentrates administered to the six patients whose alanine aminotransferase (ALT) abnormalities met the diagnostic criteria for NANBH and who later seroconverted for HCV, but it was not detected in the concentrates administered to the three patients whose ALT abnormalities failed to satisfy the diagnostic criteria and who did not seroconvert. We suggest that the use of this PCR technique to monitor clotting factor concentrates derived from pooled blood may potentially contribute to product safety.

Differential diagnosis of HTLV-I and HTLV-II infections by restriction enzyme analysis of 'nested' PCR products.

A 'nested' polymerase chain reaction (PCR) assay is described which is capable of detecting single copies of human T-cell lymphotropic virus (HTLV) in genomic DNA extracted from peripheral blood mononuclear cells (PBMCs). A single set of 'nested' oligonucleotide primers, based on the highly conserved tax/rex region of the viral genome, was able to detect both HTLV-I and HTLV-II proviral sequences in clinical samples of diverse geographical origins, from the United States, Great Britain, Japan, the Caribbean, Italy, Greece, Iraq and West Africa. Rapid discrimination between HTLV-I and HTLV-II infections was achieved by restriction enzyme analysis of unpurified second-round PCR products, even in those cases in which serological assays had failed to provide a definitive result. Over a 2-year period, a total of 53 HTLV infections (37 HTLV-I and 16 HTLV-II) were identified by this technique and complete concordance with serological typing, available in 41 cases, was observed.

Hepatitis C antibody profile and viraemia prevalence in adults with severe haemophilia.

Sera from 21 patients who had received large amounts of unheated factor VIII concentrate were tested for antibodies to the hepatitis C virus (HCV) by both commercial (Ortho C100) and 'in house' ELISAs. 'In house' assays utilized recombinant structural (core) or non-structural (replicase) HCV proteins generated by a baculovirus expression system. Antibodies to HCV were detected in 100% of the sera by the core protein based ELISA but in only 62% and 19% by the C100 and replicase based ELISAs, respectively. Hepatitis C viraemia was demonstrated in 90% of the patients by in vitro amplification of the 5' non-coding region of the HCV genome. Amplification with primer sets from two other regions of the genome proved less efficient at detecting viraemia. We conclude that the prevalence of hepatitis C infection in haemophiliacs may have been underestimated previously and that almost all HCV-infected patients have evidence of on-going viral replication.

Effect of alpha-interferon therapy on hepatitis C viraemia in community-acquired chronic non-A, non-B hepatitis: a quantitative polymerase chain reaction study.

Sera from 30 patients with community-acquired, biopsy-proven chronic non-a,non-B hepatitis (NANBH) were tested for antibodies to the C100 protein of hepatitis C virus (HCV). The 20 patients who showed reactivity in this assay were followed prospectively for 6 months, during which time seven were treated with recombinant alpha-interferon. HCV RNA was detected by "nested" polymerase chain reaction (PCR) in 19 of the 20 anti-C100-positive sera taken at the onset of the study and also in five of the ten anti-C100-negative sera. Pretreatment viraemia levels ranged from 2 x 10(3) to 2 x 10(8) HCV genomes/ml. After 6 months of interferon, elevated serum alanine aminotransferase (ALT) levels had fallen to normal in four of the seven treated patients. In each case the response to interferon was accompanied by either a disappearance of or a decline (1 log to 8 log reduction) in viraemia. HCV genome titres in the three nonresponders and in the 13 untreated anti-C100-positive patients did not change significantly over this 6 month period. These findings confirm the aetiological role of HCV in community-acquired NANBH and suggest that quantitative PCR will become a valuable technique for monitoring the antiviral effect of interferon and other experimental treatments.