Christopher J. Ring

Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement.

Abstract Background: The antiviral effect of anti-influenza drugs such as zanamivir may be demonstrated in patients as an increased rate of decline in viral load over a time course of treatment as compared with placebo. Historically this was measured using plaque assays, or Culture Enhanced Enzyme Linked Immunosorbent Assay (CE-ELISA). Objectives: to develop and characterise real time quantitative PCR (qPCR) assays to measure influenza A and B viral load in clinical samples, that offer improvements over existing methods, in particular virus infectivity assays. Study design: The dynamic range and robustness were established for the real time qPCR assays along with stability of the assay components. Cross validation of the real time PCR assays with CE-ELISA was performed by parallel testing of both serial dilutions of three different subtypes of cultured virus and a panel of influenza positive throat swab specimens. Results: the assays were specific for influenza A and B and the dynamic ranges were at least seven logs. The assay variability was within acceptable limits but increased towards the lower limit of quantification, which was 3.33 log10 viral cDNA copies/ml of virus transport medium (ten viral RNA copies/PCR). The components of the assay were robust enough to withstand extended storage and several freeze–thaw cycles. For the real time PCR assays the limit of quantification was equivalent to the virus infectivity cut off, which equates to a 93-fold increase in sensitivity. Conclusion: Well characterised real time PCR assays offer significant improvements over the existing methods for measuring the viral load of strains of influenza A and B in clinical specimens.

Cytolytic viruses as potential anti-cancer agents

The resistance of cancers to conventional therapies has inspired the search for novel strategies. One such approach, namely gene therapy, is based upon the introduction of genes such as those encoding suicide proteins, tumour suppressor proteins or cytokines into tumour cells by means of a genetic vector. The efficiency with which viruses transfer their genes from one host cell to another has led to the widespread use of viruses as genetic vectors. For safety reasons, such virus vectors are generally replication-defective but, unfortunately, this has limited the efficacy of treatment by restricting the number of cells to which the therapeutic gene is delivered. For this reason, the use of replication-competent viruses has been proposed, since virus replication would be expected to lead to amplification and spread of the therapeutic genes in vivo. The replication of many viruses results in lysis of the host cells. This inherent cytotoxicity, together with the efficiency with which viruses can spread from one cell to another, has inspired the notion that replication-competent viruses could be exploited for cancer treatment. Some viruses have been shown to replicate more efficiently in transformed cells but it is unlikely that such examples will exhibit a high enough degree of tumour selectivity, and hence safety, for the treatment of patients. Our increasing knowledge of the pathogenesis of virus disease and the ability to manipulate specific regions of viral genomes have allowed the construction of viruses that are attenuated in normal cells but retain their ability to lyse tumour cells. Such manipulations have included modifying the ability of viruses to bind to, or replicate in, particular cell types, while others have involved the construction of replication-competent viruses encoding suicide proteins or cytokines. Naturally occurring or genetically engineered oncolytic viruses based upon adenovirus, herpes simplex virus, Newcastle disease virus, poliovirus, vesicular stomatitis virus, weasles virus and reovirus have been described. The results of animal studies are encouraging and a number of viruses are now being evaluated in clinical trials.

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 prevalance 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.

Hepatitis C viraemia in United Kingdom blood donors: a multicentre study

Of 10,633 blood donations tested in three regional blood transfusion centres with two commercial first generation screening assays for antibodies to the hepatitis C virus (HCV), 65 (0.61%) were found to be repeatedly reactive in one or both assays. Five of the 65 were confirmed positive by recombinant immunoblot assay (Ortho RIBA‐2) and a further 4 were judged indeterminate. All 5 RIBA‐2 positive donations and 1 of the 4 RIBA‐2 indeterminates were shown to be viraemic by HCV‐RNA polymerase chain reaction (PCR) assays performed at three independent reference laboratories. The remaining 56 screen test reactive donations proved negative by RIBA‐2 and, with 1 exception, negative by PCR. We conclude that while first generation anti‐HCV screening assays generate a high proportion of false reactions when screening low prevalence populations, results of the RIBA‐2 confirmatory test correlate well with PCR findings and thus indirectly with both hepatitis C viraemia and infectivity.

The B cell-immortalizing functions of Epstein-Barr virus

The Epstein-Barr virus (EBV) is a human B lymphotropic herpesvirus carried in a persistent state by over 90% of the worlds population. Primary infection occurs by the oral route, generally in early childhood, and is asymptomatic. When infection is delayed until adolescence or later, infectious mononucleosis results in approximately 50% of cases. Although it has been postulated that the oropharynx provides a reservoir where circulating B cells can be infected during transit through this anatomical site, evidence has been presented suggesting that EBV can also persist in the B cell (Gratama et al., 1988; Yao et al., 1989 a, b). Cellular immune mechanisms are believed to be of the greatest importance in the suppression of infected cell proliferation, and the balance between continual infection and suppression results in a life-long carrier state (reviewed by Rickinson, 1986).

Adenoviral-mediated Cre expression effectively suppresses GlyT1 binding in the thalamic area of GlyT1 conditional knock-out mice

To properly understand the function of genes of neurological interest, in vivo manipulation in the adult is essential, particularly when the target gene is involved in brain development. Moreover, since the physiological effects of target protein may be region-specific, targeting a distinct brain region could be required to dissect these effects in specific brain locations. Infection of somatic tissues of transgenic mice bearing loxP-flanked gene sequences with a viral vector expressing Cre recombinase provides a means of allowing flexible spatio-temporal control of target gene expression. Viral vector-mediated Cre expression could be used to mediate localized gene modulation in a specific brain region. In the present study this technology was applied to the glycine transporter type-1 (GlyT1) protein which is responsible for the uptake of synaptic glycine in the forebrain and has been implicated as a therapeutic target for the treatment of schizophrenia. Since GlyT1 is widely expressed in glial cells, we employed an adenoviral-based vector (Ad5) to deliver Cre protein, due to the preferentially transduction of glial cells by adenoviral vectors in rodent brain. We show significant reduced GlyT1 binding specifically in the thalamic area of conditional GlyT1 (GlyT1c) transgenic mice injected with Ad5-Cre virus, as measured by GlyT1 autoradiography. In conclusion, we demonstrated the validity of viral vector-mediated delivery of Cre to loxP targeted transgenic mice as a novel strategy to investigate target gene function in selected subregions of the adult brain, which provides a valuable technique to investigate gene function both in normal physiology and in disease models.

Alternative Splicing Determines The Carboxy Terminus of the Epstein—Barr Virus Nuclear Antigen 5 Species Expressed in the Burkitt's Lymphoma Cell Line Daudi

The Daudi strain of Epstein-Barr virus (EBV) possesses a genomic deletion, relative to the B95-8 EBV prototype, that removes the entire Epstein-Barr virus nuclear antigen 2 (EBNA2) open reading frame (ORF) and the sequences encoding the carboxy terminus of EBNA5. Immunoblot analysis carried out in this study indicates that two species of EBNA5 (31K and 37K) are expressed in Daudi cells. Nucleotide sequence analysis of Daudi cDNA clones has confirmed that, as a consequence of the genomic deletion, exons usually appearing further downstream in EBNA messages (exons U or HF) are spliced directly onto the truncated EBNA5 ORF. Furthermore, the use of alternative splicing suggests that the two EBNA5 species expressed in Daudi cells possess different carboxy termini.