Margaret Duffy

CD4 T Helper Type 1 and Regulatory T Cells Induced against the Same Epitopes on the Core Protein in Hepatitis C Virus-Infected Persons

The factors that determine persistence or clearance of hepatitis C virus (HCV) infection are poorly understood. The CD4 T cell responses to the HCV core protein were examined in a cohort of women infected with a single genotype of HCV. CD4 T cells from HCV-infected patients secreted interferon (IFN)-gamma in response to peptides from 4 immunodominant regions of the core protein, and these responses were stronger in persistently infected women. Interleukin (IL)-10 was also produced by CD4 T cells from HCV-infected subjects in response to the same core peptides. Furthermore, HCV core-specific CD4 T cell clones secreted either IFN-gamma or IL-10 but not IL-4. These findings demonstrate that T helper type 1 and regulatory T cells are induced against the same epitopes on the core protein during HCV infection.

Dating the common ancestor of SIVcpz and HIV-1 group M and the origin of HIV-1 subtypes by using a new method to uncover clock-like molecular evolution

Attempts to estimate the time of origin of human immunodeficiency virus (HIV)‐1 by using phylogenetic analysis are seriously flawed because of the unequal evolutionary rates among different viral lineages. Here, we report a new method of molecular clock analysis, called Site Stripping for Clock Detection (SSCD), which allows selection of nucleotide sites evolving at an equal rate in different lineages. The method was validated on a dataset of patients all infected with hepatitis C virus in 1977 by the same donor, and it was able to date exactly the ‘known’ origin of the infection. Using the same method, we calculated that the origin of HIV‐1 group M radiation was in the 1930s. In addition, we show that the coalescence time of the simian ancestor of HIV‐1 group M and its closest related cpz strains occurred around the end of the XVII century, a date that could be considered the upper limit to the time of simian‐to‐human transmission of HIV‐1 group M. The results show also that SSCD is an easy‐to‐use method of general applicability in molecular evolution to calibrate clock‐like phylogenetic trees.

Expansion of innate CD5pos B cells expressing high levels of CD81 in hepatitis C virus infected liver.

BACKGROUND/AIMS Association of hepatitis C virus (HCV) with increased autoantibodies, mixed cryoglobulinaemia, non-Hodgkins B-cell lymphoma and increased peripheral innate (CD5(pos)) B cells suggests a role for B-lymphocytes in the pathogenesis of HCV-infection. METHODS Flow cytometry was used to estimate CD5(pos) B cell levels and CD81 co-expression in chronic HCV infection. Viral load was assessed using PCR. RESULTS We demonstrate expansion of innate B cells in HCV-infected liver from patients with fibrosis score less than stage II (39%, % of total B cells, P=0.002) and end stage HCV cirrhosis (20%, P<0.05) compared with normal liver (8%). Expression of CD81, a signal transducing molecule and putative HCV receptor, was significantly increased on peripheral blood CD5(pos) B cells compared with conventional B cells (P=0.0001). Higher levels of CD81 on CD5(pos) B cells were more dramatic in the liver of HCV-infected individuals. However, no significant difference was observed in the viral load of CD5(pos)CD81(High) B cells and CD5(neg)CD81(Low) B cells. CONCLUSIONS Increased expression of CD81 on innate B cells, a population that is expanded in the livers and peripheral blood of chronic HCV-infected patients, suggests a role in viral specific activation and clonal proliferation in chronic HCV infection.

Rapid molecular detection of the H275Y oseltamivir resistance gene mutation in circulating influenza A (H1N1) viruses.

Abstract In early 2008, drug susceptibility surveillance of influenza viruses in Europe revealed that some influenza A viruses (subtype H1N1) circulating during the winter season of 2007 and 2008 were resistant to the neuraminidase inhibitor, oseltamivir. This resistance arises due to a histidine to tyrosine substitution in the neuraminidase active site (H275Y in N1 nomenclature). Current methods to detect this mutation involve an end-point reverse transcription polymerase chain reaction followed by nucleotide sequencing. While accurate, this approach has the limitation of being time-consuming, labour-intensive and expensive. Herein we describe a one-step allelic discrimination assay which rapidly (2h) detects this resistance mutation. The sensitivity of the assay was as low as 10 copies per reaction and is capable of detecting the antiviral resistance mutation in a mixture of wild type H275 and mutant H275Y targets.

Site stripping based on likelihood ratio reduction is a useful tool to evaluate the impact of non-clock-like behavior on viral phylogenetic reconstructions

The site stripping for clock detection procedure was implemented in the recently developed maximum likelihood framework for estimating evolutionary rates and divergence times in measurably evolving populations. The method was used to investigate the effect of rate variability on estimating divergence times in non-clock-like trees for human immunodeficiency viruses and hepatitis C viruses. We validate our approach by comparing dated coalescent nodes in molecular phylogenies with known dates of transmission. Our method was able to rapidly recover clock-like behavior and to indicate the presence and direction of a bias when estimates of divergence times using the unstripped data were flawed.

High morbidity and mortality associated with an outbreak of influenza A(H3N2) in a psycho-geriatric facility.

In spring 2008, an influenza A subtype H3N2 outbreak occurred in a long stay psycho-geriatric ward and two wards in the intellectual disability services (IDS), part of a large psychiatric hospital. The attack rate in the index ward was 90% (18/20) for patients and 35% (7/20) for staff. It was 14% (1/7) and 17% (2/12) in the affected IDS wards for patients and 0% (0/20) and 4% (1/25) for staff. Many of the laboratory-confirmed cases did not have a fever >38 °C, a typical sign of influenza. Control measures included oseltamivir treatment for cases and prophylaxis for contacts, standard and droplet infection control precautions, active surveillance for early detection and isolation of potential cases. As a result, the outbreak did not spread throughout the hospital. Although the staff vaccination rate (10%) prior to the outbreak was low, we observed a much lower vaccine effectiveness rate in the patients (11%) than in the staff (100%) in the index ward. Vaccination of residents and staff of such facilities remains the key influenza prevention strategy.

Establishment of a national database to link epidemiological and molecular data from norovirus outbreaks in Ireland

A prospective study of norovirus outbreaks in Ireland was carried out over a 1-year period from 1 October 2004 to 30 September 2005. Epidemiological and molecular data on norovirus outbreaks in the Republic of Ireland (ROI) and Northern Ireland (NI) were collected and combined in real time in a common database. Most reported outbreaks occurred in hospitals and residential institutions and person-to-person spread was the predominant mode of transmission. The predominant circulating norovirus strain was the GII.4-2004 strain with a small number of outbreaks due to GII.2. This study represents the first time that enhanced epidemiological and virological data on norovirus outbreaks in Ireland have been described. The link established between the epidemiological and virological institutions during the course of this study has been continued and the data is being used as a source of data for the Foodborne Viruses in Europe Network (DIVINE-NET).

Kinetics of host immune responses and cytomegalovirus resistance in a liver transplant patient

Among solid organ transplant (SOT) recipients, donor‐seropositive/recipient‐seronegative (D+/R−) cytomegalovirus (CMV) status is associated with the highest risk of ganciclovir‐resistant CMV disease, which has been reported for patients receiving oral ganciclovir but not valganciclovir prophylaxis. We report a case of CMV breakthrough infection in a D+/R− liver transplant patient while he was receiving oral valganciclovir. Forty samples collected over 6 months were analyzed for the CMV viral load, lymphocyte counts, cytokine levels, and lymphocyte differentiation status. Genotypic resistance testing of the viral UL97 gene was performed when the patient failed to respond. CMV viremia occurred on day 50 post‐transplant, and 5 samples taken between days 50 and 85 showed the wild‐type UL97 genotype. The appearance of deletion 594‐595 was observed from day 114 post‐transplant. Viral loads declined when foscarnet was commenced and remained below 10,000 copies/mL when the lymphocyte count was greater than 1000/μL (P = 0.02). T cell responses revealed significant expansion of CD8+ terminal effector memory cells. CD4+ cells were largely populations of naïve and central memory cells. Circulating interleukin 10 (IL‐10) levels correlated with the viral load (P < 0.0001). Seroconversion occurred on day 230. The CMV viral load in combination with lymphocyte counts and IL‐10 may be a predictive marker for the risk of development of resistant CMV disease in D+/R− SOT patients. Liver Transpl 15:1199–1203, 2009.

Re-infection following sustained virological response with a different hepatitis C virus genotype: implications for infection control policy

We report the case of a 45-year-old haemodialysis patient who achieved a sustained virological response (SVR) following pegylated interferon therapy for hepatitis C virus (HCV) genotype 2 infection. He was subsequently cohorted with other HCV-infected dialysis patients and became re-infected with HCV genotype 3a. Epidemiological and molecular investigations identified a highly viraemic HCV genotype 3a-infected dialysis patient as the likely source of this infection. This critical incident informed a revision to local and national infection control policy regarding the dialysis management of patients who achieve an SVR following anti-viral treatment.

Management of ganciclovir-resistant cytomegalovirus retinitis in HIV infection in the era of antiretroviral therapy.

The incidence of cytomegalovirus retinitis has decreased significantly since the advent of antiretroviral therapy. However, it remains an important problem in both the developed and developing worlds. Furthermore, long-term antiviral suppression is associated with a significant increase in viral resistance. We present the case of a 46-year-old man who developed cytomegalovirus retinitis one year after being diagnosed with HIV. While he initially demonstrated an excellent clinical response to ganciclovir, his cytomegalovirus viral load remained persistently elevated. Over the subsequent years, his virus developed ganciclovir resistance with a concomitant deterioration in his visual acuity. He responded poorly to salvage therapy with foscarnet and cidofovir. This case highlights the ongoing difficulty of managing cytomegalovirus disease nearly two decades into the era of antiretroviral therapy and underlines the need to develop new treatment strategies.