HDV RNA assays: Performance characteristics, clinical utility, and challenges.

Coinfection with HBV and HDV results in hepatitis D, the most severe form of chronic viral hepatitis, frequently leading to liver decompensation and HCC. Pegylated interferon alpha, the only treatment option for chronic hepatitis D for many years, has limited efficacy. New treatments are in advanced clinical development, with one recent approval. Diagnosis and antiviral treatment response monitoring are based on detection and quantification of HDV RNA. However, the development of reliable HDV RNA assays is challenged by viral heterogeneity (at least 8 different genotypes and several subgenotypes), intrahost viral diversity, rapid viral evolution, and distinct secondary structure features of HDV RNA. Different RNA extraction methodologies, primer/probe design for nucleic acid tests, lack of automation, and overall dearth of standardization across testing laboratories contribute to substantial variability in performance characteristics of research-based and commercial HDV RNA assays. A World Health Organization (WHO) standard for HDV RNA, available for about 10 years, has been used by many laboratories to determine the limit of detection of their assays and facilitates comparisons of RNA levels across study centers. Here we review challenges for robust pan genotype HDV RNA quantification, discuss particular clinical needs and the importance of reliable HDV RNA quantification in the context of drug development and patient monitoring. We summarize distinct technical features and performance characteristics of available HDV RNA assays. Finally, we provide considerations for the use of HDV RNA assays in the context of drug development and patient monitoring.

Comparison of four commercial EBV DNA quantitative tests to a new test at an early stage of development.

BACKGROUND: Regular screening for Epstein-Barr virus (EBV) DNA using quantitative RT-PCR is recommended for early intervention in at-risk patients. Harmonization of quantitative RT-PCR assays is critical to avoid misinterpretation of results. Here, we compare quantitative results of the cobas® EBV assay to four commercial RT-qPCR assays. METHODS: The cobas EBV, EBV R-Gene, artus EBV RG PCR, RealStar EBV PCR kit 2.0 and Abbott EBV RealTime assays were compared for analytic performance using a 10-fold dilution series of EBV reference material, normalized to the WHO standard. For clinical performance, their quantitative results were compared using anonymized, leftover EBV-DNA-positive EDTA plasma samples. RESULTS: For analytic accuracy, the cobas EBV deviated -0.0097 log10 from target values. The other tests showed deviations between 0.0037 and -0.12 log10. For clinical performance, accuracy and linearity of cobas EBV data from both study sites were excellent. Bland-Altman bias and Deming regression analyses showed statistical correlation for cobas EBV to both EBV R-Gene and Abbott RealTime assays but an offset of cobas EBV to artus EBV RG PCR and RealStar EBV PCR kit 2.0. CONCLUSION: The cobas EBV showed the closest correlation to the reference material, followed closely by EBV R-Gene and Abbott EBV RealTime. Values obtained are stated in IU/mL, facilitating comparison across testing sites and potentially improving utilization of guidelines for diagnosis, monitoring, and treatment of patients.

A multi-center study comparing the analytical sensitivity and clinical concordance of three HIV-1 viral load assays.

BACKGROUND: HIV-1 viral load assays are essential tools for clinical management of people living with HIV-1. OBJECTIVES AND STUDY DESIGN: We evaluated concordance between three assays: the cobas® HIV-1 test for use on the cobas® 6800 and cobas® 8800 systems (cobas HIV-1); the COBAS® TaqMan® HIV-1 Test, v2.0 for use with the High Pure System and the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 test, v2.0. Analytical sensitivity, precision and accuracy of all three methods were assessed using the WHO 2nd International Standard for HIV-1, with concentrations from 5 to 1000 copies/mL. Accuracy and concordance were evaluated using 212 clinical specimens. Overall percent agreement (OPA) was determined using three different thresholds used as medical decision points. RESULTS: The limit of detection was below 20 copies/mL for each assay. The hit rate for each assay was 100 % for concentrations ≥ 50 copies/mL. Only the cobas HIV-1 test generated quantifiable data for all replicates at 50 copies/mL. Between 50 and 400 copies/mL, results for all assays were accurate within 0.09 log10 copies/mL, with standard deviation less than 0.14 log10 copies/mL. The mean difference between paired results in clinical specimens ranged from -0.050 to 0.107 log10 copies/mL across all assay comparisons. The OPA between pairs of assays ranged from 94.8 to 98.1% at the 50 copies/mL cutoff, and improved to a range of 97.6-99.0% at the 200 copies/mL cutoff. At the 1000 copies/mL cutoff, OPA between assays was 98.5-99.0%. CONCLUSIONS: The cobas HIV-1 assay is highly sensitive, accurate and suitable for use in clinical practice.

Classification of HIV-1 virological treatment failure using the Roche cobas plasma separation card on cobas 8800 compared to dried blood spots on Abbott RealTime HIV-1.

BACKGROUND: Measurement of HIV-1 viral load (VL) is essential for monitoring antiretroviral treatment (ART) efficacy. In remote settings, dried blood spots (DBS) may be used as the specimen type. However, cellular components in DBS not present in the gold standard specimen type, plasma, may result in low specificity i.e., over-estimation of VL results from DBS compared to plasma. The Abbott RealTime HIV-1 assay system has been reported to have improved specificity using DBS compared to other tests. A new specimen collection matrix, the cobas plasma separation card (PSC, Roche Molecular Systems), enables specimen collection from a finger prick or venous blood, using a multi-layer absorption and filtration design that results in a specimen similar to plasma. OBJECTIVES AND STUDY DESIGN: We performed a direct comparison between VL results from PSC tested with the cobas 6800/8800 assay (c8800) and DBS tested with the Abbott RealTime HIV-1 assay. RESULTS: Overall concordance between PSC and plasma around the 1000 copies/mL threshold was high (>97%). Compared to VL measured using DBS and the RealTime assay, PSC and c8800 showed improved sensitivity (96.9% vs 90.6%) and specificity (97.4% vs. 87.2%) using plasma as the reference, as there were fewer patients with VL below 1000 copies/mL in plasma in whom VL was over this threshold using PSC compared to DBS. The limit of detection for PSC was lower than for DBS (575 vs. 2314 copies/mL). CONCLUSIONS: cobas PSC represents a promising specimen type for use with the cobas 6600/8800 system in settings where plasma cannot be used.

Early Diagnosis of HIV-1 and HIV-2 Using Cobas HIV-1/HIV-2 Qualitative Test: A Novel Qualitative Nucleic Acid Amplification Test for Plasma, Serum, and Dried Blood Spot Specimens.

BACKGROUND: Nucleic acid amplification tests (NATs) minimize the time from HIV infection to diagnosis, reducing transmission during acute HIV. NATs are especially useful for diagnosing HIV in children younger than 18 months and discriminating between HIV-1 and HIV-2. METHODS: We evaluated the performance of the cobas HIV-1/HIV-2 qualitative (cobas HIV-1/2 Qual) test for use on cobas 6800/8800 Systems. The results of adult plasma and serum samples and pediatric dried blood spots were compared with those of the recomLine HIV-1 & HIV-2 Immunoglobulin G serological test and COBAS AmpliPrep/COBAS TaqMan HIV-1 qualitative test, v2.0. Genotype inclusivity and limits of detection were determined, and sensitivity on seroconversion panels was compared with that in the Bio-Rad Geenius HIV 1/2 Confirmatory Assay, Abbott ARCHITECT HIV Ag/Ab Combo serological test, and cobas TaqScreen MPX, v2.0. RESULTS: Concordance of cobas HIV-1/2 Qual test with the comparator serological test and COBAS AmpliPrep/COBAS TaqMan test was ≥99.6% with all sample types. Reactivity with all HIV genotypes was 100%. LOD in plasma samples was 14.8, 12.6, and 27.9 copies/mL for HIV-1 group M, HIV-1 group O, and HIV-2, respectively, with similar results for serum samples. LOD in dried blood spots was 255 copies/mL for HIV-1 and 984 copies/mL for HIV-2. HIV infection was detected 18.9 days and 8.5 days earlier than the confirmatory and serological assays, respectively, and at a similar time to the NAT. CONCLUSIONS: The cobas HIV-1/2 Qual test enables early and accurate diagnoses of HIV-1 and HIV-2 in adults and children across sample types. The assay could help avert transmission during acute HIV, simplify HIV diagnostic algorithms, and promote the survival of HIV-infected children.

HBV-RNA Co-amplification May Influence HBV DNA Viral Load Determination.

Despite effective hepatitis B virus (HBV)-DNA suppression, HBV RNA can circulate in patients receiving nucleoside/nucleotide analogues (NAs). Current assays quantify HBV DNA by either real-time polymerase chain reaction (PCR), which uses DNA polymerase, or transcription-mediated amplification, which uses reverse-transcriptase (RT) and RNA polymerase. We assessed the effect of RT capability on HBV-DNA quantification in samples from three cohorts, including patients with quantified HBV RNA. We compared the HBV-DNA levels by real-time PCR (cobas HBV, Roche 6800/8800; Xpert HBV, Cepheid), transcription-mediated amplification (Aptima HBV, Hologic), and real-time PCR with added RT capability (cobas HBV+RT). In the first cohort (n = 45) followed over 192 weeks of NA therapy, on-treatment HBV-DNA levels were higher with cobas HBV+RT than cobas HBV (mean difference: 0.14 log10 IU/mL). In a second cohort (n = 50) followed over 96 weeks of NA therapy, HBV-DNA viral load was significantly higher with the cobas HBV+RT and Aptima HBV compared with the cobas HBV test at all time points after initiation of NA therapy (mean difference: 0.65-1.16 log10 IU/mL). A clinically significant difference was not detected between the assays at baseline. In a third cohort (n = 53), after a median of 2.2 years of NA therapy, we detected HBV RNA (median 5.6 log10 copies/mL) in 23 patients (43.4%). Median HBV-DNA levels by Aptima HBV were 2.4 versus less than 1 log10 IU/mL in samples with HBV RNA and without HBV RNA, respectively (P = 0.0006). In treated patients with HBV RNA, Aptima HBV measured higher HBV-DNA levels than Xpert HBV and cobas HBV. Conclusion: Tests including an RT step may overestimate HBV DNA, particularly in samples with low viral loads as a result of NA therapy. This overestimation is likely due to amplification of HBV RNA and may have an impact on clinical decisions.

Utility of the new cobas HCV test for viral load monitoring during direct-acting antiviral therapy.

BACKGROUND: The COBAS AmpliPrep/COBAS TaqMan assay HCV (CAP/CTM) is widely used in clinical routine for HCV testing. Recently, the new cobas HCV test was established for high throughput testing with minimal operator intervention. As different assays may yield different quantitative/qualitative results that possibly impact treatment decisions, the aim of this study was to externally evaluate the cobas HCV test performance in comparison to CAP/CTM in a clinically relevant setting. METHODS: Serum samples were obtained from 270 patients who received direct acting antiviral therapy with different treatment regimens at two study sites (Hannover and Frankfurt) in 2016. Overall, 1545 samples (baseline, on-treatment and follow-up) were tested in parallel by both assays. RESULTS: The mean difference between cobas HCV and CAP/CTM for the quantification of HCV RNA was 0.008 log10 IU/ml HCV RNA (95% limits of agreement: -0.02-0.036) showing excellent agreement of both assays. With respect to clinical cut offs (HCV RNA detectable vs. target not detected and HCV RNA above the lower limit of quantification (LLOQ) vs.

Generating timely molecular diagnostic test results: workflow comparison of the cobas® 6800/8800 to Panther.

Background: Molecular diagnostic tests for HBV, HCV and HIV-1 and other pathogens are widely used for clinical management. Practical issues related to workflow and labor requirements need to be characterized to inform selection of the most appropriate system. Research design and methods: We compared the workflow of two high-throughput systems: cobas 6800 (Roche) and Panther (Hologic), using average mid-size laboratory test volumes for five different assays (HIV-1, HBV, HCV, HPV or TV, and CT/NG). Results: Set-up time, time to first results, time to last results, and total hands-on time for cobas 6800 was 0.40, 2.47, 7.12, and 0.98 hours, respectively; on the Panther system, these times were 0.75, 2.7, 9.1, and 1.48 hours. Fifty-seven samples had results available at the first time point on cobas 6800 compared to 5 samples on the Panther system. The Panther system required more manual steps including several with potential risks of contamination or error. The number of reagents items required was 5 for cobas 6800 and 40 for the Panther system. Conclusions: Both systems provided a high level of automation. The cobas 6800 platform had shorter start up, time to first result, time to last result and hands-on times than the Panther system.

Commutability and concordance of four hepatitis B virus DNA assays in an international multicenter study.

BACKGROUND: HBV DNA is the most important molecular marker in hepatitis B, used to determine treatment indication and monitoring. Most patients require lifelong hepatitis B virus (HBV) management, thus viral load (VL) monitoring may be performed at different laboratories, with different HBV assays, which may result in different VL results. This multicenter study compares the commutability and concordance of results from four different HBV DNA assays: CAP/CTM HBVv2, HPS/CTM HBVv2 and the new cobas 6800/8800 HBV and cobas 4800 HBV assays. METHODS: Across all four assays, HBV limit of detection (LoD) and linearity at lower concentrations were assessed using panels traceable to the World Health Organization international standard, and concordance was investigated at the important medical decision cutoffs 2000 and 20,000 IU/ml, using specimens from HBV-positive patients. RESULTS: The calculated LoD via a probit curve was 2.7 IU/ml for cobas 6800/8800 HBV, 2.8 IU/ml for cobas 4800 HBV, 9.6 IU/ml for CAP/CTM HBVv2, and 6.2 IU/ml for HPS/CTM HBVv2. The average accuracy was comparable between cobas 6800/8800 HBV, cobas 4800 HBV and CAP/CTM HBVv2 (0.04-0.05 log10 IU/ml), while a slightly lower accuracy was documented for HPS/CTM HBVv2 (-0.16 log10 IU/ml). A total of 211-245 clinical samples were used for a pairwise comparison. Mean paired log differences ranged from -0.17 log10 IU/ml to -0.01 log10 IU/ml. Coefficient of determination was over 98% for all pairs with high overall percent agreement at the 2000 and 20,000 IU/ml cutoffs (from 91.7% to 96.3%). In a subset of samples with VL±0.5 log10 to the 2000 and 20,000 IU/ml thresholds, concordance was still 72% and 82%, respectively. CONCLUSIONS: The new cobas 6800/8800 HBV and 4800 HBV assays show high accuracy in samples with low-level viremia and a high concordance with the established HBV tests, CAP/CTM HBVv2 and HPS/CTM HBVv2, at 2000 and 20,000 IU/ml. Thus, all four HBV assays have high commutability and may be used interchangeably in routine clinical practice.

The cobas® 6800/8800 System: a new era of automation in molecular diagnostics.

INTRODUCTION: Molecular diagnostics is a key component of laboratory medicine. Here, the authors review key triggers of ever-increasing automation in nucleic acid amplification testing (NAAT) with a focus on specific automated Polymerase Chain Reaction (PCR) testing and platforms such as the recently launched cobas® 6800 and cobas® 8800 Systems. The benefits of such automation for different stakeholders including patients, clinicians, laboratory personnel, hospital administrators, payers, and manufacturers are described. Areas Covered: The authors describe how molecular diagnostics has achieved total laboratory automation over time, rivaling clinical chemistry to significantly improve testing efficiency. Finally, the authors discuss how advances in automation decrease the development time for new tests enabling clinicians to more readily provide test results. Expert Commentary: The advancements described enable complete diagnostic solutions whereby specific test results can be combined with relevant patient data sets to allow healthcare providers to deliver comprehensive clinical recommendations in multiple fields ranging from infectious disease to outbreak management and blood safety solutions.

Evaluation of the COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HCV Test, v2.0 and comparison to assays used in routine clinical practice in an international multicenter clinical trial: The ExPECT study.

BACKGROUND: The COBAS(®) AmpliPrep(®)/COBAS(®) TaqMan(®) HCV Test, v2.0 (CAP/CTM2) is used for HCV RNA viral load monitoring. OBJECTIVES: The performance of the CAP/CTM2 was compared to other widely used tests, including a manual version of the assay (the COBAS(®) TaqMan(®) HCV Test, v2.0 for use with the High Pure System, HPS/CTM2) predominantly used during phase III clinical trials for the new direct acting antiviral therapies. STUDY DESIGN: Low HCV RNA level comparisons were performed across tests (Abbott Realtime HCV Test, ART; COBAS(®) AmpliPrep(®)/COBAS(®) TaqMan(®) HCV Test, v1.0, CAP/CTM1; CAP/CTM2; and HPS/CTM2) using dilutions of the 2nd HCV WHO International Standard. Additionally, the clinical performance of the CAP/CTM2 was evaluated with 421 leftover HCV RNA-positive routine clinical samples. RESULTS: All quantifiable WHO dilutions were within ±0.3log10IU/mL of the expected results across tests and the analytical sensitivity resulted in a limit of detection of 12IU/mL (95% confidence interval, 10, 15). When clinical samples were tested the results for 87% (367 of 421) of all sample comparisons were within ±0.5log10IU/mL. When low viral load results (25-3500IU/mL) were compared, values obtained by the ART assay were significantly lower (p<0.0001) than those obtained with the CAP/CTM2. CONCLUSIONS: The new CAP/CTM2 showed good accuracy with comparable sensitivity to comparator assays. The new kit is well-suited for use in the routine diagnostic laboratory, especially for accurate monitoring of patients receiving triple therapy or interferone-free regimens.

Viral latency drives 'memory inflation': a unifying hypothesis linking two hallmarks of cytomegalovirus infection.

Low public awareness of cytomegalovirus (CMV) results from the only mild and transient symptoms that it causes in the healthy immunocompetent host, so that primary infection usually goes unnoticed. The virus is not cleared, however, but stays for the lifetime of the host in a non-infectious, replicatively dormant state known as 'viral latency'. Medical interest in CMV results from the fact that latent virus can reactivate to cytopathogenic, tissue-destructive infection causing life-threatening end-organ disease in immunocompromised recipients of solid organ transplantation (SOT) or hematopoietic cell transplantation (HCT). It is becoming increasingly clear that CMV latency is not a static state in which the viral genome is silenced at all its genetic loci making the latent virus immunologically invisible, but rather is a dynamic state characterized by stochastic episodes of transient viral gene desilencing. This gene expression can lead to the presentation of antigenic peptides encoded by 'antigenicity-determining transcripts expressed in latency (ADTELs)' sensed by tissue-patrolling effector-memory CD8 T cells for immune surveillance of latency [In Reddehase et al., Murine model of cytomegalovirus latency and reactivation, Current Topics in Microbiology and Immunology, vol 325. Springer, Berlin, pp 315-331, 2008]. A hallmark of the CD8 T cell response to CMV is the observation that with increasing time during latency, CD8 T cells specific for certain viral epitopes increase in numbers, a phenomenon that has gained much attention in recent years and is known under the catchphrase 'memory inflation.' Here, we provide a unifying hypothesis linking stochastic viral gene desilencing during latency to 'memory inflation.'

Accuracy to 2nd International HIV-1 RNA WHO Standard: assessment of three generations of quantitative HIV-1 RNA nucleic acid amplification tests.

BACKGROUND: Standardization of quantitative HIV-1 tests to a global primary standard is required by regulatory authorities to ensure comparability of test results across different assays and platforms of different manufacturers. OBJECTIVES AND STUDY DESIGN: Three generations of quantitative HIV-1 tests, the COBAS(®) AMPLICOR(®) HIV-1 Monitor Test, v1.5 (HIV-1 Monitor test v1.5); the COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HIV-1 Test (HIV-1 TaqMan(®) test v1.0); and the dual-target-based COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HIV-1 Test, v2.0 (HIV-1 TaqMan(®) test v2.0), were assessed for accuracy to World Health Organization (WHO) 2nd International Standard for human immunodeficiency virus 1 (HIV-1) RNA (NIBSC code 97/650) at concentration levels below 1667 IU/mL including relevant medical decision points. RESULTS: With the 2nd WHO Standard the mean difference across all concentrations was -0.07 log(10) for the HIV-1 Monitor test v1.5; +0.12 log(10) for the HIV-1 TaqMan(®) test v1.0; and +0.09 log(10) for the HIV-1 TaqMan(®) test v2.0. Linearity, including concentrations below the claimed limit of quantitation, was demonstrated for HIV-1 TaqMan(®) test v2.0. The HIV-1 TaqMan(®) test v1.0 showed a trend towards higher quantitation at very low concentration levels and the HIV-1 Monitor test v1.5 had a tendency towards lower quantitation at low concentration levels. CONCLUSIONS: All three assays are closely traceable to the primary WHO HIV-1 RNA standard for in vitro diagnostic IVD assays. Compared to the other two assays, the HIV-1 TaqMan(®) test v2.0 showed better linearity around the limit of detection and below.

Improved HIV-1 RNA quantitation by COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 using a novel dual-target approach.

BACKGROUND: HIV-1 RNA viral load is a key parameter for reliable treatment monitoring of HIV-1 infection. Accurate HIV-1 RNA quantitation can be impaired by primer and probe sequence polymorphisms as a result of tremendous genetic diversity and ongoing evolution of HIV-1. A novel dual HIV-1 target amplification approach was realized in the quantitative COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 (HIV-1 TaqMan test v2.0) to cope with the high genetic diversity of the virus. OBJECTIVES AND STUDY DESIGN: The performance of the new assay was evaluated for sensitivity, dynamic range, precision, subtype inclusivity, diagnostic and analytical specificity, interfering substances, and correlation with the COBAS AmpliPrep/COBAS TaqMan HIV-1 (HIV-1 TaqMan test v1.0) predecessor test in patients specimens. RESULTS: The new assay demonstrated a sensitivity of 20 copies/mL, a linear measuring range of 20-10,000,000 copies/mL, with a lower limit of quantitation of 20 copies/mL. HIV-1 Group M subtypes and HIV-1 Group O were quantified within +/-0.3 log(10) of the assigned titers. Specificity was 100% in 660 tested specimens, no cross reactivity was found for 15 pathogens nor any interference for endogenous substances or 29 drugs. Good comparability with the predecessor assay was demonstrated in 82 positive patient samples. In selected clinical samples 35/66 specimens were found underquantitated in the predecessor assay; all were quantitated correctly in the new assay. CONCLUSIONS: The dual-target approach for the HIV-1 TaqMan test v2.0 enables superior HIV-1 Group M subtype coverage including HIV-1 Group O detection. Correct quantitation of specimens underquantitated in the HIV-1 TaqMan test v1.0 test was demonstrated.

A novel transmembrane domain mediating retention of a highly motile herpesvirus glycoprotein in the endoplasmic reticulum.

Gene m164 of murine cytomegalovirus belongs to the large group of 'private' genes that show no homology to those of other cytomegalovirus species and are thought to represent 'host adaptation' genes involved in virus-host interaction. Previous interest in the m164 gene product was based on the presence of an immunodominant CD8 T-cell epitope presented at the surface of infected cells, despite interference by viral immune-evasion proteins. Here, we provide data to reveal that the m164 gene product shows unusual features in its cell biology. A novel strategy of mass-spectrometric analysis was employed to map the N terminus of the mature protein, 107 aa downstream of the start site of the predicted open reading frame. The resulting 36.5 kDa m164 gene product is identified here as an integral type-I membrane glycoprotein with exceptional intracellular trafficking dynamics, moving within the endoplasmic reticulum (ER) and outer nuclear membrane with an outstandingly high lateral membrane motility, actually 100 times higher than those published for cellular ER-resident proteins. Notably, gp36.5/m164 does not contain any typical ER-retention/retrieval signals, such as the C-terminal motifs KKXX or KXKXX, and does not pass the Golgi apparatus. Instead, it belongs to the rare group of viral glycoproteins in which the transmembrane domain (TMD) itself mediates direct ER retention. This is the first report relating TMD usage of an ER-resident transmembrane protein to its lateral membrane motility as a paradigm in cell biology. We propose that TMD usage for ER retention facilitates free and fast floating in ER-related membranes and between ER subdomains.

Immune evasion proteins enhance cytomegalovirus latency in the lungs.

CD8 T cells control cytomegalovirus (CMV) infection in bone marrow transplantation recipients and persist in latently infected lungs as effector memory cells for continuous sensing of reactivated viral gene expression. Here we have addressed the question of whether viral immunoevasins, glycoproteins that specifically interfere with antigen presentation to CD8 T cells, have an impact on viral latency in the murine model. The data show that deletion of immunoevasin genes in murine CMV accelerates the clearance of productive infection during hematopoietic reconstitution and leads to a reduced latent viral genome load, reduced latency-associated viral transcription, and a lower incidence of recurrence in lung explants.

Synergism between the components of the bipartite major immediate-early transcriptional enhancer of murine cytomegalovirus does not accelerate virus replication in cell culture and host tissues.

Major immediate-early (MIE) transcriptional enhancers of cytomegaloviruses are key regulators that are regarded as determinants of virus replicative fitness and pathogenicity. The MIE locus of murine cytomegalovirus (mCMV) shows bidirectional gene-pair architecture, with a bipartite enhancer flanked by divergent core promoters. Here, we have constructed recombinant viruses mCMV-DeltaEnh1 and mCMV-DeltaEnh2 to study the impact of either enhancer component on bidirectional MIE gene transcription and on virus replication in cell culture and various host tissues that are relevant to CMV disease. The data revealed that the two unipartite enhancers can operate independently, but synergize in enhancing MIE gene expression early after infection. Kick-start transcription facilitated by the bipartite enhancer configuration, however, did not ultimately result in accelerated virus replication. We conclude that virus replication, once triggered, proceeds with a fixed speed and we propose that synergism between the components of the bipartite enhancer may rather increase the probability for transcription initiation.

The immune evasion paradox: immunoevasins of murine cytomegalovirus enhance priming of CD8 T cells by preventing negative feedback regulation.

Cytomegaloviruses express glycoproteins that interfere with antigen presentation to CD8 T cells. Although the molecular modes of action of these "immunoevasins" differ between cytomegalovirus species, the convergent biological outcome is an inhibition of the recognition of infected cells. In murine cytomegalovirus, m152/gp40 retains peptide-loaded major histocompatibility complex class I molecules in a cis-Golgi compartment, m06/gp48 mediates their vesicular sorting for lysosomal degradation, and m04/gp34, although not an immunoevasin in its own right, appears to assist in the concerted action of all three molecules. Using the L(d)-restricted IE1 epitope YPHFMPTNL in the BALB/c mouse model as a paradigm, we provide here an explanation for the paradox that immunoevasins enhance CD8 T-cell priming although they inhibit peptide presentation in infected cells. Adaptive immune responses are initiated in the regional lymph node (RLN) draining the site of pathogen exposure. In particular for antigens that are not virion components, the magnitude of viral gene expression providing the antigens is likely a critical parameter in priming efficacy. We have therefore focused on the events in the RLN and have related priming to intranodal viral gene expression. We show that immunoevasins enhance priming by downmodulating an early CD8 T-cell-mediated "negative feedback" control of the infection in the cortical region of the RLN, thus supporting the model that immunoevasins improve antigen supply for indirect priming by uninfected antigen-presenting cells. As an important consequence, these findings predict that deletion of immunoevasin genes in a replicative vaccine virus is not a favorable option but may, rather, be counterproductive.

Transactivation of cellular genes involved in nucleotide metabolism by the regulatory IE1 protein of murine cytomegalovirus is not critical for viral replicative fitness in quiescent cells and host tissues.

Despite its high coding capacity, murine CMV (mCMV) does not encode functional enzymes for nucleotide biosynthesis. It thus depends on cellular enzymes, such as ribonucleotide reductase (RNR) and thymidylate synthase (TS), to be supplied with deoxynucleoside triphosphates (dNTPs) for its DNA replication. Viral transactivation of these cellular genes in quiescent cells of host tissues is therefore a parameter of viral fitness relevant to pathogenicity. Previous work has shown that the IE1, but not the IE3, protein of mCMV transactivates RNR and TS gene promoters and has revealed an in vivo attenuation of the mutant virus mCMV-DeltaIE1. It was attractive to propose the hypothesis that lack of transactivation by IE1 and a resulting deficiency in the supply of dNTPs are the reasons for growth attenuation. Here, we have tested this hypothesis with the mutant virus mCMV-IE1-Y165C expressing an IE1 protein that selectively fails to transactivate RNR and TS in quiescent cells upon transfection while maintaining the capacity to disperse repressive nuclear domains (ND10). Our results confirm in vivo attenuation of mCMV-DeltaIE1, as indicated by a longer doubling time in host organs, whereas mCMV-IE1-Y165C replicated like mCMV-WT and the revertant virus mCMV-IE1-C165Y. Notably, the mutant virus transactivated RNR and TS upon infection of quiescent cells, thus indicating that IE1 is not the only viral transactivator involved. We conclude that transactivation of cellular genes of dNTP biosynthesis is ensured by redundancy and that attenuation of mCMV-DeltaIE1 results from the loss of other critical functions of IE1, with its function in the dispersal of ND10 being a promising candidate.