Advances towards norovirus antivirals

Human noroviruses are a leading cause of gastroenteritis worldwide, yet there are no licensed antivirals. There is an urgent need for norovirus therapeutics, particularly for chronic infections in immunocompromised individuals, but also a potential need for prophylactic use in epidemics. Continued research has led to the identification of compounds that inhibit norovirus replication in vitro and, at least in some cases, are also effective in vivo against murine norovirus. Progress has included classical approaches targeting viral proteins and harnessing the antiviral action of interferon, strategies targeting essential host cell factors, and novel strategies exploiting the high mutation rate of noroviruses.The authors declare no conflict in interest. This work was supported by the Wellcome Trust (Ref: WT097997MA)This is the final version of the article. It was first available from Oxford University Press via http://dx.doi.org/10.1093/infdis/jiv28

Norovirus replication and attenuation

Human norovirus is a major cause of viral gastroenteritis worldwide, yet there are no licensed antivirals or a vaccine. This is mainly due to the lack of an efficient cell culture system for human norovirus which has prevented full understanding of norovirus replication. Murine norovirus (MNV), which can be grown in permissive cells and manipulated by reverse genetics, now provides a model for studying norovirus replication. Here, to firstly identify the essential viral factors required for replication, the entire MNV genome was subjected to transposon-mediated insertional mutagenesis and insertion profiling. Validation of the technique and genome-wide profiling of over 2000 insertions revealed the essential regions of each protein and probed the importance of known functional motifs. Identification of tolerated insertion sites led to the generation of the first epitope-tagged noroviruses, carrying the FLAG tag in three proteins of unknown function. This facilitated SILAC-based proteomic studies to identify host cell factors involved in norovirus replication, which may represent potential antiviral targets. With the availability of a small animal model of infection, MNV also provides the opportunity to develop rational approaches to norovirus attenuation. One approach that has been successful for attenuating other RNA viruses involves harnessing the regulatory capacity of the cellular microRNA machinery to control viral replication and tropism. This approach was applied to MNV by inserting a microglial-specific microRNA target sequence into the MNV genome which successfully attenuated norovirus replication in permissive cell lines expressing the cognate microRNA. A second rational approach to attenuation was based on manipulating the rate and fidelity of the viral polymerase, which has been shown to affect RNA virus pathogenesis and can lead to attenuation. To this aim a panel of MNV polymerase mutants were engineered and characterised for their growth and fidelity and their ability to establish acute and persistent infections in vivo.Open Acces

miR-155 induction is a marker of murine norovirus infection but does not contribute to control of replication in vivo.

Background: Due to their role in fine-tuning cellular protein expression, microRNAs both promote viral replication and contribute to antiviral responses, for a range of viruses. The interactions between norovirus and the microRNA machinery have not yet been studied. Here, we investigated the changes that occur in microRNA expression during murine norovirus (MNV) infection. Methods: Using RT-qPCR-based arrays, we analysed changes in miRNA expression during infection with the acute strain MNV-1 in two permissive cell lines, a murine macrophage cell line, RAW264.7, and a murine microglial cell line, BV-2. By RT-qPCR, we further confirmed and analysed the changes in miR-155 expression in the infected cell lines, bone-marrow derived macrophage, and tissues harvested from mice infected with the persistent strain MNV-3. Using miR-155 knockout (KO) mice, we investigated whether loss of miR-155 affected viral replication and pathogenesis during persistent MNV-3 infection in vivo and monitored development of a serum IgG response by ELISA. Results: We identified cell-specific panels of miRNAs whose expression were increased or decreased during infection. Only two miRNAs, miR-687 and miR-155, were induced in both cell lines. miR-155, implicated in innate immunity, was also upregulated in bone-marrow derived macrophage and infected tissues. MNV-3 established a persistent infection in miR-155 knockout (KO) mice, with comparable levels of secreted virus and tissue replication observed as for wildtype mice. However, serum anti-MNV IgG levels were significantly reduced in miR-155 KO mice compared to wildtype mice. Conclusions: We have identified a panel of miRNAs whose expression changes with MNV infection. miR-155 induction is a marker of MNV infection in vitro and in vivo, however it does not contribute to the control of persistent infections in vivo. This finding suggests that the immune defects associated with miR-155 deletion, such as lower serum IgG levels, are also not important for control of persistent MNV-3 infection

Norovirus Polymerase Fidelity Contributes to Viral Transmission In Vivo.

Intrahost genetic diversity and replication error rates are intricately linked to RNA virus pathogenesis, with alterations in viral polymerase fidelity typically leading to attenuation during infections in vivo. We have previously shown that norovirus intrahost genetic diversity also influences viral pathogenesis using the murine norovirus model, as increasing viral mutation frequency using a mutagenic nucleoside resulted in clearance of a persistent infection in mice. Given the role of replication fidelity and genetic diversity in pathogenesis, we have now investigated whether polymerase fidelity can also impact virus transmission between susceptible hosts. We have identified a high-fidelity norovirus RNA-dependent RNA polymerase mutant (I391L) which displays delayed replication kinetics in vivo but not in cell culture. The I391L polymerase mutant also exhibited lower transmission rates between susceptible hosts than the wild-type virus and, most notably, another replication defective mutant that has wild-type levels of polymerase fidelity. These results provide the first experimental evidence that norovirus polymerase fidelity contributes to virus transmission between hosts and that maintaining diversity is important for the establishment of infection. This work supports the hypothesis that the reduced polymerase fidelity of the pandemic GII.4 human norovirus isolates may contribute to their global dominance. IMPORTANCE Virus replication fidelity and hence the intrahost genetic diversity of viral populations are known to be intricately linked to viral pathogenesis and tropism as well as to immune and antiviral escape during infection. In this study, we investigated whether changes in replication fidelity can impact the ability of a virus to transmit between susceptible hosts by the use of a mouse model for norovirus. We show that a variant encoding a high-fidelity polymerase is transmitted less efficiently between mice than the wild-type strain. This constitutes the first experimental demonstration that the polymerase fidelity of viruses can impact transmission of infection in their natural hosts. These results provide further insight into potential reasons for the global emergence of pandemic human noroviruses that display alterations in the replication fidelity of their polymerases compared to nonpandemic strains

Long-term outcomes of cardiac resynchronization therapy in adult congenital heart disease

Background and Aims: Randomized, controlled trials of cardiac resynchronization therapy (CRT) excluded patients with adult congenital heart disease (ACHD). We sought to explore long-term clinical outcomes. Methods and Results: In this single-center, observational study, events were collected from hospital records on patients with structural ACHD (sACHD) and adults with ischemic (ICM) or nonischemic (NICM) cardiomyopathy undergoing CRT. Patients with sACHD (n = 23, age: 41.6 ± 13.5 years [mean ± standard deviation]) and adults with ICM (n = 533) or NICM (n = 458) were followed-up for 4.1 years (median; interquartile range: 2.2-6.1). Total mortality was 5/23 (21.7%; 4.4 per 100 person-years) in sACHD, 221/533 (41.5%; 11.8 per 100 person-years) in ICM, and 154/458 (33.6%; 9.7 per 100 person-years) in NICM. In univariate analyses, total mortality in sACHD was lower than in ICM (hazard ratio [HR]: 0.38; 95% confidence interval [CI] 0.15-0.91), but similar to NICM (HR: 0.48, 95% CI 0.20-1.16). Cardiac mortality in sACHD was similar to ICM (HR: 0.78, 95% CI 0.32-1.92) and NICM (HR: 1.12, 95% CI 0.45-2.78). Heart failure (HF) hospitalization rates were similar to ICM (HR: 0.44, 95% CI 0.11-1.77) and NICM (HR: 0.75, 95% CI 0.18-3.08). In multivariate analyses, no differences emerged in total mortality, cardiac mortality, or HF hospitalization between sACHD and NICM or ICM, after adjustment for age, sex, New York Heart Association class, diabetes, atrial rhythm, QRS duration, QRS morphology, systemic ventricular ejection fraction, and medical therapy. Conclusion: Total mortality, cardiac mortality, and HF hospitalization after CRT in patients with sACHD was similar to adults with ICM or NICM

Targeting macrophage- and intestinal epithelial cell-specific microRNAs against norovirus restricts replication in vivo.

Until recently, our understanding of the cellular tropism of human norovirus (HuNoV), a major cause of viral gastroenteritis, has been limited. Immune cells and intestinal epithelial cells (IECs) have been proposed as targets of HuNoV replication in vivo, although the contribution of each to pathogenesis and transmission is unknown. Murine norovirus (MNV) is widely used as a surrogate model for HuNoV, as it replicates in cultured immune cells. The importance of the complete MNV immune cell tropism in vivo has not been determined. Recent work has linked replication in IECs to viral persistence in vivo. MNV provides a model to assess the relative contribution of each cell tropism to viral replication in immunocompetent native hosts. Here we exploited cell-specific microRNAs to control MNV replication, through insertion of microRNA target sequences into the MNV genome. We demonstrated the utility of this approach for MNV in vitro by selectively reducing replication in microglial cells, using microglial-specific miR-467c. We then showed that inserting a target sequence for the haematopoietic-specific miR-142-3p abrogated replication in a macrophage cell line. The presence of a target sequence for either miR-142-3p or IEC miR-215 significantly reduced viral secretion during the early stages of a persistent infection in immunocompetent mice, confirming that both cell types support viral replication in vivo. This study provides additional evidence that MNV shares the IEC tropism of HuNoVs in vivo, and now provides a model to dissect the contribution of replication in each cell type to viral pathogenesis and transmission in a native host