Long-term evolution of human seasonal influenza virus A(H3N2) is associated with an increase in polymerase complex activity.

Since the influenza pandemic in 1968, influenza A(H3N2) viruses have become endemic. In this state, H3N2 viruses continuously evolve to overcome immune pressure as a result of prior infection or vaccination, as is evident from the accumulation of mutations in the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, phylogenetic studies have also demonstrated ongoing evolution in the influenza A(H3N2) virus RNA polymerase complex genes. The RNA polymerase complex of seasonal influenza A(H3N2) viruses produces mRNA for viral protein synthesis and replicates the negative sense viral RNA genome (vRNA) through a positive sense complementary RNA intermediate (cRNA). Presently, the consequences and selection pressures driving the evolution of the polymerase complex remain largely unknown. Here, we characterize the RNA polymerase complex of seasonal influenza A(H3N2) viruses representative of nearly 50 years of influenza A(H3N2) virus evolution. The H3N2 polymerase complex is a reassortment of human and avian influenza virus genes. We show that since 1968, influenza A(H3N2) viruses have increased the transcriptional activity of the polymerase complex while retaining a close balance between mRNA, vRNA, and cRNA levels. Interestingly, the increased polymerase complex activity did not result in increased replicative ability on differentiated human airway epithelial (HAE) cells. We hypothesize that the evolutionary increase in polymerase complex activity of influenza A(H3N2) viruses may compensate for the reduced HA receptor binding and avidity that is the result of the antigenic evolution of influenza A(H3N2) viruses.

Within-host evolutionary dynamics of seasonal and pandemic human influenza A viruses in young children.

The evolution of influenza viruses is fundamentally shaped by within-host processes. However, the within-host evolutionary dynamics of influenza viruses remain incompletely understood, in part because most studies have focused on infections in healthy adults based on single timepoint data. Here, we analyzed the within-host evolution of 82 longitudinally sampled individuals, mostly young children, infected with A/H1N1pdm09 or A/H3N2 viruses between 2007 and 2009. For A/H1N1pdm09 infections during the 2009 pandemic, nonsynonymous minority variants were more prevalent than synonymous ones. For A/H3N2 viruses in young children, early infection was dominated by purifying selection. As these infections progressed, nonsynonymous variants typically increased in frequency even when within-host virus titers decreased. Unlike the short-lived infections of adults where de novo within-host variants are rare, longer infections in young children allow for the maintenance of virus diversity via mutation-selection balance creating potentially important opportunities for within-host virus evolution.

Outbreak Severity of Highly Pathogenic Avian Influenza A(H5N8) Viruses Is Inversely Correlated to Polymerase Complex Activity and Interferon Induction.

Highly pathogenic avian influenza A(H5N8) viruses first emerged in China in 2010 and in 2014 spread throughout Asia and to Europe and the United States via migrating birds. Influenza A(H5N8) viruses were first detected in the Netherlands in 2014 and caused five outbreaks in poultry farms but were infrequently detected in wild birds. In 2016, influenza A(H5N8) viruses were reintroduced into the Netherlands, resulting in eight poultry farm outbreaks. This outbreak resulted in numerous dead wild birds with severe pathology. Phylogenetic analysis showed that the polymerase genes of these viruses had undergone extensive reassortment between outbreaks. Here, we investigated the differences in virulence between the 2014-15 and the 2016-17 outbreaks by characterizing the polymerase complex of influenza A(H5N8) viruses from both outbreaks. We found that viruses from the 2014-15 outbreak had significantly higher polymerase complex activity in both human and avian cell lines than did those from the 2016-17 outbreak. No apparent differences in the balance between transcription and replication of the viral genome were observed. Interestingly, the 2014-15 polymerase complexes induced significantly higher levels of interferon beta (IFN-ß) than the polymerase complexes of the 2016-17 outbreak viruses, mediated via retinoic acid-inducible gene I (RIG-I). Inoculation of primary duck cells with recombinant influenza A(H5N8) viruses, including viruses with reassorted polymerase complexes, showed that the polymerase complexes from the 2014-15 outbreak induced higher levels of IFN-ß despite relatively minor differences in replication capacity. Together, these data suggest that despite the lower levels of polymerase activity, the higher 2016-17 influenza A(H5N8) virus virulence may be attributed to the lower level of activation of the innate immune system.IMPORTANCE Compared to the 2014-15 outbreak, the 2016-17 outbreak of influenza A(H5N8) viruses in the Netherlands and Europe was more virulent; the number of dead or diseased wild birds found and the severity of pathological changes were higher during the 2016-17 outbreak. The polymerase complex plays an important role in influenza virus virulence, and the gene segments of influenza A(H5N8) viruses reassorted extensively between the outbreaks. In this study, the 2014-15 polymerase complexes were found to be more active, which is counterintuitive with the observed higher virulence of the 2016-17 outbreak viruses. Interestingly, the 2014-15 polymerase complexes also induced higher levels of IFN-ß. These findings suggest that the higher virulence of influenza A(H5N8) viruses from the 2016-17 outbreak may be related to the lower induction of IFN-ß. An attenuated interferon response could lead to increased dissemination, pathology, and mortality, as observed in (wild) birds infected during the 2016-2017 outbreak.

Sex-specific extent and severity of white matter damage in multiple sclerosis: implications for cognitive decline.

Cognitive dysfunction is common in multiple sclerosis (MS). However, the relationship between white matter (WM) damage and cognition remains insufficiently clear. This study investigates the extent and severity of WM diffusion abnormalities in MS patients and relations with cognition. Diffusion tensor imaging scans were obtained in 131 MS patients (88 women, 6 years postdiagnosis) and 49 age-matched controls (29 women). Patient groups were equal in terms of disease duration, disability, and WM lesion volume. Fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) were compared between groups. Post hoc analyses calculated the spatial extent and severity of diffusion abnormalities to relate these to cognitive performance. In controls, 31% of WM voxels showed higher FA in men; therefore, all patient analyses were within-sex. The extent of diffusion changes was higher in male patients than in female patients for all parameters (FA: 24% in women, 53% in men), as was the severity of changes (FA: Z = -0.18 in women, Z = -0.41 in men). Especially the extent of FA abnormalities was strongly related to cognitive performance in all patients (r = -0.42, P < 0.0001). Regionally, thalamic decreases in FA were especially correlated with cognitive performance. Cognitively impaired patients showed greater extent and severity on all diffusion parameters compared to cognitively preserved patients. The WM of male patients was both more extensively and also more severely affected than that of female patients. The extent of WM FA changes, especially in the thalamus, was associated with cognitive performance in this cohort of early MS patients.

Shifting imaging targets in multiple sclerosis: from inflammation to neurodegeneration.

Classically multiple sclerosis (MS) has been regarded as an auto-immune disease of the white matter in the central nervous system leading to severe disability over the course of several decades. Current therapeutic strategies in MS are mostly based on either immune suppression or immune modulation. Although effective in decreasing relapse frequency and severity as well as delaying disease progression, MS pathology ensues nonetheless. In the last decade it became evident that gray matter pathology plays an important role in disease progression and helps explaining certain aspects of MS-related disability such as cognitive decline. Conventional MRI outcome measures commonly used in clinical trials are sufficient to demonstrate an anti-inflammatory drug-effect but lack pathological specificity and are poor to moderate predictors of disability. In this article, we review new insights in gray matter pathology and functional reorganization in MS and how these novel fields in MS research may validate and establish new MRI outcome measures, aid in the development of new therapeutic strategies for neuroprotection and neurorepair, and may lead to development of novel predictive measures of disability and disease progression in MS.