ABSTRACT:Mutations in the OPN1LW (L-) and OPN1MW (M-)cone opsin genes underlie a spectrum of cone photoreceptor defects from stationary loss of color vision to progressive retinal degeneration. Genotypes of 22 families with a range of cone disorders were grouped into three classes: deletions of the locus control region (LCR); missense mutation (p.Cys203Arg) in an L-/Mhybrid gene; and exon 3 single-nucleotide polymorphism (SNP) interchange haplotypes in an otherwise normal gene array. Moderate-to-high myopia was observed in all mutation categories. Individuals with LCR deletions or p.Cys203Arg mutations were more likely to have nystagmus and poor vision, with disease progression in some p.Cys203Arg patients. Three disease-associated exon 3 SNP haplotypes encoding LIAVA, LVAVA, or MIAVA were identified in our cohort. These patients were less likely to have nystagmus but more likely to show progression, with all patients over the age of 40 years having marked macular abnormalities. Previously, the haplotype LIAVA has been shown to result in exon 3 skipping. Here, we show that haplotypes LVAVA and MIAVA also result in aberrant splicing, with a residual low level of correctly spliced cone opsin. The OPN1LW/OPN1MW:c.532A>G SNP, common to all three disease-associated haplotypes, appears to be principally responsible for this mutational mechanism.

BackgroundTwo new subclades of influenza A(H3N2) viruses became prominent during the 2014‐2015 Northern Hemisphere influenza season. The HA glycoproteins of these viruses showed sequence changes previously associated with alterations in receptor‐binding properties. To address how these changes influence virus propagation, viruses were isolated and propagated in conventional MDCK cells and MDCK‐SIAT1 cells, cells with enhanced expression of the human receptor for the virus, and analysed at each passage.MethodsGene sequence analysis was undertaken as virus was passaged in conventional MDCK cells and MDCK‐SIAT1 cells. Alterations in receptor recognition associated with passage of virus were examined by haemagglutination assays using red blood cells from guinea pigs, turkeys and humans. Microneutralisation assays were performed to determine how passage‐acquired amino acid substitutions and polymorphisms affected virus antigenicity.ResultsViruses were able to infect MDCK‐SIAT1 cells more efficiently than conventional MDCK cells. Viruses of both the 3C.2a and 3C.3a subclades showed greater sequence change on passage in conventional MDCK cells than in MDCK‐SIAT1 cells, with amino acid substitutions being seen in both HA and NA glycoproteins. However, virus passage in MDCK‐SIAT1 cells at low inoculum dilutions showed reducing infectivity on continued passage.ConclusionsCurrent H3N2 viruses should be cultured in the MDCK‐SIAT1 cell line to maintain faithful replication of the virus, and at an appropriate multiplicity of infection to retain infectivity.

Seasonal influenza virus A/H3N2 is a major cause of death globally. Vaccination remains the most effective preventative. Rapid mutation of hemagglutinin allows viruses to escape adaptive immunity. This antigenic drift necessitates regular vaccine updates. Effective vaccine strains need to represent H3N2 populations circulating one year after strain selection. Experts select strains based on experimental measurements of antigenic drift and predictions made by models from hemagglutinin sequences. We developed a novel influenza forecasting framework that integrates phenotypic measures of antigenic drift and functional constraint with previously published sequence-only fitness estimates. Forecasts informed by phenotypic measures of antigenic drift consistently outperformed previous sequence- only estimates, while sequence-only estimates of functional constraint surpassed more comprehensive experimentally-informed estimates. Importantly, the best models integrated estimates of both functional constraint and either antigenic drift phenotypes or recent population growth.

We characterized 55 influenza A(H9N2) viruses isolated in Pakistan during 2014–2016 and found that the hemagglutinin gene is of the G1 lineage and that internal genes have differentiated into a variety of novel genotypes. Some isolates had up to 4-fold reduction in hemagglutination inhibition titers compared with older viruses. Viruses with hemagglutinin A180T/V substitutions conveyed this antigenic diversity and also caused up to 3,500-fold greater binding to avian-like and >20-fold greater binding to human-like sialic acid receptor analogs. This enhanced binding avidity led to reduced virus replication in primary and continuous cell culture. We confirmed that altered receptor-binding avidity of H9N2 viruses, including enhanced binding to human-like receptors, results in antigenic variation in avian influenza viruses. Consequently, current vaccine formulations might not induce adequate protective immunity in poultry, and emergence of isolates with marked avidity for human-like receptors increases the zoonotic risk.

In 2014 the Centre for Health Protection in Hong Kong introduced screening for influenza C virus (ICV) as part of its routine surveillance for infectious agents in specimens collected from patients presenting with symptoms of respiratory viral infection, including Influenza-Like Illness. A retrospective analysis of ICV detections up to week 26/2019 revealed persistent low-level circulation with two outbreaks having occurred in the winters of 2015-16 and 2017-18. These outbreaks occurred at the same time as, and were dwarfed by, seasonal epidemics of influenza types A and B. Gene sequencing studies on stored ICV-positive clinical specimens from the two outbreaks have shown the HE genes of the viruses to fall in two of the six recognised genetic lineages (represented by C/Kanagawa/1/76 and C/São Paulo/378/82) with there being significant genetic drift compared to earlier circulating viruses within both lineages. The location of a number of encoded amino acid substitutions in HEF glycoproteins suggests antigenic drift may also have occurred. Observations of ICV outbreaks in other countries, with some of the infections being associated with severe disease, indicates that ICV infection has the potential to have significant clinical and health care impact in humans. IMPORTANCE Influenza C virus infection of humans is common and re-infection can occur throughout life. While symptoms are generally mild, severe disease cases have been reported, but knowledge of the virus is limited as little systematic surveillance for influenza C virus is conducted and the virus cannot be studied by classical virologic methods as the virus cannot be readily isolated in laboratories. A combination of systematic surveillance in Hong Kong SAR, China and new gene sequencing methods has been used in this study to assess influenza C virus evolution and provided evidence for a two-year cycle of disease outbreaks. The results of studies like that reported here are key to developing an understanding of the impact of influenza C virus infection in humans and how virus evolution might be associated with epidemics.