The biological role of Asn-linked glycosylations of DENV-2 E protein

The envelope proteins of many viruses are glycosylated, where the glycans on the virion surface have been shown to be important, in addition to their strictly structural role, in various aspects of the virus life cycle, such as receptor-binding and escape from antibody recognition. Dengue virus (DENV) is in terms of human disease frequency the most important member of the flaviviruses, a group of small, enveloped, plus-strand RNA viruses. The four DENV serotypes differ from all other flaviviruses by the presence of two glycans on the envelope (E) protein, one linked to Asn-153, a glycosylation which is well conserved among the flaviviruses, and a second linked to Asn-67, which is unique to DENY. While the presence of glycan at Asn-153 has been shown by others to influence fusion of viral with cellular membranes, infectivity, assembly and/or release and virulence, the glycan at Asn-67 has been suggested to be critical in virus attachment to the cell surface molecule, DC-SIGN, a putative cellular receptor for DENV. In this study, I have used a reverse genetics approach to prevent glycosylation at Asn-67, Asn-153, or both carbohydrate acceptor sites, in order to investigate their physiological function(s). Single or multiple point mutation were introduced at the Asn-153 and Asn-67 glycosylation sites, respectively, on the E protein ofDENV-2 strains NGC and PUO-218, using two infectious cDNA clones. DENV -2 strain, NGC, is a laboratory-adapted virus, while strain PUO-218 is a low-passage natural isolate from Toxorhynchites splendens mosquitoes. ... I also show that removal of both glycans from the DENV-2 E protein prevents the recovery of viable virus with a stable mutant genotype (Chapters 3 and 5). Nevertheless, given the importance of strain origin and type of substitution used for deletion of the carbohydrate acceptor sites that I have demonstrated in this thesis, it is likely that the engineering of a viable DENV variant lacking both E protein glycans is feasible

Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2015-2016.

Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) assessed antiviral susceptibility of 14,330 influenza A and B viruses collected by WHO-recognized National Influenza Centres (NICs) between May 2015 and May 2016. Neuraminidase (NA) inhibition assay was used to determine 50% inhibitory concentration (IC50) data for NA inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. Furthermore, NA sequences from 13,484 influenza viruses were retrieved from public sequence databases and screened for amino acid substitutions (AAS) associated with reduced inhibition (RI) or highly reduced inhibition (HRI) by NAIs. Of the viruses tested by WHO CCs 93% were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 0.8% (n = 113) exhibited either RI or HRI by at least one of four NAIs. As in previous seasons, the most common NA AAS was H275Y in A(H1N1)pdm09 viruses, which confers HRI by oseltamivir and peramivir. Two A(H1N1)pdm09 viruses carried a rare NA AAS, S247R, shown in this study to confer RI/HRI by the four NAIs. The overall frequency of A(H1N1)pdm09 viruses containing NA AAS associated with RI/HRI was approximately 1.8% (125/6915), which is slightly higher than in the previous 2014-15 season (0.5%). Three B/Victoria-lineage viruses contained a new AAS, NA H134N, which conferred HRI by zanamivir and laninamivir, and borderline HRI by peramivir. A single B/Victoria-lineage virus harboured NA G104E, which was associated with HRI by all four NAIs. The overall frequency of RI/HRI phenotype among type B viruses was approximately 0.6% (43/7677), which is lower than that in the previous season. Overall, the vast majority (>99%) of the viruses tested by WHO CCs were susceptible to all four NAIs, showing normal inhibition (NI). Hence, NAIs remain the recommended antivirals for treatment of influenza virus infections. Nevertheless, our data indicate that it is prudent to continue drug susceptibility monitoring using both NAI assay and sequence analysis

Both E Protein Glycans Adversely Affect Dengue Virus Infectivity but Are Beneficial for Virion Release▿

The E protein of most flaviviruses is modified by Asn-linked glycosylation at residue 153/154 and in the case of the four dengue virus (DENV) serotypes by a second glycan at residue 67. However, the absence of E protein glycosylation among numerous natural isolates of different flaviviruses suggests that the glycan, per se, is not critically important in the virus life cycle. Consistent with this notion, we show that ablation of both glycans from the DENV-2 E protein reduces but does not prevent growth of the variant in mammalian and mosquito cells. We found a pronounced and opposing effect of glycan ablation on two stages of the virus growth cycle: infectivity and release. Loss of either of the two DENV E protein glycans markedly enhanced infectivity of variants for mosquito cells at the expense of efficient virion release. The variants also displayed reduced release in mammalian cells, which was more prominent for viruses lacking the Asn 67-linked glycan than for those lacking the Asn 153-linked glycan, without a marked change in infectivity. Mutations, which compensated for the defect in virus morphogenesis associated with ablation of the Asn 67-linked glycan in mammalian cells but interestingly not in mosquito cells, were identified at the glycosylation acceptor motif and a second site in E protein domain II. The dueling influences of infectivity and release on virus growth affected by the glycans may explain the plasticity in E protein glycosylation among the flaviviruses

Mutations I117V and I117M and Oseltamivir Sensitivity of Pandemic (H1N1) 2009 Viruses

Analysis of mutations I117V and I117M in the neuraminidase of influenza A pandemic (H1N1) 2009 viruses showed that I117V confers a mild reduction in oseltamivir sensitivity and has a synergistic effect of further increasing resistance when combined with H275Y. Contrary to recent reports, the I117M mutation does not alter oseltamivir sensitivity

Peramivir and laninamivir susceptibility of circulating influenza A and B viruses

Influenza viruses collected from regions of Asia, Africa and Oceania between 2009 and 2012 were tested for their susceptibility to two new neuraminidase inhibitors, peramivir and laninamivir. All viruses tested had normal laninamivir inhibition. However, 3·2% (19/599) of A(H1N1)pdm09 viruses had highly reduced peramivir inhibition (due to H275Y NA mutation) and <1% (6/1238) of influenza B viruses had reduced or highly reduced peramivir inhibition, with single occurrence of variants containing I221T, A245T, K360E, A395E, D432G and a combined G145R+Y142H mutation. These data demonstrate that despite an increase in H275Y variants in 2011, there was no marked change in the frequency of peramivir- or laninamivir-resistant variants following the market release of the drugs in Japan in 2010.Published versio

Affective dysfunction in a mouse model of Rett syndrome: therapeutic effects of environmental stimulation and physical activity

Rett syndrome (RTT) is a neurodevelopmental disorder associated with mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2) and consequent dysregulation of brain maturation. Patients suffer from a range of debilitating physical symptoms, however, behavioral and emotional symptoms also severely affect their quality of life. Here, we present previously unreported and clinically relevant affective dysfunction in the female heterozygous Mecp2tm1Tam mouse model of RTT (129sv and C57BL6 mixed background). The affective dysfunction and aberrant anxiety-related behavior of the Mecp2+ / - mice were found to be reversible with environmental enrichment (EE) from 4 weeks of age. The effect of exercise alone (via wheel running) was also explored, providing the first evidence that increased voluntary physical activity in an animal model of RTT is beneficial for some phenotypes. Mecp2+ / - mutants displayed elevated corticosterone despite decreased Crh expression, demonstrating hypothalamic-pituitary-adrenal axis dysregulation. EE of Mecp2+ / - mice normalized basal serum corticosterone and hippocampal BDNF protein levels. The enrichment-induced rescue appears independent of the transcriptional regulation of the MeCP2 targets Bdnf exon 4 and Crh. These findings provide new insight into the neurodevelopmental role of MeCP2 and pathogenesis of RTT, in particular the affective dysfunction. The positive outcomes of environmental stimulation and physical exercise have implications for the development of therapies targeting the affective symptoms, as well as behavioral and cognitive dimensions, of this devastating neurodevelopmental disorder

Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors and status of novel antivirals, 2016-2017.

A total of 13672 viruses, collected by World Health Organization recognised National Influenza Centres between May 2016 and May 2017, were assessed for neuraminidase inhibitor susceptibility by four WHO Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance Epidemiology and Control of Influenza. The 50% inhibitory concentration (IC50) was determined for oseltamivir and zanamivir for all viruses, and for peramivir and laninamivir in a subset (n = 8457). Of the viruses tested, 94% were obtained from the Western Pacific, Americas and European WHO regions, while limited viruses were available from the Eastern Mediterranean, African and South East Asian regions. Reduced inhibition (RI) by one or more neuraminidase inhibitor was exhibited by 0.2% of viruses tested (n = 32). The frequency of viruses with RI has remained low since this global analysis began (2015/16: 0.8%, 2014/15: 0.5%; 2013/14: 1.9%; 2012/13: 0.6%) but 2016/17 has the lowest frequency observed to date. Analysis of 13581 neuraminidase sequences retrieved from public databases, of which 5243 sequences were from viruses not included in the phenotypic analyses, identified 58 further viruses (29 without phenotypic analyses) with amino acid substitutions associated with RI by at least one neuraminidase inhibitor. Bringing the total proportion to 0.5% (90/18915). This 2016/17 analysis demonstrates that neuraminidase inhibitors remain suitable for treatment and prophylaxis of influenza virus infections, but continued monitoring is important. An expansion of surveillance testing is paramount since several novel influenza antivirals are in late stage clinical trials with some resistance already having been identified

Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors and status of novel antivirals, 2016-2017.

A total of 13672 viruses, collected by World Health Organization recognised National Influenza Centres between May 2016 and May 2017, were assessed for neuraminidase inhibitor susceptibility by four WHO Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance Epidemiology and Control of Influenza. The 50% inhibitory concentration (IC50) was determined for oseltamivir and zanamivir for all viruses, and for peramivir and laninamivir in a subset (n = 8457). Of the viruses tested, 94% were obtained from the Western Pacific, Americas and European WHO regions, while limited viruses were available from the Eastern Mediterranean, African and South East Asian regions. Reduced inhibition (RI) by one or more neuraminidase inhibitor was exhibited by 0.2% of viruses tested (n = 32). The frequency of viruses with RI has remained low since this global analysis began (2015/16: 0.8%, 2014/15: 0.5%; 2013/14: 1.9%; 2012/13: 0.6%) but 2016/17 has the lowest frequency observed to date. Analysis of 13581 neuraminidase sequences retrieved from public databases, of which 5243 sequences were from viruses not included in the phenotypic analyses, identified 58 further viruses (29 without phenotypic analyses) with amino acid substitutions associated with RI by at least one neuraminidase inhibitor. Bringing the total proportion to 0.5% (90/18915). This 2016/17 analysis demonstrates that neuraminidase inhibitors remain suitable for treatment and prophylaxis of influenza virus infections, but continued monitoring is important. An expansion of surveillance testing is paramount since several novel influenza antivirals are in late stage clinical trials with some resistance already having been identified