13 research outputs found

    Genetic and molecular characterisation of non-tissue culture adapted influenza B viruses detected in Singapore between 2004-2009

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    In Singapore, both influenza A and B viruses circulate in significant proportions throughout the year, with bi-annual peaks corresponding to the monsoon seasons. This statistic is not shared in most temperate countries where influenza is only prevalent during winter. Exploiting this unique situation in Singapore, we studied the characteristics of non-tissue culture adapted influenza B clinical specimens collected from SAF servicemen between the years of 2004 and 2009. A total of 81 clinical specimens were received and 66 tested positive for influenza B upon diagnostic PCR. Growth kinetics of these clinical specimens could not be studied as none of these specimens could replicate in tissue culture. Forty-six of these clinical specimens yielded sequence information for further analysis. Lineage identity of these specimens were determined through phylogenetic analysis, followed by a comparison of the amino acid sequences between the HA, NA, NB, NS1, NEP and BM2 proteins. Molecular cloning of the NB, NS1 and BM2 genes of representative strains were carried out to determine if differences in sequence would translate into phenotypic differences. Phylogenetic analysis show that majority of the specimens were re-assortants with the HA belonging to the Victoria lineage and the NA from the Yamagata lineage. Only one specimen had both these gene segments from the Yamagata lineage. This finding has resulted in us determining several vaccine mismatches over the six years in focus. The NS1 phylogenetic tree showed that seven of the specimens isolated in 2004 clustered closely to B/Lee/40 than the rest. This finding provides an interesting look into the separation of the influenza B lineages. Analysis of the amino acid sequences reveal that majority of the clinical specimens harboured a glycosylation site at position 211 of the HA protein, which was previously not dominant. No mutations conferring drug resistance were noticed among the HA and NA proteins analysed. The 7 specimens clustering with B/Lee/40 in the NS phylogenetic tree had almost identical amino acid sequences to B/Lee/40 NS1 and NEP proteins, differing from the other clinical specimens. We found an inability of these clinical specimens to replicate well in tissue culture, a trait shared with the reference strain B/Lee/40. In an attempt to exploit the differences in amino acid sequences within the clinical specimens, the NB, BM2 and NS1 genes of representative sequences were cloned into a mammalian expression vector. Basic molecular characterisation indicates that the NB protein, which has an unknown role, localised primarily in the cis-Golgi and is present throughout the Golgi. This is similar to the staining pattern displayed by the BM2 protein, suggesting a similarity in the function. SDS-PAGE analysis reveal that both these proteins are modified by the cell and further elucidation is required to determine the kinetics and the function of these modifications. The bulk of the work presented in this thesis revolves around the molecular characterisation of the influenza B NS1 protein. Two main ‘phenotypes’ were observed in respect to cellular localisation; speckled and un-speckled. Two of the strains did not display localisation with nuclear speckles while three did. The introduction of mutations as well as expression of the different domains of the protein did not clarify which residues were involved in the dynamics of this nuclear speckle interaction. Four of the specimens analysed presented a second, smaller peptide on SDS-PAGE analysis. Site-directed mutagenesis revealed that an aspartic acid residue at position 92, instead of an asparagine seems to be responsible for the presence of this smaller peptide. Even so, the mechanism yielding this peptide is still unknown since caspase and MMP inhibitors did not have an effect in its expression. Further elucidation has to be carried out to determine both the mechanism of expression as well as its function in viral replication. The inability of the clinical specimens collected in this study prompts a detailed exploration into the choice of cell-culture systems in passaging influenza B clinical specimens. This may also reflect the lack of understanding of influenza biology, which has been focused primarily on lab-adapted strains instead of naturally circulating strains. The sequence information of the analysed specimens reveal that majority of the clinical specimens sequenced in this study were re-assortants with their HA from the Victoria lineage and the NA from the Yamagata lineage. Only 1 specimen proved to have both these glycoproteins from the Yamagata lineage. Sequence analysis of the HA protein show that a glycosylation site at position 211 is now prevalent amongst circulating strains and no neuraminidase inhibitor mutations were observed in the HA and NA genes. Cloning of the NS1 gene showed that some of the specimens had a nuclear speckled localisation while other did not. The exact domain within the protein responsible for this was not determined in this study and further probing has to be carried out. A smaller peptide, p23, was expressed in 4 of the 6 cloned NS1 genes. This is the first time the expression of p23 has been documented. The amino acids at the interface of the RNA-binding domain and the linker were crucial for its expression or silencing. The exact mechanism of its expression is yet to be elucidated.DOCTOR OF PHILOSOPHY (SBS

    Inactivation and Loss of Infectivity of Enterovirus 70 by Solar Irradiation

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    Enterovirus 70 (EV70) is an emerging viral pathogen that remains viable in final treated effluent. Solar irradiation is, therefore, explored as a low-cost natural disinfection strategy to mitigate potential concerns. EV70 was exposed to simulated sunlight for 24 h at a fluence rate of 28.67 J/cm2/h in three different water matrices, namely, phosphate-buffered saline (PBS), treated wastewater effluent, and chlorinated effluent. In the presence of sunlight, EV70 decreased in infectivity by 1.7 log, 1.0 log, and 1.3 log in PBS, effluent, and chlorinated effluent, respectively. Irradiated EV70 was further introduced to host cell lines and was unable to infect the cell lines. In contrast, EV70 in dark microcosms replicated to titers 13.5, 3.3, and 4.2 times the initial inoculum. The reduction in EV70 infectivity was accompanied by a reduction in viral binding capacity to Vero cells. In addition, genome sequencing analysis revealed five nonsynonymous nucleotide substitutions in irradiated viruses after 10 days of infection in Vero cells, resulting in amino acid substitutions: Lys14Glu in the VP4 protein, Ala201Val in VP2, Gly71Ser in VP3, Glu50Gln in VP1, and Ile47Leu in 3Cpro. Overall, solar irradiation resulted in EV70 inactivation and an inhibition of viral activity in all parameters studied

    Interprofessional collaboration (or lack thereof) between faculty and learning technologists in the creation of digital learning

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    Abstract Background As digital learning becomes more prevalent and important in health professions education, learning technologists play increasingly central roles in designing and delivering learning materials. However, little is understood about the process by which learning technologists have integrated into the existing teaching and learning ecosystem, and it seems that they remain marginal and undervalued. Our aim in this paper was therefore to examine the process of interprofessional co-development of course materials as experienced by educators and learning technologists. Methods Our approach was qualitative, using individual semi-structured interviews (conducted between July 2021 to May 2022) to explore the working relationship between faculty and learning technologists. Transcripts were analysed abductively. Results We found that the attitudes of both faculty and learning technologists towards collaborating to drive digital adoption in health professions education fell into two main themes: “embrace” and “replace” – and “conflict”, which we present as a third theme. Our results revealed that faculty did not take an active and agentic role in developing their digital practices in respect of education delivery. Learning technologists positioned themselves as a resource to support faculty’s knowledge and skill gap in digital competence. There was an obvious power differential between the two groups: learning technologists lacked agency and seemed in the position of servants to faculty masters. This created barriers to effective collaboration. Conclusions By examining the process of co-development of course materials by faculty and learning technologists, we open up a space to examine the social, relational and organisational complexities associated with interprofessional collaboration in digital health professions education. Our study also has important implications for guiding educational policy to better position learning technologists to effectively collaborate with faculty and realise the potential of digital health professions education

    An Increase of Abundance and Transcriptional Activity for Acinetobacter junii Post Wastewater Treatment

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    A membrane bioreactor (MBR)-based wastewater treatment plant (WWTP) in Saudi Arabia is assessed over a five-month period in 2015 and once in 2017 for bacterial diversity and transcriptional activity using metagenomics, metatranscriptomics and real time quantitative polymerase chain reaction (RT-qPCR). Acinetobacter spp. are shown to be enriched in the chlorinated effluent. Members of the Acinetobacter genus are the most abundant in the effluent and chlorinated effluent. At the species level, Acinetobacter junii have higher relative abundances post MBR and chlorination. RNA-seq analysis show that, in A. junii, 288 genes and 378 genes are significantly upregulated in the effluent and chlorinated effluent, respectively, with 98 genes being upregulated in both. RT-qPCR of samples in 2015 and 2017 confirm the upregulation observed in RNA-seq. Analysis of the 98 genes show that majority of the upregulated genes are involved in cellular repair and metabolism followed by resistance, virulence, and signaling. Additionally, two different subpopulations of A. junii are observed in the effluent and chlorinated effluent. The upregulation of cellular repair and metabolism genes, and the formation of different subpopulations of A. junii in both effluents provide insights into the mechanisms employed by A. junii to persist in the conditions of a WWTP

    Membrane Bioreactor-Based Wastewater Treatment Plant in Saudi Arabia: Reduction of Viral Diversity, Load, and Infectious Capacity

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    A membrane bioreactor (MBR)-based wastewater treatment plant in Saudi Arabia was assessed over a nine-month period for virus removal efficiency. Viral diversity was detected using omics-based approaches. Log reduction values (LRV) of Adenoviruses (AdV) and Enteroviruses (EV) were enumerated using digital polymerase chain reaction (dPCR) and assessed for infectivity using fluorescence-based infection assays. MBR treatment was successful in reducing viral diversity. Plant viruses remained abundant in the treated effluent. Human enteric viruses were present in lower abundance than plant viruses, and were reduced by MBR at varying LRV. AdV copy numbers were reduced by 3.7-log. Infectious AdV was not detected in the effluent. EV copy numbers were reduced by 1.7-log post MBR and infectious EV decreased by an average of 2.0-log. Infectious EV was detected in the chlorinated effluent, occasionally in concentrations that approximate to its 50% infectious dose. Overall, results indicated that a MBR-based wastewater treatment plant (WWTP) effectively reduces viral diversity, viral load, and infectious capacity by up to 4-logs. These findings suggest potential concerns associated with plant and human enteric viruses for reuse events in this country. Local guidelines for assessment of treated water quality should take into consideration both infectious viral concentration and LRV

    Molecular and biochemical characterization of the NS1 protein of non-cultured influenza B virus strains circulating in Singapore

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    In this study we compared the NS1 protein of Influenza B/Lee/40 and several non-cultured Influenza B virus clinical strains detected in Singapore. In B/Lee/40 virus-infected cells and in cells expressing the recombinant B/Lee/40 NS1 protein a full-length 35 kDa NS1 protein and a 23 kDa NS1 protein species (p23) were detected. Mutational analysis of the NS1 gene indicated that p23 was generated by a novel cleavage event within the linker domain between an aspartic acid and proline at amino acid residues at positions 92 and 93 respectively (DP92-93), and that p23 contained the first 92 amino acids of the NS1 protein. Sequence analysis of the Singapore strains indicated the presence of either DP92-93 or NP92-93 in the NS1 protein, but protein expression analysis showed that p23 was only detected in NS1 proteins with DP92-93.. An additional adjacent proline residue at position 94 (P94) was present in some strains and correlated with increased p23 levels, suggesting that P94 has a synergistic effect on the cleavage of the NS1 protein. The first 145 amino acids of the NS1 protein are required for inhibition of ISG15-mediated ubiquitination, and our analysis showed that Influenza B viruses circulating in Singapore with DP92-93 expressed truncated NS1 proteins and may differ in their capacity to inhibit ISG15 activity. Thus, DP92-93 in the NS1 protein may confer a disadvantage to Influenza B viruses circulating in the human population and interestingly the low frequency of DP92-93detection in the NS1 protein since 2004 is consistent with this suggestion.Published versio

    A sustained antiviral host response in respiratory syncytial virus infected human nasal epithelium does not prevent progeny virus production

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    Respiratory syncytial virus infection was examined using a human nasal epithelial cell model. Maximum levels of shed-virus were produced at between 3 and 5 days post-infection (dpi), and the infectivity of the shed-virus was stable up to 10 dpi. The highest levels of interferon signalling were recorded at 2dpi, and infection induced a widespread antivirus response in the nasal epithelium, involving both infected cells and non-infected cells. Although these cellular responses were associated with reduced levels of progeny virus production and restricted virus spread, they did not inhibit the infectivity virus that is shed early in infection. In the clinical context these data suggest that although the host cell response in the nasal epithelium may restrict the levels of progeny virus particles produced, the stability of the shed-virus in the nasal mucosa may be an important factor in both disease progression and virus transmission.MOE (Min. of Education, S’pore)NMRC (Natl Medical Research Council, S’pore
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