Figure 1: Group photo of WHO participants and trainers for the influenza Bioinformatics workshop organized by BII, NPHL/NCID, GISAID and WHO.
BII continues to contribute strongly to regional and global influenza surveillance efforts through our tool FluSurver (http:// flusurver.bii.a-star.edu.sg/) developed by the Maurer-Stroh group. It facilitates analysis of influenza sequences, summarizing occurrence of new mutations, their geospatial and temporal context, 3D structure mapping, vicinity to structural interaction sites as well as prediction of phenotypic effects through our literature-curated database of prior known effects of influenza mutations. The FluSurver is also an integral analysis tool for GISAID, the largest influenza database known for hosting the latest outbreak sequences. As part of this collaboration, we regularly hold joint workshops with GISAID and ISIRV to train representatives of National Influenza Centres from >40 countries to use our FluSurver tool as part of the WHO surveillance network (Cape Town 2013, St. Petersburg 2014, Singapore 2014, Hong Kong 2015, Chicago 2016, Shanghai 2017, Madagascar 2018, Singapore 2019 as part of the Options X conference, Figure 1). Our in silico drug resistance findings using the FluSurver have been included in the WHO CCs’ global update on the susceptibility of human influenza viruses to neuraminidase inhibitors (Lackenby et al.) and we have been part of an international panel that reviewed viral factors in influenza pandemic risk assessment. We have also used our expertise to other infectious disease areas relevant for Singapore including detection of Norovirus in a hotel restaurant holding wedding banquets, identification of a new Coronavirus in local cave bats and of factors influencing drug susceptibility and severity in local HIV strains. We have been supporting the Ministry of Health (MOH) and the National Environment Agency (NEA) analyzing Dengue and Zika outbreaks.
Also in 2019 we published a new approach that could reduce animal studies aimed at understanding influenza virus mutations that change host specificity to adapt to replication in mammalian hosts which have been in the spotlight of government bans against gain of function experiments for concerns on safety (Lee et al.). As a safe, higher-throughput alternative, researchers from BII, Harvard and Amsterdam Medical Centre explored the possibility of using readily available passage bias data from 80,000 seasonal surveillance influenza strains shared via the GISAID initiative that were either grown in mammalian cells or eggs. Using a statistical approach to identify host adaptation sites from this data (Figure 2), we found that information from passage bias can identify the known and also provide new candidate sites for host specificity changes to aid in risk assessment for emerging strains. In other notable infectious disease work published in 2019, we identified in vivo verified drugs approved for other diseases that can be repurposed against influenza and helped characterizing intense interseasonal influenza outbreaks in Australia. Led by former PhD student Alvin Han, we also published novel methods PhyCLIP and Phydelity for parameter-free phylogenetic clustering as well as identification of transmission chains, respectively.
Figure 2: Statistical approach to identify positions relevant for host specificity by studying viruses passaged in cells of different organisms.
The group of Peter Bond is supported by an NRFfunded CRP grant which seeks to understand the mechanisms by which flaviviruses infect cells and interact with host immune factors. As part of a Singapore-wide collaborative team of researchers spanning NUS, A*STAR, Duke-NUS, and SGH, we have extended our “virtual flavivirus” models (Marzinek et al.) to show how dengue particles can “sense” the local environment to modulate their “breathing” dynamics associated with the viral life cycle, switching between smooth or bumpy shaped morphologies when transmitted from the mosquito and/or during a fever in the human host (Sharma et al.). By combining these models with cryo-electron microscopy data (Figure 3), we extended these insights to understand the process by which host antibodies inadvertently worsen dengue-induced disease by facilitating virus particle maturation (Wirawan et al), and most recently, discovered how dengue particles may “outsmart” vaccines when a patient has fever by modulating its “breathing” behavior to evade antibodies (Lim et al.). We are also interested in the healthy functioning of receptors in the human immune system (Holdbrook et al), and worked with researchers at Duke-NUS to show how interactions between such receptors and secreted dengue lipoprotein particles may increase disease severity and lead to epidemics (Chan et al.).
Figure 3: Simulations combined with cryo-electron microscopy data help to map how antibodies (orange) bind and dislodge the pr protein (white) to expose a key fusogenic region of the envelope proteins (red, yellow, blue), thereby rendering the immature dengue particle (grey transparent surface) infectious.
The new group of young PI Roland Huber analysed strains from all four serotypes of Dengue virus alongside four strains of Zika virus, which allowed us to characterise conserved structural RNA features in these viruses in collaboration with Wan Yue at GIS. Through mutational studies we could conclusively prove that genomic RNA structures in the coding region are essential for viral fitness and may prove interesting novel targets for antiviral therapy (Huber et al.). Together with the Maurer-Stroh and Bond group and NEA, he also studied Flavivirus Cross-Reactivity to Dengue Nonstructural Protein 1 Antigen Detection Assays (Tan et al).
Filoviruses utilize their VP40 viral matrix protein to drive virion assembly and budding, in part, by recruitment of specific WW domain-bearing host proteins via its conserved PPxY Late (L) domain motif. The group of Hao Fan has docked five PPxYcontaining peptides from Marburg, Ebola and angiomotin (AMOT) peptides to Yes Associated Protein (YAP), as well as a control peptide, to identify and understand the virus-host interactions (Han et al.).
Bacterial and Fungal Infections
The important long-term collaboration between the Verma group and the Beuerman group at the Singapore Eye Research Institute has focused on Defensin-derived antimicrobials for eye infections with partners from NTU, NUS and Singapore General Hospital. Besides the already available Defensins Knowledgbase (http://defensins.bii.a-star.edu.sg/), new molecules are now in pre-clinical testing and show activity against a broad spectrum of clinical strains of both gram positive and gram negative bacteria including Pseudomonas, MRSA, fungi (Fusarium, Candida) and Tb. These molecules are fast acting, stable, and do not give rise to resistance and additionally their toxicity is very low, thus characterizing them with a high therapeutic index. Some of these compounds demonstrate a high level of synergy with existing antibiotics, leading to potential reductions in dosage. We started a programme to study these and clinical trials are planned for eye infections. A spinoff has recently been setup to commercialize some of the molecules (www.sinsalabs.com).
An atomic model of the changes in the membrane associated with the MCR resistance has been created jointly by BII and SERI and has been used to design ab initio a novel molecule that was synthesized and showed activity as an excellent adjuvant to administering colistin at significantly reduced and efficacious doses towards the treatment of resistant pathogens. This has resulted in new IP, a manuscript under review and a spinoff planned for early 2020.