Outbreak.info genomic reports: scalable and dynamic surveillance of SARS-CoV-2 variants and mutations.

In response to the emergence of SARS-CoV-2 variants of concern, the global scientific community, through unprecedented effort, has sequenced and shared over 11 million genomes through GISAID, as of May 2022. This extraordinarily high sampling rate provides a unique opportunity to track the evolution of the virus in near real-time. Here, we present outbreak.info , a platform that currently tracks over 40 million combinations of Pango lineages and individual mutations, across over 7,000 locations, to provide insights for researchers, public health officials and the general public. We describe the interpretable visualizations available in our web application, the pipelines that enable the scalable ingestion of heterogeneous sources of SARS-CoV-2 variant data and the server infrastructure that enables widespread data dissemination via a high-performance API that can be accessed using an R package. We show how outbreak.info can be used for genomic surveillance and as a hypothesis-generation tool to understand the ongoing pandemic at varying geographic and temporal scales.

Urgent need for a non-discriminatory and non-stigmatizing nomenclature for monkeypox virus.

We propose a novel, non-discriminatory classification of monkeypox virus diversity. Together with the World Health Organization, we named three clades (I, IIa and IIb) in order of detection. Within IIb, the cause of the current global outbreak, we identified multiple lineages (A.1, A.2, A.1.1 and B.1) to support real-time genomic surveillance.

Variant-specific IgA protects against Omicron infection.

BACKGROUND: The emergence of rapidly evolving SARS-CoV-2 variants, coupled with waning vaccine-induced immunity, has contributed to the rise of vaccine breakthrough infections. It is crucial to understand how vaccine-induced protection is mediated. METHODS: We examined two prospective cohorts of mRNA-vaccinated-and-boosted individuals during the Omicron wave of infection in Singapore. RESULTS: We found that, individuals, who remain uninfected over the follow-up period, had a higher variant-specific IgA, but not IgG, antibody response at 1-month post booster vaccination, compared with individuals who became infected. CONCLUSIONS: We conclude that IgA may have a potential contributory role in protection against Omicron infection.

Pain Influences Neuropsychological Performance Following Electrical Injury: A Cross-Sectional Study.

OBJECTIVE: Electrical injury (EI) is a significant, multifaceted trauma often with multi-domain cognitive sequelae, even when the expected current path does not pass through the brain. Chronic pain (CP) research suggests pain may affect cognition directly and indirectly by influencing emotional distress which then impacts cognitive functioning. As chronic pain may be critical to understanding EI-related cognitive difficulties, the aims of the current study were: examine the direct and indirect effects of pain on cognition following EI and compare the relationship between pain and cognition in EI and CP populations. METHOD: This cross-sectional study used data from a clinical sample of 50 patients with EI (84.0% male; Mage = 43.7 years) administered standardized measures of pain (Pain Patient Profile), depression, and neurocognitive functioning. A CP comparison sample of 93 patients was also included. RESULTS: Higher pain levels were associated with poorer attention/processing speed and executive functioning performance among patients with EI. Depression was significantly correlated with pain and mediated the relationship between pain and attention/processing speed in patients with EI. When comparing the patients with EI and CP, the relationship between pain and cognition was similar for both clinical groups. CONCLUSIONS: Findings indicate that pain impacts mood and cognition in patients with EI, and the influence of pain and its effect on cognition should be considered in the assessment and treatment of patients who have experienced an electrical injury.

Peroxidation of tetronic 1107 reduces protein chaperone effect.

To enhance the stability of protein therapeutics, pharmaceutical companies have long used various copolymer surfactants as excipients. They act to stabilize proteins by adhering to the hydrophobic surface of the protein preventing denaturation and aggregation. However, some commonly used excipients possess polyoxyalkylene chains that are susceptible to oxidative degradation while in aqueous solution. We postulate that oxidation reactions involving the hydrophobic domains reduce the surfactant's ability to stabilize the native protein structure. We investigated the effect of UV (λ = 254 nm) radiated poloxamine T1107 (T1107) on its ability to disaggregate DTT denatured hen egg-white lysozyme (HEWL). Peroxidation of UV irradiated T1107 was analyzed using FTIR spectroscopy, the Fe+2to Fe+3ion reduction assay method, and1H NMR. Our results indicate that increased UV irradiation led to structural changes in T1107, specifically the addition of a carbonyl on the formate group. The structural change decreased T1107's ability to disaggregate HEWL thus supporting our hypothesis. These results indicate that peroxide content is an important parameter to control in polyoxyalkylene-based excipients.

Hidden realities of electrical injuries.

ABSTRACT: The clinical spectrum of electrical injury ranges from the absence of any external physical signs to severe and life-threatening trauma. This article discusses the fundamental concepts and misunderstandings surrounding electrical injuries and the best practices for evaluation and treatment.

Higher rates of carbapenem-related seizures in older hospitalised adults.

Clinical experience suggests higher occurrence of carbapenem-associated seizures in the elderly than what is reported in the available literature (range between 0.2% and 0.7%). An audit of 1345 patients with age 60 years or older, who received imipenem, ertapenem or meropenem during their acute hospitalisation found 32 (2.4%) subjects developed seizures. Subjects with more than one central nervous system disorders were 11.6 times more likely to develop seizures (odds ratio 11.61, P < 0.001) and subjects with prior history of seizures is associated with four times greater risks (odds ratio 4.02, P = 0.005). Physicians should exercise caution when prescribing carbapenems in elderly, especially those with known epilepsy and a high number of intracranial pathologies.

Electromuscular Incapacitation Current Induced Neuromuscular Tissue Injury.

Electrical stun devices (ESDs) serve a basic role in law enforcement and provide an alternative to lethal options for target control by causing electromuscular incapacitation (EMI). A fundamental concern is the adverse health consequences associated with their use. The capability of EMI electric field pulses to disrupt skeletal muscle cells (i.e. rhabdomyolysis) was investigated over the operational range commonly used in commercial EMI devices. Functional and structural alteration and recovery of muscle and nerve tissue were assessed. In an anesthetized swine model, the left thigh was exposed to 2 min of electrical pulses, using a commercially available ESD or a custom-made EMI signal power amplifier. Serum creatinine phosphokinase (CPK), troponin, aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) levels were monitored intermittently for 6 h post-EMI exposure. A standard external cardiac defibrillator served as a positive control. Muscle and nerve tissue histology adjacent to the EMI contacts were examined. Post-EMI shock skeletal muscle function was evaluated by analyzing the compound muscle action potentials (CMAPs) of the rectus femoris muscle. Maximal energy cardiac defibrillator pulses resulted in rhabdomyolysis and marked elevation of CPK, LDH, and AST 6 h post-shock. EMI field pulses resulted in the animals developing transient acidosis. CMAP amplitudes decreased approximately 50% after EMI and recovered to near-normal levels within 6 h. Within 6 h post-EMI exposure, blood CPK was mildly increased, LDH was normal, and no arrhythmia was observed. Minimal rhabdomyolysis was produced by the EMI pulses. These results suggest that EMI exposure is unlikely to cause extremity rhabdomyolysis in normal individuals. Bioelectromagnetics. © 2020 Bioelectromagnetics Society.

Early transmission patterns of coronavirus disease 2019 (COVID-19) in travellers from Wuhan to Thailand, January 2020.

We report two cases of coronavirus disease 2019 (COVID-19) in travellers from Wuhan, China to Thailand. Both were independent introductions on separate flights, discovered with thermoscanners and confirmed with RT-PCR and genome sequencing. Both cases do not seem directly linked to the Huanan Seafood Market in Hubei but the viral genomes are identical to four other sequences from Wuhan, suggesting early spread within the city already in the first week of January.

Dynamic Convergent Evolution Drives the Passage Adaptation across 48 Years' History of H3N2 Influenza Evolution.

Influenza viruses are often propagated in a diverse set of culturing media and additional substitutions known as passage adaptation can cause extra evolution in the target strain, leading to ineffective vaccines. Using 25,482 H3N2 HA1 sequences curated from Global Initiative on Sharing All Influenza Data and National Center for Biotechnology Information databases, we found that passage adaptation is a very dynamic process that changes over time and evolves in a seesaw like pattern. After crossing the species boundary from bird to human in 1968, the influenza H3N2 virus evolves to be better adapted to the human environment and passaging them in embryonated eggs (i.e., an avian environment) leads to increasingly stronger positive selection. On the contrary, passage adaptation to the mammalian cell lines changes from positive selection to negative selection. Using two statistical tests, we identified 19 codon positions around the receptor binding domain strongly contributing to passage adaptation in the embryonated egg. These sites show strong convergent evolution and overlap extensively with positively selected sites identified in humans, suggesting that passage adaptation can confound many of the earlier studies on influenza evolution. Interestingly, passage adaptation in recent years seems to target a few codon positions in antigenic surface epitopes, which makes it difficult to produce antigenically unaltered vaccines using embryonic eggs. Our study outlines another interesting scenario whereby both convergent and adaptive evolution are working in synchrony driving viral adaptation. Future studies from sequence analysis to vaccine production need to take careful consideration of passage adaptation.

Amphiphilic copolymers reduce aggregation of unfolded lysozyme more effectively than polyethylene glycol.

Certain amphiphilic block copolymers are known to prevent aggregation of unfolded proteins. To better understand the mechanism of this effect, the optical properties of heat-denatured and dithiothreitol reduced lysozyme were evaluated with respect to controls using UV-Vis spectroscopy, transmission electron microscopy (TEM) and circular dichroism (CD) measurements. Then, the effects of adding Polyethylene Glycol (8000 Da), the triblock surfactant Poloxamer 188 (P188), and the tetrablock copolymer Tetronic 1107 (T1107) to the lysozyme solution were compared. Overall, T1107 was found to be more effective than P188 in inhibiting aggregation, while PEG exhibited no efficacy. TEM imaging of heat-denatured and reduced lysozymes revealed spherical aggregates with on average 250-450 nm diameter. Using CD, more soluble lysozyme was recovered with T1107 than P188 with ß-sheet secondary structure. The greater effectiveness of the larger T1107 in preventing aggregation of unfolded lysozyme than the smaller P188 and PEG points to steric hindrance at play; signifying the importance of size match between the hydrophobic region of denatured protein and that of amphiphilic copolymers. Thus, our results corroborate that certain multi-block copolymers are effective in preventing heat-induced aggregation of reduced lysozymes and future studies warrant more detailed focus on specific applications of these copolymers.

Discovery of Influenza A Virus Sequence Pairs and Their Combinations for Simultaneous Heterosubtypic Targeting that Hedge against Antiviral Resistance.

The multiple circulating human influenza A virus subtypes coupled with the perpetual genomic mutations and segment reassortment events challenge the development of effective therapeutics. The capacity to drug most RNAs motivates the investigation on viral RNA targets. 123,060 segment sequences from 35,938 strains of the most prevalent subtypes also infecting humans-H1N1, 2009 pandemic H1N1, H3N2, H5N1 and H7N9, were used to identify 1,183 conserved RNA target sequences (≥15-mer) in the internal segments. 100% theoretical coverage in simultaneous heterosubtypic targeting is achieved by pairing specific sequences from the same segment ("Duals") or from two segments ("Doubles"); 1,662 Duals and 28,463 Doubles identified. By combining specific Duals and/or Doubles to form a target graph wherein an edge connecting two vertices (target sequences) represents a Dual or Double, it is possible to hedge against antiviral resistance besides maintaining 100% heterosubtypic coverage. To evaluate the hedging potential, we define the hedge-factor as the minimum number of resistant target sequences that will render the graph to become resistant i.e. eliminate all the edges therein; a target sequence or a graph is considered resistant when it cannot achieve 100% heterosubtypic coverage. In an n-vertices graph (n ≥ 3), the hedge-factor is maximal (= n- 1) when it is a complete graph i.e. every distinct pair in a graph is either a Dual or Double. Computational analyses uncover an extensive number of complete graphs of different sizes. Monte Carlo simulations show that the mutation counts and time elapsed for a target graph to become resistant increase with the hedge-factor. Incidentally, target sequences which were reported to reduce virus titre in experiments are included in our target graphs. The identity of target sequence pairs for heterosubtypic targeting and their combinations for hedging antiviral resistance are useful toolkits to construct target graphs for different therapeutic objectives.

Inter-Species Host Gene Expression Differences in Response to Human and Avian Influenza A Virus Strains.

Low pathogenic avian influenza (LPAI) viruses are a source of sporadic human infections and could also contribute to future pandemic outbreaks but little is known about inter-species differences in the host responses to these viruses. Here, we studied host gene expression signatures of cell lines from three species (human, chicken, and canine) in response to six different viruses (H1N1/WSN, H5N2/F59, H5N2/F118, H5N2/F189, H5N3 and H9N2). Comprehensive microarray probe set re-annotation and ortholog mapping of the host genes was necessary to allow comparison over extended functionally annotated gene sets and orthologous pathways. The annotations are made available to the community for commonly used microarray chips. We observe a strong tendency of the response being cell type- rather than virus-specific. In chicken cells, we found up-regulation of host factors inducing virus infectivity (e.g., oxysterol binding protein like 1A (OSBPL1A) and Rho GTPase activating protein 21 (ARHGAP21)) while reducing apoptosis (e.g., mitochondrial ribosomal protein S27 (MRPS27)) and increasing cell proliferation (e.g., COP9 signalosome subunit 2 (COPS2)). On the other hand, increased antiviral, pro-apoptotic and inflammatory signatures have been identified in human cells while cell cycle and metabolic pathways were down-regulated. This signature describes how low pathogenic avian influenza (LPAI) viruses are being tolerated and shed from chicken but potentially causing cellular disruption in mammalian cells.

Estimating the fitness advantage conferred by permissive neuraminidase mutations in recent oseltamivir-resistant A(H1N1)pdm09 influenza viruses.

Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide.

South-east Asian Zika virus strain linked to cluster of cases in Singapore, August 2016.

Zika virus (ZIKV) is an ongoing global public health emergency with 70 countries and territories reporting evidence of ZIKV transmission since 2015. On 27 August 2016, Singapore reported its first case of local ZIKV transmission and identified an ongoing cluster. Here, we report the genome sequences of ZIKV strains from two cases and find through phylogenetic analysis that these strains form an earlier branch distinct from the recent large outbreak in the Americas.

Genetic signatures of HIV-1 envelope-mediated bystander apoptosis.

The envelope (Env) glycoprotein of HIV is an important determinant of viral pathogenesis. Several lines of evidence support the role of HIV-1 Env in inducing bystander apoptosis that may be a contributing factor in CD4(+) T cell loss. However, most of the studies testing this phenomenon have been conducted with laboratory-adapted HIV-1 isolates. This raises the question of whether primary Envs derived from HIV-infected patients are capable of inducing bystander apoptosis and whether specific Env signatures are associated with this phenomenon. We developed a high throughput assay to determine the bystander apoptosis inducing activity of a panel of primary Envs. We tested 38 different Envs for bystander apoptosis, virion infectivity, neutralizing antibody sensitivity, and putative N-linked glycosylation sites along with a comprehensive sequence analysis to determine if specific sequence signatures within the viral Env are associated with bystander apoptosis. Our studies show that primary Envs vary considerably in their bystander apoptosis-inducing potential, a phenomenon that correlates inversely with putative N-linked glycosylation sites and positively with virion infectivity. By use of a novel phylogenetic analysis that avoids subtype bias coupled with structural considerations, we found specific residues like Arg-476 and Asn-425 that were associated with differences in bystander apoptosis induction. A specific role of these residues was also confirmed experimentally. These data demonstrate for the first time the potential of primary R5 Envs to mediate bystander apoptosis in CD4(+) T cells. Furthermore, we identify specific genetic signatures within the Env that may be associated with the bystander apoptosis-inducing phenotype.

Influenza A(H5N8) Virus Similar to Strain in Korea Causing Highly Pathogenic Avian Influenza in Germany.

Highly pathogenic avian influenza (H5N8) virus, like the recently described H5N8 strain from Korea, was detected in November 2014 in farmed turkeys and in a healthy common teal (Anas crecca) in northeastern Germany. Infected wild birds possibly introduced this virus.

Influenza viruses with B/Yamagata- and B/Victoria-like neuraminidases are differentially affected by mutations that alter antiviral susceptibility.

OBJECTIVES: The burden of disease due to influenza B is often underestimated. Clinical studies have shown that oseltamivir, a widely used neuraminidase inhibitor (NAI) antiviral drug, may have reduced effectiveness against influenza B viruses. Therefore, it is important to study the effect of neuraminidase mutations in influenza B viruses that may further reduce NAI susceptibility, and to determine whether these mutations have the same effect in the two lineages of influenza B viruses that are currently circulating (B/Yamagata-like and B/Victoria-like). METHODS: We characterized the effect of 16 amino acid substitutions across five framework residues and four monomeric interface residues on the susceptibility to four different NAIs (oseltamivir, zanamivir, peramivir and laninamivir). RESULTS: Framework residue mutations E117A and E117G conferred highly reduced inhibition to three of the four NAIs, but substantially reduced neuraminidase activity, whereas other framework mutations retained a greater level of NA activity. Mutations E105K, P139S and G140R of the monomeric interface were also found to cause highly reduced inhibition, but, interestingly, their effect was substantially greater in a B/Victoria-like neuraminidase than in a B/Yamagata-like neuraminidase, with some susceptibility values being up to 1000-fold different between lineages. CONCLUSIONS: The frequency and the effect of key neuraminidase mutations on neuraminidase activity and NAI susceptibility can differ substantially between the two influenza B lineages. Therefore, future surveillance, analysis and interpretation of influenza B virus NAI susceptibility should consider the B lineage of the neuraminidase in the same manner as already occurs for different influenza A neuraminidase subtypes.

Antigenic drift of the pandemic 2009 A(H1N1) influenza virus in A ferret model.

Surveillance data indicate that most circulating A(H1N1)pdm09 influenza viruses have remained antigenically similar since they emerged in humans in 2009. However, antigenic drift is likely to occur in the future in response to increasing population immunity induced by infection or vaccination. In this study, sequential passaging of A(H1N1)pdm09 virus by contact transmission through two independent series of suboptimally vaccinated ferrets resulted in selection of variant viruses with an amino acid substitution (N156K, H1 numbering without signal peptide; N159K, H3 numbering without signal peptide; N173K, H1 numbering from first methionine) in a known antigenic site of the viral HA. The N156K HA variant replicated and transmitted efficiently between naïve ferrets and outgrew wildtype virus in vivo in ferrets in the presence and absence of immune pressure. In vitro, in a range of cell culture systems, the N156K variant rapidly adapted, acquiring additional mutations in the viral HA that also potentially affected antigenic properties. The N156K escape mutant was antigenically distinct from wildtype virus as shown by binding of HA-specific antibodies. Glycan binding assays demonstrated the N156K escape mutant had altered receptor binding preferences compared to wildtype virus, which was supported by computational modeling predictions. The N156K substitution, and culture adaptations, have been detected in human A(H1N1)pdm09 viruses with N156K preferentially reported in sequences from original clinical samples rather than cultured isolates. This study demonstrates the ability of the A(H1N1)pdm09 virus to undergo rapid antigenic change to evade a low level vaccine response, while remaining fit in a ferret transmission model of immunization and infection. Furthermore, the potential changes in receptor binding properties that accompany antigenic changes highlight the importance of routine characterization of clinical samples in human A(H1N1)pdm09 influenza surveillance.

Tamoxifen Upregulates Collagenase Gene Expression in Human Dermal Fibroblasts.

Background: Tamoxifen is a known inhibitor of fibroblast transforming growth factor beta biosynthesis and wound scar formation. Tamoxifen is also known to be an estrogen antagonist and protein kinase C (PKC) inhibitor. Cells treated with tamoxifen and other PKC/calmodulin inhibitors depolymerize their membrane focal adhesion complexes and cytoskeletal protein structures. These effects result in substrate detachment, cell shape rounding, and upregulation of collagenase synthesis and extracellular matrix degradation. The purpose of our study was to test the hypothesis that tamoxifen treatment of human foreskin fibroblasts results in alteration of cytoskeletal protein organization, cell detachment and rounding, and increased collagenase synthesis similar to known PKC/calmodulin inhibitors such as H-7. Methods: We characterized the effects of PKC/calmodulin inhibitors tamoxifen and H-7 on human dermal fibroblast morphology, cytoskeletal protein organization, and collagenase gene expression in monolayer culture and within collagen gels. Results: We found that fibroblasts responded to tamoxifen by initiation of actin filament depolymerization followed by alteration from spindle to spheroidal shapes. This change in cell shape led to increased collagenase synthesis in cells treated with either tamoxifen or H-7 compared with controls. There was also a 23% increase of hydroxyproline release from tamoxifen-treated fibroblast-populated collagen matrices. Conclusions: Tamoxifen may reduce scarring by inhibiting fibroblast PKC/calmodulin activity, which down-regulates pro-fibrotic transforming growth factor beta signaling and upregulates collagenase production. These effects mimic those of the known PKC/calmodulin inhibitor H-7. Overall, these findings suggest that tamoxifen and its analogues are promising agents for clinical investigation as small molecule regulators of fibrosis and scarring disorders.