GeneRaMeN enables integration, comparison, and meta-analysis of multiple ranked gene lists to identify consensus, unique, and correlated genes.

High-throughput experiments often produce ranked gene outputs, with forward genetic screening being a notable example. While there are various tools for analyzing individual datasets, those that perform comparative and meta-analytical examination of such ranked gene lists remain scarce. Here, we introduce Gene Rank Meta Analyzer (GeneRaMeN), an R Shiny tool utilizing rank statistics to facilitate the identification of consensus, unique, and correlated genes across multiple hit lists. We focused on two key topics to showcase GeneRaMeN: virus host factors and cancer dependencies. Using GeneRaMeN 'Rank Aggregation', we integrated 24 published and new flavivirus genetic screening datasets, including dengue, Japanese encephalitis, and Zika viruses. This meta-analysis yielded a consensus list of flavivirus host factors, elucidating the significant influence of cell line selection on screening outcomes. Similar analysis on 13 SARS-CoV-2 CRISPR screening datasets highlighted the pivotal role of meta-analysis in revealing redundant biological pathways exploited by the virus to enter human cells. Such redundancy was further underscored using GeneRaMeN's 'Rank Correlation', where a strong negative correlation was observed for host factors implicated in one entry pathway versus the alternate route. Utilizing GeneRaMeN's 'Rank Uniqueness', we analyzed human coronaviruses 229E, OC43, and SARS-CoV-2 datasets, identifying host factors uniquely associated with a defined subset of the screening datasets. Similar analyses were performed on over 1000 Cancer Dependency Map (DepMap) datasets spanning 19 human cancer types to reveal unique cancer vulnerabilities for each organ/tissue. GeneRaMeN, an efficient tool to integrate and maximize the usability of genetic screening datasets, is freely accessible via https://ysolab.shinyapps.io/GeneRaMeN.

The genomic landscape of swine influenza A viruses in Southeast Asia.

Swine are a primary source for the emergence of pandemic influenza A viruses. The intensification of swine production, along with global trade, has amplified the transmission and zoonotic risk of swine influenza A virus (swIAV). Effective surveillance is essential to uncover emerging virus strains; however gaps remain in our understanding of the swIAV genomic landscape in Southeast Asia. More than 4,000 nasal swabs were collected from pigs in Cambodia, yielding 72 IAV-positive samples by RT-qPCR and 45 genomic sequences. We unmasked the cocirculation of multiple lineages of genetically diverse swIAV of pandemic concern. Genomic analyses revealed a novel European avian-like H1N2 swIAV reassortant variant with North American triple reassortant internal genes, that emerged approximately seven years before its first detection in pigs in 2021. Using phylogeographic reconstruction, we identified south central China as the dominant source of swine viruses disseminated to other regions in China and Southeast Asia. We also identified nine distinct swIAV lineages in Cambodia, which diverged from their closest ancestors between two and 15 B.P., indicating significant undetected diversity in the region, including reverse zoonoses of human H1N1/2009 pandemic and H3N2 viruses. A similar period of cryptic circulation of swIAVs occurred in the decades before the H1N1/2009 pandemic. The hidden diversity of swIAV observed here further emphasizes the complex underlying evolutionary processes present in this region, reinforcing the importance of genomic surveillance at the human-swine interface for early warning of disease emergence to avoid future pandemics.

TMEM41B and VMP1 modulate cellular lipid and energy metabolism for facilitating dengue virus infection.

Transmembrane Protein 41B (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two ER-associated lipid scramblases that play a role in autophagosome formation and cellular lipid metabolism. TMEM41B is also a recently validated host factor required by flaviviruses and coronaviruses. However, the exact underlying mechanism of TMEM41B in promoting viral infections remains an open question. Here, we validated that both TMEM41B and VMP1 are essential host dependency factors for all four serotypes of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43), but not chikungunya virus (CHIKV). While HCoV-OC43 failed to replicate entirely in both TMEM41B- and VMP1-deficient cells, we detected diminished levels of DENV infections in these cell lines, which were accompanied by upregulation of the innate immune dsRNA sensors, RIG-I and MDA5. Nonetheless, this upregulation did not correspondingly induce the downstream effector TBK1 activation and Interferon-beta expression. Despite low levels of DENV replication, classical DENV replication organelles were undetectable in the infected TMEM41B-deficient cells, suggesting that the upregulation of the dsRNA sensors is likely a consequence of aberrant viral replication rather than a causal factor for reduced DENV infection. Intriguingly, we uncovered that the inhibitory effect of TMEM41B deficiency on DENV replication, but not HCoV-OC43, can be partially reversed using exogenous fatty acid supplements. In contrast, VMP1 deficiency cannot be rescued using the metabolite treatment. In line with the observed phenotypes, we found that both TMEM41B- and VMP1-deficient cells harbor higher levels of compromised mitochondria, especially in VMP1 deficiency which results in severe dysregulations of mitochondrial beta-oxidation. Using a metabolomic profiling approach, we revealed distinctive global dysregulations of the cellular metabolome, particularly lipidome, in TMEM41B- and VMP1-deficient cells. Our findings highlight a central role for TMEM41B and VMP1 in modulating multiple cellular pathways, including lipid mobilization, mitochondrial beta-oxidation, and global metabolic regulations, to facilitate the replication of flaviviruses and coronaviruses.

Unravelling ionic liquid solvent effects for a non-polar Cope rearrangement reaction.

The impact of ionic liquids (ILs) on polar reactions is well recognised, however the impact of ILs on non-polar reactions is less well understood or explored. Pericyclic Cope rearrangements are highly concerted, exhibit minimal charge localisation and pass through an uncharged but well-defined transition state, and thus provide a good mechanism for exploring the impact of IL polarizability on chemical reactivity. Recently, a 10× rate enhancement has been observed for the Cope rearrangement of 3-phenyl-1,5-hexadiene in the IL 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [C4C1im][NTf2] compared to benzene. In this work we undertake a DFT based computational study (B3LYP-D3BJ/6-311+G(d,p) and M06-2X-D3/6-311+G(d,p)) of the Cope rearrangement of 3-phenyl-1,5-hexadiene and 3-propyl-hexa-1,5-diene in molecular solvents (acetonitrile, benzene and ethanol) and the IL [C4C1im][NTf2] using the SMD solvation model. The impact of benzene and [C4C1im][NTf2] on the Cope rearrangement of 3-phenyl-1,5-hexadiene is studied in more detail and we provide insight into the reason for the rate enhancement in an IL. The volume of activation is evaluated and the potential impact of 'solvent pressure' is discussed. We identify two potential mechanisms for volume effects to contribute to the rate enhancement. Solvent association energies are evaluated at the DLNPO-CCSD(T) level. Specific solvent interactions are explored through atomic partial charge, molecular orbital and bond critical point analysis, as well as via non-colvalent interaction (NCI) plots, electrostatic potential (ESP) differences and density difference Δρ(r) plots. We find that the cation and anion together form an extensive van der Waals pocket in-which the transition state (TS) sits. Electron density within the TS is anisotropically polarised via a 'push-pull' effect due to the dual cation-anion nature of the IL, stabilising the TS relative to benzene. We also provide experimental evidence that these effects are generalisable to other ILs. Overall, our aim is to provide a deeper moleuclar level understanding of the impact of ILs on non-polar reactions.

Divergent evolutionary trajectories of influenza B viruses underlie their contemporaneous epidemic activity.

Influenza B viruses have circulated in humans for over 80 y, causing a significant disease burden. Two antigenically distinct lineages ("B/Victoria/2/87-like" and "B/Yamagata/16/88-like," termed Victoria and Yamagata) emerged in the 1970s and have cocirculated since 2001. Since 2015 both lineages have shown unusually high levels of epidemic activity, the reasons for which are unclear. By analyzing over 12,000 influenza B virus genomes, we describe the processes enabling the long-term success and recent resurgence of epidemics due to influenza B virus. We show that following prolonged diversification, both lineages underwent selective sweeps across the genome and have subsequently taken alternate evolutionary trajectories to exhibit epidemic dominance, with no reassortment between lineages. Hemagglutinin deletion variants emerged concomitantly in multiple Victoria virus clades and persisted through epistatic mutations and interclade reassortment-a phenomenon previously only observed in the 1970s when Victoria and Yamagata lineages emerged. For Yamagata viruses, antigenic drift of neuraminidase was a major driver of epidemic activity, indicating that neuraminidase-based vaccines and cross-reactivity assays should be employed to monitor and develop robust protection against influenza B morbidity and mortality. Overall, we show that long-term diversification and infrequent selective sweeps, coupled with the reemergence of hemagglutinin deletion variants and antigenic drift of neuraminidase, are factors that contributed to successful circulation of diverse influenza B clades. Further divergence of hemagglutinin variants with poor cross-reactivity could potentially lead to circulation of 3 or more distinct influenza B viruses, further complicating influenza vaccine formulation and highlighting the urgent need for universal influenza vaccines.

Effects of a major deletion in the SARS-CoV-2 genome on the severity of infection and the inflammatory response: an observational cohort study.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with a 382-nucleotide deletion (∆382) in the open reading frame 8 (ORF8) region of the genome have been detected in Singapore and other countries. We investigated the effect of this deletion on the clinical features of infection. METHODS: We retrospectively identified patients who had been screened for the ∆382 variant and recruited to the PROTECT study-a prospective observational cohort study conducted at seven public hospitals in Singapore. We collected clinical, laboratory, and radiological data from patients' electronic medical records and serial blood and respiratory samples taken during hospitalisation and after discharge. Individuals infected with the ∆382 variant were compared with those infected with wild-type SARS-CoV-2. Exact logistic regression was used to examine the association between the infection groups and the development of hypoxia requiring supplemental oxygen (an indicator of severe COVID-19, the primary endpoint). Follow-up for the study's primary endpoint is completed. FINDINGS: Between Jan 22 and March 21, 2020, 278 patients with PCR-confirmed SARS-CoV-2 infection were screened for the ∆382 deletion and 131 were enrolled onto the study, of whom 92 (70%) were infected with the wild-type virus, ten (8%) had a mix of wild-type and ∆382-variant viruses, and 29 (22%) had only the ∆382 variant. Development of hypoxia requiring supplemental oxygen was less frequent in the ∆382 variant group (0 [0%] of 29 patients) than in the wild-type only group (26 [28%] of 92; absolute difference 28% [95% CI 14-28]). After adjusting for age and presence of comorbidities, infection with the ∆382 variant only was associated with lower odds of developing hypoxia requiring supplemental oxygen (adjusted odds ratio 0·07 [95% CI 0·00-0·48]) compared with infection with wild-type virus only. INTERPRETATION: The ∆382 variant of SARS-CoV-2 seems to be associated with a milder infection. The observed clinical effects of deletions in ORF8 could have implications for the development of treatments and vaccines. FUNDING: National Medical Research Council Singapore.

Robust dengue virus infection in bat cells and limited innate immune responses coupled with positive serology from bats in IndoMalaya and Australasia.

Natural reservoir hosts can sustain infection of pathogens without succumbing to overt disease. Multiple bat species host a plethora of viruses, pathogenic to other mammals, without clinical symptoms. Here, we detail infection of bat primary cells, immune cells, and cell lines with Dengue virus. While antibodies and viral RNA were previously detected in wild bats, their ability to sustain infection is not conclusive. Old-world fruitbat cells can be infected, producing high titres of virus with limited cellular responses. In addition, there is minimal interferon (IFN) response in cells infected with MOIs leading to dengue production. The ability to support in vitro replication/production raises the possibility of bats as a transient host in the life cycle of dengue or similar flaviviruses. New antibody serology evidence from Asia/Pacific highlights the previous exposure and raises awareness that bats may be involved in flavivirus dynamics and infection of other hosts.

Avian Influenza A(H9N2) Virus in Poultry Worker, Pakistan, 2015.

Avian influenza A(H9N2) virus isolated from a poultry worker in Pakistan in 2015 was closely related to viruses detected in poultry farms. Observed mutations in the hemagglutinin related to receptor-binding affinity and antigenicity could affect cross-reactivity with prepandemic H9N2 vaccine strains.

Avian influenza viruses in humans: lessons from past outbreaks.

BACKGROUND: Human infections with avian influenza viruses (AIV) represent a persistent public health threat. The principal risk factor governing human infection with AIV is from direct contact with infected poultry and is primarily observed in Asia and Egypt where live-bird markets are common. AREAS OF AGREEMENT: Changing patterns of virus transmission and a lack of obvious disease manifestations in avian species hampers early detection and efficient control of potentially zoonotic AIV. AREAS OF CONTROVERSY: Despite extensive studies on biological and environmental risk factors, the exact conditions required for cross-species transmission from avian species to humans remain largely unknown. GROWING POINTS: The development of a universal ('across-subtype') influenza vaccine and effective antiviral therapeutics are a priority. AREAS TIMELY FOR DEVELOPING RESEARCH: Sustained virus surveillance and collection of ecological and physiological parameters from birds in different environments is required to better understand influenza virus ecology and identify risk factors for human infection.

The effective rate of influenza reassortment is limited during human infection.

We characterise the evolutionary dynamics of influenza infection described by viral sequence data collected from two challenge studies conducted in human hosts. Viral sequence data were collected at regular intervals from infected hosts. Changes in the sequence data observed across time show that the within-host evolution of the virus was driven by the reversion of variants acquired during previous passaging of the virus. Treatment of some patients with oseltamivir on the first day of infection did not lead to the emergence of drug resistance variants in patients. Using an evolutionary model, we inferred the effective rate of reassortment between viral segments, measuring the extent to which randomly chosen viruses within the host exchange genetic material. We find strong evidence that the rate of effective reassortment is low, such that genetic associations between polymorphic loci in different segments are preserved during the course of an infection in a manner not compatible with epistasis. Combining our evidence with that of previous studies we suggest that spatial heterogeneity in the viral population may reduce the extent to which reassortment is observed. Our results do not contradict previous findings of high rates of viral reassortment in vitro and in small animal studies, but indicate that in human hosts the effective rate of reassortment may be substantially more limited.