Assembly and Evolution of Poxviruses.

Poxvirus assembly has been an intriguing area of research for several decades. While advancements in experimental techniques continue to yield fresh insights, many questions are still unresolved. Large genome sizes of up to 380 kbp, asymmetrical structure, an exterior lipid bilayer, and a cytoplasmic life cycle are some notable characteristics of these viruses. Inside the particle are two lateral bodies and a protein wall-bound-biconcave core containing the viral nucleocapsid. The assembly progresses through five major stages-endoplasmic reticulum (ER) membrane alteration and rupture, crescent formation, immature virion formation, genome encapsidation, virion maturation and in a subset of viruses, additional envelopment of the virion prior to its dissemination. Several large dsDNA viruses have been shown to follow a comparable sequence of events. In this chapter, we recapitulate our understanding of the poxvirus morphogenesis process while reviewing the most recent advances in the field. We also briefly discuss how virion assembly aids in our knowledge of the evolutionary links between poxviruses and other Nucleocytoplasmic Large DNA Viruses (NCLDVs).

Synthesis of N2-trans-isosafrole-dG-adduct Bearing DNAs and the Bypass Studies with Human TLS Polymerases κ and η.

Safrole is a natural product present in many plants and plant products, including spices and essential oils. During cellular metabolism, it converts to a highly reactive trans-isosafrole (SF) intermediate that reacts with genomic DNA and forms N2-SF-dG and N6-SF-dA DNA adducts, which are detected in the oral tissue of cancer patients with betel quid chewing history. To study the SF-induced carcinogenesis and to probe the role of low fidelity translesion synthesis (TLS) polymerases in bypassing SF adducts, herein, we report the synthesis of N2-SF-dG modified DNAs using phosphoramidite chemistry. The N2-SF-dG modification in the duplex DNA does not affect the thermal stability and retains the B-form of helical conformation, indicating that this adduct may escape the radar of common DNA repair mechanisms. Primer extension studies showed that the N2-SF-dG adduct is bypassed by human TLS polymerases hpolκ and hpolη, which perform error-free replication across this adduct. Furthermore, molecular modeling and dynamics studies revealed that the adduct reorients to pair with the incoming nucleotide, thus allowing the effective bypass. Overall, the results indicate that hpolκ and hpolη do not distinguish the N2-SF-dG adduct, suggesting that they may not be involved in the safrole-induced carcinogenicity.

Far-UVC (222 nm) irradiation effectively inactivates ssRNA, dsRNA, ssDNA, and dsDNA viruses as compared to germicidal UVC (254 nm).

Ultraviolet-C (UVC) irradiation is being used as an effective approach for the disinfection of pathogenic viruses present in air, surfaces, and water. Recently, far-UVC radiation (222 nm) emitted by KrCl* (krypton-chloride) excimer lamps have been recommended for disinfecting high-risk public spaces to reduce the presence and transmission of infectious viruses owing to limited human health exposure risks as compared to germicidal UVC (254 nm). In this study, the UVC inactivation performances of individual filtered KrCl* excimer lamp (222 nm) and germicidal UVC lamp (254 nm) were determined against four viruses, bacteriophages MS2, Phi6, M13, and T4, having different genome compositions (ssRNA, dsRNA, ssDNA and dsDNA, respectively) and shapes (i.e., spherical (Phi6), linear (M13), and icosahedral (MS2 and T4)). Here, the disinfection efficacies of filtered KrCl* excimer lamp (222 nm) and germicidal UVC lamp (254 nm) were evaluated for highly concentrated virus droplets that mimic the virus-laden droplets released from the infected person and deposited on surfaces as fomites. Filtered KrCl* excimer (222 nm) showed significantly better inactivation against all viruses having different genome compositions and structures compared to germicidal UVC (254 nm). The obtained sensitivity against the filtered KrCl* excimer (222 nm) was found to be in the order, T4 > M13 > Phi6 > MS2 whereas for the germicidal UVC (254 nm) it was T4 > M13 > MS2 > Phi6. These results provide a strong basis to promote the use of filtered KrCl* excimer lamps (222 nm) in disinfecting contagious viruses and to limit the associated disease spread in public places and other high-risk areas.

Giant Virus Global Proteomics Innovation: Comparative Evaluation of In-Gel and In-Solution Digestion Methods.

With their unusually large genome and particle sizes, giant viruses (GVs) defy the conventional definition of viruses. Although most GVs isolated infect unicellular protozoans, such as amoeba, studies in the last decade have established their much wider prevalence infecting most eukaryotic supergroups and some giant viral families with the potential to be human pathogens. Their complexity, almost autonomous life cycle, and enigmatic evolution necessitate the study of GVs. The accurate assessment of GV proteome is a veritable challenge. We have compared the coverage of global protein identification using different methods for GVs isolated in Mumbai, Mimivirus Bombay (MVB), Powai Lake Megavirus (PLMV), and Kurlavirus (KV), along with two previously studied GVs, Acanthamoeba polyphaga Mimivirus (APMV) and Marseillevirus (MV). Our study shows that the simultaneous use of in-gel and in-solution digestion methods can significantly increase the coverage of protein identification in the global proteome analysis of purified GV particles. Combining the two methods of analyses, we identified an additional 72 proteins in APMV and 114 in MV compared with what have been previously reported. Similarly, proteomes of MVB, PLMV, and KV were analyzed, and a total of 242 proteins in MVB, 287 proteins in PLMV, and 174 proteins in KV were identified. Our results suggest that a combined methodology of in-gel and in-solution methods is more efficient and opens up new avenues for innovation in global proteome analysis of GVs. Future planetary health research on GVs can benefit from consideration of a broader range of proteomics methodologies as illustrated by the present study.

An integrated method for targeted Oxford Nanopore sequencing and automated bioinformatics for the simultaneous detection of bacteria, fungi, and ARG.

AIMS: The use of metagenomics for pathogen identification in clinical practice has been limited. Here we describe a workflow to encourage the clinical utility and potential of NGS for the screening of bacteria, fungi, and antimicrobial resistance genes (ARGs). METHODS AND RESULTS: The method includes target enrichment, long-read sequencing, and automated bioinformatics. Evaluation of several tools and databases was undertaken across standard organisms (n = 12), clinical isolates (n = 114), and blood samples from patients with suspected bloodstream infections (n = 33). The strategy used could offset the presence of host background DNA, error rates of long-read sequencing, and provide accurate and reproducible detection of pathogens. Eleven targets could be successfully tested in a single assay. Organisms could be confidently identified considering ≥60% of best hits of a BLAST-based threshold of e-value 0.001 and a percent identity of >80%. For ARGs, reads with percent identity of >90% and >60% overlap of the complete gene could be confidently annotated. A kappa of 0.83 was observed compared to standard diagnostic methods. Thus, a workflow for the direct-from-sample, on-site sequencing combined with automated genomics was demonstrated to be reproducible. CONCLUSION: NGS-based technologies overcome several limitations of current day diagnostics. Highly sensitive and comprehensive methods of pathogen screening are the need of the hour. We developed a framework for reliable, on-site, screening of pathogens.

Simultaneous microbial capture and nucleic acid extraction from wastewater with minimal pre-processing and high recovery efficiency.

The COVID-19 pandemic accelerated research towards developing low-cost assays for automated urban wastewater monitoring assay that can be integrated into an environmental surveillance system for early warning of frequent disease outbreaks and future pandemics. Microbial concentration is one of the most challenging steps in wastewater surveillance, due to the sample heterogeneity and low pathogen load. Keeping in mind the requirements of large-scale testing in densely populated low- or middle-income countries (LMICs), such assays would need to be low-cost and have rapid turnaround time with high recovery efficiency. In this study, two such methods are presented and evaluated against commercially available kits for pathogen detection in wastewater. The first method utilizes paper dipsticks while the second method comprises of a PTFE membrane filter (PMF) integrated with a peristaltic pump. Both methods were used to concentrate and isolate nucleic acids from different microbes such as SARS-CoV-2, pepper mild mottle virus (PMMoV), bacteriophage Phi6, and E. coli from wastewater samples with minimal or no sample pre-processing. While the paper dipstick method is suitable for sub-milliliter sample volume, the PMF method can be used with larger volumes of wastewater sample (40 mL) and can detect multiple microbes with recovery efficiency comparable to commercially available kits.

Human Translesion Synthesis Polymerases polκ and polη Perform Error-Free Replication across N2-dG Methyleugenol and Estragole DNA Adducts.

The secondary metabolites of polypropanoids, methyleugenol (MEG), and estragole (EG), found in many herbs and spices, are commonly used as food flavoring agents and as ingredients in cosmetics. MEG and EG have been reported to cause hepatocarcinogenicity in rodents, human livers, and lung cells. The formation of N2-dG and N6-dA DNA adducts is primarily attributed to the carcinogenicity of these compounds. Therefore, these compounds have been classified as "possible human carcinogens" by the International Agency for Research on Cancer and "reasonably anticipated to be a human carcinogen" by the National Toxicology Program. Herein, we report the synthesis of the N2-MEG-dG and N2-EG-dG modified oligonucleotides to study the mutagenicity of these DNA adducts. Our studies show that N2-MEG-dG and N2-EG-dG could be bypassed by human translesion synthesis (TLS) polymerases hpolκ and hpolη in an error-free manner. The steady-state kinetics of dCTP incorporation by hpolκ across N2-MEG-dG and N2-EG-dG adducts show that the catalytic efficiencies (kcat/Km) were ∼2.5- and ∼4.4-fold higher, respectively, compared to the unmodified dG template. A full-length primer extension assay demonstrates that hpolκ exhibits better catalytic efficiency than hpolη. Molecular modeling and dynamics studies capturing pre-insertion, insertion, and post-insertion steps reveal the structural features associated with the efficient bypass of the N2-MEG-dG adduct by hpolκ and indicate the reorientation of the adduct in the active site allowing the successful insertion of the incoming nucleotide. Together, these results suggest that though hpolκ and hpolη perform error-free TLS across MEG and EG during DNA replication, the observed carcinogenicity of these adducts could be attributed to the involvement of other low fidelity polymerases.

Biochemical Reconstitution of the Mimiviral Base Excision Repair Pathway.

Viruses are believed to be the obligate intracellular parasites that only carry genes essential for infecting and hijacking the host cell machinery. However, a recently discovered group of viruses belonging to the phylum nucleocytovirocota, also known as the nucleo-cytoplasmic large DNA viruses (NCLDVs), possess a number of genes that code for proteins predicted to be involved in metabolism, and DNA replication, and repair. In the present study, first, using proteomics of viral particles, we show that several proteins required for the completion of the DNA base excision repair (BER) pathway are packaged within the virions of Mimivirus as well as related viruses while they are absent from the virions of Marseillevirus and Kurlavirus that are NCLDVs with smaller genomes. We have thoroughly characterized three putative base excision repair enzymes from Mimivirus, a prototype NCLDV and successfully reconstituted the BER pathway using the purified recombinant proteins. The mimiviral uracil-DNA glycosylase (mvUDG) excises uracil from both ssDNA and dsDNA, a novel finding contrary to earlier studies. The putative AP-endonuclease (mvAPE) specifically cleaves at the abasic site created by the glycosylase while also exhibiting the 3'-5' exonuclease activity. The Mimivirus polymerase X protein (mvPolX) can bind to gapped DNA substrates and perform single nucleotide gap-filling followed by downstream strand displacement. Furthermore, we show that when reconstituted in vitro, mvUDG, mvAPE, and mvPolX function cohesively to repair a uracil-containing DNA predominantly by long patch BER and together, may participate in the BER pathway during the early phase of Mimivirus life-cycle.

Amoebal Tubulin Cleavage Late during Infection Is a Characteristic Feature of Mimivirus but Not of Marseillevirus.

Mimivirus and Marseillevirus infections of Acanthamoeba castellanii, like most other viral infections, induce cytopathic effects (CPE). The details of how they bring about CPE and to what extent and how they modify the host cytoskeletal network are unclear. In this study, we compared the rearrangement of the host cytoskeletal network induced by Mimivirus and Marseillevirus upon infection. We show that while both Mimivirus and Marseillevirus infections of A. castellanii cells cause retraction of acanthopodia and depolymerization of the host actin filament network, the Mimivirus infection also results in characteristic cleavage of the host tubulin, a phenomenon not previously reported with any intracellular pathogens. Furthermore, we show that the amoebal tubulin cleavage during Mimivirus infection is a post-replicative event. Because time-lapse microscopy showed that Mimivirus infection leads to the bursting of cells, releasing the virus, we hypothesize that tubulin cleavage together with actin depolymerization during the later stages of Mimivirus assembly is essential for cell lysis due to apoptotic/necrotic cell death. We also characterize the Mimivirus-encoded gp560, a Zn metalloprotease, however, the purified gp560 protein was unable to cleave the commercially available porcine brain tubulin. While protein synthesis is essential for causing the morphological changes in the case of Mimivirus, the proteins which are packaged in the viral capsid along with the genome are sufficient to induce CPE in the case of Marseillevirus. IMPORTANCE In general, intracellular pathogens target the cytoskeletal network to enable their life cycle inside the host. Pathogen-induced changes in the host cell morphology usually accompany global changes in the cytoskeleton resulting in cytopathic effects. While viruses have been shown to use the host actin cytoskeleton for entry and transport during early infection, the role of microtubules in the viral life cycle is only beginning to emerge. Here, we show that the giant viruses Mimivirus and Marseillevirus both induce depolymerization of the actin filament, Mimivirus also causes a characteristic cleavage of tubulin not previously reported for any intracellular pathogen. Because tubulin cleavage occurs late during infection, we hypothesize that tubulin cleavage aids in cell death and lysis rather than establishing infection. The different strategies used by viruses with similar host niches may help them survive in competition.

Development of a PNA-DiSc2 based portable absorbance platform for the detection of pathogen nucleic acids.

We report the development of a portable absorption (PortAbs)-based pathogen nucleic acid detection system using peptide nucleic acid (PNA) and a cyanine dye, DiSc2(5). When the dye binds to the PNA-DNA hybrid, it results in a characteristic ∼110 nm shift in the dye absorbance, which we measure using PortAbs. The protocol involves amplification of the target DNA, PNA-DNA hybridization and dye complexing steps followed by absorption measurement. The system is built using a broad-spectrum photodiode whose output is amplified and then measured by a high resolution (24 or 32 bit) analog-to-digital converter. The excitation pulses of light are delivered by a color-changing LED. The sequence of excitation, measurement and display of results are all controlled by an embedded Raspberry-Pi board (or alternatively a laptop). At higher concentrations of the target amplicon (∼200 ng), the color change can be detected visually. At lower concentrations, PortAbs outperforms a plate reader and can detect target DNA as low as 30 ng or approximately 10 nM which is at least 10 fold better than previously reported studies. We validate the methodology using SARS-CoV-2 clinical samples containing about 1000 copies of the viral RNA and show that the entire workflow takes about 90 min. The cost of the complete standalone system is less than INR 40 000 (approx. 500 USD).

Whole-genome sequencing of presumptive MDR-TB isolates from a tertiary healthcare setting in Mumbai.

OBJECTIVES: Whole-genome sequencing (WGS) of Mycobacterium tuberculosis (MTB), proven to be a better alternative when compared with the combined sensitivity and specificity of all other modalities for diagnosis of tuberculosis (TB), aids epidemiological surveillance investigations by combining the current research with diagnostics. This study was conducted to identify and resolve operational challenges in performing WGS-based drug resistance testing (DRT) for MTB in a TB culture and drug susceptibility testing (DST) laboratory. Three critical, non-redundant steps for WGS-based DRT were tested: viz. DNA extraction, high-throughput paired-end next-generation sequencing (NGS), and genomic analysis pipeline for automated reporting of WGS-based DRT. METHODS: DNA was extracted from 100 liquid culture isolates on a mycobacterial growth indicator tube (MGIT) using DNEASY Ultraclean Microbial Kit (Qiagen, USA) as per the manufacturer's instructions. Illumina paired-end sequencing was performed. All analysis steps were automated using custom python scripts, requiring no intervention. Variant calling was performed as per the World Health Organization (WHO) technical guide. RESULTS: The number of cultures resistant to rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin was 89, 88, 35, 67, and 73, respectively. Resistance to amikacin, kanamycin, and capreomycin was found in 15, 17, and 15 cultures, respectively. Seventy cultures were resistant to fluoroquinolones, four were resistant to ethionamide, and 12 were resistant to linezolid. Six cultures were resistant to only one of the 18 drugs tested. Seventy-five cultures were resistant to more than three anti-TB drugs. One culture was resistant to 13 of the 18 anti-TB drugs tested for this study. The maximum number of variants were observed in the rpoB gene (n = 93, 93%), wherein the Ser450Leu was the predominant mutation (n = 68, 73%). Ser315Thr was the most common variant (n = 86, 97%) that encoded resistance to isoniazid. The Lys43Arg variant encodes resistance to streptomycin and was the third most predominant variant (n = 65, 89%). In addition to the high levels of resistance observed in the dataset, we also observed a high proportion of Beijing strains (n = 63, 63%). CONCLUSION: Compared with results from routine diagnostics based on the 'Guidelines on Programmatic Management of Drug-Resistant TB (PMDT) in India', none of the samples had DST available for all 18 drugs. This represents a gap in PMDT guidelines. The WGS-DRT must be considered as the primary DST method after a sample is flagged rifampicin-resistant by cartridge-based nucleic acid amplification testing (CBNAAT). With several research studies currently underway globally to identify novel variants associated with drug resistance and classifiy their minimum inhibitory coefficients, WGS-DRT presents a scalable technology that updates analytical pipelines, relegating the need for changing microbiological protocols.

Longer amplicons provide better sensitivity for electrochemical sensing of viral nucleic acid in water samples using PCB electrodes.

The importance of monitoring environmental samples has gained a lot of prominence since the onset of COVID-19 pandemic, and several surveillance efforts are underway using gold standard, albeit expensive qPCR-based techniques. Electrochemical DNA biosensors could offer a potential cost-effective solution suitable for monitoring of environmental water samples in lower middle income countries. In this work, we demonstrate electrochemical detection of amplicons as long as [Formula: see text] obtained from Phi6 bacteriophage (a popular surrogate for SARS-CoV-2) isolated from spiked lake water samples, using ENIG finish PCB electrodes with no surface modification. The electrochemical sensor response is thoroughly characterised for two DNA fragments of different lengths ([Formula: see text] and [Formula: see text]), and the impact of salt in PCR master mix on methylene blue (MB)-DNA interactions is studied. Our findings establish that length of the DNA fragment significantly determines electrochemical sensitivity, and the ability to detect long amplicons without gel purification of PCR products demonstrated in this work bodes well for realisation of fully-automated solutions for in situ measurement of viral load in water samples.

MMP modulated differentiation of mouse embryonic stem cells on engineered cell derived matrices.

Stem cell differentiation is dictated by the dynamic crosstalk between cells and their underlying extracellular matrix. While the importance of matrix degradation mediated by enzymes such as matrix metalloproteinases (MMPs) in the context of cancer invasion is well established, the role of MMPs in stem cell differentiation remains relatively unexplored. Here we address this question by assaying MMP expression and activity during differentiation of mouse embryonic stem cells (mESCs) on mouse embryonic fibroblast (MEF) derived matrices (MEFDMs) of varying stiffness and composition. We show that mESC differentiation into different germ layers is associated with expression of several MMPs including MMP-11, 2, 17, 25 and 9, with MMP-9 detected in cell secreted media. Different extents of softening of the different MEFDMs led to altered integrin expression, activated distinct mechanotransduction and metabolic pathways, and induced expression of germ layer-specific markers. Inhibition of MMP proteolytic activity by the broad spectrum MMP inhibitor GM6001 led to alterations in germ layer commitment of the differentiating mESCs. Together, our results illustrate the effect of MMPs in regulating mESC differentiation on engineered cell derived matrices and establish MEFDMs as suitable substrates for understanding molecular mechanisms regulating stem cell development and for regenerative medicine applications.

The coevolution of large and small terminases of bacteriophages is a result of purifying selection leading to phenotypic stabilization.

Genome packaging in many dsDNA phages requires a series of precisely coordinated actions of two phage-coded proteins, namely, large terminase (TerL) and small terminase (TerS) with DNA and ATP, and with each other. Despite the strict functional conservation, TerL and TerS homologs exhibit large sequence variations. We investigated the sequence variability across eight phage types and observed a coevolutionary framework wherein the genealogy of TerL homologs mirrored that of the corresponding TerS homologs. Furthermore, a high purifying selection observed (dN/dS«1) indicated strong structural constraints on both TerL and TerS, and identify coevolving residues in TerL and TerS of phage T4 and lambda. Using the highly coevolving (correlation coefficient of 0.99) TerL and TerS of phage N4, we show that their biochemical features are similar to the phylogenetically divergent phage λ terminases. We also demonstrate using the Surface Plasma Resonance (SPR) technique that phage N4 TerL transiently interacts with TerS.

The Phage T4 Antiholin RI Has a Cleavable Signal Peptide, Not a SAR Domain.

Holin/endolysin-mediated lysis of phage T4 of Escherichia coli is tightly regulated by the antiholins RI and RIII. While regulation by the cytoplasmic RIII plays a minor role, the periplasmic antiholin RI binds tightly to the holin T and is believed to directly sense periplasmic phage DNA from superinfections as a trigger for the inhibition of lysis. RI has been reported to contain a non-cleavable signal peptide that anchors the protein to the membrane. Lysis is believed to be induced at some stage by a membrane depolarization that causes a release of RI into the periplasm without cleavage of the signal anchor. For the current model of phage lysis induction, it is thus a fundamental assumption that the N-terminal trans-membrane domain (TMD) of RI is such a signal anchor release (SAR) domain. Here we show that, in contrast to previous reports, this domain of RI is a cleavable signal peptide. RI is processed and released into the periplasm as a mature protein, and inactivation of its signal peptidase cleavage site blocks processing and membrane release. The signal peptide of RI can also mediate the normal translocation of a well-characterized Sec substrate, PhoA, into the periplasm. This simplifies the current view of phage lysis regulation and suggests a fundamentally different interpretation of the recently published structure of the soluble domains of the RI-T complex.

Label-Free Detection of Escherichia coli from Mixed Bacterial Cultures Using Bacteriophage T4 on Plasmonic Fiber-Optic Sensor.

Consumption of water contaminated with pathogenic bacteria is a major cause of water-borne diseases. To address this challenge, we have developed a novel and sensitive sensing scheme for the rapid detection of bacteria (Escherichia coliB40) on a fiber-optic platform using bacteriophage (T4) as a bio-recognition element. The novelty of our sensing scheme is that instead of bacteriophages, bacteria (analyte) were first captured on the sensing surface and then the sensing surface was subjected to bacteriophages for specific detection of bacteria. The sensor was subjected to 100 to 107 cfu/mL of E. coliB40 spiked in a lake water matrix, and the least concentration of bacteria that could be easily detected was found to be 1000 cfu/mL. The control studies were performed with nonhost bacteria Pseudomonas aeruginosa. Bacteriophage T4, being specific to its host E. coliB40, did not interact with P. aeruginosa captured on the sensing probe, giving a negligible nonspecific response. Due to the specificity of bacteriophages to its host bacteria, it is possible to use this scheme to carry out the detection of specific bacteria in a mixed sample (containing a combination of bacteria) using bacteriophages specific to it. The sensor was able to detect E. coliB40 (target bacteria) even in the presence of a very high concentration (1000 times higher) of P. aeruginosa (nontarget bacteria).

Scaling relation between genome length and particle size of viruses provides insights into viral life history.

In terms of genome and particle sizes, viruses exhibit great diversity. With the discovery of several nucleocytoplasmic large DNA viruses (NCLDVs) and jumbo phages, the relationship between particle and genome sizes has emerged as an important criterion for understanding virus evolution. We use allometric scaling of capsid volume with the genome length of different groups of viruses to shed light on its relationship with virus life history. The allometric exponents for icosahedral dsDNA bacteriophages and NCDLVs were found to be 1 and 2, respectively, indicating that with increasing capsid size DNA packaging density remains the same in bacteriophages but decreases for NCLDVs. We argue that the exponents are largely shaped by their entry mechanism and capsid mechanical stability. We further show that these allometric size parameters are also intricately linked to the relative energy costs of translation and replication in viruses and can have further implications on viral life history.

Coevolutionary and Phylogenetic Analysis of Mimiviral Replication Machinery Suggest the Cellular Origin of Mimiviruses.

Mimivirus is one of the most complex and largest viruses known. The origin and evolution of Mimivirus and other giant viruses have been a subject of intense study in the last two decades. The two prevailing hypotheses on the origin of Mimivirus and other viruses are the reduction hypothesis, which posits that viruses emerged from modern unicellular organisms; whereas the virus-first hypothesis proposes viruses as relics of precellular forms of life. In this study, to gain insights into the origin of Mimivirus, we have carried out extensive phylogenetic, correlation, and multidimensional scaling analyses of the putative proteins involved in the replication of its 1.2-Mb large genome. Correlation analysis and multidimensional scaling methods were validated using bacteriophage, bacteria, archaea, and eukaryotic replication proteins before applying to Mimivirus. We show that a large fraction of mimiviral replication proteins, including polymerase B, clamp, and clamp loaders are of eukaryotic origin and are coevolving. Although phylogenetic analysis places some components along the lineages of phage and bacteria, we show that all the replication-related genes have been homogenized and are under purifying selection. Collectively our analysis supports the idea that Mimivirus originated from a complex cellular ancestor. We hypothesize that Mimivirus has largely retained complex replication machinery reminiscent of its progenitor while losing most of the other genes related to processes such as metabolism and translation.

Immunoinformatics approach for a novel multi-epitope subunit vaccine design against various subtypes of Influenza A virus.

Vaccination is the best strategy for the control and prevention of contagious diseases caused by Influenza A viruses. Extraordinary genetic variability and continual evolvability are responsible for the virus having survival and adaptation to host cell immune response, thus rendering the current influenza vaccines with suboptimal effectiveness.Therefore, in the present study, using a novel immunoinformatics approach, we have designed a universal influenza subunit vaccine based on the highly conserved epitopic sequences of rapidly evolving (HA), a moderately evolving (NP) and slow evolving (M1) proteins of the virus. The vaccine design includes 2 peptide adjuvants, 26 CTL epitopes, 9 HTL epitopes, and 7 linear BCL epitopes to induce innate, cellular, and humoral immune responses against Influenza A viruses. We also analyzed the physicochemical properties of the designed construct to validate its thermodynamic stability, hydrophilicity, PI, antigenicity, and allergenicity. Furthermore, we predicted a highly stable tertiary model of the designed subunit vaccine, wherein additional disulfide bonds were incorporated to enhance its stability. The molecular docking and molecular dynamics simulations of the refined vaccine model with TLR3, TLR7, TLR8, MHC-I and MHC-II showed stable vaccine and receptors complexes, thus confirming the immunogenicity of the designed vaccine. Collectively, these findings suggest that our multi-epitope vaccine construct may confer protection against various strains of influenza A virus subtypes, which could prevent the need for annual reformulation of vaccine and alleviate disease burden.

Upregulation of miR-101 during Influenza A Virus Infection Abrogates Viral Life Cycle by Targeting mTOR Pathway.

Micro RNAs (miRNAs) are a class of small non-coding single-stranded RNA, which play an important role in modulating host-Influenza A virus (IAV) crosstalk. The interplay between influenza and miRNA interaction is defined by a plethora of complex mechanisms, which are not fully understood yet. Here, we demonstrate that in IAV infected A549 cells, a synchronous increase was observed in the expression of mTOR up to 24 hpi and significant downregulation at 48 hpi. Additionally, NP of IAV interacts with mTOR and modulates the levels of mTOR mRNA and protein, thus regulating the translation of host cell. RNA sequencing and qPCR analysis of IAV-infected A549 cells and NP transfected cells revealed that miR-101 downregulates mTOR transcripts at later stages of infection. Ectopic expression of miR-101 mimic led to a decrease in expression of NP, a reduction in IAV titer and replication. Moreover, treatment of the cells with Everolimus, a potent inhibitor of mTOR, resulted in an increase of miR-101 transcript levels, which further suppressed the viral protein synthesis. Collectively, the data suggest a novel mechanism that IAV stimulates mTOR pathway at early stages of infection; however, at a later time-point, positive regulation of miR-101 restrains the mTOR expression, and hence, the viral propagation.