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International audience; The search for inhibitors of SARS-CoV-2 viral replication depends on the understanding of the events taking place at different molecular levels during the viral infection. The macro-molecular level focuses on the interactions among viral and host proteins, while the micro-molecular level focuses on the different biochemical modifications that occur to one or more amino acids on proteins. A hybrid approach for modeling the SARS-CoV-2 viral replication in the micro-and macro-molecular levels is presented in this paper. The proposed approach combines two domains which complement one another, ontology engineering and discrete event system specification (DEVS) modeling.In this approach, biological knowledge at the micro-level of the viral system is capitalized and inferred by ontological models, while the dynamic behavior of SARS-CoV-2 molecular mechanisms and their different state changes in time are modeled by DEVS models. We illustrate the proposed approach through the modeling and simulation of the ribosome, a key molecule of the host cell that all viruses compete for, including the SARS-CoV-2.

Biophysical separation promises label-free, less-invasive methods to manipulate the diverse properties of live cells, such as density, magnetic susceptibility, and morphological characteristics. However, some cellular changes are so minute that they are undetectable by current methods. We developed a multiparametric cell-separation approach to profile cells with simultaneously changing density and magnetic susceptibility. We demonstrated this approach with the natural biophysical phenomenon of Plasmodium falciparum infection, which modifies its host erythrocyte by simultaneously decreasing density and increasing magnetic susceptibility. Current approaches have used these properties separately to isolate later-stage infected cells, but not in combination. We present biophysical separation of infected erythrocytes by balancing gravitational and magnetic forces to differentiate infected cell stages, including early stages for the first time, using magnetic levitation. We quantified height distributions of erythrocyte populations—27 ring-stage synchronized samples and 35 uninfected controls—and quantified their unique biophysical signatures. This platform can thus enable multidimensional biophysical measurements on unique cell types., Deshmukh et al. combine microscale magnetic levitation with minute density and magnetic susceptibility differences to enhance biophysical separation of cells. They demonstrate the feasibility of this approach on cells infected with malaria parasites, which simultaneously decrease host cell density and increase its magnetic susceptibility.

International audience; Benefiting from the recent advances of synchrotron X-ray nanoprobes, we demonstrate three-dimensional (3D) correlative nano-imaging on malaria-infected human red blood cells. By combining X-ray fluorescence to-mography and phase contrast nanotomography on the same cell with sub-100 nm pixel size, we establish a routine workflow from the data acquisition, data processing to tomographic reconstruction. We quantitatively compare the elemental volumes obtained with different reconstruction methods, the total variation minimization giving the most satisfactory results. We reveal elemental correlations in different cell compartments more reliably on reconstructions as opposed to 2D projections. Finally, we determine for the first time the 3D mass fraction maps of multiple elements at sub-cellular level. The estimated total number of Fe atoms and the total mass of the red blood cell show very good agreement with previously reported values.

We used a combination of DOP-PCR with high throughput sequencing (HTS) to study infected cell cultures over time to assess the feasibility of using this technique to provide a read-out other than cytopathic effect in cell culture infectivity assays. Because DOP-PCR primers feature a short constant sequence at their 3' terminus, the procedure yields a reproducible representational library of products from a given PCR template, including viral nucleic acids. Using SV40- and MVM-infected cultures harvested at different times, we show that the number of viral matches among DOP-PCR products parallels the quantity of virus as shown by real-time PCR, and further show that HTS analysis of specific DOP-PCR products that increase in quantity over time could be used to identify the infecting virus with a sensitivity similar to that of typical cell-culture assays that rely on cytopathic effect.

Hematological diseases are blood-related diseases which include leukemia, leucopenia, hemophilia, etc. In which leukemia is severe hematological disease and death rate is very high and hence need proper and faster diagnosis. Leukemia is the formation of giant nuclei or immature increase of leukocytes or WBCs; hence, the need of extracting the shape features and other symptoms must be investigated. This paper suggested and experimented a technique for separating or segmenting white blood cell stain images of leukemia-affected patients. The successful segmentation of an infected cell divides the images into nucleus and outer fluid part cytoplasm, since these two regions contain features that correlate to different forms of leukemia. The algorithm checks and experiments 250 images, and the performance validation using cross-validation techniques shows the nucleus segmentation accuracy as 94% and cytoplasm segmentation accuracy as 78%, respectively.

Malaria is still an infectious disease that causes high mortality in endemic regions. It is thought that it will maintain importance in the future, especially due to people travelling from African countries where malaria is endemic to its eradicated regions. Therefore, rapid and accurate diagnosis is a critical step in the effective treatment of malaria and reducing mortality rates. This paper provides a malaria diagnosis system using an artificial neural network approach with SURF (Speeded Up Robust Features) method that helps the clinicians to predict and locate infected cell with malaria on the sample thin blood smear image. The performance of the proposed neural network and local image feature extraction technique SURF were analyzed statistically and presented in this paper. The network was trained using only 45 infected thin blood smear images and was then tested with 200 (100 infected and 100 non-infected) unseen images. The experimental results showed that the proposed system identified the malaria parasite with 93% accuracy, 86% sensitivity and 100% specificity.

Sequencing individual HIV-proviruses and their adjacent cellular junctions can elucidate mechanisms of infected cell persistence in vivo. Here, we present a high throughput microfluidic method to sequence entire proviruses in their native integration site context. We used the method to analyze infected cells from people with HIV on suppressive antiretroviral therapy, demonstrating >90% capture and sequencing of paired proviral genomes and integration sites. This method should enable comprehensive genetic analysis of persistent HIV-infected cell reservoirs, providing important insights into the barriers to HIV cure.

To make new infectious particles, all viruses must manipulate host cell metabolism to secure sufficient availability of biomolecules and energy-a phenomenon now known as metabolic reprogramming. Numerous observations of this has already been made for a range of viruses with each type of virus seemingly applying its own unique tactics to accomplish this unifying goal. In this light, metabolic reprogramming of the infected cell is largely beneficial to the virus and not to the host. On the other hand, virus-induced metabolic reprogramming represents a transformed self with distorted cellular and extracellular levels of distinct metabolites and metabolic by-products. This review briefly outlines current knowledge of virus-induced metabolic reprogramming, discusses how this could be sensed by the infected host to initiate anti-viral programs, and presents examples of innate anti-viral mechanisms of the host that target the availability of biomolecules to block viral replication.

Mycoplasma contamination of cell line cultures is a common, yet often undetected problem in research laboratories. Many of the existing techniques to detect mycoplasma contamination of cultured cells are time-consuming, expensive, and have significant drawbacks. Here, we describe a mycoplasma detection system that is useful for detecting multiple species of mycoplasma in infected cell lines. The system contains three dye-labeled detection aptamers that can specifically bind to mycoplasma-infected cells and a dye-labeled control aptamer that minimally binds to cells. With this system, mycoplasma-contaminated cells can be detected within 30 min by using a flow cytometer, fluorescence microscope, or microplate reader. Further, this system may be used to detect mycoplasma-contaminated culture medium. This study presents an novel mycoplasma detection model that is simple, rapid, inexpensive, and sensitive.

This article examines three aspects of antivirals, such as hydroxychloroquine, chloroquine, and remdesvir, as they might relate to the treatment of a viral infection such as COVID-19: (i) the use of vaporization for the delivery of antivirals, with the bulk constituents having mild antiviral efficacy; (ii) the application of a marine natural product extract as opposed to a single molecule as an antiviral agent; and (iii) a counter intuitive approach to formulation that is, in part, based on delivering multiple species that fall into three categories: building blocks for the virus to accelerate replication; an energy source for the infected cell to boost its immune response; and the species that antagonize or provide toxicity to the virus.

A critical step in the life cycle of a virus is spread to a new target cell, which generally involves the release of new viral particles from the infected cell which can then initiate infection in the next target cell. While cell-free viral particles released into the extracellular environment are necessary for long distance spread, there are disadvantages to this mechanism. These include the presence of immune system components, the low success rate of infection by single particles, and the relative fragility of viral particles in the environment. Several mechanisms of direct cell-to-cell spread have been reported for animal viruses which would avoid the issues associated with cell-free particles. A number of viruses can utilize several different mechanisms of direct cell-to-cell spread, but our understanding of the differential usage by these pathogens is modest. Although the mechanisms of cell-to-cell spread differ among viruses, there is a common exploitation of key pathways and components of the cellular cytoskeleton. Remarkably, some of the viral mechanisms of cell-to-cell spread are surprisingly similar to those used by bacteria. Here we summarize the current knowledge of the conventional and non-conventional mechanisms of viral spread, the common methods used to detect viral spread, and the impact that these mechanisms can have on viral pathogenesis.

Malaria causes a serious health problem in the tropical and subtropical regions of the globe. In many cases, the consequences of this disease are fatal. Therefore, a simple, fast, accurate and affordable diagnostic system for the early detection of this disease is necessary for the timely administration of antimalarial drugs.The malarial parasite, during its intra-erythrocyte development, processes a significant amount of hemoglobin, which in this case turns into a hem form called hemozoin. Hemozoin and hemoglobin have different molar extinction coefficients at certain optical wavelengths, hence, light absorption and an optoacoustic signal (OAS) from the infected cell will be different from that of a healthy cell. The paper describes the developed theoretical model intended for studying the influence of intra-erythrocyte malarial parasite development on optoacoustic signals. The OAS were calculated based on the models of healthy and infected blood modeled on the basis of a 3D model.The simulated OAS were analyzed in the temporal and frequency domains to obtain signs of infection at various stages. The calculated OAS spectra have different amplitude levels, which indicates that the optoacoustic method can be useful for differentiating various intraerythrocyte stages of the malarial parasite. The carried out modeling and the results obtained allow us to continue working on the creation of an optoacoustic flow cytometer.

The presence of cells latently infected with HIV is currently considered to be a major barrier to viral eradication within a patient. Here, we consider birth-death-immigration models for the latent cell population in a single patient, and present analytical results for the size of this population in the absence of treatment. We provide results both at steady state (viral set point), and during the non-equilibrium setting of early infection. We obtain semi-analytic results showing how latency-reversing drugs might be expected to affect the size of the latent pool over time. We also analyze the probability of rare mutant viral strains joining the latent cell population, allowing for steady-state and dynamic viral populations within the host.

Transmissible spongiform encephalopathies (TSEs) are a group of lethal neurodegenerative diseases involving the structural conversion of cellular prion protein (PrP(C)) into the pathogenic isoform (PrP(Sc)) for which no effective treatment is currently available. Previous studies have implicated that a polymeric molecule with a repeating unit, such as pentosane polysulfate and polyamidoamide dendrimers, exhibits a potent anti-prion activity, suggesting that poly-(amino acid)s could be a candidate molecule for inhibiting prion propagation. Here, by screening a series of poly-(amino acid)s in a prion-infected neuroblastoma cell line (GT(FK)), we identified poly-L-His as a novel anti-prion compound with an IC(50) value of 1.8 µg/mL (0.18 µM). This potent anti-prion activity was specific to a high-molecular-weight poly-L-His and absent in monomeric histidine or low-molecular-weight poly-L-His. Solution NMR data indicated that poly-L-His directly binds to the loop region connecting Helix 2 and Helix 3 of PrP(C) and sterically blocks the structural conversion toward PrP(Sc). Poly-L-His, however, did not inhibit prion propagation in a prion-infected mouse when administered intraperitoneally, suggesting that the penetration of blood-brain barrier and/or the chemical stability of this polypeptide must be addressed before its application in vivo. Taken together, this study revealed the potential use of poly-L-His as a novel treatment against TSEs. (203 words)

In this paper we propose and analyze an HCV dynamics model taking into consideration the cure of infected hepatocytes and antibody immune response. We incorporate both virus-to-cell and cell-to-cell transmissions into the model. We incorporate a distributed-time delay to describe the time between the HCV or infected cell contacts an uninfected hepatocyte and the emission of new active HCV. We show that the solutions of the proposed model are nonnegative and ultimately bounded. We derive two threshold parameters which fully determine the existence and stability of the three steady states of the model. Using Lyapunov functionals, we established the global stability of the steady states. The theoretical results are confirmed by numerical simulations.

This paper proposes and analyzes an adaptive immunity HIV infection model. The model describes the interaction between healthy CD 4 + T cells, silent (latent) infected cells, active infected cells, free HIV particles, Cytotoxic T lymphocytes (CTLs) and antibodies. The healthy CD 4 + T cells can be infected when they are contacted by one of the following: (i) free HIV particles, and this is known as virus-to-cell (VTC) transmission (ii) silent infected cells, and we call this mode of infection as silent HIV-infected cell-to-cell (CTC) transmission, and (iii) active infected cells, and we call this mechanism active HIV-infected CTC transmission. The incidence rates of the healthy CD 4 + T cells with free HIV particles, silent infected cells, and active infected cells are given by general functions. Moreover, the production/proliferation and removal/death rates of all compartments are represented by general functions. The model is an improvement of the existing HIV infection models which have neglected the incidence between the silent infected cells and healthy CD 4 + T cells. We first show that the model is well-posed. Then, we show that the model has five equilibria and their existence are governed by five threshold parameters. Under a set of conditions on the general functions and the threshold parameters, we have proven the global asymptotic stability of all equilibria by using Lyapunov’s method. We have illustrated the theoretical results via numerical simulations. We have studied the effect of CTC transmission on the dynamical behavior of the system. We have shown that inclusion of CTC transmission decreases the concentration of the healthy CD4 + T cells and increases the concentrations of the infected cells and free HIV particles.

Efforts to cure HIV-1 infection require better quantification of the HIV-1 reservoir, particularly the clones of cells harboring replication-competent (intact) proviruses, termed repliclones. The digital droplet PCR assays commonly used to quantify intact proviruses do not differentiate among specific repliclones, thus the dynamics of repliclones are not well defined. The major challenge in tracking repliclones is the relative rarity of the cells carrying specific intact proviruses. To date, detection and accurate quantification of repliclones requires in-depth integration site sequencing. Here, we describe a simplified workflow using integration site-specific qPCR (IS-qPCR) to determine the frequencies of the proviruses integrated in individual repliclones. We designed IS-qPCR to determine the frequencies of repliclones and clones of cells that carry defective proviruses in samples from three donors. Comparing the results of IS-qPCR with deep integration site sequencing data showed that the two methods yielded concordant estimates of clone frequencies (r = 0.838). IS-qPCR is a potentially valuable tool that can be applied to multiple samples and cell types over time to measure the dynamics of individual repliclones and the efficacy of treatments designed to eliminate them.

Characteristics of E. coli O157:H7-specific infection bacteriophages (O157 coliphages) and broad-host-range bacteriophages for other E. coli serotypes (broad-host coliphages) were compared. The burst sizes of the two groups ranged from 40 to 176 PFU/infected cell. Distributions of the virulence factors stx1, stx2, ehxA, and saa between the two groups were not differentiated. Broad-host-range coliphages showed lower stability at 70°C, in relation to O157 coliphages. However, O157 coliphages showed high acid and ethanol tolerance by reduction of only 22% and 11% phages, respectively, under pH 3 and 70% ethanol for 1 h exposure. Therefore, these results revealed that the O157 coliphages might be more stable under harsh environments, which might explain their effective infection of the acid-tolerant E. coli O157:H7.

Background and Hypothesis: High-risk human papillomavirus type-16 and -18 cause approximately 70% of cervical and 60% of oropharyngeal cancers. While the majority of HPV infections are transient and resolve on their own, some infections persist and progress to cancer. The in vivo and in vitro high-risk HPV transformation ability works to immortalize primary human keratinocytes resulting from the activities of the viral oncoproteins E6 and E7. A well-studied effect of E6 is the degradation of tumor suppressor gene p53 via binding to the ubiquitin ligase E6AP. Additionally, E6 interacts with the cellular calcium-binding protein E6BP, but the functional significance remains unknown. Here we studied the effects of E6BP knockout in normal human and HPV-infected cell lines. Experimental Design or Project Methods: CRISPR-Cas9 system was used to knockout E6BP in HPV-human cervical cancer cell lines: HeLa (high-copy HPV18) and SiHa (low-copy HPV16) as well as human keratinocyte HaCaT (mutant p53) and p53-null lung carcinoma H1299 cell lines. Cells were transfected with a puromycin-resistant CRISPR*Cas9 control plasmid (pLentiv2) and one that carries a guide RNA against E6BP (pLentiv2-E6BP) and selected with puromycin. RFP-GFP, which does not carry a puromycin resistance, was transfected into cells to establish transfection efficiency and to serve as the control for the puromycin. After successful selection, polyclonal populations of pLentiv2 control and pLentiv2-E6BP cells were screened for E6BP protein levels. pLentiv2-E6BP cell populations with reduced E6BP protein were serial diluted to generate monoclonal cell lines. Clones were then screened E6BP protein expression, and low expression clones were further processed for DNA sequencing of the E6BP gene editing site. Additionally, the effects of E6BP knockout in Siha and HeLa cells on p53, p21, and E6AP protein expression was studied by immunoblot. Results: Monoclonal pLentiv2-E6BP cell lines for H1299 and HaCaT cells were established and as well as polyclonal pLentiv2-E6BP HeLa and SiHa cell populations. Preliminary results indicate that E6BP knockout in HeLa and Siha cells reduces the p53 and p21 levels compared to pLentiv2 control cells. Additionally, E6AP protein expression was not affected by E6BP knockout in the polyclonal SiHa cells. Conclusion and Potential Impact: These preliminary results suggest that E6BP levels may influence p53 and p21 levels in HPV cells. It is of particular importance to further investigate if low levels of E6BP contribute to a worse outcome during HPV induced carcinogenesis.

It has been isolated a lytic bacteriophage specific toEscherichia coli, which can infect at least one different bacterial group. Phage ФGF1 was isolated from a wastewater treatment plant. It is resistant to the effect of chloroform and is stable at 40 and 50 °C. In addition, it is stable in the range of pH 5-8. Its host range is wide, infecting even strains from another genus such asShigella. The one-step growth curve yielded a short latent period of 15 minutes and a burst size of 85 PFU per infected cell. Under the electron microscope, this phage presents the C3 morphotype, extremely rare among members of the Podoviridae family. Phage ФGF1 shows some characteristics that could be considered useful in biocontrol applications againstE. coli. Keywords: Bacteriophage,Escherichia coli, morphotype C3,Podoviridae.IMPORTANCEWastewater throughout the world is a heavy carrier of potential pathogens that live in their environment along with other biological agents, such as bacteriophages, which play a controlling role of the bacterial populations there, as in soil. The description of the diversity of such bacteriophages is of paramount importance since they could be used to intentionally reduce or remove those pathogens from that environment. Our work describes a bacteriophage that lives primarily in this type of water.

Persistent infection with human papillomavirus (HPV) initiates ~5% of all human cancers, and particularly cervical and oropharyngeal cancers. HPV vaccines prevent HPV infection, but do not eliminate existing HPV infections. Papillomaviruses induce hyperproliferation of epithelial cells. In this review we discuss how hyperproliferation renders epithelial cells less sensitive to immune attack, and impacts upon the efficiency of the local immune system. These observations have significance for the design of therapeutic HPV cancer immunotherapies.

Growing evidence suggests the outcome of Mycobacterium tuberculosis (Mtb) infection is established rapidly after exposure, but how the current tuberculosis vaccine, BCG, impacts early immunity is poorly understood. Here we found that murine BCG immunization promotes a dramatic shift in infected cell types. While alveolar macrophages (AM) are the major infected cell for the first two weeks in unimmunized animals, BCG promotes the accelerated recruitment and infection of lung infiltrating phagocytes. Interestingly, this shift is dependent on CD4 T cells, yet does not require intrinsic recognition of antigen presented by infected AM. Mtb-specific T cells are first activated in lung regions devoid of infected cells, and these events precede vaccine-induced reduction of the bacterial burden, which occurs only after the co-localization of T cells and infected cells. Understanding how BCG alters early immune responses to Mtb provides new avenues to improve upon the immunity it confers.

Advances in biotechnology have led to the development of a number of biological therapies for the treatment of diverse human diseases. Since these products may contain or are made using human or animal (e.g. cattle) derived materials, it is crucial to test their safety by ensuring the absence of infectious agents; specifically prions, which are highly resilient to elimination and produce fatal diseases in humans. Many cases of iatrogenic Creutzfeldt-Jakob disease have been caused by the use of biological materials (e.g. human growth hormone) contaminated with prions. For this reason, it is important to screen cells and biological materials for the presence of prions. Here we show the utility of the Protein Misfolding Cyclic Amplification (PMCA) technology as a screening tool for the presence of human (vCJD) and bovine (BSE) prions in a human cell therapy product candidate. First, we demonstrated the sensitivity of PMCA to detect a single cell infected with prions. For these experiments, we used RKM7 cells chronically infected with murine RML prions. Serial dilutions of an infected cell culture showed that PMCA enabled prion amplification from a sample comprised of only one cell. Next, we determined that PMCA performance was robust and uncompromised by the spiking of large quantities of uninfected cells into the reaction. Finally, to demonstrate the practical application of this technology, we analyzed a human cell line being developed for therapeutic use and found it to be PMCA-negative for vCJD and BSE prions. Our findings demonstrate that the PMCA technology has unparalleled sensitivity and specificity for the detection of prions, making it an ideal quality control procedure in the production of biological therapeutics.

Eating food that has been contaminated with bacteria called Listeria monocytogenes can result in life-threatening infections. The bacteria first invade the epithelial cells that line the small intestine. After this, L. monocytogenes can move from one host cell to another, which allows the infection to reach other organs. Most studies into how L. monocytogenes infections spread have focused either on how single bacterial cells move from one host cell to the next, or on how millions of bacteria damage host tissues. Little was known about the intermediate steps of an infection, where the bacteria start to colonize the small intestine. To investigate, Ortega et al. recorded videos of L. monocytogenes spreading between epithelial cells grown on a glass coverslip, and developed computer simulations to try to reproduce how the bacteria spread. This revealed that the bacteria do not all move in the same way. Instead, less than 1% of the bacteria move in ‘steps’ that are up to 10 times longer than those taken by the others. Ortega et al. named these bacteria ‘pioneers’. Ortega et al. propose that the pioneers form long protrusions that allow them to spread directly from an infected cell to a non-neighboring cell. By taking these large steps, the pioneers may increase the chances that the bacteria will cause a long-lasting infection. Future research will be needed to answer further questions about the pioneers. For example, how do the pioneer bacteria differ from the majority of bacterial cells? Would targeting anti-bacterial treatments at pioneers make it easier to treat infections? It also remains to be seen if other types of bacteria also show this pioneer behavior.

Lionfish (Pterois volitans) are a fish that live in the Indo-Pacific region. Today, Lionfish have an increasing population and invaded up to the Atlantic Ocean. The invasion of Lionfish was not accompanied by the presence of predators, thus causing damage to the ecosystem and food chain. One of the control efforts that possible be done is the utilization of venomous spines of Lionfish that contains Phospholipase A2 known to have antiretroviral activity. So it has the potential to be used as an alternative treatment of HIV/AIDS infection. This research aims to increase the purity and specific activity of Phospholipase A2 from venomous spines of Lionfish in the Java Sea, Indonesia. Phospholipase A2 isolated from Lionfish venom from the waters of the Java Sea with Salting-out method using Ammonium Sulfate on 20% saturation level and the addition of 0.5 mL of Caprylic Acid by heating 60 °C for 20 minutes has the highest enzymes activity and purity. The specific activities reaching 6.48 units/μg, safe tested on human cells with 28.36 ppm inhibition measured with LC50 and also can inhibit infected cell growth with 98.13% inhibition degree.

Virus particles, 'virions', range in size from nano-scale to micro-scale. They have many different shapes and are composed of proteins, sugars, nucleic acids, lipids, water and solutes. Virions are autonomous entities and affect all forms of life in a parasitic relationship. They infect prokaryotic and eukaryotic cells. The physical properties of virions are tuned to the way they interact with cells. When virions interact with cells, they gain huge complexity and give rise to an infected cell, also known as 'virus'. Virion-cell interactions entail the processes of entry, replication and assembly, as well as egress from the infected cell. Collectively, these steps can result in progeny virions, which is a productive infection, or in silencing of the virus, an abortive or latent infection. This book explores facets of the physical nature of virions and viruses and the impact of mechanical properties on infection processes at the cellular and subcellular levels.

PhageFISH uses the power of fluorescence in situ hybridization to monitor intracellular phage infections at single cell level. It combines host cell identification via rRNA probes and phage identification via phage-specific gene probes, allowing for the quantification of the infected cell fraction and the discrimination between infection stages. This book chapter covers all aspects of the procedure, from phage probe design and synthesis, to the phageFISH protocol itself, to microscopy and image analysis.

SARS-CoV accessory protein Orf8b is involved in suppressing interferon-mediated immune response of the infected cell and this might lead to supposition that the corresponding protein 2019-nCoV Orf8 shares the same role. But the tertiary structures of these proteins are still unknown, and the primary structures demonstrate very low homology and different calculating parameters. This time they both are affected by stabilizing selection and in natural viral populations do not tend to be deleted. The question whether in this case very different proteins could share the same function rises from the present data.

The search and development of anti-HIV drugs is currently one of the most urgent tasks of pharmacological studies. In this work, a quantitative structure–activity relationship (QSAR) model based on some new norm indexes, was obtained to a series of more than 150 HEPT derivatives (1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine) to find their pEC 50 (the required effective concentration to achieve 50% protection of MT-4 cells against the cytopathic effect of virus) and pCC 50 (the required cytotoxic concentration to reduce visibility of 50% mock infected cell) activities. The model efficiencies were then validated using the leave-one-out cross validation (LOO-CV) and y-randomization test. Results indicated that this new model was efficient and could provide satisfactory results for prediction of pEC 50 and pCC 50 with the higher R train 2 and the higher R test 2 . By using the leverage approach, the applicability domain of this model was further investigated and no response outlier was detected for HEPT derivatives involved in this work. Comparison results with reference methods demonstrated that this new method could result in significant improvements for predicting pEC 50 and pCC 50 of anti-HIV HEPT derivatives. Moreover, results shown in this present study suggested that these two absolutely different activities pEC 50 and pCC 50 of anti-HIV HEPT derivatives could be predicted well with a totally similar QSAR model, which indicated that this model might have the potential to be further utilized for other biological activities of HEPT derivatives.

Hepatitis C Virus (HCV) promotes lipid droplet (LD) formation and perturbs the expression of the LD associated PAT proteins ADRP and TIP47, to promote its own lifecycle. HCV enhances TIP47 and suppresses ADRP by displacing it from LD surface in infected cell models. We have previously shown that suppression of TIP47 by miR-148a and miR-30a decreased intracellular LDs and HCV RNA. Thus, this study aimed at examining whether this microRNA-mediated suppression of HCV would limit HCV-dependent displacement of ADRP from LDs. ADRP expression was examined in 21 HCV-infected liver biopsies and 9 healthy donor liver tissues as well as in HCV-infected Huh7 cells using qRT-PCR. miR-148a and miR-30a expression was manipulated using specific oligos in JFH-1 infected, oleic acid treated cells, to study their impact on ADRP expression using qRT-PCR, and immunofluorescence microscopy. Intracellular HCV RNA was assessed using qRT-PCR. ADRP is down regulated in patients as well as HCVcc-JFH-I infected cell models. Forcing the expression of both miRNAs induced ADRP on the mRNA and protein levels. This study shows that HCV suppresses hepatic ADRP expression in infected patients and cell lines. Forcing the expression of miR-148a and miR-30a limits the suppressive effect of HCV on ADRP. J. Med. Virol. 89:653-659, 2017. © 2016 Wiley Periodicals, Inc.

Cytomegalovirus (CMV) infection occurs frequently in young children, who, when infected, are then a major source of transmission. Oral CMV shedding by 14 infants with primary infection was comprehensively characterized using quantitative polymerase chain reaction weekly for ≥9 months. Three phases of oral shedding were identified: expansion, transition, and clearance. Viral expansion occurred over a median of 7 weeks, with a median doubling time of 3 days. During the transition phase, expansion slowed over a median of 6 weeks before peak viral load was reached. Clearance was slow (22-day median half-life), and shedding did not resolve during observation for any infant. Mathematical modeling demonstrated that prolonged oral CMV expansion is explained by a low within-host reproduction number (median, 1.63) and a delayed immune response that only decreases the infected cell half-life by 44%. Thus, the prolonged oral CMV shedding observed during primary infection can be explained by slow viral expansion and inefficient immunologic control.

The host antiviral programs that are initiated following viral infection form a dynamic and complex web of responses that we have collectively termed as “the antiviral landscape”. Conventional approaches to studying antiviral responses have primarily used reductionist systems to assess the function of a single or a limited subset of molecules. Systems biology is a holistic approach that considers the entire system as a whole, rather than individual components or molecules. Systems biology based approaches facilitate an unbiased and comprehensive analysis of the antiviral landscape, while allowing for the discovery of emergent properties that are missed by conventional approaches. The antiviral landscape can be viewed as a hierarchy of complexity, beginning at the whole organism level and progressing downward to isolated tissues, populations of cells, and single cells. In this review, we will discuss how systems biology has been applied to better understand the antiviral landscape at each of these layers. At the organismal level, the Collaborative Cross is an invaluable genetic resource for assessing how genetic diversity influences the antiviral responses. Whole tissue and isolated bulk cell transcriptomics serves as a critical tool for the comprehensive analysis of antiviral responses at both the tissue and cellular levels of complexity. Finally, new techniques in single cell analysis are emerging tools that will revolutionize our understanding of how individual cells within a bulk infected cell population contribute to the overall antiviral landscape.

Virology Virus RNA within a cell is either packaged into progeny viral particles for release (“leave strategy”) or serves as a template for replication (“stay strategy”). To test the implications of leave versus stay strategies for transmission and replication efficiency, Iwanami et al. selected hepatitis C virus (HCV) strains with differing virion release characteristics. A series of culture experiments showed that a strain isolated from a fulminant hepatitis patient that transmits efficiently packages a higher proportion of RNA into particles (leave strategy). Conversely, a virus strain engineered to maximize productivity favors retention of RNA (stay strategy). A model constructed using data from time courses of viral production, infectivity of progeny, and infected cell numbers provides insights into viral decay dynamics and possible mechanisms of action of anti-HCV drugs. PLOS Biol. 18 , e3000562 (2020).

OBJECTIVES: Exposure to air pollution has been associated with throat discomfort, cough, pulmonary disease, and even lung cancer. We developed a botanical extract-based blend with the goal of ameliorating some of the adverse effects of airborne pollutants. This blend consisted of broccoli seed (BS, Brasssica oleracea) extract, Luo Han Guo (LH G, Siraitia grovenorii) extract, and Honeysuckle Flower Bud (HFB, Lonicera japonica) extract. BS extract is rich in Sulforaphane glucosinolate and is reported to promote healthy inflammatory balance as well as to regulate oxidative stress and DNA-damaging substances. LHG has been used as a traditional medicine for the treatment of pharyngitis, pharyngeal pain, as well as an antitussive remedy in China. HFB is a traditional herbal medicine widely used in East Asia as an anti-inflammatory and anti-oxidative agent. METHODS: In order to evaluate the effectiveness of this putative airborne pollutant defense blend, human alveolar basal epithelial cells in adenocarcinoma (A549) were treated by exposing total culture medium (TCMed, to 0.02 mg/mL of haze particles (HP). Gene expression of inflammatory factors IL-1β, IL-8, TNF-α, INF-γ, and cell tight junction proteins were detected by qPCR at 0.5, 1, 2, and 6H when treated with 0.02 mg/mL HP TCMed alone (group H), 0.02 mg/mL HP TCMed + 5 mg/mL of LHG + HFB blend (LHG: HFB 1:10) +0.01 mg/mL of L-sulforaphane (group HT). RESULTS: We found that gene expression of IL-1β, IL-8 and TNF-α were significantly decreased in HT at 1, 2, and 6 H treatment as compared to the F group. Gene expression of tight junction protein OCLN and ZO-1, which is a scaffolding protein, were significantly increased in HT as compared to the F group. CONCLUSIONS: This botanical extract blend regulates inflammatory factors and balances cell transmembrane stability in a primary cell haze infection model and therefore may be useful as a preventive formulation to help alleviate the adverse effects of airborne pollutants. FUNDING SOURCES: Nu Skin Enterprises.

This study targeting the characterization of new lytic phages and studying their impact on Staphylococcus aureus biofilm and growth in milk. Two lytic phages (vB_SauM_ME18 & vB_SauM_ME126) were isolated using 40 Multidrug-Resistant (MDR) S. aureus isolates. Four isolates (10%) were susceptible to phages. Phages were related to Family Myoviridae, had icosahedral heads and long contractile tails. The genome size is 20 kbp for both phages and DNA was digested with hinf I, hind III and hae III. Phages had a latent period (~15 min), rise period (~30 min) and burst size (114 PFU/infected cell for ME18 and 140 PFU/infected cell for ME126). There was a significant reduction in biofilm formation and reduction in the established biofilm biomass in phage treated S. aureus compared with controls (P