Towards a Mathematical Model for the Viral Progression in the Pharynx

In this work, a comprehensive model for the viral progression in the pharynx has been developed. This one-dimension model considers both Fickian diffusion and convective flow coupled with chemical reactions, such as virus population growth, infected and uninfected cell accumulation as well as virus clearance. The effect of a sterilizing agent such as an alcoholic solution on the viral progression in the pharynx was taken into account and a parametric analysis for the effect of kinetic rate parameters on virus propagation was made. Moreover, different conditions caused by further medical treatment, such as a decrease in virus yield per infected cell, were examined. It is shown that the infection fails to establish by decreasing the virus yield per infected cell. It is believed that this work could be used to further investigate the medical treatment of viral progression in the pharynx.

Label-Free Detection of Human Coronaviruses in Infected Cells Using Enhanced Darkfield Hyperspectral Microscopy (EDHM)

Human coronaviruses (HCoV) are causative agents of mild to severe intestinal and respiratory infections in humans. In the last 15 years, we have witnessed the emergence of three zoonotic, highly pathogenic HCoVs. Thus, early, and accurate detection of these viral pathogens is not only essential for preventing transmission but also for the timely treatment and monitoring of drug resistance. Herein, we applied enhanced darkfield hyperspectral microscopy (EDHM), a novel non-invasive, label-free diagnostic tool for rapid and accurate identification of two strains of HCoVs, i.e., OC43 and 229E. The EDHM technology allows collecting the optical image with both spectral and spatial details in a single measurement without direct contact between the specimen and the sensor. Thus, it can provide the direct mapping of spectral signatures specific for a given viral strain in a complex biological milieu. Our study demonstrated distinct spectral patterns for HCoV-OC43 and HCoV-229E virions in the solution, which can serve as distinguishable parameters for their differentiation. Furthermore, spectral signatures for both HCoV strains in the infected cells displayed a considerable peak wavelength shift compared to the uninfected cell samples indicating that the EDHM is applicable to detect and differentiate between HCoV infected and uninfected cells.

Transcriptome-wide N6-methyladenosine modification profiling of long non-coding RNAs during replication of Marek’s disease virus in vitro

Background The newly discovered reversible N6-methyladenosine (m6A) modification plays an important regulatory role in gene expression. Long non-coding RNAs (lncRNAs) participate in Marek’s disease virus (MDV) replication but how m6A modifications in lncRNAs are affected during MDV infection is currently unknown. Herein, we profiled the transcriptome-wide m6A modification in lncRNAs in MDV-infected chicken embryo fibroblast (CEF) cells. Results Methylated RNA immunoprecipitation sequencing results revealed that the lncRNA m6A modification is highly conserved with MDV infection increasing the expression of lncRNA m6A modified sites compared to uninfected cell controls. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that lncRNA m6A modifications were highly associated with signaling pathways associated with MDV infection. Conclusions In this study, the alterations seen in transcriptome-wide m6A occurring in lncRNAs following MDV-infection suggest this process plays important regulatory roles during MDV replication. We report for the first time profiling of the alterations in transcriptome-wide m6A modification in lncRNAs of MDV-infected CEF cells.

HIV cell-to-cell spread slows evolution of drug resistance

Many enveloped viruses such as HIV have evolved to transmit by two infection modes: cell-free infection and cell-to-cell spread. Cell-to-cell spread is highly efficient as it involves directed viral transmission from the infected to the uninfected cell. In contrast, cell-free infection relies on chance encounters between the virion and cell. Despite the higher efficiency of cell-to-cell spread, there is substantial transmission by cell-free infection in conjunction with cell-to-cell spread. A possible reason is that cell-free infection offers a selective advantage by increasing sensitivity to factors interfering with infection, hence accelerating evolution of resistance relative to cell-to-cell spread alone. Here we investigated whether a combination of cell-free infection and cell-to-cell spread confers a selective advantage in experimental evolution to an antiretroviral drug. We maintained HIV infection using coculture of infected with uninfected cells in the face of moderate inhibition by the reverse transcriptase inhibitor efavirenz. We tested the effect on the rate of drug resistance evolution of replacing one coculture infection cycle with an infection cycle involving cell-free infection only, and observed earlier evolution of drug resistance mutations to efavirenz. When we increased selective pressure by adding a second reverse transcriptase inhibitor, emtricitabine, infection with the cell-free step consistently evolved multidrug resistance to both drugs and was able to replicate. In contrast, infection without a cell-free step mostly failed to evolve multidrug resistance. Therefore, HIV cell-to-cell spread decreases the ability of HIV to rapidly evolve resistance to inhibitors, which is conferred by cell-free infection.Author summaryCell-to-cell spread of HIV differs from cell-free, diffusion-based HIV infection in that viral transmission is directed from the infected to the uninfected cell through cellular interactions. Cell-to-cell spread has been recognized as a highly efficient infection mode that is able to surmount inhibition by antibodies and antiretroviral drugs. However, the effect of HIV cell-to-cell spread on the rate of evolution of viral resistance to infection inhibitors has not been studied. Here we used experimental evolution to investigate the effect of cell-to-cell spread versus cell-free infection on the emergence of drug resistance mutations to one or a combination of antiretroviral drugs. We found that replacing one infection cycle in experimental evolution with cell-free infection, where the filtered supernatant from infected cells, but not the cellular fraction, is used as the viral source, results in more rapid evolution of resistance. The consequences are that multidrug resistance consistently evolves with a cell-free viral cycle, but not when infection is solely by coculture of infected and uninfected cells. A possible consequence is that in environments where HIV cell-to-cell spread may predominate and some residual viral replication occurs in the face of ART, the emergence of drug resistance mutations would be delayed.

Hepatozoon pyramidumi sp. n. (Apicomplexa: Adeleorina) from the blood of Echis pyramidum: morphology and SSU rDNA sequence

Hepatozoon pyramidumi sp. n. is described from the blood of the Egyptian saw-scaled viper, Echis pyramidum, captured from Saudi Arabia. Five out of ten viper specimens examined (50%) were found infected with Hepatozoon pyramidumi sp. n. with parasitaemia level ranged from 20-30%. The infection was restricted only to the erythrocytes. Two morphologically different forms of intraerythrocytic stages were observed; small and mature gamonts. The small ganomt with average size of 10.7 × 3.5 μm. Mature gamont was sausage-shaped with recurved poles measuring 16.3 × 4.2 μm in average size. Infected erythrocytes were hypertrophied; their nuclei were deformed and sometimes displaced from their central position in the normal uninfected cell. Merogonic stages were observed in the lung endothelial cell and the liver parenchyma cells. Mature meront was 17.8 × 13.6 µm and contained banana-shaped merozoites with average size of ~15 × 2 µm. Phylogenetic analysis based on the SSU rDNA sequence clustered Hepatozoon pyramidumi sp. n with previously sequenced Hepatozoon spp., most of them infected reptilian hosts without geographic consideration. The morphological and molecular comparison with closely related species proved the taxonomic uniqueness and novelty of the present form.

Synergistic Activity of Mobile Genetic Element Defences in Streptococcus pneumoniae

A diverse set of mobile genetic elements (MGEs) transmit between Streptococcus pneumoniae cells, but many isolates remain uninfected. The best-characterised defences against horizontal transmission of MGEs are restriction-modification systems (RMSs), of which there are two phase-variable examples in S. pneumoniae. Additionally, the transformation machinery has been proposed to limit vertical transmission of chromosomally integrated MGEs. This work describes how these mechanisms can act in concert. Experimental data demonstrate RMS phase variation occurs at a sub-maximal rate. Simulations suggest this may be optimal if MGEs are sometimes vertically inherited, as it reduces the probability that an infected cell will switch between RMS variants while the MGE is invading the population, and thereby undermine the restriction barrier. Such vertically inherited MGEs can be deleted by transformation. The lack of between-strain transformation hotspots at known prophage att sites suggests transformation cannot remove an MGE from a strain in which it is fixed. However, simulations confirmed that transformation was nevertheless effective at preventing the spread of MGEs into a previously uninfected cell population, if a recombination barrier existed between co-colonising strains. Further simulations combining these effects of phase variable RMSs and transformation found they synergistically inhibited MGEs spreading, through limiting both vertical and horizontal transmission.

Synaptic transmission may provide an evolutionary benefit to HIV through modulation of latency

Transmission of HIV is known to occur by two mechanisms in vivo: the free virus pathway, where viral particles bud off an infected cell before attaching to an uninfected cell, and the cell-cell pathway, where infected cells form virological synapses through close contact with an uninfected cell. It has also been shown that HIV replication includes a positive feedback loop controlled by the viral protein Tat, which may act as a stochastic switch in determining whether an infected cell enters latency. In this paper, we introduce a simple mathematical model of HIV replication containing both the free virus and cell-cell pathways. Using this model, we demonstrate that the high multiplicity of infection in cell-cell transmission results in a suppression of latent infection, and that this modulation of latency through balancing the two transmission mechanisms can provide an evolutionary benefit to the virus. This benefit increases with decreasing overall viral fitness, which may provide a within-host evolutionary pressure toward more cell-cell transmission in late-stage HIV infection.

Remote Activation of Host Cell DNA Synthesis in Uninfected Cells Signaled by Infected Cells in Advance of Virus Transmission

ABSTRACTViruses modulate cellular processes and metabolism in diverse ways, but these are almost universally studied in the infected cell itself. Here, we study spatial organization of DNA synthesis during multiround transmission of herpes simplex virus (HSV) using pulse-labeling with ethynyl nucleotides and cycloaddition of azide fluorophores. We report a hitherto unknown and unexpected outcome of virus-host interaction. Consistent with the current understanding of the single-step growth cycle, HSV suppresses host DNA synthesis and promotes viral DNA synthesis in spatially segregated compartments within the cell. In striking contrast, during progressive rounds of infection initiated at a single cell, we observe that infection induces a clear and pronounced stimulation of cellular DNA replication in remote uninfected cells. This induced DNA synthesis was observed in hundreds of uninfected cells at the extended border, outside the perimeter of the progressing infection. Moreover, using pulse-chase analysis, we show that this activation is maintained, resulting in a propagating wave of host DNA synthesis continually in advance of infection. As the virus reaches and infects these activated cells, host DNA synthesis is then shut off and replaced with virus DNA synthesis. Using nonpropagating viruses or conditioned medium, we demonstrate a paracrine effector of uninfected cell DNA synthesis in remote cells continually in advance of infection. These findings have significant implications, likely with broad applicability, for our understanding of the ways in which virus infection manipulates cell processes not only in the infected cell itself but also now in remote uninfected cells, as well as of mechanisms governing host DNA synthesis.IMPORTANCEWe show that during infection initiated by a single particle with progressive cell-cell virus transmission (i.e., the normal situation), HSV induces host DNA synthesis in uninfected cells, mediated by a virus-induced paracrine effector. The field has had no conception that this process occurs, and the work changes our interpretation of virus-host interaction during advancing infection and has implications for understanding controls of host DNA synthesis. Our findings demonstrate the utility of chemical biology techniques in analysis of infection processes, reveal distinct processes when infection is examined in multiround transmission versus single-step growth curves, and reveal a hitherto-unknown process in virus infection, likely relevant for other viruses (and other infectious agents) and for remote signaling of other processes, including transcription and protein synthesis.

Cell Sorting-Assisted Microarray Profiling of Host Cell Response to Cryptosporidium parvum Infection

ABSTRACT To study the transcriptional response of mammalian cells to infection with the intracellular apicomplexan parasite Cryptosporidium parvum, infected and uninfected cells were recovered from C. parvum-infected cell monolayers. This approach, which contrasts with a more conventional experimental design that compares infected to uninfected cell monolayers, enabled the identification of functional categories of genes that are differentially transcribed as a direct consequence of the presence of intracellular parasites. Among several categories of upregulated genes, glycoprotein metabolism was significantly overrepresented. To investigate whether these transcriptional changes affected the composition of the surface of infected cells, cells were probed with fluorescently labeled lectins. Among a panel of seven lectins, soybean agglutinin, which recognizes N-acetyl-d-galactosamine, generated the largest difference in fluorescence between infected and uninfected cells. The origin of the fluorescent signal emitted by infected cells was further investigated and attributed to the overexpression of glycoprotein on the surface of infected cells, as well as the presence of glycoprotein located in the proximity of intracellular parasites.

Sharing the burden: antigen transport and firebreaks in immune responses

Communication between cells is crucial for immune responses. An important means of communication during viral infections is the presentation of viral antigen on the surface of an infected cell. Recently, it has been shown that antigen can be shared between infected and uninfected cells through gap junctions, connexin-based channels, that allow the transport of small molecules. The uninfected cell receiving antigen can present it on its surface. Cells presenting viral antigen are detected and killed by cytotoxic T lymphocytes. The killing of uninfected cells can lead to increased immunopathology. However, the immune response might also profit from killing those uninfected bystander cells. One benefit might be the removal of future ‘virus factories’. Another benefit might be through the creation of ‘firebreaks’, areas void of target cells, which increase the diffusion time of free virions, making their clearance more likely. Here, we use theoretical models and simulations to explore how the mechanism of gap junction-mediated antigen transport (GMAT) affects the dynamics of the virus and immune response. We show that under the assumption of a well-mixed system, GMAT leads to increased immunopathology, which always outweighs the benefit of reduced virus production due to the removal of future virus factories. By contrast, a spatially explicit model leads to quite different results. Here we find that the firebreak mechanism reduces both viral load and immunopathology. Our study thus shows the potential benefits of GMAT and illustrates how spatial effects may be crucial for the quantitative understanding of infection dynamics and immune responses.