Within-host spatial dynamics of viruses and defective interfering particles

Defective-interfering (DI) viruses arise spontaneously by deletion mutations. The shortened genomes of the DI particles cannot replicate unless they coinfect a cell with a wild-type virus. Upon coinfection, the DI genome replicates more quickly and outcompetes the wild type. The coinfected cell produces mostly DI viruses. At the population level, the abundances of DI and wild-type viruses fluctuate dramatically under some conditions. In other cases, the DI viruses appear to mediate persistent infections with relatively low levels of host cell death. This moderation of viral damage has led some to suggest DI particles as therapeutic agents. Previous mathematical models have shown that either fluctuation or persistence can occur for plausible parameter values. I develop new mathematical models for the population dynamics of DI and wild-type viruses. My work extends the theory by developing specific predictions that can be tested in the laboratory. These predictions, if borne out by experiment, will explain the key processes that control the diversity of observed outcomes. The most interesting prediction concerns the rate at which killed host cells are replaced. A low rate of replacement causes powerful epidemics followed by a crash in viral abundance. As the rate of replacement increases, the frequency of oscillations increases in DI and wild-type viral abundances, but the severity (amplitude) of the fluctuations declines. At higher replacement rates for host cells, nearly all cells become infected by DI particles and a low level of fluctuating, wild-type viremia persists.     

Viral reproductive strategies: How can lytic viruses be evolutionarily competitive?

Viral release strategies can be roughly classified as lytic (the ones that accumulate inside the host cell and exit in a burst, killing the cell), and budding (the ones that are produced and released from the host cell gradually). Here we study the evolutionary competition between the two strategies. If all the parameters, such as the rate of viral production, cell life-span and the neutralizing capacity of the antibodies, were the same for lytic and budding viruses, the budding life-strategy would have a large evolutionary advantage. The question arises what makes lytic viruses evolutionarily competitive. We propose that it is the different removal capacity of the antibodies against budding and lytic virions. The latter exit the cell in a large burst such that the antibodies are "flooded" and a larger proportion of virions can escape the immune system and spread to new cells. We create two spatial models of virus-antibody interaction and show that for realistic parameter values, the effect of antibody flooding can indeed take place. We also argue that the lytic life cycle, including a relatively large burst-size, has evolved to promote survival in the face of antibody attack. According to the calculations, in the absence of efficient antibodies, the optimal burst size of lytic viruses would be only a few virus particles, as opposed to the observed 10(2)-10(5) viral particles. Similarly, there is an evolutionary pressure to extend the life-span as a response to antibody action.     

Different applications of virus‐like particles in biology and medicine: Vaccination and delivery systems

Virus‐like particles (VLPs) mimic the whole construct of virus particles devoid of viral genome as used in subunit vaccine design. VLPs can elicit efficient protective immunity as direct immunogens compared to soluble antigens co‐administered with adjuvants in several booster injections. Up to now, several prokaryotic and eukaryotic systems such as insect, yeast, plant, and E. coli were used to express recombinant proteins, especially for VLP production. Recent studies are also generating VLPs in plants using different transient expression vectors for edible vaccines. VLPs and viral particles have been applied for different functions such as gene therapy, vaccination, nanotechnology, and diagnostics. Herein, we describe VLP production in different systems as well as its applications in biology and medicine. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 113–132, 2016.     

Epidemiology of insect-transmitted plant viruses: modelling disease dynamics and control interventions

Abstract. Plant viruses are an important constraint to crop production world‐wide. Rarely have plant virologists, vector entomologists and crop specialists worked together in search of sustainable management practices for viral diseases. Historically, modelling approaches have been vector‐based dealing with empirical forecasting systems or simulation of vector population dynamics. More recently, epidemiological models, such as those used in human/animal epidemiology, have been introduced in an attempt to characterize and analyse the population ecology of viral diseases. The theoretical bases for these models and their use in evaluating control strategies in terms of the interactions between host, virus and vector are considered here. Vector activity and behaviour, especially in relation to virus transmission, are important determinants of the rate and extent of epidemic development. The applicability and flexibility of these models are illustrated by reference to specific case studies, including the increasing importance of whitefly‐transmitted viruses. Some outstanding research and methodological issues are considered.     

In vivo egress of an alphaherpesvirus from axons

Many alphaherpesviruses establish a latent infection in the peripheral nervous systems of their hosts. This life cycle requires the virus to move long distances in axons toward the neuron's cell body during infection and away from the cell body during reactivation. While the events underlying entry of the virion into neurons during infection are understood in principle, no such consensus exists regarding viral egress from neurons after reactivation. In this study, we challenged two different models of viral egress from neurons by using pseudorabies virus (PRV) infection of the rat retina: does PRV egress solely from axon terminals, or can the virus egress from axon shafts as well as axon terminals? We took advantage of PRV gD mutants that are not infectious as extracellular particles but are capable of spreading by cell-cell contact. We observed that both wild-type virus and a PRV gD null mutant are capable of spreading from axons to closely apposed nonneuronal cells within the rat optic nerve after intravitreal infection. However, infection does not spread from these infected nonneuronal cells. We suggest that viral egress can occur sporadically along the length of infected axons and is not confined solely to axon terminals. Moreover, it is likely that extracellular particles are not involved in nonneuronal cell infections. Taking these together with previous data, we suggest a model of viral egress from neurons that unifies previous apparently contradictory data.     

Cell culture and infection system for hepatitis C virus

Hepatitis C virus (HCV) infection causes chronic liver disease and is a worldwide health problem. Despite ever-increasing demand for knowledge on viral replication and pathogenesis, detailed analysis has been hampered by a lack of efficient viral culture systems. We isolated HCV genotype 2a strain JFH-1 from a patient with fulminant hepatitis. This strain replicates efficiently in Huh7 cells. Efficient replication and secretion of recombinant viral particles can be obtained in cell culture by transfection of in vitro-transcribed full-length JFH-1 RNA into Huh7 cells. JFH-1 virus generated in cell culture is infectious for both naive Huh7 cells and chimpanzees. The efficiency of viral production and infectivity of generated virus is substantially improved with permissive cell lines. This protocol describes how to use this system, which provides a powerful tool for studying viral life cycle and for the construction of antiviral strategies and the development of effective vaccines. Viral particles can be obtained in 12 days with this protocol.     

Therapeutic cell engineering: designing programmable synthetic genetic circuits in mammalian cells

Cell therapy approaches that employ engineered mammalian cells for on-demand production of therapeutic agents in the patient’s body are moving beyond proof-of-concept in translational medicine. The therapeutic cells can be customized to sense user-defined signals, process them, and respond in a programmable and predictable way. In this paper, we introduce the available tools and strategies employed to design therapeutic cells. Then, various approaches to control cell behaviors, including open-loop and closed-loop systems, are discussed. We also highlight therapeutic applications of engineered cells for early diagnosis and treatment of various diseases in the clinic and in experimental disease models. Finally, we consider emerging technologies such as digital devices and their potential for incorporation into future cell-based therapies.     

Production of complex viral glycoproteins in plants as vaccine immunogens

Plant molecular farming offers a cost‐effective and scalable approach to the expression of recombinant proteins which has been proposed as an alternative to conventional production platforms for developing countries. In recent years, numerous proofs of concept have established that plants can produce biologically active recombinant proteins and immunologically relevant vaccine antigens that are comparable to those made in conventional expression systems. Driving many of these advances is the remarkable plasticity of the plant proteome which enables extensive engineering of the host cell, as well as the development of improved expression vectors facilitating higher levels of protein production. To date, the only plant‐derived viral glycoprotein to be tested in humans is the influenza haemagglutinin which expresses at ~50 mg/kg. However, many other viral glycoproteins that have potential as vaccine immunogens only accumulate at low levels in planta. A critical consideration for the production of many of these proteins in heterologous expression systems is the complexity of post‐translational modifications, such as control of folding, glycosylation and disulphide bridging, which is required to reproduce the native glycoprotein structure. In this review, we will address potential shortcomings of plant expression systems and discuss strategies to optimally exploit the technology for the production of immunologically relevant and structurally authentic glycoproteins for use as vaccine immunogens.     

Production of hepatitis C virus lacking the envelope-encoding genes for single-cycle infection by providing homologous envelope proteins or vesicular stomatitis virus glycoproteins in trans

Hepatitis C virus (HCV) infection is a major worldwide health problem. The envelope glycoproteins are the major components of viral particles. Here we developed a trans-complementation system that allows the production of infectious HCV particles in whose genome the regions encoding envelope proteins are deleted (HCVΔE). The lack of envelope proteins could be efficiently complemented by the expression of homologous envelope proteins in trans. HCVΔE production could be enhanced significantly by previously described adaptive mutations in NS3 and NS5A. Moreover, HCVΔE could be propagated and passaged in packaging cells stably expressing HCV envelope proteins, resulting in only single-round infection in wild-type cells. Interestingly, we found that vesicular stomatitis virus (VSV) glycoproteins could efficiently rescue the production of HCV lacking endogenous envelope proteins, which no longer required apolipoprotein E for virus production. VSV glycoprotein-mediated viral entry could allow for the bypass of the natural HCV entry process and the delivery of HCV replicon RNA into HCV receptor-deficient cells. Our development provides a new tool for the production of single-cycle infectious HCV particles, which should be useful for studying individual steps of the HCV life cycle and may also provide a new strategy for HCV vaccine development.     

Production methods for viral particles

Viral particles and virus-like particles (VLPs) or capsids are becoming important vehicles and templates in bio-imaging, drug delivery and materials sciences. Viral particles are prepared by infecting the host organism but VLPs are obtained from cells that express a capsid protein. Some VLPs are disassembled and then re-assembled to incorporate a material of interest. Cell-free systems, which are amenable to manipulating the viral assembly process, are also available for producing viral particles. Regardless of the production system employed, the particles are functionalized by genetic and/or chemical engineering. Here, we review various methods for producing and functionalizing viral particles and VLPs, and we discuss the merits of each system.     

大肠杆菌表达的病毒样颗粒疫苗

重组病毒样颗粒是病毒衣壳蛋白外源表达的重要形式,形态结构与天然病毒高度相似,位于纳米尺度的大小易于被免疫系统识别,可激发机体产生保护性免疫反应,且不含有病毒基因,因此,是一种理想的疫苗形式,也是基于结构进行疫苗设计的重要结构载体。目前已上市的乙型肝炎疫苗、人乳头瘤病毒疫苗和戊型肝炎疫苗等基因工程疫苗均采用病毒样颗粒形式。大肠杆菌表达系统被广泛用于基因工程药物的生产,具有安全性好、生产周期短、易于放大生产等优点,在病毒样颗粒疫苗应用上具有良好前景。本文综述了利用大肠杆菌研制戊型肝炎疫苗和人乳头瘤病毒疫苗的进展,特别是这些病毒样颗粒疫苗的表达及组装、表位结构特征和临床试验结果。     

Yellow Fever virus NS3 plays an essential role in virus assembly independent of its known enzymatic functions

In flaviviruses it has been proposed that there is a coupling between genome replication and virion assembly and that nonstructural proteins are involved in this process. It was previously reported that mutations in yellow fever virus (YFV) nonstructural protein NS2A blocked production of infectious virus and that this block could be released by a suppressor mutation in NS3. Here, based on studies using a YFV replicon-based trans-packaging system as well as full-length YFV cDNA, we report that mutation of a conserved tryptophan at position 349 in the helicase domain of NS3 blocks production of infectious virus particles, revealing an as-yet-unknown role for NS3 in virus assembly. Mutation of tryptophan 349 to alanine (W349A) had no effect on viral replication, as demonstrated by wild-type levels of viral RNA amplification and protein expression in W349A-transfected cells. Although release of infectious virus was not detected, release of capsidless subviral particles was not blocked. The assembly defect in W349A could be trans-complemented inefficiently using BHK-REP cells (a cell line containing persistently replicating YFV replicon RNA). trans-complementation was also demonstrated by supplying wild-type NS2B-3 or NS3 protein alone as well as by supplying inactive NS2B-3 protein, indicating that this function of NS3 in virus assembly was independent of its known enzymatic functions.     

Dynamic flux balance analysis of batch fermentation: effect of genetic manipulations on ethanol production

In silico optimization of bioethanol production from lignocellulosic biomasses is investigated by combining process systems engineering approach and systems biology approach. Lignocellulosic biomass is an attractive sustainable carbon source for fermentative production of bioethanol. For enhanced ethanol production, metabolic engineering of wild-type strains—that can metabolize both hexose and pentose sugars or microbial consortia consisting of substrate-selective microbes—may be advantageous. This study presents a detailed in silico analysis of bioethanol production from glucose-xylose mixtures of various compositions by batch mono-culture and co-culture fermentation of specialized microbes. Dynamic flux balance models based on available genome-scale reconstructions of the microorganisms have been used to analyze bioethanol production, and the maximization of ethanol productivity is addressed by computing optimal aerobic–anaerobic switching times. Effects of ten metabolic engineering strategies that have been suggested in the literature for ethanol overproduction, have been evaluated for their efficiency in enhancing the ethanol productivity in the context of batch mono-culture and co-culture processes.     

Identification and quantitation of vesivirus 2117 particles in bioreactor fluids from infected Chinese hamster ovary cell cultures

The prevention of adventitious agent contamination is a top priority throughout the entire biopharmaceutical production process. For example, although viral contamination of cell banks or cell cultures is rare, it can result in serious consequences (e.g., shutdown and decontamination of manufacturing facilities). To ensure virus free production, numerous in vivo and in vitro adventitious agent assays and biophysical characterizations such as electron microscopy are conducted on cell banks, raw materials, process materials, and drug substances throughout the manufacturing process. Molecular assays such as PCR and other nucleotide‐based techniques are also routinely used for screening and identification of any viral agents. However, modern techniques in protein identification of complex protein mixtures have not yet been effectively integrated throughout the industry into current viral testing strategies. Here, we report the identification and quantitation of Vesivirus 2117 particles in bioreactor fluid from infected Chinese hamster ovary cell cultures by global protein sequencing using mass spectrometry in combination with multi‐dimensional liquid‐chromatography. Following mass spectrometric data acquisition and rigorous data analysis, six virus specific peptides were identified. These peptides were fragments of two structural proteins, capsid protein pre‐cursor (four unique peptides) and small structural protein (two unique peptides), from the same species: Vesivirus 2117. Using stable heavy isotope‐labeled peptides as internal standards, we also determined the absolute concentration of Vesivirus particles in the bioreactor fluid and the ratio of two capsid proteins (VP1:VP2) in the particles as approximately 9:1. The positive identification of Vesivirus 2117 was subsequently confirmed by RT‐PCR. Biotechnol. Bioeng. 2013; 110: 1342–1353. © 2012 Wiley Periodicals, Inc.     

Suitability of a generic virus safety evaluation for monoclonal antibody investigational new drug applications

Biologics produced from CHO cell lines with endogenous virus DNA can produce retrovirus-like particles in cell culture at high titers, and other adventitious viruses can find their way through raw materials into the process to make a product. Therefore, it is the industry standard to have controls to avoid introduction of viruses into the production process, to test for the presence of viral particles in unclarified cell culture, and to develop purification procedures to ensure that manufacturing processes are robust for viral clearance. Data have been accumulated over the past four decades on unit operations that can inactivate and clear adventitious virus and provide a high degree of assurance for patient safety. During clinical development, biological products are traditionally tested at process set points for viral clearance. However, the widespread implementation of platform production processes to produce highly similar IgG antibodies for many indications makes it possible to leverage historical data and knowledge from representative molecules to allow for better understanding and control of virus safety. More recently, individualized viral clearance studies are becoming the rate-limiting step in getting new antibody molecules to clinic, particularly in Phase 0 and eIND situations. Here, we explore considerations for application of a generic platform virus clearance strategy that can be applied for relevant investigational antibodies within defined operational parameters in order to increase speed to the clinic and reduce validation costs while providing a better understanding and assurance of process virus safety.     

Structural basis of the herpesvirus M3-chemokine interaction

Viruses have been fighting the immune systems of their hosts for millions of years and have evolved evasion strategies to ensure their survival. Viruses can teach us efficient mechanisms to control the immune system, and this information can be used to design new strategies of immune modulation that we might apply to diminish immunopathological responses that cause human diseases. Large DNA viruses, such as poxviruses and herpesviruses, encode proteins that are secreted from infected cells, bind cytokines and neutralize their activity. A subgroup of these viral proteins binds chemokines, a complex family of cytokines that control the recruitment of cells to sites of infection and inflammation. One of the major unresolved questions in the field was to understand how these viral secreted proteins bind chemokines with high affinity, despite having no amino acid sequence similarity to the host chemokine receptors, which are seven-transmembrane-domain proteins that cannot be engineered as soluble proteins.     

The effect of delay in viral production in within-host models during early infection

ABSTRACT

Delay in viral production may have a significant impact on the early stages of infection. During the eclipse phase, the time from viral entry until active production of viral particles, no viruses are produced. This delay affects the probability that a viral infection becomes established and timing of the peak viral load. Deterministic and stochastic models are formulated with either multiple latent stages or a fixed delay for the eclipse phase. The deterministic model with multiple latent stages approaches in the limit the model with a fixed delay as the number of stages approaches infinity. The deterministic model framework is used to formulate continuous-time Markov chain and stochastic differential equation models. The probability of a minor infection with rapid viral clearance as opposed to a major full-blown infection with a high viral load is estimated from a branching process approximation of the Markov chain model and the results are confirmed through numerical simulations. In addition, parameter values for influenza A are used to numerically estimate the time to peak viral infection and peak viral load for the deterministic and stochastic models. Although the average length of the eclipse phase is the same in each of the models, as the number of latent stages increases, the numerical results show that the time to viral peak and the peak viral load increase.     

Strategy for minimization of excess sludge production from the activated sludge process

Increased attention has been given to minimization of sludge production from activated sludge process since environmental regulations are being more and more stringent in relation to excess sludge disposal. In a biological process, the more organic carbon utilized in carbon dioxide production, the fewer sludge produced, and vice versa. This paper, therefore, reviews strategies developed for minimization of excess sludge production, such as oxic-settling-anaerobic process, high dissolved oxygen process, uncoupler-containing activated sludge process, ozonation-combined activated sludge process, control of sludge retention time and biodegradation of sludge in membrane-assisted reactor. In these modified activated sludge processes, excess sludge production can be reduced by 20-100% without significant effect on process efficiency and stability. It is expected that this paper would be helpful for researchers and engineers to develop novel and efficient operation strategy to minimize sludge production from biological systems.     

Persistence of viruses and DNA in soil

With growing environmental concerns over the use of chemical pesticides for insect control in both agriculture and forestry, increased emphasis is being placed on the development of alternative, biological pesticides such as genetically modified baculoviruses. Before the large-scale use of genetically modified viruses (GMV) can be realized, fate of GMV and their DNA in soil should be investigated. There are a number of factors that have the potential to affect persistence of both wild-type and genetically modified viruses and their DNA in soil. In this mini-review, the persistence of viral particles and DNA in soil is examined with particular emphasis on baculoviruses.