Estimating numbers of infectious units from serial dilution assays

The paper concerns the design and analysis of serial dilution assays to estimate the infectivity of a sample of tissue when it is assumed that the sample contains a finite number of indivisible infectious units such that a subsample will be infectious if it contains one or more of these units. The aim of the study is to estimate the number of infectious units in the original sample. The standard approach to the analysis of data from such a study is based on the assumption of independence of aliquots both at the same dilution level and at different dilution levels, so that the numbers of infectious units in the aliquots follow independent Poisson distributions. An alternative approach is based on calculation of the expected value of the total number of samples tested that are not infectious. We derive the likelihood for the data on the basis of the discrete number of infectious units, enabling calculation of the maximum likelihood estimate and likelihood-based confidence intervals. We use the exact probabilities that are obtained to compare the maximum likelihood estimate with those given by the other methods in terms of bias and standard error and to compare the coverage of the confidence intervals. We show that the methods have very similar properties and conclude that for practical use the method that is based on the Poisson assumption is to be recommended, since it can be implemented by using standard statistical software. Finally we consider the design of serial dilution assays, concluding that it is important that neither the dilution factor nor the number of samples that remain untested should be too large.

Display of a Maize cDNA library on baculovirus infected insect cells

Background: Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica ( AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. Results: We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABPI), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. Conclusion: The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication

In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G(2)/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G(2) regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G(2)/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the Go phase or asynchronously replicating cells. Our data suggested that IBV induces a G(2)/M phase arrest in infected cells to promote favorable conditions for viral replication.

Mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in RNA binding by using surface plasmon resonance

Phosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphospborylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5' end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.

Evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus

As an immunogen of the coronavirus, the nucleoprotein (N) is a potential antigen for the serological monitoring of infectious bronchitis virus (IBV). In this report, recombinant N protein from the Beaudette strain of IBV was produced and purified from Escherichia coli as well as Sf9 ( insect) cells, and used for the coating of enzyme-linked immunosorbent assay ( ELISA) plates. The N protein produced in Sf9 cells was phosphorylated whereas N protein from E. coli was not. Our data indicated that N protein purified from E. coli was more sensitive to anti-IBV serum than the protein from Sf9 cells. The recombinant N protein did not react with the antisera to other avian pathogens, implying that it was specific in the recognition of IBV antibodies. In addition, the data from the detection of field samples and IBV strains indicated that using the recombinant protein as coating antigen could achieve an equivalent performance to an ELISA kit based on infected material extracts as a source of antigen(s). ELISAs based on recombinant proteins are safe ( no live virus), clean ( only virus antigens are present), specific ( single proteins can be used) and rapid ( to respond to new viral strains and strains that cannot necessarily be easily cultured).

Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus

The nucleolus is a dynamic subnuclear structure involved in ribosome subunit biogenesis, cell cycle control and mediating responses to cell stress, among other functions. While many different viruses target proteins to the nucleolus and recruit nucleolar proteins to facilitate virus replication, the effect of infection on the nucleolus in terms of morphology and protein content is unknown. Previously we have shown that the coronavirus nucleocapsid protein will localize to the nucleolus. In this study, using the avian infectious bronchitis coronavirus, we have shown that virus infection results in a number of changes to the nucleolus both in terms of gross morphology and protein content. Using confocal microscopy coupled with fluorescent labelled nucleolar marker proteins we observed changes in the morphology of the nucleolus including an enlarged fibrillar centre. We found that the tumour suppressor protein, p53, which localizes normally to the nucleus and nucleolus, was redistributed predominately to the cytoplasm.

The coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus

The coronavirus nucleoprotein (N) has been reported to be involved in various aspects of virus replication. We examined by confocal microscopy the subcellular localization of the avian infectious bronchitis virus N protein both in the absence and in the context of an infected cell and found that N protein localizes both to the cytoplasmic and nucleolar compartments.

Amphioxus type I keratin cDNA and the evolution of intermediate filament genes

We report the cloning of an intermediate filament (IF) cDNA from the cephalochordate amphioxus that encodes a protein assignable to the type I keratin group. This is the first type I keratin reported from an invertebrate. Molecular phylogenetic analyses reveal that amphioxus also possesses a type II keratin, and that the genes encoding short-rod IF proteins underwent different patterns of duplication in vertebrates and their closest relatives, the cephalochordates. Extensive IF gene duplication and divergence may have facilitated the origin of new specialised cell types in vertebrates.

Livestock infectious disease and climate change: a review of selected literature

It is indisputable that climate is an important factor in many livestock diseases. Nevertheless, our knowledge of the impact of climate change on livestock infectious diseases is much less certain.Therefore, the aim of the article is to conduct a systematic review of the literature on the topic utilizing available retrospective data and information. Across a corpus of 175 formal publications,limited empirical evidence was offered to underpin many of the main arguments. The literature reviewed was highly polarized and often inconsistent regarding what the future may hold. Historical explorations were rare. However, identifying past drivers to livestock disease may not fully capture the extent that new and unknown drivers will influence future change. As such, our current predictive capacity is low. We offer a number of recommendations to strengthen this capacity in the coming years. We conclude that our current approach to research on the topic is limiting and unlikely to yield sufficient, actionable evidence to inform future praxis. Therefore, we argue for the creation of a reflexive, knowledge-based system, underpinned by a collective intelligence framework to support the drawing of inferences across the literature.

A complex system perspective on the emergence and spread of infectious diseases: integrating economic and ecological aspects

The emergence and spread of infectious diseases reflects the interaction of ecological and economic factors within an adaptive complex system. We review studies that address the role of economic factors in the emergence and spread of infectious diseases and identify three broad themes. First, the process of macro-economic growth leads to environmental encroaching, which is related to the emergence of infectious diseases. Second, there are a number of mutually reinforcing processes associated with the emergence/spread of infectious diseases. For example, the emergence and spread of infectious diseases can cause significant economic damages, which in turn may create the conditions for further disease spread. Also, the existence of a mutually reinforcing relationship between global trade and macroeconomic growth amplifies the emergence/spread of infectious diseases. Third, microeconomic approaches to infectious disease point to the adaptivity of human behavior, which simultaneously shapes the course of epidemics and responds to it. Most of the applied research has been focused on the first two aspects, and to a lesser extent on the third aspect. With respect to the latter, there is a lack of empirical research aimed at characterizing the behavioral component following a disease outbreak. Future research should seek to fill this gap and develop hierarchical econometric models capable of integrating both macro and micro-economic processes into disease ecology.

How the double spherules of infectious bronchitis virus impact our understanding of RNA virus replicative organelles

Powered by advances in electron tomography, recent studies have extended our understanding of how viruses construct "replication factories" inside infected cells. Their function, however, remains an area of speculation with important implications for human health. It is clear from these studies that whatever their purpose, organelle structure is dynamic (M. Ulasli, M. H. Verheije, C. A. de Haan, and F. Reggiori, Cell. Microbiol. 12:844-861, 2010) and intricate (K. Knoops, M. Kikkert, S. H. Worm, J. C. Zevenhoven-Dobbe, Y. van der Meer, et al., PLOS Biol. 6:e226, 2008). But by concentrating on medically important viruses, these studies have failed to take advantage of the genetic variation inherent in a family of viruses that is as diverse as the archaea, bacteria, and eukaryotes combined (C. Lauber, J. J. Goeman, M. del Carmen Parquet, P. T. Nga, E. J. Snijder, et al., PLOS Pathog. 9:e1003500, 2013). In this climate, Maier et al. (H. J. Maier, P. C. Hawes, E. M. Cottam, J. Mantell, P. Verkade, et al., mBio 4:e00801-13, 2013) explored the replicative structures formed by an avian coronavirus that appears to have diverged at an early point in coronavirus evolution and shed light on controversial aspects of viral biology.

Animal infectious disease and climate change: a selected review

It is indisputable that climate is an important factor in many livestock diseases. Nevertheless, our knowledge of the impact of climate change on livestock infectious diseases is much less certain. Therefore, the aim of the article is to conduct a systematic review of the literature on the topic utilizing available retrospective data and information. Across a corpus of 175 formal publications, limited empirical evidence was offered to underpin many of the main arguments. The literature reviewed was highly polarized and often inconsistent regarding what the future may hold. Historical explorations were rare. However, identifying past drivers to livestock disease may not fully capture the extent that new and unknown drivers will influence future change. As such, our current predictive capacity is low. We offer a number of recommendations to strengthen this capacity in the coming years. We conclude that our current approach to research on the topic is limiting and unlikely to yield sufficient, actionable evidence to inform future praxis. Therefore, we argue for the creation of a reflexive, knowledge-based system, underpinned by a collective intelligence framework to support the drawing of inferences across the literature.

Climate change and infectious disease: time for a new normal?

At present, there is a clarion call for action on climate change across the global health landscape. At the recent WHO-sponsored conference on health and climate (held in Geneva, Switzerland, on Aug 27–29, 2014) and the UN Climate Summit (New York, USA, on Sept 23, 2014), participants were encouraged to act decisively to change the current trajectory of climate disruption. Health inequalities, including those related to infectious diseases, have now been pushed to centre stage. This approach represents a step-change in thinking. But as we are urged toward collective action, is it time to rethink our approach to research, especially in relation to climate change and infectious disease?