Tools for quantitative analysis: The Al Yergey perspective.

The development of a reliable quantitative method for a specific application requires consideration of several important principles. Although mass spectrometry has become the "gold standard" for sensitive and precise multicomponent quantification, it does not necessarily follow that all quantitative data generated by mass spectrometry are either precise or accurate. Depending on the nature of the sample and the goal of the assay, the endeavor can be challenging. With attention to some critical concerns, valid results can be obtained; without attention to these concerns, results can be misleading and, in some instances, invalid. For almost 10 years, beginning in 1996, the authors of this article, together with their friend and colleague Al Yergey, taught an American Society for Mass Spectrometry (ASMS) Short Course on the principles of quantitative mass spectrometry. In this Special Feature, we revisit and update some of these principles. We pay special attention to the contributions of Al Yergey to the course that not only enriched our own understanding of quantitative analysis but also had a measurable impact on the entire field of mass spectrometry.

Risk assessment for recrudescence of avian influenza in caged layer houses following depopulation: the effect of cleansing, disinfection and dismantling of equipment.

Following an outbreak of highly pathogenic avian influenza virus (HPAIV) in a poultry house, control measures are put in place to prevent further spread. An essential part of the control measures based on the European Commission Avian Influenza Directive 2005/94/EC is the cleansing and disinfection (C&D) of infected premises. Cleansing and disinfection includes both preliminary and secondary C&D, and the dismantling of complex equipment during secondary C&D is also required, which is costly to the owner and also delays the secondary cleansing process, hence increasing the risk for onward spread. In this study, a quantitative risk assessment is presented to assess the risk of re-infection (recrudescence) occurring in an enriched colony-caged layer poultry house on restocking with chickens after different C&D scenarios. The risk is expressed as the number of restocked poultry houses expected before recrudescence occurs. Three C&D scenarios were considered, namely (i) preliminary C&D alone, (ii) preliminary C&D plus secondary C&D without dismantling and (iii) preliminary C&D plus secondary C&D with dismantling. The source-pathway-receptor framework was used to construct the model, and parameterisation was based on the three C&D scenarios. Two key operational variables in the model are (i) the time between depopulation of infected birds and restocking with new birds (TbDR) and (ii) the proportion of infected material that bypasses C&D, enabling virus to survive the process. Probability distributions were used to describe these two parameters for which there was recognised variability between premises in TbDR or uncertainty due to lack of information in the fraction of bypass. The risk assessment estimates that the median (95% credible intervals) number of repopulated poultry houses before recrudescence are 1.2 × 104 (50 to 2.8 × 106), 1.9 × 105 (780 to 5.7 × 107) and 1.1 × 106 (4.2 × 103 to 2.9 × 108) under C&D scenarios (i), (ii) and (iii), respectively. Thus for HPAIV in caged layers, undertaking secondary C&D without dismantling reduces the risk by 16-fold compared to preliminary C&D alone. Dismantling has an additional, although smaller, impact, reducing the risk by a further 6-fold and thus around 90-fold compared to preliminary C&D alone. On the basis of the 95% credible intervals, the model demonstrates the importance of secondary C&D (with or without dismantling) over preliminary C&D alone. However, the extra protection afforded by dismantling may not be cost beneficial in the context of reduced risk of onward spread.

A qualitative risk assessment of cleansing and disinfection requirements after an avian influenza outbreak in commercial poultry.

1. During an avian influenza (AI) outbreak in the United Kingdom, the joint aim of the poultry industry and the Government is to eliminate and prevent the spread of infection, through control measures based on the current European Union (EU) Council Directive (2005/94/EC). An essential part of these measures is the cleansing and disinfection (C&D) of infected premises.2. This risk assessment assessed the differences in re-infection in a repopulated flock if the EU Directive is interpreted to permit secondary C&D to be undertaken either with or without dismantling complex equipment. The assessment estimated the probability of virus survival on different types of equipment in a depopulated contaminated poultry house before and after preliminary and secondary C&D procedures. A risk matrix spreadsheet tool was used to carry out the assessment and concluded that, provided secondary C&D is carried out with due diligence (i.e. carried out to a defined code of practice as agreed by both industry and policymakers), the risk of re-infection from equipment is negligible, both with and without dismantling complex equipment in all farm types considered.3. By considering the equipment types individually, the assessment identified those areas of the house which may still contain viable virus post-preliminary C&D and on which attention should be focussed during secondary C&D. The generic risk pathway and matrix spreadsheet tool have the potential to be used for other pathogens and species, given appropriate data.

Livestock Health and Food Chain Risk Assessment.

The EUFORA fellowship programme 'Livestock Health and Food Chain Risk Assessment' was proposed by the Animal and Plant Health Agency (APHA), a British governmental institution responsible for safeguarding animal and plant health in the UK. The working programme, which was organised into four different modules, covered a wide range of aspects related to risk assessment including identification of emerging risks, risk prioritisation methods, scanning surveillance, food production exposure assessment and import risk assessment of animal and human infectious diseases. Over the course of the year, the Fellow had the opportunity to work for international projects with experts in these disciplines. This allowed for significant opportunities to 'learn-by-doing' the methods and the techniques that are employed to assess animal health and food safety risks. Moreover, he consolidated his knowledge by attending several training courses and academic lessons, submitting scientific papers to peer-reviewed journals and conferences, giving presentations and using modelling software.

Could Bat Cell Temperature and Filovirus Filament Length Explain the Emergence of Ebola Virus in Mammals? Predictions of a Thermodynamic Model.

The host reservoir of Zaire ebolavirus (EBOV) remains elusive. One suggestion is that EBOV emerges in mammals when the precursor virus jumps from mayflies (or other riverine insects) to insectivorous bats. However, this does not fit with the current view that filoviruses cannot infect arthropods. Here, it is first argued that the evidence that arthropods are refractory is not definitive. Second, it is proposed that a combination of filovirus filament length and the high temperature (~42°C) experienced by an insect virus ingested by a flying bat, together with the large number of insects eaten by bats (e.g. during an ephemeral mass emergence of mayflies), facilitate jumping the species barrier. The length of a filovirus filament is related to the number of genome copies (GC). Predictions from a preliminary thermodynamic model developed here suggest that filament length could greatly affect EBOV infectivity to mammalian cells with infectivity peaking for filaments of a certain length. Importantly, the infectivity to mammals of even short filaments may be more than one million-fold higher than that for the single GC virion. Third, it is proposed that at the high temperature within the bat, the phospholipid phosphatidylserine in the virus envelope promotes filament formation through fusion of single GC particles within the ingested insect, thus hugely increasing their infectivity to bats. Forth, according to the thermodynamic model, increasing the temperature from 27°C (insect cell temperature at average air temperature in Guinea, West Africa) to 42°C (bat) could increase the affinity of the filaments for bat cells by 1-2 orders of magnitude, while having no effect on the binding affinity of the single GC virions. The thermodynamic model developed here is supported by the counterintuitive observation that high glycoprotein densities on the EBOV surface reduce its infectivity in contrast to other viruses such as HIV.

The challenge of using experimental infectivity data in risk assessment for Ebola virus: why ecology may be important.

Analysis of published data shows that experimental passaging of Zaire ebolavirus (EBOV) in guinea pigs changes the risk of infection per plaque-forming unit (PFU), increasing infectivity to some species while decreasing infectivity to others. Thus, a PFU of monkey-adapted EBOV is 10(7) -fold more lethal to mice than a PFU adapted to guinea pigs. The first conclusion is that the infectivity of EBOV to humans may depend on the identity of the donor species itself and, on the basis of limited epidemiological data, the question is raised as to whether bat-adapted EBOV is less infectious to humans than nonhuman primate (NHP)-adapted EBOV. Wildlife species such as bats, duikers and NHPs are naturally infected by EBOV through different species giving rise to EBOV with different wildlife species-passage histories (heritages). Based on the ecology of these wildlife species, three broad 'types' of EBOV-infected bushmeat are postulated reflecting differences in the number of passages within a given species, and hence the degree of adaptation of the EBOV present. The second conclusion is that the prior species-transmission chain may affect the infectivity to humans per PFU for EBOV from individuals of the same species. This is supported by the finding that the related Marburg marburgvirus requires ten passages in mice to fully adapt. It is even possible that the evolutionary trajectory of EBOV could vary in individuals of the same species giving rise to variants which are more or less virulent to humans and that the probability of a given trajectory is related to the heritage. Overall the ecology of the donor species (e.g. dog or bushmeat species) at the level of the individual animal itself may determine the risk of infection per PFU to humans reflecting the heritage of the virus and may contribute to the sporadic nature of EBOV outbreaks.

Q fever through consumption of unpasteurised milk and milk products - a risk profile and exposure assessment.

Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii which is endemic in cattle, sheep and goats in much of the world, including the United Kingdom (UK). There is some epidemiological evidence that a small proportion of cases in the developed world may arise from consumption of unpasteurised milk with less evidence for milk products such as cheese. Long maturation at low pH may give some inactivation in hard cheese, and viable C. burnetii are rarely detected in unpasteurised cheese compared to unpasteurised milk. Simulations presented here predict that the probability of exposure per person to one or more C. burnetii through the daily cumulative consumption of raw milk in the UK is 0·4203. For those positive exposures, the average level of exposure predicted is high at 1266 guinea pig intraperitoneal infectious dose 50% units (GP_IP_ID50 ) per person per day. However, in the absence of human dose-response data, the case is made that the GP_IP_ID50 unit represents a very low risk through the oral route. The available evidence suggests that the risks from C. burnetii through consumption of unpasteurised milk and milk products (including cheese) are not negligible but they are lower in comparison to transmission via inhalation of aerosols from parturient products and livestock contact.

Pathways for entry of livestock arboviruses into Great Britain: assessing the strength of evidence.

The emergence of bluetongue virus and Schmallenberg virus in Great Britain (GB) during the last decade has highlighted the need for understanding the relative importance of the various pathways of the entry of livestock arboviruses so as to help focus surveillance and mitigation. This study summarizes what is known for the main routes of entry and assesses the strength of the current evidence for and against. Entry through infected arthropod vectors is considered at the level of each life cycle stage for tick-, biting midge- and mosquito-borne viruses, and while there is evidence that this could happen through most tick and mosquito stages, strong evidence that only exists for entry through adult midges. There is also strong evidence that entry through immature midge stages could not happen. The weight of supporting evidence is strongest for importation of viraemic livestock including horses. While there is some indication of a common pathway for midge-borne viruses from sub-Saharan Africa to GB via Continental Europe, other factors such as maternal transmission in dogs and sheep need to be considered in the light of recent findings.

Applications of omics approaches to the development of microbiological risk assessment using RNA virus dose-response models as a case study.

The last decade has seen a huge increase in the amount of 'omics' data available and in our ability to interpret those data. The aim of this paper was to consider how omics techniques can be used to improve and refine microbiological risk assessment, using dose-response models for RNA viruses, with particular reference to norovirus through the oral route as the case study. The dose-response model for initial infection in the gastrointestinal tract is broken down into the component steps at the molecular level and the feasibility of assigning probabilities to each step assessed. The molecular mechanisms are not sufficiently well understood at present to enable quantitative estimation of probabilities on the basis of omics data. At present, the great strength of gene sequence data appears to be in giving information on the distribution and proportion of susceptible genotypes (for example due to the presence of the appropriate pathogen-binding receptor) in the host population rather than in predicting specificities from the amino acid sequences concurrently obtained. The nature of the mutant spectrum in RNA viruses greatly complicates the application of omics approaches to the development of mechanistic dose-response models and prevents prediction of risks of disease progression (given infection has occurred) at the level of the individual host. However, molecular markers in the host and virus may enable more broad predictions to be made about the consequences of exposure in a population. In an alternative approach, comparing the results of deep sequencing of RNA viruses in the faeces/vomitus from donor humans with those from their infected recipients may enable direct estimates of the average probability of infection per virion to be made.

Entry of H5N1 highly pathogenic avian influenza virus into Europe through migratory wild birds: a qualitative release assessment at the species level.

AIMS: To estimate qualitatively the probabilities of release (or entry) of Eurasian lineage H5N1 highly pathogenic avian influenza (HPAI) virus into Great Britain (GB), the Netherlands and Italy through selected higher risk species of migratory water bird. METHODS AND RESULTS: The probabilities of one or more release events of H5N1 HPAI per year (Pre(lease)) were estimated qualitatively for 15 avian species, including swans, geese, ducks and gulls, by assessing the prevalence of H5N1 HPAI in different regions of the world (weighted to 2009) and estimates of the total numbers of birds migrating from each of those regions. The release assessment accommodated the migration times for each species in relation to the probabilities of their surviving infection and shedding virus on arrival. Although the predicted probabilities of release of H5N1 per individual bird per year were low, very low or negligible, Pre(lease) was high for a few species reflecting the high numbers of birds migrating from some regions. Values of Pre(lease) were generally higher for the Netherlands than for GB, while ducks and gulls from Africa presented higher probabilities to Italy compared to the Netherlands and GB. CONCLUSIONS: Bird species with high values of Pre(lease) in GB, the Netherlands and Italy generally originate from within Europe based on data for global prevalence of H5N1 between 2003 and 2009 weighted to 2009. Potential long-distance transfer of H5N1 HPAI from North Asia and Eurasia to GB, the Netherlands and Italy is limited to a few species and does not occur from South-East Asia, an area where H5N1 is endemic. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach accommodates biogeographical conditions and variability in the estimated worldwide prevalence of the virus. The outputs of this release assessment can be used to inform surveillance activities through focusing on certain species and migratory pathways.

Epidemic threats to the European Union: expert views on six virus groups.

In recent years, several animal disease epidemics have occurred within the European Union (EU). At the 4th Annual Meeting of the EPIZONE network (7-10 June 2010, St. Malo, France), an interactive session was run to elicit the opinions of delegates on a pre-defined list of epidemic threats to the EU. Responses from over 190 delegates, to questions relating to impact and likelihood, were used to rank six virus groups with respect to their perceived threat now (2010) and in 2020. The combined opinions of all delegates suggested that, from the pre-selected list of virus groups, foot-and-mouth disease and influenza are currently of most concern. Delegates thought that influenza would be less of a threat and zoonotic arboviruses would be more of a threat in 2020. Although the virus group rankings should not be taken as definitive, the results could be used in conjunction with experimental and field data, by scientists, policy-makers and stakeholders when assessing and managing risks associated with these virus groups.

Horizon scanning for emergence of new viruses: from constructing complex scenarios to online games.

Horizon scanning techniques can be developed to identify novel routes and sources for the emergence of viruses in the medium to long term. Central to horizon scanning is prediction of the complex scenarios through which viruses could emerge before they occur. One approach involves 'spidergrams' in which complex scenarios are generated by combining factors randomly selected from different categories of events. Spidergrams provide a framework for how different factors could interact, irrespective of the virus, and also enable testing of combinations not previously considered but which would be 'tested' in nature by a virus. The emergence of viruses through new routes is often related to changes, for example, in environmental and social factors, and the Internet will undoubtedly be used to identify long-term trends for consideration. In addition, online games may provide horizon scanners with suggestions for new routes and strategies that could be used by emerging viruses.

Impact of climate change on risk of incursion of Crimean-Congo haemorrhagic fever virus in livestock in Europe through migratory birds.

AIMS: To predict the risk of incursion of Crimean-Congo haemorrhagic fever virus (CCHFV) in livestock in Europe introduced through immature Hyalomma marginatum ticks on migratory birds under current conditions and in the decade 2075-2084 under a climate-change scenario. METHODS AND RESULTS: A spatial risk map of Europe comprising 14 282 grid cells (25 × 25 km) was constructed using three data sources: (i) ranges and abundances of four species of bird which migrate from sub-Saharan Africa to Europe each spring, namely Willow warbler (Phylloscopus trochilus), Northern wheatear (Oenanthe oenanthe), Tree pipit (Anthus trivialis) and Common quail (Coturnix coturnix); (ii) UK Met Office HadRM3 spring temperatures for prediction of moulting success of immature H. marginatum ticks and (iii) livestock densities. On average, the number of grid cells in Europe predicted to have at least one CCHFV incursion in livestock in spring was 1·04 per year for the decade 2005-2014 and 1·03 per year for the decade 2075-2084. In general with the assumed climate-change scenario, the risk increased in northern Europe but decreased in central and southern Europe, although there is considerable local variation in the trends. CONCLUSIONS: The absolute risk of incursion of CCHFV in livestock through ticks introduced by four abundant species of migratory bird (totalling 120 million individual birds) is very low. Climate change has opposing effects, increasing the success of the moult of the nymphal ticks into adults but decreasing the projected abundance of birds by 34% in this model. SIGNIFICANCE AND IMPACT OF THE STUDY: For Europe, climate change is not predicted to increase the overall risk of incursion of CCHFV in livestock through infected ticks introduced by these four migratory bird species.

Assessing the impact of climate change on disease emergence in freshwater fish in the United Kingdom.

A risk framework has been developed to examine the influence of climate change on disease emergence in the United Kingdom. The fish immune response and the replication of pathogens are often correlated with water temperature, which manifest as temperature ranges for infection and clinical diseases. These data are reviewed for the major endemic and exotic disease threats to freshwater fish. Increasing water temperatures will shift the balance in favour of either the host or pathogen, changing the frequency and distribution of disease. A number of endemic diseases of salmonids (e.g. enteric red mouth, furunculosis, proliferative kidney disease and white spot) will become more prevalent and difficult to control as water temperatures increase. Outbreaks of koi herpesvirus in carp fisheries are likely to occur over a longer period each summer. Climate change also alters the threat level associated with exotic pathogens. The risk of viral haemorrhagic septicaemia (VHSV), infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) declines as infection generally only establishes when water temperatures are less than 14°C for VHSV and IHNV and 17°C for SCVC. The risk of establishment of other exotic pathogens (epizootic haematopoietic necrosis and epizootic ulcerative syndrome) increases. The spread of Lactococcus garvieae northwards in Europe is likely to continue, and thus is more likely to be both introduced and become established. Measures to reduce the threat of exotic pathogens need to be revised to account for the changing exotic diseases threat. Increasing water temperatures and the negative effects of extreme weather events (e.g. storms) are likely to alter the freshwater environment adversely for both wild and farmed salmonid populations, increasing their susceptibility to disease and the likelihood of disease emergence. For wild populations, surveillance and risk mitigation need to be focused on locations where disease emergence, as a result of climate change, is most likely.

Assessing the impact of climate change on vector-borne viruses in the EU through the elicitation of expert opinion.

Expert opinion was elicited to undertake a qualitative risk assessment to estimate the current and future risks to the European Union (EU) from five vector-borne viruses listed by the World Organization for Animal Health. It was predicted that climate change will increase the risk of incursions of African horse sickness virus (AHSV), Crimean-Congo haemorrhagic fever virus (CCHFV) and Rift Valley fever virus (RVFV) into the EU from other parts of the world, with African swine fever virus (ASFV) and West Nile virus (WNV) being less affected. Currently the predicted risks of incursion were lowest for RVFV and highest for ASFV. Risks of incursion were considered for six routes of entry (namely vectors, livestock, meat products, wildlife, pets and people). Climate change was predicted to increase the risk of incursion from entry of vectors for all five viruses to some degree, the strongest effects being predicted for AHSV, CCHFV and WNV. This work will facilitate identification of appropriate risk management options in relation to adaptations to climate change.