Several methods exist to cost hospital contacts when estimating the cost effectiveness of a new intervention. However, the implications of choosing a particular approach remain unclear. We compare the use of the three main diagnosis-related group (DRG)-based national unit costs in England to determine whether choice of approach can impact on economic evaluation results.A cost-utility model was developed to compare secondary fracture prevention models of care for hip fracture patients, using data from large primary and hospital care administrative datasets in England. A healthcare and personal social services payer perspective was adopted, and utilities were informed by a meta-regression. Hospital resource use was valued using three DRG-based unit costs, and regression-based costing models were developed using data from 13,906 patients to inform the model health states.Finished consultant episode (FCE)-level reference costs resulted in the highest costs on admission (£9075) and in the year of the fracture . Relative to FCE-level costs, spell-level tariffs led to the lowest total hospital care costs per patient within 1 year of fracture (− £3691) compared with spell-level reference costs (− £2106). At a £20,000/quality-adjusted life-year threshold, using spell-level reference costs or spell-level tariffs, the introduction of a nurse-led fracture liaison service model of care was the cost-effective alternative. However, using FCE-level reference costs, usual care was the cost-effective option.Our results show that, conditional on the set of national unit costs adopted, the costs of hip fracture may vary considerably and different decisions may be reached regarding the introduction of new healthcare interventions.The online version of this article (10.1007/s40273-018-0673-y) contains supplementary material, which is available to authorized users. There are several methods to cost hospital contacts when estimating the cost effectiveness of a new intervention. These can range from local micro-costing approaches to the use of diagnosis-related group (DRG)-based costs, which group patients according to their diagnosis and procedure codes as recorded in healthcare administrative records. In England, DRGs are called healthcare resource groups (HRGs) and the National Institute for Health and Care Excellence (NICE) recommends their use to cost hospital resource utilisation and inform economic evaluations . HoweverA hospital spell, or hospital admission, comprises the total continuous stay of a patient using a hospital bed in the same hospital. During a hospital spell, a patient may receive medical care from one or more consultants. Time spent in the care of one consultant is called an FCE, and a hospital spell may contain one or more FCEs. Since 1997-98, reference cost data has been collected in England for all public-funded healthcare services (i.e. the NHS). Reference costs represent the cost of providing one unit of care in a given financial year and allow comparisons across hospital providers at the level of diagnosis, treatment and procedures , 3. RefeTariffs for admitted patient activity are paid at spell level not FCE level as the Department of Health in England considers spells to be a more robust measure of hospital activity than FCEs. Spell-level costs were first collected in 2011–2012 alongside FCE-level costs . These sThe analyst is therefore faced with three potential sources of unit costs for a given financial year to apply in an economic evaluation or cost-of-illness study. The implications of choosing a particular source of HRG-based unit costs when conducting costing studies and economic evaluations remain unclear. For example, the costs of a disease may be considerably underestimated or overestimated depending on which source of unit costs is used. Also, an intervention may be judged to be cost effective at a given willingness-to-pay threshold when a particular set of unit costs is used but not another.We aim to address this gap in knowledge using hip fractures as a case study. Hip fractures are a major public health problem with significant patient morbidity and mortality and were estimated to cost £2–3 billion annually in health and social care costs in the UK , 6. We eWe developed a cohort transition model (Markov model) to estimate the lifetime costs, quality-adjusted life-years (QALYs) and cost effectiveness of three models of care for patients with a hip fracture admitted to an NHS hospital in England: (1) introduction of an orthogeriatrician (OG)-led service which focuses on achieving optimal recovery after hip fracture; (2) introduction of a nurse-led fracture liaison service (FLS) which focuses on secondary fracture prevention; and (3) standard post-hip fracture care (without the introduction and/or expansion of the OG and FLS models of care).®  to simulate the natural history, quality of life and costs of individuals with an index hip fracture across health states representing history of index hip fracture, second hip fracture, major non-hip fracture(s)  requiring hospitalisation, living in patient’s own home or in a care home, and death (within 30 days post-hip fracture or within a year) . We used an iterative process to define the model involving discussions with clinical experts and epidemiologists, supplemented by a literature review of economic models in the area. An annual cycle length was adopted, the model was run until all individuals were dead (lifetime) and half-cycle correction was performed [The cost-utility model is described in detail elsewhere . Brieflyerformed . All cosModel inputs were derived from two main sources: Hospital Episode Statistics (HES) records and Clinical Practice Research Datalink (CPRD) records. The HES dataset comprised hospital records for 33,152 patients older than 60 years who had had an emergency hospital admission with a primary International Classification of Diseases, 10th revision (ICD-10)  diagnosiQuality-of-life estimates were derived from a meta-regression, using a linear mixed-effects model, of 32 studies  reporting preference-based quality of life . All modHRG4 2014-15 Payment Grouper and HRG4 + 2014-15 Reference Costs Grouper) [HES data captures all hospital NHS patient care, as well as care for private patients treated in NHS hospitals and care delivered by treatment centres (including private providers) funded by the NHS. For each FCE, it contains anonymised patient administrative information , diagnosis ICD-10) and procedure codes . For the hospital cost analysis, we used HES data from April 2009 to March 2013 (4 years of data) comprising admitted patient care records, hospital outpatient activity (available from April 2003), adult critical care data (available from April 2008), and accident and emergency (A&E) attendances (available from April 2007). For each HES dataset, we derived two sets of 2014/2015 HRGs, one corresponding to the tariffs and one corresponding to the reference costs using specific Grouper software ( and procGrouper) . The basGrouper) . The GroSpell-level tariffs 2014/2015 FCE-level reference costs 2014/2015 [Spell-level reference costs 2014/2015 [We matched the derived sets of HRGs at FCE and spell level to three sets of unit costs to convert hospital resource utilisation into 2014–2015 tariffs and costs:When the Grouper software produced an error code for the HRG at FCE or spell level (e.g. UZ01Z), we valued these HRGs using the weighted average of all HRGs (weights from FCE and spell activity as reported in the unit cost databases) by type of admission . If the length of stay exceeded the defined trimpoints for a given HRG, the cost of each additional bed day was added to the FCE-level costs or spell-level tariffs. Excess bed days were already included in the spell-level costs.Spell-level reference costs are only collected for admitted patient care data  so we valued other types of hospital activity  using the FCE-level reference costs dataset . In the Total hospital costs per patient were aggregated into annual amounts for the purposes of the analysis. We estimated the hospital costs of index fracture, hospital costs in the year of fracture and total annual hospital costs of incident hip fractures for the UK. The latter was estimated by multiplying the hospital costs in the year of fracture by the incidence of hip fracture in the UK  . The HESp < 0.05). Model fit was assessed using Pregibon’s Link test and different family and link functions were compared using Akaike’s information criterion.Generalised linear models (GLMs) were used to predict annual hospital costs by health state. We estimated separate models for hospitalisation costs  and non-hospitalisation costs (A&E and outpatient consultations). The following covariates were examined: sex, current age, age at hip fracture (first and second), living in a care home , 30-day mortality following hip fracture, 1-year mortality following hip fracture, second hip fracture, major non-hip fracture requiring hospitalisation, history of second hip fracture, and history of major non-hip fracture. Covariates had to have a frequency of at least 100 patients to be considered for analysis, and were included in the final model if they were found to be statistically significant  and spell-level tariffs . In contrast, index admissions with two FCEs were valued £2850 (p < 0.001) and £3922 (p < 0.001) higher using FCE-level reference costs than using spell-level reference costs and spell-level tariffs, respectively. Index admissions with three or more FCEs were £7308 (p < 0.001) and £7778 (p < 0.001) higher if valued using FCE-level reference costs than using spell-level reference costs and spell-level tariffs, respectively. Using spell-level tariffs resulted in the lowest costs of index admissions. Table A15 in the Electronic Supplementary Material reports the top five HRGs in our dataset for index admissions with one FCE according to the three sets of unit costs.Table The total annual hospital costs associated with all incident hip fractures in the UK were estimated to vary between £813 million (spell-level tariffs) and £1099 million (FCE-level reference cost) per year depending on the source of unit costs used Fig. .Fig. 2ToBetween 2009 and 2013, there were 19,644 patient-years of data available in the HES dataset to estimate the hospital cost equations. Tables A5–A14 in the Electronic Supplementary Material report the regression models for hospitalisation  and non-hospitalisation costs (A&E and outpatient) based on the three sets of national unit costs. Overall, the direction of the coefficients was consistent across the three sets of unit costs. However, the magnitude and statistical significance of the covariates varied depending on the unit costs used.For example, hospitalisation costs in year of second hip fracture were significantly associated with death within 30 days of hip fracture using FCE-level reference costs and spell-level tariffs but not with spell-level reference costs . Conditional on hospitalisation and death, living in a care home was significantly associated with higher hospitalisation costs if HRGs were valued using reference costs at FCE-level or national tariffs, but the association was no longer significant using reference costs at spell level .The average age of a male patient with a hip fracture, not living in a care home, was 81.4 years with a Charlson co-morbidity index (CCI) score of 1.9. Table For our male cohort, the most effective model of care was the introduction of an OG, followed by the introduction of an FLS and then usual care. On average, when compared with usual care, FLS and OG-led models of care resulted in an additional 0.10 and 0.13 QALYs gained (discounted), respectively. At a £20,000/QALY threshold, using spell-level reference costs or spell-level tariffs, the introduction of a nurse-led FLS model of care was the cost-effective alternative. The probability that this was the cost-effective option was estimated at 53% , of which £25 billion concerned admitted patient care. Spell-based reference costs captured £25 billion in the same period , 19. TheThis is the first study to evaluate the implications of choosing different HRG-based national unit costs when conducting costing studies and to then use the resulting cost estimates within economic evaluations. A key strength of our study is that we were able to utilise a large linked dataset of hospital and clinical practice records that allowed us to ensure that observed differences were precisely estimated. Our work builds on two earlier related studies. The first study, by Geue et al. , compareThe second study, by Thorn et al. , evaluatOur work moves this literature forward by using linked data on a variety of hospital activities (not just acute care) over a long time period for a large sample to investigate the impact of choice of costing approach on economic evaluation results. For this reason, we believe that our findings are broadly generalisable to other costing studies and economic evaluations in an English NHS context. Several limitations of our study should, however, be noted. It would have been useful to contrast the results against those obtained using a micro-costing approach. However, given the scale of the exercise and the available dataset, this was not judged to be appropriate or feasible. Furthermore, our aim was to replicate what is most likely to occur in practice when a researcher needs to cost a disease or perform an economic evaluation. Spell-based reference costs only concern admitted patient care and, as a result, non-admitted patient care  were valued using the FCE-based reference costs. Spell-based tariffs were missing for some HRGs as they are subject to local prices to be contracted between commissioners and providers ; we usedWe hope that the results of our study will be informative for analysts who need to select a set of HRG-based unit costs for a future costing study or economic evaluation. The appropriate set of unit costs to use will vary depending on the analytical perspective that is adopted; hence, it is not possible to make an overall recommendation regarding this analytical decision. If the broad aim of a study is to inform resource allocation, analysts may prefer to apply reference costs as these are likely to be the best proxy for opportunity costs. Alternatively, if a study is being conducted from the perspective of a hospital provider, tariffs may be the more appropriate choice. It should also be noted that the Department of Health favour the use of FCE-based reference costs rather than spell costs . Health As the availability of large administrative records increases it becomes important to ensure that such data are analysed appropriately and the methods used are documented fully. Our results show that, conditional on the set of national unit costs adopted, the cost of diseases may vary considerably and different policy decisions may be made regarding the introduction of new healthcare interventions. The variability in cost estimates may impair healthcare planning and any misallocation of scarce healthcare resources may ultimately lead to suboptimal patient health outcomes, reducing population health.The data underlying the analyses in this paper cannot be made publicly available as this is not permitted by the data supplier, NHS Digital.Supplementary material 1 (DOCX 228 kb)Below is the link to the electronic supplementary material.


Anas platyrhynchos) in eastern Kazakhstan.An avian influenza virus strain, A/mallard/Balkhash/6304/2014 (H1N1), was isolated during a wild bird monitoring study in Kazakhstan in 2014. The virus was isolated from a wild mallard duck ( Anas platyrhynchos) in eastern Kazakhstan. Here, we present the near-complete genome sequence of the virus.An avian influenza virus strain, A/mallard/Balkhash/6304/2014 (H1N1), was isolated during a wild bird monitoring study in Kazakhstan in 2014. The virus was isolated from a wild mallard duck ( Orthomyxoviridae family, genus Influenzavirus A (Avian influenza virus (AIV) is a single-stranded segmented negative-sense RNA virus belonging to the avirus A . Continuavirus A . H1N1 isavirus A .The A/mallard/Balkhash/6304/2014 (H1N1) virus strain was isolated from a cloacal swab sample from a wild mallard duck after inoculation into 10-day-old embryonated chicken eggs. Allantoic fluid was checked for the presence of hemagglutinating agents by using a hemagglutination assay with chicken red blood cells . In totaThis study documents the isolation of AIV during a global spread of postpandemic swine H1N1 influenza virus, and it was interesting to compare a local avian strain with human pandemic strains. A BLAST search of hemagglutinin and neuraminidase gene sequences revealed their similarity at the nucleotide level to those of AIV strains isolated in Caucasus and Russia . The dedThe internal genes at the nucleotide level were 98 to 99% similar to those of viral strains isolated in Mongolia, the Czech Republic, China, and Bangladesh . PhylogeMK463931 to MK463938. Raw sequence reads are available under BioProject accession number PRJNA525314.The complete genome sequence of A/mallard/Balkhash/6304/2014 (H1N1) is available at GenBank under the accession numbers


Viruses decided that a Special Issue on viral recombination was appropriate.Recombination between and within virus genomes is being increasingly recognized as a major driver of virus evolution. Virus evolution can lead to changes in virus pathogenesis and virus ecology. The ubiquity of high-throughput sequencing of multiple virus isolates has revolutionized data acquisition, thus the editors of This Special Issue contains papers on a variety of viruses, both from the host and genome perspectives. There are manuscripts describing five ssRNA viruses, four ssDNA viruses, and one dsDNA virus, all of which undergo or have undergone varying amounts of recombination. Manuscripts in this Special Issue discuss recombination in two plant viruses and six animal viruses, as well as recombination in a group of viruses discovered by metagenomic analysis, and one virus with an unknown host that is apparently derived from a recombination between DNA and RNA viruses. Two manuscripts describe recombination in the RNA foot and mouth disease viruses (FMDV) in wildlife, Ferretti et al. discuss FMDV in a very narrow host range where recombination seems to dominate , and LasRecombination is not limited to RNA viruses; both ds and ssDNA viruses are known to undergo recombination. A novel set of fluorescent HSV-1 viruses is presented by Law et al.  and is uI hope that this collection of diverse manuscripts describing recombination in a variety of viruses will lead to a better understanding of virus genome diversity generated by recombination. Clearly there are many aspects of virus recombination that are not included herein. These papers provide an indication of the widespread nature of recombination in virus genomes. The next steps are to understand the mechanism of the recombination processes and determine the role of recombination in pathogenesis and virus evolution.


Viruses evolve rapidly and unpredictably, challenging the effectiveness of disease diagnostics. To help control outbreaks and understand their origins, the first step is often isolating viruses from infected samples for characterization. We demonstrate that multiple emerging virus strains can be simultaneously enriched and optically detected in only a few minutes without using any labels. A portable platform that captures viruses by their size, coupled to Raman spectroscopy, resulted in successful virus identification with 90% accuracy in real time directly from clinical samples. Furthermore, this viable enrichment process enables further culturing and characterization by electron microscopy and deep sequencing. This microplatform is an effective disease-monitoring system and broadens virus surveillance by enabling real-time virus identification. 2 EID50/mL (50% egg infective dose per microliter), with a virus specificity of 90%. After enrichment using the device, we demonstrated by sequencing that the abundance of viral-specific reads significantly increased from 4.1 to 31.8% for parainfluenza and from 0.08 to 0.44% for influenza virus. This enrichment method coupled to Raman virus identification constitutes an innovative system that could be used to quickly track and monitor viral outbreaks in real time.Emerging and reemerging viruses are responsible for a number of recent epidemic outbreaks. A crucial step in predicting and controlling outbreaks is the timely and accurate characterization of emerging virus strains. We present a portable microfluidic platform containing carbon nanotube arrays with differential filtration porosity for the rapid enrichment and optical identification of viruses. Different emerging strains (or unknown viruses) can be enriched and identified in real time through a multivirus capture component in conjunction with surface-enhanced Raman spectroscopy. More importantly, after viral capture and detection on a chip, viruses remain viable and get purified in a microdevice that permits subsequent in-depth characterizations by various conventional methods. We validated this platform using different subtypes of avian influenza A viruses and human samples with respiratory infections. This technology successfully enriched rhinovirus, influenza virus, and parainfluenza viruses, and maintained the stoichiometric viral proportions when the samples contained more than one type of virus, thus emulating coinfection. Viral capture and detection took only a few minutes with a 70-fold enrichment enhancement; detection could be achieved with as little as 10 Viruses are the most abundant biological entities on earth , more th2912In virus surveillance, collected samples are subjected to a series of time-consuming steps, such as ultracentrifugation and cell culture, to enrich virus particles or amplify virus titers , 21–23. 21Existing technologies, such as immune-based  and moleWe have previously shown that viruses can be captured between aligned carbon nanotubes, without the use of specific labels, when their sizes match the intertubular distance (ITD) . In our SI Appendix, Fig. S1). In our previous study, we demonstrated that the CNxCNTs had better biocompatibility than the pristine CNTs  arrays, decorated with gold (Au) nanoparticles to separate, enrich, and detect fluidic human samples by direct Raman-based spectroscopic identification 3•9H2O, on micromolds with herringbone patterns. Then, Fe-based precursors were transferred on a Si/SiO2 substrate by stamping the side of the micromolds covered with precursors. During spin-coating and stamping, the concentration and location of the precursor were controlled and defined. As a result, herringbone-shaped precursor was applied and patterned on a substrate. The stamped precursor arrays were then used as catalytic substrates to grow arrays of aligned CNxCNTs using CVD in the presence of benzylamine   .3)3•9H2O] 3•9H2O] B, and th)3•9H2O] , 33. We )3•9H2O] D, measur)3•9H2O] . Thus, t)3•9H2O] .6 phonons is absorbed or emitted through Raman (inelastic) scattering. This weak efficiency dramatically limits the signal intensity of Raman spectra, thus constraining its application. However, studies have shown that Au nanoparticles enhance localized plasmon resonance, which can further enhance the signal of Raman scattering up to ∼108. Interestingly, this Raman signal enhancement enables the detection down to a single-molecule level (approximately picomolar) . Results of the UV-Vis measurements indicate that the Au/CNxCNT arrays have a strong localized surface plasmon resonance . We characterized the sensitivity and uniformity of the SERS detection using a reference Raman dye, Rhodamine 6G (R6G) . The results from all of the characterizations showed that the Au/CNxCNTs arrays provide uniform, consistent, and sensitive (10−10 M) SERS signals across different ITDs.To optically detect the captured viruses using a label-free approach, we functionalized the CNxCNTs with Au nanoparticles to enable SERS A. Raman comolar) , 36. Precomolar) –39. We oSI Appendix, Fig. S4 A and B). To capture and separate each subgroup of silica particles in the mixture, we assembled a microdevice with 3 zones of aligned CNxCNT arrays having 400-, 140-, and 25-nm ITDs to match their sizes. The efficiency of the size-based capture was calculated under different flow rates by measuring the fluorescent intensities of the flow through and normalizing by the initial intensities. After capture, strong fluorescent signals of individual subgroups of silica particles were detected in the CNxCNT array zones. The ITDs matched the silica bead sizes  were captured , thus demonstrating the size range of the CNxCNT arrays.After growing the CNxCNT arrays and fabricating microfluidic devices, we characterized the size-based capture by using different sizes of fluorescently labeled silica particles A. We mixad sizes B, illustad sizes B. Under ad sizes . NotablySI Appendix, Fig. S4D). These results demonstrate that the VIRRION can selectively capture different sizes of silica particles from a mixture, even at low flow-through pressure when using a syringe.We repeated the capture by reloading the microfluidic device into the same VIRRION via a manual push through a syringe to test whether the capture efficiency could be improved. The results indicated that the capture efficiency increased by ∼10% after the second capture . We assembled a VIRRION with an ITD of 100 nm to match the average size of the AIV. We manually pushed 5 mL of the supernatant from virus culture through a syringe containing 104 EID50/mL (50% egg infective dose per microliter) H5N2 into a VIRRION. We then performed an immunofluorescence assay on the VIRRION to detect the captured AIV in situ by using an AIV H5 subtype-specific monoclonal antibody. Strong fluorescent signals were detected on the CNxCNT arrays . Under SEM and TEM, we clearly observed virus particles captured , which is an RNA virus A. The avT arrays B, while captured A and 4B.2 EID50/mL . We applied a principal-component analysis (PCA) to classify all viral Raman spectra. Our results indicate that different strains cluster separately  . Since H7N2 and H5N2 have similar Raman spectra  and reovirus. We first collected multiple Raman spectra after VIRRION capture of these viruses to build a simple database. Surface enhancement of the Raman scattering happens at a so-called “hot spot,” i.e., the 1-nm region between 2 adjacent gold nanoparticles. Thus, when viruses are trapped between the Au/CNxCNT arrays, the only Raman signal from the virus in the vicinity of the hot spot is enhanced. We recorded Raman signals and averaged over 100 spectra for each strain to generate a reliable average fingerprint for each virus. Each virus strain has a distinct fingerprint that was still distinguishable at concentrations as low as ∼10EID50/mL C. It is Note S1) . To deveNote S1) E 41–44)–442 EID5 spectra C, there 6 EID50/mL, which is similar to controls where the virus is propagated in the same cell line in a culture flask. We obtained a higher virus titer, ∼107 EID50/mL, when we propagated captured viruses using embryonated chicken eggs (ECEs). To do that, we disassembled the VIRRION, collected the Au/CNxCNT with embedded viruses, and directly inoculated these into ECE for AIV propagation. For both cell culture and ECE propagation, we also confirmed viral replication by Dot-ELISA . These results clearly show that VIRRION maintains viability of the viruses during capture and makes direct virus isolation on-chip possible.After capturing and identifying H5N2 virus particles, we propagated the virus in cell culture directly on the VIRRIONs. Notably, during culture, the host cells attached and proliferated on the Au/CNxCNT arrays. The virus-like particles were observed on the surface of the cells, indicating effective replication and production of virus progeny B. To conot-ELISA   , an essential housekeeping gene that is conserved in all eukaryotic cells. We used H5N2 spiked-in samples and extracted total RNA from the VIRRION after capture. We then characterized the enrichment by measuring the ratio of the copy number of host 18S rRNA to the virus hemagglutinin (HA) gene of H5N2 by RT-qPCR (ig. S7B) , 45. Befig. S7B) G.9 . We compared viral reads of the HA gene before and after enrichment. Interestingly, before enrichment, the H5N2:H7N2 ratio based on HA viral reads was 1:7.8, where 344 reads were from H5N2 and 2,693 reads from H7N2. After enrichment, the ratio was relatively well preserved at 1:6.5, but with an increase in viral reads . There appeared to be a more than 3-fold enrichment for both viruses. However, this difference in enrichment efficiencies maintained the stoichiometric proportion of viruses present in the original samples. Moreover, the complete genome of both strains was sequenced with an average coverage of 519× for H5N2 and 599× for H7N2. In addition, after enrichment, the host (chicken) reads decreased from 68.44% (reads) of the total reads  to 34.2%  of the total reads . These results indicate that the VIRRION can minimize artifacts during enrichment. This feature is important for virus diversity profiling.We further analyzed the captured viruses by NGS to characterize the captured virus population without prior knowledge of the strains –22, 46. 9 , into viSI Appendix, Figs. S9 and S10) . We extracted nucleic acid on-chip from the VIRRIONs and confirmed by RT-qPCR what viruses were captured . Their Raman spectra fingerprints were determined and recorded . Compared to conventional detections, such as ELISA or PCR, our results indicate that VIRRION can be successfully used to detect specific viruses within several minutes after collecting clinical samples.Acute respiratory infections are the third most common cause of death worldwide and responsible for 4 million deaths each year, which is 7% of all deaths annually . Most cland S10) –58. We arecorded A. The PCrecorded B demonstSI Appendix, Figs. S13 and S14 and Table S1). The percentage of virus-specific reads increased from 0.08 to 0.44% for influenza A and from 4.1 to 31.8% for HPIV 3. Genome coverage also increased following enrichment. The percentage of the influenza A genome increased from 33% before enrichment to 48% and 57% following the first and second enrichment steps, respectively. The percentage of the HPIV 3 genome increased from 22% before enrichment to 81% and 77% after the first and second enrichment steps. The average genome coverage increased from 8.8× to 15.9× for influenza, and 363× to 2,040× for HPIV 3. We were also able to assemble large portions of each viral genome. No influenza A contigs greater than 500 bp were identified following assembly in the unenriched sample. However, 4 contigs matching the influenza A genome were found in both enrichment steps with an average of 590 bp. They are 681-bp matches to PB1 (MH540988.1), 622-bp matches to PB1 (MH845979.1), 570-bp matches to NA (LC409054.1), and 553-bp matches to PB2 (MH540997.1). The entire genome was assembled in each of the HPIV 3 samples. The results of coverage and variant analyses were summarized in SI Appendix, Fig. S12. The NGS results supported that VIRRION can enrich different viruses directly from patient swab samples. Unfortunately, no rhinovirus-related reads were detected by NGS after enrichment. We suspect that this is because the virus titer is extremely low even after VIRRION enrichment, as determined from PCR results .After label-free capture and detection, we processed the isolated nucleic acid for NGS and determined that after VIRRION capture, the influenza A and HPIV 3 viruses were significantly enriched 3•9H2O  using a mixture (1:1) of DI water and toluene . To perform stamping, a PDMS-coated mold was placed on a silicon substrate covered with a thin film of liquid iron precursors for 30 s. Then, the PDMS stamp was transferred and applied on a fused silica substrate for 10 min in order to ensure iron precursors were transferred and dried. More detailed information regarding the materials and methods are available in SI Appendix. All of the raw NGS data have been deposited in the Sequence Read Archive (SRA) at the National Center for Biotechnology Information under submission number SUB5200307.Precursor solutions were prepared by diluting Fe (NOSupplementary File


West Nile virus (WNV) is a mosquito-borne flavivirus, first detected in the Western Hemisphere in 1999 and spread across North America over the next decade. Though endemic in the most populous areas of North America, few studies have estimated the healthcare costs associated with WNV. The objective of this study was to determine direct healthcare costs attributable to WNV illness in Ontario, Canada.We conducted a cost-of-illness study on incident laboratory confirmed and probable WNV infected subjects identified from the provincial laboratory database from Jan 1, 2002 through Dec 31, 2012. Infected subjects were linked to health administrative data and matched to uninfected subjects. We used phase-of-care methods to calculate costs for 3 phases of illness: acute infection, continuing care, and final care prior to death. Mean 10-day attributable costs were reported in 2014 Canadian dollars, per capita. Sensitivity analysis was conducted to test the impact of WNV neurologic syndromes on healthcare costs.One thousand five hundred fifty-one laboratory confirmed and probable WNV infected subjects were ascertained; 1540 (99.3%) were matched to uninfected subjects. Mean age of WNV infected subjects was 49.1 ± 18.4 years, 50.5% were female. Mean costs attributable to WNV were $1177  for acute infection, $180  for continuing care, $11,614  for final care - acute death, and $3199  for final care - late death. Expected 1-year costs were $13,648, adjusted for survival. Three hundred seventeen infected subjects were diagnosed with at least one neurologic syndrome and greatest healthcare costs in acute infection were associated with encephalitis .WNV is associated with increased healthcare resource utilization across all phases of care. High-quality studies are needed to understand the health system impact of vector-borne diseases and evaluate the cost effectiveness of novel WNV interventions. West Nile virus (WNV) is a mosquito-borne flavivirus, detected in the Western Hemisphere in 1999 that spread across North America over the next decade . From 20Approximately 20% of WNV infections are symptomatic . CommonlThough endemic in the most populous areas of North America, few studies have estimated the healthcare costs associated with WNV –18. AmonWe conducted an incidence-based matched cohort study to estimate costs attributable to WNV in Ontario, Canada from the healthcare payer perspective . This study was approved by the Ontario Agency for Health Protection and Promotion  institutional Ethics Review Board. Data analysis was performed using SAS 9.3 .A cohort of incident infected subjects was established from all laboratory confirmed and probable cases of WNV illness from January 1, 2002 through December 31, 2012 from Public Health Ontario Laboratory (PHOL) data by applying the laboratory case definitions . This WNEach infected subject was matched to three uninfected subjects from the general population on age ± 1 year, sex, ±30 days index date for healthcare resource use (randomly assigned date ±30 days from the index date for the infected), and propensity score , the last 100 days would be allocated as final care, the first 90 days would be acute infection, and the remaining time (270 days) would be allocated as continuing care. For subjects observed for less than 90 days (index date to death date was less than the acute infection phase length), the entire observation period would be allocated as final care.Costs were calculated using person-level costing methods established by ICES . All dirFees associated with distinct healthcare service categories were used to derive stratified costs by utilization  for hospitalization after the index date and mortality were estimated using a modified Poisson regression with robust error variances  and adjuTo test the impact of severe WNV syndromes on healthcare costs, costs and RRs for hospitalization and mortality were estimated for individuals with WNND syndromes .Neurologic syndromes were identified from ICD-10-CA  diagnostic codes and Ontario Health Insurance Plan (OHIP) codes used by physicians for billing in the province of Ontario ±30 days from index date, with input from clinical and WNV content experts. If a subject was diagnosed with > 1 neurologic syndrome, they were classified to the more severe clinical syndrome .n = 11) were younger than matched, infected subjects  and more likely to reside rurally.From 2002 through 2012, 1551 incident, laboratory confirmed and probable WNV infected subjects were identified from the PHOL dataset and individually linked to health administrative data. 1540 99.3%) infected subjects were matched to uninfected subjects. Balance was assessed and all standardized differences were below 0.10, indicating good balance  subjects were each matched to three uninfected subjects who also died. Mean age at death was 70.9 ± 14.5 years, 104 (64.2%) were male, and 3 (1.9%) resided rurally. Nearly all standardized differences were below 0.10 indicating good balance between matched infected and uninfected subjects who died.Median duration of follow-up for infected subjects was 3.6 years. 547 (35.3%) subjects were admitted to hospital within 30 days of index date .Expected cumulative mean attributable one-year and two-year costs were $13,648 and $20,124, respectively, adjusted for survival. No significant difference for RR of hospitalization within 30 days after the index date or all-cause mortality between WNV infected compared to uninfected subjects was observed  , $507  for continuing care, $4588  for final care - acute death, $5164  for final care - late death , $254  in continuing care, $4827  in final care - acute death, and $4639  in final care - late death , followed by AFP , and then meningitis   for those with WNND syndromes compared to non-WNND infected subjects, adjusted for age and sex. Adjusted RR for all-cause mortality was 1.9  for infected subjects with WNND syndromes compared to non-WNND infected subjects  compared to the remaining subjects with non-WNND infections. Due to the low incidence of AFP syndromes, we were unable to draw reliable estimates on the attributable mortality costs of patients with AFP. Notably, previous case reports on patients with AFP from WNV infections found physical recovery was incomplete after 1 year of follow-up, suggesting the potential for long-term costs associated with WNV sequelae , 32. ForIn conclusion, by applying population-based methods to estimate the costs of WNV from the healthcare payer perspective, we found that healthcare resource utilization is elevated across all phases of care attributable to WNV infections and WNND syndromes. Though current evidence supports that WNND results rarely from WNV infections, a large proportion of WNV infected individuals who interface with the healthcare system in Ontario have severe neurologic conditions temporally associated with WNV infection, requiring increased healthcare resource use predominantly from hospitalization and physician services. High costs of treatment underscore the importance of evaluating the cost effectiveness of WNV and other mosquito-borne disease prevention through vector control strategies  and novel vaccinations to inform healthcare resource allocation in Ontario.Additional file 1: Figure S1. Panel A shows the mean total costs of WNV infected subjects (n = 1551) 360 days after index date. Panel B shows the mean total cost of all WNV infected subjects who died during the observation period (n = 163) Additional file 2: Table S1. Baseline characteristics of WNV infected subjects matched ±30 days from index date (n = 1540)


The term “virosphere” describes both the space where viruses are found and the space they influence, and can extend to their impact on the environment, highlighting the complexity of the interactions involved. Studying the biology of viruses and the etiology of virus disease is crucial to the prevention of viral disease, efficient and reliable virus diagnosis, and virus control. Electron microscopy (EM) is an essential tool in the detection and analysis of virus replication. New EM methods and ongoing technical improvements offer a broad spectrum of applications, allowing in-depth investigation of viral impact on not only the host but also the environment. Indeed, using the most up-to-date electron cryomicroscopy methods, such investigations are now close to atomic resolution. In combination with bioinformatics, the transition from 2D imaging to 3D remodeling allows structural and functional analyses that extend and augment our knowledge of the astonishing diversity in virus structure and lifestyle. In combination with confocal laser scanning microscopy, EM enables live imaging of cells and tissues with high-resolution analysis. Here, we describe the pivotal role played by EM in the study of viruses, from structural analysis to the biological relevance of the viral metagenome (virome). Since the recognition of viruses as the causative agents of disease in the last decades of the nineteenth century  is a good initial step in virus diagnosis for several reasons Huger, . The metThe hallmark of TEM is its “open view” nature, since it provides an immediate overview of actual status, discerning amount and shape of virus(es) present, including the unexpected  is based on the same serological principles as ELISA, and can be used for further virus identification during routine TEM diagnosis Figure . IEM hasBased on its ability to recognize contaminations, rapid application of TEM is essential for quality control of reference material or of virus preparations used for antibody production or directly for vaccination. Prompt production of reliable and comparable results is essential for efficient diagnostics during unexpected regional virus outbreaks or epidemics, making TEM an integral part of the protocols followed by national reference laboratories. Laue and Möller  recentlyDue to the potentially serious consequences of viral outbreaks, rigorous, and universally recognized training of personnel dealing with virus identification is crucial.Training in Diagnostic Electron Microscopy (DEM) of human and veterinary infectious diseases can be obtained in an annual External Quality Assurance (EQA) scheme prepared by the Robert Koch Institute.Comprehensive studies of virus biology and host responses require a combination of molecular biology, biochemical analyses and EM —one of the very first objects to be seen in an electron microscope  and advances in image processing have extended the resolution limit of electron cryo-microscopy (cryo-EM) into the atomic range  subunit as being required for virus assembly —a positive-sense, single-stranded RNA plant virus—and other members of the order Advances in cryo-EM have revealed near-atomic structures of rod-shaped and flexible filamentous plant viruses. In contrast to the right-handed helical organization of the CPs of rod-shaped Tobamovirus  assembled by the International Committee on Taxonomy of Viruses (ICTV) classifies 4853 virus species covering all host phyla. The NCBI database records 7512 sequenced viral genomes from all kingdoms. Viral abundance extrapolated from studies on the virosphere is estimated at 10Caulimoviridae, representing dsDNA viruses, or Bromoviridae comprising multipartite positive ssRNA viruses, is still unknown. In the case of filoviruses—enveloped negative sense ssRNA viruses—the three different shapes and virion lengths observed have been assigned to different numbers of encapsidated viral genomes (Booth et al., Caulimoviridae (Geijskes et al., High-resolution EM in combination with generation of mutants of infectious viruses provides a powerful tool for the detection and study of structural aberrations and their impact on virus replication and evolution. The biological relevance of the coexistence of isometric and bacilliform particles, as occurs in the family The ubiquitous nature, high mobility and genetic versatility of viruses makes them ideal for mediating horizontal DNA transfer. As demonstrated in recent years, a combination of molecular, next-generation sequencing and EM technologies has shown that viruses can encapsidate host nucleic acids corresponding to their genome composition (Ghoshal et al., International efforts by virologists have already established platforms for direct communication and scientific exchange (Gould et al., KR-P, KF, KH, and RK contributed to writing and editing the manuscript including figures. KR-P wrote sections for introduction and outlook. KR-P, KF, and RK wrote the sections on routine diagnosis and functional virology and KH described cryo EM. All authors approved the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Foot-and-mouth disease virus (FMDV) is an important livestock pathogen that is often described as the greatest constraint to global trade in animal products. The present study utilized a standardized pig-to-cow contact exposure model to demonstrate that FMDV infection of cattle initiates in the nasopharyngeal mucosa following natural virus exposure. Furthermore, this work confirmed the role of the bovine nasopharyngeal mucosa as the site of persistent FMDV infection in vaccinated and nonvaccinated cattle. The critical output of this study validates previous studies that have used simulated natural inoculation models to characterize FMDV pathogenesis in cattle and emphasizes the importance of continued research of the unique virus-host interactions that occur within the bovine nasopharynx. Specifically, vaccines and biotherapeutic countermeasures designed to prevent nasopharyngeal infection of vaccinated animals could contribute to substantially improved control of FMDV. The pathogenesis of foot-and-mouth disease virus (FMDV) in cattle was investigated through early and late stages of infection by use of an optimized experimental model for controlled contact exposure. Time-limited exposure of cattle to FMDV-infected pigs led to primary FMDV infection of the nasopharyngeal mucosa in both vaccinated and nonvaccinated cattle. In nonvaccinated cattle, the infection generalized rapidly to cause clinical disease, without apparent virus amplification in the lungs prior to establishment of viremia. Vaccinated cattle were protected against clinical disease and viremia; however, all vaccinated cattle were subclinically infected, and persistent infection occurred at similarly high prevalences in both animal cohorts. Infection dynamics in cattle were consistent and synchronous and comparable to those of simulated natural and needle inoculation systems. However, the current experimental model utilizes a natural route of virus exposure and is therefore superior for investigations of disease pathogenesis and host response. Deep sequencing of viruses obtained during early infection of pigs and cattle indicated that virus populations sampled from sites of primary infection were markedly more diverse than viruses from vesicular lesions of cattle, suggesting the occurrence of substantial bottlenecks associated with vesicle formation. These data expand previous knowledge of FMDV pathogenesis in cattle and provide novel insights for validation of inoculation models of bovine FMD studies.IMPORTANCE Foot-and-mouth disease virus (FMDV) is an important livestock pathogen that is often described as the greatest constraint to global trade in animal products. The present study utilized a standardized pig-to-cow contact exposure model to demonstrate that FMDV infection of cattle initiates in the nasopharyngeal mucosa following natural virus exposure. Furthermore, this work confirmed the role of the bovine nasopharyngeal mucosa as the site of persistent FMDV infection in vaccinated and nonvaccinated cattle. The critical output of this study validates previous studies that have used simulated natural inoculation models to characterize FMDV pathogenesis in cattle and emphasizes the importance of continued research of the unique virus-host interactions that occur within the bovine nasopharynx. Specifically, vaccines and biotherapeutic countermeasures designed to prevent nasopharyngeal infection of vaccinated animals could contribute to substantially improved control of FMDV. Aphthovirus, family Picornaviridae), is a contagious livestock disease of global concern (Foot-and-mouth disease (FMD), caused by FMD virus  associated with the appearance of characteristic vesicular lesions in the oral cavity and in the interdigital clefts and coronary bands of the feet , 4. MortFMDV is infectious in low doses and can be transmitted by both direct and indirect mechanisms, including movement of infected animals as well as mechanical transfer by contaminated vehicles and fomites. Cattle are specifically sensitive to FMDV infection via the respiratory route , 5. EarlHistorically, challenge of cattle during experimental FMDV studies has been done through intraepithelial injection on the dorsal surface of the tongue . This inSeveral investigations have used contact exposure to experimentally infect cattle with FMDV , 20; howThis study reports the outcome of FMDV infection in cattle after time-limited, direct contact exposure to FMDV-infected pigs. Six vaccinated and six nonvaccinated cattle were monitored and sampled through 35 days after contact exposure for the objective of investigating FMDV infection dynamics, as well as the occurrence and characteristics of FMDV persistence. Additionally, 12 nonvaccinated cattle were euthanized for tissue harvest during early infection (6 to 72 h postexposure [hpe]) for temporo-anatomic mapping of FMDV in bovine tissues during the acute phase of infection .The pigs that were used as virus donors during contact exposure were infected through heel bulb inoculation. The dynamics of infection were highly synchronous across all 12 pigs. All pigs were viremic, as determined by detection of FMDV RNA in serum, and were shedding detectable quantities of FMDV RNA in oropharyngeal and nasal secretions at 24 h postinoculation (hpi) . The firA total of 18 nonvaccinated cattle were included in the investigations . Twelve Six vaccinated cattle were included in the investigation for monitoring of FMDV infection dynamics and persistence following natural virus exposure. None of these cattle developed any clinical signs of FMD, and there was no detection of FMDV RNA in serum. However, all six cattle were subclinically infected, based upon detection of FMDV in secretions and/or tissues. Declining levels of FMDV RNA were detected in oral and nasal secretions in all animals from the end of contact exposure at 1 dpe until approximately 4 to 6 dpe  and C. STwelve nonvaccinated cattle were euthanized during the early phase of infection (6 to 72 hpe) for determination of the temporo-anatomic progression of infection following natural virus exposure. The most consistent detection of FMDV in tissues prior to the onset of viremia occurred in the nasopharyngeal tissues. Specifically, infectious FMDV was isolated from the dorsal soft palate and/or the dorsal nasopharynx of all four cattle that were euthanized at 6 to 12 hpe . AdditioAll four cattle that were euthanized at 24 hpe were viremic, but had not yet developed any clinical signs of FMD. In these animals, moderate to large quantities of FMDV RNA were consistently detected in the nasopharyngeal tissues, concurrent with isolation of virus. Smaller quantities of FMDV RNA were detected in the tongue and oropharyngeal epithelium and oropharyngeal tonsils . InfectiThe four cattle that were euthanized at 48 and 72 hpe were viremic and had characteristic vesicular lesion in the oral cavity and/or on one or two feet. During this more advanced stage of infection, FMDV was isolated from all sampled tissues , interprAll 6 nonvaccinated cattle and 5 out of 6 vaccinated cattle that were monitored through to the persistent phase of infection were FMDV carriers. Infectious virus and FMDV RNA were consistently detected in nasopharyngeal tissues  from the persistently infected carriers, regardless of vaccination status . One nas+ cells (presumptive antigen-presenting cells) was seen in close proximity to FMDV-infected areas. At 48 to 72 hpe, substantial FMDV replication was detected in epithelial crypts of the palatine tonsils . Similarly, viral RNA quantities in air samples declined in all rooms during the first 24 h after removal of the infected pigs from the rooms (2 dpe) . From 2 Samples obtained from cattle and pigs in room A were subjected to FMDV deep sequence analysis by next-generation sequencing (NGS). This experimental cohort included 3 pigs that were all euthanized at 72 h postinoculation (hpi), as well as 6 cattle, of which 2 were euthanized for tissue collection at each of 6, 24, and 72 hpe  .p-distance = 0.002; 13 nucleotides [not shown]). However, the frequency distribution of subconsensus viral variants provided insights into FMDV pathogenesis events.It was not possible to identify definitive transmission chains based on FMDV consensus sequence phylogenetic analysis due to the high similarity between consensus sequences (maximum 147 (end of the G-H loop) and asparagine to aspartate at 3A136—were detected at the consensus level in at least one sample from all 3 donor pigs and were also present at high frequencies in all sampled cattle. Of the remaining synonymous changes, three  occurred in at least one sample from every animal in the study.The original inoculum used to infect the pigs contained a highly diverse viral population. Consequently, during early infection, viral populations in pig-derived samples had abundant synonymous and nonsynonymous single nucleotide polymorphisms (SNPs) compared to the inoculum consensus sequence . A singlVesicular lesions obtained from cattle during the clinical phase of disease (72 hpe) contained viral populations that were substantially less diverse  than samples obtained from nasopharyngeal tissues at corresponding time points. Pulmonary tissue samples contained virus populations of mixed characteristics: these were either highly diverse, and thus, similar to nasopharyngeal tissues , or cloMultiple previous works have investigated FMDV pathogenesis in cattle. However, differences in study design, specifically with regard to methods used for virus exposure, have led to various findings. The objective of the current investigation was to utilize a natural but controlled system of direct contact exposure to achieve a detailed understanding of the progression of FMDV infection in cattle, from primary infection through systemic generalization to establishment of persistent infection.The experimental model included inoculation of donor pigs with a cattle-derived FMDV strain. Infected pigs were subsequently moved into clean rooms housing naive or vaccinated recipient cattle, at a time point that corresponded to the early clinical phase of FMD in the pigs. Initiating the contact exposure at a time at which the pigs were known to be shedding large quantities of FMDV  and limiThe onset and progression of FMD in exposed cattle were rapid and consistent. The first detection of viremia in nonvaccinated cattle occurred as early as 18 h after the start of contact exposure. Similarly, the majority of nonvaccinated cattle were viremic at 24 hpe, and all had vesicular lesions in the mouth or on at least one foot at 48 hpe. This very rapid disease progression is similar to that which occurs after conventional tongue inoculation of cattle with virulent FMDV strains , 22. AddSimilar to previous investigations, vaccination with an adenovirus-vectored FMD A vaccine 14 days prior to virus exposure conferred complete protection against clinical FMD and viremia , 12. AllAnalysis of tissue samples from early and persistent stages of disease by immunomicroscopy confirmed localization of both primary and persistent FMDV infection to specialized regions of epithelium of the nasopharyngeal mucosa overlying the mucosa-associated lymphoid tissue (MALT). This finding confirms that the simulated natural, aerosol , 23, andDespite the identical microanatomic locations of virus associated with primary versus persistent FMDV infection, there were notable differences in histopathological characteristics. Consistent with previous investigations , 10, priViral RNA quantities detected in air samples in room C, with 6 nonvaccinated cattle, increased from 2 to 3 dpe, after removal of the pigs, suggesting that the infected cattle contributed to airborne virus during the clinical phase of disease. Contrastingly, the quantities of viral RNA detected in room D, with 6 vaccinated cattle, decreased continuously after removal of the pigs from the room. Despite this, infectious FMDV was isolated as late as 7 dpe in filters from both rooms. The combination of sequence similarity between pig samples and air samples and previous observations confirming that in a similar setting, vaccinated and protected cattle did not shed detectable virus into the air (unpublished data), suggests that the airborne virus in the room of the vaccinated cattle may to a large extent have been derived from the pigs.Analysis of the frequency of FMDV genome substitutions below and above the consensus level provided interesting insights into the complexities of FMDV pathogenesis during early infection. This part of the investigation included only a limited subset of study animals and was limited to unpassaged sample material to avoid introduction of artifacts occurring during viral amplification in cell culture. Overall, there was a consistent trend that viruses obtained from vesicular lesions of cattle were highly homogenous, approaching clonality, whereas the viruses obtained from nasal swabs, air, and most tissues had highly heterogenous virus populations (swarms). This finding is consistent with previous reports that concluded that FMDV samples from vesicular epithelium were less diverse than esophagopharyngeal scrapings, suggesting within-host bottleneck events in association with formation of FMDV vesicles , 28. HerThe virus samples obtained from the inoculated pigs were highly diverse, reflecting the high diversity found in the original virus inoculum. In contrast to cattle-derived samples, a similar level of diversity was seen in samples from the site of primary virus replication (tonsil swabs), as in vesicular epithelium (coronary band lesions) in the pigs. This suggests a lesser extent of bottleneck events within the pigs compared to the more extensive effects that occurred as part of the process of transmission, adaptation, and pathogenesis within a new host species. Additionally, the species-specific virus populations may represent differential species-specific viral fitness defined by host selective pressures.The different levels of within-animal virus diversity across the different cattle may be suggestive of different mechanisms of virus transmission or the occurrence of bottlenecks associated with transmission from one host to another. For example, the striking homogeneity of virus samples obtained from animal 16-28 suggests that infection in this animal was likely founded by a narrow population. In contrast to this, the greater diversity of viruses in animals 16-27 and 16-29 suggests that these animals were likely exposed to different or more diverse founding virus populations. The highly synchronous infection dynamics across the cattle suggests that all animals received a comparable challenge dose. However, the differences in the diversity of within-host virus populations suggest that there may still have been qualitative differences in how these animals acquired the primary infection.The primary output of this investigation was confirmation of the importance of the bovine nasopharyngeal mucosa as the site of both primary and persistent FMDV infection after natural contact exposure. This confirms the output of many previous studies that reached similar conclusions using simulations of natural infection. Additionally, natural virus exposure did not cause substantial FMDV replication in the lower respiratory tract prior to establishment of viremia. The standardized experimental system of time-limited exposure to FMDV-infected pigs provided a stringent and consistent virus challenge across different cohorts of animals, providing a useful tool for FMDV pathogenesis studies. The output of the FMDV sequence analyses demonstrated that FMDV quasispecies compositions differ substantially between primary and secondary replication sites. The current findings provide novel insights on the within- and between-host evolution of FMDV, which are being explored in greater detail in ongoing investigations within our laboratory.24 Cruzeiro that has been characterized in detail in previous pathogenesis studies  biocontainment facilities at the Plum Island Animal Disease Center, New York. All procedures were performed in compliance with an experimental protocol approved by the Institutional Animal Care and Use Committee (protocol 231.03-14-R). Cattle were Holstein heifers weighing approximately 150 to 200 kg when delivered. Pigs were castrated Yorkshire males weighing approximately 25 to 30 kg. Pigs and cattle were housed in separate isolation rooms before virus challenge.5 BTID50 (50% infectious doses titrated in bovine tongue epithelium), diluted in minimal essential medium (MEM) to a total volume of 0.4 ml. At 48 h postinoculation (hpi) of the pigs, the pigs were moved into different isolation rooms that housed the cattle. After 24 h of cohabitation, the pigs were removed from the rooms and euthanized. During each contact exposure trial, 3 pigs were housed together with 6 cattle in a room with an available floor area of 27 m2, a total air volume of approximately 135 m3, and 28 complete air exchanges per hour. The study included replicate challenge trials performed in four separate rooms. Rooms A and B housed nonvaccinated cattle that were included in investigations of FMDV pathogenesis during early stages of infection. These animals were euthanized at predetermined time points between 6 and 72 h postexposure (hpe). Cattle in rooms C and D were kept for 35 days for the objective of investigating FMDV persistence. Among these animals, there were 6 vaccinated and 6 nonvaccinated individuals. During contact exposure of cattle in rooms C and D, 3 vaccinated and 3 naive cattle were housed together with 3 donor pigs to ensure uniform exposure conditions for all cattle. Vaccinated and nonvaccinated cattle were subsequently separated and moved into different rooms after 24 h of cohabitation with the infected pigs: nonvaccinated in room C and vaccinated in room D.The study included a total of 24 cattle and 12 pigs. Pigs were inoculated through intraepithelial injection in the heel bulb of one foot as previously described . Each piFMDV infection dynamics and virus shedding in pigs were monitored through collection of blood, nasal, and oropharyngeal (OP) swabs at 24-h intervals from 0 to 72 hpi. Swabs from pigs were immersed in 2 ml of MEM with 25 mM HEPES. The progression of clinical FMD in pigs was recorded using a cumulative scoring system as previously described , 33. SamFor the objective of evaluating tissue distribution of FMDV during early infection, two nonvaccinated cattle were euthanized for tissue harvest at 6, 12, 48, and 72 hpe, and four animals were euthanized at 24 hpe. For evaluation of tissue distribution during the persistent phase of infection, six vaccinated and six nonvaccinated cattle were euthanized at 35 dpe. A standardized necropsy procedure with collection of 18 to 22 distinct tissue samples  and 2 waAir sampling was performed using a model 1000 air pump developed by the Program Executive Office for Chemical Biological Defense (PEO-CBD), fitted with an original DFU filter assembly holding two separate Lockheed Martin polyester filter discs  as previously described . The airTC) values were converted to RNA copies per milliliter or milligram using an equation derived from analysis of serial 10-fold dilutions of in vitro-synthesized FMDV RNA of known concentration. The equations of the curve of RNA copy numbers versus TC values were further adjusted for average mass of tissue samples and specific dilutions used during processing of samples. The qRT-PCR results reported in 10 GCN/µl for all animals sampled at each time point. Results for analysis of air filters are reported as TC values.Two aliquots of each tissue sample collected at necropsy were thawed and individually macerated in tissue culture medium, using a TissueLyser bead beater  and stainless steel beads . Air filters were cut into quarters and disrupted using a similar approach as for tissue samples, but with washed glass beads . The air filter “homogenate” was subsequently centrifuged to extract the fluid that had been absorbed by the filter. Total RNA was extracted from tissue macerates, air filter supernatants, serum, swabs, and OPF samples using Ambion’s MagMax-96 viral RNA isolation kit  on a King Fisher-96 magnetic particle processor . Extracted RNA was analyzed using quantitative real-time reverse transcription PCR (qRT-PCR), targeting the 3D region of the FMDV genome  with forAliquots of macerated tissue samples, air filter supernatants, and TTE-treated probang samples were cleared from debris and potential bacterial contamination by centrifugation through Spin-X filter columns . Samples were subsequently analyzed for infectious FMDV through virus isolation (VI) on LFBK-αvβ6 cells , 42, folAfter screening of tissue samples for FMDV RNA and infectious virus by qRT-PCR and VI, respectively, detection of antigen in cryosections by immunohistochemistry (IHC) and multichannel immunofluorescence (MIF) was performed as previously described , 24. Sli24-specific primers with the SuperScriptIII kit per the manufacturer’s instructions (Thermo Fisher Scientific). Amplicons were quantified using the Qubit 2.0 fluorometer, and NGS libraries were constructed using the Nextera XT DNA library kit  and run on a NextSeq 500. Reads were quality trimmed and mapped to the inoculum consensus sequence using CLC Genomics Workbench v9.5 (Qiagen). Full ORF consensus sequences were subjected to a minimum coverage of 10 reads with quality scoring/vote used for ambiguous base calls. Consensus sequences were aligned using MUSCLE . This experimental cohort included 3 pigs that were all euthanized at 72 hpi, as well as 6 cattle, of which 2 were euthanized for tissue collection at each of 6, 24, and 72 hpe . g MUSCLE , implemeg MUSCLE , and a pMH746921, and animal and air samples, MH559773 to MH559806. NGS data for animal and air samples are available at the Sequence Read Archive with sample identifiers SAMN09580658 to SAMN09580694.FMDV consensus sequence data are available at GenBank with the following sample identification numbers: inoculum,


Central Nervous System (CNS) infections are one of the most critical problems in public health, as frequently patients exhibit neurologic sequelae. Usually, CNS pathologies are caused by known neurotropic viruses such as measles virus (MV), herpes virus and human immunodeficiency virus (HIV), among others. However, nowadays respiratory viruses have placed themselves as relevant agents responsible for CNS pathologies. Among these neuropathological viruses are the human respiratory syncytial virus (hRSV), the influenza virus (IV), the coronavirus (CoV) and the human metapneumovirus (hMPV). These viral agents are leading causes of acute respiratory infections every year affecting mainly children under 5 years old and also the elderly. Up to date, several reports have described the association between respiratory viral infections with neurological symptoms. The most frequent clinical manifestations described in these patients are febrile or afebrile seizures, status epilepticus, encephalopathies and encephalitis. All these viruses have been found in cerebrospinal fluid (CSF), which suggests that all these pathogens, once in the lungs, can spread throughout the body and eventually reach the CNS. The current knowledge about the mechanisms and routes used by these neuro-invasive viruses remains scarce. In this review article, we describe the most recent findings associated to neurologic complications, along with data about the possible invasion routes of these viruses in humans and their various effects on the CNS, as studied in animal models. Respiratory diseases caused by viral agents are one of the most critical problems in public health, as every year they are responsible for high rates of morbidity and mortality, mainly of young children, the elderly and immunocompromised individuals  and the subsequent pathology have been more studied in infection caused by Japanese encephalitis virus (JEV), Varicella-Zoster virus (VZV), measles virus (MV) and human immunodeficiency virus (HIV), among others  and that it can also infect sensory neurons of the lungs, as reported in culture , B (IBV), C (ICV) and D (IDV) viruses belong to the Associations to respiratory pathologies in IV infections have been described, with neurological complications in both children and adults  showed that neurological complications incidence increased, when compared to H1N1 pandemics and remarkably IAV was isolated post-mortem from the brain of a patient , BetaCoV (BCoV), DeltaCoV (DCoV) and GammaCoV  that will eventually be cleaved into 15 or 16 proteins (Desforges et al., Despite HCoV capacities to infect CNS, it has been recently characterized, its presence in human CNS-related samples date back as early as 1980, where the first detection of this virus was performed in autopsy of patients with MS (Burks et al., The first case of SARS-CoV infection with neurological manifestations was reported the year 2003 in a 59-year-old woman (Hung et al., A case report of HCoV-OC43 detection in nasopharyngeal and CSF samples from a child patient was performed the year 2004 (Yeh et al., A recent report—performed the year 2016 by Li et al. (HCoV capacity to reach CNS after the nasal infection has been described previously in mice, particularly for HCoV-OC43 (St-Jean et al., Mice studies are mainly performed with MHV, a virus that belongs to the BCoV genus and is genetically related to HCoV-OC43; likewise, the disease at CNS as elucidated by both viruses are similar, as they both induce demyelination (Jacomy and Talbot, As described so far, CoVs are respiratory viruses that exhibit neurotropic capacities that not only allows them to achieve latency and avoid the immune response of the host, but also have neurological implications that can complicate the disease associated to its infection. Although their mechanisms and routes to reach the CNS have not been elicited yet, the detection of either viral proteins or genetic material in this issue has been confirmed thoroughly, branching the researcher’s goals into acquiring new insights regarding this topic. So far, epidemiological reports have allowed to achieve this, but further work in animal models is required to fully comprehend the mechanisms that CoV uses to reach CNS and to achieve more suitable treatments to resolve this viral infection without an exacerbated disease.Mononegavirales order, Pneumoviridae family and the Metapneumovirus genus (Amarasinghe et al., The hMPV is a new virus first reported the year 2001 in Netherlands (van den Hoogen et al., In humans, there is a handful of reports associated with encephalitis and hMPV-infections. The first approach to this topic was a report described the year 2003 in China (Peiris et al., From this point on, several cases were described where hMPV-infection was related to encephalitis, and in some cases, the detection of genetic material in different CNS samples was detected. The year 2007, the death of a 6-month-old girl was reported 9 days after her admission to a healthcare unit (Hata et al., On the same line, the year 2012 the first report associated with the hMPV genetic material in CSF was published (Sánchez Fernández et al., More cases in which status epilepticus was reported—a single long-lasting seizure or several seizures in a specific time range- were associated with hMPV. A 3.5-year-old girl with hMPV respiratory disease was reported the year 2013 (Niizuma et al., Most recently, cases of adults with acute encephalitis and hMPV detection have been described. The first report was published the year 2015 in Australia, in a 47-year-old man (Fok et al., Remarkably, no studies have been performed describing the ability of hMPV to cause CNS damage in mice. However, it has been described that hMPV can persist in the lung of mice after acute infection (Alvarez et al., hMPV remains a viral agent that needs to be thoroughly characterized, as although most of its respiratory-related pathogenesis has been taken to spotlight, its capacity to achieve latency in CNS cells is yet to be elicited. Although there are extensive case reports that indicate neurological manifestations associated to hMPV-infection in humans, further studies are required in mice models to characterize this disease. Considering that this virus was first described only 17 years ago, there is plenty of work to be done in order to fully comprehend the impact that this virus may be playing in CNS-related pathologies.Respiratory viruses are the leading cause of bronchiolitis and pneumonia throughout the world, affecting varying ranges of ages, but being more aggressive in children, elderly and immunocompromised individuals. Most prominent respiratory viruses are hRSV, IV, CoV and hMPV. Remarkably, extra-pulmonary symptoms have been described in these viruses, highlighting their capacity to cause neurological complications. Febrile seizures, loss of consciousness, convulsion, ataxia, status epilepticus, encephalitis, myelitis, neuritis and MS are among the several extra-pulmonary symptoms that have been described. Case reports of children, elderly and even adults exhibiting these symptoms have been described throughout the years for all of these viruses, bringing to spotlight the urgency to describe their neuroinvasive capacity. Moreover, the detection of genetic material and even viral proteins in CNS samples, such as CSF or brain, is a recurrent fact described in several case reports.Several ways to achieve CNS have been described, including transneural and hematogenous pathways. However, the specific mechanisms responsible for dissemination of each of these viruses with neurotropic capacities into the CNS, have not been thoroughly characterized yet. For instance, hRSV has been reported to reach CNS via the hematogenous pathway, although other routes cannot be discarded entirely. IV can reach the brain via the transneural route, mainly by retrograde axonal transport, reaching first vagal and trigeminal nerves. Likewise, CoV has been described to reach the brain via olfactory bulb, spreading from this point onward into the CNS and the periphery. Since hMPV is an emerging virus, no studies have been performed regarding its capacity to reach CNS, so further data for this virus is required.So far, several mice models have been established for every one of these viruses, allowing the acquisition of new data and the development of new insights of their neurotropic capacities and their neurological manifestations. However, further researches are still required, as many aspects of these CNS pathologies remain unknown. These studies will add new approach edges to a topic that urgently needs to be characterized, as many children may currently be exhibiting these symptoms and are not being treated properly, as the respective viral infection may not be diagnosed.All authors listed have made substantial, direct and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nipah virus (NiV) infection is a newly emerging zoonosis that causes severe disease in humans. Nipah virus is one of the lesser studied of the WHO emerging pathogens for which research is a priority. Survival and persistence data is important for risk management and understanding the hazard of the virus for laboratory and health care workers that may work with the virus and we present some initial findings on the survival of Nipah virus in blood and tissue culture media under different conditions. The titre of Nipah virus in blood or media at two different temperatures and exposed or sealed to the atmosphere was measured every day for three days and after a week. Nipah virus was very stable in blood in closed tubes held at room temperature with minimal decay over seven days. Decay was observed in all the other conditions tested and was more rapid in samples exposed to the atmosphere. Persistence data is useful for safety planning and risk management. Nipah virus (NiV) infection is a newly emerging zoonosis that causes severe encephalitic and respiratory disease in humans. NiV infection has a high human case fatality rate estimated to be between 40 and 75% . NiV is NiV is one of the lesser studied of the WHO emerging pathogens  and whil6 TCID50/mL. NiV-M was titrated by 50% tissue culture infectious dose (TCID50) assay to enumerate viable virus. Briefly, NiV-M was added to the first column on a 96-well cell culture plate of confluent Vero C1008 cells. Serial ten-fold dilutions were made across the plate for nine dilutions. Plates were incubated at 37°C and 5% CO2 for 7 days and then observed under a microscope for cytopathic effects. Each column of 8 wells of cells was scored for the presence or absence of CPE and the 50% end-point calculated by the methods of Reed & Muench [50 assay being performed. Each experiment was repeated on three separate occasions from the same initial starting stock of NiV-M. Samples were titrated immediately and were not frozen and thawed prior to enumeration. All work with NiV was performed in Dstl’s Biosafety Level 4/Advisory Committee for Dangerous Pathogens Level 4 laboratory.Nipah virus-Malaysia (NiV-M) was kindly provided by P. Rollin, CDC. At CDC it was derived from the “199901924” isolate and had undergone 2 passages in Vero-E6 cells and one in RK13 cells. At Dstl it was passaged a further two times in Vero C1008 cells to produce a working stock of 1 × 1050/mL in TCM and 1.78 × 103 TCID50/mL in blood. At 30°C in sealed tubes there was more of a drop off in titre indicating lower survival compared to the lower temperature. In open tubes there was a rapid drop off in titre in blood and TCM at 30°C and a more gradual decrease in titre in TCM at room temperature. No NiV-M was detectable after a week after storage at 30°C in either matrix  or no v of 37°C  or pigs  of 37°C ) has bee of 37°C  and in h of 37°C . If NiV Future work could expand the environmental conditions, surfaces and matrices tested, such as higher temperatures and altered relative humidity and/or survival on metal surfaces or on materials and/or survival in other bodily fluids. We would also like to perform the same experiments with a Bangladesh NiV (NiV-B) isolate (if available) to compare the two types. We also plan to investigate the aerosol survival of NiV to further help in risk assessment and understanding the factors that might be involved in transmission. This preliminary data can also be expanded to look at disinfection and inactivation of NiV in relevant matrices which will be important for understanding the hazard and informing risk assessments and safety decisions.


Human infections with vaccinia virus (VACV), mostly from laboratory accidents or contact with infected animals, have occurred since smallpox was eradicated in 1980. No recent cases have been reported in China. We report on an outbreak of VACV from occupational exposure to rabbit skins inoculated with VACV. Orthopoxvirus [OPV]) is used as a lyophilized live virus vaccine against smallpox, variola virus . We conducted real-time PCR to screen for OPV  to isolate the virus in positive samples, which showed characteristic cytopathic effects, including cell rounding, loss of adherence, and cellular debris 48 hours postinfection. We extracted viral DNA using the QIAamp MinElute Virus Spin Kit   and constructed a phylogenetic tree with MEGA5 (https://www.megasoftware.net). To establish the relationship between the illness and exposure, we collected residue from the pulverizer blade and outlet and from the surrounding floor. We used real-time PCR to detect VACV-specific nucleic acids in these specimens. We amplified the hemagglutinin gene A56R and sequenced it using an Applied Biosystems 3130 Genetic Analyzer . All sequences showed 100% identity match with each other and had 99.6% identity match with isolate VACV-MNR-76 (GenBank accession no. DQ792504) . UnfortuEpidemiologic and molecular data implicated VACV as the etiology of this outbreak, the source of which was rabbit skins inoculated with smallpox vaccine. The phylogenetic tree revealed a close relationship between the isolated strains and the TianTan strain , a highlCase-patients 3, 4, and 5 received prior smallpox vaccination and had less severe clinical manifestations than the other 2 case-patients, indicating that vaccination might have reduced illness severity but failed to protect them from infection (VACV is commonly used in research settings and the biopharmaceutical industry. Functional studies on extracts from rabbit skins inoculated with VACV are common in China (Additional information on outbreak of vaccinia virus infection from occupational exposure, China, 2017