Apostichopus japonicas) in the CMNV host range investigation. The amplicons of RT-nPCR from sea cucumber were sequenced, and its sequences showed 100% identity with the RNA-dependent RNA polymerase gene of the original CMNV isolate. Histopathological analysis revealed pathologic changes, including karyopyknosis and vacuolation of the epithelial cells, in the sea cucumber intestinal tissue. The extensive positive hybridization signals with CMNV probe were shown in the damaged epithelial cells in the in situ hybridization assay. Meanwhile, transmission electron microscopy analysis revealed CMNV-like virus particles in the intestine epithelium. All the results indicated that the sea cucumber, an Echinodermata, is a new host of CMNV. This study supplied further evidence of the wide host range of CMNV and also reminded us to pay close attention to its potential risk to threaten different aquaculture animal species.Cross-species transmission of emerging viruses happens occasionally due to epidemiological, biological, and ecological factors, and it has caused more concern recently. Covert mortality nodavirus (CMNV) was revealed to be a unique shrimp virus that could cross species barrier to infect vertebrate fish. In the present study, CMNV reverse transcription-nested PCR (RT-nPCR)-positive samples were identified from farmed sea cucumber ( For most viruses, the virus–receptor interactions determine viral host range and therefore constitute the interspecies barrier of viral infection, eventually leading to the strong host specificity of a virus ,2. HowevNodaviridae is composed of two genera, Alphanodavirus and Betanodavirus [Alphanodaviruses were mostly isolated from insects and their host range also appears to be restricted to insects [Culex tritaeniorhynchus) [Costelytra zealandica (Coleoptera: Scarabaeidae), is capable of replicating in many species of plants, including chenopodium Chenopodium hybridum, barley Hordeum vulgare, and tobacco Nicotiana tabacum [Betanodavirus mainly infects larvae, juvenile or adult marine fish, and is different from Alphanodavirus [odavirus . Alphano insects , except  insects ,13. NoV hynchus) ; howeverhynchus) ,15. FHV, tabacum ,16. Therodavirus ,18,19.Penaeus vannamei, Penaeus chinensis, Marsupenaeus japonicus, and Penaeus monodon, and VCMD outbreaks in shrimp farms represent a significant threat to the shrimp culture industry [Parathemisto gaudichaud, amphipod Corophium sinense Zhang, and the ghost crab Ocypode cordimandus) and several fish species  are also natural hosts of CMNV. And the host range spanning crustaceans and fish illustrates the capacity of CMNV to spread crossing the species barrier [Covert mortality nodavirus (CMNV) is an alphanodavirus first isolated from shrimp with viral covert mortality disease (VCMD) in China and later also found in Thailand and Ecuador ,21,22,23 barrier ,25,26.Holothuroidea, which belongs to the invertebrate Echinodermata. Apostichopus japonicus is the sea cucumber species with the largest social demand, and its aquaculture has become an emerging marine industry [in situ hybridization (ISH), histopathology, and transmission electron microscopy (TEM). Our study provides significant novel insights into the new natural host discovery of CMNV.Sea cucumber (Holothuians) is a common marine invertebrate , and it industry ,29,30. IP. vannamei collected from polyculture ponds of shrimp and sea cucumber was infected with CMNV. Considering the capacity of CMNV cross-species infection and the needs of further exploration of its host range, four sea cucumber individuals   a,b were re pond) c was selThe total RNA of intestinal tissues was extracted from four sea cucumber individuals using RNAiso Plus Reagent  according to the manufacturer’s instructions. The tissues samples were first homogenized in RNAiso Plus, and then trichloromethane was added into the homogenate for protein degeneration. Finally, isopropanol was used to get total RNA from liquid supernatant obtained by centrifugation. The concentration and purity of purified RNA were measured by Nanodrop 2000 .The total RNA from the intestinal tissue of sea cucumber was used as a template for RT-PCR analysis. First, the first-step PCR amplification was conducted by using the template of 1 μL total RNA (concentration of the template was 100–200 ng/μL) and a PrimeScript One Step RT-PCR Kit  with the primer sets of CMNV-F1/R1  according to the recommended procedures, and the annealing temperature was 52 °C. Using the first-step RT-PCR products as templates, the second-step PCR was carried out using a TaKaRa Ex Taq Kit  with the primer sets of CMNV-D-F1/R1 , and the annealing temperature was 52 °C. The expected CMNV target fragments of the first and second rounds of the PCR amplifications were 619 bp and 413 bp amplicons from the CMNV RNA-dependent RNA polymerase (RdRp) gene, respectively. Then, the amplicons were resolved by 2% agarose gel electrophoresis for 0.5 h.https://www.ncbi.nlm.nih.gov/, accessed on 4 February 2021). Then, the phylogenetic tree, based on 25 relevant RdRp protein sequences retrieved from the GenBank database .The amplicons (413 bp) from the second step of the RT-nPCR of sea cucumber were sent for commercial sequencing to Sangon Biological Engineering  Co. Ltd. The obtained 413 bp RdRp gene fragment sequences were subjected to multiple sequence alignment by the online software of BLASTn  were first preserved in 2.5% glutaraldehyde for 24 h at 4 °C, and then further fixed in 1% osmium tetroxide for 2 h. Finally, the tissues were embedded in plastic resin [To detect the presence of CMNV particles in sea cucumber, the intestinal tissues  in the RT-nPCR assay a. In addAlphanodavirus, and that members from Betanodavirus were clustered into the other independent branch  from the second-step PCR showed that the CMNV sequences of sea cucumber shared 100% identity with the known CMNV RdRp gene (GenBank no. KM112247). Phylogenetic analysis revealed that the deduced amino acid sequences of CMNV RdRp from sea cucumber samples were clustered closely into the embranchment of the primordial CMNV isolate b. Additit branch .Purple positive hybridization signals of CMNV probe were widely present in the intestine of CMNV RT-nPCR-positive sea cucumber c, and esTEM analysis was used to further confirm the CMNV infection in the sea cucumber. TEM micrographs of the ultrathin sections of the intestinal tissue of sea cucumber revealed the presence of massive CMNV-like virus particles of about 28–32 nm in the intestinal villus epithelium cells a–d.Mugilogobius abei and Japanese flounder Paralichthys olivaceus [Carassius auratus [Recently, the phenomenon of cross-species transmission of a few viruses has negatively affected areas such as human life, animal husbandry, and aquaculture and, meanwhile, also piqued the concern of scholars ,39,40. Nlivaceus ,25, as wMugilogobius abei infected with CMNV [CMNV-positive individuals were found in the farming sea cucumber by RT-nPCR. Thus, a further investigation was performed to confirm the infection of CMNV in sea cucumber. The histopathological analysis revealed that obvious pathological lesions, including karyopyknosis and vacuolation of the epithelial cells, occur in the sea cucumber intestinal tissue, and these lesions are similar to the intestinal lesions of gobiid fish ith CMNV . Consistith CMNV ,42,43, aVibrio harveyi [Vibrio splendidus [Lactococcus garviaeae [During the past decade, the rapid and disorderly expansion of sea cucumber aquaculture was companied by various diseases that caused high mortality of sea cucumbers, resulting in grievous economic losses to the culture industry of sea cucumber ,45. Freq harveyi , Vibrio lendidus , and Lacarviaeae , while sarviaeae ,50,51. HIt is known that sea cucumber, a scavenger, is the pivotal component of the marine environment , playingOverall, the results of this study demonstrated that CMNV is a broad host range virus that deserves close attention to its potential risk to infect more aquaculture animal species. Considering the key ecological function of sea cucumber in the marine environment and the high economic value of this aquatic species, the potential negative impact of the spread and prevalence of CMNV in sea cucumber on the marine ecosystem and social economy should be of concern. The present study contributes to further investigation of the viral pathogen of diseased sea cucumber.


It has always seemed likely that viruses originated early in the history of life. However, until the identification of endogenous viral elements (EVEs), there was little if any direct evidence for most virus groups ever having existed in the distant past . EVEs arBornaviridae) are a poorly understood group of single-stranded negative sense RNA viruses  that infect vertebrates. Until 2015 the family contained only one genus (Orthobornavirus); however, recent progress in sampling viral diversity has led to the establishment of two novel genera (Carbovirus and Curtervirus) (Bornaviruses (family ervirus) . The proervirus) . The asservirus) ] of zoonervirus) .A zoonotic transfer event involving a novel bornavirus was recently recorded in Germany following the deaths of three men from progressive encephalitis. Strikingly, all three were also breeders of exotic squirrels, and this epidemiological connection soon led investigators to identify the pathogen responsible—variegated squirrel bornavirus 1 (VSBV-1) . RetrospInformation archived in EVE sequences can reveal the long-term evolutionary history of viruses, leading to a more complete understanding of their biology. The distribution and diversity of bornavirus EVEs have been investigated previously, but no previous study approaches the scale of that performed by Kawasaki et al. They searched the genome sequences of 969 eukaryotic species for bornaviral EVEs and identified hundreds of unique loci. Most of these bornavirus “fossils” are highly degraded and fragmentary, making it difficult to do meaningful comparative analyses. However, by artfully combining high-throughput computational approaches with careful human oversight, Kawasaki et al. tease out the evolutionary connections between contemporary bornaviruses and the extinct “paleoviruses” represented by EVE sequences, thereby reconstructing a detailed picture of bornavirus evolution . NumerouOf all the kinds of inferences that can be extracted from EVE sequence data, age calibrations based on the detection of orthologous EVEs in related species are among the most reliable, since they are derived in part from an orthogonal data source (the fossil record of host species) . HoweverFor an EVE sequence to arise, DNA derived from a virus genome has to become integrated into the genome of an infected germline cell. Current evidence indicates that in the case of bornaviruses and other mononegaviruses, integration usually occurs via LINE1-mediated retrotransposition: Viral messenger RNA (mRNA) becomes associated with a LINE1 protein complex, which leads to it being reverse-transcribed into DNA and integrated into the nuclear genome . A telltThe frequency with which bornavirus EVEs occur in certain vertebrate groups raises questions about their potential functional roles as co-opted or exapted host genes. Previous work has found evidence that some have antiviral functions, and conceivably bornaviral EVEs could be involved in other biological processes –15. PracThe PNAS study illustrates some of the limitations of nonretroviral EVE data. Even though bornavirus-derived EVEs are quite common in some groups, they are sparsely and patchily distributed overall. This can make it quite difficult to interpret the relationship between EVE distribution and diversity and long-term virus–host interactions. For example, the absence of EVEs from some species groups might superficially seem to imply the historical absence of virus, but other factors impacting the distribution of EVEs need to be considered. These are not restricted to the biological aspects of virus replication that increase the likelihood of germline integration —all of which could potentially vary greatly among virus strains and host species—but also encompass a broad range of factors influencing the frequency with which integrated EVE sequences become fixed in the germline. Straightforward population genetics laws dictate that the vast majority of EVEs will be eliminated from the gene pool within a few host generations. Consequently, factors impacting the likelihood of fixation  could potentially have played an important role in shaping EVE distributions, and this may distort our impressions.While EVE-based studies have limitations, there is no denying their fundamental impact on our understanding of virus evolution. Increasingly, this is being reflected in studies of contemporary viruses as date calibrations allow species diversity and biological properties to be placed into evolutionary context , 16. In


Pneumococcal disease contributes significantly to childhood morbidity and mortality and treatment is costly. Nigeria recently introduced the pneumococcal conjugate vaccine (PCV) to prevent pneumococcal disease. The aim of this study is to estimate health provider and household costs for the treatment of pneumococcal disease in children aged <5 years (U5s), and to assess the impact of these costs on household income.We recruited U5s with clinical pneumonia, pneumococcal meningitis or pneumococcal septicaemia from a tertiary level hospital and a secondary level hospital in Kano, Nigeria. We obtained resource utilisation data from medical records to estimate costs of treatment to provider, and household expenses and income loss data from caregiver interviews to estimate costs of treatment to households. We defined catastrophic health expenditure (CHE) as household costs exceeding 25% of monthly household income and estimated the proportion of households that experienced it. We compared CHE across tertiles of household income (from the poorest to least poor).Of 480 participants recruited, 244 had outpatient pneumonia, and 236 were hospitalised with pneumonia (117), septicaemia (66) and meningitis (53). Median (IQR) provider costs were US$17 (US$14–22) for outpatients and US$272 (US$271–360) for inpatients. Median household cost was US$51 (US$40–69). Overall, 33% of households experienced CHE, while 53% and 4% of the poorest and least poor households, experienced CHE, respectively. The odds of CHE increased with admission at the secondary hospital, a diagnosis of meningitis or septicaemia, higher provider costs and caregiver having a non-salaried job.Provider costs are substantial, and households incur treatment expenses that considerably impact on their income and this is particularly so for the poorest households. Sustaining the PCV programme and ensuring high and equitable coverage to lower disease burden will reduce the economic burden of pneumococcal disease to the healthcare provider and households. Children <5 years have the highest incidence of pneumonia and invasive pneumococcal disease (IPD) and Nigeria bears the largest burden in sub-Saharan Africa.Pneumococcal conjugate vaccine (PCV) was introduced in Nigeria in 2016 to reduce the burden of pneumococcal disease.PCV is currently subsidised through Gavi (the Vaccine Alliance) financial support and Nigeria will transition to full self-financing in a few years.There is no contextual evidence in Nigeria on economic burden of IPD to the health system and society that can support longer term investments in PCV when Gavi co-financing terminates.Treatment of one hospitalised episode of pneumococcal disease cost on average, US$300 to the provider, and US$83 to the household with significant variation by clinical syndrome and level of care.Overall, one-third of the households encountered costs that were catastrophic .Burden of catastrophic health expenditure varied by household income tertile ranging from 4% in the least poor households (highest income tertile) to 53% in the poorest households (lowest income tertile).Despite the short illness duration, pneumococcal disease syndromes result in huge economic costs to providers and households.Sustaining the PCV programme and achieving high PCV coverage has the potential of saving resources at both provider and household level.Households are at risk of further impoverishment from catastrophic expenses associated with treatment of pneumococcal disease.This risk can also be significantly reduced by PCV.Introduction of the pneumococcal conjugate vaccine (PCV) has significantly reduced the global burden of pneumococcal disease.With >1 million pneumococcal disease cases resulting in nearly 50 000 deaths among U5s in 2015, Nigeria has the highest burden of pneumococcal disease in sub-Saharan Africa (sSA).14Financing of healthcare in Nigeria is via multiple and largely uncoordinated channels.20 21Ahead of Nigeria completing the transition to full self-financing of PCV in 2028,The objectives of this study are to: (1) to estimate the provider costs of outpatient and inpatient clinical pneumonia, and inpatient pneumococcal septicaemia and meningitis; (2) to estimate the household costs of hospitalised clinical pneumonia, and pneumococcal meningitis and bacteraemia; and (3) to assess the economic impact to households of hospitalisation with clinical pneumonia, and pneumococcal septicaemia and meningitis among U5s in Kano, northern Nigeria.2.25This was a cross-sectional study conducted at the two largest paediatrics units in Kano, Kano State in northern Nigeria—Aminu Kano Teaching Hospital (AKTH) and Murtala Muhammed Specialist Hospital (MMSH)—which serve an overlapping catchment population. AKTH is a Federal Government Teaching Hospital and MMSH is a State Government Secondary Hospital. Kano is the capital city of Kano state and covers approximately 8 out of the 44 local government areas (LGAs) of the state. However, catchment population of both hospitals includes other LGAs outside the city and neighbouring states. The description of the hospitals is shown in Children were recruited prospectively and were eligible if aged 1–59 months, presented to AKTH or MMSH and had at least one of three possible diagnoses of interest. These were (1) clinical pneumonia, (2) pneumococcal septicaemia and (3) pneumococcal meningitis. We excluded children that died during admission.We used the formula belowWhere: n=minimum sample size.Z=the standard normal deviate for the desired confidence level .σ=SD deviation of respective mean costs reported from previous studies.8 9e=precision or smallest desirable margin of error allowable for estimation of the respective costs.Sample sizes of 100, 50 and 30 were expected to provide a cost estimate for outpatient clinical pneumonia, inpatient pneumonia and septicaemia and meningitis based on SD (±precision) of US$5 (±US$1), US$21 (±US$6) and US$33 (±US$12), respectively.8 9 27We recruited outpatient pneumonia cases and interviewed caregivers on the day of diagnosis. For inpatient pneumonia cases, eligible children admitted from 08:00 to 16:00 were recruited on the day of admission; those admitted from 16:00 to 08:00 the next morning were enrolled on the next day. For septicaemia and meningitis cases, participants were recruited when confirmatory laboratory results were available. We collected data between January and October 2020. For each hospital, we recruited a volunteer nurse not directly involved in clinical care to collect data. We used structured quantitative tools adapted from a similar study in The Gambia for data collection.9We extracted resource use data such as length of hospital stay, type and quantity of medications and intravenous fluids used, laboratory investigations and other specialised services including blood transfusion and use of oxygen from patients’ records, case folders, prescriptions and laboratory request forms. We obtained unit costs of hospital resources, for example, medication, fluids used from the respective hospitals see .10.1136/bmjgh-2021-007080.supp1Supplementary dataSociodemographic characteristics, OOP costs, non-medical expenses, productivity time loss, household income and sources of finances used to pay the treatment costs were obtained through caregiver interviews. Additional data on household income and sources of finances used to pay the treatment costs were also collected.We collected provider costs, and direct and indirect household costs.Provider costs included costs of direct healthcare services, that is, costs of medications, laboratory investigations, intravenous fluids, oxygen, blood transfusion and inpatient bed-day. We used full costs for drugs and only applied dose-specific costs if the drug was re-useable and the residuals amounted to another full dose. For instance, for a re-useable drug, if a unit dose was 1000 mg and 500 mg was administered, the cost per dose would be half of the unit cost. We obtained cost of oxygen from the nursing unit, and for blood transfusion, we used previously published costs.28The inpatient bed-day is the daily stay cost or the ‘hotel’ component and comprises costs of food, personnel and utilities. We used the average 2020 admission charge for AKTH and assumed admission costs at MMSH to be 60% of AKTH since admission is ‘free’ to patients at MMSH. Studies have found up to 60%–70% differences in bed-day costs between tertiary-level and secondary-level hospitals.8 9We collected direct healthcare costs to households which are user fees related to consultations, investigations and medications incurred from date of admission to date of discharge. Non-healthcare costs were the costs of transportation, accommodation and feeding incurred during admission (from date of admission to date of discharge) by main and accompanying caregiver. The accompanying caregiver was defined as any household member that assisted the main caregiver with care of the patient during the admission. Preadmission costs data were collected either on the day of recruitment or on the earliest convenient day for the caregiver, while data on costs incurred over the course of admission were obtained at or close to discharge.We collected data on caregiver’s income and productivity time loss due to time away from their usual activities owing to illness.Data were analysed using Stata V.15.1 (Stata Corp LP). All costs were converted to US$ using average 2020 conversion rates 1 US$=360.5 NGN .29We summed up the components of respective cost categories for provider costs and direct household costs. We estimated indirect costs using the human-capital approach (HCA) by estimating income lost by caregiver(s) due to absence from work per day spent caring for the child. Self-employed caregivers were asked to give an estimate of daily earnings while those on monthly salary were asked to state their monthly wage from which daily income was calculated. Indirect costs were then calculated as daily income multiplied by the number of days taken off from work.We present costs from the health provider and household’s perspectives along with their components separately as means with SD, and medians with IQR. We used Kruskal-Wallis test to assess differences in costs between disease categories and Wilcoxon rank-sum test to compare costs between the two hospitals.25) and also explored impact at 10% (CHE10) and 40% (CHE40) thresholds. We used multivariable logistic regression models to identify factors associated with CHE25. Independent variables that were associated with CHE25 at significance level p=0.1 were sequentially added to the model and kept if they were significantly associated with cost (p<0.05) or changed effects of included variables. Excluded variables were then re-introduced to check if they further changed the effect sizes of included variables. Adjusted ORs and p values from the likelihood ratio test (LRT) are reported.We evaluated the impact of health expenditures on available household resources by assessing direct, indirect and total costs as respective proportions of household income. We used household income to categorise households into tertiles from the poorest (tertile 1) to least poor (tertile 3). We used Kruskal-Wallis tests to compare costs as a proportion of household income across household income tertiles.We conducted one-way sensitivity analyses of provider costs by varying the source of bed-day costs. We used the average cost per inpatient bed-day for tertiary and secondary facilities in Nigeria from the WHO-CHOICE after accounting for inflation and adjusting to 2020 rates.Patients were not directly involved in the design, conduct, reporting or dissemination plans of this research.Overall, 480 out of 495 caregivers of eligible children consented to be interviewed. A total of 480 children  were enrolled see . Of thesThe median provider cost for outpatient pneumonia was US$17 (IQR: 14–22), and was higher in AKTH  compared with MMSH . Overall, costs for outpatient clinic visit, medications and investigations accounted for 43%, 37% and 20% of provider costs. The median costs for laboratory investigations were higher in AKTH US$7 (IQR: 0–8) compared with MMSH US$0 . Medications costs were similar between the two hospitals. Median expenses on seeking care elsewhere prior to index presentation were US$9 (IQR: 0–13) in AKTH and US$8 (IQR: 5–13) in MMSH (p=0.70).The respective median/mean provider costs, as shown in Median household income was similar between disease categories but was significantly higher  for those presenting to AKTH  compared with MMSH . Majority of caregivers  reported using a combination of current income and savings to cover expenses. Only about 3% reported using other sources such as borrowing, asking relatives or selling assets to cover expenses.Median direct household costs as shown in Median indirect costs were lowest for inpatient pneumonia compared with meningitis or septicaemia, as shown in The poorest households spent a median of 25% of their monthly income directly on treatment costs and lost an additional 8% from loss of caregiver time, compared with 13% of income and 6% of caregiver time for the least poor households (data not shown). Treatment costs as fractions of monthly household income were inversely related to household income tertiles see .25 at different threshold cut-off values across household income levels. CHE was substantial, and at 10% threshold nearly all households across all income levels encountered CHE. CHE increased with decreasing household income level. This inverse relationship is further illustrated in 25, while having ≥3 U5 children and higher indirect costs lowered the odds of CHE25 , meningitis or septicaemia, seeking care at a private hospital prior to admission and higher provider costs were associated with increased odds of CHEHE25 see .Provider costs were sensitive to source of hospital (bed-day) costs. Provider costs were between 38% and 40% lower across all disease categories and between the hospitals when the WHO-CHOICE estimates were used  comparedIndirect costs were also sensitive to approach used as costs were higher for all conditions in both hospitals with the WTP  comparedIn this study, we estimated the costs of treatment of clinical pneumonia, septicaemia and meningitis in Nigerian children as well as the economic impact of these costs on households. Costs varied by hospital as they were higher in the tertiary hospital (AKTH) for all disease categories. Provider costs also varied by illness and were highest for meningitis irrespective of hospital. Costs to households were similar between the hospitals but highest for meningitis and lowest for pneumonia. The economic impact to households was considerable with total costs to households ranging between 25% and 37% of monthly household income for 5–7 days of hospitalisation. One-third of the households incurred CHE at 25% threshold and the poorest households bore the greatest burden of CHE.Although provider costs are likely to vary across the country and between hospitals, when applied to the global burden of disease and the proportions of the different pneumococcal disease syndromes, our cost estimates translate to annual provider costs of >US$110 million, that is, ~9% of Nigeria’s 2020 health budget.For each IPD category and hospital in our study, ‘hospital stay’ accounted for the largest proportion of provider costs. This is similar to findings in diverse settings across Africa (Kenya and The Gambia), Asia (India and Vietnam) and South America.39We found substantial household costs ranging from US$44 for pneumonia to US$72 for meningitis. These figures are higher than reported for malaria in Nigeria, which ranged from ~US$7 for outpatients to ~US$10 for hospitalised cases25 in one-third of households. We note the differences in CHE in ours compared with other studies in Nigeria. Although several studies analysed nationally-representative surveys, their findings differ from ours because they do not address a specific illness, target chronic conditions and may have limited applicability to our study setting due to subnational differences in healthcare seeking behaviour.10, the cost of treatment costs had significant burden on households regardless of their income. At higher thresholds, our results are similar to others including those that used household income as a measure of available resources like we did.18 43 44 46 47Costs incurred during treatment had considerable economic impact to households particularly for large households dependent on little income. With large numbers of economic dependents per household, that is, non-income earning household members, a 1-week illness of one child, resulted in CHE25 almost fourfold. This suggests that the benefits of the contributory scheme are yet to reach this target population, possibly due to lack of awareness and unwillingness to pre-pay for CBHI.21Unplanned treatment expenses are likely to affect other household expenditures. OOP payment for healthcare can provide obstacles for treatment access particularly to the poorest, skewing treatment seeking towards only those that can afford to pay.25. OOP payment from available household resources is the predominant way of financing healthcare in Nigeria. However, other non-health expenditures  are financed with the same household resources as healthcare, and if substantial, can reduce resources ‘available’ for health expenditure.Having a salaried job, ≥3 children U5 in the household and higher indirect costs (HCA) reduced the odds of CHEThis study has some limitations. First, the costs here do not account for full costs to the provider because capital costs are excluded due to challenges in accessing such data. Second, we collected data on household costs on admission and close to or at discharge to limit bias because admission duration was short. However, there is still a risk of recall bias. Second, costs exclude children who died during admission and may have incurred costs from higher resource use due to more severe disease. Third, we only estimated time loss for the primary caregiver in hospital which does extend to fathers or other household members. Another limitation is the use of monthly income rather than household (or non-food) expenditure to assess CHE which may not identify the different ways of health financing. However, we believe current income reflects current resources and captures the current household capacity to pay for expenses of treatment given the short-term duration of illness. That majority of caregivers reported using both current income and savings to cover healthcare expenses supports our decision to use current income rather than long-term household asset. Lastly, subnational differences in household incomes may limit generalisability of our findings, particularly the costs to household cost and its economic impact. However, some components of provider costs such as bed-day costs are not likely to differ at the tertiary hospital level because these hospitals are directly funded by the Federal Government. Additionally, because many states also offer subsidised health services to children at the secondary, our findings may be generalisable to such settings.Our analyses illustrate the treatment costs of pneumococcal disease to providers and households in Nigeria. Hospitalisation particularly at tertiary level is associated with substantial costs to both the provider and households. Households incur expenses prior to diagnosis and incur substantial direct and indirect costs that has significant impact on their incomes.Our findings have important implications for policy. First, it is evident that the PCV programme, by averting disease, can free up scarce resources for the health sector to divert to competing health problems, reduce unexpected expenditures and CHEs and increase resources within household for savings and essential non-health expenditures. So, it is essential to achieve and maintain high PCV coverage levels to reduce this financial burden, especially for the poorer households. Second, due to higher cost of providing care at the tertiary level, strengthening lower levels of care to provide early treatment will also significantly reduce provider costs and reduce strain on the health sector resources. Finally, the current mechanisms for financing health expenditures are inadequate to protect households from catastrophic expenses. Given that OOP payments were mainly driven by medication costs, the state government when declaring ‘free’ health services should have a realistic plan for uninterrupted supply of drugs and other essential commodities/consumables to ensure that the families are not obliged to pay for these OOP.


A superior analgesic method in perioperative pain-management of patients receiving total knee arthroplasty is the subject of controversial debate. Although higher cost-efficiency is claimed for the local infiltration analgesia (LIA), there is a lack of data on its costs compared to peripheral nerve block anaesthesia (PNBA). The goal of this study was to investigate the differences in immediate perioperative costs between the LIA and PNBA in treatment of patients receiving total knee arthroplasty.The comparison was conducted based on a randomized controlled clinical trial examining 40 patients with elective, primary total knee arthroplasty . The analysis included surgical case costs, anaesthesiological case costs, material, costs of postoperative opioid requirements and catheter review visits for patients receiving PNBA.p = 0.851). While there was no statistically significant difference in surgical case costs, the anaesthesiological costs were lower with the LIA procedure . Material costs in the LIA group were 4.18€/patient and 94.64€/patient with the PNBA. Costs for postoperative opioid requirements showed no statistically significant difference between the two procedures.The overall mean costs for the LIA-group were 4328.72€ and 4368.12€ for the PNBA  and registered with data safety authorities. Study patients provided written informed consent to participate in the trial. Study registry: ClinicalTrials.gov,  The local infiltration analgesia (LIA) and peripheral nerve block anaesthesia (PNBA) are techniques in the postoperative pain-management of patients undergoing major orthopaedic surgery such as total knee replacement.PNBA – a regional anaesthesia procedure with/without catheters - is seen as an effective method in postoperative pain-management of endoprosthesis implantation . The LIAHowever, research-based discussion on the preferred method remains controversial , 5. SomeDue to this ambivalent data, there is a rising interest in economic analyses complementing clinical and more patient-orientated aspects of determining which procedure is preferable . PreviouClinicalTrials.gov, NCT03114306) [The study is a secondary analysis of a randomised controlled trial (RCT) published by Kastelik et al. (study registry: 3114306) . The bac3114306) .The main subject of the secondary analysis at hand was the difference in overall case costs between LIA and PNBA procedures.All patients received postoperative analgesia following a standardised protocol of opioids , dipyrone and non-steroidal anti-inflammatory drugs . For additional analgesic treatment in case of acute pain scores on the numeric rating scale (NRS) > 6, patients received 10 mg of oral morphine.During the LIA procedure the surgeon injects 150 ml Ropivacain 0.2% into the periarticular structures, thereby blocking the sensitive nerve endings of the knee. The targeted structures are the subcutaneous and periarticular soft tissues as well as the joint capsule. LIA is often administered during the orthopaedic implantation of endoprosthesis, immediately before suture .For PNBA, ultrasound-guided sub-gluteal block of the sciatic nerve as well as the adductor canal block with anaesthesia of the saphenous nerve were performed in the primary study . Both neTo calculate the overall case costs for each procedure we summed up the surgical case costs, anaesthesiological case costs, postoperative opioid requirements, material costs and costs of catheter review visits (only in PNBA procedure). The specification for the times used for the calculations was taken from Bauer et al. on perioperative process times and indicators  and is sSurgical case costs were made up of the price per minute for personnel , operational infrastructure and material (33.77€) and were multiplied by the incision to suture time (O8-O10 ). The exAnaesthesiological case costs consisted of the price per minute for personnel (doctor and anaesthesiology nurse), infrastructure and material (10.28€) and were multiplied by the total time of anaesthesia (beginning of the anaesthesiologist’s presence with the patient until end of patient monitoring by the anaesthesiologist in the operating room (OR) or similar (A5-A9) ).Material costs Tables  and 2 weOpioid costs for the treatment of acute postoperative pain included the individual and average dosages of tilidine/naloxone retard and oral morphine 10 mg tablets [Catheter review-visits for patients receiving the PNBA were calculated with 6.25€ per visit based on the assumption of a before-tax income of 25€/h of an anaesthesiological nurse and an average time per visit of 15 min.Total costs were obtained by the summation of the above variables. All costs and prices were taken from the expenses of the Charité–Universitätsmedizin Berlin at the time the study was conducted.The statistical analysis of our data was conducted in IBM SPSS 25. Descriptive data was summarised by mean and standard deviation or median depending on statistical distribution. The t-test for independent samples was used to test for statistical significance of parametric data. Data for opioid requirement for postoperative acute pain as well as for induction costs were analysed using the Mann-Whitney-U-Test. For all statistical analysis a 5% two-sided alpha level was applied. A case number analysis was not conducted for this secondary end-point as this was a descriptive non-confirmatory supporting analysis. The statistical graphic was originally produced with SPSS 25 revised as vector graphics using Affinity Designer 1.8.6. Images were designed to be accessible for people with anomalous trichromacy, dichromacy and monochromacy.p = 0.132).Mean surgical case costs within the OR for the LIA procedure were 2933.81€ (SD ± 463.94€). For the PNBA mean costs were 2690.67€ (SD ± 532.67€). There was no statistically significant difference between the two procedures (p = 0.048).Mean anaesthesiological case costs for the LIA were 1370.26€ (SD ± 209.54€) and 1542.45€ (SD ± 313.12€) for the PNBA. This difference was statistically significant . Mean costs of cumulative postoperative oral morphine per patient were 1.46€ (SD ± 2.08€) for LIA and 1.59€ for PNBA (SD ± 2.08€) (p = 0.646).There was no statistically significant difference in the cumulative use of postoperative opioids. Mean costs of cumulative postoperative tilidine/naloxone per patient were 19.01€ (SD ± 2.44€) for LIA and 17.22€ (SD ± 4.85€) for PNBA (Mean costs for catheter review-visits amounted to 21.56€ per patient (SD ± 4.74€) and were added to the PNBA-group only, as outlined above.p = 0.851). Figure Overall, the mean costs for the LIA procedure were 4328.72€ SD ± 644.14€) per patient compared to 4368.12€ (SD ± 677.24€) for the PNBA procedure. This difference was not statistically significant (4€ per pap = .758) [The authors of the primary study reported no significant postoperative complications that may have had an impact on costs with either procedure and there was no significant difference in total hospital length of stay between the two groups ( = .758) . There wOur analysis shows that there seems to be no statistically significant difference in immediate perioperative costs between the two analgesic procedures in our hospital. Indeed, both concepts seem to provide fast-track mobilisation and allowed early in-hospital recovery after surgery and ambulation maintaining high patient comfort with comparable costs (patient-related outcome measure).Although the results show that the anaesthesiological case costs were significantly lower for the LIA procedure, this was primarily due to the prolonged anaesthesia induction time for the PNBA  and willKastelik et al. reported a longer mean incision to suture time in the LIA group  which, aMaterial costs were considerably lower in the LIA group, which was consistent with existing research . Other tOne objective of the development of the LIA was to reduce risk of postoperative complications such as venous thrombosis or nosocomial infection  achievedMobilisation to stand was successfully achieved in the recovery room in 90% of LIA and 95% of PNBA patients in the primary study . This maN = 40), which was powered for the primary endpoint of mobilization. Thus it may be insufficient to draw conclusive evidence towards economical differences between the two procedures without further research. However, due to the randomization in the study design risk of confounding bias stays relatively small. Further research should expand on these issues and potentially include other postoperative costs such as physical rehabilitation and overall hospital length of stay.Limitations of our work may include the restricted transferability of cost structures of a university hospital like the Charité to other institutions on a national and international level. The surgical case costs as the largest cost factor may differ depending on the surgeon’s experience in the handling of the LIA and the overall procedure of TKA. Another limitation may be the relatively small sample size (This study expands on the financial evaluation for determining a preferred procedure in perioperative pain management for patients receiving TKA. Although we were able to describe in detail lower anaesthesiological case costs with the LIA, there was no difference found between the overall case costs for LIA and PNBA. Therefore, based on our analysis we can conclude that the organisational structure and process optimization are the key to reduce costs for either procedure.It should be noted that the determination of an analgesic procedure for patients receiving knee replacement surgery should always rely primarily on medical indications and should therefore be chosen by carefully evaluating individual risk factors and benefits for each individual patient. Economic factors should only be seen as secondary arguments for the individual but can play a considerable role in the effective allocation of resources and organisational planning.


Mosquito-borne viruses cause diseases of great public health concern. Arboviral disease case distributions have complex relationships with socioeconomic and environmental factors. We combined information about socio-economic  and environmental  characteristics with reported human cases of arboviral disease in the counties of Alabama, USA, from 2007–2017. We used county level data on West Nile virus (WNV), dengue virus (DENV), chikungunya virus (CHIKV), Zika virus (ZIKV), California serogroup virus, Eastern equine encephalitis virus, and Saint Louis encephalitis virus to provide a detailed description of their spatio-temporal pattern. We found a significant spatial convergence between incidence of WNV and poverty rate clustered in the southern part of Alabama. DENV, CHIKV and ZIKV cases showed a different spatial pattern, being mostly located in the northern part, in areas of high socioeconomic status. The results of our study establish that poverty-driven inequities in arboviral risk exist in the southern USA, and should be taken into account when planning prevention and intervention strategies. Culex spp. mosquitoes, which are known for thriving in contaminated water sources and sewage overflow. Conversely, cases of DENV, CHIKV, and ZIKV, arboviruses primarily transmitted by Aedes spp. mosquitoes, were reported in areas of high socioeconomic status. These findings suggest differential distribution of arboviruses relevant to human health in Alabama, and that poverty in the southern US is a significant factor that should be considered when planning WNV prevention and intervention strategies.Mosquito-borne arboviruses like West Nile virus (WNV), dengue virus (DENV), chikungunya virus (CHIKV), Zika virus (ZIKV), California serogroup virus (CSV), Eastern equine encephalitis virus (EEE), and Saint Louis encephalitis virus (SLE) are on the rise globally. Socioeconomic and environmental conditions have played a role in directing in this expansion by creating conditions ideal for mosquito vectors and transmission. In this study, we used 10 years (2007–2017) of county level human arboviral case data from the US state of Alabama to better understand the roles socioeconomics (poverty rate) and environmental  conditions may play in driving patterns of arboviral disease in the southern US. We found a significant association between poverty rate and incidence of WNV, an arbovirus primarily transmitted by  Globally, infectious diseases are recognized as leading causes of mortality for the world’s poor . The assAedes aegypti, a main vector of several arboviral diseases, including CHIKV, Zika (ZIKV), and dengue (DENV) fever, has been detected in Alabama after a 26-year absence [Aedes aegypti and Ae. albopictus [Studies investigating the association between socio-economic factors and mosquito-borne infectious diseases in the USA have reported contrasting results. While middle income neighborhoods show higher transmission risk of West Nile virus (WNV) in the northern USA ,7, in th absence . The stabopictus ,16, whicbopictus , making bopictus ,19.Here, we use publicly available data from 2007–2017 to examine socio-ecological associations between human incidence of mosquito-borne arboviral disease, county level indicators of poverty, and mosquito vector distributions. Our aim was to evaluate whether an association between poverty level and mosquito-borne diseases occurs in the southeastern USA, specifically Alabama.This paper analyzes human cases of seven arboviral diseases reported in Alabama’s counties from 2007–2017 : California serogroup virus (CSV), Eastern equine encephalitis virus (EEEV), Saint Louis encephalitis virus (SLEV), West Nile virus (WNV), DENV, ZIKV, and CHIKV. We used cases officially reported by each Alabama county during that time period. Cases are reported by county of patient’s residence and not necessarily by county of disease acquisition. Detailed case definition is described in the https://www.census.gov). We used the all-age poverty rate from the year 2007 to 2017 calculated by the Small Area Income and Poverty Estimates (SAIPE) Program (https://www.census.gov/programs-surveys/saipe.html). The SAPEI Program applies estimates the number of people living below the poverty threshold at the county level for all the USA using regression models based on census data. The poverty threshold is set by the Census Bureau based on family size and age of the members (https://www.census.gov/topics/income-poverty/poverty/guidance/poverty-measures.html). Population data included population of all ages from the year 2007 to the year 2017. To account for environmental factors, we downloaded land use data at county level from the USA Department of Agriculture (https://www.ers.usda.gov/data-products/major-land-uses/). From these data, we extracted the fraction of each county’s territory covered by woodland, crops, water bodies, and urban areas. Data on rainfall was provided by Emory University, consisting of a multi-band raster on the monthly mean of daily accumulated rainfall from 2007–2017 at 30 km of resolution. We collected data on flooding events from the Federal Emergency Management Agency’s (FEMA) through the OpenFEMA platform (https://www.fema.gov/media-library/collections/). We included flooding events in our analyses because—if occurring—they could increase the mosquito breeding sites and contribute to an increase in the risk of arboviral infection for humans [Ae. aegypti predicted presence for Alabama was extracted from the raster map obtained from the Malaria Atlas Project’s data repository  [Data on poverty, and population at the county level were obtained from the USA Census Bureau (as.org/)  , predicted presence of Ae. aegypti (VECTOR), and spatial autocorrelation (SPAT). Model formula:We also performed a GLM to investigate the relationship of poverty rate (POOR), predicted presence of Ae. aegypti presence map already accounted for them.Given the high number of non-reporting counties (68.7%) and the heterogeneity of DENV, ZIKV, and CHIKV case counts, we have modeled these cases using a zero-inflated negative binomial as the selected distribution. Environmental factors were not included in the model because we assumed that the Gi*(d) local statistic was used to identify hot-spots of WNV incidence in Alabama [The spatial autocorrelation was accounted in both the GLMs by using the spatial Gaussian Markov random field (GMRF) models . Spatial Alabama . The spa Alabama . Signifi Alabama . A VIF = Alabama . The geo Alabama .A total of 324 arboviral disease cases were reported in Alabama from 2007–2017 . Among tGi*(d) identified several hot-spots in those counties  rely on passive surveillance and reporting from local physicians and county health departments. Actual incidence of disease is likely under-reported, with detection and reporting of neuroinvasive diseases being considered more consistent and complete than that of non-neuroinvasive diseases. Diagnostic testing approaches and the potential lack of a differential diagnosis may be another factor impacting reported case rates. Another limitation of our study is that we did not include entomological data in the WVN regression models. The study relied on the environmental characteristics of the counties to capture the variability of the abundance of The high numbers of WNV cases in Alabama and their clear association with poverty are indicative of a need for a paradigm shift in the control of arboviral disease, specifically WNV, in the southern USA. The Gulf states are home to over 15 million people living below the poverty line, more than anywhere else in the nation. However, with regards to arboviral disease control, many of these states do not receive the adequate resources proportionate to the disease risk and burden. In high income countries, populations living in poverty, like those in the US Gulf states, are highly vulnerable to arboviral infection, as well as to other neglected tropical diseases (NTDs): howeverS1 Appendixwww.usgs.gov and data from the paper written by Kraemer and colleagues [https://malariaatlas.org/) processed with the R software [The map was created using available county boundaries downloaded from lleagues  (availabsoftware .(DOCX)Click here for additional data file.


Viruses exhibit a diverse array of strategies for infecting host cells and for virion release after replication. Cell exit strategies generally involve either budding from the cell membrane or killing the host cell. The conditions under which either is at a selective advantage is a key question in the evolutionary theory of viruses, with the outcome having potentially important impacts on the course of infection and pathogenicity. Although a plethora of external factors will influence the fitness of either strategy; here, we focus just on the effects of the physical properties of the system. We develop theoretical approaches to assess the effects of the time delays between initial infection and virion release. We show that the length of the delay before apoptosis is an important trait in virus evolutionary dynamics. Our results show that for a fixed time to apoptosis, intermediate delays lead to virus fitness that is lower than short times to apoptosis — leading to an apoptotic strategy — and long times to apoptosis — leading to a budding strategy at the between-cell level. At fitness minima, selection is expected to be disruptive and the potential for adaptive radiation in virus strategies is feasible. Hence, the physical properties of the system are sufficient to explain the existence of both budding and virus-induced apoptosis. The fitness functions presented here provide a formal basis for further work focusing on the evolutionary implications of trade-offs between time delays, intracellular replication and resulting mutation rates. Viruses have evolved to infect a diverse range of hosts, from bacteria to vertebrates. For viruses infecting protozoan or animal cells, virions can exit infected cells either by crossing the cell membrane — herein referred to as budding — or by killing the cell . LeavingA key question in the evolutionary theory of viruses is under what conditions is budding, killing the host cell or lysogeny at a selective advantage? This question has been addressed in some detail for lytic and lysogenic phages . In addiMost phages are transmitted either by killing the host cell or by lysogeny, but some can be secreted across the host envelope without killing the cell. As such, most of the theoretical work has focused on the former two strategies. However, viruses that infect animal or protozoan cells can either exit the cell by killing the host cell or by budding. This brings an extra dimension to the evolution of the latent period — should a virus inhibit cell death for as long as possible and exit cells by budding only? Few studies have addressed the evolution of virion release strategies for viruses other than phage. Some viruses that infect cells lacking a cell wall can also incorporate into host genetic material, but here we focus on budding and virus-induced cell death.There are many ways viruses can control cell death , and theWhile virus-induced cell death is generally associated with non-enveloped viruses, such as picornaviruses, evidence shows that some non-enveloped virus-cell combinations can result in viral exit by traversing the cell membrane . FurtherFurthering work in this area for viruses of vertebrates, There is some evidence, however, that budding viruses have lower viral replication rates compared with apoptotic viruses. For example, In addition to variation in intracellular replication rates, the time to a virus either releasing mature virions by budding from a cell or the time to inducing apoptosis are likely two important parameters influencing the evolution of either strategy. The delay between cell infection and mature virion production is well documented, frequently referred to as the ‘eclipse phase’ . Bonachevice versa for viruses that predominantly bud , infected cells (I) and virions (V) as state variables:λ) is zero and the apoptosis rate (α) and virus yield at apoptosis (γ) are non-zero then the model reflects an apoptotic infection where virus kills the cell and virions are released only on apoptosis. If α and γ are zero and λ is non-zero, this reflects a budding infection with virions leaving the cell via budding only and virus not inducing apoptosis.At two extremes, if the budding rate (2.2τ) and a time delay before virus budding can occur . The case is similar for the terms including the virus budding rate (λ) and yield at apoptosis (γ).In this model, λ) or the yield at apoptosis (γ) and the apoptosis rate (α), to zero to represent either of the virion release strategies. For the model including delays, variations of λ and τ) and yield (γ) are removed to represent a purely budding strategy. These two model variations are provided in For the model without delays, we can set either the budding rate  when the underlying dynamics are more complex  and free living virus (V), termed the Jacobian. The dominant eigenvalue of this matrix is a measure of virus fitness—equivalent to the per capita net growth rate. Note that this is not the same as the basic reproduction number. For simple systems, the equivalence of this interpretation with the basic reproduction number can be shown , were equivalent. We set α = 1/24 hours−1 (apoptosis rate) for the model without delays, and where appropriate, set τ = 24 hours (fixed time to apoptosis) and τ′ = 1 hour (budding delay) for the model with delays. The resident virus budding rate (λ) was set to 100 hours−1. The values for the virus yield at apoptosis (γ) obtained from the invasion analysis were divided by the average (1/α), or fixed (τ), time to apoptosis and subsequently by the resident virus budding rate (λ) to get a relative virion production rate necessary for invasion by an apoptotic virus.For the evolutionary invasion analysis, we assume for both viruses β), budding rate (λ), yield at apoptosis (γ) and the apoptosis rate (α), within the ranges given in α) and the probability of infection, there is no combination of other parameter values, within the ranges used, that could result in a fitness equivalent to that achieved for higher values of these parameters. Conversely, relatively high fitness values could be obtained even when the budding rate (λ) is low (γ) increases as the apoptosis rate (α) decreases, assuming that the longer the cell is alive the more virions can be produced, fitness is no longer constrained by the apoptosis rate and the effects of the virus budding rate and yield at apoptosis are similar  is low . However similar .β) and the virus decay rate (Vμ) are equivalent between an apoptotic and budding virus, then the virus budding rate (λ), compared with the apoptosis rate (α) and yield at apoptosis (γ), relative to the cell death rate (Cμ), will determine evolutionary outcomes.These results highlight that if both the virus yield and apoptosis rate can be maximised, there are conditions under which an apoptotic virus could be at an evolutionary advantage. In addition, assuming the probability of infection . The relative amount of virus produced per unit time by infected cells required for invasion would initially decline, but then increase (Including a budding delay and fixed time to apoptosis in the model  gives siincrease .τ′) and time to apoptosis (τ) dominate the outcome of the virus fitness function relative to other parameter values  for some combinations of parameter values—particularly a short budding delay (τ′) relative to average cell lifespan (1C/μ) and a budding rate (λ) sufficient to contribute more to transmission as the apoptosis delay increases  and budding rate (λ), the full derivation of the virus invasion analysis  for the virus to spread under long budding delays.To explore this fitness minimum further, and the interaction between the time delays  as a function of the virus budding rate (λ) shows that as the time to apoptosis increases, greater investment in virus yield (γ) is required to endure positive fitness (λ) increases, less investment in virus yield at apoptosis is required to ensure positive fitness (Solving this expression for the virus yield at apoptosis ( fitness . However fitness . This trHere, we have developed theoretical approaches to understand the interplay between apoptosis, budding and time delays on the evolution of virus replication strategies. Viruses cannot immediately leave host cells. Several steps of genome replication and assembly must be carried out before mature virions are produced. This results in a delay between infection and virus release. We have shown that the length of this delay is likely an important trait in virus evolutionary dynamics, for viruses that can either leave host cells by budding or killing the host cell.Our results show that intermediate times to apoptosis lead to virus fitness that is lower than short times to apoptosis—leading to an apoptotic strategy—and long times to apoptosis—leading to a budding strategy at the between-cell level. At the between-cell level, trade-offs arise from the physical properties of the virus system. While the role of time delays on destabilizing dynamics in biological systems is well established , the evoWhile there exists a body of theoretical work with respect to phage evolutionary dynamics , includiThere are, of course, a plethora of external factors not accounted for in our relatively simple models of virus infection that will undoubtedly contribute to determining the relative fitness of either strategy in a given context. For viruses infecting multi-cellular organisms, cell type, in addition to immune responses, will be particularly important to consider. Infections of multi-cellular organisms therefore present a greater difficulty for modeling than chemostat systems of bacteria and phage. Our intention here was, however, to provide a general foundation for further work that would introduce trade-offs in the parameters in addition to the effects of external factors.With respect to immunity, While virus release by apoptosis may be at an advantage if apoptotic bodies containing virus go undetected by the immune system before they are taken up by susceptible cells , virusesOther, related, extensions to the analysis presented here would be to introduce trade-offs in the parameters that feature in the virus fitness functions, arising from intracellular replication dynamics. For example, an increase in the rate of intracellular replication can lead to earlier apoptosis . While bIncreases in the intracellular replication rate likely also have implications for mutation rates, leading to trade-offs in the amount of viable virus produced, the time to apoptosis as well as the evolutionary potential of a virus. For example, for positive-sense single-stranded RNA viruses, two extremes of virus replication within cells have been described and the effect on replication and mutation rates quantified . StampinWhether there is a general trend for apoptotic viruses to replicate geometrically remains to be quantified, but it provides a mechanistic explanation why some viruses can have higher intracellular replication rates, which may initiate cell death processes at earlier time points. The interplay between time delays, replication and mutation rates therefore have consequences for the evolutionary rates determined by different viral strategies. If apoptotic strategies arise because of geometric replication, an additional advantage may be generation of greater viral diversity and exploration of the fitness landscape.in vitro experiments. For the evolutionary invasion analyses, we assume that the system is at a steady state before invasion by a mutant virus. This approach could be extended by relaxing the assumption that the system is at a steady state.The theoretical approaches developed here provide a formal definition of virus fitness at the cellular level and could be used to generate hypotheses and inform the design of Our analysis has considered two extremes for modeling the virus within-cell latent period. We acknowledge that there is more likely to be an intermediate between these two models, with the time to budding or apoptosis varying between individual cells. However, our work serves as a basis for future analyses of infection strategies common to RNA viruses infecting multi-cellular organisms and similar to veab039_Supplementary_DataClick here for additional data file.


We show that the resistance in those lines is not a restriction of long-distance movement but due to preventing virus unloading from the phloem into parenchyma cells for replication, thus restricting the virus to the phloem cells only. When DSC 167 and DSC 260 were compared for virus invasion, only a low CBSV signal was found in phloem tissue of DSC 167, indicating that there is no replication in this host, while the presence of intense hybridization signals in the phloem of DSC 260 provided evidence for virus replication in companion cells. In neither of the two lines studied was there evidence of virus replication outside the phloem tissues. Thus, we conclude that in resistant cassava lines, CBSV is confined to the phloem tissues only, in which virus replication can still take place or is arrested.Cassava brown streak disease (CBSD) is a destructive disease of cassava in Eastern and Central Africa. Because there was no source of resistance in African varieties to provide complete protection against the viruses causing the disease, we searched in South American germplasm and identified cassava lines that did not become infected with the cassava brown streak viruses. These findings motivated further investigations into the mechanism of virus resistance. We used RNAscope Manihot esculenta Crantz) that strongly affects the quality and yield of this important food crop’s storage roots. Tuberous roots of virus-infected plants show brownish necrotic areas varying in extent, from a few corky patches visible only on a few tubers to extensive necrosis resulting in the total destruction of the tuberous roots. The disease is caused by cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (U/CBSV), both being distinct species of the genus Ipomovirus in the family Potyviridae [Cassava brown streak disease (CBSD) is the most devastating disease of cassava , we previously found no evidence for virus replication and neither of the two viruses U/CBSV were traceable in leaves or tuberous roots . This ob® in situ hybridization (ISH), a highly sensitive detection method that was recently developed in our laboratory for virus studies in cassava [Plants utilize diverse defense mechanisms to restrict virus replication and accumulation in infected tissues as well as to counteract viral cell-to-cell or long-distance movement ,10. Gene cassava ,15 and wThe African cassava varieties Albert, Namikonga and TMS 96/0304 and the South American cassava germplasm accessions DSC 167  and DSC 260  were propagated and kept under glasshouse conditions between 26 °C to 32 °C. The virus isolates, CBSV-Mo83  and UCBSV-Ke125 , were maintained in the cassava variety TMS 96/0304 and further propagated through stem cuttings. Virus infections were established in cassava plants essentially as described in our early study [To study virus movement in the accession DSC 167, scions  from DSC 167 were side grafted onto healthy TMS 96/0304 rootstocks. Once DSC 167 branches had formed, virus infections were initiated in the chimeric plant by grafting two CBSV-Mo83-infected buds into the lower portion of the stem of the susceptible TMS 96/0304 rootstock. To investigate virus translocation across DSC 167, scions of a healthy TMS 96/0304 were side grafted onto the DSC 167 branch of the chimeric plant TMS 96/0304-DSC 167 .To study virus movement in DSC 260, scions  from TMS 96/0304 were first side grafted onto a healthy DSC 260 rootstock, and allowed to grow and develop into branches in a few weeks. Then, virus infections were initiated by grafting two CBSV-Mo83-infected buds into the branch of the susceptible TMS 96/0304.−ΔΔCt method [Total RNA from cassava leaves and stems was extracted using an RNA extraction kit , essentially following the manufacturer’s protocol. RNA purity, quantity and integrity were analyzed in a NanoDrop UV/Vis spectrophotometer . The accumulation of CBSV-Mo83 was measured in a one-step TaqMan qRT-PCR assay  with primers, probes and reaction conditions as described before . Cycle tt method  with COXFor virus localization in cassava tissues, the protocol for fixation, embedding and sectioning described by Munganyinka et al.  was follv/v) and pure xylene. Xylene was then replaced with paraffin by incubating the samples in mixtures of xylene/paraffin  and finally in pure paraffin, at 60 °C for 1 h each step. Samples were then transferred into Peel-A-Way molds (Sigma-Aldrich) and kept at RT. Prior to sectioning, paraffin blocks were cooled at 4 °C and sections of approximately 12 µm in thickness were cut using a Microm HM 355 rotary microtome (Thermo Fisher Scientific). Sections were separated from paraffin ribbons, relaxed in a water bath at 37 °C, placed on Superfrost Plus slides (Thermo Fischer Scientific), dried overnight at RT and fixed by baking for 1 h at 60 °C. Directly after baking, sections were deparaffinized by incubating two times in xylene for 5 min each, followed by ethanol washing and storage in the dark.For formalin-fixed paraffin embedding (FFPE), 2 steps of infiltration into 95% and 100% ethanol for 30 min each preceded the substitution of ethanol with xylene. This was achieved by stepwise incubation of the explants at RT for 45 min in ethanol/xylene mixtures with decreasing portions of ethanol  for 30 min each, followed by two steps of incubation in PBS for 15 min at RT. Cryoprotection steps were conducted to prevent structural damages from crystal formation during freezing. To this effect, PBS was complemented with 10%, 20% and 30% sucrose and samples immerged in the solution under vacuum at 4 °C for 3.5 h each step. Embedding was carried out in Optimal Cutting Temperature (OCT) compound . Samples were first transferred to filter paper to absorb excess sucrose from the tissue surfaces, immediately placed into OCT compound and gently mixed. Next, the samples were placed in Peel-A-Way molds complemented with fresh OCT compound and then plunged in liquid nitrogen for several minutes prior to storage at −20 °C. For cryo-sectioning, blocks were placed in a cryostat  with a specimen temperature set to −20 °C and chamber temperature of −21 °C ± 2 °C. Sections between 15–20 µm in thickness were obtained, immediately placed on Superfrost Plus and Superfrost Plus Gold slides (Thermo Fischer Scientific) and subsequently baked for 1 h at 60 °C to optimize sections attachment.® ISH assay was performed as described [® 2.5 HD Detection Reagent-RED kit (cat. no. 322360) from Advanced Cell Diagnostics . Prior to hybridization, slides were baked for 30 min at 60 °C followed by cooling at RT for 20 min, after which a hydrophobic barrier was drawn around the sections using an ImmEdge hydrophobic barrier pen ; in the case of samples from cryo-sectioning, two additional washing steps with PBS for 5 min each were included directly after the baking step. Next, the sections were treated with hydrogen peroxide provided with the kit for 10 min at RT, to prevent degradation by endogenous peroxidases, followed by washing with distilled water. Directly after, leaf and stem sections were incubated for 15 min in target retrieval buffer warmed up at a temperature in the range of 98–102 °C, while graft-junctions and tuberous roots were incubated in the range of 80–85 °C for 20 min and 5 min, respectively. Tissue sections were dried overnight at RT and treated with the “Protease Plus” solution provided with the kit at 40 °C for 15 min in the case of leaf and stem sections, and for 10 min in the case of tuber sections. Hybridization was performed by incubating the CBSVs-specific probes  at 40 °C for 2 h in the hybridization solution provided, followed by washing the sections for 15 min with 2 buffer exchanges. Serial steps of incubation in the provided “AMP” kit reagents were conducted, after which the signal was developed by addition of the Fast-Red substrate, resulting in a red chromogenic signal after processing by the alkaline phosphatase linked to the probes. Slides were subsequently washed in water, counterstained with 50% Gill’s hematoxylin (Sigma-Aldrich) for 2 min and rinsed several times in distilled water. Slides were baked at 60 °C for 45 min, briefly submerged in xylene, covered with EcoMount mounting media  and 24 × 40 mm microcover glasses, and air-dried for at least 10 min at RT prior to examination at the microscope.The RNAscopeescribed  using thw/v) toluidine blue O  for 1 min and a fourth stain for starch was in Lugol solution  for 5 min. All staining steps were followed by rinsing in water to remove excess stain. After drying at RT, sections were examined under the microscope.Leaves, petioles, stems and tuberous roots sections were used for histological examination and understanding the anatomy of cassava tissues. Cassava sections (12–70 µm in thickness) were stained with FCA  for 2 min and washed with 100% ethanol. Further staining was in 1% Safranin O (Sigma-Aldrich), and 0.1% Astrablue for 1 min. A third stain was in 0.1 %, an Axioscope-A1  and an Axioscope-2 Plus (Zeiss). The “D” setting of the modulator disk was occasionally used to improve the acquisition of the signal.The infection process of CBSV-Mo83 in the chimera plant TMS 96/0304-DSC 167 was followed. Symptoms on leaves were observed 14 days post inoculation (dpi) in the susceptible rootstock TMS 96/0304 followed by the occurrence of brown streaks visible on the stem close to the graft intersection between TMS 96/0304 and the DSC 167 at 20 dpi. In contrast, leaves and stem of the resistant DSC 167 remained symptomless during the entire 5-6 months of observations in accordance with our previous study [At the graft intersection area, severe necrosis of the TMS 96/0304 tissues was noticed A and a hA next bioassay step gave further proof that long-distance virus movement in the resistant DSC 167 was not impaired . In thes® ISH using a probe against the CBSV-Mo83 P1 gene. Examination of TMS 96/0304 sections revealed strong signals distributed throughout the tissues as red dots or clusters of dots in external and internal phloem cells . In contrast, CBSV was only detected in the external phloem of the resistant DSC 167. This was also shown for cucumber mosaic virus (CMV), potato leaf roll virus (PLRV) and pepper mottle virus (PepMoV) using both external and internal phloem for directed movement ,26. In a® ISH clearly demonstrated that in tissue sections of both DSC 167 and DSC 260 the virus was effectively contained in the phloem. In the resistant cassava DSC 260, despite being absent in leaves, CBSV was able to translocate from the roots through the stem of DSC 260 to infect a TMS 96/0304 scion and this long-distance movement was in both directions, in the external and internal phloem. Furthermore, compared to only traces of virus visible by RNAscope® ISH in DSC 167, there was a considerable amount of virus in the phloem of DSC 260 stem and root tuber sections. Thus, from the density of hybridization signals, it can be assumed that virus replication in DSC 260 phloem companion cells happens. Consequently, inhibition of phloem unloading is a likely explanation for the resistance observed in the immune DSC 167 and the resistant DSC 260 alike.Resistance to long-distance movement is achieved either by preventing the virus from entering the sieve element-companion cells (SE-CC) (loading) or by preventing the virus from exiting the SE-CC complex to enter into sink tissues (unloading) ,30. In oArabidopsis sp., Pseudomonas syringae caused infections in young plants by suppressing the salicylic acid SA pathway. Upon maturation, bacterial infections declined rapidly as a result of age-related resistance responses [Resistance against plant diseases is the result of an interaction between host (genotype) and pathogen  modulated by developmental stages of the host and the environment . The casesponses . Similaresponses ,34. HoweHypersensitivity (HR) indicates an incompatible plant/virus interaction. After virus recognition and primary replication, localized necrotic lesions develop in inoculated leaves to restrict the virus to the point of entry and prevent virus movement and invasion to neighboring cells. In systemic hypersensitive reactions (SHR), the signal spreads along with the virus and more cells eventually become necrotized ,35,36. TIn this study, we provide an explanation for the resistance identified in some South American cassava germplasm lines against the viruses causing CBSD. While this is a decisive step towards the development of U/CBSV resistant varieties, there are still many open questions around our observation. In both cassava lines, the virus infections are restricted to the phloem cells, but only in DSC 260 does virus accumulation indicate virus replication. Are there two discrete mechanisms acting, inhibition of replication and phloem restriction? What is the specific function of the internal phloem in this virus resistance? Structural differences and functions for these phloem types were shown in pumpkin. The proteins PP1 and PP2 were found in both phloem types but only the extrafascicular phloem released mature proteins, despite both mRNAs occurring in both phloem ,41. It w


The importance of virus disease outbreaks and its prevention is of growing public concern but our understanding of virus transmission routes is limited by adequate sampling strategies. While conventional swabbing methods provide merely a microbial snapshot, an ideal sampling strategy would allow reliable collection of viral genomic data over longer time periods. This study has evaluated a new, paper-based sticker approach for collection of reliable viral genomic data over longer time periods up to 14 days and after implementation of different hygiene measures. In contrast to swabbing methods, which sample viral load present on a surface at a given time, the paper-based stickers are attached to the surface area of interest and collect viruses that would have otherwise been transferred onto that surface. The major advantage of one-side adhesive stickers is that they are permanently attachable to a variety of surfaces. Initial results demonstrate that stickers permit stable recovery characteristics, even at low virus titers. Stickers also allow reliable virus detection after implementation of routine hygiene measures and over longer periods up to 14 days. Overall, results for this new sticker approach for virus genomic data collection are encouraging, but further studies are required to confirm anticipated benefits over a range of virus types. However, regardless of the viral pathogen, virus detection and determination of transmission routes are still poorly understood, largely because of inadequate sampling and monitoring tools. Until now the gold standard for virus detection, especially for infectious viruses, has been the plaque assay6. However, for verification purposes, growth-dependent assays are cost and time intensive and are not suitable for all viruses7. Consequently, there has been a drive for other, growth-independent detection methods for rapid virus detection8.The importance of virus disease outbreaks is of growing public concern. While viral epidemics and pandemics caused by Ebola, MERS-CoV and the current SARS-CoV-2 are most likely temporary events10. However, swab sampling is still accepted as the method of choice11. Extensive surface sampling is not only relevant for detecting viral pathogens, but also for the demonstration of contamination routes, contamination sources and for monitoring persistent viruses in different environments14.In general, virus detection in the food industry and healthcare settings is mostly performed by molecular methods, especially polymerase chain reaction (PCR) techniques16. Although swabbing methods are easy to perform, much effort has been expended to obtain more than merely a momentary, microbial snapshot from surfaces. Primarily, the sampling time point is not trivial as flushing, cleaning and disinfection can blur the actual viral load in multiple ways17. Since hygiene measures are essential to minimize pathogen transmission, ideally sampling approaches must overcome this limitation, which is essentially information loss17. Moreover, long-term virus monitoring strategies have likewise been neglected. Resolution of these major limitations requires novel, innovative sampling approaches to enable virus traceability and, simultaneously, increased safety.As viruses are able to endure in bacterial biofilms and remain infectious over long periods, their persistence in food facilities, hospitals and other institutions has been suspected to be a recurring cause of disease outbreaks18 demonstrated the advantages of this sticker technique for detection and monitoring of bacterial pathogens on surfaces, including door handles. However, its utility for virus surface sampling has yet to be investigated. The main advantage of this new technique is its simplicity, as shown in Fig. This study focused on the evaluation of a new paper-based sticker approach for detection of viruses on surfaces over extended time periods. Recently Bobal and Witte et al.19, this study focuses on evaluation of a new sampling approach that is both simple and based on existing material—the plain paper-based sticker. Both microbiological and molecular biological methods have been used to evaluate the benefits of this new paper-based sampling material for virus collection. As to this date no comparable sampling and monitoring approach to the recently invented paper-based sticker exists, we had to compare the routine cotton swabs and the sticker approach to each other, although a direct comparison is unsuitable, due to the fact that the new sticker approach will collect viral particles that would be otherwise have been transferred onto that entire surface of interest. In order to ensure an adequate evaluation of the new paper-based sticker, a comparison of a well-known sampling material was necessary. As the cotton swab is one of the best-known sample materials, it was chosen to clearly define the benefits of the new sticker sampling method and to determine its limitations. However, a clear separation of both methods is important, as their application and relating thereto-informative value strongly related to the question and issue posed by the user.Until now virus transmission routes have been difficult to track due to a lack of adequate sampling strategies. Most of these employ swabs for virus uptake, which merely capture a momentary snapshot, and which are strongly influenced by hygiene measures that contribute to lost microbial information. Although a momentary snapshot of a surface is sufficient for virus detection, this type of sampling does not allow an all-encompassing recording of data over a longer period of time. So far novel long-term surface monitoring strategies and sampling materials for virus data collection are being rarely described6. The quantity of infectious virus particles (plaque forming units (PFU)/100 µL) of artificially contaminated stickers and swabs was determined. Results presented in Table 8 and 107 PFU/100 µL); results presented in Fig. 21. As the binding strength of a virus depends on its isoelectric point, the interaction between virus and sampling material must be determined for each virus individually21. Alternatively, poor recovery can be a consequence of the inactivating effect of the sampling material itself; inactivation of virus particles on filter papers has already been demonstrated by Picard-Meyer and Cliquet22. Nevertheless, the interactions of virus P100 and sticker material was beyond the scope of this study.We initially compared the paper sticker sampling methods for infectious virus particles using plaque assays, the contemporary diagnostic gold standard4 PFU/100 µL /100 µL and the viral load was determined using qPCR. The same protocol was used for the swabs, which were directly compared. As shown in Fig. 2 PGN/100 µL23. These outcomes are highly relevant as they demonstrate that stickers have comparable sensitivity to the current virus sampling system.To investigate the broad range linearity of detection of this new approach, the stickers were spike-inoculated with virus concentrations ranging from 1024 have demonstrated the efficacy of paper-based filters for preservation of viral RNA, we investigated the long-term characteristics of stickers and whether they were suitable for collection of viral genomic matter over longer time periods. Stickers were inoculated with a known virus suspension and the detectable genomic viral load after 0, 1, 3, 7 and 14 days was determined. Results are presented in the supplement Figure As Cardona-Ospin et al.As the paper-based stickers exhibited good linear and long-term monitoring properties, we quantified viral genomic material after implementation of various surface hygiene measures. Sterile ceramic tiles were used as experimental surfaces. Stickers were applied to the tiles and surfaces contaminated with known concentrations of virus solution. After a drying phase of at least 1 h, the surfaces were separately cleaned or disinfected with a variety of techniques. Five different hygiene measures were compared to cover as many routine procedures as possible, representing those commonly applied in food environments, hospitals and public places. Results presented in Fig. 25. Overall, results indicate that the sticker approach was not associated with information losses that might be attributed to common hygiene measures. Further, results indicate that sticker sampling offers new opportunities for the identification of viral transmission hotspots and could provide further insight into viral transmission routes not available through “snapshot” swabbing approaches.Considering the use of virus-inactivating treatments and electrostatic binding of virus particles to the paper stickers, recontamination events associated with the sticker itself can be excluded. However, confirmation will necessitate further investigations, using a range of viruses26. Plates with confluent lyses were overlaid with 5 mL TSB or saline-magnesium (SM) buffer  and shaken for about 3 h at room temperature or overnight at 4 °C. Afterwards the medium was separated and centrifuged at 8000 rpm for 2 min. Finally, the supernatant was filtered (0.02 μm), aliquoted and stored at − 20 °C.Bacterial virus P100, purchased as PhageGuard Listex P100 solution  was used in this study. For enumeration and propagation of phage P100 the related bacterial strain L. monocytogenes EGDe (ATCC BAA-679) was used. The bacteria were grown overnight at 37 °C in tryptone soya broth (TSB) with 0.6% (w/v) yeast extract . After overnight incubation, the bacterial culture was ten-fold diluted in fresh medium and incubated at 37 °C for 3–4 h to obtain a maximum number of viable cells in the logarithmic growth phase (log phase). For propagation of virus stock, virus solutions were used for plaque assay, according to the protocol of Kropinski et al.10–108 PFU/100 µL. The investigated range of about 108–104 PFU/100 µL was based on a study of Herzog et al.27, which investigated virus P22 recovery efficiencies over a broad range of virus concentrations. Virus titers of artificially contaminated stickers, swabs and positive control were determined using the Small Drop Plaque Assay28. An overnight culture of L. monocytogenes EGDe was tenfold diluted in SM-buffer28 and subsequently a tenfold serial dilution from the aqueous-phase was prepared in bacteria-containing buffer. After 30 min of incubation at 37 °C, 20 μL of each dilution were dropped onto a tryptone soya agar (TSA)-plate and incubated overnight at the strain-respective temperature of 37 °C. The next day plaques were counted and compared with untreated, positive controls. All plaque assay experiments were repeated at least twice in duplicate.Virus P100 was used at stock concentrations of approximately 1029. All experiments included a positive control (untreated phage-solution) and were repeated at least twice in duplicate or individually on three different days.Each sticker was detached with sterile tweezers and transferred into a 1.5-mL Eppendorf tube and overlaid with 200 µL sterile water and thoroughly vortexed. The same procedure was performed with each swab sample and all positive and negative controls. Subsequently the DNA of each sample was isolated using the commercially available NucleoSpin tissue kit  in accordance with the user manual 30 and qPCR assays were performed using the non-specific DNA-dye EvaGreen Dye . One qPCR reaction of 25 µL final volume contained 2.5 µL of 10 × reaction buffer , 1.2 µM of probe EvaGreen Dye, 200 µM of each dNTPs  and 1 U of Taq DNA polymerase . Each P100 primer (forward: AGCAGAGTTTGAAAAAATTGATGACTAC and reverse: TCGTCATGCGTGTTTCTATGC), purchased from Eurofins Genomics , was inserted to reach a final concentration of 500 nM. All qPCR assays were performed in an Mx3000 real-time PCR thermocycler  with cycling conditions of 2 min at 94 °C, 45 × 30. The average efficiency of this qPCR was 99.6% and the average Rsq was 0.997. All qPCR experiments were conducted as a PCR-duplicate and included a negative amplification control as well as a standard series for calculation of a standard curve, whereas the standard curve was used for quantification of the viral nucleic acids.For determination of the viral load (PGN/100 µL), the isolated samples were analyzed with qPCR. The primers for virus P100 qPCR were chosen according to Fister et al.18. Subsequently stickers were artificially contaminated or attached to the surface of interest for further experiments. Stickers and swabs were then contaminated with 5 µL virus solution and dried for at least 1 h. For the positive control the equi-volume of virus solution was transferred directly to 200 µL water and DNA isolation was performed as described above .Commercially available text-marking stickers  were applied to an aseptic Petri dish 100 mm × 15 mm  using sterile tweezers. The transferred stickers were sterilized with UV-C radiation for at least 15 min, as previously described by Bobal and Witte et al.Different test conditions were chosen for the surface experiments. Each sterile ceramic tile (5 cm × 5 cm) was artificially contaminated with 5 µL virus solution, whereas the sticker was applied onto the tile before contamination. After a drying phase of at least 1 h, the surfaces were treated with different cleaning and disinfection treatments: (NTC) without washing, (I) flush with water, (II) flush with soapy water, (III) wiped with Mikrozid, (IV) cleaned with BUDENAT ALKASEPT D 445 and wiped with Mikrozid, and (V) flushed with soapy water and wiped with Mikrozid. Unwashed tiles were used as positive, untreated controls. For the flush treatments, (I) with water and (II) soapy water, the tiles were flushed twice with 1 mL liquid, whereas the soapy water tile was additionally rinsed with 0.5 mL distilled water. The soapy water was prepared by diluting the household cleaner Exact AC  in water to provide concentrations commonly used for surface cleaning (0.001–0.002% as described in the product data sheet). For treatment (III), disinfection of surfaces, Mikrozid AF liquid  was applied by wiping. Treatment IV), cleaning with BUDENAT ALKASEPT D 445 and wiped with Mikrozid, was provided as BUDENAT ALKASEPT D 445 , which is recommended as a high alkaline disinfectant cleaning agent for surface cleaning in food environments. For this treatment 1 mL of a 5% BUDENAT ALKASEPT D 445 solution was added to the ceramic tile and incubated for 30 min. Subsequently the tiles were rinsed with 0.5 mL water and wiped with Mikrozid. The last treatment, treatment (V), comprised a combined treatment with treatments (II) and (III). After surface treatments the previously applied sticker was further treated as described in 2.3 Viral DNA Extraction. The swab sample was taken by moistening the cotton swab with 100 µL SM-Buffer and afterwards the moistened swab was rubbed over the whole surface for 10 s. Afterwards the samples were treated as described in 2.3 Viral DNA Extraction.Recoveries from all experiments, determination of the PFU/100 µL (plaque assay) and the PGN/100 µL (qPCR) results were compared with those from the relevant positive controls. Further, sticker results were compared with corresponding swab results. Each experiment was repeated at least twice in duplicate, enabling calculation of average recovery and standard errors to account for variation of the data across means. Data were analysed using the Kruskal–Wallis test of independent samples from stickers and swabs .18, but its application to virus monitoring has remained uninvestigated. Single-sided adhesiveness means that stickers are attachable almost anywhere, including previously “difficult to access” areas and collect viral particles that would have otherwise been transferred onto the surface. Paper-based stickers also exhibit characteristics particularly suitable for long-term surface monitoring of viruses, unlike to swabs, which are made for the determination of actual viral load on a surface. These include a good linear binding capacity in terms of viral particles and viral DNA for up to 14 days, and resilience to routine cleaning and disinfection. Stable recovery rates of viruses from surfaces have also been demonstrated after various cleaning and disinfection measures without information loss. It is also unlikely that the stickers will have fomite properties as bound viruses remained non-infectious. In the future, various improvement options are to be tested in order to guarantee stable virus recoveries. While initial studies raise optimism for a new approach to surface virus monitoring, further studies are required to ascertain its suitability across a range of viruses, including enveloped and RNA viruses.Current knowledge of virus transmission routes and virus persistence is limited by imperfect viral particle detection and long-term monitoring strategies. Consequently, this study has evaluated a new and easy-to-use sampling material, the paper-based sticker. This sticker has already been investigated in respect of bacterial samplinghttps://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020182924.This article is intended to discuss, but not modify, Patent Application Publication No. WO 2020/182924 A1, titled ‘A Novel Sampling Method for Long-Term Monitoring of Microbes’, for the purpose of increasing the accessibility of the scientific content contained therein. Inventors: Martin Bobal, Anna Witte, Patrick Mester and Peter Rossmanith. Current Applicant: Merck Patent GmbH . Filed: 14 March 2019. Published: 17 September 2020. This patent application is in no way limited by the publication of this article. Readers interested in the specific intellectual property covered by this patent should review the patent at the link provided below. Readers may also contact the authors or Merck Patent GmbH for licensing information. Supplementary Information 1.