The transmissibility and pandemic potential of influenza viruses depends on their ability to efficiently replicate and be released from an infected host, retain viability as they pass through the environment, and then initiate infection in the next host. There is a significant gap in knowledge about viral properties that enable survival of influenza viruses between hosts, due to a lack of experimental methods to reliably isolate viable virus from the air. Using a novel technique, we isolate and characterise infectious virus from droplets emitted by 2009 pandemic H1N1-infected ferrets. We demonstrate that infectious virus is predominantly released early after infection. A virus containing a mutation destabilising the haemagglutinin (HA) surface protein displayed reduced survival in air. Infectious virus recovered from droplets exhaled by ferrets inoculated with this virus contained mutations that conferred restabilisation of HA, indicating the importance of influenza HA stability for between-host survival. Using this unique approach can improve knowledge about the determinants and mechanisms of influenza transmissibility and ultimately could be applied to studies of airborne virus exhaled from infected people. Influenza viruses can transmit through the air between two hosts. For virus to successfully transmit through the air, it must be exhaled from an infected donor in sufficient quantities and retain infectiousness in the air. These aspects of transmission are poorly understood due to a paucity of methods for quantifying infectious virus from airborne particles. Using a novel technique of virus plaque isolation from depositing airborne droplets, we show that ferrets infected with an airborne transmissible influenza virus exhaled a peak of infectious virus early after infection. We demonstrate the importance of virion stability for the retention of infectivity as virus travels through the air. Our findings highlight the fate of infectious virus outside the respiratory tract as an important parameter for understanding influenza transmission. Risk assessing an influenza virus for sustained human-to-human transmission is key to understanding its pandemic potential and the threat to public health. Animal Infectious virus is released into the air from an infected donor into particles of different sizes–5. PractVirus infectivity in the air is likely to be dependent on the constituents of respiratory secretions at the time of emission and molecular properties of the infecting viral strain. One viral property demonstrated to facilitate transmission in the ferret model is stability of the haemagglutinin (HA) surface protein–10. SomePrevious studies have attempted quantification of influenza virus from respiratory droplets and/or aerosols using bioaerosol samplers or impactors and assaying for viral nucleic acid–4,12–15.Within the IVTT, droplets collected are likely to be of a certain minimum size in order to settle the short distance (~5cm) onto cell culture plates during the 10-minute sampling period. To obtain an estimate of particle sizes depositing, we adopted a model for the airflow, motion and evaporation of particles in the IVTT see . The reain vitro experiments using nebulised virus are that airborne particles will not necessarily display the same size distribution nor have the same composition of salts/proteins/lipids as from respiratory secretions and thus virus may display a different lifespan to that occurring in an in vivo setting. We therefore focussed our efforts on detection of infectious virus emitted by infected ferrets. In three independent experiments, two ferrets were intranasally inoculated with pH1N1 virus (Eng/09). We[The limitations of g/09). We–26 and og/09). We,28, haveThere was a clear peak in airborne infectious virus on day 2 for all ferrets. This correlated with peak nasal wash titre in some ferrets but in others the timing was discordant. For donors #1 and #4, peak virus shedding in the nasal wash was on day 3 and 4 respectively when a low number of plaques were detected in the IVTT, but an order of magnitude less than was detected on day 2 . On otheWe next sought to implement our methodology to investigate how virus viability in airborne droplets might vary between influenza viruses with different pH stability. A pH stable HA surface protein was previously demonstrated to facilitate successful transmission of virus through the air between ferrets–10. InflWe generated recombinant mutants of pH1N1 with HA mutations that alter its pH stability. Mutation Y7H (Y17H in H3 numbering) was previously shown to destabilise Eng/09 HA, increasing the pH of fusion to 5.9, a level similar to highly pathogenic avian H5N1 viruses that do not transmit via the airborne route. Mutation E21K (E31K in H3 numbering) was previously shown to stabilise Eng/09 HA, decreasing pH of fusion to 5.3, typical of a transmissible human seasonal virus. Both viruses readily formed plaques on MDCK cells and grew with similar replication kinetics in multicycle replication assays in MDCK cell monolayers. Details on the generation and analysis of these viruses have been reported previously.4 PFU and two ferrets (donors #3 and #4) were infected with 106 PFU. Based on our previous work that identified replicative differences between Y7H and E21K in primary airway cells[We inoculated 4 ferrets each with either Y7H or E21K pH1N1 mutants and sampled using the IVTT on days 1 to 4 p.i. Two ferrets (donors #1 and #2) in each group were infected with 10way cells, we pred4 PFU of Y7H compared to those infected at the higher dose or with E21K. Despite comparable levels of viral replication in the nasal tract, more plaques were recovered from air emitted by ferrets infected with the more stable E21K virus  than by the four ferrets infected with Y7H virus   analysis, other than Y7H donor #3 that had an increased AUC and a higher peak nasal wash titre . Howeverplaques) .Deep sequencing was performed on populations of virus obtained by nasal washing of infected ferrets on days 1–4 p.i and also on the viral inoculums (Eng09/E21K and Eng09/Y7H). Individual infectious virus plaques isolated from air emitted by infected ferrets using the IVTT were picked and Sanger sequencing of the viral HA and M genes was performed.In samples isolated from E21K-infected ferrets, there were no new mutations detected in nasal virus compared to the viral inoculum (at >5% frequency by deep sequencing). In a selection of individual exhaled virus plaques collected on IVTT culture plates, there were no new mutations detected upon Sanger sequencing of HA or M genes. In contrast, in samples isolated from Y7H-infected ferrets, HA mutations were detected in both airborne and nasal virus that were not present in the inoculum virus.The greatest number of virus plaques (n = 15) isolated from droplets emitted by a Y7H-infected ferret using the IVTT was from donor #3. In 14 out of 15 of these unique virus plaques, HA mutations were detected on Sanger sequencing,. These mutations included either a reversion at position 7 (H7Y) (in 10/15 plaques) or additional mutations located around the HA stem  . InteresIn both donor #2 and donor #4, there was also evidence of survival of airborne virus with a pH stabilised phenotype and corresponding genotypic changes  Figs. ThInterestingly, in the nasal wash of Y7H-infected ferrets, the mutations detected in exhaled plaques were present as minority populations including some existing as low frequency variants (<5% frequency)  Figs. ThAll of the pH-stabilised plaques isolated from virus shed into the air carried mutations that were present in nasal washes, albeit detected as a minority population in several cases. Several other mutations were present at various frequencies in nasal wash of Y7H donor animals that were not present in the plaques isolated from air, perhaps because these mutations were not adequately stabilising to preserve virus viability in droplets or that due to sampling constraints these particular mutants were not detected as viral plaques. Taken together, our data show that virus mutants with more stable HA arose during replication in the ferret nasal tract and that increased pH stability can improve retention of infectivity in airborne droplets.6 PFU) was nebulised into the IVTT  was nebulised into the IVTT (4 PFU) was nebulised into the IVTT  after droplets are released from the URT. Others et al.[et al.14 also detected a five times higher amount of viable virus in particles >4.7μm despite the most frequent size of exhaled particle being <1μm. Our technique was unsuccessful in collecting viral genome copies emitted by pH1N1-infected ferrets, likely because quantities collected in 10 minutes are below the limit of detection by RT-qPCR. To overcome this limitation, a longer ferret exposure time would be necessary to increase virus in the IVTT to a measureable quantity. However, because ferrets are introduced into the chamber while fully conscious , a maximum breath collection of 10 minutes is applied for animal welfare reasons. We did note ferret-to-ferret variability in the quantities of airborne infectious virus detected, which has also been observed in previous studies on ferrets[As with all methods employed to isolate influenza virus from the air, there are limitations to the design of the IVTT that must be considered. The IVTT is uniquely designed as a straightforward and sensitive method to sample infectious virus from airborne droplets depositing on cell culture plates but the ability to collect infectious virus from small aerosols is less certain. Interestingly, emerging data demonstrates that droplets play a central role in transmissibility between ferrets. Zhou et al. demonstrn ferrets and human ferrets. It is pAlthough ferrets are considered the best available model for influenza transmission, these studies will never truly represent human infection. The duration of human contagiousness following primary infection with influenza and the relative contribution of different routes of transmission and particle sizes remain critical unknowns in the field. Employing the IVTT technique to sample from naturally or experimentally infected humans might provide valuable information on contagious virus shedding. In ferrets, others have shown that infection with airborne transmissible viruses result in more viable exhaled virus than those that do not transmit and obseOverall, the novel methodology and findings described here highlight the fate of infectious virus outside the respiratory tract as an important parameter for understanding influenza transmission. Direct measurements of airborne infectious virus and characterisation of their genotype and phenotype can significantly enhance the utility and quality of information obtained from ferret transmission studies.2.Madin-Darby Canine Kidney (MDCK) cells (ATCC) were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) (Gibco-Life technologies) supplemented with 10% foetal bovine serum (FBS) , 1% penicillin and streptomycin (Gibco- Life technologies) and 1% nonessential amino acids (Sigma-Aldrich) at 37°C with 5% COde novo synthesis (GeneArt) from published sequence data. E21K and Y7H point mutations were introduced into a pPol1 plasmid containing the HA of Eng09 using QuikChange site-directed mutagenesis kit (Stratagene) according to manufacturer’s instructions. Viruses were generated using a 12 plasmid reverse genetics system as previously described[The prototypic 2009 pandemic H1N1 (pH1N1) virus A/England/195/2009 (Eng09) was derived using reverse genetics by described and prop4 PFU), Eng09/E21K or Eng09/Y7H virus (104 or 106 PFU as stated) diluted in phosphate buffered saline (PBS) (0.1 ml per nostril). All animals were nasal washed daily, while conscious, by instilling 2 ml PBS into the nostrils and the expectorate collected in modified 250 ml centrifuge tubes. Infectious virus was titrated immediately by plaque assay of the nasal wash on MDCK cells.Animal studies were conducted as previously described. Female in vitro experiments. All experiments were conducted with minimal-to-no UV light exposure.The influenza virus transmission tunnel (IVTT) was designed in house and manufactured by EMMS systems . The system consists of a bias flow pump (EMMS) connected to a 37.5cm (height) x 25cm (diameter) ferret chamber. The ferret chamber is connected to the IVTT (a 100cm (length) x 18cm (width) x 9cm (height) half-cylindrical clear acrylic exposure tunnel) by a 1.5 cm (diameter) aperture allowing free passage of air. Sentinel cell plates are centred at 30, 60 and 90 cm from the tunnel opening and can be accessed via drawers from the side of the IVTT. Air is channelled from the exit port of the IVTT toward a downflow bench or microbiological safety cabinet filter to provide a low level draw. For air sampling, an SKC Biosampler (SKC Inc.) is connected to the exit port of the IVTT, with air pulled through at a rate of 12.5L/min. The ferret chamber is replaced by a 10cm (height) x 9cm (diameter) nebulisation chamber (EMMS) attached to an Aerogen Pro nebuliser (Aerogen), which generates droplets with a volume mean diameter (VMD) of 4 to 6μm, for 2 prior to the addition of a semi-solid agarose overlay medium containing 2.5μg/mL of amphotericin B and 1μg/mL TPCK treated trypsin and incubated for a further 3 days to allow for formation of viral plaques. Between exposures for each donor ferret, sentinel cells were replaced and the collection chamber and cell tunnel cleaned to remove any surface deposited virus. Ferrets were nasal washed after IVTT exposures had been carried out. Temperature and humidity were monitored throughout experiments and observed to be 21+/-1°C and 50+/-10% respectively. The IVTT was disinfected after use with a 1% virucidal disinfectant solution. All experiments were conducted at containment level 2.Infected ferrets were placed into the ferret chamber for 10 minutes per exposure. Ferrets were not sedated during the exposure period and therefore, for well being, exposure was restricted to 10 minutes. Airflow of 7.5L/minute was introduced using the bias flow pump via three ports into the ferret chamber (2.5L/minute into each port), which connects to the IVTT via a 1.5cm diameter opening during the exposure period. Analysis of the impact of air flowing through a 1.5cm opening at 7.5L/minute found no suggestion that particle distribution would be altered or particle breakup would occur based on calculations of the Ohnesgorge and Weber numbers. Sentinel 6-well plates containing a confluent monolayer of MDCK cells overlaid with 0.5mL overlay medium were added to the tunnel for the duration of the exposure period. Overlay medium contained a final concentration of 1xMEM, 0.21% bovine serum albumin (BSA) V, 1mM L-glutamate, 0.15% sodium bicarbonate, 10mM HEPES, 1x penicillin-streptomycin  and 0.01% dextran DEAE (Sigma). The plates were incubated for one hour at 37°C, 5% CO2 prior to the addition of semi-solid agarose overlay medium containing 1μg/mL TPCK treated trypsin and incubated for a further 3 days to allow formation of viral plaques. Viral RNA or tracer DNA were collected from1120μL PBS supplemented with 0.375% BSA-V placed in the central space between the wells of the three 6-well IVTT culture plates. Where air sampling was undertaken, after the 10 minute collection window, air remaining in the IVTT tunnel was pulled through a SKC Biosampler (SKC Inc.) at a rate of 12.5L/minute , for a further 10 minutes. Air samples were collected into 15mL PBS and quantified immediately by plaque assay on MDCK cells. Temperature and humidity were monitored throughout experiments and were 21+/-1°C and 50+/-10% respectively. All experiments were performed with the IVTT placed within a class II biological safety cabinet at containment level 2. The IVTT was disinfected after use with a 1% virucidal disinfectant solution.Viruses (and/or plasmid DNA) under study were diluted in PBS and nebulised using the Aerogen Pro nebuliser into the nebuliser chamber attached to the IVT tunnel. Airflow at a rate of 1L/minute was introduced via two ports into the nebuliser chamber, which connects to the IVTT. Sentinel MDCK cells overlaid with 0.5mL overlay medium were exposed for 10 minutes per nebulisation and after exposure the plates were incubated for one hour at 37°C, 5% CO8 to 2x100 copies/μL.Real-time quantitative PCR (RT-qPCR) was used to quantify the amount of plasmid DNA or viral RNA deposited on each culture plate and from an air sample collected using a SKC Biosampler. Viral RNA was extracted using the QIAamp viral RNA mini it (Qiagen) from 140μL samples and eluted into 40μL water. RT-qPCR of the viral M gene or DNA plasmid was performed using AgPath-ID One-Step RT-PCR Reagents (Thermo Fisher Scientific) with specific primers and probes. Primer N, at a distance x. By integrating the plaque density over definite limits we formed an estimate for the total number of plaques, P, within a given area, A:x1 and x2 represent axial distances along the tube, and Lz is the spanwise width.Viral plaque counts were taken from 6-well plates centred at 30cm (plate 1), 60cm (plate 2) and 90cm (plate 3). Plaque counts from the 6 wells were divided by the measured surface area to obtain an estimate for the viral plaques per square metre, or plaque density, at that recorded point. As the results were taken in triplicate, this then allowed for a set of upper, lower and mean values at each site. The decay in plaque numbers between subsequent sites was somewhere between linear and exponential across all ferrets. An exponential or linear regression fit was adopted accordingly to model the viral plaque densities, Airflow into the IVTT was modelled as a laminar round jet, and a LViral plaques were picked using a Gilson pipette and propagated on MDCK cells. Viral RNA was isolated using QIAamp viral RNA mini kit (Qiagen) and reverse transcribed using SuperScript III reverse transcriptase (Invitrogen) with random hexamers. The HA or M gene was amplified using KOD polymerase (Thermo Fisher Scientific) with specific primers (sequences available on request) and Sanger sequenced.Whole genome sequencing of influenza A was performed using total RNA extracted directly from 150μL of ferret nasal wash samples using Easymag, according to manufacturer instructions. Multisegment reverse transcription-PCR (M-RTPCR) was used2, 0.5mM MgCl2) adjusted to pH 5.5 or 7.0 in triplicate and incubated for 15 minutes at 37°C. The buffer was inactivated with a 10-fold dilution in DMEM and samples titrated by plaque assay on MDCK cells.Each virus was mixed with MES buffer . A p value <0.05 was considered significant.All work was approved by the local genetic manipulation (GM) safety committee of Imperial College London, St. Mary’s Campus (centre number GM77), and the Health and Safety Executive of the United Kingdom. Animal work was performed under a United Kingdom Home Office License, PPL 70/7501 in accordance with the approved guidelines, under the Animals (Scientific Procedures) Act 1986 (ASPA).S1 Fig7 PFU of Eng/09 virus and 1x109 copies of plasmid DNA diluted in 100 μL PBS were nebulised into the IVTT simultaneously and cell culture plates exposed for 10 minutes, in triplicate. Infectious virus was collected as plaque forming units (PFU) on MDCK cell culture plates (white bars). Plasmid DNA and viral RNA were collected into 1120μL PBS supplemented with 0.375% BSA-V place in the central space of the 6 well plates and quantified by real-time quantitative PCR (black and grey bars). After the 10 minute collection window, air was sampled into 15mL PBS with a SKC Biosampler connected to the end of the IVTT for a further 10 minutes and subjected immediately to plaque assay for infectious virus and real-time quantitative PCR for DNA and viral RNA. Error bars show standard deviation of three independent experiments.2x10(PDF)Click here for additional data file.S2 Fig4 PFU in 0.1mL PBS pH1N1 Eng/09 virus. On day 2 post inoculation, the number of exhaled infectious viral plaques detected on IVTT plate 1 , 2  and 3  for each of the six donor ferrets was counted.Six ferrets were intranasally inoculated with 10(PDF)Click here for additional data file.S3 FigThe Y7H virus inoculum was interrogated for the presence of any low frequency mutations. Data on the amino acid present in the positions at which mutations were detected in IVTT plaques exhaled by Y7H-infected ferrets is shown.(PDF)Click here for additional data file.S4 Fig(A) Upper panel: No virus was detected in the IVTT from Y7H-infected ferret donor #1 on days 1 to 4. Lower panel: Nasal wash from days 1 to 4 was next-generation sequenced and variants present at >5% frequency are shown in the bar graph: H7Y orange, R54G blue, S289N green and D19N in HA2 purple. In each of the bar graphs, the proportion of virus in nasal wash with sequence encoding the amino acid as in the parental virus (Y7H) is shown in grey. (B) HA mutations are modelled on a HA monomer using Pymol molecular visualization tool (PDB: 4jtv). H1 numbering using the mature HA sequence is used throughout[(PDF)Click here for additional data file.S5 Fig(A) Upper panel: Virus emitted by Y7H-infected ferret donor #2 on days 1 to 4 was collected as plaques picked from plates 1 (30cm), 2 (60cm) and 3 (90cm) of the IVTT and viral RNA extracted. The haemagglutinin (HA) gene was Sanger sequenced. HA mutations identified in viral plaques are represented by the colours orange V19I, blue I57F, green T241I and purple L98M-HA2. Lower panel: The proportion of corresponding HA mutations on days 1 to 4 in the nasal wash was determined by next-generation sequencing. In each of the bar graphs, the proportion of virus in nasal wash with sequence encoding the amino acid as in the parental virus (Y7H) is shown in grey. (B) HA mutations are modelled on a HA monomer using Pymol molecular visualization tool (PDB: 4jtv). H1 numbering using the mature HA sequence is used throughout. HA1 is (PDF)Click here for additional data file.S6 Fig(A) Upper panel: Virus emitted by Y7H-infected ferret donor #4 on days 1 to 4 was collected as picked plaques from plates 1 (30cm), 2 (60cm) and 3 (90cm) of the IVTT and viral RNA extracted. The haemagglutinin (HA) gene was Sanger sequenced. HA mutations identified in viral plaques are represented by the colours orange H7Y, blue V16I, green T61P in HA2. Lower panel: The proportion of corresponding HA mutations at positions 7 and 16 in HA1 and 61 in HA2 detected on days 1 to 4 in the nasal wash was determined by next-generation sequencing. No additional mutations were detected in the nasal wash at >5% frequency. In each of the bar graphs, the proportion of virus in nasal wash with sequence encoding the amino acid as in the parental virus (Y7H) is shown in grey. (B) HA mutations are modelled on a HA monomer using Pymol molecular visualization tool (PDB: 4jtv). H1 numbering using the mature HA sequence is used throughout. HA1 is (PDF)Click here for additional data file.


Evidence suggests that pigs seroconvert after experimental exposure to Zika virus and are potential sentinels. We demonstrate that pigs are also susceptible to natural Zika virus infection, shown by the presence of antibodies in domestic pigs in Yucatan, Mexico. Zika virus RNA was detected in 5 species of mosquitoes collected inside pigpens. Pigs are susceptible to experimental Zika virus infection , Valladolid (159 km east), and Xkalakdzonot (155 km southeast). Each village maintained herds of Yucatan black hairless pigs as a food source for residents. We visited each site 1–3 times during 2018 and 2019, and no pigs were sampled more than once. An unusually high number of porcine fetal deaths occurred in Xmatkuil and Xkalakdzonot several weeks before our initial visits. The stillborn pigs displayed signs of mummification but no apparent neurologic malformations, according to their owners. During each visit, we searched human-made structures and vegetation for resting mosquitoes, which were collected by manual aspiration.>90% reduction in the number of plaques (PRNT90). For etiologic diagnosis, the PRNT90 antibody titer to the respective virus was required to be >4-fold that of other flaviviruses tested.Serum samples were assayed by plaque-reduction neutralization test (PRNT) using dengue virus (DENV) serotype 1 (strain Hawaii), DENV serotype 2 (strain NGC), DENV serotype 3 (strain H-87), DENV serotype 4 (strain 241), Ilheus virus , St. Louis encephalitis virus (strain TBH-28), West Nile virus (strain NY99–35261–11), and Zika virus (strain PRVABC59). Serum specimens were initially screened at a dilution of 1:20 by using Zika virus. Positive samples were further diluted, then assayed using all 8 viruses. Titers were expressed as the reciprocal of serum dilutions yielding <50 according to species, sex, date, study site, and location within the study site. Mosquitoes were transported in RNAlater  to Iowa State University, then homogenized by using mortars and pestles. Total RNA was extracted by using Trizol Reagent  and tested for Zika virus RNA by using reverse transcription PCR and Sanger sequencing using primers that amplify a 667-nt region of the envelope protein gene.Mosquitoes were transported alive to the arbovirus laboratory at the Universidad Autonoma de Yucatan and sorted into pools of 90 titers ranged from 40 to 320 . Thirty-eight (12.8%) pigs were positive for flavivirus-specific antibodies. Thirteen (4.8%) pigs were seropositive for Zika virus, 1 (0.3%) pig was seropositive for West Nile virus, and 24 (8.1%) pigs had antibodies to an undetermined flavivirus. Zika virus PRNT0 to 320 . Eleven >1 engorged mosquito, were positive for Zika virus sequence, and all consisted of mosquitoes collected inside pigpens in Xmatkuil (Genbank accession nos. MT309004–309008). One pool each of the following mosquito species tested positive: Aedes aegypti, Ae. taeniorhynchus, Culex lactator, Cx. nigripalpus, and Cx. thriambus. All sequences were identical and differed from the positive control, an isolate from the state of Chiapas, Mexico, in 2016 (Genbank accession no. KX446950.2) in 1 nucleotide position, a C→T substitution at genomic position 1893.The entomologic investigation yielded 1,870 mosquitoes of 8 species that were sorted into 190 pools. Of these, 381 mosquitoes were collected inside pigpens, and >50% were engorged . MosquitAe. aegypti, Ae. taeniorhynchus, Cx. lactator, Cx. nigripalpus, and Cx. thriambus mosquitoes that were temporally and spatially associated with pigs seropositive for this virus. The role of Culex spp. mosquitoes in Zika virus transmission has been debated, but the consensus among the arbovirus community is that they are inefficient vectors  on the same date . We speculate that these pigs were infected with Zika virus just before our visit and that some mosquitoes then bit them, without virus disseminating from the midguts of We provide additional evidence that pigs produce neutralizing antibodies upon Zika virus exposure and are potential sentinels. This information will be useful for investigators and public and veterinary health personnel conducting surveillance in Zika virus–endemic areas where pigs are common and usually raised outdoors. One limitation of our study is that pig farmers were not tested for evidence of flavivirus infection. Future studies should investigate whether those persons are at increased risk for Zika disease.


Infection is a major cause of cancers. We estimated the economic burden of cancers attributable to infection in 2014 in Korea, where cancer causing infection is prevalent, but the economic burden of it has never been examined. Cancer patients were defined as those having made medical claims as recorded by the National Health Insurance Service, which is a mandatory insurance for all citizen. We multiplied the costs by the population-attributable fraction for each type of cancer. The study included direct and indirect costs, where direct costs comprised direct medical and non-medical costs of inpatients and outpatients, while indirect costs were estimated by identifying future income loss due to premature death, productivity loss during hospitalization/outpatient visits, and job loss. In 2014, there were 100,054 infection-related cancer patients, accounting for 10.7% of all Korean cancer cases for that year. Direct costs of cancers associated with infection stood at nearly USD 676.9 million, while indirect costs were much higher at USD 2.57 billion. The average expenditure of a typical patient was USD 32,435. Economic burden of cancers attributable to infection is substantial in Korea, accounting for 0.23% of the national gross domestic product and 1.36% of national healthcare expenditure in 2014. Cancer places an enormous burden on society regardless of countries’ development. Overall, the numbers of new cancer cases and deaths reported worldwide in 2012 were 14.1 and 8.2 million, respectively . In 2015Clonorchis sinensis (C. sinensis), Epstein Barr virus (EBV), human immunodeficiency virus (HIV), and Kaposi sarcoma-associated herpes virus (KSHV) have been categorized as Group 1 human carcinogens by the International Agency for Research on Cancer (IARC) [A significant number of infectious agents, namely helicobacter pylori (HP), hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), r (IARC) . With rer (IARC) . Thereafr (IARC)  and 2.2 r (IARC) , constitr (IARC) .In 2007, the estimated proportions of all cancers in Korea attributable to infection were 25.1% and 16.8% for cancer incidence in men and women, respectively . In addiTo date, several cost-of-illness studies have emphasized the large burden incurred by specific types of cancer and groups of cancers linked to risk factors such as smoking . HoweverClonorchis sinensis, EBV, HIV, and KSHV/HIV. The current study used the population-attributable fraction (PAF), based on previous research conducted in Korea [The list of infection-causing agents includes HP, HBV, HCV, HPV, in Korea .The total economic cost of cancer due to infection was measured by multiplying the cost of each cancer site by the respective PAF, as described in the following formula:Health insurance claims data provided by the National Health Insurance Service (NHIS) were analyzed. NHIS insurance is a mandatory single-payer system that provides benefits for medical services. All South Korean citizens must either enroll in the NHIS (97% of the entire population) or receive medical aid (3%) . The NHIThis study used anonymous secondary data and was exempted from the Institutional Review Board of the National Cancer Center, Korea . The requirement for obtaining consent was waived.We conducted a cost-of-illness study to assess the economic burden of cancer due to infection based on a prevalence-based approach aiming at existing and newly diagnosed patients in 2014. Major cancers attributable to infection were selected with regards to the IARC Monograph Evaluation Carcinogenic Risks to Humans 2012 . InfectiThe total economic burden of infection-related cancers included direct and indirect costs.Direct costs are costs incurred during medical treatment and consist of direct medical and nonmedical costs. Medical claim records from the 2014 NHIS were used to obtain direct medical care costs, and existing and newly diagnosed patients with infection-related cancer were targeted. The non-covered medical costs were estimated at 19.9% of the total direct medical costs . Direct Formula:DC = direct costs; j = age; i = gender; y = cancer typeIP = total treatment duration for inpatients of i and j in NHIS dataOP = total treatment duration for outpatients of i and j in NHIS dataα = the ratio of noncoverage and coverage rates IV = duration of inpatient visits (in days)OV = duration of outpatient visits (in days)TIV = cost of one-way trip to hospital for inpatientsTOV = cost of one-way trip to hospital for outpatientsCGR = caregivers’ average wage per dayC = utilization rateOV’ = durations of outpatient visits (in days) for those aged ≥65 yearsPAF = population-attributable fractionIndirect costs included future income loss (FIL) because of premature death, productivity loss (PL) due to inpatient or outpatient hospital visits, and job loss (JL) after cancer diagnosis. A human capital approach was applied to the estimate FIL by calculating the income each person would have earned during the period from earlier death to normal life expectancy for the age cohort. A human capital approach based on the assumption that future income acts as a substitute for future productivity was used to determine future income loss, but it does provide an accurate representation in some cases . FIL folFormula:FIL = future income lossi = sex; j = age; t = age at deathy = cancer typek = 1, 2, …, n (n is the difference between age of death and life expectancy of the age cohort)D = number of deathsDPAF = death population-attributable fractionsYW = yearly wage by i and jE = employment rates by i and jr = discount ratePL = productivity lossi = sex; j = age; y = cancer typeIV = number of inpatient visitsOV = number of outpatient visitsE = employment rateDW = daily wagePAF = population-attributable fractionJL = job lossN = number of prevalent cancer casesi = sex; j = age; y = cancer typeYW = yearly wageE = employment rateL = job loss ratePAF = population-attributable fractionSensitivity analysis was performed to examine productivity loss following premature death, with 0% and 5% annual discount rates. Furthermore, sensitivity analysis was stratified according to noninsured healthcare costs and the upper and lower bounds of the 95% confidence interval for the PAF. All analyses were performed using SAS version 9.2  and Microsoft Excel 2013. All costs were converted from KRW to USD based on the exchange rate in 2014 (1 USD = 1091.85 KRW) .There were approximately 100,054 patients with infection-related cancer in Korea in 2014, accounting for almost 10.7% of all cancer cases that year in which no remarkable differences in the figures for males and females were recorded, with relatively equal numbers of patients  of each sex . OverallThe estimated number of deaths with infection-related cancer was 13,380 deaths, accounting for 13.4% of prevalent cases in that year .The economic burden of cancer attributable to infection in 2014 was USD 3.25 billion, and the majority of the total indirect cost was incurred for HBV, HP, and HCV. The direct cost of cancer attributable to infection in Korean adults aged ≥20 years was estimated at approximately USD 676.9 million . The estIn contrast, the indirect cost of cancer attributable to infection in Korean adults aged ≥20 years was approximately USD 2.57 billion . RegardlRegarding cancer types, the costs incurred for hepatocellular carcinoma accounted for the largest proportion of the total cost (USD 1.9 billion), followed by uterine cervical cancer (USD 285 million) and cholangiocarcinoma , followed by job loss (24.0%) and direct medical costs (18.9%) .The results of the sensitivity analysis performed to examine different discount rates showed that the economic burden was USD 3.69 billion with a discount rate of 0% and USD 3.04 billion with a discount rate of 5%. The sensitivity analysis involving the lower and upper bounds of PAF showed that the economic burden ranged from USD 2.68 billion using the lower bound to USD 4.14 billion with the upper bound .The economic burden of cancers attributed to infection in Korea in 2014 was approximately USD 3.25 billion, and indirect costs were 3.8 times higher than direct costs. Indirect costs were significantly more burdensome and 2–3 times higher than direct costs reported in previous studies ,24. FurtRegarding direct costs, HBV, HP, and HCV accounted for the majority of infection-related cancers in men, while women’s direct costs were incurred for HPV, HBV, and HP. Trends for indirect costs incurred for the top three infectious agents were similar for both sexes. Overall, HBV accounted for the greatest economic burden of morbidity and mortality resulting from infection-related cancers, followed by HP and HCV. The costs incurred by men for infection-related cancers were four times higher than those incurred by women.The current study included the cost of job loss as an essential part of indirect costs. Although several Korean studies involving economic burden estimation did not examine income lost through unemployment following cancer diagnosis, job loss should be considered carefully because of the high proportion of the total cost attributable to this factor. The current results suggested that 23.96% of the overall infection-related cancer costs were attributable to job loss. The annual decrease in the mortality rate since 2002, combined with the improvement of 5-year survival rates in cancer patients in Korea , raises From the results of our study, a combination of productivity loss and job loss burden accounts for a substantial fraction of the total infection-related cancer costs 27.8%). There is also other research placing an importance on the burden of productivity loss and job loss in terms of cancer costs. In a study on the economic burden of cancer survivorship among adults in the US , indirec.8%. TherIn our study, we selected patients based on the special code V193 claimed by medical providers for inpatient stays and outpatient visits. The main reason for this was that, in accordance with the policy of expanding benefit coverage, special codes are provided for patients with severe diseases, such as cancer and myocardial infarction, to reduce the substantial healthcare expenses that could be incurred. In contrast, with the exception of one study that estimated the economic burden of metabolic syndrome-related cancers  and usedMeasurement of the economic burden of cancers associated with infection has not received a considerable attention in developed countries, because these types of cancer are uncommon in highly industrialized nations, with the proportion of cases fluctuating between 5% and 10% , while nThe study was subject to several limitations. The first was the potential overestimation of productivity loss based on the human capital approach used to measure future income loss, as this method has been criticized for its assumption that workers cannot be replaced, even if unemployment is significantly high. However, the human capital approach is one of the most widely used methods in the estimation of indirect costs of diseases. In addition, medical costs could have been underestimated, because the outpatient pharmaceutical costs of cancer-specific treatment could not be verified via the NHIS claim data, and the cost of alternative and complementary medicine was excluded because of an absence of data. Furthermore, cancer costs attributed to infection could have been underestimated, as only primary diagnoses were included in the study. Despite these limitations, this was the first study to focus on the estimation of the economic burden of infection-related cancers in Korea, using NHIS claim data for the entire Korean population. Further investigation on economic burden of infection-specific cancer for evaluating impact of intervention programs of infection control and prevention is needed.In the current study, we calculated the economic cost of cancers attributed to infection in Korea in 2014, using a representative dataset from the NHIS, which reflects the entire Korean population. Despite a gradually decreasing trend in the incidence of infection, the results showed a substantial economic burden resulting from infection-related cancers, and this is expected to increase in the near future because of population growth and the time lag between infection and cancer development. These results could assist policymakers to reinforce and promote infection prevention programs and early treatment for infectious diseases nationwide. In addition, the findings could provide a useful baseline for further research examining the effectiveness of ongoing preventive policies in Korea.


Aedes canadensis, Coquillettidia perturbans and Culiseta melanura mosquitoes infected with the EEE virus  than with Culiseta morsitans, Aedes vexans, Culex pipiens-restuans, Anopheles quadrimaculatus or Anopheles punctipennis . The estimated relative risk of a case in a year in which the virus was detected vs. not detected was 14.67 for Ae. canadensis, 6.38 for Cq. perturbans and 5.50 for Cs. morsitans. In all 5 years with a case, Cs. melanura with the virus was detected. In no year was there a case in the absence of Cs. melanura with the virus. There were 18 years with no case in the presence of Cs. melanura with the virus. Such observations may identify the time of increased risk, and when the methods may be used to prevent or reduce exposure to vector mosquito species in this geographic region.From 1971 to 2012, in New York State, years with human Eastern equine encephalitis (EEE) were more strongly associated with the presence of  Eastern equine encephalitis (EEE) neuro-invasive disease can be severe, with symptoms including seizures, paralysis, coma and death . EEE hasCuliseta melanura (Coquillett) is a vector that maintains a life cycle of the virus, through birds [2.The mosquito species gh birds , 7. In Ngh birds . Such hagh birds . The twoIn 1959, occasional testing of mosquitoes for arboviruses, including the EEE virus, began on Long Island in New York State , in the Occurrences of human cases , 13, 15 http://www.ncbi.nlm.nih.gov/pubmed/). These data were originally obtained by the members of this Department of Health. One of the authors has been present since the beginning of data collection.Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed . Data on2), Oneida (3140 km2), Onondaga (2020 km2) and Oswego (3398 km2), located in a central geographic region of New York State having a centre point at a coordinate of 43.2° north and 75.8° west. Cases of EEE in humans and horses have been documented within a 3600 km2 area in these counties [2) and 2000 hectares (20 km2) [The study area consisted of the four contiguous counties of Madison . Standard nomenclature for mosquito genus and species was used [Aedes canadensis  was used, in preference to a recently proposed renaming to Ochlerotatus canadensis  [Detection of the EEE virus in mosquitoes in New York State was obtained from published literature. For the study period 1971–2012, published articles of surveillance for the EEE virus in mosquitoes covered the years: 1971 , 1970–19wetlands . Among an detail . In thesn detail . For thiwas used –34. The heobald) .P value was calculated.The hypothesis that the presence of an infected species of mosquito was associated with the presence of a case in a human was evaluated using Fisher's exact test . This teAn alternative hypothesis tested was that the proportion of years with human cases would be higher when the virus was present in a species of mosquito than the corresponding proportion of years with human cases when the virus was absent in a species of mosquito. Relative risk was used to quantify the strength of the association between years in which a human case was present with the presence of the virus in the mosquito species. Relative risk was defined as the probability of a human case occurring in a year in which the virus was present for the listed species divided by the probability of a human case occurring in a year in which the virus was absent for the listed species.http://www.sas.com).Statistical Analysis Software (SAS) was used , Coquillettidia perturbans  (P = 0.03) and Cs. melanura (P = 0.03)  (P = 0.05), Aedes vexans (Meigen) (P = 0.40), Culex pipiens-restuans complex of Culex pipiens Linnaeus and Culex restuans Theobald (P = 0.33), Anopheles quadrimaculatus Say (P = 1.00) or Anopheles punctipennis (Say)   . There wct test) .Table 2vs. not detected, in a specific mosquito species, was 14.67 for Ae. canadensis, 6.38 for Cq. perturbans and 5.50 for Cs. morsitans. There was no year during which a case was found in a human when the virus was absent in Cs. melanura  of mosquito poolings with the EEE virus were collected in those two counties. Flight distance maximums, for termined . A flightermined . Flight ectively . The lonThis study has the limitation of being dependent on reporting of cases by doctors, hospitals, commercial laboratories, agency laboratories and departments of health of counties and cities , 46. DurSampling error can occur, because the sample sizes customarily allowed or accepted and published by laboratories have been 10–100 individual mosquitoes. If the prevalence of the virus in a population of a species of mosquito was one in 1 000 or one in 10 000, then nine of 10 samples, referred to here as poolings or pools, may have no virus detected. In such instances, the results may lead to a conclusion that virus was not present in that population, when in actuality it was present at a level below the limit of detection in that circumstance.Cx. pipiens-restuans, Ae. vexans, An. quadrimaculatus or An. punctipennis should not be misinterpreted to imply that these species could not transmit the virus to humans in other circumstances.This study applies to one region of New York State and is not necessarily generalisable to other geographic areas of the state or country. The laboratory that tested for the virus in mosquitoes set limits to the number of poolings of mosquitoes that each county could send for testing. Each county made the decision on what species of mosquitoes to send for testing, based in part on the species of viruses of concern for public health or vector control. Therefore, not all species of mosquitoes could be sent to the laboratory for testing. The absence of a statistically significant association between the presence of cases in humans and the virus in This information may be useful for public health officials deciding whether or not to initiate methods to suppress vectors, for individuals using personal protection to reduce exposure to mosquitoes and for clinicians to include EEE in the differential diagnosis of encephalitis.


Tobacco product waste (TPW) is one of the most ubiquitous forms of litter, accumulating in large amounts on streets, highways, sidewalks, beaches, parks, and other public places, and flowing into storm water drains, waste treatment plants, and solid waste collection facilities. In this paper, we evaluate the direct and indirect costs associated with TPW in the 30 largest U.S. cities. We first developed a conceptual framework for the analysis of direct and indirect costs of TPW abatement. Next, we applied a simulation model to estimate the total costs of TPW in major U.S. cities. This model includes data on city population, smoking prevalence rates, and per capita litter mitigation costs. Total annual TPW-attributable mean costs for large US cities range from US$4.7 million to US$90 million per year. Costs are generally proportional to population size, but there are exceptions in cities that have lower smoking prevalence rates. The annual mean per capita TPW cost for the 30 cities was US$6.46, and the total TPW cost for all 30 cities combined was US$264.5 million per year. These estimates for the TPW-attributable cost are an important data point in understanding the negative economic externalities created by cigarette smoking and resultant TPW cleanup costs. This model provides a useful tool for states, cities, and other jurisdictions with which to evaluate a new economic cost outcome of smoking and to develop new laws and regulations to reduce this burden.  Tobacco product waste (TPW) is one of the most ubiquitous forms of litter, accumulating in substantial quantities on streets, highways, sidewalks, beaches, parks, and other public places, and flowing into storm water drains, waste treatment plants, and solid waste collection facilities ,2,3,4. TMore than 249 billion cigarettes were consumed in the United States in 2017 . While mAn “externality” occurs whenever the activities of one economic agent affect the activities of another agent in ways that are not taken into account by the operation of the market. When these activities are harmful to one of the economic agents, and the harmed agent is not compensated for the harm, the cause of the harm is typically referred to as a “negative externality” ,15,16. TTPW generally originates and accumulates in places where people smoke. As smoking becomes increasingly banned or restricted in restaurants, bars, and public areas such as parks and airports, the locations in which smoking takes place may have become more conducive to discarding cigarette butts ,32. WhenSmoked cigarette butts are the main focus of this analysis, but we note the enormous increase in sales of electronic nicotine delivery systems (ENDS) beginning in 2007 in the United States. These products contain varying amounts of plastics, nicotine, flavorings, and lithium batteries that create additional waste. The tobacco industry and smaller manufacturers have diversified production of these products and have already been able to market some of them with US Food and Drug Administration (FDA) approval, including heat-not-burn products (IQOS). Producers will be submitting for pre-market approval additional tobacco products including ENDS, smokeless tobacco, and various other forms of nicotine and non-nicotine containing products. Currently, we are unable to estimate the total impact such products may have on waste streams, but FDA’s Environmental Assessment on IQOS estimates that modest switching among people who smoke to primarily dual use of the product will result in an increased weight of discarded "sticks" equivalent to 74,100,000 more butts per year (over the previously stated estimate of 249 billion butts per year in 2017) . FurtherTo estimate the total TPW cost, we combine (1) indirect costs attributable to the accumulated TPW prior to abatement and (2) the direct costs attributable to deterrence and abatement. In order to fully evaluate the externalities of TPW abatement, we first present a conceptual framework that describes the ways in which TPW generates direct and indirect costs to cities and municipalities. Next, based on this conceptual framework and existing data, we develop an online simulation model to estimate total direct and indirect TPW costs for the 30 largest U.S. cities.n = 8), the average annual per-capita costs of litter cleanup were $12.54. The Kier study focuses mainly on waste that may enter a coastal or marine environment, which may be an underestimate of total litter. We increased this estimate in our study by 25%, which resulted in a direct annual cost estimate of $15.68 per person. When adding this amount to the indirect costs that might be associated with TPW, there is an estimated annual cost between $20 and $30 per person.Estimating a per capita cost for cities could help planning agencies determine how best to manage the costs of TPW and decide whether there are best practices to help cut costs. There have been very few studies performed to estimate TPW costs. However, a 2012 report by Kier Associates for the U.S. Environmental Protection Agency provides a useful model for combining several concepts discussed here to estimate TPW costs . The resAs a result of smoking behaviors, TPW accumulates in public places. It is picked up either mechanically or manually as part of organized cleanup efforts; what is not picked up remains stationary or migrates into public storm water and sewage systems . MigratiNicotine has been used for more than a century as a pesticide and was banned for such use by the United States Environmental Protection Agency in 2014 . It is fIn addition to the effects of TPW toxicity to the aquatic biome, environmental “cleanliness” plays an important role in the demand for tourism and as a quality of life issue ,47,48,49A growing body of research has shown that cigarette butts have the potential to harm humans and animals ,6. CigarCities and municipalities devote considerable resources to litter deterrence and abatement. Deterrence includes the posting of signage indicating the fines associated with littering or the harm caused to the environment, and may in some cases include other means through which to increase public awareness, such as public service announcements on billboards and local radio/television broadcasting. Deterrence also includes law enforcement in areas prone to littering and the issuing of summons when law enforcement officials directly observe littering behavior. Littering behavior, however, has still proved to be extremely difficult to address through deterrence alone . Thus, cAbatement tasks typically include the following: provision and management of disposal receptacles , mechanical street sweeping, mechanical power washing, manual power washing, manual cleanup, storm drain clean out, and water treatment processes. It should be noted that mechanical street sweeping is not intended solely to collect street litter but is also intended to clear the streets of organic debris  that would otherwise accumulate on the street and migrate to storm drains. There are also administrative tasks associated with the management and execution of each of these activities. Direct costs generally include costs related to labor , equipment and supplies, transportation and fuel, and landfill fees. Administrative costs are also considered direct costs, but such costs are mostly in the form of labor. Mechanical street sweeping and mechanical power washing have relatively high equipment and labor costs (vehicle operators and maintenance), whereas manual power washing and manual cleanup are mainly labor costs. Storm water clean out is mainly labor costs, though specialized equipment may be used in some cases. Water treatment system clean out may in some cases be mechanical  but may also require a non-trivial labor component. Law enforcement is associated with high labor costs, though the proportion of law enforcement activities attributable to litter deterrence is likely to be very small in most cities and municipalities. Fuel costs can be considerable, especially for the mechanical street sweeping and power washing equipment. Fuel costs are also incurred in transporting litter to landfills, and landfills charge fees based on the weight of the trash deposited.https://rminator.shinyapps.io/TPW1/. The flowchart for the simulation is shown in The Monte Carlo simulation was used in this study, as exact information regarding some variables (such as per capita litter cleanup costs) is not available. Using known data mixed with probability distributions and sensitivity analysis allows for estimating the total cost ranges for cleanup by city. The simulation was written in R  with R SB), and a visitor adjustment factor (V).After a pseudo-random number seed is set for replication, the number of iterations (between 100 and 2000) is set by the user in Step A. This allows for improving the interval estimates based on specific user requirements. Three other parameters are set in Step A, the deterrence/abatement goal (α), the base cleanup cost per person . For example, a city may tolerate 10% of existing and accumulated litter, which implies that its goal (α) will be to deter and/or abate 90% of existing and accumulated litter. In step A, the user estimates a global value for α between the range of 0.5 and 1.0.B).The third user entry is the deterrence/abatement estimate per capita. The simulation model estimates the total TPW-attributable costs for these 30 largest U.S. cities based on total population residing within city boundaries . BaselinV provides between −10% and +10% of a population adjustment.The last user-flexible parameter provides an option to estimate increases or decreases based on visitor nights in the city. Specifically, some large city visitor populations might increase the overall number of daily residents in those cities. Other cities may actually have fewer full-time residents due to seasonal tourism . To accoj is the number of iterations . Index k loops over the cities . An N × 30 matrix holds the results of the total costs, which are then analyzed post-hoc.Step B provides the indices necessary for the simulation. Index kPOP) from 2017 derived from the U.S. Census Bureau [kPREV) are from the County Health Rankings data provided by the Centers for Disease Control and Prevention BRFSS dataset [kPARITY) are from the Bureau of Economic Analysis [The data for the simulation are read in step C. Population data for each city  differ widely, meaning that the proportion of litter waste attributable to TPW varies widely. To account for this factor, an index factor was used in the simulation. The mean prevalence rate for all cities in the study (0.147) calculated by dividing the total number of smokers in all cities (SMOKERS) by the population became the denominator to scale each individual prevalence rate as in Equation (1). .City prevalence rates  information for 2017 . BEA datkPCC, is a straightforward calculation from the previous steps. Equation (3) shows this calculation. The total cost estimate per person from Step E is adjusted for the contribution of TPW waste specified as λ and the municipality goal for deterrence or abatement α.The final cost per capita, kTC. Total costs are calculated from Equation (3) by multiplying by the population (kPOP) and adjusting for visitor effects on the population (V). V is designed for evaluating a single city based on its seasonal tourism. Equation (4) is the resultant estimate for city total costs.The final calculation in the simulation provides the estimate for total cost for each city, A combination of methods was used for verification and validation. First, the simulation was initially written in Excel. Those results were evaluated after improvement and migration to R to establish convergent validity. Secondarily, post-hoc results were evaluated for prima facia validity. For verification, the definition of total cost as direct plus indirect cost is well established, and these are the components used.https://rminator.shinyapps.io/TPW1/. Raw data for the simulation are available online at A pairs plot (scatterplot matrix) with histograms on the diagonal and bivariate boxplots on the lower triangle shows the distributions and relationships among variables . The uppN = 2000 simulation, α = 0.9 deterrence/abatement goal, V = 0% adjustment for visitors, and B = $23 base per person cleanup cost. Location statistics and measures of center by city for this simulation are shown in For this analysis, we evaluated a simulation with the following parameters: p = 0.05 level (Notched boxplots perform a median test, where notches that do not overlap are statistically different at the 05 level . The minTo date, there have been no studies specifically designed to assess the indirect costs of TPW. These may include environmental degradation, defined as any change or disturbance to the environment perceived to be deleterious or undesirable. It is also defined by the United Nations as "the reduction of the capacity of the environment to meet social and ecological objectives and needs .” In theThe cost pathways for ecosystem impact are less clear, and, as discussed in the conceptual framework, the literature on the association between TPW, ecosystem impact, and human health is relatively new and developmental ,66,67,68This study presents the first estimates of the substantial negative economic externalities imposed on large U.S. communities by TPW. In this paper, we have further explored some of the issues that arose in our earlier work on tobacco product waste in San Francisco . In the This study has some important limitations. First, we use a simulation model to estimate TPW costs, and there are tradeoffs associated with simulation models. The model uses the best available aggregate data and point estimates, but it is not a perfect substitute for a comprehensive survey of TPW costs within each of the U.S. cities. Second, the model is not sensitive to variation in the propensity to litter; the main source of intra-city variation in TPW costs is smoking prevalence, although it is possible that the propensity to litter varies by city or region. Littering is a crime and crime rates vary among cities and regions, and littering rates are likely to vary similarly. Unfortunately, we were not able to identify a reliable and accurate data source on littering propensity. Finally, some cities may be more efficient than others in their cleanup efforts, and this variation in efficiency could result in cost differentials that are not reflected in our model.As mentioned in the introduction, this model does not include any additional or reductions in TPW costs associated with ENDS and other novel tobacco product use. These may only represent a small percentage of total TPW at present, but it will be important to include in future models the effects of substitution or additional costs associated with these products. Further, in addition to the effects of nicotine mentioned, the lithium in these devices could contribute toxicity to the environment, but this impact is still poorly studied .Nevertheless, economic estimates of the costs of TPW to taxpayers and communities are an important avenue of research that can raise the level of interest in the heretofore poorly managed waste stream of TPW. EPR and PS approaches to TPW costs may have substantial impacts on its prevention, mitigation, and reduction, as these efforts will require tobacco product producers to take responsibility for all or most of the community economic burden due to TPW. EPR/PS approaches may include litter fees and advanced recycling program fees, take back programs such as those for paint and electronics, and litigation for cost recovery ,29,71. T


Lampyris noctiluca) are best known for emission of green light by their larvae and sexually active adult females. However, both their DNA and RNA viruses remain unknown. Glow-worms are virologically interesting, as they are non-social and do not feed as adults, and hence their viral transmission may be limited. We identified viral sequences from 11 different virus taxa by the RNA-sequencing of two Finnish populations of adult glow-worms. The viruses represent nine different virus families and have negative, positive, or double-stranded RNA genomes. We also found a complete retroviral genome. Similar viral sequences were found from the sequencing data of common eastern firefly of North America, a species belonging to the same family (Lampyridae) as that of the common glow-worm. On average, an individual glow-worm had seven different RNA virus types and most of them appeared to establish a stable infection since they were found from glow-worms during two consecutive years. Here we present the characterization of load, prevalence, and interactions for each virus. Most of the glow-worm RNA viruses seem to be transmitted vertically, which may reflect the biology of glow-worms as non-social capital breeders, i.e., they invest stored resources in reproduction.The common glow-worms (The online version of this article (10.1007/s11262-019-01724-5) contains supplementary material, which is available to authorized users. Lampyris noctiluca) is a nocturnal beetle, whose larvae and sedentary females emit highly visible green light. Larva glow is a warning signal for distastefulness [The common glow-worm , were observed to be transmitted vertically, that is, from parent to offspring [Glow-worm populations seem to be isolated from each other due to local differences in their emergence time and females’ inability to fly. This lack of conspecific contact makes glow-worms epidemiologically interesting. Larvae are predators feeding mainly on slugs and snails, and at early stages the larvae may forage together . Individffspring .We identified viral sequences from two Finnish glow-worm populations and studied their presence in different life stages and tissues in Finnish and English glow-worms. We identified 11 RNA viruses from adult glow-worms, four positive-stranded, three negative-stranded, three double-stranded, and one retrovirus. Most of the viruses formed stable infections as they were obtained from glow-worms collected in two different years. The glow-worm viruses are found in egg tissue as well as unfed larvae, which suggests that most of them may be transmitted vertically from parents to offspring.N = 12) hatched one month after egg-laying and were kept alive, unfed for 2 weeks. Online Resource 1 shows a map of sample collection sites. All the insects were stored in RNAlater until RNA extraction.The data consists of several cohorts: (1) Adult female and male glow-worm samples used for RNA-sequencing were collected from two Finnish populations: Nine females and four males from Konnevesi ; and eight females and eight males from Hanko . (2) Adult female samples, used for qPCR analyses, were collected in June and July, 2018, from two populations, four females from Äänekoski , and five females from Tvärminne Zoological Station . (3) Laboratory-reared larvae from different mothers were used for qPCR analyses. Mothers of three of these larvae were collected from southern Finland  and mated in 2016, and three larvae with unknown relations were collected from England . These six larvae were reared and kept in the laboratory for two years and fed with organic cat food and terrestrial and aquatic snails collected from southern Finland. (4) Two-week-old larval offspring of two parental glow-worms (collected and mated in June 2018) were obtained from Tvärminne Zoological Station. The larvae . The RNA samples were sent to BGI Tech Solutions for poly-A selected and strand-specific library preparation using Illumina TruSeq Stranded mRNA Library Prep Kit and RS-122–2101 kit. The libraries were sequenced with Illumina HiSeq4000 producing 100 bp paired-end reads. Samples from 2018 were isolated with Trizol (ThermoFisher). cDNA was prepared using 500 ng total-RNA with ReverAid H Minus First Strand cDNA synthesis kit (ThermoFisher), as per the manufacturer’s protocol.e-value < 10–10 and culling limit 1) against invertebrate virus protein sequences using NCBI virus protein RefSeq . Contigs from individual data were translated to protein sequences with Transdecoder (github.com/TransDecoder), and only the longest of protein coding sequence over 160 aa was selected for search with Blastp against the invertebrate virus proteins. Virus positive contigs were pooled and aligned with each other with CAP3 and searched using Blastx, as above. Virus contigs were manually checked for complete or nearly complete viral genomes using NCBI ORFfinder (www.ncbi.nlm.nih.gov/orffinder/). All the identified viral sequences were seeded into NOVOplasty [The raw RNA-seq reads from the 2017 samples were trimmed for adapters and low-quality nucleotides (Q < 20) and filtered for reads less than 36 nt in length. The trimmed reads were pooled and assembled using Trinity v2.3.2 with in silico normalization . ContigsVOplasty  with theVOplasty  so that VOplasty .NCBI Blastp (nr) was used to identify possible domains and identical protein sequences: identity describes, which percent of characters in the query sequence is identical with the target sequence and coverage describes how much of the query sequence is covered by the target sequence. HHpred  was usedSimilar virus sequences to glow-worm viruses were searched with Blastx from other insects (taxid:6960) from NCBI’s transcriptome shotgun assembly sequence database (TSA). We set Blastx bit score, which takes into account the alignment length, mismatches and gaps, above 200 for positive hits.Phylogenies were reconstructed from the RdRP amino acid sequences of each virus and 19 most similar hits in Blastp, RSA TSA hits Table , and virqPCR was performed with virus-specific primers (Online Resource 2) using EvaGreen (Solis BioDyne) kit, as per manufacturers’ protocol. Standards for qPCR were created with virus-specific primers using Phusion enzyme (ThermoFisher). The PCR products were isolated with GeneJET gel extraction kit (ThermoFisher). Only the results with over 10 viral sequences were analyzed further.www.R-project.orf/). Pairwise analysis of virus reads was done by Spearman’s correlation using multiple testing p-value correction.All statistical analyses were performed in R v3.5.0 . The strand-specific data indicated that all identified positive-strand RNA viruses were replicating in the glow-worms. However, as the production of the sequencing library involved poly-A enrichment that biases the ratios of the virus transcripts, the strand-specific data were not analyzed further.We identified 11 RNA viruses by whole-transcriptome sequencing of 29 adult glow-worms. The samples were from two Finnish populations collected in 2017, four males (heads) and eight females (lanterns) from Konnevesi Research Station and eight males (heads) and nine females (lanterns) from Hanko (Online Resource 1). The tissues were chosen because they are important in sexual signaling. Viral sequences were assembled from pooled sample with the Trinity assembler . However, only fraction of the glow-worms had complete virus sequences without gaps and thus the analysis produced only descriptive information. When considering only southern population females that had at least two complete virus sequences per group, highest frequency of variable sites were with iflaviruses (LnoIV1: 0.0097 variable sites/genome size/sample number and LnoIV2: 0.0152), and lowest with flavivirus LnoFV1 (0.00097) and partitivirus LnoPV1 (0.0010). Frequency of variable sites for partitivirus LnoPLV2 was 0.0016 variable sites/genome size/sample number.L. noctiluca flavivirus 1 (LnoFV1) is 18,917 nt long and encodes a 6152 aa polyprotein. Blastp search results indicated protein sequence similarity to the putative polyprotein of Drosophila flavivirus, Takaungu virus . HHpred found sites in the LnoFV1 sequence similar to Serine protease NS3/RNA helicase/hydrolase of classical swine fever virus  and methyltransferase and RNA-dependent RNA polymerase (RdRP) of Japanese encephalitis virus . Both swine fever virus and Japanese encephalitis virus are flaviviruses. On the basis of the phylogenetic analysis, LnoFV1 was most similar to Drosophila flavi-like viruses, Takaungu virus and Hermitage virus  (Online Resource 5) [Flaviviruses are positive-strand RNA viruses and include notorious arboviruses with mosquito  and tick vectors . ource 5) . RNAfoldource 5) .Lampyris noctiluca iflavirus 1 (LnoIV1) is 10,339 nt long and codes for a polyprotein of 3134 aa. Blastp indicates polyprotein similarity (47% coverage and 28% identity) to a hypothetical protein of Hubei picorna-like virus 31  from odonata mix [e-value = 1.6e-23, 1AYM_2), coat protein of iflavirus Deformed wing virus , capsid protein of iflavirus Slow bee paralysis virus , ATPase of enterovirus 2C , and RdRP of human polio virus 1 . All the HHpred identified viruses belonged to the Picornavirales order. Based on the phylogenetic analysis LnoIV1 was most closely related to unclassified bat Rolda virus  (Online Resource 6). Stable RNA structures, which could function as an internal ribosomal entry site (IRES), were identified by RNAfold on the first 729 nucleotides.Iflaviruses are insect-infecting positive-strand RNA viruses coding for a single polyprotein. nata mix . AccordiLampyris noctiluca iflavirus 2 (LnoIV2) is 10,428 nt long and codes a polyprotein of 3132 aa. Blastp search shows polyprotein similarity (73% coverage and 26% identity) to unclassified bat virus polyprotein of Rolda virus . According to HHpred, the polyprotein had sites similar to the capsid proteins of Slow bee paralysis virus  and Deformed wing virus , ATPase of enterovirus 2C , and RdRP of human poliovirus 1 . All HHpred identified viruses belong to the Picornavirales order. According to phylogenetic analysis, LnoIV2 was most similar to Hubei picorna-like virus 31  from odonata mix [L. noctiluca iflavirus 1 and 2 is 29.5% with 33% coverage. Thus, the sequence identity at the amino acid level between the capsid proteins is less than 90% and the viruses represent separate new species according to the International Committee on Taxonomy of Viruses (ICTV).nata mix   [682,186) , 25, sil682,186) , mites  .Lampyris noctiluca macula-like virus 1 (LnoMLV1) is 6744 nt long with two open reading frames (ORFs). The longer ORF codes for a protein of 1920 aa, which was similar to an RdRP and a hypothetical protein of Bombyx mori Macula-like virus  according to Blastp search. The best hit by HHpred was a replicase of Tomato mosaic virus , and several shorter hits to viruses of the Tymoviridae family. Shorter ORF, coding for a protein of 231 aa, was similar (98% coverage and 45% identity) to a coat protein of Bee Macula-Like virus 2 , according to Blastp search. HHpred showed that the ORF was similar to plant tymovirus desmodium yellow mottle virus capsid protein . Species demarcation criteria for Maculavirus genus is by ICTV less than 90% identity in capsid protein. Based on phylogenetic analysis, LnoMLV1 was most similar to Bee Macula-Like virus 2  (Online Resource 7).Lampyris noctiluca bunya-like virus 1 (LnoBLV1). The 2182 aa long protein was most similar to Hubei insect virus 1 RdRP  and several bat bunyaviruses  according to Blastp search. HHpred showed that the protein was similar to RNA polymerase L (5AMR_A) of bunyavirus “la Crosse”. Phylogenetic analysis placed LnoBLV1 closest to hypothetical protein of Smoketree sharpshooter  was found in three parts. The longest transcript of 6218 nt codes for a protein of 1924 aa, which according to Blastp search, is similar to Hubei rhabdo-like virus 3  RdRP from Coleoptera mix (99% coverage and 34% identity). According to HHpred, the protein was similar to vesicular stomatitis virus L polymerase . The second transcript of 2571 nt encodes a protein of 603 aa. Blastp search indicated the similarity of the protein to Hubei rhabdo-like virus 3 hypothetical protein 1 (80% coverage and 23% identity), and HHpred analysis showed that the protein was somewhat similar to fusion glycoprotein of Hendra virus , which suggests that the protein codes for an envelope. The smallest transcript of 1743 nt codes for a protein of 426 aa, which Blastp search found most similar to putative glycoprotein of Hubei rhabdo-like virus 3 (39% coverage and 28% identity), and HHpred showed a weak similarity to p40 nucleoprotein of Borna disease virus  from the Mononegavirales order, to which the rhabdoviruses belong. Thus, the smallest transcript most probably codes for a capsid protein. Based on the phylogenetic analysis, LnoRLV1was most similar to Hubei rhabdo-like virus 3  isolated from a beetle mix [Rhabdoviruses are monopartite negative-strand RNA viruses. etle mix  . According to Blastp search, the 693 amino acids protein was similar to putative glycoprotein of Hubei chuvirus-like virus 3 (91% coverage and 49% identity) and HHpred showed similarity to the envelope glycoprotein of human herpesvirus 1 , which suggests that the ORF codes for an envelope protein. The 407 aa protein was similar to a hypothetical protein of Hubei chuvirus-like virus 3 (Blastp: 95% coverage and 37% identity), and HHpred showed protein structural similarity to p40 nucleoprotein of Borna disease virus  suggesting that the ORF may code for a coat protein. For the shortest protein of 115 aa, neither Blastp nor HHpred found any significantly similar proteins. According to phylogenetic analysis, LnoCLV1 was most similar to Hubei chuvirus-like virus 3  (Online Resource 9), which has been isolated from Odonata mix and has a monopartite genome [circular . LnoCLV1e genome .Lampyris noctiluca partiti-like virus 1 (LnoPLV1) genome was 1462 nt long and codes for a protein of 377 aa. According to Blastp search, the protein is similar to RdRP of Hubei partiti-like virus 31 . HHpred showed that the protein sequence was similar to RdRP of negative-strand RNA virus, Thosea asigna virus . In phylogenetic analysis LnoPLV1 was closest to Hubei partiti-like virus 31 (Online Resource 10) isolated from spider mix. Only the RdRP genome segment was identified in the Hubei partiti-like virus 31 [Partitiviruses are small bipartite double-stranded RNA viruses coding for RdRP and capsid proteins. We could only find the RdRP coding segment for two partitivirus-like viruses identified from the glow-worms. virus 31 .Lampyris noctiluca partiti-like virus 2 (LnoPLV2) genome segment was 1461 nt long and encoded a 436 aa protein. Blastp search showed similarity to RdRP of Hubei partiti-like virus 51 , which was closest in the phylogenetic analysis also (Online Resource 10). Hubei partiti-like virus 51 was identified from Chinese land snail mix, and only RdRP segment was found, as observed in LnoPLV2, HHpred showed that LnoPLV2 RdRP is most similar to Mengo virus RdRP . Mengo virus belongs to the Picornaviridae family. LnoPLV1 and LnoPLV2 are separate species as their had only 28.6% identity over RdRP residues (57.6% coverage) and were only distantly related by phylogenetic analysis (Online Resource 10).L. noctiluca totivirus-like virus 1 (LnoTLV1) genome is 4761 nt long and contains two ORFs coding for proteins of 834 and 693 aa. As seen in Blastp search, the longer ORF was similar to hypothetical protein 3 of Hubei toti-like virus 16 . HHpred found the longer ORF similar to RdRP of foot-and-mouth disease virus . According to Blastp search, the shorter ORF was similar to hypothetical protein 2 of Hubei toti-like virus 16 (99% coverage and 31% identity), whereas HHPred found no similar protein sequences. Hubei toti-like virus 16 is isolated form spiders [Totiviruses are double-stranded RNA viruses without an envelope. L. noctiluca errantivirus Diaphorina citri  and ORF B of errantivirus Trichoplusia ni TED virus  according to Blastp search. HHpred identified two sites similar to reverse transcriptase/ribonuclease H p80 of moloney murine leukemia virus  and to integrase of human spumaretrovirus . The 508 aa protein sequence was similar to several predicted insect proteins  and envelope protein of Drosophila melanogaster , according to Blastp search. HHpred found protein similarity to fusion protein of newcastle disease virus . Blastp search found the shortest ORF of 317 aa to be similar to ORF A of Trichoplusia ni TED virus (70% coverage and 33% identity). According to ICTV, species of errantiviruses have generally less than 50% identity in their Gag protein. HHpred showed that the protein was similar to gag protein of rous sarcoma virus . In phylogenetic analysis, LnoErV1 was found to be similar to uncharacterized proteins from several insects, such as, kissing bug, the old world swallowtail butterfly, fruit fly, louse, termite, ant, and silkworm, and transposons from the cabbage looper moth and red flour beetle, indicating that these insects have similar endogenous retroviruses (Online Resource 11).Errantiviruses are endogenous retroviruses of insects. Photinus pyralis) shared five similar virus sequences  with glow-worms. The western tarnished plant bug (Lygus hesperus) shared four similar virus sequences , and the mediterranean fruit fly (Ceratitis capitata) and sharpshooter (Cuerna arida) shared three similar viruses . All the insect hosts that had LnoIV1-like virus sequences also had LnoIV2-like virus.We used NCBI’s transcriptome shotgun assembly sequence database (TSA) to search for similar virus sequences in other insects. We set Blastx bit score, which takes into account the alignment length, mismatches and gaps, above 200 for positive hits. We found no similar viruses from insect transcriptomes for LnoFV1, LnoPLV2, or LnoTLV1 , LnoPLV1 , and LnoPLV2 . LnoFV1 load levels were higher in Hanko than those in Konnevesi population and partiti-like viruses were almost exclusively found in the Hanko population.Viral RNA read amounts for samples collected in 2017 were analyzed for each glow-worm individually. Before the analysis, we removed poly-A-tails from the virus sequences. Only reads with a properly paired mate read mapping to the virus sequence were analyzed and the results were standardized by virus length and all clean reads of the sample .Viral load variation was analyzed for each virus separately Fig. b. The pop-value = 0.036) and existence of the two partiti-like viruses correlated positively in the individuals .Glow-worms, irrespective of their gender or population, had seven different virus types, on average. A minimum of two and at the most nine virus types were observed Fig. c. We stuWe studied the virus loads by qPCR of glow-worm samples collected in 2018. Four female samples from Äänekoski and five female samples from Tvärminne were separated into head , body (B), and eggs (E). The females were most probably unmated since they were still glowing when they were collected. Egg samples were scraped from body cavity and may have contained other tissues such as fat .Additionally, three laboratory-reared larvae from southern Finland (Hanko) and three from southern England (Princes Risborough) were divided into head (H) and body (B) sections. Twelve isolated and unfed 2-week-old larvae from a single southern Finnish mother were split into two groups with six larvae in each (2wL1 and 2wL2) to yield enough RNA for experiment. All the viruses characterized from the 2017 samples, except LnoMLV1, were found from the 2018 female samples and their eggs Fig. a, indicaN = 55, t = − 1.12, p-value = 0.27). However, the egg tissue had significantly lesser viruses than those in heads  or bodies . We excluded FN3 sample . The common glow-worm and the eastern firefly are from the same Lampyridae family, which may explain why they share similar viruses. Yet, they do not occur in the same continent: eastern firefly is found in North America and common glow-worm only in Eurasia [Interestingly, we found five similar viruses from RNA-sequencing data of another glowing insect: the common eastern firefly Supplementary file2 (DOCX 12 kb)Supplementary file3 (DOCX 1705 kb)Supplementary file4 (DOCX 13 kb)Supplementary file5 (PDF 23 kb)Supplementary file6 (PDF 64 kb)Supplementary file7 (PDF 34 kb)Supplementary file8 (PDF 40 kb)Supplementary file9 (PDF 41 kb)Supplementary file10 (PDF 38 kb)Supplementary file11 (PDF 31 kb)Supplementary file12 (PDF 27 kb)Below is the link to the electronic supplementary material.