Hospitalizations for ambulatory care sensitive conditions are commonly used to evaluate primary health care performance, as the hospital admission could be avoided if care was timely and adequate. Previous evidence indicates that avoidable hospitalizations carry a substantial direct financial burden in some countries. However, no attention has been given to the economic burden on society they represent. The aim of this study is to estimate the direct and lost productivity costs of avoidable hospital admissions in Portugal.Hospitalizations occurring in Portugal in 2015 were analyzed. Avoidable hospitalizations were defined and their associated costs and years of potential life lost were calculated. Direct costs were obtained using official hospitalization prices. For lost productivity, there were estimated costs for absenteeism and premature death. Costs were analyzed by components, by conditions and by variations on estimation parameters.The total estimated cost associated with avoidable hospital admissions was €250 million , corresponding to 6% of the total budget of public hospitals in Portugal. These hospitalizations led to 109,641 years of potential life lost. Bacterial pneumonia, congestive heart failure and urinary tract infection accounted for 77% of the overall costs. Nearly 82% of avoidable hospitalizations were in patients aged 65 years or older, therefore did not account for the lost productivity costs. Nearly 84% of the total cost comes from the direct cost of the hospitalization. Lost productivity costs are estimated to be around €40 million.The age distribution of avoidable hospitalizations had a significant effect on costs components. Not only did hospital admissions have a substantial direct economic impact, they also imposed a considerable economic burden on society. Substantial financial resources could potentially be saved if the country reduced avoidable hospitalizations. Ambulatory care sensitive conditions (ACSCs) are health conditions for which hospital admission could be prevented by timely and adequate ambulatory care , 2. HospDirect costs of admissions for ACSCs were estimated in previous studies in Ireland , the UniOf the limited number of studies that estimated the costs of avoidable hospitalizations, none of them included estimations of costs for lost productivity, lost wages and premature death. Although the findings of existing studies already indicate that avoidable hospitalizations create a substantial direct financial burden on health expenditures, considering lost productivity costs can further illustrate how much economic pressure such hospitalizations present.Analysis of the economic burden on society associated with avoidable hospitalizations provides valuable information for planning of health services and allocation of resources. Moreover, as such hospitalizations could have potentially been avoided in the ambulatory care setting; this analysis indicates the potential of saving costs by reducing hospitalizations for ACSCs. The aim of this study was to estimate direct and lost productivity costs of hospitalizations for ACSCs in Portugal. This country provides universal health care, with services financed primarily through tax payments. The primary health care is intended to be the first point of contact of users with the health system.This study used the hospitalization data base provided by the Portuguese Central Administration of the Health System for the year 2015. A total of 1,000,670 hospitalizations were registered in continental Portugal in 2015. For each hospital admission, the inpatient data used in this study were age, sex, principal and secondary diagnosis (according to ICD-9-CM code), diagnosis-related groups (DRGs), length of stay and reason for end of the hospitalization.The definition of which hospitalizations were for ACSCs was determined according to the AHRQ methodology, which uses the codes of principal and secondary diagnosis, for different versions of the ICD . This liThe costs associated with hospitalizations for ACSCs were both direct and from a socioeconomic perspective, incurred by lost productivity. The identification of direct costs was done in the total of hospitalizations. The valuation was done according to the number of hospital admissions classified as avoidable. For the monetization of direct costs, official hospital inpatient admission prices were used as proxy for costs. Prices are defined by DRGs and severity of the condition, according to values published by the Ministry of Health . The priE. coli infections [Lost productivity costs were identified for absenteeism and premature death. The valuation was based on to the length of stay and years of potentially productive life lost for avoidable hospitalizations. In order to monetize productivity losses, the human capital approach was applied. In this methodology, the value to society of potentially lost production (either due to absenteeism or premature death) is estimated by market wages , 16. Thefections , visual fections  and roadfections , to nameFor all hospitalizations for ACSCs, the estimated absenteeism cost was estimated as the value of days of productivity lost, calculated as the length of stay multiplied by the daily wage , takFor avoidable hospitalizations in which the patient died in the hospital, premature deaths are understood as potentially preventable hospitalization-related deaths that occur at working age have a cost of lost productivity that extends to retirement age. The estimated cost of the lost productivity for such premature death was also calculated. In this case, the years of potentially productive life lost were calculated as the difference between the age of the patient and the retirement age (considered 65 years old), and multiplied by the annual mean local wage. Monthly wage, unemployment and labor force participation were specific according to the gender of the patient for both absenteeism and premature death, to reflect the gender differentials in the labor market. These data were obtained at the municipal level from Statistics Portugal (SP).The methodology used for lost productivity costs only accounts for the working-age population. While it has to be acknowledged that retired people contribute to the production of the country, this was not monetized in this study. For equity, the impact of avoidable hospitalizations in the whole population was taken into consideration. Therefore, this impact was considered illness burdens instead of lost productivity costs. Years of potential life lost (YPLL) were calculated to quantify the burden imposed on society by avoidable hospital admissions. YPLL were calculated to represent the non-financial impact these hospitalizations present. For hospitalizations that ended in intra-hospital death, YPLL were calculated as the difference between the age of the patient and the patient’s life expectancy based on their age and gender. The data source was SP.Both crude and standardized hospitalization rates were calculated. Age and sex-standardized hospitalization rates were calculated using the direct method, taking as a reference the 2015 European population prospect from the United Nations Population Division . DescripFor direct, lost productivity and total costs, cost per person and per hospitalization were calculated. For the direct and total costs per person, the population aged 18 or over of each country was the denominator. For the cost of lost productivity per person, the denominator was people aged 18 to 64 years, as people aged 65 years or older were considered not part of the workforce and therefore did not have associated lost productivity costs.Sensitivity analysis was done according to variations in the variables retirement age and age limit, presented as percent variation from base-case values. The retirement age used as the base-case was 65 years, for consistency and to facilitate comparison. Variations were made according to the respective official retirement ages for basic and minimum pensions in Portugal in 2015, which was 66 years.Some other methodologies that identify hospitalizations for ACSCs limit the inclusion of older age groups , 28, as Table Hospitalizations for ACSCs were disproportionally concentrated in older-age groups (> 65 years), accounting for more than 80% of them. The mean age of patients hospitalized for ACSCs was 76 years (SD = 14.5) and the mean length of stay was 10 days (SD = 11.2). Women accounted for 52% of avoidable hospital admissions. Around 13.5% of the hospitalizations attributable to ACSCs ended with intra-hospital death. The most frequent cause of hospital admission was pneumonia (35.5%), followed by congestive heart failure (22.8%) and urinary tract infections (17.1%). Avoidable hospitalizations led to 109,641 YPLL.Table Table Table Hospitalizations for ACSCs involve high costs to both the health system and to individuals, and this situation is cause for concern, since these episodes are potentially avoidable. The cost of each avoidable hospitalization was estimated at €2515 for Portugal, indicating that substantial health resources could be saved by reducing the number of avoidable hospitalizations. In 2015, the total budget of public hospitals in Portugal was €4299 million [This study is the first to provide estimates of lost productivity costs associated with avoidable hospitalizations. Therefore, it is unfeasible to contrast the overall findings of this study with the existing literature. It is possible, however, to compare the direct costs estimated on previous studies with the ones found here. The previous study in Portugal that also used the AHRQ methodology found that, between 2000 and 2007, the mean yearly direct costs amounted to €200 million , which iIt is important to emphasize that the choice of methodology used to select ACSCs codes leads to differences in the estimation of costs. The two studies in France and Ireland used different ACSC identification methodologies that included several conditions not considered in this study, such as vaccine-preventable conditions, angina, nutritional deficiencies and cellulitis, among others , 8. The The human capital approach employed in this study attributes higher values of lost productivity to younger people, due to the higher number of potentially productive years lost. Patients over the age of 65 accounted for 81% of the 99,000 hospitalizations for ACSC. Nonetheless, lost productivity due to absenteeism and premature death represented 16% of the total estimated cost. Studies produced in other countries analyzed lost productivity costs for some conditions (avoidable or not), including diabetes , asthma When comparing frequencies and costs between genders, results show that although 52% of the avoidable hospitalizations occurred among women, they represented 46% of total costs. As each gender represented nearly half of all intra-hospital deaths, the difference can be seen both in the higher death rates among younger age groups for men, and their higher mean salaries and labor force participation, when compared to women. Therefore, lost productivity was the driver for men accounting for a higher share of total costs. Previous studies on costs for lost productivity for different conditions that analyzed gender differences also found higher costs for men –18.The exclusion of people aged 75 years or older in the sensitivity analysis showed the relevance of age distributions to the cost composition. As direct costs account for a high share of total costs in Portugal, the estimation of total costs in this case became less than half of what was estimated on the base-case (from €250 million to €68 million). Concerning the variations in the retirement age, it is important to note that the average effective age of retirement in different countries varies from what is defined as official retirement age. In Portugal, there is the possibility of retiring early or continuing to work after retirement age. In fact, data from 2014 shows that, among the population between 65 and 69 years in Portugal, around 20% were still working .Concerning cost distribution by condition, acute conditions were responsible for more than half of all avoidable hospital admissions and associated costs. Previous studies showed that distribution between avoidable conditions varied substantially between countries, with different conditions accounting for the largest proportion of total costs –8.To our knowledge, this is the first study to estimate both direct and lost productivity costs associated with avoidable hospital admissions. Its strengths include the use of population data and a transparent and reproducible methodology. However, there are limitations in this study according to the method applied to estimate costs. The direct costs were estimated according to prices defined with the purpose of reimbursing hospitals . These values do not reflect actual costs of an inpatient admission. Official prices are good proxy for real costs, are useful for health managers as they represent values reimbursed, allow the use of the same values for patients treated in different hospitals but with the same disease and have been used in other studies of cost estimation of avoidable hospitalizations , 6, 36.When estimating lost productivity costs, the human capital measures potential lost productivity, instead of actual losses. The costs associated with premature death are not limited to the year of 2015, but also include the future lost potential such events represent. This methodology does not take into consideration the time it would require to replace a worker, how long it takes for the patient to return to work after discharge and the possibility of the patient not returning to the labor market due to being declared permanently incapable of work. There was no available information to calculate individually the patients experience after the discharge, as well as no indication if this population behaves the same as the average of hospitalized individuals for all causes. Costs were estimated using a standardized 30-day month; different approaches of estimation for daily wages provides different results; to consider only the working days in a month would require detailed information about the work of the patient, which were not available. Furthermore, the estimation for premature death does not take into consideration whether the death of the patient occurred after leaving the hospital, as it could still be associated with an ACSC. Therefore, the criteria selected might lead to underestimation of productivity loss.There are other methods to estimate productivity loss costs, such as the friction cost method. The costs estimated by this method are expected to be lower, as it depends on the time organizations need to restore the initial production level in the absence of a worker . EstimatThe age distribution of avoidable hospitalizations had a significant impact on cost components in Portugal. One of the main findings of this study was that, although these hospital admissions had a substantial direct economic impact, they also imposed a considerable economic burden on society. Despite the methodological limitations on the estimation of costs, results indicate that substantial financial resources and YPLL could potentially be saved if the country reduced hospitalizations for ACSCs. Effective primary health care in the dimensions of accessibility, prevention and promotion are important to achieve such reduction. Reducing the number of avoidable hospitalizations can contribute to reduced hospital care use and alleviate healthcare-related financial pressures both on the state and on society, with positive results for the population.


The pDNA/MHA/MCH-modified genosensors are proven to improve the hybridization efficiency of a synthetic 160-mer target DNA (160mtDNA) with a 140-mer electrode side overhang as compared to other MHA/MCH ratio-modified genosensors. The MHA(0.04 mM)/MCH(1 mM)-modified genosensors also present good hybridization efficiency with the extracted DENV serotype 1 (DENV1) RNA samples, having the same electrode side overhangs with the 160mtDNA, showing a low detection limit of 20 plaque forming units (PFU)/mL, a linear range of 102–105 PFU/mL and good selectivity for DENV1. The pDNA density-controlled method has great promise to construct sensitive genosensors based on the hybridization of extracted DENV nucleic acids.Developing rapid and sensitive diagnostic methods for dengue virus (DENV) infection is of prime priority because DENV infection is the most prevalent mosquito-borne viral disease. This work proposes an electrochemical impedance spectroscopy (EIS)-based genosensor for the label-free and nucleic acid amplification-free detection of extracted DENV RNA intended for a sensitive diagnosis of DENV infection. A concentration ratio of 0.04 mM 6-mercaptohexanoic acid (MHA) to 1 mM 6-mercapto-1-hexanol (MCH) was selected to modify thin-film gold electrodes as a link to control the coverage of self-designed probe DNA (pDNA) at a density of 4.5 ± 0.4 × 10 The epidemic of dengue, malaria, chikungunya, yellow fever, West Nile virus and Japanese encephalitis, notorious diseases transmitted by mosquitoes , have bePresently, many strategies have been developed to detect DENV based on the measurement of specific biomarkers of DENV infection, such as soluble non-structural protein 1 (NS1) ,10,11, D2CdSnS4/O2/Si ) and potassium hexacyanoferrate(II) trihydrate (K4[Fe(CN)6]) 3H2O were purchased from Showa Chemical . All chemicals were of reagent grade and were used without further purification. All solutions were prepared with water purified through a Milli-Q system. Sodium phosphate dibasic, sodium phosphate monobasic dihydrate, tris(hydroxymethyl)aminomethane (Tris), N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), MHA, MCH, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-(N-morpholino) ethanesulfonic acid (MES), potassium chloride (KCl), hexaammineruthenium(III) chloride and bovine serum albumin (BSA) were purchased from Sigma-Aldrich . Potassium hexacyanoferrate(III) (K2-(CH2)6 group at the 5′ terminus. In order to realize the effect of electrode side overhang on the hybridization efficiency, a 160-mer target DNA, designated as 160mtDNA, was synthesized according to the last 160-mer sequence from 3′ terminus of DENV1 RNA. The sequence of all tDNA and pDNA is shown in A full-length sequence of DENV1  was obtained from GenBank of National Center for Biotechnology Information (NCBI). Compared with DENV2 , DENV3  and DENV4 , a 20-mer specific sequence, found in DENV1 RNA by a CLC Sequence Viewer ), was identified as target DNA (designated as 20mtDNA) with the sequences of 5′-GTACCCTGGTGGTAAGGACT-3′. The 3′ terminus of the 20mtDNA has a 140-mer distance away from the 3′ terminus of full-length DENV1 RNA. The corresponding pDNA is completely complementary to the 20mtDNA with the sequences of 5′-AGTCCTTACCACCAGGGTAC-3′ and is modified with the NH30-nm Ti and 200-nm Au thin films were patterned as a rectangular electrode on a cleaned glass substrate by lift-off microfabrication techniques. The working area of electrodes was defined as a disk shape with 1.6 mm in diameter by a 7 μm-thick negative photoresist, SU8-3010 . After cleaning the surface of the Au electrodes, the electrodes were dipped in the mixture of MHA and MCH with concentration-varied ratio for 4 h to form a binary SAM. The preparation of genosensor is shown in The surface coverage of MHA/MCH binary SAM can be quantified via a one-electron reductive path in an alkaline solution (pH ≥ 11) . The red2. The pDNA coverage immobilized at the MHA/MCH-modified electrodes can be calculated by the amount of positively electroactive molecules adsorbed with the negatively charged phosphate group of DNA backbone −43.1 and saturated at the 105 PFU/mL. The Ret value of pre-hybridized genosensors was 8.5 ± 0.2 kΩ, implying the calculated limit of detection (LOD) was about 20 PFU/mL (S/N > 3). Generally, 1 PFU of DENV is equivalent to 600–1000 copies of virus particles in Dr. Perng’s laboratory, which depends on the virus strains, culture conditions and the stored time of the virus [2@APTES-GO/Pt genosensors [Ret. The ΔRet ratio of 104 PFU/mL obtained in the calibration curve was different from that presented in the time-lapse hybridization, partly attributed to the different amount of complementary fragments of extracted RNA and the different hybridizing procedures. As expected, it was difficult to obtain the same copy number of complementary fragments in each extracting procedure. Furthermore, in the previous study [he virus ,37. The osensors . The lowus study  the genous study  and Darwus study , while tRet ratio (56.4 ± 7.9%) for DENV1 is significantly larger than that for DENV2 (11.4 ± 3.4%), DENV3 (17.4 ± 2.3%) and DENV4 (14.7 ± 2.9%) (p < 0.05 by student t-test). Moreover, the ΔRet ratio value for the mixture of DENV1+2, DENV1+3 and DENV1+4 was, respectively, 58.4 ± 4.4%, 60.3 ± 5.0% and 57.8 ± 2.0%. The calculated selectivity (= DENV1/DENV mixture) was in the range of 93.5% to 97.6%, indicating the good selectivity of the self-designed pDNA-immobilized genosensors to DENV1. Furthermore, real-time PCR method was used to identify the specificity of self-designed pDNA as shown in ® FAST qPCR Master Mix (2X) kit. The self-designed pDNA was used as a specific backward primer, and the sequence of 5′-GGTTAGAGGAGACCCCTCCC-3′ was used as a universal forward primer, which is suitable for all DENVs. The result shows that the threshold cycle for DENV1 is much smaller than that for DENV2, 3 and 4, implying that the self-designed pDNA is very specific to DENV1. Moreover, The selectivity test of genosensors is shown in 2 to 105 PFU/mL and good selectivity for DENV1. This study demonstrates that the combination of well-controlled pDNA density and the self-designed pDNA sequence capable of reducing the electrode side overhang length of extracted RNA makes the detection of extracted DENV1 RNA feasible. As expected, this method has great promise to construct label-free EIS-based genosensors for the optimal hybridization of extracted nucleic acids without the process of nucleic acid amplification. The ratio-varied MHA/MCH binary SAM can be used as a link to control the density of immobilized pDNA. In particular, the pDNA/MHA(0.04 mM)/MCH-modified genosensors improved the hybridization efficiency for 160 mtDNA having 140-mer electrode side overhangs via the EIS analysis. The genosensors designed in this study could detect extracted DENV1 RNA after 20 min hybridization with a low LOD of 20 PFU/mL, good detection linearity from 10


We analyzed 2 clusters of 12 patients in Vietnam with severe acute respiratory syndrome coronavirus 2 infection during January–February 2020. Analysis indicated virus transmission from a traveler from China. One asymptomatic patient demonstrated virus shedding, indicating potential virus transmission in the absence of clinical signs and symptoms. During the past 2 months, emergence of 2019 novel coronavirus disease (COVID-19) has caused global public health concern  were sent to Wuhan, China, for technical training for ≈2 months. On January 17, 2020, they returned to Vietnam via a flight from Wuhan to Guangzhou, China, followed by a flight from Guangzhou to Hanoi, Vietnam. During January 21–27, 2020, fever and cough developed in 6 of those travelers. Real-time PCR confirmed SARS-CoV-2 infection in all 6 travelers. Virus isolation and next-generation sequencing were performed for samples that were positive for viral RNA. The remaining 2 employees that had been on the same flight were quarantined, but real-time PCR indicated that they were negative for viral RNA. Patients were hospitalized at the National Hospital of Tropical Diseases, Thanh Hoa Provincial Hospital, and Tam Dao District Hospitals in Vinh Phuc Province, where they were closely monitored in isolated wards and followed up. Patient throat swab samples were sent to the Institute of Hygiene and Epidemiology, Hanoi, Vietnam, for laboratory diagnosis. The patients provided consent to have their details shared.Among those tested for viral RNA by SARS-CoV–specific reverse transcription PCR, results were negative for 155 persons who had been in close contact with the 6 SARS-CoV–positive travelers and 1,092 persons exhibiting clinical signs, including cough and fever . We moniClinical signs, including fever and cough, were demonstrated by 11 patients an average of 9.9 (± 5.4) days after travel or close contact with patients, indicating that the incubation period was 1–2 weeks after exposure . In thesAlthough close-contact transmission of SARS-CoV-2 between family members has been identified (Virus shedding patterns detected during study of severe acute respiratory syndrome coronavirus 2 shedding by travelers, Vietnam, 2020.


Scientific Reports 10.1038/s41598-019-54714-w, published online 08 January 2020Correction to: The original version of this Article contained errors.After publication of the Article, it came to the Authors attention that when other sequence alignment tools are used, no recombination signal is identified. The Authors speculate that the positive identification of recombination events reported in the Article may have been caused by the highly variable non-coding region between M and F, which is hard to align. The Authors consider this identification to therefore be a bioinformatics error. Article was changed as follows to reflect this.In the Results, ‘Recombination analysis’M gene (3406–4888 bp) and 3ʹ UTR of the F gene (4892–7306 bp), a putative recombination event was observed in the complete genome (4607–5425 nts) of Pakistan-origin strain of SRMV. With a probability of MC value of 2.357 E−22, this event was found between a recombinant Pakistani strain  and Indian strains   and 3ʹ UTR of the F gene (4892–7306 bp), apparently a putative recombination event was observed in the complete genome (4607–5425 nts) of Pakistan-origin strain of SRMV but it is attributed to bioinformatics errors. Therefore, no recombination was found in the current study.”“Lying between 5ʹ UTR of the Additionally, Figure 6 and Table 10 were removed. The original figure and table are reproduced below.In the Discussion,46. Beside rare occurrence of recombination in negative sense RNA viruses particularly SRMV, an analysis for the detection of recombination event/s is recommended as a standard component of every phylogenetic analysis to serve an important quality-control function to weed out laboratory and analytical errors47. We found recombination events among Pakistani- and Indian-origin strains which further highlight the co-existence of similar SRMV strains along with its transboundary nature of transmission48. Indicating a high resolution of prediction, the observed putative recombination event was statistically significant and was identified by more than five recombination detection algorithms. Such an interference of Indian strains as major and minor parents for Pakistan-originated recombinant strain highlight its potential to cross international borders48. Similar finding has previously been observed for another RNA virus (Yellow leaf virus) from Pakistan and India49. Potnetial reason for such a sharing of genetic material could be speculative and may be attributed to an increased disease incidence rate and frequent disease outbreaks near borderline of these countries50,51. Though potnetial occurrence of homologous recombination in some of the negative sense RNA viruses is low52, it is not surprising because sporadic recombination in various negative-sense RNA viruses such as Hantavirus53,54, ambisense arenaviruses55,56, Newcastle disease viruses57,58 and morbilliviruses (e.g. canine distemper virus59 and measles virus60) has been evidenced. Hence, an emergence of viral variants could be anticipated that may differ antigenically and serologically and therefore may have consequences in terms of failure in diagnostics and vaccine efficacy.”“The occurrence of recombination events is considered a significant source of genetic diversity for RNA virusesnow reads:46. Occurrence of recombination in negative-sense RNA viruses is extremely rare, still analysis for the detection of recombination event/s is recommended as a standard component of every phylogenetic analysis to serve an important quality-control function to weed out laboratory and analytical errors47. In the current study, no recombination event was found.”“The occurrence of recombination events is considered a significant source of genetic diversity for RNA virusesAs a result, the following References were removed and are listed below:Global J of Med Res (2018).Seyoum, B. & Teshome, E. Major Transboundary Disease of Ruminants and their Economic Effect in Ethiopia. Plant Pathol67(1), 221–32 (2018).Elsayed, A. I., Boulila, M., Odero, D. C. & Komor, E. Phylogenetic and recombination analysis of sorghum isolates of Sugarcane yellow leaf virus. et al. Molecular characterization of peste-des-petits ruminants virus (PPRV) isolated from an outbreak in the Indo-Bangladesh border of Tripura state of North-East India. Vet Microbiol174(3-4), 591–5 (2014).Muthuchelvan, D. et al. Evaluation of risk factors for peste des petits ruminants virus in sheep and goats at the Wildlife-Livestock Interface in Punjab Province, Pakistan. BioMed Res Int (2016).Aziz-ul-R. J Gen Virol84(10), 2691–703 (2003).Chare, E. R., Gould, E. A. & Holmes, E. C. Phylogenetic analysis reveals a low rate of homologous recombination in negative-sense RNA viruses. et al. Genetic interaction between distinct Dobrava hantavirus subtypes in Apodemus agrarius and A. flavicollis in nature. J Virol77(1), 804–809 (2003).Klempa, B. J Virol75(23), 11803–11810 (2001).Sironen, T., Vaheri, A. & Plyusnin, A. Molecular evolution of Puumala hantavirus. Arenaviridae). Virology283(2), 161–166 (2001).Charrel, R. N., de Lamballerie, X. & Fulhorst, C. F. The Whitewater Arroyo virus: natural evidence for genetic recombination among Tacaribe serocomplex viruses (family Virology304(2), 274–281 (2002).Archer, A. M. & Rico-Hesse, R. High genetic divergence and recombination in Arenaviruses from the Americas. Virus Res151(1), 45–53 (2010).Zhang, R., Wang, X., Su, J., Zhao, J. & Zhang, G. Isolation and analysis of two naturally-occurring multi-recombination Newcastle disease viruses in China. Virology371(1), 54–60 (2008).Han, G. Z., He, C. Q., Ding, N. Z. & Ma, L. Y. Identification of a natural multi-recombinant of Newcastle disease virus. et al. Homologous recombination is a force in the evolution of canine distemper virus. PloS one12(4), e0175416 (2017).Yuan, C. BMC Evol Biol5(1), 52 (2005).Schierup, M. H., Mordhorst, C. H., Muller, C. P. & Christensen, L. S. Evidence of recombination among early-vaccination era measles virus strains. Consequently, References 48–57 were incorrectly listed as References as 61–70.These changes do not affect the overall conclusions of the Article. This has now been corrected in the PDF and HTML versions of the Article.


Amnoonviridae, a family within the order Articulavirales comprising segmented negative-sense RNA viruses. We sought to identify additional viruses within the Amnoonviridae through total RNA sequencing (meta-transcriptomics) and data mining of published transcriptomes. Accordingly, we sampled marine fish species from both Australia and China and discovered several segments of two new viruses within the Amnoonviridae, tentatively called Flavolineata virus and Piscibus virus, respectively. In addition, by mining vertebrate transcriptome data, we identified nine additional virus transcripts matching to multiple genomic segments of TiLV in both marine and freshwater fish. These new viruses retained sequence conservation with the distantly related Orthomyxoviridae in the RdRp subunit PB1, but formed a distinct and diverse phylogenetic group. These data suggest that the Amnoonviridae have a broad host range within fish and that greater animal sampling will identify additional divergent members of the Articulavirales.Tilapia lake virus (TiLV) has caused mass mortalities in farmed and wild tilapia with serious economic and ecological consequences. Until recently, this virus was the sole member of the  Amnoonviridae are a recently described family of segmented and enveloped negative-sense RNA viruses associated with disease in fish. Until recently, the Amnoonviridae comprised only a single species, tilapinevirus or tilapia lake virus (TiLV) [Oreochromis niloticus and Oreochromis niloticus x O. aureus hybrid). As the second most farmed fish globally [The s (TiLV) ,2, whichglobally  and an iglobally , tilapiaglobally ,6. Whileglobally , whetherAmnoonviridae are members of the order Articulavirales that also includes the Orthomyxoviridae [Amnoonviridae, the Orthomyxoviridae, and closely related but unclassified orthomyxo-like viruses, infect a broad range of host species comprising both invertebrates and vertebrates. Notably, a divergent member of the Amnoonviridae, Lauta virus, was recently identified in an Australian gecko [Lauta virus and TiLV suggests that the former may even constitute a new genus within the Amnoonviridae, with the long branches throughout the Articulavirales phylogeny likely indicative of very limited sampling.The oviridae  that arean gecko , stronglAmnoonviridae might be present in a wider range of vertebrate taxa, we screened for their presence using a meta-transcriptomic analysis of marine fish sampled in Australia and China, combined with data mining of published transcriptomes.To help address whether the Rhombosolea tapirina, Platycephalus bassensis, Platycephalus speculator, Trachurus declivis, Trachurus novaezelandiae, Scorpaena papillosa, Pristiophorus nudipinnis, Pentaceropsis recurvirostris and Meuschenia flavolineata. Fish were caught via repeated research trawls on the fisheries’ training vessel, Bluefin, following the methodology outlined in [Fish samples were collected from the Bass Strait , Australia, in November 2018. The fish species collected included lined in . Ten indProscyllium habereri, Urolophus aurantiacus, Rajidae sp., Eptatretus burgeri, Heterodontus zebra, Dasyatis bennetti, Acanthopagrus latus, Epinephelus awoara, Conger japonicus, Siganus canaliculatus, Glossogobius circumspectus, Halichoeres nigrescens and Boleophthalmus pectinirostris. Liver samples from each species were pooled and stored in a −80 °C freezer. The procedures for sampling and sample processing were approved by the ethics committee of the National Institute for Communicable Disease Control and Prevention of the China CDC.As well as Australia, we analysed the transcriptome data derived from a previous study of the viromes of fish sampled from the South China Sea . For thaFor RNA extraction, frozen tissue was partially thawed and submerged in lysis buffer containing 1% ß-mercaptoethanol and 0.5% Reagent DX before tissues were homogenized together with TissueRupture . The homogenate was centrifuged to remove any potential tissue residues, and RNA from the clear supernatant was extracted using the Qiagen RNeasy Plus Mini Kit. RNA was quantified using NanoDrop . RNA isolated from the Australian samples was pooled for each host species, whereas RNA isolated from the Chinese samples was pooled from all species , resultiLauta virus obtained from NCBI GenBank. The sequences retrieved were then aligned with those generated here using MAFFT (v7.4) employing the E-INS-i algorithm. Ambiguously aligned regions were removed using trimAl (v.1.2) [Sequencing reads were first quality trimmed then assembled de novo using Trinity RNA-Seq (v.2.11.0) . The ass (v.1.2) . To esti (v.1.2) , selecti (v.1.2) , and usiFlavolineata virus in the yellow-striped leatherjacket collection, 10μl of extracted RNA was transcribed into cDNA using SuperScript® VILO™ reverse transcriptase . PCR amplification was performed using Platinum™ II Hot-Start PCR Master Mix (2X)  and 3 sets of primers .To further confirm the presence of  primers  designedAmnoonviridae we screened de novo transcriptome assemblies available at the NCBI Transcriptome Shotgun Assembly (TSA) database (https://www.ncbi.nlm.nih.gov/genbank/tsa/). Amino acid sequences of Flavolineata virus, Piscibus virus and TiLV were queried against the assemblies using the translated Basic Local Alignment Search Tool (tBLASTn) algorithm. We restricted the search to transcriptomes within the Vertebrata (taxonomic identifier: 7742). Putative virus contigs were subsequently queried using BLASTx against the non-redundant virus database.To identify additional novel vertebrate viruses within the New viruses identified in this study are tentatively named by drawing from the names of their host species.Piscibus virus, see Bioproject PRJNA418053 , and for Flavolineata virus, see Bioproject: PRJNA667570. Alignments with new virus transcripts are available at https://github.com/jemmageoghegan/Amnoonviridae-in-fish.Sequencing reads are available at the NCBI Sequence Read Archive (SRA). For Amnoonviridae, tentatively named Flavolineata virus, in a sequencing library of 10 pooled individuals of yellow-striped leatherjacket (Meuschenia flavolineata) sampled from the Bass Strait off the coast of Tasmania, Australia. No amnoonviruses were identified in the remaining eight fish species. We identified a complete, highly divergent protein in which a Diamond BLASTx analysis revealed 37% amino acid identity to TiLV segment 1, characterized as the PB1 subunit , with a GC composition of 48.2% and a standardised abundance of 0.00004% of the total non-rRNA library. The presence of Flavolineata virus was further confirmed in the unpooled samples using RT-PCR  sampled in the South China Sea as described previously [−15) to Flavolineata virus using a custom database including the known members of the family Amnoonviridae. In addition, a comparison to the NCBI nr database showed that Piscibus virus had 48.5% amino acid similarity  to the PB1 subunit of the TiLV RdRp. The GC composition of the assembled sequence was 49.2%, and it had a standardized abundance of 0.0001% of the total non-rRNA library. Despite the limited contig length for Piscibus virus, such that its status as a bona fide novel virus will need to be confirmed with additional sequencing, we did identify conserved motifs within the PB1 subunit (see below).An additional novel member of the eviously . SpecifiAmnoonviridae, we screened de novo transcriptome assemblies available at NCBI’s TSA database. In doing so, we identified nine further potentially novel viruses in fish matching segments 1–4 of TiLV  from marine (Lepidonotothen nudifrons and Chionodraco hamatus) and freshwater ecosystems  (Relatives of the  asotus) . All vir asotus) . NotablyArticulavirales (Amnoonviridae (with 83% bootstrap support). The original member of this virus family, TiLV, grouped with Flavolineata virus, Piscibus virus, Dolomieu virus, Namensis virus and Hamatus virus in one clade. The second fish virus clade comprised the newly identified Stewartii virus, Plagiostomus virus, Przewalskii virus, Asotus virus 1 and Asotus virus 2. Lauta virus, identified in a native Australian gecko, appears to form a distinct lineage, suggestive of a separate genus. This phylogenetic analysis clearly illustrates the diversity of these viruses within both marine and freshwater fish, with no apparent host taxonomic structure  across the order avirales . This retructure .Articulavirales including the Orthomyxoviridae . Not only does the identification of these new viruses greatly increase the phylogenetic diversity in this newly identified group of viruses, but it may also provide insight into the potential origins and host range of TiLV, a virus that has major economic and ecological impacts on fisheries and aquaculture.Through both sampling marine fish and mining publicly available sequence data, we discovered 11 new viruses, all of which are the closest genetic relatives of TiLV. These viruses fall within the Amnoonviridae. While we only found new viruses in fish and no other vertebrate classes, it is important to note that fish comprise 44% of currently available vertebrate transcriptomes (as of September 2020). With the expansion of these databases, it is likely we will identify additional highly divergent viruses within the Amnoonviridae and hence of the Articulavirales as a whole. The discovery of these 11 viruses invites further research into the true diversity and evolutionary origins of the Amnoonviridae.The viruses discovered here were highly divergent in sequence, likely limiting our ability to detect all genome segments present in the data. Nevertheless, sequence conservation within segment 1 across the entire taxonomic order strongly supports the inclusion of these new viruses within the


In 2016/2017, H5N8 highly pathogenic avian influenza (HPAI) virus of the Goose/Guangdong lineage spread from Asia to Europe, causing the biggest and most widespread HPAI epidemic on record in wild and domestic birds in Europe. We hypothesized that the wide dissemination of the 2016 H5N8 virus resulted at least partly from a change in tissue tropism from the respiratory tract, as in older HPAIV viruses, to the intestinal tract, as in low pathogenic avian influenza (LPAI) viruses, allowing more efficient faecal-oral transmission. Therefore, we determined the tissue tropism and associated lesions in wild birds found dead during the 2016 H5N8 epidemic, as well as the pattern of attachment of 2016 H5N8 virus to respiratory and intestinal tissues of four key wild duck species. We found that, out of 39 H5N8-infected wild birds of 12 species, four species expressed virus antigen in both respiratory and intestinal epithelium, one species only in respiratory epithelium, and one species only in intestinal epithelium. Virus antigen expression was association with inflammation and necrosis in multiple tissues. The level of attachment to wild duck intestinal epithelia of 2016 H5N8 virus was comparable to that of LPAI H4N5 virus, and higher than that of 2005 H5N1 virus for two of the four duck species and chicken tested. Overall, these results indicate that 2016 H5N8 may have acquired a similar enterotropism to LPAI viruses, without having lost the respirotropism of older HPAI viruses of the Goose/Guangdong lineage. The increased enterotropism of 2016 H5N8 implies that this virus had an increased chance to persist long term in the wild waterbird reservoir. Avian influenza causes major economic damage to the poultry industry, as well as welfare issues to the poultry involved. For example, the global highly pathogenic avian influenza (HPAI) virus epidemic of the subtype H5N8 in 2014/2015 led to the death or culling of over 50 million birds in seven countries in Asia, Europe, and North America . In addiThe Goose/Guangdong lineage of H5 HPAI virus (originating from H5N1 HPAI virus A/Goose/Guangdong/1/1996) has persisted in poultry populations in parts of South-East Asia at least since 2003, and has circulated between poultry and wild birds, allowing continual virus evolution as well as reassortment of H5 HPAI virus with other avian influenza (AI) viruses  was detected in the gastro-intestinal epithelium of three species: Eurasian magpie, Canada goose ( cygnus) –35  that were naturally infected with 2016 H5N8, compared to the more limited virus antigen expression in the intestinal epithelium of wild birds infected with earlier viruses of the Goose/Guangdong lineage (this study). Based on virus attachment, 2016 H5N8 attached better than H5N1 to intestinal epithelium in three of four duck species tested. Based on these results, we accept the alternate hypothesis that 2016 H5N8 is more enterotropic than H5N1.Enterotropism of HPAI virus is a novel phenomenon in wild birds. According to our review, infection of epithelial cells of the gastro-intestinal tract was reported in only three or four species , none of them ducks. In contrast to HPAI virus, LPAI virus is well known to be enterotropic in wild birds. In fact, the epithelium of intestine and cloacal bursa were the only tissues that expressed LPAI virus antigen in naturally infected mallards and black-headed gulls , 9. An iIn addition to gaining enterotropism, 2016 H5N8 retained respirotropism. Infection of the respiratory tract is considered to be the main source of HPAI virus excretion from the oropharynx in wild birds . In the Besides in respiratory tract and intestinal tract, 2016 H5N8 antigen also was expressed in other tissues, including in particular brain, liver, lung, heart, and pancreas. Infection in most of these tissues was associated with both necrosis and inflammation, and it is likely that these 2016 H5N8-associated lesions were fatal to the birds in our study. The character and severity of these lesions were similar to those caused by H5N1 infection, with the exception of intestinal lesions, which are not present in H5N1 infection . HoweverBased on experimental infections in different species of ducks, 2014 H5N8 from the USA and from the Netherlands is less virulent than H5N1 , 15. HowFrom the results of this study, we can conclude that 2016 H5N8 has a tropism for both digestive tract and respiratory tract of at least four wild bird species  based on virus antigen detection in naturally infected birds. This means that 2016 H5N8 mirrors the enterotropism of LPAIV, without having lost the respirotropism of older viruses of the Goose/Guangdong lineage, like H5N1. How the H5 viruses of the Goose/Guangdong lineage will evolve in wild birds in future is not clear. One possibility is that they become completely enterotropic, like LPAIV in mallards and black-headed gulls. Another possibility is that they retain tropism for both digestive and respiratory tracts. It is conceivable that such a dual tropism provides maximum flexibility for the virus to adapt to multiple species of wild and domestic birds, depending on the ecological niche where the virus happens to be.The implications of increased tropism for the digestive tract is that relatively more virus is excreted from the cloaca and contaminates the water bodies on which wild waterbirds reside. Indirect transmission via contaminated water, up to several weeks after the infected birds have left the water body, is a key factor for the maintenance of LPAI in wild waterbirds . If indiAdditional file 1. Expression of avian influenza virus antigen in cell types of other organs than those of respiratory and gastro-intestinal tracts. Number of birds expressing influenza virus antigen in a cell type of an organ.Additional file 2. Frequency and distribution of gross lesions associated with virus antigen expression in carcasses of wild birds. Number of birds with gross lesions in different organs.Additional file 3. Frequency and distribution of histological lesions associated with virus antigen expression in carcasses of wild birds. Number of birds with histological lesions in different organs.Additional file 4. Literature review of articles in English from the PubMed database. Literature reporting influenza virus antigen expression in the gastro-intestinal epithelium.Additional file 5. Common and scientific name of additional bird species mentioned in Additional file 4. Common and scientific names of the birds mentioned.


Arising from Niknafs et al. Nature Communications 10.1038/s41467-019-13100-w (2019)KrasG12D and Trp53R172H mutations (KPC model). As an additional aspect, the study reports frequent homozygous deletions at the Nlrp1 locus, which are interpreted as a somatic driver event in pancreatic cancer. We observed that the origin of this Nlrp1 alteration is strain-specific germline variation, having profound impact on the interpretation of its biological relevance. Beyond this specific locus, we show that strain-specific germline variation is a general confounder of genome analyses in mouse models of cancer.Niknafs et al. describe evolutionary trajectories in pancreatic cancer using mouse models with engineered 1, we also observed frequent changes at the Nlrp1 locus in our own cohorts of KPC mice. However, Nlrp1 changes were invariably associated with a series of unusual characteristics. First, the deletion encompasses the exact same genomic region on chromosome 11 in all affected cancers  as well as through re-analysis of publicly available datasets . Somatic acquisition of absolutely identical homozygous deletions in different cancers, models, entities, and laboratories is rather unlikely. Third, we observed Nlrp1 locus alterations only in mouse models with engineered mutant or floxed Trp53 alleles (Trp53ENG). More specifically, Nlrp1 locus alterations were only detected in heterozygous Trp53ENG tumors, but never in mice, which were crossed to Trp53ENG homozygosity .In line with Niknafs et al.ers Fig. . These iNLRP1. In the mouse reference genome (based on strain C57BL/6J) the Nlrp1 locus comprises three related genes: Nlrp1a, Nlrp1b, and Nlrp1c-ps. Importantly, Trp53 and the Nlrp1 locus are separated by only 1.5 Mb on chromosome 11, causing tight genetic linkage between both loci. The genetically engineered Trp53 allele was generated on a 129S-related background (Trp53ENG-129S). We examined the Nlrp1 locus in 129S genomes (Nlrp1129S) and found that parts of the C57BL/6J sequence have no genomic alignment in the 129S reference assembly5 . Thus, the origin of the Nlrp1 locus deletion is not somatic acquisition followed by selection during tumor evolution, but a pre-existing strain-specific germline variant.These seeming inconsistencies prompted us to examine the locus in detail. Humans have only one gene at this locus, ors Fig.  and all Trp53ENG-129S allele is genetically linked to the Nlrp1129S locus , we interrogated the status of the second allele in the germline. This consideration is important, because we kept the mice on a mixed 129S;C57BL/6J background . Our analysis revealed two important findings, which explain the genesis of Nlrp1 locus deletions in cancer: First, Nlrp1 locus deletions in cancer were only observed in mice, whose second haplotype is of C57BL/6J origin  manifests as a focal homozygous deletion  in tumors from mice crossed to Trp53ENG homozygosity  or other more rare scenarios  why a single somatic event  whyios Fig.  and (3) see Fig. .Nlrp1129S variant biologically relevant and thus selected for during tumor evolution? Several lines of evidence suggest that interpretation of the Nlrp1129S variant as a cancer driver requires further functional validation: First, the pre-existing Nlrp1129S variant does not arise through somatic mutation . LOH at the locus is explained by the tight genetic linkage of Nlrp1129S to Trp53ENG-129S. Second, in our own cohorts of several hundred KrasG12D-driven mouse pancreatic cancers, there are no Nlrp1 locus deletions without associated LOH of mutant Trp53ENG. Third, large transposon-based pancreatic cancer gene discovery screens performed on a mixed 129S;C57BL/6J genetic background did not find common insertions in the wild-type, hemizygous Nlrp1C57BL/6J allele11. Fourth, in human pancreatic cancer, isolated, deep NLRP1 deletions that spare TP53, have been only reported in 1/109 cases in one study . In fact, re-analysis of raw-sequencing data with manual inspection of the NLRP1 locus in PDA_078 did not confirm the presence of an isolated NLRP1 deletion in our hands.Is the Nlrp1 locus, our findings highlight an important—and so far underappreciated—confounder of mouse cancer genome analysis. The basis of this confounder is the widespread use of inbred mice with extensive inter-strain haplotype variation5. Typical experimental cohorts are derived from few generations of crosses involving few different inbred strains, with inheritance of large strain-specific haplotype blocks. Thus, when genetically linked to a cancer gene undergoing LOH, any strain-specific deletion/insertion variant will appear as a somatically acquired CNV in related tumors. Importantly, this CNV will be hugely recurrent in the cohort. Backcrossing of mice/alleles to a single genetic background can substantially reduce the amount of strain-specific germline haplotypes. However, this confounder can persist in direct genomic proximity to engineered alleles (which are bred/genotyped for) even after extensive backcrossing , but also a somatically acquired cancer gene alteration. For example, in mouse pancreatic cancer cohorts we observed Skint locus deletions with equivalent characteristics to Nlrp1 alterations: (1) identical genomic deletion coordinates in unrelated tumors, (2) corresponding strain-specific locus variation in the germline, and (3) genetic linkage to a cancer gene (Cdkn2a) undergoing LOH in the tumor. As for Nlrp1, the Skint locus deletion is present in the germline of 129S-related mouse strains but not in C57BL/6J  of Technische Universität München, Regierung von Oberbayern and the UK Home Office.Data and conclusions of this commentary are based on the systematic genetic analysis of our own cohort of over 1000 mouse cancers derived from a variety of distinct mouse models covering different cancer entities. This large cohort of mouse cancers comprise primary cell cultures as well as tissues and was characterized by a series of methods, including array comparative genomic hybridization (aCGH), whole-exome sequencing (WES), long-read sequencing and/or quantitative insertion-site sequencing (QiSeq). Animal experiments, primary mouse pancreatic cancer culture preparation, and maintenance, gDNA isolation were performed as described in detail before1 were analyzed by using a workflow adapted to the analysis of mouse cancer sequencing data which we described elsewhere in detail14 (source code: https://github.com/roland-rad-lab/MoCaSeq). In brief, reads were trimmed using Trimmomatic 0.38. BWA-MEM 0.7.17 was used to align reads to the mouse reference genome GRCm38.p6 . Picard 2.20.0 and GATK 4.1.0.0 were used for postprocessing . For LOH analyses from WES data, germline SNP calling was performed with Mutect2 which removes the vast majority of sequencing artifacts. The high number of pseudogenes and segmental duplications in the mouse genome (as compared to the human genome) increases the chance of read mis-mapping. To avoid ambiguous SNP positions resulting from mis-mapping, only reads with a mapping quality of 60 were included in LOH analyses14. For CNV detection in mouse and human pancreatic cancers, we used CopywriteR 2.6.1.216 which is based on the analysis of “off-target” reads. “Off-targets” (such as intronic reads), which represent ~20% of all reads in typical WES data sets , are not affected by variation in capture efficiencies. CopywriteR outperforms algorithms based on the analysis of “on-target” reads  for CNV calling from human and mouse WES data16.Raw WES data of mouse pancreatic cancers from our cohorts and from Niknafs et al.1, Maser et al.2, Foijer et al.3, Foijer et al.4, Cutler et al.6, Mueller et al.13 and our own KPC cohort was analyzed using Agilent Genomic Workbench software version 7.0.4.0. Importantly, the identical aCGH array  was used in all studies, allowing for the direct comparison of strain-specific germline variation at Nlrp1 and Skint loci across all mouse cancer cohorts/genomes.aCGH data from Niknafs et al.map-ont). Read coverage was extracted using bam-read count .Long-read sequencing libraries were prepared using the Oxford Nanopore LSK109 kit . A total of 400 ng of library was loaded on a promethION flowcell and run for 72 h on a promethION beta sequencer . Base-calling was performed on the promethION compute unit’s GPU using guppy 3.2.8 basecaller. The resulting FASTQ file was mapped to the reference genome GRCm38.p6 using Minimap2 (option Further information on research design is available in the Reporting Summary


In the case of each DPW, >6 log10 removal/inactivation of virus was observed, with limited (EBOV/Mak) or no (VSV) virus transfer observed. The efficacy of wipes for decontaminating high-touch environmental surfaces spiked with EBOV/Mak or VSV is discussed. In summary, removal of EBOV/Mak and VSV using wipes was extensive in this study. In the absence of a sufficient concentration and contact time of an appropriate microbicidal active in DPW (such as the AHP- and QAC-based DPW tested), transfer of a low, albeit significant (from an infectious unit/infectious dose perspective), quantity of infectious virus from the inoculated surface to a secondary surface was observed. In the case of Ebola virus, it is essential that a DPW with an appropriate microbicidal active, following the appropriate contact time, be used to prevent unintended transfer of infectious virus to a clean secondary surface (as observed in negative control /J-Cloth). Otherwise, there exists the possibility of dissemination of Ebola virus and the associated risk of transmission of Ebola virus disease.Disinfectant pre-soaked wipes (DPW) containing activated hydrogen peroxide (AHP) or quaternary ammonium compounds (QAC) were tested using ASTM E2967-15 to determine removal, transfer, and inactivation of Ebola virus Makona variant (EBOV/Mak) and vesicular stomatitis virus (VSV) from contaminated stainless steel prototypic environmental surfaces. The infectious virus-contaminated carriers were subjected to wiping in the Wiperator per the standard. Following the use of negative control (J-Cloth)-, AHP-, or QAC-based wipes, recovery of residual infectious virus was assayed. In the case of the J-Cloth wipes (negative control), although removal of virus from inoculated carriers was extensive i.e., ~99% (1.9–3.5 log In tMaillard  to incluMaillard –5. For tStaphylococcus aureus  from stainless steel surfaces. Our interest in this study was to evaluate two enveloped viruses, each of which was expected to be inactivated by the microbicidal active used in the DPW, while each test virus was expected to be removed, but not inactivated, by the control wipe without microbicidal active. In addition, VSV has been used as a surrogate for the Ebola virus in previous studies . The twoe family . Vesiculs family . The res2 in Dulbecco's modified Eagle medium  supplemented with 10% fetal bovine serum  and 10 units/ml penicillin/streptomycin (Gibco). Ebola virus Makona variant  was obtained from a clinical isolate and biotechnologically engineered to express green fluorescent protein (GFP). Vesicular stomatitis virus (VSV) stocks were prepared from a reverse genetics construct  were maintained at 37°C/5% COonstruct , 17 and 10 TCID50/ml by the Reed-Muench procedure  and the supernatant was aliquoted and stored at −70°C until needed. Working stock virus titers were determined, on the basis of viral CPE in Vero E6 cells, to be in excess of 8.8 log10 TCID50/ml by the Reed-Muench procedure  was aspirated from the T-175 flask and replaced with 5 ml of diluted virus stock (MOI 0.01) overlaid for 1 h at 37°C ± 5% COrocedure .The “J-Cloth,” a representative cloth material that has been used in previous studies of this type , 8 was u“AHP” wipes consisted of J-Cloth squares impregnated with a 1:40 solution of accelerated hydrogen peroxide (AHP). Sterile J-Cloths (4 × 4 cm) were prepared in the same manner as mentioned above. A single sterile J-Cloth was placed into a sterile Petri plate with 320 μl of prepared AHP added to the wipe. The impregnated wipe was immediately placed overtop the sterile O-ring  and, a s“QAC” wipes consisted of a ready-to-use commercially available wipe impregnated with a quaternary ammonium compound . Each of the QAC wipe lots examined were tested at the end of their stated expiry dates. The QAC wipes were used fresh on the day of each assay. Prior to use in an assay, a container containing QAC wipes was inverted for 10 s five times. Using sterile forceps, three QAC wipes were removed and deposited into a sterile plastic Petri dish (150 × 15 mm). Additional QAC wipes were removed from the same container and sixteen 4 × 4 cm squares were excised from the center of four QAC wipes using sterilized forceps and scissors. Several other QAC wipes were removed from the QAC wipe container and overlaid onto the excised squares to keep the test squares moist. A plastic lid was placed on top of the Petri dish, and the dish was sealed with Parafilm and transported to the biosafety hood in the testing laboratory. Prior to efficacy testing, sealed Petri dishes were inverted as described above and the wipe squares were removed using sterile forceps and loaded onto the Wiperator Boss . ExcisedPrior to evaluating the efficacy of wipes for decontaminating infectious virus (Ebola virus or VSV), a neutralization assay was performed to evaluate the interaction of neutralizers and disinfectants, cytotoxicity to the Vero E6 cells used for reporting GFP/CPE, and impacts on the recovery of infectious virus from the surfaces post-test. Combinations of neutralizers, disinfectants, and viruses were evaluated.50 per mL) of VSV or EBOV/Mak was also prepared with only 10 μl inoculum used for the appropriate test conditions requiring live virus. All neutralization controls were performed in replicates of three over a single experiment. Neutralization controls included the following:On the day of neutralization assay, several neutralizing reagents  were prepared fresh. In addition, a low titer virus stock  to 10−3. Cells were scored for cytotoxicity 14 days post-inoculation.The neutralizer being evaluated was ten-fold serially diluted in VCM and 50 μl were added to Vero E6 cells in replicates of five for each dilution from 100 to 10−3). For each dilution, 50 μl were added to an 80% confluent Vero E6 cell monolayer in replicates of five and monitored over 14 days for CPE. Three technical replicates were used for each test wipe.Microbicidal active-impregnated wipes or DMEM impregnated wipes were loaded onto the Wiperator and a sterile stainless steel coupon was exposed to the wipe for 5 s. The exposed stainless steel coupon was placed into 1 ml of neutralizer, mixed by pipetting, and finally 10-fold serially diluted in VCM (100 (neat) to 10−3. Cells were scored for viral CPE 14 days post-inoculation.The positive virus control was prepared by adding 10 μl of diluted low titer virus to 990 μl of VCM. The positive control was 10-fold serially diluted in VCM and 50 μl of each dilution were added to Vero E6 cells in replicates of five for each dilution from 100 (neat) to 10−3. Cells were scored for viral CPE 14 days post-inoculation.To account for the effect of the neutralizer acting on the virus, 10 μl of low titer virus were added to 990 μl of neutralizer. The neutralizer virus control samples were 10-fold serially diluted in VCM and 50 μl were added to Vero E6 cells in replicates of five for each dilution from 100 (neat) to 10−3. Cells were scored for viral CPE 14 days post-inoculation.To account for the ability for the neutralizer to affectively mitigate the effects of the disinfectant active tested, a prepared active impregnated wipe was loaded onto the Wiperator and the sterile stainless steel carrier was exposed to the active wipe for 5 s. The “wiped” stainless steel carrier was placed into 1 ml of neutralizer, the liquid was mixed by pipetting, and 10 μl of low-titer virus were added. The neutralization disinfectant virus control sample was incubated for 10 min at room temperature after which it was 10-fold serially diluted in VCM with 50 μl added in replicates of 5 added to Vero E6 cells for each dilution from 10Efficacy testing of wipes  was perfUsing sterile forceps, sterile microbicide-impregnated active wipes or sterile wipes impregnated with DMEM were removed from the Petri dishes, loaded onto the Wiperator Boss, and secured in place with a large O-ring . Loaded During the evaluation of possible neutralizing agents, it was determined that 100% virus culture medium (VCM) added to the AHP or QAC wipe dilutions prior to introduction of the EBOV/Mak or VSV in tripartite soil load  preventeremoval was estimated by the difference in titer of infectious virus recovered from the dried positive control carrier and that recovered from the inoculated carrier following use of the wipe by the Wiperator at the various wiping times evaluated. Extent of virus transfer was expressed as a percentage of the infectious virus estimated (on the basis of recovery from the dried positive control condition) to be on the inoculated control carrier that was recovered from the secondary carrier after the 5-s transfer wiping step. The amount of virus remaining on the wipe itself was not measured, therefore complete mass balances for the spiking viruses were not calculable. As a result, inactivation could not be quantitatively measured, but was implied by the absence of viral recovery from the inoculated carriers following wiping for 5, 15, 30, or 60 s, or from the secondary carrier following the 5-s infectious virus transfer wiping step.The results obtained in carrier testing of a single lot of AHP-impregnated wipes and three lots of QAC-impregnated wipes and of the negative control (J-Cloth) wipes are displayed in All testing involving EBOV/Mak was performed at room temperature within a Class II BSC in the BSL-4 laboratories of Public Health Agency of Canada at the Canadian Science Center for Human and Animal Health, Winnipeg, Manitoba operated by Government of Canada. The results of the Wiperator study performed to evaluate efficacy of AHP wipes and QAC wipes for decontaminating EBOV/Mak-contaminated carriers are displayed in 10 TCID50/mL for the 5, 15, 30, and 60 s wiping times from an initial titer of ~6.6 log10 TCID50/mL. The log10 removal of EBOV/Mak from the inoculated carrier for each wiping time ranged from ~2.5 to ~3.5 log10, with a minimal increase in removal observed with increasing wiping time  to a secondary carrier. The EBOV/Mak titers recovered from the secondary carrier were found to range from ~3.9 to ~2.3 log10 TCID50/mL; n = 3 replicates per time point) was recovered from the inoculated carrier after each wiping time  following transfer from the inoculated carriers wiped for 5 and 30 s, respectively. The virus was detected in 3 replicates out of the 9 evaluated from the inoculated carriers wiped for 5 s and in a single replicate from the inoculated carriers wiped for 30 s. No infectious EBOV/Mak was transferred from the inoculated carriers wiped for 15 or 60 s. These results indicate that the AHP wipe inactivated residual infectious EBOV/Mak virus that was removed during the wiping of the inoculated carriers at these times.In the transfer step, we examined the potential for transfer of virus removed from the inoculated carrier (and suspended within the AHP wipe) to a secondary carrier. The infectious EBOV/Mak titers recovered from the secondary carriers were ~0.9 log10 TCID50/mL; n = 9 replicates) was recovered from three inoculated carriers out of the total of nine replicates evaluated after 5 s wiping time . After 15 and 60 s wiping, no infectious virus was recovered from the inoculated carriers.Following wiping of the EBOV/Mak-inoculated carrier with the QAC wipe, a low titer of infectious EBOV/Mak  was transferred from the inoculated carrier wiped for 5 s . No infectious EBOV/Mak was recovered from the inoculated carriers wiped for 15, 30, or 60 s. These results indicate that the QAC wipe inactivated EBOV/Mak virus that was removed during the wiping of the inoculated carriers.In the transfer step, we examined the potential for transfer of virus removed from the inoculated carrier (and suspended within the QAC wipe) to a secondary carrier. A low titer of infectious EBOV/Mak  to a secondary carrier. The VSV titers recovered from the secondary carrier were found to range from ~3.8 to ~3.3 log10 TCID50/mL, estimated on the basis of the value for the dried positive control carrier).Following wiping of the VSV-inoculated carrier with the AHP wipe, no infectious VSV was recovered from the inoculated carrier after the 5, 15, 30, or 60 s wiping times . This inSince no infectious VSV was recovered from the secondary carriers, it is presumed that any VSV removed from the inoculated carriers was inactivated during the transfer process or subsequent to being transferred onto the secondary carriers.10 TCID50/mL, estimated on the basis of the value for the dried positive control carrier).Following wiping of the VSV-inoculated carrier with the QAC wipe, no infectious VSV was recovered from the inoculated carrier after 5, 15, 30, or 60 s wiping time . This inSince no infectious VSV was recovered from the secondary carriers, it is presumed that any VSV removed from the inoculated carriers was inactivated during the transfer process or subsequent to being transferred onto the secondary carriers.Disposable disinfectant pre-soaked wipes (DPW) are commonly used for reducing pathogen loads on HITES in healthcare and home settings , 2. Thesdecontamination by Sattar and Maillard  are known to be relatively less susceptible to microbicides. Differences in surface composition of the differing microbes also may play a role in their ability to attach to a surface.A few publications have described the use of ASTM-2967-15 and the Wiperator instrument for evaluating the removal, transfer, and inactivation of bacterial pathogens by DPW . These sTo our knowledge, the first use of ASTM-2967-15 to evaluate viral removal, transfer, and inactivation using DPW was reported by Wesgate and Maillard , and it In the present study, the ASTM-2967-15 method was used to evaluate the performance of DPW containing one of two actives, AHP and QAC, vs. a control wipe (J-Cloth impregnated with DMEM). The model viruses used (EBOV/Mak and VSV) were enveloped viruses that were expected to be readily inactivated by the two active formulations tested. The negative control wipe was employed to help parse the contributions of removal and inactivation, as the negative control wipe was expected to remove, but not inactivate the viruses. The results obtained using the negative control wipe in the present study might also be expected when a DPW impregnated with an active ingredient with limited efficacy for the target pathogen is used. An example of an inappropriate active might be a detergent-impregnated DWP used for decontaminating a surface containing a non-enveloped virus  load.10) of virus from a surface contaminated with over 6 log10 of virus is insufficient from a public health standpoint, considering the low infectious dose for this virus. In addition, the spread of even a low percentage  of the Ebola virus from an inoculated surface to a new surface could represent a significant opportunity of transmitting Ebola virus disease.The results reported herein quantitatively demonstrate the reduction of EBOV/Mak and VSV (each suspended in tripartite organic load) from a stainless steel carrier surface by wiping action and, in the absence of sufficient inactivating activity, the transfer of infectious virus from the inoculated carrier to a secondary surface. In a real-world situation, the use of a wipe having no or limited microbicidal activity would therefore be expected to afford some removal, albeit at the price of simply moving a portion of the contaminant from one location on the surface to another . For EboOn the other hand, the present results demonstrate that a wipe impregnated with a sufficient concentration of an appropriate microbicidal active (in this case exemplified by AHP or QAC), given sufficient contact time, will remove virus from and inactivate remaining virus on a contaminated surface. Transfer of infectious virus from the inoculated surface to a secondary surface also is limited by the inactivating activity of the DPW. In additional studies involving EBOV/Mak and VSV, we have obtained similar efficacy data for DPW containing a variety of microbicidal actives, including 70% ethanol, 1% sodium hypochlorite, and a product containing 5% of a dual QAC .As observed previously , 5, 7–10In summary, removal of EBOV/Mak and VSV using wipes was extensive in this study. In the absence of a sufficient concentration and contact time of an appropriate microbicidal active in DPW (such as the AHP- and QAC-based DPW tested), transfer of a low, albeit significant (from an infectious unit/infectious dose perspective), quantity of infectious virus from the inoculated surface to a secondary surface was observed. In the case of Ebola virus, it is essential that a DPW with an appropriate microbicidal active, following the appropriate contact time, be used to prevent unintended transfer of infectious virus to a clean secondary surface (as observed in negative control /J-Cloth). Otherwise, there exists the possibility of dissemination of Ebola virus and the associated risk of transmission of Ebola virus disease. Disinfectant wipes pre-soaked with appropriately formulated microbicidal actives should be useful during outbreaks of Ebola virus as well as other highly contagious emerging viruses such as SARS-CoV-2 now causing pandemic COVID-19.The original contributions presented in the study are included in the article/MI, TC, and ST conceived and designed the experiments. RN and TC analyzed the data. RN, MI, JR, TC, CR, and SK each contributed to the writing of the manuscript. All authors reviewed and approved the manuscript.JR and MI are employed by RB, who also provided partial funding support for this study. RN provided data analysis and manuscript preparation as a paid consultant employed by RMC Pharmaceutical Solutions, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hovenia dulcis (Rhamnaceae family). Based upon their genomic information, specific primers were synthetized and used in RT-PCR assays to identify plants hosting the viral sequences. The larger contig was tentatively named as Hovenia dulcis-associated virus 1 (HDaV1), and it exhibited low nucleotide and amino acid identities with Picornavirales species. The smaller contig was related to insect-associated members of the Dicistroviridae family but exhibited a distinct genome organization with three non-overlapping open reading frames (ORFs), and it was tentatively named as Hovenia dulcis-associated virus 2 (HDaV2). Phylogenetic analysis using the amino acid sequence of RNA-dependent RNA polymerase (RdRp) revealed that HDaV1 and HDaV2 clustered in distinct groups, and both viruses were tentatively assigned as new members of the order Picornavirales. HDaV2 was assigned as a novel species in the Dicistroviridae family. The 5′ ends of both viruses are incomplete. In addition, a nucleotide composition analysis (NCA) revealed that HDaV1 and HDaV2 have similarities with invertebrate-infecting viruses, suggesting that the primary host(s) of these novel virus species remains to be discovered.In a systematic field survey for plant-infecting viruses, leaf tissues were collected from trees showing virus-like symptoms in Brazil. After viral enrichment, total RNA was extracted and sequenced using the MiSeq platform (Illumina). Two nearly full-length picorna-like genomes of 9534 and 8158 nucleotides were found associated with  Natural forest ecosystems and cultivated forest plantations are responsible for covering around 30% of the entire surface of the Earth . The ecoCarlavirus, Orthotospovirus, Potyvirus, and Tymovirus , 1 µL of M-MLV enzyme (200 U/µL), 1 µL of RNase OUT (40 U/µL)  1 µL of 100 mM DTT, and 4 µL of RNAse-free water were added to, in a total volume of 20 µL. Samples were incubated at 37 °C for 60 min and 15 min at 70 °C. The PCR assays were performed in a total volume of 12.5 µL. The reaction was composed by 8.0 µL of DNAse-free water, 1.25 µL of buffer 10X, 0.4 µL of MgCl2 (50 Mm), 0.25 µL of dNTP (10 mM), 0.25 µL reverse primer (10 µM), 0.25 µL of forward primer (10 µM), 0.1 µL of Taq DNA polymerase (500 U/µL) , and 2 µL of cDNA. The PCR parameters were as follows: initial denaturation of 94 °C for 2 min followed by 34 cycles of denaturation (94 °C for 30 s), annealing (58 °C for 45 s), and extension (72 °C for 1 min). A final extension step (72 °C for 7 min) was employed. The correct size PCR products were identified by gel electrophoresis (1%), gel-purified, cloned into pGEM-T Easy Vector , and Sanger sequenced at CNPH .Based on the assembled contigs, a set of specific primers were designed to determine the presence or absence of each of the five viruses and used to assay each sample by RT-PCR . The totThe 3′ end of the genomes was amplified using the 3′ RACE, as described by ,29. BriePicornavirales. The amino acid sequences were aligned using ClustalW [Phylogenetic analyses were carried out with RNA-dependent-RNA-polymerase (RdRP) protein sequences from members belonging to the order ClustalW  implemenClustalW . The maxClustalW  implemenClustalW . Branch ClustalW . The genClustalW . The accPicornavirales were retrieved from the NCBI/GenBank (https://www.ncbi.nlm.nih.gov/). In viruses with bipartite genomes, the sequences of the components were concatenated, and these were considered as the complete genome. A Linear Discriminant Analysis (LDA) was performed to identify the most likely host species of the viruses reported in the present work. Dinucleotide frequencies for each sequence were determined using the program simple sequence editor (SSE), version 1.3 [www.R-project.org) (version 3.4.2) [The nucleotide composition analysis (NCA) method was used to infer the most likely virus host in this study . For NCAsion 1.3 . LDA wasn 3.4.2) , implemeH. dulcis showing virus-like symptoms   were related to unclassified members of Picornavirales and Dicistroviridae, the remaining three contigs with small sizes  and relatively low coverage (≤50 reads) were related to Secoviridae members. The presence of these putative novel viruses was investigated by RT-PCR and Sanger sequencing in all samples individually. All sixty samples were negative to the Secoviridae related contigs; therefore, they were not further investigated. The two large contigs were detected only in one H. dulcis leaf sample. After 3′ RACE, five additional nucleotides from 3′-terminal sequence of the larger contig were recovered, resulting in a final contig with 9534 nts plus the poly(A) tail. Moreover, the minor contig was increased in 32 nts, resulting in a final contig with 8158 nts plus the poly(A) tail.As part of a field survey for plant-infecting viruses, samples from a variety of plant species were collected in 2014 at NOVACAP Nursery, located in Brasília-DF, Brazil. This nursery is responsible for the production of tree seedlings for urban reforestation purposes. We processed and sequenced one pool of samples containing viral enriched RNA from 60 plants, including two plants of lorosis) a. After Picornavirales.The nearly full-length genome of 9534 nts (assembled from 8669 reads) displayed two non-overlapping ORFs b. The fiPicornavirales members indicated that it shares the highest degree of nucleotide identity (71%) with the Darwin bee virus 6 isolate NT-8 (9123 nts) (MG995696), a yet unclassified Picornavirales member reported infecting honey bees (Apis mellifera) [Hovenia dulcis-associated virus 1 (HDaV1) is proposed for this virus.Pairwise identity comparisons of this contig sequence with those of representative llifera) . Additiollifera) . The namDicistroviridae. To further investigate this result, we performed a new HTS using total RNA from H. dulcis leaves, and no insertion was observed in the reads mapped in this genomic region. However, only a limited number of reads (226 reads) mapped to the HDaV2 genome.Interestingly, the contig of 8158 nts (assembled from 689 reads) presented three non-overlapping ORFs of 3758 nts (ORF1), 1446 (ORF1b) and 2571 nts (ORF2) b. Since Picornavirales. A hairpin structure was predicted at nucleotide positions 8124–8158  (BaDV-2) and Bemisia associated dicistrovirus 1 (BaDV-1) (MH459180) [Dicistroviridae, which we have tentatively named Hovenia dulcis-associated virus 2 (HDaV2).The pairwise identity comparisons indicated that it shares the highest degree of nucleotide identity with two viruses reported in Picornavirales confirmed the BLASTx and pairwise identity results  and representative viruses in the order  results . HDaV1 ses order . The genoviridae .Picornavirales were used, and four pre-defined categories of hosts were used for NCA , plants, and vertebrates). As shown in A nucleotide composition analysis (NCA) was performed in an attempt to identify the most likely host(s) of the HDaV1 and HDaV2. A total of 278 sequences from the order H. dulcis seedlings grown under nursery conditions, confirming that the viral enrichment protocol followed by HTS is a sensible and economic strategy for discovering new viruses, even with the dilution effect of sample-pooling. Based upon the genomic organization and phylogenetic analyzes, these two viruses were tentatively classified as novel viral species within the order Picornavirales. We proposed the names Hovenia dulcis-associated virus 1 (HDaV1) and Hovenia dulcis-associated virus 2 (HDaV2).In a systematic field survey for plant-infecting viruses, leaf tissues were collected from trees showing virus-like symptoms in Brazil. Two putative new ssRNA+ viruses were found in leaves of Picornavirales harbors viruses with ssRNA+ genomes, spherical particles with a diameter around 30 nm, distinct genomic organization and segmentation (mono or bipartite), as well as distinct host organisms  [Dicistroviridae, Iflaviridae, Marnaviridae, Secoviridae, Picornaviridae, and Polycipiviridae) [Picornavirales members, including the genomic organization with conserved regions with HEL/PRO/RdRp motifs [Dicistroviridae, with three non-overlapping ORFs.The order ebrates) . Currentviridae) ,46. HDaVp motifs . HoweverBemisia tabaci genes, which was the only insect observed during our surveys. Moreover, dual tropism (invertebrates/plants) have already been described in both invertebrate-infecting viruses or plant-infecting viruses [H. dulcis leaf samples. For instance, Rhopalosiphum padi virus—RhPV  is commonly reported infecting aphids, which are well-characterized plant-pests. In this context, plants might also serve as secondary hosts or reservoirs for RhPV, contributing to its horizontal transmission in aphids [Nepovirus, family Secoviridae) is a main example, which was found causing systemic infection in Apis mellifera [Vigna unguiculata (L.) Walp) as well as in mammalian cell culture lines has been demonstrated, providing evidence of a virus that can infect hosts of distinct kingdoms [Secoviridae, but they were not detected in any plant within the pool, probably due to their relatively low coverage or due to index hopping, which may result in the assignment of sequencing reads to the wrong index during demultiplexing [Importantly, HDaV1 and HDaV2 clustered with invertebrate-infecting viruses, suggesting that they might be (i) invertebrate-infecting viruses derived from some undetected invertebrate that was contaminating our plant samples, (ii) they are bona fide yet unknown plant-infecting viruses, or (iii) they can infect both invertebrates and plants. The presumed relationships with insect-infecting viruses were supported by the NCA results, which grouped HDaV1 and HDaV2 with picornaviruses infecting invertebrate and “other” hosts rather than those infecting either vertebrates or plants . Therefo viruses ,48,49,50n aphids ,52,53. Iellifera ,47,48. Rkingdoms . We alsoiplexing .H. dulcis allowed us to speculate that the two new viruses described here could have a close relationship with this plant, even though, no viral movement protein was identified in the HDaV1 and HDaV2 genomes. In addition, no other plant sampled in the same area was found to be positive for either HDaV1 or HDaV2, reinforcing the hypothesis that these viruses might be exclusively associated with either H. dulcis or with some yet unidentified arthropod pest of this plant species. In this context, further biological assays should be performed to elucidate the interaction among H. dulcis and both viruses.Therefore, the virus detection exclusively in leaf samples of


Culex annulirostris Skuse mosquito, an important vector of pathogenic arboviruses in Australia. Mosquitoes were collected from three sites in the Kimberley region of Western Australia. Unbiased high-throughput sequencing (HTS) revealed the presence of 16 novel viral sequences that share less than 90% identity with known viruses. None were closely related to pathogenic arboviruses. Viruses were distributed unevenly across sites, indicating a heterogeneous Cx. annulirostris virome. Polymerase chain reaction assays confirmed HTS data and identified marked variation between the virus prevalence identified at each site.Metagenomics revealed an impressive breadth of previously unrecognized viruses. Here, we report the virome of the  Mosquito-borne viral diseases pose a persistent challenge to global public health. Flaviviruses, such as yellow fever, dengue, Zika, Japanese encephalitis, and West Nile (WNV), and alphaviruses including Ross River and chikungunya cause significant morbidity and mortality in humans ,2,3. EacComprehensive arboviral surveillance was conducted in Western Australia (WA) since 1972 , pooled,Culex annulirostris mosquitoes collected from three sites located in the Kimberley region in the far north of WA that are representative of locations where MVEV and WNV (Kunjin) are most regularly detected in surveillance programs and where human cases are most frequent [Cx. annulirostris was chosen, as it is the major vector of the human pathogenic flaviviruses in this region, as well as a significant vector for pathogenic alphaviruses.Here, we describe the virome of frequent . Prior sfrequent ,16. Tech2-baited traps [Cx. annulirostris were trapped at the three sites used in this study , and incubated at room temperature for 45 min. Total nucleic acid was extracted from pools using the MagMax Express-96 automated platform  with modifications [Mosquitoes were individually washed three times using 750 µL of phosphate-buffered saline to remove environmental nucleic acids. Three 5-mm stainless-steel beads  and 750 µL of cold virus transport medium (in-house formulation ) were adications . NucleicCulex genome, Cx. quinquefasciatus,  reference sequence NZ_AAWU00000000.1) were subtracted using Bowtie2 . Assemb. Contig protocol as described above. Complementary DNA was prepared from TNA using SuperScript III (Invitrogen) and random hexamer primers. PCR screening primers were designed using assembled viral sequences obtained from HTS data . All PCRhttp://tree.bio.ed.ac.uk/software/figtree) for annotation.RNA-dependent RNA polymerase (RdRp) sequences were utilized for phylogenetic analyses. Representative protein sequences were aligned using MUSCLE in Geneious 10.2.3  and expoConsensus viral genomic sequences were deposited in GenBank under accession numbers MT498812 to MT498834. Illumina sequence data were deposited in GenBank under BioProject identifier (ID) PRJNA632594. Illumina sequence data for Jogalong virus (accession number MN133813) were reported previously and are available under BioProject ID PRJNA590265.Culex annulirostris was the most frequently captured mosquito species during the 2017–2018 season in the Kimberley region of WA [on of WA . Mosquiton of WA .−10 and 1 × 10−3, respectively.A total of 341 million reads were obtained from a single lane of sequencing (excluding controls). An average of 10.5 million reads per mosquito pool, 2.4 million reads per negative control and 16.9 million for the positive control remained following primer trimming, quality-filtering, and host subtraction. Assembly of reads generated an average of 233,411 contigs  and 2.7 million unique singletons per mosquito pool; 1.74% of these sequences shared identity with viral sequences using MegaBLAST and BLASTX similarity alignments with minimum E-value cutoffs of 1 × 10Cx. annulirostris revealed one single-stranded DNA (ssDNA), five positive-sense single-stranded RNA (ssRNA), four negative-sense ssRNA, and six double-stranded RNA (dsRNA) viral sequences  shared identity with viruses typically associated with vertebrates. All other viruses shared phylogenetic relationships with invertebrate, fungal, or plant viruses.Analysis of 300 equences . SequencPodargus strigoides (tawny frogmouth) for the sequence, tentatively named Jogalong virus, were previously described [We obtained sequences distantly related to hepaciviruses from a single pool from the Parry’s Creek site. Phylogenetic analysis, PCR prevalence, genome annotation, and potential host association with escribed .Cx. inatomii from South Korea   and Cx. (Korea) . Aside f (Korea) .Virgaviruses also possess positive-sense ssRNA genomes and belong to the same insect-infecting alphavirus-like superfamily as negeviruses . An 11,1Iflaviridae that was sequenced from water striders, Tetragnatha maxillosa (spiders) and an arthropod mix in China [Sequences related to picornaviruses were identified in a single pool from Fitzroy Crossing. The genome for Fitzroy Crossing iflavirus 1 (FCIflaV1) encodes a single 8718-nt open reading frame (2904 aa) and includes a 5′ nontranslated (NTR) region of 736 nt and a 139 nt 3′ NTR at each terminus. FCIflaV1 shares greatest similarity (48%) with Hubei arthropod virus 1, a member of family in China   and Parry’s Creek phasivirus 1 (PCPhasV1) share 87.5% and 88.3% aa identity in the RdRp protein, respectively, with Badu phasivirus (BADUV), an insect-specific bunyavirus identified in Cx. annulirostris and Cx. sitiens (Weidermann) mosquitoes [Cx. annulirostris phasiviruses from WA reveal 91%, 88%, and 89% identity across the RdRp, glycoprotein, and capsid genes, respectively. The distribution of the phasiviruses across the three trapping sites was varied. BrPhasV1 was detected in 59% of mosquitoes in Broome, whereas it was rarely detected at the Fitzroy Crossing (2%) or Parry’s Creek (3%) sites , China [Three phenuiviruses representing two genera, squitoes  (Table 3%) sites . Convers%) sites . Phylogepp., USA ), and Wu), China ) , is highly divergent and shares just 33% aa identity with rice grassy stunt virus across the conserved RdRp protein . Tenuivinuivirus .Cx. pipiens from Sweden [Cx. globocoxitis (Dobrotworsky) from southern regions of WA, Wilkie qin-like virus, was only distantly related (32% aa identity in RdRp) to FCqinV1  [Six double-stranded RNA viruses were identified from across all trapping sites. Sequences share sequence similarity with reoviruses (Broome reo-like virus 1), totiviruses , chrysoviruses (Broome chryso-like virus 1), and partitiviruses (Broome partiti-like virus 1). Broome reo-like virus 1 forms a sister clade to fijiviruses, but shares just 37% identity with Sanxia reo-like virus 1—an unclassified sequence identified in water strider from China  Figure ; Table 3n Sweden   . Fitzroydentity)   from mosquitoes found at all three sites. BrDenV1 shares just 28% aa identity with Haematobia irritans densovirus . Phyloge viruses . The BrDCx. annulirostris mosquitoes to assess the distribution across the three collection sites  using individual mosquitoes. We confirmed the presence of each viral sequence in individual mosquitoes sourced from sites where HTS data indicated the presence of a virus sequence. We detected viral sequences by PCR in mosquitoes from pools on six occasions where HTS did not indicate the presence of the virus. This is likely due to several factors, including a dilution effect when pooling mosquitoes for HTS analysis and superior sensitivity of PCR versus unbiased HTS.Three viruses screened by PCR were sparsely distributed with low abundance. Jogalong virus (0.3%), Fitzroy Crossing iflavirus 1 (0.3%), and Fitzroy Creek tenui-like virus 1 (0.7%) were detected at single sites in two or fewer mosquitoes . The twoArbovirus discovery and surveillance saw three major generational shifts over the last six decades. The use of suckling mice in the 1970s and 1980s led to the first wave of arbovirus discovery, including the isolation of Kununurra and Kimberley viruses from the northwest of WA . A shiftCx. annulirostris mosquito is associated with the transmission of several important Australian arboviruses, including MVEV, Ross River virus, and WNV (Kunjin). To further investigate the complete virome of Cx. annulirostris, we performed HTS on 300 mosquitoes collected from across three sites in northern Western Australia. A diverse collection of 16 novel viral sequences was recovered, representing 11 distinct classified and unclassified viral families. We did not recover any sequences with homology to known human or mammalian pathogens. One sequence, Jogalong virus, shares identity with vertebrate-associated hepaciviruses, and we presented evidence elsewhere that this incidental discovery may be due to contamination from a blood meal taken from the tawny frogmouth (Podargus strigoides) [The igoides) . HoweverCulex species, from the southwest of WA recovered an impressive array of RNA viruses, but none that share identity with known pathogens [Culex mosquitoes from California, USA, also failed to identify any known animal or human arboviruses despite the recovery of 56 mosquito-associated viral genomes [Cx. annulirostris virome, but our study and the unbiased metagenomic studies described above highlight the limitations of HTS for the purposes of surveillance for human and animal pathogenic arboviruses. Increased mosquito pool sizes and incorporation of positive selection systems to offset the impact of significant dilution effects may yield improved data for that purpose. Conversely, it does suggest that the detection of any pathogenic arboviruses in a mosquito population indicates an increased risk to human and animal health.Previous studies also showed that pathogenic viruses are rarely detected by HTS of mosquito samples ,40,42; tathogens . A metagCx. annulirostris at each collection site. Nine to 10 viruses were detected at each site, but the composition of the virome demonstrated variation. Aside from four viruses that were present across all three sites, seven viral sequences were confined to a single site and the remaining five viruses were confined to two sites. Phasivirus sequences that were 80% similar to Badu phasivirus and 90% similar to each other were identified from all sites. However, Broome phasivirus 1 was highly prevalent (59%) in mosquitoes sampled from Broome, whereas Parry’s Creek phasivirus was most prevalent (16%) in Parry’s Creek. Interestingly, each virus was either absent or rarely detected at the other site, while both phasiviruses were present in Fitzroy Crossing (located between Broome and Parry’s Creek), albeit in low prevalence (less than 4% for both viruses). These data indicate that even closely related viruses are distributed unevenly in Cx. annulirostris from across the north of WA. Differences between the local environments from each site may contribute to this unevenness [Cx. annulirostris populations at each site suggest that there is minimal movement of mosquitoes across the remote Kimberley region of WA, as we would expect increased virus sharing and homogeneity if this were the case. However, longitudinal studies will be required to further investigate this finding.We identified differences between the viromes obtained from evenness . TrappinCulex species from the south of WA [Cx. annulirostris-associated viruses identified in this study. Further virome analyses of other mosquito species from the same regions investigated in this study will be required to confirm this finding.We did not identify any viral sequences that share greater than 90% amino-acid identity across conserved RdRp proteins to known viruses. The mosquito taxon was proposed as playing an important role in virome structure , but we th of WA  shared lArbovirus surveillance historically relied on pooling mosquitoes in order to determine the viral content at a community level. The proportion of viruses recovered from these mosquito pools is known as the infection rate . Pooling