Computers store numbers in two mutually incompatible ways: little-endian or big-endian. They differ in the order of bytes within representation of numbers. This ordering is called endianness. When two computer systems, programs or devices communicate, they must agree on which endianness to use, in order to avoid misinterpretation of numeric data values.We present Endicheck, a dynamic analysis tool for detecting endianness bugs, which is based on the popular Valgrind framework. It helps developers to find those code locations in their program where they forgot to swap bytes properly. Endicheck requires less source code annotations than existing tools, such as Sparse used by Linux kernel developers, and it can also detect potential bugs that would only manifest if the given program was run on computer with an opposite endianness. Our approach has been evaluated and validated on the Radeon SI Linux OpenGL driver, which is known to contain endianness-related bugs, and on several open-source programs. Results of experiments show that Endicheck can successfully identify many endianness-related bugs and provide useful diagnostic messages together with the source code locations of respective bugs.


Canis lupus) of around 2 years found dead by a hunter in the province of Avellino, Campania Region. Necropsy showed pathological findings consistent with encephalitis and gastroenteritis. Organs were analysed by microbiological and molecular investigations following standard procedures to ascertain the possible cause of death. Real‐time PCR revealed the presence of PRV in the brain and of canine parvovirus 2b in organs like intestine, liver, brain, kidney and pancreas. Death probably occurred very shortly after SHV‐1 infection in an animal already weakened by parvovirosis.Pseudorabies virus (PRV) or suid herpesvirus 1 (SHV‐1) is the causative agent of Aujeszky's disease, a highly contagious viral infection which causes neurological fatal illness in mammals other than suids. Here we report a case of a young wolf ( We describe a case of wolf dead since infected by pseudorabies virus and canine parvovirus, with pseudorabies attributable to wild boar meat ingestion. Since this virus has been recently described as responsible for human encephalitis, its presence in wild animals must be taken in consideration for the circulation of the virus in the environment. The virus is a member of the Herpesviridae family, subfamily Alphaherpesvirinae  of around 2 years was found dead by a hunter in the province of Avellino, Campania Region and referred for necropsy to our laboratories. SHV‐1 positive control, strain ‘Kaplan’ was kindly given by Friederich‐Loeffler Institut, Federal Research Institute for Animal Health, Federal Republic of Germany. Parvovirus strains, a, b and c used as positive controls, were kindly given by University of Bari Aldo Moro, Italy.In December 2018, a young wolf  was homogenized by Tissue Lyser (Qiagen). Nucleic acid extraction was carried from 400 µl of organ homogenate by using a Qiasimphony automated extraction system (Qiagen) with the DSP Virus/Pathogen Midi kit (Qiagen) according to the manufacturer's instructions. Nucleic acids were eluted in 60 µl of elution buffer containing 40 unit/µl RNase inhibitor (Promega) and immediately analysed using real‐time PCR. A strain of SHV‐1 isolated in our laboratory from the brain of a farm dog, was also analysed for phylogenetic analysis purposes. The dog, dead with AD in 2012, lived in a pig farm in the province of Sorrento (Campania Region). Viral DNA was extracted from cell culture (RK13 cells) supernatant (400 µl) as indicated above.2.4DNA viruses  were investigated by real‐time PCR using a Quantitect Real time PCR detection kit (Qiagen). RNA viruses like: canine adenovirus 1 and 2, morbillivirus  and canine coronavirus were investigated by real‐time reverse transcription PCR using AGPATH reaction kit (Thermo Fisher Scientific). All the reactions were carried with primers (Tema Ricerca) and probes (Thermo Fisher Scientific) specific for the virus tested on a Quantstudio5 system (Thermofisher) with protocols routinely used in our laboratory. In particular, SHV‐1 was identified as described by Ma et al. . Canine 2.5In order to characterize the strains of suid herpesvirus, 5 µl of nucleic acids underwent PCR to amplify an 804‐bp fragment of the pseudorabies gC gene  and phylogenetic analysis was carried out using the Jalview and MEGA 7 softwares.Amplicons were sequenced by capillary electrophoresis as previously described  were positive for the presence of canine parvovirus (CPV), identified by multiplex PCR further investigation as CPV strain 2b. Any attempt to cell isolate the pseudorabies virus from the samples was unsuccessful probably due to not enough quantity of virus or to the bad condition of the material used.3.3Sus scrofa in 2011  exhibited 100% homology with a strain identified in a 4www.cfsph.iastate.edu). Results of the sequencing analysis surprisingly showed that the strain identified in the present study  was more similar to strains identified in different years in other regions (Abruzzi 1993 and Emilia Romagna 2014) with respect to the strain isolated from the Sorrento farm dog in 2012 and analysed in the present study for phylogenetic purposes since belonging to the same region. Our results clearly indicate that the difference observed cannot be ascribed to the year of isolation (considering the similarity between strain of 2018 and strain of 1993), nor to the geographical distance, since even if the two cases here reported come from different areas of the same Region ; these places are, however, closer with respect to the other regions to which strains KP862620.1 and GQ259112 belonged.Pseudorabies virus has been detected in many domestic and wild mammals (Moreno et al., The difference observed can be most likely explained with the diverse source of contamination: while farm dog was surely contaminated by pigs, wolf was probably infected ingesting wild boar meat. As a matter of fact, wild boar population in Italy is very abundant and wolves consume their meat as a usual part of their diet (Verpoest et al., The authors declare that they have no conflicts of interest.Maria Grazia Amoroso: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Supervision; Writing‐original draft; Writing‐review & editing. Denise Di Concilio: Formal analysis; Investigation; Validation. Nicola D'Alessio: Conceptualization; Formal analysis; Funding acquisition; Writing‐original draft. Vincenzo Veneziano: Conceptualization; Funding acquisition; Supervision; Visualization. Giorgio Galiero: Conceptualization; Data curation; Supervision; Writing‐review & editing. Giovanna Fusco: Project administration; Resources; Supervision; Validation.


Although tuberculosis (TB) patients often incur high costs to access TB-related services, it was unclear beforehand whether the implementation of universal health coverage (UHC) in Indonesia in 2014 would reduce direct costs and change the pattern of care-seeking behaviour. After its introduction, we therefore assessed TB patients’ care-seeking behaviour and the costs they incurred for diagnosis, and the determinants of both.In this cross sectional study, we interviewed adult TB patients in urban, suburban, and rural districts of Indonesia in July–September 2016. We selected consecutively patients who had been treated for TB in primary health centers for at least 1 month until we reached at least 90 patients in each district. After establishing which direct and indirect costs they had incurred during the pre-diagnostic phase, we calculated the total costs (in US Dollars). To identify the determinants of these costs, we applied a general linear mixed model to adjust for our cluster-sampling design.P = 0.119) and among those with a low educational level . Visiting a private clinic as the first contact also led to more visits  and higher costs than first visiting a Primary Health Centre, both in terms of direct costs  and total costs .Ninety-three patients of the 282 included in our analysis (33%) first sought care at a private clinic. The preference for such clinics was higher among those living in the rural district (aOR 1.88, 95% CI 0.85–4.15, Despite UHC, high costs of TB seeking care remain, with direct medical costs contributing most to the total costs. First seeking care from private providers tends to lead to more pre-diagnostic visits and higher costs. To reduce diagnostic delays and minimize patients’ costs, it is essential to strengthen the public-private mix and reduce the fragmented system between the national health insurance scheme and the National TB Programme. Studies in Asian countries suggest that the private healthcare sector has the potential to play an important role in national and global tuberculosis (TB) control –4. Many Jaminan Kesehatan Nasional, JKN), to ensure people’s access to healthcare. This insurance scheme is managed by the Social Security Agency for Health  and covers all essential care services, including TB diagnostic tests through healthcare providers that are linked to the BPJS-K [In 2014, Indonesia started implementing a universal health coverage (UHC) scheme, also called the Indonesian National Health Insurance scheme , which are national level government boards that are responsible for TB control. Private providers that are linked to the BPJS-K are therefore also not necessarily linked to the NTP. Responding to this problem, the NTP launched a guideline of TB care to coordinate the care with the BPJS-K . AlthougNevertheless, there is no evidence available on the costs incurred during the pre-diagnostic phase of TB and TB patients’ care seeking behaviour since the implementation of UHC in Indonesia. It is therefore unclear to which extent UHC has saved patients from high costs during this phase. The aims of this study are to assess the costs incurred before diagnosis by patients seeking care for TB after the implementation of UHC in Indonesia, and to assess care-seeking behaviour of TB patients in this period on the basis of the first contact facility and the number of healthcare visits.To assess patients’ TB care-seeking behaviour and the costs they incurred during the pre-diagnostic phase, we conducted a cross-sectional study that was a part of a larger study on measuring catastrophic costs in Indonesia . In thisWe included all subjects who met the inclusion criteria of our main study, which were adult patients who had received TB treatment in a PHC for at least 1 month. We excluded extra-pulmonary cases, as these are diagnosed using different methods that may result in bias on pre-diagnostic costs.2.p.q/d2) with assumptions of a power of 0.90 and an error of 0.05. With a ratio of TB patients of 1:1:1 in each district, we required 90 patients per district. In each district, we included all the PHCs that were linked to the Indonesian NTP in our sampling framework, and then randomly selected five PHCs per district. We chose only PHCs since most of TB patients were treated in PHCs. In each PHC, we selected consecutively adult TB patients until we reached at least 90 patients per district. If the sample size was not reached, we randomly selected additional PHCs until we obtained at least 90 patients per district. In total, 19 PHCs were included in our study.The sample size and sampling methods in this study followed that of our larger study, which assumed an incidence of catastrophic costs due to TB of 20–30% . We usedAll patients were interviewed by four medical students and six public health graduates we had recruited and trained for the purpose. As a part of our main study on measuring catastrophic costs, the interview was conducted during the TB treatment phase – 48% of patient in their intensive phase and 52% of patients in their continuous phase . To inteRetrospectively, each respondent listed each healthcare facility he or she had visited between developing TB-related symptoms  and the establishment of their TB diagnosis . We alsomantri . We then assessed whether the first contact facility was associated with the district , household income level (poor/non-poor household), educational level (low/middle-high), health insurance status (being covered/not being covered by the BPJS-K scheme), and formal employment (yes/no).First, to assess patients’ care-seeking behaviour, we established the nature of their first contact facility, i.e., a primary health centre (PHC); a private clinic (whether a solo practice or part of a multiple practice); a public hospital; a private hospital; or ‘other’, such as a pharmacy, a practitioner of alternative medicine, or a Second, we calculated the number of healthcare visits made during the pre-diagnostic phase. To assess the determinants of the number of visits, we also included first contact as an independent variable, together with the other independent variables.Third, we asked patients the details of all the types of cost they had incurred in each facility visited during the pre-diagnostic phase, i.e., direct medical costs, direct non-medical costs, and indirect costs related to seeking care for their TB-related symptoms. The definitions of cost items used in this study conformed with the WHO handbook on TB costs survey . The dirData were double-entered into Microsoft Excel 2010 and EpiInfo version 7  and then exported to IBM SPSS 21 for data analysis. Number of visits and costs data were displayed as means and their 95% confidence intervals (CIs) while categorical variables as numbers and proportions (%). To examine the determinants of the patients’ first contact facility, the number of visits, and their costs during the pre-diagnostic phase, we applied the generalized linear mixed models (GLMMs) with random effects to adjust for our cluster sampling design (19 PHCs), since we selected the PHCs randomly. We assessed the goodness of fit for models by examining the Akaike corrected and the Bayesian information criterion provided by SPSS. To analyse the determinants of the first health-service contact, we estimated crude odds ratios (cORs), adjusted odds ratios (aORs), and their 95% CIs with a target distribution of multinomial regression. To analyse the determinants of number of visits and costs incurred by patients and their family, we established crude and adjusted GLMM regression coefficients (β) and their 95% CIs with a target distribution of linear regression.Before the interview, we provided all respondents with a written and oral explanation of the study. Only those who had signed an informed consent form were interviewed. Before the study, we received ethical clearance from the Ethical Committee at the Faculty of Medicine of Universitas Indonesia–Cipto Mangunkusumo Hospital, Jakarta, Indonesia (No. 416/UN2.F1/ETIK/VI/2016).We included the interviews of 282 patients. Whereas a PHC had been the first point of diagnostic care-seeking for 45% of them, 33% had first sought care at a private clinic, 11% at a public hospital, 7% at a private hospital, and 4% at a pharmacy, practitioner of alternative medicine, or other healthcare provider. . Seeking inital care at a private provider rather than a PHC was also greater among patients with a low educational level than among those with a middle to high educational level . Despite the borderline statistical significance of the result, multivariable analysis showed that the odds of seeking care at a private clinic rather than at a PHC was higher in the rural district  and in patients with low education level . There were no statistically significant differences regarding first contact preferences for the following: between patients with insurance and without insurance; between patients living in a poor household and a non-poor household; and between patients with and without formal work.Table P < 0.001 for private clinics and aβ 1.77, 95% CI 0.97–2.57, P < 0.001 for other health care providers). (Table The average number of visits during the pre-diagnostic phase was 2.56 95% CI 2.41–2.71). Patients who first sought care at private clinics and other health care providers had more visits until their diagnosis was confirmed than patients who first visited a PHC  and of total costs . Patients who visited a private hospital as the first contact also incurred statistically significantly higher direct costs  and total costs  than those visiting a PHC as the first contact. The direct medical costs incurred by patients who first sought care at private clinics  and private hospitals  were significantly higher than those who first sought care at PHCs , while travel costs between private providers and PHCs did not differ significantly . No other variables determined the indirect costs.The average total cost incurred during the pre-diagnostic phase was USD 22 95% CI 18–26). It consisted mainly of direct costs .


Transmission of SARS-CoV-2 leading to COVID-19 occurs through exhaled respiratorydroplets from infected humans. Currently, however, there is much controversy over whetherrespiratory aerosol microdroplets play an important role as a route of transmission. Bymeasuring and modeling the dynamics of exhaled respiratory droplets, we can assess therelative contribution of aerosols to the spreading of SARS-CoV-2. We measure sizedistribution, total numbers, and volumes of respiratory droplets, including aerosols, byspeaking and coughing from healthy subjects. Dynamic modeling of exhaled respiratorydroplets allows us to account for aerosol persistence times in confined public spaces. Theprobability of infection by inhalation of aerosols when breathing in the same space canthen be estimated using current estimates of viral load and infectivity of SARS-CoV-2. Thecurrent known reproduction numbers show a lower infectivity of SARS-CoV-2 compared to, forinstance, measles, which is known to be efficiently transmitted through the air. In linewith this, our study of transmission of SARS-CoV-2 suggests that aerosol transmission is apossible but perhaps not a very efficient route, in particular from non-symptomatic ormildly symptomatic individuals that exhibit low viral loads. Respiratory droplets form the most important carrier of SARS-CoV-2 virions and may infecthumans by direct inhalation or indirectly through hand or object contact. During the currentCOVID-19 pandemic, numerous explosive local outbreaks, so-called super-spreading events, inpublic spaces or health care settings have raised concerns of aerosol transmission ofSARS-CoV-2. Aerosols, or microdroplets, are formed and exhaled during loud speaking,singing, sneezing, and coughing. As infected persons  may have none or mildsymptoms, an aerosol transmission route of SARS-CoV-2 may have tremendous impact on healthcare strategies to prevent the spreading of COVID-19 in public spaces. Importantly,SARS-CoV-2 viral particles have been detected in microdroplets, which may spread in exhaledair during breathing, talking, singing, sneezing, or coughing by an infectedindividual.Microdroplets form aerosol clouds, which have a relatively long airborne time,Next to virus and host factors, this type of viral transmission through aerosols dependsstrongly on droplet properties and behavior.We measure size distributions of droplets in aerosols released when speaking or coughingusing laser diffraction  and consistently find a double-peaked dropsize distribution for coughing, and a single-peak drop size distribution for speech, whichcan be described by a distribution corresponding to a normal liquid spraying process,Using a precision balance, the volumes of saliva/mucus produced by the high emitter whencoughing or speaking into a small plastic bag were measured by weighing before and after asingle cough or saying “Stay Healthy” for ten times.µm and 2 mm can be measured.Speaking and coughing is done directly into the laser beam, and data acquisition is donein the “fast acquisition” mode so that there is no dead time and the drop sizedistribution is measured before evaporation. For coughing, the volumetric distributionmeasured using laser diffraction shows that on average, 98% ± 1% of the volume of thespray is contained in the large drops (100 μm–1000 μm).For the small aerosol droplets, this amounts to ∼20 × 106 microdropletsproduced in a single cough and ∼7 × 106 for speech. For COVID-19, thus fromsymptomatic patients, the viral RNA load in the undiluted oral fluid or sputum has beenfound to be 104–106 copies/ml.11 copies/ml have been reported,6 copies/ml in respiratorysamples in our primary analysis. The total number of virus particles present in the totalvolume of only the microdroplets is then 104, implying that only one in 2000aerosol droplets contains a virus particle.Size distributions of droplets from aerosols released when speaking or coughing weremeasured using laser diffraction employing Malvern Spraytech with a 300 mm lens. In thisconfiguration, drop sizes between 0.2 F = mg) and air drag, from which it follows that a droplet with a radius of 5μm will take 9 min to reach the ground from an initial height of 1.5 m.This time will even increase by the evaporation of the liquid phase of the droplet. Sputumdroplets are known to consist for 1%–10% of their volume of solid solutes.µm in radius, the contraction to the solid core having half of theoriginal droplet size  happens within a second inquiescent air with a relative humidity (RH) of 50%,The persistence of these aerosol droplets in the air is of the greatest concern regardingcommunity transmission of SARS-CoV-2 in public spaces. This airborne time is governed byevaporation and gravity-driven sedimentation toward the floor. The latter can be explainedby balancing the forces of gravity  yielding the same droplet size distribution as droplets from atypical cough. To achieve this, we use a mixture of 1% glycerol and 99% ethanol; within asecond, ethanol evaporates, yielding polydisperse non-evaporating droplets of glycerolwith a median mass aerodynamic diameter (MMAD) of 5 ± 3 If these aerosol droplets are a vector of transmission for the SARS-CoV-2 virus, how thenumber of droplets decreases as a function of time will have a significant influence onthe potential airborne transmission of SARS-CoV-2. To predict the evolution in the numberof microdroplets, the evaporation and sedimentation can be accounted for to calculate thenumber of airborne aerosol particles with knowledge of the initial droplet sizedistribution.md(t), is givenbyR(t) is the radius of the droplet,Dva is the diffusivity of water vapor inair, and C is the water vaporconcentration along direction r. Assuming that the droplets aresufficiently spaced and that the relative humidity of the air in which they are fallingthrough does not change, the final term can be written asr =R(t)] is given by the equilibrium vapor pressure,ρvap, of the environment and, very far awayfrom the droplet surface , is given by the product of the RH of theenvironment and ρvap, resultinginmd, of thedroplet at any time is given byρs is the density of the solid found inhuman mucus/saliva  from Ref. ρw is the density of liquidwater. Differentiating Eq. R(t) =R0,R0 of each droplet is half of the initial sizeR(t = 0) and corresponds to an initial density of∼1080 kg/m3 for the water–solute mixture. µm droplet. Within 1 s, the evaporation of the smallmicro-droplets is complete, resulting in a solid core.The evaporation of a spherical droplet in an environment with a known relative humidity(RH) can be evaluated using the diffusion model presented and validated in Ref. h(R(t), t), throughwhich the droplet has fallen where ρa is thedensity of air and g is the acceleration due to gravity. By solving Eqs. N(R(t = 0)), initially in the volumecan be obtained. The evolution in the total number of particles for each size class isthen directly given by h(R(t), t) toalways be smaller than the system height hsysin which dispersion experiments are made and the computational domain height in which thesedimentation and evaporation of the droplets are calculated. The total number of dropletsin the system, Ntotal, at any timet is then the discrete summation of this number over all particlesizes, n, for whichh(R(t), t) <hsys.Due to the fact that the evaporation occurs quickly, the dominant mode of decline insuspended droplets is sedimentation. As we will show below, the exponential decay in thenumber of drops that we observe can be quantitatively accounted for by taking only thesedimentation of already evaporated droplets into account. At all times, the droplets areassumed to be vertically falling at their terminal velocities described by Stokesflow,No of drops with diameterD, and in view of the experimental results, it is reasonable to assumethat the decrease in time will be exponential: α being an empirical constant independent of the droplet diameterD. A good estimate is α ≅ρg/18ηh, with h being a typicalsedimentation height. The life time of a micro-droplet is then characterized by theexponent in Eq. tlife ≡1/αD2.In case of droplets with a varying size distribution, we collect the different dropletsizes and obtain 3 unventilated space that could represent, e.g., a restroom. The dropproduction by coughing was found to be very similar for six out of the seven emitters. Wefind peak values of 1.18 ± 0.09 × 103 pixels that light up in the field of viewof our laser sheet (21 × 31 cm2). This directly corresponds to the volume ofemitted droplets;4 lit up pixels, more than an order of magnitudelarger. This then allows us to estimate how many virus particles one would inhale while inside aroom where an infected person coughed a single time. The highest probability of infectionoccurs when a person enters a poorly ventilated and small space where a high emitter hasjust coughed and inhales virus-carrying droplets. We model coughing in our 2 × 2 × 2m6 copies/ml ofsaliva.3  and a normal respiratory rate of ∼16 inhalations/min.Based on these numbers and the earlier measured volume and drop size, we can calculatethe amount of virus inhaled by a person entering and staying in the same room where aninfected person produced the droplets as a function of entrance delay and residence time.As detailed above, the calculation assumes a viral load of 7 × 10Ninf, needs to be considered to translatethese findings into risk of infection. This obviously also depends on factors such as thevulnerability/susceptibility of the host; in addition, as detailed in Ref. Ninf ∼ 100–1000, which corresponds to ∼10PFU to 100 PFU where PFU denotes plaque forming units, the standard way of expressingwhether a virus is infectious or not.Ninf ∼ 100), we findthat our unventilated 2 × 2 × 2 m3 space contaminated by a single cough isrelatively safe for residing times less than 12 min due to the low virus content of theaerosol particles. Additionally, the maximal number of inhaled viral copies by a personentering the room after the high emitter has coughed is ∼120 ± 60, where the error margincomes from variation in relative volume of small and large drops produced by a cough. Ifthe infected person is a regular emitter, the probability of infecting the next visitor ofthe confined space by means of a single cough for any delay or residence time is thereforerather low. For speech, due to the low volumes emitted, this probability is even smaller.Nevertheless, prolonged speaking produces very large numbers of aerosols that could resultin droplet accumulation to levels far higher than that in coughing or sneezing, therebyleading to an increased risk. Our small non-ventilated room can be looked upon as a“worst-case”: in better ventilated, large rooms, aerosols become diluted veryrapidly.The number of virus particles needed to infect a single individual,Our dynamic modeling of transmission of SARS-CoV-2 in confined spaces suggests that aerosoltransmission is not a very efficient route, in particular from non-symptomatic or mildlysymptomatic individuals that are likely to have low virus content in their saliva. Highlyinfected people having a large viral load in their saliva and superspreaders producing lotsof aerosols are likely far more dangerous. Comparing aerosol transmission to othertransmission routes, it is useful to realize that the large droplets that are believed to beresponsible for direct and nosocomial infections may contain about 500 virus particles perdroplet and are thus likely to also be very important in a mixed transmission model.A limitation to our study is that we cannot easily take changes in virus viability insidemicrodroplets into account, which depend on the local microenvironment of the aerosol gasclouds as produced under different circumstances.The data that support the findings of this study are available from the correspondingauthor upon reasonable request. Part of this study included measurements from subjects,which were approved by a local ethical committee (AMC2020_098/NL73585.018.20).


Betacoronavirus) in Cynopterus brachyotis, commonly known as the lesser dog-faced fruit bat. Using a capture-enrichment sequencing platform, the full-length genome of the bat CoV was sequenced and found to be closely related to the bat coronavirus HKU9 species found in Leschenault’s rousette discovered in the Guangdong and Yunnan provinces.Bats are important reservoirs and vectors in the transmission of emerging infectious diseases. Many highly pathogenic viruses such as SARS-CoV and rabies-related lyssaviruses have crossed species barriers to infect humans and other animals. In this study we monitored the major roost sites of bats in Singapore, and performed surveillance for zoonotic pathogens in these bats. Screening of guano samples collected during the survey uncovered a bat coronavirus ( Infectious diseases continue to be a major threat to modern society and coronaviruses (CoVs) are one of the most notable virus families responsible for recent, highly pathogenic viral disease outbreaks. Over the last two decades, major outbreaks of deadly CoVs have been reported in humans and livestock, including severe acute respiratory syndrome (SARS) in 2003 , Middle Nidovirales, and the subfamily Orthocoronavirinae can be divided into four genera, Alphacoronavirus, Betacoronavirus, Deltacoronavirus and Gammacoronavirus, based on antigenic reactivity and/or genome sequences . CoVs in general can cause disease in a variety of domestic and wild animals, as well as in humans, whereas alpha- and betacoronaviruses predominantly infect mammals. SARS-CoV and MERS-CoV belong to the genus Betacoronavirus, under the subgenera Sarbecovirus and Merbecovirus, respectively  and three major roost sites were also identified  for periodic guano sampling. During sampling, groundsheets were placed under the roosting bats and guano samples were collected from the groundsheets and put into universal transport media.In addition, opportunistic rectal swabs were collected from bats that were accidentally caught in mist nets during the NParks bird-ringing programme or provided by the Wildlife Reserve Singapore (WRS). Any bat carcasses found in a nature reserve (SBWR) or donated by pest control companies were sent to the laboratory for testing. Guano samples and rectal swabs were placed in universal transport media and bat carcasses were kept refrigerated before processing in the laboratory. Bat carcasses were swabbed in the oral and rectal cavities before dissection. Tissues  were then harvested.The geographical distribution of bat roosts in Singapore was plotted in QGIS 3.2.3  using the basemap of Singapore downloaded from Onemap . Project coordinate system WGS 84 projection (EPSG:4326) was used for plotting.A total of 107 guano samples, 14 rectal swabs and 6 bat carcasses, collected over a period of 3 years, were screened for the presence of CoVs , JapanesThe primer sequences for the detection of coronavirus, JEV , rabies-2. MERS-CoV (accession number: NC_019843) was propagated and amplified in Vero B4 cells in DMEM supplemented with 5 % FBS at 37°C and 5 % CO2. PaKi cells C. brachyrus HKU9  and dilirus HKU9 .As for any probe-based enrichment NGS strategy, our probe library is designed to detect both known CoVs and novel CoVs genetically related to known members of the family. We believe that the same approach is equally applicable to both outbreak responses and active surveillance programmes, such as the US PREDICT project  and the Click here for additional data file.


Polymyxa betae. Although BNYVV is considered the causal agent of rhizomania, additional viruses frequently accompany BNYVV in diseased roots. In an effort to better understand the virus cohort present in sugar beet roots exhibiting rhizomania disease symptoms, five independent RNA samples prepared from diseased beet seedlings reared in a greenhouse or from field-grown adult sugar beet plants and enriched for virus particles were subjected to RNAseq. In all but a healthy control sample, the technique was successful at identifying BNYVV and provided sequence reads of sufficient quantity and overlap to assemble > 98% of the published genome of the virus. Utilizing the derived consensus sequence of BNYVV, infectious RNA was produced from cDNA clones of RNAs 1 and 2. The approach also enabled the detection of beet soilborne mosaic virus (BSBMV), beet soilborne virus (BSBV), beet black scorch virus (BBSV), and beet virus Q (BVQ), with near-complete genome assembly afforded to BSBMV and BBSV. In one field sample, a novel virus sequence of 3682 nt was assembled with significant sequence similarity and open reading frame (ORF) organization to members within the subgenus Alphanecrovirus . Construction of a DNA clone based on this sequence led to the production of the novel RNA genome in vitro that was capable of inducing local lesion formation on leaves of Chenopodium quinoa. Additionally, two previously unreported satellite viruses were revealed in the study; one possessing weak similarity to satellite maize white line mosaic virus and a second possessing moderate similarity to satellite tobacco necrosis virus C. Taken together, the approach provides an efficient pipeline to characterize variation in the BNYVV genome and to document the presence of other viruses potentially associated with disease severity or the ability to overcome resistance genes used for sugar beet rhizomania disease management.“Rhizomania” of sugar beet is a soilborne disease complex comprised of beet necrotic yellow vein virus (BNYVV) and its plasmodiophorid vector,  The increasing globalization of food and other commodities has resulted in greater exposure of crops to historical and emerging pests and diseases, including viruses ,2. WithiBenyviridae, genus Benyvirus). Like most plant viruses, BNYVV is a positive-sense RNA virus. The genome of BNYVV is divided among 4 or 5 segments (designated RNAs 1 to 5), the roles of which, during infection, have been the source of intense investigation over the past 30 years [p25 gene on RNA 3, implicated in the breaking of monogenic resistance provided by the Rz1 gene [Polymyxa betae, plays a role in disease aggressiveness, and the degree to which other known, and possibly unknown, viruses in the sugar beet root infection court positively or negatively impact disease.Sugar beet contributes nearly half of the sucrose produced and consumed within the United States of America (USA) . “Rhizom30 years ,10,11,1230 years . Recent Rz1 gene ,15,16,17Rapid and accurate genetic analysis of known and potential pathogens is a fundamental goal of disease management programs as the genetic constitution of a pathogen population may be crucial in determining their disease-causing potential. The advent of “next-generation sequencing” (NGS) technologies for DNA and RNA analysis, combined with the increasing power of computational platforms, has removed a long-standing roadblock in rapid, cost-effective, population-scale sequence generation and genome examination ,19. In cIn the present study, samples from diseased sugar beet seedlings and adult plants were enriched for viruses and subjected to RNAseq analyses. Reads were assembled to produce genomic segments, or in some cases, open reading frames (ORFs), which were used to identify potential relatedness of the viral genomes obtained to existing viral sequences in public databases. Based on the derived BNYVV sequence of RNAs 1 and 2 and that of a previously uncharacterized necrovirus, infectious cDNA clones were developed, validating the integrity of the approach. With the additional discovery of potentially novel satellite viruses, the results confirm the usefulness of the method in assessing the spectrum of viruses present in sugar beet plants exhibiting rhizomania disease.Three soil samples (S1–S3) and one root sample (S6) were obtained from the sugar beet production areas of the Red River Valley of North Dakota and Minnesota and southern Minnesota by agriculturalists of the Min-Dak Farmers Cooperative  and Southern Minnesota Beet Sugar Cooperative  in 2018 . Two soiTo recover BNYVV from infested soil samples, we followed the methodology described by Weiland et al. . Briefly4 pH 5.2 (3 mL buffer per g fresh weight of tissue). Each homogenized sample was transferred as 1.0 mL aliquots to several 1.5 mL microcentrifuge tubes, each containing 0.4 mL of chloroform, and the contents mixed well and then centrifuged at 16,000× g at 4 °C for 18 min. Supernatants were transferred to new tubes, adjusted to 1% NaCl and 8% PEG 8000, and incubated on ice for 10 min. Following a centrifugation step for 18 min at 16,000× g at 4 °C, the supernatant was removed and discarded. The whitish pellet was resuspended in 0.4 mL of cold 0.1 M NaPO4 pH 5.2 and the sample extracted with 0.1 mL of chloroform. The aqueous phase from the extraction was precipitated once again by the addition of NaCl to 1% and PEG 8000 to 8%, and samples were incubated on ice overnight. The following day, the samples were centrifuged at 4 °C for 18 min at 16,000× g. Care was taken to remove to the extent possible all PEG supernatant from the small pellet. RNA was extracted from the pellet, as previously described in Weiland and Edwards [Efforts were made to enrich for virus particles using standard PEG precipitation of crude extracts . Briefly Edwards . The intRNAseq libraries (150 bp insert size) were prepared and sequenced (pair-end 100 bp) by BGI Americas  or Admera  using the Illumina Highseq 4000 sequencing platform. Customized bioinformatics was also provided by each company. Briefly, low-quality reads and adaptor sequences were removed. For each sample, short reads were de novo assembled with different k-mer sizes in parallel. Reads were subsequently mapped back to the assembled contigs for validation. The best assembly was chosen based on contig N50 and mapping rate. Standard BLAST queries were used to verify or postulate the identification of novel viruses discovered in this work and for confirming the accuracy and completeness of genome assemblies.To identify known sugar beet viruses, high-quality filtered reads from each sample were mapped to reference genome sequences of BNYVV (GeneBank assembly accession: GCF_000854885.1), BSBMV (GeneBank assembly accession: GCA_002867265.1) and BBSV (GeneBank assembly accession: GCF_000855285.1). Mapping was done using default parameters (except length fraction = 0.8) in CLC genomics workbench v 8.0 . Any reads (paired- and single-end reads) mapped to a given viral genome were extracted for de novo assembly in CLC genomics using default parameters . A near-complete genome assembly of BNYVV, BSBMV, and BSBV was obtained from the majority of the samples. Assembly of sequences from Sample 3 also suggested the presence of a novel virus with sequence characteristics of plant Alphanecroviruses and potential satellite viruses.https://blast.ncbi.nlm.nih.gov) searched against publicly available BNYVV sequences to inspect the percentage identity (homology) between them. Nucleic acid consensus sequences for BNYVV isolates collected as part of this work were designated according to sample number. Assembled sequences of RNAs 1 to 4 of BNYVV were used to infer ORF locations. The analysis of sequence relatedness between RNAs 1 and 2 from this study  and those in GenBank was performed using BLAST. Due to the larger number and wider geographic distribution of available sequences for BNYVV RNAs 3 and 4, genes p25 and p31  were analyzed. Multiple sequence alignment of p25 sequences from 52 strains and for p31 from 46 strains were carried out using MUSCLE v3.8.31 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC390337/). The phylogenetic relationships were inferred using Randomized Axelerated Maximum Likelihood (RAxML) v8.2.9 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3998144/). Our RAxML analysis utilized rapid bootstrap analysis to search for the best-scoring ML tree with the number of bootstrap iterations determined at runtime using the extended majority-rule consensus tree criterion , and the “GTRGAMMA” model of nucleotide substitutions. The best-scoring ML tree for each gene was visualized in FigTree v1.4.4 (https://github.com/rambaut/figtree). Orthologous genes from BSBMV were tested for inclusion in the analysis to serve as an outgroup, but the sequences were too divergent, a similar conclusion as previously reported [The de novo generated BNYVV RNA sequences were BLASTn (BamHI-T7 promoter (25 nt) and polyA60-HindIII-BamHI (72 nt) sequences were incorporated on the 5′ end of the first fragment and the 3′ end of the fourth fragment, respectively, by PCR. Subsequently, these amplicons were used as templates in overlap extension PCR (BamHI restrictions sites. The full-length BNYVV RNA 2 cDNA clone (4610 nt genome) was synthesized by Genewiz and delivered in a pSMART-BAC  vector flanked by two BamHI restriction sites. The RNA 1 and RNA 2 clones were linearized by BamHI and HindIII restriction enzymes, respectively, and used to produce capped, polyadenlylated RNA using methods previously described [Chenopodium quinoa followed the procedure of Petty et al. [Based on the data obtained in the present work from RNAseq and conserved sequences at the 5′- and 3′-termini in BNYVV genomes from across the globe (Table 2), clones of RNA 1 and 2 were designed for construction. For BNYVV RNA 1 cDNA, four synthetic overlapping fragments were generated by Genewiz  and delivered as discrete fragments in vector pUC57, which were used as a template to produce four PCR amplicons  conditions, using primers indicated below, were as outlined in Edwards et al. . AmplifiEco R1 restriction enzyme and transcribed in vitro, as described previously, for the generation of infectious RNA of the Betanecrovirus BBSV [C. quinoa with the synthetic RNA derived from clones pBvANV #7 and 10 also followed the methods of Weiland et al. [C. quinoa leaves employed the same methods as noted above for the detection of BNYVV CP but using a TNV-A detection kit .Full-length clone construction for the novel Alphanecrovirus was initiated by generation of a genome-length amplicon using primers MDB-2460 and MDB-2462  in whichrus BBSV . Inoculad et al. . ELISA aThe use of polyethylene glycol in virus precipitation has been employed in virus purification for decades  and provApproximately 60,000,000 RNAseq reads were obtained per sample . Using BAnalysis of the BNYVV genomes obtained in the study indicated low-level variation in the sequence of RNAs 3 and 4 from different sampling locations, but no significant variation between sampling sites for RNAs 1 and 2. The most variable region of the BNYVV genome worldwide is the noted “tetrad region” encoded on RNA3 between nucleotides 645 and 656 . Application of RNAseq to the enriched Sample S3 resulted in greater than 50% of the total reads being assigned to this RNA. Agarose gel electrophoresis of the 1.2 kb RNA suggested that it was a mixture of two isoforms, an observation corroborated by RNAseq reads that were able to be assembled into two related variants , ection”; , our stuTombusviridae for its replication [Third, the validity of the approach was illustrated by the discovery of a potential novel virus of sugar beet along with a novel satellite virus. The presence of small satellite viruses in sugar beet had not been reported in the USA prior to this study and was unexpected. Analysis of the putative satellite virus genome revealed two variants of a closely-related sequence, both encoding proteins with similarity to themselves and with weak similarity to that encoded by satellite MWLMV, a satellite virus requiring a member of the lication . The RNATombusviridae. The sequence revealed features suggestive of a recombinant virus with OMMV/OLV-1 and TNV-A as donor parent viruses (C. quinoa (Alphanecrovirus within the family Tombusviridae, which we propose to be named BvANV-1. Although Liu et al. [Further strengthening the validity of this approach in novel virus discovery in sugar beet, the sample in which the satellite virus was present also harbored a previously undocumented variant sequence of TNV-A, possessing a genome organization and gene similarities aligning it with plant Alphanecroviruses within the family  viruses . The ORF viruses . Since R viruses  yielded . quinoa . As the u et al.  previousA second satellite virus detected in Sample S4 in this study was more closely related to satellite tobacco necrosis virus C, an entity likely associated with helper virus TNV-D or a related Betanecrovirus. Interestingly, Sample S4 harbors BBSV, a well-documented member of that virus subgenus, the US isolate of which was characterized in our laboratory previously ,32. AlthThe NGS approach for detecting known and novel viruses described here and elsewhere provides clear advantages over prior methods of assessment. With reduced bias in the viral genomes targeted, the technique allows for the detection of the presence of unanticipated viruses as compared to more common cloning and sequencing strategies. Moreover, sequence reads for all viruses present are generated simultaneously instead of detecting them within separate, sequential analyses. Standard BLAST alignment can then be employed to detect both known and novel viruses, and more sophisticated applications can detect novel virus agents based on likelihood analysis . Neverth


Trichoplusia ni  were treated with the biopesticide AfMNPV baculovirus in a dosage response assay. Treated soybean had, on average, a 6-fold increase in virus activity compared with the other crops. Leaf trichomes on soybeans were not found to be responsible for the observed increase of insecticidal activity. Three flavonoid compounds  were uniquely found only in the soybean crop, and were not detected in cotton, cabbage, or green bean plant matter. The individual flavonoid compounds did not cause T ni. mortality in no-virus assays when incorporated into artificial insect diet. The combination of the three flavonoid compounds at leaf level concentrations significantly increased baculovirus activity in diet incorporation assays. When the daidzein, genistein, and kaempferol were added to artificial diet, at 3.5–6.5 × leaf level concentrations, virus activity increased 1.5, 2.3, and 4.2-fold for each respective flavonoid. The soybean flavonoid compounds were found to synergistically improve baculovirus activity against T. ni.Four crop plants known to be hosts for the lepidopteran  The development of microbial-based biopesticides represents the augmentation of biological control for insect pests of plants and is increasingly viable as a commercial control tactic. The use of naturally occurring biological control agents allows growers to precisely target a pest population2. Research on this topic often focuses on the interaction specifically between the pathogen and target insect, such as the control efficacy provided by the application of a biopesticide. However, plants primarily protect themselves through the production of chemical defense compounds, which may alter aspects of this pest/pathogen interaction3. In a natural forest ecosystem, plant chemistries contribute to complex interactions depending on many parameters of the particular ecosystem, such as the density of oak trees in a forest4. Although comparatively less complex, reports of interactions among crop, pest, and pathogen vary for monoculture cropping systems. The susceptibility of Helicoverpa zea to Helicoverpa zea nucleopolyhedrovirus was found to be greater when fed on soybean rather than cotton, however, no specific plant chemistry was identified5. In okra and tomato, induced plant defense chemistries are correlated with reduced baculovirus infection of the lepidopteron Heliothis virescens (Fabricius), however induced systemic acquired resistance in cotton foliage had no effect on baculovirus infection of the lepidopteron Helicoverpa armigera (Hübner)7.Biological control has been defined as the use of predators, parasites, or pathogens to manage crop pests in a highly specific and environmentally friendly methodAnticarsia gemmatalis Hübner9. Another baculovirus isolated from Anagrapha falcifera Kirby,  can also be used as a biological control of key lepidopteran pests10. During preliminary research on formulation development with AfMNPV, we observed that larval mortality of lepidopteran Trichoplusia ni Hübner was substantially and unexpectedly high on soybean leaves treated with only AfMNPV, as compared with larvae feeding on virus treated cotton, cabbage, and the closely related green bean. This observation prompted evaluations to determine whether differences in plant morphology or chemistry significantly interacted with baculovirus infection.A naturally occurring baculovirus was isolated and incorporated in an integrated pest management system in Brazil, which successfully controlled the soybean defoliating lepidopteran velvet bean caterpillar, T. ni when baculovirus was applied to soybeans by (1) determining the comparative AfMNPV activity against T. ni for treatments applied to soybean, cotton, cabbage, and green bean, (2) determine whether physical leaf structures such as trichomes (pubescence) affect insecticidal activity of the virus, (3) determine flavonoid composition of leaf tissues, (4) determine whether identified flavonoids affect AfMNPV activity against T. ni in flavonoid-diet assays.The objective of this investigation was to determine the cause of increased viral activity against Genistein (98%), kaempferol (99%), trifluroacetic acid (99%), and formic acid (98%) were purchased from Sigma, St. Louis, MO. Daidzein (98%) was purchased from Organic Technologies, Coshocton, OH. Dimethyl sulfoxide , methanol (99.8%), trifluroacetic acid was purchased from ThermoFisher, Waltman, MA. Deionized (DI) water was used for the preparation of all diets.T. ni (Hübner) (Lepidoptera: Noctuidae) from a colony maintained on an artificial diet  at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL (NCAUR), were used for all bioassays. Standard protocol for preparing two liters of GPLD diet consisted of autoclaving 38 g of agar  in 1.760 L of DI water at 250 °C for 20 minutes. The liquid agar was blended with 288 g of GPLD powder in a blender  for approximately 5 minutes. Diet mixtures of GPLD were stirred by a IKA RW 20 digital stand mixer  for approximately 2 minutes before dispensing about 3.3 mL of the molten diet treatment into each cup . The insects used to start this colony were obtained from USDA-ARS, Biological Control Insect Research Laboratory, Columbia, MO before 1995.Neonate A. falcifera (Kirby) (Lepidoptera: Noctuidae), commonly known as celery looper. Subsequent baculovirus stock was produced in vivo at the USDA NCAUR via cabbage loopers following the modified procedures outlined in Behle et al.11. Briefly, AFMNPV was propagated by infecting third instar T. ni and harvesting larval corpses. Collected larvae were mixed with 0.5% SDS, homogenized, and filtered through cheese cloth. The filtrate was then centrifuged at 5000 rpm (5 °C) for 10 min, the supernatant was removed, and the pellet was resuspended in de-ionized water. Virus stock was then frozen for storage. The baculovirus stock utilized in this experiment was produced in May 2017 and contained 4.8 × 108 occlusion bodies per milliliter (OB mL−1), determined microscopically using a hemacytometer. Baculovirus stock was frozen for long term storage and refrigerated at 5 °C for short term storage (up to 4 weeks).Stocks of the baculovirus provided by Thermo Trilogy  were originally isolated from Glycine max: Soybean, (Proharvest A15U B146P), Phaseolus vulgaris: green bean (‘Savannah’), Gossypium hirsutum: cotton (‘DES 607’), and Brassica oleracea: cabbage (‘Bravo F1’) were grown in a greenhouse for approximately 5 weeks in 110 mm square plastic pots . The potting mix used in the greenhouse was Redi-Earth  fertilized with Osmocote Classic (14-14-14) and Micromax Micronutrients  per packaging recommendations.Treatments were applied to plants with a research track sprayer  through a spray jet 80015 nozzle  programmed to deliver 36 mL of each treatment per pass at 179 kPa, equivalent to 340 L/Ha. The plants were positioned 50 cm below the nozzle and centered in the spray chamber.T. ni  were treated with AfMNPV in a dosage response assay. Five or more virus concentrations were prepared by 1:3 serial dilutions with water for application through the spray chamber as described above. Virus concentrations were mixed to provide application rates between 1.3 × 109 and 1.0 × 1012 OB Ha−1. Rates applied to soybean required lower virus application rates compared with the other crops to in an effort to center the response near 50% larval mortality. After treatment, the spray residue was allowed to dry before five circular disks were cut from treated leaves for each crop by application rate combination. Leaf disks were placed individually in petri dishes with about 20 neonates, and sealed petri dishes were incubated in the dark at 28 °C for a 24 h feeding period. After feeding, six live larvae were randomly selected from each leaf disk and transferred to individual cups with GPLD for a total of 30 larvae crop × application rate combination. These insects were incubated for an additional six days, then evaluated for mortality. A control (no virus application) was included for each crop to assess handling mortality, which typically averaged less than 10%. Experiments were replicated three or more times on different days for a minimum of 90 larvae exposed for each crop × application rate combination. Insect mortality data were subjected to probit analysis  to determine LC50 values .This experiment was conducted to evaluate the impact of leaf pubescence on the activity of the virus treatments applied to soybean plants by comparing shaved with un-shaved plants. Shaved plants were prepared by carefully removing the leaf pubescence from the upper leaf surface with a razor blade just prior to virus treatment. Following the techniques described above, greenhouse grown soybean plants (shaved and un-shaved) were treated with either a water control or a single LCLeaf tissue was excised, and sputter coated with Au particles: cotton, cabbage, green bean, and soybean (with and without trichomes) were analyzed. The coated leaf tissue was then examined using a JEOL 6400 V scanning electron microscope to evaluate differences in trichome density .Greenhouse grown plant leaf samples were freeze-dried and subsequently ground to a fine powder. Weighed samples of dried leaf powder  were placed in a vial and extracted with 3 mL of a methanol:DMSO (1:1) solvent. The samples were typically sonicated for 30 minutes and allowed to stand overnight at room temperature. The extract was filtered through a 0.45 µM nylon 66 filter for HPLC analysis.et al., 2018 and Riddick et al., 2018, on a Shimadzu LC-20 HPLC system 14. The column used was an Inertsil ODS-3 reverse phase C-18 column . For phenolic compound analysis, the initial conditions were 20% methanol with 0.25% trifluroacetic acid and 80% water with 0.25% trifluroacetic acid, at a flow rate of 1 mL min−1. The column was held at the initial conditions for 2 minutes, then developed to 100% methanol with 0.25% trifluroacetic acid in a linear gradient over 55 minutes. Five-point standard curves for the evaluation of the concentration of the identified flavonoids in soybean leaves were prepared with commercially purchased kaempferol, genistein, and daidzein for the determination of extinction coefficients at 280 and 340 nm.HPLC analysis was conducted following the protocol described in Dowd et al., 2018 and Riddick et al., 2018, leaf extraction samples were run on an Thermo Electron LTQ Orbitrap Discovery Mass Spectrometer–a linear ion trap (LTQ XL) MS, coupled to a high precision electrostatic ion trap (Orbitrap) MS with a high energy collision (HCD) cell with an Ion Max electrospray ionization (ESI) source, and a Thermo Scientific ACCELA series HPLC system  all running under Thermo Scientific Xcalibur 2.1.0.1140 LC-MS software 14. For phenolic analysis, the initial solvent system was 10% methanol verses water with 0.25% formic acid at a flow rate of 0.25 mL per minute. After injection the column was developed with a linear gradient to 100% methanol over 50 to 60 min. The software package was set to collect mass data between 100–2000 AMUs. Generally, the most significant sample ions generated under these conditions were [M-1]− and [M + HCOO]−.Following the protocol described in Dowd Six mass spec “events” were programed to run in sequence in the MS detection scheme. (1) LTQ(IT)-MS full scan m/z 150 to 2000. (2) LTQ(IT)-MS set to trap the most abundant ion above a threshold of 500 units and perform CID at 35% energy, with the resulting ions being detected by the IT-MS. 3) FT-MS (Orbitrap) full scan m/z 150 to 2000. (4) Mass-dependent MS/MS on the most abundant ion trapped by the IT-MS in Event 1 and perform HCD at 25% energy with the resulting fragmentation ions being detected by the FT-MS. (5) Mass-dependent MS3 on the most abundant fragment ion generated from Event 2 and perform HCD at 25% energy with the resulting fragmentation ions being detected by FT-MS. (6) Mass-dependent MS3 on the most abundant fragmentation ion generated from Event 2 and perform CID at 35% energy with the resulting ions being detected by IT-MS.For the evaluation of Xcalibur accurate mass data by the Cerno BioScience LLC MassWorks 5.0.0.0 software the FTMS was set to collect spectra at a resolution of 7500 and a range of m/z of 100 to 2000 and then were evaluated by self-Calibrating Line-shape Isotope Profile Search, which enhances formula ID accuracy.15. Two controls were used for the virus overlay experiment, a no additive control and a 500 µL DMSO solvent control. The no additive control treatment was made with an additional 500 µL of water. Prepared diet treatments were cooled at room temperature to solidify and stored in refrigeration at 5 °C until use.Artificial insect diet treatments containing genistein, daidzein and kaempferol were prepared to compare the effect of three flavonoids identified above on the insecticidal activity of the baculovirus. Treatments were prepared by incorporating the flavonoids into the insect diet using the same GPLD diet used for colony maintenance. Six diet treatments were prepared by mixing flavonoids in 100 mL insect diet at concentrations intended to approximate the dry weight concentrations of the flavonoids found in the soybean leaves Table . Geniste4 OB mL−1 to 4.1 × 102 OB mL−1 concentrations. The stock virus preparation was counted under a microscope using a hemocytometer to verify the virus concentration. Each of the baculovirus concentrations plus a water control were applied to a set of 5 cups  for each flavonoid incorporated diet preparation. Baculovirus treatments (0.1 mL per cup) were spread along the surface of the diet with a glass stir rod. After drying, approximately 12 T. ni neonates were added to each cup, and the cups were incubated for 24 hours in a dark incubator at 28 °C. To mimic the leaf assay above, larvae were allowed to feed for 24 hours, then 6 larvae from each cup were transferred individually to fresh cups containing GPLD, without the flavonoids, providing 30 insects exposed to each flavonoid × virus concentration. These larvae were incubated at 28 °C for 6 days, then evaluated for mortality. Each experiment was replicated at least 3 times on different days, using replicated diet preparations and different cohorts of insects. To determine whether flavonoid interactions were synergistic or additive the following equation was used with dosage response data (Eq. 1):18.Insecticidal activity for the baculovirus in the presence of the flavonoids was evaluated by a dosage-response assay using a virus overlay exposure technique. Diet incorporated with genistein, kaempferol, daidzein, and a mixture of the three flavonoids  were surface treated with 5 concentrations of baculovirus. Five concentrations of baculovirus were made by 1:3 serial dilutions to provide 3.3 × 10A second experiment, following the method stated above for virus overlay of incorporated diet, was conducted to evaluate the effect of higher concentrations of the individual flavonoids. The amounts of the flavonoids added to 100 mL GPLD were: 0.0186 g daidzein, 0.0836 g kaempferol, and 0.0186 g genistein. Experiments were repeated 3 times on different days as before.T. ni larvae19, virus LC50 rates were observed to vary widely across multiple plant species. Applications of AfMNPV were found to be more active when applied to soybean plants as compared to applications on cabbage, cotton, or green bean plants. The observed difference among these crop plants suggested that a component of the soybean plants may increase T. ni susceptibility to baculovirus or may enhance the baculovirus activity in some capacity. Thoroughly replicated experiments revealed that the Af baculovirus treatments were far more toxic to larvae that fed on treated soybeans as compared with the other three crops  genistein, kaempferol, and daidzein.This observed difference in activity among crop plants suggested a potential interaction between leaf morphology or chemical composition which was synergistic with Each crop had differing degrees of leaf pubescence, cabbage lacking any and soybean having the most pubescence among the species tested Fig. . To dete−1 dry leaf, respectively . The highest LC50 value  was measured for the GPLD diet control preparation, which was not significantly different from the DMSO or individual flavonoid treatments.Incorporating the three flavonoids into the artificial insect diet was intended to mimic the crop leaves and overlaying the surface of the diet with a virus treatment mimicked a spray application. Flavonoids were incorporated into the diet at the dry weight concentrations found in the soybean leaves. The virus free flavonoid diets did not cause a significant increase in larval mortality; all virus free control diets had <5% morality. Adding the flavonoids to the diet did significantly increase the insecticidal activity of the virus, but only for the combination treatment Table . The LC550 = 628 OB mL−1) of the virus 4.2 × compared with diet alone (LC50 = 2668 OB mL−1) or 3.9 × DMSO diet controls (LC50 = 2431 OB mL−1). The high concentration of genistein (estimated at 6.5 × greater than leaf concentrations) improved virus potency about 2.3 × compared to the control diet, with an LC50 at 1196 OB mL−1. The high concentration of daidzein (estimated at 6.5 × greater than leaf concentrations) improved the insecticidal activity of the virus 1.5 × compared to the control diet, with an LC50 at 1757 OB mL−1. High doses of daidzein, kaempferol, and genistein were found to synergistically improve virus activity, with co-toxicity factors of approximately 20, 60, and 150, respectively. The virus free high concentration flavonoid diets did not cause a significant increase in larval mortality; all virus free control diets had <5% morality.Incorporating higher concentrations of genistein, daidzein, and kaempferol into insect diet improved the insecticidal activity of the virus overlay treatment Table . The higT. ni. Of the four crop species tested, soybean not only enhanced viral activity, but also had the greatest amount of leaf pubescence (trichomes); cabbage was entirely glabrous, or lacking trichomes  increasing resistance to insect attack. For T. ni, it had been previously observed that the presence of trichomes on soybean leaves decreased feeding and increased mortality with trichome density; the removal of the trichomes on the youngest fully expanded leaves resulted in a significant increase in T. ni feeding22. In this experiment, T. ni feeding on the youngest fully expanded soybean leaves did not display any increased mortality when compared between shaved and unshaved leaves without a virus treatment . Additionally, there was no significant differences in viral activity  in the T. ni fed on shaved vs. unshaved leaves that were treated with virus (pr > t = 0.52). Therefore, the trichomes of soybean do not play a significant role in the increased activity of AfMNPV against T. ni.AfMNPV treated soybean had, on average, a 6-fold increase in virus activity compared with the other crops. Three flavonoid compounds  were uniquely found only in the soybean crop, and were not detected in cotton, cabbage, or green bean plant matter. The soybean flavonoid compounds were found to synergistically improve baculovirus activity against mes Fig. . Leaf pu23. As such, analytical efforts concentrated on defining the amount and types of flavonoids present in soybean and either not present, or to a reduced extent, in the other crops. When comparing the flavonoid composition of the four crop species, three flavonoid compounds were produced only by soybean: genistein, kaempferol, and daidzein.After determining trichomes did not affect virus activity, the chemical composition of each crop was analyzed particularly looking for differences in flavonoid concentration. Flavonoid compounds are known to be involved in numerous biochemical processes including defense functions against insect pests26. Kaempferol is a flavonoid which falls into the subcategory of flavonol, and is also found to be abundant in soybean leaves27. However, green bean (Phaseolus vulgaris L.) had no detectible genistein, kaempferol, or daidzein, despite being a legume in the same family as soybean (Fabaceae). Isoflavonoids are often produced by plants in response to biotic stress and can be utilized to inhibit pathogen growth and help protect plant tissues from insect herbivory30. Flavonoids play a highly complex role in plant-insect interactions that goes beyond simple broad spectrum defense mechanisms and are often highly species specific23. Furthermore, these compounds are involved in a myriad of biochemical functions including inducers of nodule formation and symbiosis between soybean roots and bacteria31.The isoflavonoids genistein and daidzein are typically abundant in soybean and other legumes and are of great dietary interest as bioactive agentsAs these compounds were only found in soybean, each was selected and incorporated into artificial diet. At leaf level concentrations, none of these compounds interacted with the virus treatment to improve insecticidal activity. However, the combination treatment had a significantly greater virus activity. This observation may have been the result of the overall increase in flavonoid content in the combination treatment. Experiments using higher concentrations of the genistein and kaempferol also improved virus activity, supporting the theory that the observed affect was concentration dependent.32. Genistein and daidzein vary only in their number of hydroxyl groups, while kaempferol has additional hydroxyls and the second phenyl ring attached at the ether carbon of the heterocyclic ring  has been observed to be anti-apoptotic, inhibiting reactive oxygen species production, chronic inflammation, auto-aggressive immune responses, and cell apoptosis (except in osteoclasts) in mammalsT. ni. Genistein and kaempferol did increase virus activity (2.3–4.2 ×) at higher concentrations of the individual flavonoid compounds and had a synergistic interaction with the baculovirus. The smaller increase in virus activity observed in the daidzein diets (1.5 ×) may be due to daidzein having the lowest anti-oxidant and phytoestergenic activity, as compared with genistein and kaempferol42. Daidzein can often produce distinctly different physiological and cell signaling responses despite a minor structural difference with genistein44.The combined leaf level concentrations of flavonoids had a significantly increased lethal dose response (approximately 2-fold), but only partially accounts for the 6-fold increase in virus activity observed for soybean. Increasing the concentrations of the flavonoids genistein and kaempferol well above the physiological concentrations in the artificial diet resulted in increased virus activity. Importantly, even at the highest concentrations of flavonoid incorporation there was no increase in larval mortality in the no-virus controls. Therefore, the flavonoids are not acting as broad-spectrum defense compounds against the Further studies are underway to compare various soybean cultivars and identify potential genotypic variability. Comparisons will be performed to further elucidate the soybean components responsible for the synergistic increase in baculovirus activity. The significant interactions between plant flavonoids, entomopathogenic baculovirus, and increased insecticidal activity suggests a potential plant breeding objective to improve plant insect resistance concurrent with an integrated pest management system.


Sulfolobus Spindle-shaped Virus (SSV) system has become a model for studying thermophilic virus biology, including archaeal host-virus interactions and biogeography. Several factors make the SSV system amenable to studying archaeal genetic mechanisms  as well as virus-host interactions in high temperature acidic environments. Previously, we reported that SSVs exhibited differential infectivity on allopatric vs. sympatric hosts. We also noticed a wide host range for virus strain SSV9 . For decades, SSVs have been described as “non-lytic” double-stranded DNA viruses that infect species of the genus Sulfolobus and release virions via budding rather than host lysis. In this study, we show that SSVs infect hosts representing more than one genus of the family Sulfolobaceae in spot-on-lawn “halo” assays and in liquid culture infection assays. Growth curve analyses support the hypothesis that SSV9 virion release causes cell lysis. While SSV9 appears to lyse allopatric hosts, on a single sympatric host, SSV9 exhibits canonical non-lytic viral release historically reported SSVs. Therefore, the nature of SSV9 lytic-like behavior may be driven by allopatric evolution. The SSV9-infected host growth profile does not appear to be driven by multiplicity of infection (MOI). Greater stability of SSV9 vs. other SSVs  in high temperature, low pH environments may contribute to higher transmission rates. However, neither higher transmission rate nor relative virulence in SSV9 infection seems to alter replication profile in susceptible hosts. Although it is known that CRISPR-Cas systems offer protection against viral infection in prokaryotes, CRISPRS are not reported to be a determinant of virus replication strategy. The mechanisms underlying SSV9 lytic-like behavior remain unknown and are the subject of ongoing investigations. These results suggest that genetic elements, potentially resulting from allopatric evolution, mediate distinct virus-host growth profiles of specific SSV-host strain pairings.The  Reduced model viral systems have been used extensively to study fundamental properties of virus evolution  system has become a popular model for studying thermophilic archaeal virus biology and virus-host biogeography. Several factors make this system ideal for studying virus-host infections in crenarchaea . First, endemic populations of SSV hosts from the family Sulfolobaceae exhibit biogeographic structure such that there is a positive correlation between genetic distance among strains  and geographic distance among various sites from which strains have been isolated  shaped viruses  rather than the gross lysis of host cells and cell death in liquid culture or clear plaques on host lawns—both of which result from lytic replication. Recently, it was suggested that SSV9 can induce a state of dormancy, empty cells, and eventual host death in a sympatric host; however, the mechanism for this proposed dormancy is unclear and there does not seem to be any cell lysis , often featuring diffuse boundaries  Kamchatka, Russia  in contrast to the turbid diffusely-bound halos characteristic of all other SSVs  form turbid areas of growth inhibition or Sulfolobaceae, host growth profiles in single-virus/single-host infection assays using three distinct SSVs  were evaluated. Liquid culture and spot-on-lawn “halo” assays were used to verify productive infection by a given SSV. To assess the relative susceptibility of different hosts to a given SSV and to elucidate conspicuous differences in host growth profiles during the infection, both allopatric and sympatric hosts were infected. Each specific virus-host pairing was conducted under different culture conditions to evaluate the impacts of distinct conditions on growth dynamics. For example, different multiplicities of infection (MOI) were tested and end-point assays (in liquid culture) were conducted to measure virus and host dynamics in parallel. In addition to evaluating halo “phenotypes” in plate-based assays, small-scale liquid culture assays were conducted to gauge the relative amount of cellular debris emerging from different virus-host pairings. Assays to assess the relative stability of different SSVs under conditions that simulate the natural Sulfolobales habitats were also performed to determine if virion stability plays a role in successful transmission and between strain differences in virion production.To test the hypothesis that SSV9 lyses susceptible hosts within the family Sulfolobus strains stored at −80°C were partially thawed on ice. For most trials, 100 μL of infected cell suspension were added to 30 mL of YS media (pH 3.2) in a 125 mL Erlenmeyer flask  between 0.4 and 0.6, 3.0 mL of cell suspension was used to inoculate 600 mL of pre-heated fresh media in a 1.0 L baffled flask.Once liquid culture reached an optical density  to pellet the cell mass while leaving virus in suspension. The supernatant was decanted and filtered through a 0.45 μm vacuum PES filter system. The filtrate was concentrated using 10 kDa Centricon Plus-70 spin concentration tubes  to produce ~3.0 mL concentrated SSV suspension.These cultures were incubated at 78°C/70 RPM until reaching an ODspot-on-lawn “halo” assays and liquid culture infection assays within 2–3 weeks of harvesting. Dilutions of viral stocks were used as inocula.TEM was used to confirm the presence of virions and virus particle count was measured using electrospray ionization/mass spectrometry or serial dilution halo assays (see below). Virus was stored at 4°C and used in Sulfolobus strains stored at −80°C were used to establish 30 mL cultures in YS (or T or TS) media, as described above. Uninfected Sulfolobus cultures were either used to prepare host lawns for halo assays  per protocols in Wick and McCubbin . A mixtuThis specialized ESI/MS is designed to detect intact virus particles , thus, a discrepancy is expected. In some cases, SSVs exhibited dual peaks or shouldered peaks in the ESI/MS analyses. Such spectral features may be due to different conformations of the same SSV or, potentially, due to the presence of pleomorphic SSVs. Under ESI/MS, SSVs exhibit characteristic peaks between 46.1 and 61.5 nm for purified virus suspensions with major peaks typically appearing at 46.1, 47.8, and 49.6 within the ±4 nm system tolerance.600 = 0.4–0.6, 500 μL of cell suspension were added to 4.5 mL of a 78°C mixture of equal parts 1.0% w/v Gelrite®  and 2-fold concentrated YS medium. The 5 mL mixture was spread on pre-warmed Gelrite® plates (1% w/v in medium) and allowed to solidify for 15 min at room temperature followed by a 20 min incubation at 78°C. 1.0 μL of each viral suspension was spotted onto the prepared plate in labeled areas. Depending upon host strain, plates were incubated for 3–9 days at 78°C. Successful infection was scored by the formation of a visible halo of growth inhibition on the host lawn. Triton X-100 (0.05% v/v) was used as a positive control and sterile water was used as a negative control. Initial tests also used a 2 μL spotting of ultrafiltrate (10 kDa MWCO) to ensure that effects were not from other proteinaceous toxins  but from SSV infection.Halo assays were performed  for various time intervals. Virus was then harvested as detailed above and ESI was used for virus particle counts. Growth curves from SSV-Sulfolobus cultures were fit to Logistic, modified Logistic , Gompertz, modified Gompertz , and other growth models  = ex, where e is Euler's number. A represents the amplitude or peak growth value in the given environment, which corresponds to stationary phase and the maximum carrying capacity; r is the intrinsic growth rate coefficient; y0 is the initial population size; μ is the maximum slope of the growth curve, and t is time. For equations in which the maximum specific growth rate (μmax) was not a fit parameter; it was calculated by taking the derivative of y(t) evaluated at a software-derived point of maximum growth using parameter estimates for A, r, and y0.3.0 mL of uninfected t = 0) to the time-point delineating the end of the exponential growth phase of the uninfected host growth curve . This process was used for each data set resulting in successful fits to Equations (1)–(4) (above) as well as other growth models (not shown here). The second method for calculating AUC values was through Riemann sums, also known as the trapezoidal approximation method  parameter, which undermines the robustness of the AUC value as a unifying metric of growth curve changes.Parameter estimates were derived in R 3.6.1  using curve fitting functions of easynls, growthcurver, and grofit packages :−t/τ represents the exponential decay of the oscillation amplitude whereby A is the driving amplitude and 1/τ is a decay rate constant. In the second term, ω is the frequency variable. The average growth curve of SSV9-infected cultures was fit to a damped sinusoidal function simply because this function best described the cyclical bursts in growth that had increasing smaller amplitude and which were each followed by rapid declines in host population . However, for SSV9 replication, a Gaussian function was used to fit the dataset.tc is the time at which the center of the peak occurs; and, w represents the width of the peak function at one-half the peak amplitude. The “goodness-of-fit” of these data sets  to the sinusoidal and Gaussian, respectively, prompted their use. For the sinusoidal fit, the adjusted r2 value was 0.90  of the vp1 gene fragment (138-bp) cloned into a TOPO TA pCR2.1 plasmid (Invitrogen). Copy number in each standard was calculated using formula:NA is Avogadro's number, and 1 × 109 is used to convert units to nanograms. Each qPCR reaction was performed on a Rotor-Gene Q  and consisted of 1× Green/ROX qPCR Master Mix , 3 pmol of each primer, 1 uL of DNA template, and nuclease-free water to a final volume of 20 mL. Three technical replicates were performed of each standard and samples. The qPCR cycle parameters were as follows: 98°C for 2 min, followed by 40 cycles of 98°C for 5 s and 20 s at 60°C. A melt curve analysis was performed after each run from 65 to 95°C in 0.5°C increments at 2 s intervals, to ensure specific amplification of a single target, and no primer dimer formation. The qPCR amplicons were resolved in 2% agarose gels to assess amplicon sizes for target-specific amplification. Reaction mixtures with sterile water (no-template DNA) or pCR2.1 DNA without insert served as a negative controls to control for false positives. Assays showed amplification efficiencies of 100% ± 10% , consistency across replicate reactions, and linear standard curves (R2 > 0.970). The absolute quantification method was used to calculate the number of viral genomes per mL of culture.SYBR green-based qPCR was used to quantify the number of SSV9 genomes for inocula and end-point infection. Two PCR primers were employed: SSV9F (5′-GTGAAGCGACCAACATAGGTGCAA-3′) and SSV9R (5′-GTTGCGTTTGTACCGGTTACGCTA-3′)—targeting the single-copy gene −1); serial dilution plaque-like assays in units of halo-forming units per milliliter (hfu mL−1); virus-like particle (VLP) counts using transmission electron micrographs (VLP mL−1); and/or, quantitative polymerase chain reaction (qPCR) in terms of the total number of viral genomes per milliliter (vg mL−1). For infection assays, end-point titers were also determined by one or more of these methods. ESI/MS and plate assays provided the most consistent results.To ensure that virus infection was a cause of host growth decline, presence of virus was determined by one (or more) of four distinct methods: electrospray ionization/mass spectrometry (ESI/MS) in units of virus particles per milliliter  resulted in comparable virion yields as shown by electrospray ionization/mass spectrometry (ESI/MS) spectra . Verification of the presence of SSV virions in virus suspensions was accomplished by transmission electron microscopy  verification that a given SSV strain is able to infect a specific strain of the family Sulfolobaceae  across multiple susceptible hosts.Sulfolobus strain Gθ  is slowed and the peak cell density upon entering the stationary phase  is reduced compared to uninfected control (max and area-under-the curve (AUC) when compared to controls. Despite SSV2 and SSV3 infections exhibiting slightly different host growth profiles (VR) between strains on a given host was generated.Liquid culture assays using SSV1, SSV2, SSV3, SSV8, and SSV10 infecting the universally susceptible host strain  control . Individprofiles  with higprofiles . Measureprofiles  for the profiles . Using t2 values for the Gompertz of 0.9875 and 0.9775, respectively. Employing Riemann Sums or alternative models  does not significantly alter relative virulence (VR) for SSV1 and SSV8 . Therefore, in subsequent trials, SSV1 and SSV8 were chosen as comparators for SSV9.Using a Gompertz model and AUC as a measure of percent inhibition (PI), SSV1 exhibits the lower PI (8.57%) while SSV8 has a significantly higher PI (28.44%) with RSSV8 see . RegardlSulfolobus strain Gθ infected with SSV9 did not grow with Gompertzian-like ; S444 ; and, S437 . With the exception of SSV1 infection of strain HVE 10/4, all host growth curves show significantly reduced Nasymp, AUC, and/or μmax for SSV1 and SSV8 when compared to uninfected controls. However, when each host was infected with SSV9, a distinct host growth profile emerged. Specifically, Gompertzian Models failed to adequately represent the SSV9 infection data. Instead, non-Gompertzian cyclical spike patterns are consistently observed in host growth. Low R2 values (<0.5) emerged for both individual traces and averaged growth curves for SSV9  were typical for all other SSVs infecting these same hosts , SSV8 , and SSV9  were used to infect susceptible hosts: S200  is observed for all SSV8 infections on s of MOI . A posits of MOI . AmplituSulfolobus strain Gθ infected with either SSV8 or SSV9 at MOI = 1  at higher observation frequency (every 4 h) were conducted with  MOI = 1 . Two fla MOI = 1  through SSV9-infected Sulfolobus strain Gθ showed a distinct pattern from the Gθ-SSV8 infection. SSV9-infected growth peaked (18 HPI) followed by a rapid drop in cell density . The decIt was also noted that the growth inhibition did not result in an optical density value of zero. Instead, cell density stabilized at approximately one-third the average peak value. Furthermore, SSV9 titer (via ESI/MS) decreased substantially see .To determine if SSV9 lytic replication was dependent upon host strain allopatry, two isolates of Sulfolobales derived from volcanic hot springs in Kamchatka, Russia were also tested as plausible hosts for SSV9.9 VP mL−1 indicating productive infection (Liquid culture infection assays confirm that strain MU is resistant to infection  and thatnfection . The mosnfection .R2 = 0.934), indicating that SSV9 exhibits a non-lytic replication profile on a sympatric host, Sulfolobus sp. strain GV , while the GV-SSV9 trial shows a significant PI (>0.20) with a convincing Gompertz fit  characteristic of other SSVs.Previously published work demonstrated that some Sulfolobales are completely resistant to infection by well-characterized SSVs  while others are susceptible to subset or all of these SSVs in Growth curve analysis provides insights into the nature of virus-host infection dynamics that are not resolvable through plate-based halo assays. Using host growth curve analyses, it is not only possible to determine percent inhibition and relative virulence of a different SSVs on a host see  but it iAlthough it can be argued that a highly unstable virus strain could rapidly breakdown yielding a false negative for infection, TEM see , 3 and sVR) by calculating percent inhibition (PI) of each SSV on a given host strain .Archetypal SSV infection see  induces (not shown), the curve still fit a Gompertz Model with high coefficient of determination (R2 = 0.87) and did not show a cyclic growth profile. Furthermore, virus particle count at the end of a 20:1 viral concentrate trial (not shown) was 2-fold greater than the initial inoculum, indicating that the depression in the growth curve was not due to lysis-from-without  allow replicates to be harvested periodically during the course of infection to monitor changes in virus titer in parallel to host growth . Samplinalso reported in Quemin et al., Another report suggests that group II chaperonin complexes may form highly stable networks of chaperonin complex filaments at the intracellular surface of membrane  within the same area —were challenged with SSV9, unanticipated dynamics emerged.Although SSV9 lytic behavior does not appear to be dependent upon MOI and seems to be an inherent property of the SSV9 replication strategy, all susceptible hosts initially tested were quasi-sympatric strain MU was not susceptible to SSV9 in spot-on-lawn assays. Moreover, in liquid culture assays with strain MU, there were no significant differences in Nasymptote, μmax, or AUC between the SSV9-infected and MU uninfected controls . The full impact of this taxonomic reorganization, especially with regard to biogeography and physiological differences is being assessed. Interestingly, Sulfurisphaera tokodaii , there is also the question of whether a switch in replication strategy over evolutionary timescales  is part of a more generalizable pattern of attenuation in the virus-host coevolutionary “arms race” . CD performed the plate assays and conducted the SSV thermal stability and adsorption studies, and also did the virus titering using serial dilution plaque assays. CS and RC fit the data to mathematical models and determined NThe 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.