How to prevent the flushing-induced plume without changing people’s daily habits?Enlightened by thoughts of redesigning the restroom, this article provides a redesignedtoilet using liquid-curtain-based strategy and verifies its advantages from thecomputational fluid dynamics. Two favorable effects are spotted: (1) the liquid curtaincan suppress the upward virus particles (only 1% viruses can be lifted out of the toilet)and (2) the flow distribution caused by the liquid curtain can deliver virus particlesinto the sewage efficiently. In December 2019, COVID-19 pandemic first broke out in Wuhan, China. COVID-19 is caused by ahighly infectious novel coronavirus “SARS-CoV-2.”Putting the toilet lid down before flushing can be an effective method to prevent the plumecaused by the flushing of a toilet. However, a male urinal does not have a lid or otherbarriers which are able to prevent the virus transmission, and people do not always have thehabit of putting the toilet lid down before flushing. Therefore, “how to preventflushing-induced virus transmission without changing people’s daily habits?” is an urgentpublic concern that needs to be responded to. Enlightened by the report regarding rethinkingthe design of restrooms,supplementary material for the reason to choose this velocity). For detailedmodel establishment, simulation assumption, and boundary conditions, refer to our previouswork.supplementarymaterial for the time-varying distribution of pressure for another explanation ofrestraining the plume.In conclusion, a redesigned toilet equipped with a liquid-curtain can effectively impedeupward movement of the virus particle. Two favorable effects are identified: (1) the ejectedhorizontal liquid-curtain can suppress the upward movement of the particles; (2) the downwardvelocity distribution caused by the curtain impact can deliver these particles into the sewagepipe and wash them away. Numerical data show only 1% particles can be lifted out of theredesigned toilet. In addition, such a liquid-curtain-based strategy can also be applied tomale urinals to prevent virus transmission.supplementary material for reasons to select the velocity of inlet 2 (2.6m/s) and time-varying distribution of pressure during flushing with the liquidcurtain.See the The data that support the findings of this study are available from the correspondingauthor upon reasonable request.


The National Civil Protection Directorate promptly implemented strict measures to limit the virus transmission, which have decreased the number of daily new cases from almost 100 at the peak of the epidemic to below 10. Bringing the epidemic under control, which means that the basic reproduction number (R0) is below 1, transmission is controlled, and there is an ample number of beds and respirators available in hospitals, created the conditions for the restrictions to be eased. Although this has brought relief to the population and the hard-hit economy, it is impossible to predict further developments beyond the devastating effect on the economy. The efforts of countless laboratories around the world have resulted in an unprecedented accumulation of knowledge on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Yet, many aspects of the virus biology, transmission, and treatment remain elusive.Since the outbreak of atypical pneumonia cases in the Wuhan region, China, in late 2019, the virus causing what is now called COVID-19 has rapidly spread around the globe. As of late May 2020, more than 5.5 million people have been infected and almost 350 000 have died. The first case of infection in Croatia was identified on February 25, 2020 . In addition, we retrieved partial sequences, covering 50% to 84% of the viral genome, for the remaining 14 samples, which can be used for partial genomic analyses. The main question is what the virus genome sequences can be used for. There are several important applications.Briefly, we obtained 33 RNAs from the Institute of Public Health in Rijeka collected as part of routine diagnostics between March 22 and April 10, 2020. In all samples, SARS-CoV-2 had been confirmed by RT-qPCR (generously provided by Neven Sučić) before sequencing. The samples of adequate quality  were used for next-generation-sequencing (NGS) on an Illumina platform  at Ruđer Bošković Institute. Our NGS approach was based on an in-house designed panel, consisting of 173 amplicons, covering the complete SARS-CoV-2 genome. We obtained high-quality whole genome sequences for seven samples, with average sequence coverage between 6000 and 12 000 × . The sequences were instantly deposited in GISAID, the main SARS-CoV-2 genomes open-source database  carrying specific mutations. These are named clade S , G , and V . Six of our genomes belong to the clade G, and one genome is still under investigation. Nonetheless, our preliminary results indicate multiple virus entries and, as expected, close similarity to the viruses sequenced in Italy and Austria (Third, there are many reasons to further monitor the virus mutation dynamics. For example, some virus regions mutate at a significantly higher rate than others, indicating a strong selective pressure. Indeed, the greatest number of mutations has been identified within the S gene, the main viral antigen. This is relevant for the selection of antigens or novel drug targets in vaccine and treatment development, respectively. The virus could also possibly show a population-specific adaption, which is immensely interesting but has to be confirmed by large virus genome studies . Tracingin vitro, showing that some mutations might impact the virulence of SARS-CoV-2 (Forth, the COVID-19 pandemic is fraught with contradictions. On one hand, older age and chronic/pre-existing medical conditions are well-documented risks for the disease severity, and on the other, some younger, previously healthy, individuals develop severe illness and even die. In addition, a significant number of individuals infected with SARS-CoV-2 with mild symptoms or no symptoms at all were identified – ie, seemingly healthy people may inadvertently spread the disease. Such puzzling contradictions might be explained by differences in the virulence and pathogenicity of the circulating strains, and thus, it is mandatory to delve into the core of the viral genome to the last nucleotide to confidently answer these questions. Indeed, recently, Yao et al have investigated a number of patient-derived isolates RS-CoV-2 .In summary, the COVID-19 pandemic is currently waning in many European countries, including Croatia. Hopefully, the rest of the world will follow the same trend. Many lessons have been learned, and by far more wait to be addressed. Thus, a thorough analysis of global measures to mitigate the pandemic will hopefully help to create advanced procedures to tackle future challenges. We strongly believe that our effort in designing the sequencing platform will contribute to a better preparedness for a possible second SARS-CoV-2 wave or a similar disaster.Acknowledgment We thank Filip Rokić, Lovro Trgovec-Greif, and Neven Sučić for the preparation of samples and sequencing analysis, and overall contribution to the project.


Liao ning virus (LNV) is a member of the genus Seadornavirus, family Reoviridae and has been isolated from kinds of vectors in Asia and Australia. However, there are no systematic studies describe the molecular genetic evolution and migration of LNVs. With the development of bioinformatics, viral genetic data combining the information of virus isolation time and locations could be integrated to infer the virus evolution and spread in nature.Here, a phylogenetic and phylogeographic analysis using Bayesian Markov chain Monte Carlo simulations was conducted on the LNVs isolated from a variety of vectors during 1990–2014 to identify the evolution and migration patterns of LNVs.The results demonstrated that the LNV could be divided into 3 genotypes, of which genotype 1 mainly composed of LNVs isolated from Australia during 1990 to 2014 and the original LNV strain (LNV-NE97–31) isolated from Liaoning province in northern China in 1997, genotype 2 comprised of the isolates all from Xinjiang province in western China and genotype 3 consisted the isolates from Qinghai and Shanxi province of central China. LNVs emerged about 272 years ago and gradually evolved into three lineages in the order genotype 1, genotype 2 and genotype 3. Following phylogeographic analysis, it shows genotype 1 LNVs transmitted from Australia  to Liaoning province  in Northeast Asian continent then further spread across the central part of China to western China .LNVs were initially isolated from Liaoning province of China in the Northeast Asia, however, the present study revealed that LNVs were first appeared in Australia in the South Pacific region and transmitted to mainland China then rapidly spread across China and evolved three different genotypes. The above results suggested that LNV had the characteristics of long-distance transmission and there were great genetic diversity existed in the LNV population. Notably, current information of 80 strains of LNVs are limited. It is of great importance to strengthen the surveillance of LNVs to explore its real origin in nature and monitoring of the LNVs’ population variation and maintain vigilance to avoid LNV breaking through the species barrier and further clarify its relationship to human and animal infection. Liao ning virus (LNV) belongs to the genus Seadornavirus of the family Reoviridae [Seadornavirus comprises 3 species, Banna virus (BAV), Kadipiro virus (KDV) and LNV [Seadornaviruses have a genome composed of 12 segments of double-stranded RNA, which decreases with the relative molecular weight during gel electrophoresis, and is named 1–12 segments [oviridae . The gen and LNV –3. The SSeadornaviruses could be carried by a variety of blood-sucking insects including mosquitoes, ticks, and midges and even have been isolated from pigs, cattle and patients with fever and encephalitis [Seadornavirus, is an emerging pathogen that causes human viral encephalitis [Seadornaviruses may well be a group of newly discovered viruses close related to human and animal diseases [It is reported that the phalitis –10. Studphalitis . Therefodiseases , 4.Aedes dorsalis) in Liaoning province, northeast China in 1997 [culex and Aedes) in Shanxi province and Qinghai province in China [Culex, Anopheles, Mansonia and Aedes) in Australia in 2016 [Seadornaviruses  [LNV was initially isolated from mosquitoes  .Japanese encephalitis virus (JEV) [Zika virus (ZIKV) [Middle East respiratory syndrome coronavirus (MERS-CoV) [Ebola virus (EBOV) [Previous researches have reported on the molecular genetic evolution of LNV isolated in China and Australia , 7, 11. us (JEV) , 17,idens (ZIKV) , inferenERS-CoV)  and Ebols (EBOV) . TherefoCulex (n = 64), genus Aedes (n = 10), genus Ochlerotatus (n = 3),unidentified mosquitoes (n = 3). The LNV isolation sites included the Liaoning province, Shanxi province, Qinghai province and Xinjiang province in China as well as New South Wales, Northern Territory, Queensland and Western Australia in Australia. The geographical distribution extends from Australia  to mainland China   and was indicated as effective sample size (ESS>200), and the maximum clade credibility (MCC) tree was constructed using TreeAnnotator (http://beast.community/treeannotator) with 10% burn-in.The 10th segment sequence database of LNV was analyzed using Bayesian Markov chain Monte Carlo (MCMC) method . The GTRIn order to clarify the geographical dispersal history of LNVs, the bayesian stochastic search variable selection (BSSVS) was used to provide evidence for statistically supported diffusion between state variables under BEAST software package . This meSequence similarity and nucleotide base composition analysis were performed using the CLUSTALX software , BioEditThe crystal structure of the cell attachment protein VP9 of BAV (PDB 1w9z) was selected as the best template for the homology modeling. The YASARA software , PyMol s− 3 s/s/y   approach Fig. . The posThe skyline plot of the LNV population dynamics is shown in Fig. The Sequence analyses of 80 strains of LNV isolated from different vectors and locations during 1990–2014 revealed that the level of nucleotide similarity varied from 70.4 to 100% with an average value of μ = 92.6% while the amino acid similarity ranged from 75.0 to 100% with an average value μ = 94.6%  between the three genotypes of LNV, we analyzed the amino acids encoded by the VP10 genes derived from the previously mentioned LNV isolates. The results revealed that the ORF of VP10 was 753 nucleotides in length and encoded 250 amino acids. The amino acid sequences of genotype 1 were significantly different from those of the rest two genotypes while the genotype 2 and 3 shared a great similarity in amino acid sequences. The detailed results were that a total of 20 common amino acid differences identified between genotype 1 to genotype 2 and 3 , while the remaining thirteen were located in the loop region . These differences have some effect on the structure and electrostatic properties of VP10 between the different genotypes. In particular, the residue at position 64, 65, 131, 132, 167, 171, 207, 209 and 219.There were great differences existed the R group of the amino acid and the electrical polarity of the mentioned 9 mutations, resulting in significant differences in structure and charge at these sites between the LNVs  [Culex, Anopheles, Mansonia and Aedes) collected from 1988 to 2014 in Australia in the southern hemisphere. The isolation sites included New South Wales, Northern Territory, Queensland and Western Australia, almost across the entire Australian continent. Thus, it is obvious that the geographical distribution and the vectors of LNV are wide and variable in Australia. What is more, the initial LNV isolate in Australian was obtained from mosquitoes collected in 1988, predating the first Chinese LNV isolate which was obtained in 1997 [During the arboviruses survey in China in 1997, two virus strains (LNSV-NE9731 and LNV-NE9712) were obtained from us (LNV) . Since tus (LNV) . A more  in 1997 , 11.Several phylogenetic analysis have been conducted on the LNV’s nucleotide sequences, previously. The LNVs isolated in China could be divided into 3 evolutionary branches. The LNSV-NE9731 strain isolated in Liaoning province in 1997 formed an independent evolutionary branch while another Liaoning isolates named LNSV-NE9712 clustered together with the isolates from Qinghai province and Shanxi province that formed a branch. All of the Xinjiang isolates grouped together formed the largest evolutionary branch , 33. Allseadornaviruses BAV, whose tMRCA is 105 years ago [The time-scaled evolutionary analysis of LNV revealed the branching of the lineages occurred in the following order:genotype 1 emerged 73 years ago , the genotype 2 at 46.5 years ago  and genotype 3 at 25.3 years ago  Thus, genotype 1 is the oldest lineage and the genotype 3 is the youngest one , West Nile virus (WNV), yellow fever virus (YFV) and JEV [Seadornavirus, was also observed a higher AU content [Human Immunodeficiency virus (HIV) to avoid recognition by the innate immune system of host cell [Additionally, there was a very interesting finding that the AU content was relative high in VP10 of LNV, whereas the AU content was also high within the LNV’s whole viral genome (data not shown). These results were consistent with the prior studies wherein A and U frequencies were higher than C and G frequencies for avian  and JEV –39. Besi content . It has ost cell . HoweverThis may be the reasons why LNVs have been continuously isolated from 12 species of mosquitoes belonging to four genera in different geographical and climatic regions in Australia, during the last 30 years , 12. HowAedes aegypti of African origin, reminding us that LNVs was not only limited to China in Asia and Australia in the South Pacific region but may well be extended to Africa and even posing a high risk of spreading to new areas such as Central Asia and Europe. It is well known that genetic mutation such as recombination or reassortment can easily occur in segmented RNA virus. For example, BAV which also belongs to the Seadornavirus, was originally discovered in southern China. However, several novel variants of BAV have been found, such as the BALV isolate from Hungary [Mangshi virus from southern China [Seadornavirus that can replicate in mammalian cell lines and cause fatal haemorrhagic symptoms in mice [In this study, the evolution analysis of LNVs revealed that the virus belongs to the emerging virus group. In particular, it was suggested that the genotype 1 LNVs were a group of segmented double-strand RNA virus with extremely adaptability, which have a large group of vectors, a high rate of genetic variation and apparent active transmissibility that can adapt to different geographical environments over a quite long distance. Recently, the genome information of LNV was reported to be detected in  Hungary  and the rn China . Conside in mice , it migh


The aim of this study was to determine prognostic factors for medical and productivity costs, and return to work (RTW) during the first two years after trauma in a clinical trauma population.2 (explained variance) and c-statistics (discrimination).This prospective multicentre observational study followed all adult trauma patients (≥18 years) admitted to a hospital in Noord-Brabant, the Netherlands from August 2015 through November 2016. Health care consumption, productivity loss and return to work were measured in questionnaires at 1 week, 1, 3, 6, 12 and 24 months after injury. Data was linked with hospital registries. Prognostic factors for medical costs and productivity costs were analysed with log-linked gamma generalized linear models. Prognostic factors for RTW were assessed with Cox proportional hazards model. The predictive ability of the models was assessed with McFadden RA total of 3785 trauma patients  responded to at least one follow-up questionnaire. Mean medical costs per patient  and mean productivity costs per patient  varied widely. Prognostic factors for high medical costs were higher age, female gender, spine injury, lower extremity injury, severe head injury, high injury severity, comorbidities, and pre-injury health status. Productivity costs were highest in males, and in patients with spinal cord injury, high injury severity, longer length of stay at the hospital and patients admitted to the ICU. Prognostic factors for RTW were high educational level, male gender, low injury severity, shorter length of stay at the hospital and absence of comorbidity.Productivity costs and RTW should be considered when assessing the economic impact of injury in addition to medical costs. Prognostic factors may assist in identifying high cost groups with potentially modifiable factors for targeted preventive interventions, hence reducing costs and increasing RTW rates. Trauma is considered an important public health problem. Almost 80,000 patients  were admitted to a hospital due to an injury in 2017 in the Netherlands ) with 95% Confidence Interval (CI) were reported for the three models.Prognostic factors for medical costs and productivity costs were assessed with log-linked gamma generalized linear models (GLM). The predictive ability was measured with explained variance . The period of absenteeism (weeks) was set as time variable and RTW  as the dependent variable. The proportional hazards assumption (the ratio of the hazards for patients was constant over time) was checked visually with Kaplan-Meier curves. Hazard Ratios (HR) and 95% CI were reported. The predictive ability of the model was assessed with the C-statistic (a measure of goodness of fit for binary outcomes). A p-value of <0.05 was considered statistically significant.. Responders had a mean age (SD) of 64.2 (18.9) years and 1911 (50.5%) were female . The median ISS (IQR) was 5 (4–9) and the median (IQR) length of stay at the hospital was 4 (2–8) days.A total of 3785 trauma patients  completed at least one follow-up questionnaire on health care use and RTW in the context of the BIOS-study Responders (n = 3785) were more often admitted to the intensive care unit (ICU), and were more severely injured, according to the ISS (median [IQR] 5 –9 and 5 Table 2), of which medical costs comprised 75% and productivity costs comprised 25%. Highest mean total costs and medical costs per patient based on injury classifications were found in patients with spinal cord injury , followed by patients with severe abdominal injury  . Patients with ISS>15 also showed high mean medical and high mean productivity costs per respondent .Mean total costs per respondent were €12,970   times higher in patients aged ≥75 compared to patients aged 18–44. Pelvic injury, tibia, complex foot or femur fracture, severe head injury, severe abdominal injury, spinal cord injury or stable vertebral fracture/disc injury were associated with increased medical costs. Besides, female gender, higher ISS and ≥2 comorbidities were prognostic factors for higher medical costs compared to male gender, low ISS and no comorbidities respectively. McFadden R2 of the multivariable model was 31,5%.Mean medical costs per patient were €9,710 . All variables showed significant associations with medical costs in the univariable GLM . Mean productivity costs were also high in patients who were admitted due to occupational injury  and in patients with ISS>15 .Average productivity costs per respondent were €9,000  within the working population. Highest mean productivity costs per patient were found in patients with spinal cord injury  and pelvic injury  . In contrast with the univariable GLM, lower age showed to be associated with higher productivity costs in the multivariable GLM. Prognostic factors for increased productivity costs were male gender, low educational level, stable vertebral fracture/disc injury, higher ISS, higher LOS, occupational injury and admission to the ICU in the multivariable GLM . McFadden R2 of the multivariable model was 19,8%.Number of comorbidities, pre-injury health status and age showed not to be associated with productivity costs in the univariable GLM . At 12 months after injury, 76% (n = 841) of the patients with known outcome returned to work and 6% (n = 69) did not return to work.Within the working age population  a total of 1,236 patients (63%) reported to have paid employment prior to injury, of which 131 patients (11%) had a missing value for time to event. For patients with known outcome (n = 1105), median survival weeks was 8 (IQR: 2.5–17.7). A total of 180 patients were censored due to loss to follow-up . Prognostic factors for RTW in the multivariable analyses were higher age , male gender , higher educational level  and patients with a sport injury  . Injury classifications with decreased probability on RTW were tibia, complex foot or femur fracture  and stable vertebral fracture/disc injury . Besides, higher ISS and longer LOS showed a decreased probability on RTW. C-statistic of the multivariable Cox-regression model was 0.700 .Age, gender, number of comorbidities and pre-injury health status showed no significant association with RTW in the univariable Cox regression analyses This study explored prognostic factors for medical costs, productivity costs and RTW after trauma during 24 months of follow-up. A stable vertebral fracture or disc injury and higher ISS were independently associated with higher medical costs, higher productivity costs and longer period of absenteeism at work. Although female gender and higher age were prognostic factors for higher medical costs, they were also both associated with lower productivity costs after adjustment for confounding. A total of 5% of the patients with paid employment did not return to work at 12 months after injury. Important prognostic factors for RTW were higher educational level, higher age, low ISS and low LOS.In line with previous studies, higher age and female sex were associated with higher medical costs,37. No cFemale sex is a prognostic factor for higher medical costs, but is also associated with lower productivity costs. The lower productivity costs might be explained by the fact that women more often work part-time compared to men in the Netherlands.A Dutch study on the economic burden of injury reported the highest health care costs for hip fracture patients 10. Although our study showed that hip fractures were in the top 5 of high medical costs , several other injury classifications were considerably more expensive. However, the prevalence of hip fractures in the injury cohort is high, so this could have more impact on health care consumption and costs compared to the relatively low prevalence of more costly injuries .A previous study on RTW in major trauma patients also found comorbidities, pre-injury disability, and presence of spinal cord injury as prognostic factors for no RTW . They alPrevious research found an association between unemployment and poor recovery ,42. ThisThe proportion of RTW in our study is higher compared to previous studies ,45. ThisA strength of this study is the large study population, including short-term (1 week) and long-term (two years). This study design allows to calculate costs and RTW in detail and could give an overview of costs and RTW in the total clinical trauma population. Furthermore, to our knowledge, this was the first attempt to develop prediction models for the total trauma population for both medical as productivity costs and also RTW. Productivity costs were based on the friction cost method. This method does not discriminate between patients who are 90 days absent at work or 2 years absent at work. Although there are no extra productivity costs, it does affect society and the patient. Therefore, a strength of this study is the addition of RTW (in days) up to 2 years after injury as outcome.An important limitation in this study is that educational level is not taken into account for the calculation of productivity costs. Although different average wages were used for males and females, no distinction was made between average wages of the different educational levels. The analyses showed that high educational level was a prognostic factor for RTW, indicating that patients with high educational level were sooner back at work after trauma compared to patients with low educational level. However, because the wages per hour were similar to patients with low educational level, the significant result of high educational level as prognostic factor for lower productivity costs probably only indicates this shorter period of absenteeism at work instead of actually lower productivity costs. In addition, the proportion of patients with low educational level was higher in our study population, compared to the Dutch working population (29% vs 20%) . This coIn addition, costs due to presenteeism were not taken into account, resulting in a probable underestimation of the total productivity costs. Future research should take into account both absenteeism and presenteeism.Another limitation of this study is the use of self-reported questionnaires which are prone to recall-bias. Patients were asked about care consumption and RTW over the period between the previous questionnaire and the current questionnaire. This period increased from 1 week for the first questionnaire to 12 months for the last follow-up questionnaire.Next, the primary aim of the BIOS-study is to determine physical and psychological outcome after injury. This means that only survivors of an injury were included in the BIOS-study. Patients who did not survive until one week after trauma were excluded and costs were not calculated .Furthermore, although the analyses were conducted in a large study population, only 39% of the total trauma population completed the necessary questionnaires. Patients who were fully recovered were probably more likely to be lost to follow-up. Differences between responders and non-responders indicate that more severely injured patients participated. This non-response bias could result in an overestimation of both medical and productivity costs.Last, injury severity in our study population was probably low in contrast to other trauma populations. However, in the Dutch trauma registry, all patients who were admitted to the hospital are included, regardless of the ISS. In contrast with the 5% severely injured in the total Dutch trauma population, our cohort included more severely injured patients (7%). CompareThe findings of this study showed high medical costs, high productivity costs and long absenteeism from work. An intervention to help patients to earlier return to work after sustaining a trauma could reduce productivity costs and decrease the period of absenteeism at work. Considering the differences in prognostic factors for medical costs and productivity costs, it is important that medical costs and productivity costs are both taken into account and analysed separately. The identified prognostic factors in this study were all easy to detect patient and injury characteristics. This enables health practitioners and policy makers to inform patients and induce prevention interventions to reduce medical costs and productivity costs.Although many prognostic factors resulted in both higher medical costs and higher productivity costs, some factors showed differential effects. Productivity costs and RTW should be considered when assessing the economic impact of injury in addition to medical costs. Prognostic factors may assist in identifying high cost groups with potentially modifiable factors for targeted preventive interventions, hence reducing costs and increasing RTW rates.S1 Fig(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Of the approximately 1,100 known plant viruses, about one-third are DNA viruses that are vectored by insects. Plant virus infections often induce cellular and molecular responses in their insect vectors, which can, in many cases, affect the spread of viruses. However, the mechanisms underlying vector responses that affect virus accumulation and transmission are poorly understood. Here, we examined the role of virus-induced apoptosis in the transmission of begomoviruses, a group of single-stranded plant DNA viruses that are transmitted by whiteflies and cause extensive damage to many crops worldwide. We demonstrated that virus infection can induce apoptosis in the insect vector conferring protection to the virions from degradation, leading to enhanced viral accumulation and transmission to host plants. Our findings provide valuable clues for designing new strategies to block the transmission of insect-vectored plant viruses, particularly plant DNA viruses. Begomovirus within the family Geminiviridae, which are transmitted by whiteflies of the Bemisia tabaci species complex in a persistent manner, we revealed that virus-induced apoptosis in insect vectors can facilitate viral accumulation and transmission. We found that infection with tomato yellow leaf curl virus activated the apoptosis pathway in B. tabaci. Suppressing apoptosis by inhibitors or silencing caspase-3 significantly reduced viral accumulation, while the activation of apoptosis increased viral accumulation in vivo. Moreover, the positive effect of whitefly apoptosis on virus accumulation and transmission was not due to its cross talk with the autophagy pathway that suppresses begomovirus infection in whiteflies. We further showed that viral replication, rather than the viral coat protein, is likely the critical factor in the activation of apoptosis by the virus. These novel findings indicate that similarly to many animal and a few plant RNA viruses, plant DNA viruses may activate apoptosis in their insect vectors leading to enhanced viral accumulation and transmission.Apoptosis is generally considered the first line of defense against viral infection. However, the role of apoptosis in the interactions between plant viruses and their insect vectors has rarely been investigated. By studying plant DNA viruses of the genus IMPORTANCE Of the approximately 1,100 known plant viruses, about one-third are DNA viruses that are vectored by insects. Plant virus infections often induce cellular and molecular responses in their insect vectors, which can, in many cases, affect the spread of viruses. However, the mechanisms underlying vector responses that affect virus accumulation and transmission are poorly understood. Here, we examined the role of virus-induced apoptosis in the transmission of begomoviruses, a group of single-stranded plant DNA viruses that are transmitted by whiteflies and cause extensive damage to many crops worldwide. We demonstrated that virus infection can induce apoptosis in the insect vector conferring protection to the virions from degradation, leading to enhanced viral accumulation and transmission to host plants. Our findings provide valuable clues for designing new strategies to block the transmission of insect-vectored plant viruses, particularly plant DNA viruses. Begomovirus, family Geminiviridae, infect hundreds of plant species, including many important crops, and cause devastating damage to agricultural production in tropical and subtropical regions  increased significantly in TYLCV-infected whiteflies  to verify our findings further, and the effector caspase-3 was taken as the candidate gene for silencing. After the whiteflies had fed on double-stranded RNA (dsRNA) for 2 days, their survival was not affected, while the expression of markably  and B. W reduced  and D, a reduced . Consistiteflies . Our datTo investigate the role of apoptosis in the transmission of TYLCV by whiteflies, we fed whiteflies on virus-infected tomato plants and then immediately treated them with either Z-DEVD-FMK or PAC-1. We monitored the viral retention in whiteflies after they had fed on either of the two chemicals. TYLCV infection was not markedly affected by suppressing or enhancing apoptosis at 0 day . HoweverTo investigate the key viral factor(s) that activates whitefly apoptosis, we infected whiteflies with another two begomoviruses: tomato yellow leaf curl China virus (TYLCCNV), which replicates in whiteflies after a short-interval virus acquisition by the insects on virus-infected tomato, and papaya leaf curl China virus , which does not replicate in whiteflies . The lev10.1128/mSystems.00433-20.3FIG S3FIG S3, TIF file, 0.8 MB.TYLCCNV-induced apoptosis in whitefly. (A) Detection of TYLCCNV CP and cleaved-caspase-3 using Western blot analysis in nonviruliferous whiteflies and whiteflies that fed on TYLCCNV-infected tomato plants for 24 h and then transferred to feed on cotton plants for 120 h. ACTB is the control for loading. (B) Detection of PalCuCNV CP and cleaved-caspase-3 using Western blot analysis in nonviruliferous whiteflies and whiteflies that fed on PalCuCNV-infected tomato for 24 h and then transferred to feed on cotton plants for 120 h. ACTB is the control for loading. Download Copyright © 2020 Wang et al.2020Wang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00433-20.4FIG S4t test). Download FIG S4, TIF file, 0.8 MB.Virus increase following induction of apoptosis in PalCuCNV-infection whiteflies. Whiteflies were placed to feed on PalCuCNV-infected tomato plants for 24 h for virus acquisition, treated with apoptosis inducer PAC-1 or untreated using artificial diet for 12 h, and then transferred to feed on cotton plants for 120 h before being collected for analysis. (A) Images of guts of whiteflies that were treated with the apoptosis inducer PAC-1 or untreated, dissected, fixed, and labeled with TUNEL to show apoptosis. For treated and untreated whiteflies, 10 midguts were dissected, respectively, and representative images are shown. Bar, 100 μm. (B) Proportions of apoptotic cells (number of TUNEL-positive cells/number of DAPI-positive cells) in midguts. (C) Immunoblot analysis of PalCuCNV CP in whiteflies treated with PAC-1 or untreated. ACTB is the control for loading in Western blots. (D) PalCuCNV genomic DNA measured by qPCR in nonviruliferous whiteflies and whiteflies treated with PAC-1. In panels B and D, data are mean ± SD, and different letters above the two columns in each of the diagrams indicate significant difference  were significantly lower in whiteflies infected by PalCuCNV and mPalCuCNV than in whiteflies infected by TYLCV or mTYLCV  showed no significant changes between the treated and control whiteflies   and examficantly  and H. Sficantly  and J, sficantly  and CP  of the Bemisia tabaci whitefly complex was reared on cotton plants (Gossypium hirsutum L. cv. Zhemian 1793) in insect-proof cages at 26 ± 1°C, 16 h of light, and 8 h of darkness. Cotton plants were sown into pots and were cultivated to the 7- to 8-true-leaf stage for experiments unless otherwise indicated.The cryptic species MEAM1 (mtAM282874.1), TYLCCNV isolate Y10 (GenBank accession no. AJ319675.1), and PalCuCNV isolate HeNZM1 (GenBank accession no. FN256260.1) were agroinoculated into tomato (Solanum lycopersicum L. cv. Hezuo903) at the 3- to 4-true-leaf stage as in our previous study . Total RNA was extracted from the whiteflies using the SV total RNA isolation system . RNA was reverse transcribed to cDNA using a cDNA synthesis kit . qPCR was performed on the CFX96 Real-Time PCR Detection System  with SYBR green detection . Primers used in this work are listed in 10.1128/mSystems.00433-20.5TABLE S1Table S1, DOCX file, 0.01 MB.qRT-PCR primers, related to Materials and Methods. Download Copyright © 2020 Wang et al.2020Wang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10) and TYLCCNV coat protein (8D10) were provided by the Institute of Biotechnology, Zhejiang University. Commercial antibodies to cleaved-caspase-3, ATG8, SQSTM1, cytochrome c, and ACTB (beta-actin) were purchased from Cell Signaling Technology .Samples were prepared using radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors and analyzed by Western blotting as previously described . Antibodin situ cell death detection kit . For apoptotic cell death analyses, 10 whitefly midguts and 10 salivary glands were dissected in TBS  and fixed in 4% paraformaldehyde for 2 h at room temperature. The tissues were blocked with 5% bovine serum albumin (BSA) in TBS with 0.1% Tween 20 (TBST) and then incubated with the TUNEL reagents (TdT enzyme/dUTP ratio = 1:10) for 6 h. After washing three times in TBS, the tissues were mounted in Fluoroshield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI)  and analyzed under a Zeiss LSM710 confocal microscope . The number of apoptotic cells  was counted, and experiments were performed in triplicate.TUNEL staining was performed using the 4 in phosphate buffer for 2 h. After dehydration using increasing ethanol levels, midguts were transferred to acetone for 20 min and then embedded in LX-112, and sections were stained with uranyl acetate and alkaline lead citrate. Images were observed under a Hitachi H-7650 TEM.For transmission electron microscopy (TEM) analyses, 200 midguts obtained from whiteflies were immediately fixed with 2.5% glutaraldehyde in phosphate buffer  for 4 h and then fixed with 1% OsOWhitefly guts were dissected in TBS  and fixed in 4% paraformaldehyde for 2 h at room temperature. Ten midguts were washed three times in TBS, blocked in 5% milk in TBST (TBS buffer with 0.05% Tween 20), and subsequently incubated with primary antibody. The guts were then washed twice in TBST and incubated with secondary antibody diluted in TBST for 2 h at room temperature. After washing three times in TBST, the samples were mounted in Fluoroshield mounting medium with DAPI  and imaged using a Zeiss LSM710 confocal microscope . All treatments were replicated three times.Apoptosis inhibitor Z-DEVD-FMK  and inducer PAC-1  were used to investigate the effect of apoptosis on TYLCV infection. The inhibitor and the inducer were dissolved in dimethyl sulfoxide (DMSO). First, 600 adult whiteflies at 3 to 5 days postemergence were fed on virus-infected tomato plants for 24 h to acquire the virus and divided into three groups to be fed with a diet of 10 μM Z-DEVD-FMK with 15% sucrose, 10 μM PAC-1 with 15% sucrose, and 15% sucrose containing 0.1% DMSO, respectively, in cylindrical containers for 12 h. Next, the three groups of whiteflies were transferred to feed on cotton plants for 120 h and then collected for Western blot analysis, immunofluorescence, or qPCR. All treatments were replicated 3 times.caspase-3 dsRNA was used as the treatment group and the sucrose diet containing 200 ng/μl dsgfp was used as a negative control. First, 400 adult whiteflies at 3 to 5 days postemergence were fed on virus-infected tomato plants for 24 h and then were fed with dscaspase-3 or dsgfp in cylindrical containers for 48 h, respectively. Subsequently, the two groups of whiteflies were transferred to feed on cotton plants for 120 h before being collected for Western blot analysis, immunofluorescence, and qPCR assays. All treatments were replicated 3 times.The dsRNA was synthesized using the AmpliScribe T7-Flash transcription kit  with specific primers. For RNA interference, the sucrose with 200 ng/μl dsFirst, 400 newly emerged (3 to 5 days old) whiteflies were fed on the mutant TYLCV or mutant PalCuCNV-infected tomato for 24 h and then transferred onto cotton plants for 120 h. Next, those viruliferous whiteflies were collected for Western blot analysis and immunofluorescence. All treatments were replicated 3 times. The two CP mutant viruses were prepared as described in a previous study , and a 1First, 600 adult whiteflies at 3 to 5 days postemergence were fed on the virus-infected tomato for 24 h to acquire the virus, and then the viruliferous whiteflies were collected into three groups of 200 each and fed in a rearing tube with a diet of 15% sucrose containing 10 μM Z-DEVD-FMK, 10 μM PAC-1, or DMSO for 12 h for the two treatments and control, respectively.To examine virus retention by whiteflies in each of the three treatments (2 treatments plus a control), female whiteflies of each of the three groups, as treated above, were immediately transferred to feed on a cotton plant. On 0, 5, and 10 days after transfer onto cotton, 25 whiteflies were collected from each of the three treatments, and their TYLCV genomic DNA was assessed individually using PCR.To examine TYLCV transmission by whiteflies in the three treatments, the three groups of whiteflies as treated above were transferred to feed on cotton plants for 120 h. Female whiteflies were then transferred and enclosed using clip cages to feed on the third leaf of an uninfected tomato plant at the 3-true-leaf stage for TYLCV transmission. For each of the three treatments, 25 viruliferous female whiteflies were inoculated individually to 25 plants. After 48 h, the whiteflies were removed, and the plants were sprayed with imidacloprid (50 mg/liter) to kill the eggs. After a further 25 days, the leaves of each tomato plant were collected and subjected to qPCR assays of virus infection and virus abundance in the plant as normalized with tomato actin.Apoptosis inhibitor Z-DEVD-FMK  and autophagy inhibitor 3-MA  were dissolved in DMSO. First, 800 adult whiteflies at 3 to 5 days postemergence were fed on the virus-infected tomato plants for 24 h and then were divided into four groups to be fed with a diet of 15% sucrose containing 10 μM Z-DEVD-FMK (for 12 h), DMSO (for 12 h), 1 μM 3-MA (for 24 h), and DMSO (for 24 h), respectively. Next, the four groups of whiteflies were transferred to feed on cotton plants for 120 h and then collected for Western blot analysis, immunofluorescence, and qPCR. The whole experiment was replicated 3 times.t test was applied for the analysis when there were only two treatments. Percentage data were arcsine square-root transformed to be used in the analysis, but the original data of percentages are presented. The percentage data of tomato plants showing typical symptoms of TYLCV infection were analyzed with Mann-Whitney U-tests because each of the three treatments had only one replicate. Differences were judged significant when P was <0.05.For all data of gene expression, percentages of apoptotic cells, the abundance of virus DNA, percentages of mortality, the proportion of whiteflies or plants with virus DNA, and percentages of virus-infected plants, one-way analysis of variance (ANOVA) was applied for the analysis followed by Bonferroni test when there were ≥3 treatments, and Student’s two-tailed


The treatment strategies for residual deformity of Perthes disease are not established.This is a case report of a 15-year-old boy. He developed right Perthes disease  when he was 10 years old and underwent varus femoral osteotomy of the right side. At 12 years of age, he developed left Perthes disease  and underwent varus femoral osteotomy of the left side. Postoperatively, he was treated with partial weight bearing of the left leg with crutches. At 15 years, range of motion (ROM) of his left hip was markedly limited at 30° flexion, 10° abduction, 70° external rotation, and −20° internal rotation, and he was having difficulty maintaining a sitting position.Stulberg group V was noted on plain radiography. Computed tomography (CT) showed collapse of the load-bearing part of the femoral head on the coronal plane, but the ball-shape was maintained in the posterior femoral head on sagittal and transverse sections.Valgus-flexion-internal rotation osteotomy was performed to improve ROM.Left hip ROM improved to 90° flexion, 20° abduction, 50° external rotation, and 40° internal rotation immediately after the surgery. He was able to sit 10 months postoperatively but was left with a limp and limited ROM in the left hip at 60° flexion. Chondroplasty was performed during the plate removal surgery at 10 months postoperatively, which improved hip flexion to 100° immediately after the surgery. The patient was left with limited ROM of 60° flexion of the left hip at the final observation.Femoral osteotomy to improve ROM could be an option for residual deformity of Perthes disease. •Treatment strategies for residual deformity of Perthes disease after skeletal maturity are not established.•Soft tissues must also be altered to achieve ROM.•Valgus-flexion-internal rotation osteotomy was performed as salvage operation for residual deformity of Perthes disease and improved ROM. Hinge aPatient has provided informed consent for publication of the case.All procedures performed in this study were in accordance with ethical stanndards of the Ethics Committee of Nagasaki University Graduate School of Biomedical Sciences . This study was conducted and reported in line with SCARE 2018 criteria [2Case: A 15-year-old boy.Medical and family history: Nothing particular to note.History of present illness: Pain in the right hip and right Perthes disease was detected at 10 years of age . Varus femoral osteotomy was performed on the right side.Left hip pain and left Perthes disease was detected at 12 years of age  . Varus fThe patient walked on crutches postoperatively to relieve load on the left leg. Limited ROM of the left hip was evident at 15 years of age. He limped and had difficulty sitting but did not have pain when he was referred to our department.Physical findings: The patient's height was 165 cm, weight 75 kg, and body mass index 27.5. Limited ROM of the left hip: His ROM in the left hip was 30° flexion, 10° abduction, 70° external rotation, −20° internal rotation, and Drehmann sign-positive . His hipImaging findings: Stulberg group V was noted on plain radiography . Poor coPreoperative plan: Salvage operation was planned to improve ROM. 30° flexion osteotomy for the restricted flexion  a, 30° iOperative findings: The adductor longus and gracilis muscles were completely cut through a medial approach in the lateral decubitus position. Next, 10 × 10-cm resection of the fascia lata was performed through a lateral approach. Twenty degrees valgus, 30° flexion and 30° internal rotation osteotomy and distal advancement of the greater trochanter were performed according to the preoperative plan . The opePostoperative course: ROM improved from flexion 30°to90°, abduction 10°to20°, external rotaion 70° to 50°and internal rotation −20° to 40° immediately after the surgery. The limp persisted, but walking was possible at 10 months postoperatively. Plain radiography showed that collapse of the femoral head's load-bearing surface was not progressing. However, the limited ROM of the left hip remained at 60° flexion.The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper.


N-isopropylacrylamide) (PNIPAAm) were combined to prepare a semi-interpenetrating network (IPN) hydrogel with temperature sensitivity. Calcium chloride was used as cross-linking agent, the non-toxigenic Aspergillus flavus spores were successfully encapsulated as biocontrol agents by the method of ionic gelation. Characterization of the hydrogel was performed by Fourier-transform infrared spectroscopy (FTIR), scanning electron micrograph (SEM), and thermogravimetry analysis (TGA). Formulation characteristics, such as entrapment efficiency, beads size, swelling behavior, and rheological properties were evaluated. The optical and rheological measurements indicated that the lower critical solution temperature (LCST) of the samples was about 29–30 °C. TGA results demonstrated that the addition of kaolin could improve the thermal stability of the semi-IPN hydrogel. Morphological analysis showed a porous honeycomb structure on the surface of the beads. According to the release properties of the beads, the semi-IPN hydrogel beads containing kaolin not only have the effect of slow release before peanut flowering, but they also can rapidly release biocontrol agents after flowering begins. The early flowering stage of the peanut is the critical moment to apply biocontrol agents. Temperature-sensitive hydrogel beads containing kaolin could be considered as carriers of biocontrol agents for the control of aflatoxin in peanuts. Starch, alginate, and poly( Aspergillus flavus and Aspergillus parasiticus, has attracted extensive attention. The peanut plant has the characteristics of aboveground blooming and underground fruiting. The pods are in direct contact with soil fungi, and soil is the main source of mold that infects peanuts [Aflatoxin contamination of peanuts, caused by the invasion of toxigenic strains of  peanuts . Biologi peanuts ,3.Previous studies have shown that sterilized grains such as rice and barley can be used as biocontrol carriers ,4. TheseTrichoderma [Natural polysaccharides have attracted widespread interest as materials for encapsulation and controlled release . Starch choderma , and thechoderma , soprotuchoderma . In a prchoderma . The relN-isopropylacrylamide) (PNIPAAm) is a typical temperature-responsive polymer, exhibiting a temperature-dependent volume phase transition near the lower critical solution temperature (LCST) in aqueous solution [2+/PNIPAAm hydrogel with thermosensitivity and established a release kinetic model [Smart hydrogels, which can undergo abrupt changes or phase transitions when encountering external stimulation, such as temperature, pH, and light, have attracted considerable attention ,20,21. Asolution ,28. As Psolution . At tempsolution ,31,32. Tsolution . Julianaic model . While tPNIPAAm gel is known to be thermoresponsive at an LCST of 32 °C . IPN hydThis work aimed to develop a formulation to achieve the temperature-triggered release of biocontrol agents at a suitable time, thereby achieving biological control of aflatoxin. Semi-IPN hydrogel beads based on starch, alginate, and PNIPAAm were prepared for the delivery of biocontrol fungus spores. The ingredients in the carrier were biocompatible and biodegradable and served as the carbon source for the growth and sporulation of the biocontrol agents after moisture adsorption. To the best of our knowledge, this is the first example of the use of a temperature-responsive bead as an intelligent carrier for the controlled release of biocontrol agents aimed at aflatoxin contamination. The swelling behavior of these semi-IPN beads was investigated as a function of temperature, pH, and ionic strength. Fourier-transform infrared spectroscopy (FTIR), thermogravimetry analysis (TGA), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and rheological analysis were used to characterized the beads with different formulations, and the temperature-triggered release behavior of the biocontrol agents was examined. N-isopropylacrylamide (NIPAAm), soluble starch (maize starch), Tween 20, and sodium alginate (SA) were obtained from Aladdin Biochemical Technology . The viscosity of SA  was 200 ± 20 mPa∙s. Ammonium persulfate (APS) and N,N,Nʹ,Nʹ-tetramethylethylenediamine (TEMED) were supplied by Shanghai Chemical Reagent Co., Ltd. . Calcium chloride, potassium bromide, and phosphate buffer solution (PBS) were purchased from Sino Pharm Chemical Reagent Co., Ltd. . Dichloran 18% glycerol (DG18) agar was provided by Beijing Aoboxing biotech Co., Ltd. Kaolin was purchased from Xincheng Fine Chemical Co., Ltd. . Nontoxic A. flavus strains (H 4–5) were isolated from our laboratory in Changchun, China. Ultrapure water was used in the experiment. All chemicals were used without further purification. The isolated nontoxic strains were grown and maintained on DG18 agar. After 2 weeks of incubation at 30 °C in the dark, the spores were removed by lightly scraping the medium and were suspended in aqueous 0.2% Tween 20. The density of the spore suspensions was determined by a hemocytometer and adjusted as needed .PNIPAAm was obtained through radical polymerization using NIPAAm as a monomer, APS as an initiator, and TEMED as a catalyst. Specifically, an aqueous solution was prepared by dissolving 35.3 mmol of NIPAAm and 1.35 mmol of APS in 45 mL of water. Then, nitrogen was purged into the solution for 30 min to remove oxygen. After that, 0.267 mmol of TEMED was added. The polymerization lasted for 6 h at room temperature (20 °C) . After tv/v spore suspensions were added. After stirring for 30 min until becoming uniform, the dispersion was added dropwise by a 50 mL syringe (needle diameter of 1.2 mm), from a 30 cm height, into 100 mL of 2% w/v CaCl2 solution under constant stirring. Thus, the spherical beads were formed and maintained in the CaCl2 solution until completely crosslinked. After that, the beads were washed with ultrapure water to remove excess ions, air-dried overnight, and then vacuum-dried to constant weight at 40 °C. The characteristics of the beads are shown in The quantitative starch, alginate, and kaolin were dissolved in hot water by magnetic stirring . After fThe molecular weight (Mn) and polydispersity (PDI) of the synthesized PNIPAAm were measured by a Waters 1525 gel permeation chromatograph (GPC)  equipped with microstyragel columns and a 2414 differential refractometer detector. The eluant was tetrahydrofuran (THF), and the flow rate was 1 mL/min. The calibration curve was obtained using monodisperse polystyrene as the standard.Thirty thoroughly dried beads were taken from each formulation, and their diameters were determined by a Vernier caliper. FTIR, SEM, and TGA were used to characterize the different formulations.The FTIR spectra of the samples were measured using a Nexus670 FTIR spectrometer  by the conventional KBr disk tablet method.TGA was performed using a SHIMADZU DTG-60H thermal analyzer  at a heating rate of 10 °C/min in the range of 20–700 °C under a nitrogen atmosphere, with a flow rate of 50 mL/min.The surface morphology of the hydrogels was observed by SEM using a HITACHI S-4800 . Samples were dissolved in ultrapure water at room temperature, quickly frozen in liquid nitrogen, and then freeze-dried  for 48 h. The lyophilized hydrogels were fixed on a copper plate and sputtered with gold to avoid charging effects.DSC measurements  were used to determine the LCST of the semi-IPN hydrogel beads. First, all of the beads were swollen in PBS for 24 h at room temperature. Then, DSC analysis of the swollen beads was carried out from 20 to 40 °C at a rate of 1 °C/min and under a nitrogen flow rate of 20 mL/min. Temperature and heat flow were calibrated using a pure indium standard at the same ramp rate.The absorbance of the semi-IPN hydrogels at 480 nm was determined using a UV–vis spectrophotometer . The hydrogels were placed in a cuvette in a temperature-controlled box. The temperature was raised from 25 to 40 °C, and each sample was allowed to stand for 15 min before measuring the absorbance.Ws and Wd are the weight of the swollen and dried beads, respectively. The experiment was repeated three times for each group.The swelling properties of the samples were measured gravimetrically. The weighed dried beads were immersed in PBS (pH 6.0) separately at 25, 30, and 35 °C. After 12 h, the swollen beads were taken out and weighed after removing the excess water on the surfaces with filter paper. The swelling ratio (SR) was obtained by the following expression:−1 ≤ 0.8) at 25 °C. The effect of pH on the swelling properties of the beads was studied by varying the pH of the medium. The weighed dried beads were immersed in 20 mL of PBS . The method of obtaining the swelling ratio was the same as the above-described method.To investigate the ionic strength dependence of the swelling of the beads, the quantitative beads were placed in 20 mL of NaCl aqueous solutions at different ionic strength values (0.1 ≤ I/mol L−1) and test time (1 min) were the same for all measurements.The rheological properties of the samples were determined in aqueous solutions using a dynamic shear rheometer . The aqueous samples prior to crosslinking were gently placed on the surface of the lower Peltier plate, and the upper plate declined until it reached a 1000 μm gap distance. A solvent trap was used to minimize evaporation during all measurements. Temperature sweep experiments from 25 to 40 °C were carried out at a scan rate of 1 °C/min. The shear rate (10 sw/v) and then gradient diluted in 0.85% w/v sodium chloride solution. On DG18 agar, 100 μL of aliquots was inoculated for 3–5 days at 28 ± 2 °C. The results are expressed in terms of the colony-forming units per gram of beads. The quantity of spores released after the dissolution of beads divided by the total number of spores applied was the entrapment efficiency of spores.The colony-forming unit (CFU/g) method was used to evaluated the entrapment efficiency of the spores. Specifically, 0.1 g of beads of each formulation was dissolved in 10 mL of sodium citrate solution  in 50 mL of PBS. The pH of the PBS was adjusted to 6.0 to be consistent with that of peanut soil. The solution media were placed in the dark and maintained at 25, 30, and 35 °C. The density of the spores in the release medium was determined by plate counting. The sample (200 μL) was periodically taken out and serially diluted in PBS before plating. Aliquots of 100 μL of each gradient concentration were inoculated on DG18 agar for 3–5 days at 28 ± 2 °C, and the number of colonies of t, k is the release rate constant, and n is the diffusion exponent of the release system. In terms of the release mechanism, for normal Fickian diffusion, n = 0.5; for case II diffusion, n = 1.0; and for non-Fickian, n = 0.5–1.0.The release kinetics of the spores from the beads was analyzed by an empirical model according to the cumulative release data versus time ,41.(2)Mp < 0.05.Triplicate experiments were performed, results are expressed as mean ± standard deviation (SD), and significance was analyzed using Duncan’s multiple comparison tests in SPSS Statistics 20.0 . The criterion for significance was p < 0.05). Similar results have been previously reported [The characterization of the synthesized PNIPAAm was obtained from the GPC test, and the data showed that the average molecular weight and PDI were 26,156 and 1.8, respectively. The composition of the sample beads is shown in reported .PNIPAAm hydrogels undergo a phase change near the LCST in aqueous solution, and the phase state of semi-IPN hydrogels at different temperatures can be expressed by the transmittance of light. The changes in transmittance with rising temperature were recorded from 25 to 40 °C using a UV–vis spectrophotometer, as shown in −1 was present owing to O-H stretching vibrations. This broad peak may come from hydroxyl groups in G and M residues of the alginate chain [−1 of SAP13 and SAPK2 corresponded to the stretching of CH3 groups in PNIPAAm. The asymmetric stretching vibration of COO– groups in the alginate network was manifested at the peak near 1640 cm−1 [−1 of SAP13 and SAPK2 could be assigned to the N–H bending of the amide group, which was related to PNIPAAm [−1 was due to the C–O stretching of alginate structural units [−1 of SAPK2 was due to the Si–O bending modes that belonged to kaolin [The results of the FTIR analysis of SA, SAP13, and SAPK2 are presented in te chain . The abs640 cm−1 . The pea PNIPAAm , and thial units . In parto kaolin .−1, are shown in The thermal characteristics of the samples were investigated by thermogravimetry. The TGA and derivative thermogravimetry (DTG) curves of pure components  and hydrogel samples with various compositions, obtained at a heating rate of 10 °C minNormally, thermal decomposition entails moisture loss, dehydration, and decomposition processes . The dehAccording to the thermogravimetric analysis of the pure components, the temperatures of the maximum rate of mass loss were 240, 297, 498, and 401 °C for alginate, starch, kaolin, and PNIPAAm, respectively. Correspondingly, the temperatures of the maximum rate of mass loss of SA, SAP13, and SAPK2 hydrogel beads were 252, 275, and 301 °C. The results showed that the temperatures of the maximum rate of weight loss of PNIPAAm and kaolin were much higher than those of alginate and starch. Consequently, SAPK2 had a higher decomposition temperature than SAP13, and SAP13 had a higher decomposition temperature than SA.Compared with the SA hydrogel, the DTG curves of SAP13 and SAPK2 hydrogels were more complicated due to there being more interactions between the components. For all samples, the remaining residues at the end were 36.78%, 18.89%, 84.54%, 2.97%, 19.32%, 4.89%, and 14.57%, for alginate, starch, kaolin, PNIPAAm, SA, SAP13, and SAPK2, respectively. Compared with SAP13, it was obvious that there was more residue of the hydrogel containing kaolin (SAPK2), which was due to the presence of oxides, carbonates, and silicates . In addiAt high magnification (1000× magnification), the honeycomb structure on the surface of SAP13 and SAPK2 could be noticed. Dumitriu’s research revealed a porous morphology with a honeycomb-like structure of PNIPAAm/alginate hydrogels crosslinked with N, N’-methylenebisacrylamide (BIS), which is consistent with our results . In contAs is shown in A statistical analysis was performed to analyze the effect of the composition on the swelling ratio, which is shown in −1 at pH 7.0 and room temperature. As shown in The effect of ionic strength on the swelling behavior of the beads was investigated in NaCl aqueous solutions with a range of 0.1–0.9 mol L– at pH > 3.2, and this could cause high anion–anion repulsion, which in turn resulted in a higher swelling capacity. This type of research was also studied by others, who had similar findings [The pH dependence of the swelling behavior of the beads was studied at room temperature at various pH values between 5.0 and 7.0 . The obtfindings ,33,54.The rheological properties of the samples were investigated by monitoring the storage modulus (G’) and loss modulus (G’’) as a function of temperature . The G’’Considered together, the transmittance and viscosity results indicate that the formulations containing PNIPAAm were stable at normal temperature but dissociated above the LCST (close to the temperature of the flowering stage of peanuts). As such, these formulations may be suitable carriers for biocontrol agents.The release of biocontrol agents from the beads requires two processes. Solvents penetrate the network of polymers and swell, then the biocontrol agents diffuse along the aqueous pathways to the outside of the beads. In this work, the dried and loaded samples were immersed in PBS (pH 6.0) at 25, 30, and 35 °C in the dark to simulate the environment of peanut growth. The release profiles are shown in As is shown in −1; when the temperature rose to 35 °C, the number was higher. In comparison, there were no spores released from SA beads at the same time. Moreover, the addition of kaolin could obviously delay the release of spores from the semi-IPN hydrogel beads at 25 °C. The number of spores released from SAPK1, SAPK2, and SAPK3 beads was an order of magnitude lower than that released from SAP13 within 12–120 h. The retarded release with the increasing kaolin content may have been due to better interaction between kaolin and other composites [Raoultella planticola Rs-2 in alginate-based formulations containing clay. They found that the swelling and release rate decreased with increasing bentonite content, which can be attributed to the strong interactions between bentonite and sodium alginate [According to mposites . Resultsmposites . He. Y aalginate . In addialginate . Furtherk and the diffusional exponent n were evaluated from the slope and intercept of the plot of ln (tM/M0) versus ln t. The values of n and k are shown in As for the release mechanism, the release kinetic constant The release profile of the samples showed that the release of spores from the semi-IPN hydrogel beads was sustainable and controllable with good thermosensitivity. Particularly, the addition of kaolin could meet the characteristics of early application of biocontrol agents and burst release after the temperature was above 30 °C, which is close to the temperature of peanut flowering.Thermosensitive hydrogel beads were successfully prepared by combining PNIPAAm with starch–alginate hydrogel. The biocontrol agents of non-aflatoxigenic spores were entrapped in the beads, as confirmed by the determination of entrapment efficiency. The morphology of the semi-IPN hydrogel showed a porous honeycomb structure on the surface of the beads. The measurement of thermosensitive behavior and rheological properties indicated that the semi-IPN hydrogel demonstrated an obvious phase transformation around 29–30 °C, and this LCST is close to the initial temperature of peanut flowering. The temperature change at flowering was identified as the main trigger mechanism for hydrogel dissolution and biocontrol agent release. The beads based on the semi-IPN hydrogel with kaolin could serve as carriers of biocontrol agents.


Phlebovirus. These findings highlight the need to control this potentially emerging virus to help safeguard public health.We isolated 17 viral strains capable of causing cytopathic effects in mammalian cells and death in neonatal mice from sand flies in China. Phylogenetic analysis showed that these strains belonged to the genus  Phlebovirus belongs to the order Bunyavirales, family Phenuiviridae (The genus PM–7:00 AM) in Wuxiang County , Shanxi Province, China, using Wentaitai MM200 traps . We classified all specimens according to their morphology under ice bath conditions and stored them in liquid nitrogen until laboratory testing (https://www.takarabiomed.com.cn) . We observed a large number of virus particles in ultrathin sections of brain tissue of neonatal mice under electron microscopy; the virus particles were uniform spherical particles with diameters of 80–100 nm  and then inoculated it into neonatal mice (0–100 nm  (9). https://www.dnastar.com) for nucleotide sequence splicing, MEGA version 6.0 (https://www.megasoftware.net) for phylogenetic analysis, and Meg alignment (DNAStar) for homology analysis , medium (M), and small (S) segment genes . We usedThe complete genome of the SXWX1813-2 virus contains 3 segments . The L sThe nucleotide and amino acid sequences of SXWX1813-2 were compared with those of other phleboviruses. L, M, and S segment genes showed the strongest homology with those of Toros virus (TORV) and Corfou virus (CFUV) . For thePhlebovirus genus. Further analysis showed that SXWX1813-2 is closely related to viruses isolated from sandflies in Turkey (TORV) and Greece (CFUV), forming independent branches. (Phylogenetic analysis showed that SXWX1813-2 belongs to the mosquito- and sand fly–borne virus group of the ranches. , panel ADermacentor nuttalli ticks collected in Xinjiang, China. Our findings indicate that both sand flies and ticks serve as vectors for phleboviruses, including WUXV, in China. In 2011, severe fever with thrombocytopenia syndrome virus, a tickborne virus known to cause fever and hemorrhaging, was reported in China (Phlebovirus CFUV is listed as a tentative species and TORV as an unclassified virus (To date, both CFUV (Ph. chinensis is the dominant sand fly species in China and serves as the primary vector of Leishmania in this country (Ph. chinensis sand flies, suggesting that the species can also serve as a vector for phleboviruses in China. This finding also suggests the possibility of co-infection with Leishmania and phleboviruses. Our finding of phlebovirus in sand flies in China suggests new challenges for controlling a potentially emerging virus to help safeguard public health. Additional information on emerging sand fly–borne phlebovirus in China.


Neurospora crassa is used as a model organism for genetics, developmental biology and molecular biology. Remarkably, it is not known to host or to be susceptible to infection with any viruses. Here, we identify diverse RNA viruses in N. crassa and other Neurospora species, and show that N. crassa supports the replication of these viruses as well as some viruses from other fungi. Several encapsidated double-stranded RNA viruses and capsid-less positive-sense single-stranded RNA viruses can be experimentally introduced into N. crassa protoplasts or spheroplasts. This allowed us to examine viral replication and RNAi-mediated antiviral responses in this organism. We show that viral infection upregulates the transcription of RNAi components, and that Dicer proteins  and an Argonaute (QDE-2) participate in suppression of viral replication. Our study thus establishes N. crassa as a model system for the study of host-virus interactions.The filamentous fungus  Neurospora crassa is a model organism for the study of various biological processes, but it is not known to be infected by any viruses. Here, Honda et al. identify RNA viruses that infect N. crassa and examine viral replication and RNAi-mediated antiviral responses, thus establishing this fungus as a model for the study of host-virus interactions.The fungus  Like viruses of other host kingdoms, fungal viruses have different types of genomes, although they largely are double-stranded (ds) and single-stranded (ss) positive-sense (+) RNA viruses1. Mycoviruses are increasingly discovered in fungi by the conventional detection of dsRNAs, a sign of RNA virus infection4, via high-throughput sequencing of transcripts and/or dsRNA7, and in silico mining of publicly available transcriptomic data9. Fungal viruses commonly have no extracellular route for entry with the exception of an encapsidated ssDNA virus10, and are generally difficult to experimentally introduce into their host fungi extracellularly, which often impedes the progress of this kind of study. Only for some mycoviruses have protocols been developed for the experimental introduction or inoculation. Virion transfection is frequently used for encapsidated dsRNA viruses such as reoviruses11, totiviruses12, megabirnaviruses13, and partitiviruses14. For several capsidless (+)ssRNA viruses, transfection with in vitro-synthesized full-length genomic RNA16 or transformation with full-length cDNA18 are available. Protoplast fusion-based virus horizontal transmission was shown to be useful between virus-infected donors and many recipients for any viruses, i.e., encapsidated and capsidless viruses20.Fungal viruses or mycoviruses are omnipresent in all major groups of fungi, and the majority of them show asymptomatic and frequently mixed infectionsNeurospora crassa (family Sordariaceae) is a filamentous ascomycete used as a research material for the one-gene–one-enzyme hypothesis by Beadle and Tatum in 194121. This fungus has served as a model eukaryotic multicellular organism for genetics, developmental biology, and molecular biology. In particular, circadian rhythm-based physiological regulation22, RNA interference (RNAi) post-transcriptional gene silencing24, and DNA methylation-mediated epigenetic control25 have been pioneered by researchers using N. crassa. The advantages of this fungus over other organisms, especially over other filamentous fungi, include the public availability of a number of biological and molecular tools, biological tractability26, i.e., rapid vegetative growth and ease of sexual mating, shared techniques, a well-annotated genome sequence27, and single-gene knockout (KO) collection28 available from the Fungal Genetics Stock Center (FGSC) (http://www.fgsc.net)29. Surprisingly, despite these merits, this fungus is not utilized in virological studies.31. RNAi pathways in fungi were deciphered for the first time in N. crassa32. The known pathways in N. crassa operate at the the post-transcriptional level, and are largely divided into two groups: meiotic and mitotic RNAi depending on which stage RNAi occurs in. The second group is further categorized into two: quelling and qiRNA -mediated silencing depending on how dsRNA is generated34. N. crassa has three RNA-dependent RNA polymerase (RDR), two Dicer (Dicer-like DCL), and two Argonaute (Argonaute-like AGL) proteins. Two vegetative RNAi pathways share players, while aberrant RNA is generated from repetitive chromosomal regions and the UV-damaged nuclear genome, respectively. Although RNAi in some fungi has been shown to act as an antiviral defense-system response as in the case of animals and plants, its role in antiviral response has not been demonstrated in N. crassa. Choudhary et al. demonstrated the transcriptional induction of RNAi pathway and other putative antiviral genes by transgenic expression of dsRNA, suggesting that the RNAi pathway may act as part of the viral defense mechanism in this fungus35. In this sense, a phytopathogenic ascomycete, Cryphonectria parasitica , has been used to dissect antiviral RNAi in the past decade. Of four RDRs, two Dicer, and four Argonaute genes, one Dicer (dcl2) and one Argonaute (agl2) gene play major roles in antiviral RNAi37. Of note is that the key genes are transcriptionally upregulated upon virus infection, for which a general transcriptional activator SAGA (Spt–Ada–Gcn5 acetyltransferase) complex and DCL2 (positive-feedback player) are essential39.RNAi, also known as RNA silencing, occurs in a wide variety of eukaryotic organisms, including animals and plants. The basic process of RNAi involves dsRNA that is recognized and diced by Dicer into small RNAs. These small RNAs then are incorporated into the effector Argonaute complex for repression of the target gene expression at transcriptional and post-transcriptional levelsN. crassa and two other Neurospora species N. intermedia and N. discreta, and experimental introduction into the N. crassa standard strain of several viruses originally isolated from other fungi using three different methods. Utilizing the biological resources and molecular tools of N. crassa led to interesting insights into how virus/host interactions, particularly antiviral RNAi/viral counter-RNAi, are regulated. Collectively, this study establishes a foundation for the study of virology in the model organism N. crassa.Here, we demonstrate the detection of diverse RNA viruses from Neurospora species, we first chose six wild N. intermedia isolates from Bogor, Indonesia  and performed a small-scale screen by a conventional method. The presence of dsRNA of ~8 kbp and 5 kbp was observed in the FGSC #2559 and FGSC #5099, respectively ssRNA viruses , one dsRNA virus (partitivirus), and one uncategorized RNA virus, were detectable in each data set for 15 out of 134 different N. crassa field-collected strains , termed Neurospora crassa fusarivirus 1 (NcFV1)  from the wild Floridan isolate JW35 (DDBJ/EMBL/GenBank accession #: LC530176 and LC530177)  Fig. . NcFV1-J77) Fig. .Table 1LNeurospora discreta strain FGSC #8579 , which is mostly used as a standard strain, has recently been suggested to carry a fusarivirus, Neurospora discreta fusarivirus 1 (NdFV1) using the publicly available transcriptomic data SRR15397739. Similarly, we took an in silico approach and detected not only NdFV1 but also many other virus-like sequences: another fusarivirus and four partitiviruses, termed Neurospora discreta fusarivirus 2 (NdFV2), Neurospora discreta partitivirus 1–4 (NdPV1–4), and two mitoviruses (capsidless (+)ssRNA viruses) and an endornavirus [a capsidless (+)ssRNA virus] (Table s] Table .Neurospora spp. showed that (1) viral RdRP was more conserved than the P2 protein , (2) NcFV1, NiFV1, and NdFV2 belong to the same clade (I) and share larger genome size, while NdFV1 contains a coiled-coil domain in P2 and belongs to another clade (II), and (3) N. crassa fusariviruses appeared to be separated into three subgroups, NcFV1A, B, and C in clade I, based on the phylogenetic relationship and amino acid sequence identity of RdRP and P2  able to replicate in N. crassa43 45 isolated from the white root rot fungus  and mycoreovirus 1 11 isolated from the chestnut blight fungus (C. parasitica). Using purified preparation, the two viruses were transfected into spheroplasts of the standard strain of N. crassa. Figure To elucidate whether R. necatrix host than in the newly expanded N. crassa host. This is not so surprising for multisegmented, multiparticulate viruses such as partitiviruses in which each genomic segment is packaged separately46. This is different from reoviruses that are multisegmented, monoparticulate viruses in which a set of the multiple genomic segments are packaged in a single particle. Indeed, similar phenomena were observed earlier in natural and experimental host fungi after virion transfection47. In contrast, the MyRV1 pattern was indistinguishable from the original infection of C. parasitica48. It should be noted that, unlike RnPV2, MyRV1 showed unstable infection during the subculture of transfected colonies.An interesting difference was noted; the ratio of dsRNA1:dsRNA2 was smaller in the natural 20, because virion transfection was not applicable. This method was tested for the putative capsidless RNA virus NiFV1 (a fusarivirus) originally hosted by N. intermedia  that harbored a nourseothricin (NTC)-resistance gene. After protoplast fusion, recipient colonies were selected on potato-dextrose agar (PDA) supplemented with NTC (PDA–NTC). Several of them tested positive for NiFV1 and repeatedly anastomosed with the virus-free, NAT-resistant strain of N. crassa. The genetic background of NiFV1-infected recipients was shown to be N. crassa by the observation that the recipient could anastomose with the original isogenic recipient strain. Interestingly, NiFV1 dsRNA-replicative form accumulated much less in N. crassa than in the original host, N. intermedia when normalized to starting mycelia . Among these, fusariviruses appeared to prevail in Neurospora spp.  from the original host strains, NcFV1-infected FGSC #10950 (JW60), and NcPV1-infected FGSC #3975 (JW35), by coculture. The helper-5 strain has been used for forcing and resolving heterokaryons  and its derivatives. Hereafter, these three viruses were largely utilized in subsequent deeper analyses of virus/host interactions.Among N. crassa strain with deletions of genes related to two RNAi pathways, mitotic silencing  in the vegetative stage and meiotic silencing by unpaired DNA 34, or other function  was prepared and infected by NcFV1. Consequently, the double mutant (Δdcl-1/2) showed much greater susceptibility to NcFV1 than the single dcl mutants or wild type, indicating the redundant function of the two Dicers against this virus  and triple (Δqde-1/ΔSad-1/Δrrp-3) deletion mutants, respectively. No or slightly elevated (~1.9-fold) accumulation of NcFV1 dsRNA and ssRNA was observed in the double Argonaute mutant  compared to the qde-2 single mutant (Δqde-2) showing that QDE-2, but not SMS-2, predominantly functions in the antivirus RNAi against this virus. Similarly, NcFV1 accumulated in a triple rdr mutant (Δqde-1/ΔSad-1/Δrrp-3) at a level similar to that in their single-deletion mutants, suggesting no involvement of these rdr genes in the antiviral RNAi.We tested for enhancement of NcFV1, NcPV1, and RnPV2 accumulation a total of 14 single- and two double- and one-triple deletion mutant of the standard ely Fig. . The tworus Fig. . To examrrp-3, Δdcl-2, and Δqde-2 showed only ~1.4-fold or <3.7-fold increased accumulation levels of NcPV1 genomic dsRNA or its transcripts compared with any other single-deletion mutants  analysis using the available N. crassa RNA-seq data , NCU07036 (3′–5′ exonuclease), NCU04472 (RNA helicase), NCU09495 (set-6), and NCU00947 (unknown function)  followed by RT-qPCR. Dimethylation of histone H3 Lys 4 (H3K4me2) and Ser 5 phosphorylation of the RNA polymerase II C-terminal domain (Pol II S5P-CTD) are well-characterized as transcriptionally activated markers. As expected, significant and moderate accumulation of H3K4me2 and Pol II S5P-CTD was observed at the ely Fig. . Howeverent Fig. . RnPV2 lcus Fig. . Alternaqde-2. This downregulation may result from a counterdefense response targeting QDE-2 directly or indirectly by NcFV1.The results prompted us to examine their protein accumulation levels upon virus infection. We created strains expressing FLAG-octapeptide-tagged DCL-2, QDE-2, or RRP-3 from their endogenous loci and infected them by NcFV1, NcPV1, and RnPV2. Western blotting revealed that DCL-2 and RRP-3 were strikingly elevated upon virus infection Fig. . SurprisN. crassa upon virus infection.Taken together, these combined results clearly show transcriptional and post-transcriptional regulation of key genes of the antiviral RNAi in N. crassa as a virus model host for studying virus/host interactions or virology in this well-studied model organism, based on three achievements. First, we report the discovery of diverse RNA viruses from different isolates of N. crassa and other Neurospora spp.  and proposed as the family “Fusariviridae”, were detected in different Neurospora spp. of different geographical origins such as the United States (Louisiana), Haiti, and Indonesia, suggesting its widespread nature in members of the genus Neurospora 58. These two fusariviruses appear to differ in genome organization and gene expression strategy. FgV1 has four open-reading frames (ORFs) and the three downstream ORFs are expressed via subgenomic RNAs, while RnFV1 has only two ORFs and the downstream ORF is not likely to be expressed via subgenomic RNAs but via an unknown mechanism. All fusariviruses identified from Neurospora spp. resemble RnFV1 in terms of the 2-ORF genome organization. Another difference from FgV1 was detected in host factor requirement. A Fusarium graminearum (family Nectriaceae) gene product, hexagonal peroxisome (Hex1) protein, was identified as necessary for efficient FgV1 replication and normal symptom induction. Hex1 is a major component of the Woronin body, a peroxisome-derived organelle that fills the septal pore under hyphal wounding stress and prevents the extension of wound-induced damage to neighboring cells60. In this study, we examined the possible effects of deletion of the orthologous hex-1 gene of N. crassa on NcFV1 replication. However, no discernable effect was observed ssRNA viruses that are distantly related to hypoviruses . Quelling corresponds to cytoplasmic mitotic transgene RNAi in other eukaryotes62. N. crassa has two Dicer (dcl-1 and dcl-2), three RDR  and two Argonaute genes 63. Transgene-induced RNAi in N. crassa requires two Dicers , one RDR (qde-1), one Argonaute , and RecQ DNA helicase (qde-3)50. However, what genes are required for antiviral RNAi in N. crassa was unknown. We have shown an essential role of qde-2-encoded Argonaute (QDE-2) in defense against NcFV1. This study clearly indicated the redundant functional role of DCL-1 and DCL-2 in antiviral RNAi as reported for quelling50, that is, enhanced virus replication was only observed in the double-deletion mutant of dcl-1 and dcl-2, but not in the respective single-deletion mutants 66 and F. graminearum57  and two Argonautes (FgAGO1 and FgAGO2) function redundantly in antiviral RNAi68. Note that FgAGO1 is homologous to N. crassa qde-2 and C. parasitica agl2.N. crassa, dsRNA induces transcriptional elevation of RNAi-related genes such as dcl-2, qde-2, and rrp-335. Taking three approaches, we confirmed that these genes are also induced upon virus infection 39. Comparison of RNAi regulation between the two fungi reveals interesting conservation and differences. It is likely that dsRNA, regardless of viral or host origin, can trigger transcriptional induction, but not its small RNAs, suggesting that the dicing activity of Dicers is not required for the induction39. In contrast, an interesting difference was observed in the degree of induction: dcl2 of C. parasitica was induced more highly than that in N. crassa, i.e., ~40-fold vs. ~8-fold, whereas the induction of rrp-3 and its ortholog rdr4 are comparably induced in the two fungi, i.e., 20–30-fold. However, C. parasitica RDR4 seems to be not fully functional due to a nonsense mutation in any of the three alternatively splicing variants of transcripts69. Thus, the biological significance of high transcriptional induction of rdr4 in C. parasitica or rrp-3 in N. crassa remains elusive. In C. parasitica, no redundancy was found in Dicer in antiviral RNAi, and dcl2 and agl2 transcript levels were increased 10- to 40-fold upon virus infection70. By contrast, two Dicer genes, dcl-1 and dcl-2, in N. crassa played redundant roles ssRNA hypovirus71. Thus, the failure of NcPV1 and RnPV2 to accumulate more in dcl-1/2 and qde-2 mutants than in the wild-type strain is not surprising and suggests their evolution of ways to evade antiviral RNAi.Different patterns between the accumulation of the partitiviruses (NcPV1 and RnPV2) and NcFV1 in an array of mutant red Fig. . Such anred Fig. . It was N. crassa73. Some plant fungal diseases such as chestnut blight are targets of biological control using viruses infecting the pathogenic fungi so-called “virocontrol”75. Filamentous fungi have multilayered antiviral defense impairing virocontrol: RNAi working at the cellular level and vegetative incompatibility functioning at the population level65. Better understanding of antiviral defense vs. viral counterdefense and fine-tuning of expression of associated genes are prerequisite for their successful virocontrol. In this regard, further studies using the N. crassa/viruses, e.g., aiming at exploring antivegetative incompatibility responses evoked by viruses76, should contribute to virocontrol of phytopathogenic fungi.Collectively, this study has opened up an avenue in modern virology, and shall accelerate its advance with available molecular tools and biological resources. This has great impact on studies with viruses of other fungi, particularly plant pathogenic ascomycetous fungi that share many homologous genes with N. crassa ssRNA virus (NcFV1) in ssa Fig. . A plante-2 Fig. . It is aN. crassa strains were purchased from the Fungal Genetics Stock Center (FGSC) (http://www.fgsc.net) and Neurospora protocols available in the website were used unless otherwise mentioned. Full names and accession numbers of mycoviruses detected in this and previous studies are provided in Table R. necatrix strains W57 (infected by RnPV2) and C. parasitica strain 9B21 (infected by MyRV1) were described earlier78. These fungal strains were grown on DifcoTM PDA plates for maintenance unless otherwise mentioned.The fungal strains tested in this study are summarized in Table Neurospora knock-in (KI) and KO strains  were prepared by the method of Eusebio-Cope et al.48 as it is generally applicable to protoplast preparation of ascomycetous fungi. Briefly, liquid cultures of N. crassa were harvested and incubated in a cell-wall digestion solution containing β-glucronidase and lysing enzyme (Sigma-Aldrich). Protoplasts (0.5 × 107 cells in 200 μl) were mixed with purified virus particles in the presence of polyethylene glycol (PEG) and Ca2+ ion.Virion transfection was performed as described by Hillman et al.N. intermedia (FGSC #2559) and N. crassa as described by Shahi et al.20. For this purpose, the N. crassa standard strain (74-OR23-1VA) was transformed by the NTC (nourseothricin)-resistance gene and used for subsequent screening. An equal number of protoplasts (~2 × 105) from the two fungal strains were fused with the aid of PEG/CaCl2. The protoplast fusants were grown regenerated on the regeneration media for 1 day and subsequently on overlaid top agar containing 30 µg/ml NTC for 2–3 days for screening the N. crassa recipient. Protoplast regenerants resistant to NTC were transferred into PDA plates containing 30 µg/ml NTC and incubated for 1 day before detecting NiFV1. After confirming the presence of NiFV1 by the one-step colony PCR method80, NiFV1-positive colonies were anastomosed with the original nontransformed recipient N. crassa strain. A mycelial plug taken from the recipient side was again anastomosed with the original recipient. This hyphal fusion step was repeated three times.Protoplast fusion was pursued between N. crassa helper-5 strain  was created as a donor strain for virus horizontal transfer to a series of recipient strains. Specifically, the virus-free helper-5 strain and the virus-infected N. crassa wild strain  were cocultured into a slant of Vogel’s sucrose medium containing 10 μg/ml pantothenic acid and 25 μg/ml histidine. Subsequently, the virus-infected heterokaryon was forced by a passage into a slant of minimal medium containing 1.5 μg/ml benomyl and then the virus-infected helper-5 strain was purified through microconidia preparation on SC medium containing 1 mM iodoacetate. Similarly, the standard wild-type strain (74-OR23-1VA) and the derived KO and KI strains were infected by forcing heterokaryon with the virus-infected helper-5 strain and then were extracted by spreading onto a plate of Vogel’s FGS medium containing 5 μM FUDR (5-fluorodeoxyuridine) and 1 mM uracil after microconidia preparation. A schematic diagram for the manipulation was described in Supplementary Fig. The virus-infected 48. Specifically, Neurospora strains were grown with shaking in Vogel’s minimal medium N at 32 °C for 2 days and were harvested by filtration. Tissues were transferred into 22-ml screw-capped tubes  containing 0.40.6-mm zirconia beads, 450 µl of 2× STE, 50 µl of 10% SDS, and 300 µl of phenol/chloroform, and were extracted by using the Minilys homogenizer (Bertin Instruments) for 30 s at maximum speed. After centrifugation at 15,000 rpm for 5 min at 4 °C, 500 µl of the upper phase was transferred into a 1.5-ml microtube and 50 µl of 3 M sodium acetate and 400 µl of isopropanol were added. The concentrations of total RNA were measured by using the Qubit RNA Assay Kit (Thermo Fisher), and the qualities were confirmed by agarose gel electrophoresis. For dsRNA purification, equal amounts of total RNA were dissolved in 420 µl of 1× STE after isopropanol precipitation, then incubated in a 65 °C water bath for 15 min, and quickly chilled on ice. Then, 80 µl of ethanol were added as described by Okada et al.81 to purify dsRNA. Complementary DNA (cDNA) was prepared by ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo), and quantitative PCR was performed three times using THUNDERBIRD Probe qPCR Mix (Toyobo) and a LightCycler 96 system (Roche Diagnostics) with specific primers summarized in Supplementary Table The total RNA and dsRNA fractions were modified by the method of Eusebio-Cope and SuzukiN. crassa genes were prepared by genomic PCR and used in northern blotting according to the manufacturer’s instructions. See Supplementary Table Dioxigenin-labeled DNA probes for 82, the reads were mapped to genomic DNA, rDNA, mtDNA, and tRNA of the standard N. crassa strain 74-OR23-1VA with Bowtie283 to obtain unmapped reads. Unmapped reads were then assembled de novo with Trans-ABySS (https://www.bcgsc.ca/resources/software/trans-abyss) into contigs. To create local fungal virus database, potential fungus was extracted from current virus database (NCBI: txid10239). The candidates of fungal viral sequences were selected from the contigs by a BlastX search  using the local fungal virus database.RNA-seq data were obtained from NCBI. After quality trimming by trimommaticN. intermediate  as described by Chiba et al.44 The dsRNA preparation (17.8 ng/µl) was subjected for cDNA library construction using the TruSeq Stranded Total RNA LT Sample Prep  and then next-generation sequencing in Illumina technology . Raw data for this project were deposited in NCBI Sequence Read Archive (SRA) under accession No. DRR248874. The cDNA library construction and deep-sequencing analysis were performed by Macrogen Japan, Ltd. A total of 56,528,326 paired-end reads  were assembled into 8415 contigs  using de novo assembler of CLC Genomics Workbench . These contigs were subsequently used as queries for a local BLAST search against the RefSeq annotated viral‐genome database.The dsRNA-enriched fractions were obtained from petri dish-grown mycelia of a strain of 84. The following antibodies were used: anti-H3K4me2 antibody , anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody , anti-alpha-tubulin antibody , and anti-FLAG antibody . RT-qPCR experiments were performed two times using FAST SYBR Green master kit (KAPA) with the listed primers (Supplementary Table N. crassa strain (74-OR23-1VA) was engineered such that rrp-3-Flag, dcl-2-Flag, or qde-2-Flag was knocked in. Alpha-tubulin detected by Western blotting with anti-alpha-tubulin antibody was used as a loading control.We followed the procedures of the ChIP assay and Western blotting as described previously85. Based on the results of in silico detection of virus-candidate sequences, we choose ten virus-infected strains  and ten highly likely virus-free strains  and compared their levels of gene expression.DEG analysis was performed by the standard HISAT-StringTie-Ballgown pipelinehttp://mafft.cbrc.jp/alignment/server/index.html) with default parameters86. Maximum-likelihood phylogenetic tree analyses were generated using RAxML-NG87 with 1000 bootstrap replicates and the specific model selected by ModelTest-NG88, and were visualized using the graphical viewer FigTree (http://tree.bio.ed.ac.uk/software/figtree/). SNPs were identified using the variant calling and core genome alignment program Snippy (https://github.com/tseemann/snippy) with the N. crassa wild strain RNA-seq reads and the standard 74-OR23-1VA strain (annotation NC12_fixed) genome assembly. The core SNP alignment was used for phylogenetic analysis as mentioned above.The sequences of mycovirus used in this study were obtained from the NCBI website and are summarized in Supplementary Table Commercially unavailable fungal strains and reagents are available from the authors upon reasonable request, through a material transfer agreement with Okayama University or Fukui University.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


During 2017–2018, Barmah Forest virus was recovered from mosquitoes trapped in military training areas in Australia and from a soldier infected at 1 of these areas. Phylogenies of the nucleotide sequences of the envelope glycoprotein gene E2 and the 3′ untranslated region suggest that 2 lineages are circulating in eastern Australia.  Alphavirus, family Togaviridae. Other viruses in this genus include chikungunya virus, RRV, Sindbis virus, and Eastern and Western equine encephalitis viruses. BFV was first isolated in 1974 from Culex annulirostris mosquitoes trapped near Barmah Forest, northern Victoria, Australia  is the second most common cause of human arboviral disease in Australia, after Ross River virus (RRV) , we detected BFV IgG and IgM, but not RRV IgG and IgM, in convalescent serum samples collected 23, 28, and 38 days after onset of symptoms in the patient. After inoculating 100 µL of the serum into cultures of C6/36 mosquito cells and 2 subsequent passages in this cell line, we did not detect infectious virus in the acute-phase serum sample collected on the day of symptom onset. However, we detected BFV RNA, but not RRV RNA, using a quantitative reverse-transcription PCR assay of RNA extracted from 140 µL of the acute-phase sample using a QiaAMP Viral RNA Mini Kit . BFV E2 RNA was present at 4.2 ×106 copies/mL, with a forward primer 8985F (5′-AGTGTGGCAGTACAACTCCCAAT-3′) corresponding to genome position 8985–9006 and a reverse primer (5′-AAGGCACATGGATCTTTCCTTTC-3′) corresponding to genome position 9036–9058. During a retrospective investigation of the outbreak, using PanBio ELISA kits . The PCR amplifications were performed using pfu DNA polymerase , which has 3′–5′ exonuclease proofreading activity. The PCR amplicons were separated by agarose gel electrophoresis and recovered using a rapid gel extraction system (QIAGEN). Sequencing was performed at the Australian Genome Research Facility as described elsewhere (https://www.geneious.com). The human isolate was named SWBTA40/2017. Two strains of BFV (TullyA/2017 and WBTA/2018) isolated from mosquitoes collected in the Tully and Wide Bay military training areas in Queensland, eastern Australia, in 2017 and 2018  and synthesized by Sigma , 8835 (U®C), 9108 (A®G), and 9427 (U®C), resulting in nucleotide divergence of 0.32% , as well as in the process of budding of alphavirus envelope proteins from host cell membranes  suggests that BFV may warrant more detailed virological surveillance. Given the relatively recent detection of BFV in western Australia (