The recent Ebola virus disease (EVD) epidemic was one of the most severe public health emergencies in modern times. The economic impact of epidemics has mostly been analysed at the macroeconomic level. Conversely, we aimed to estimate the economic costs of preventive measures of the epidemic to an extractive firm, ArcelorMittal (AM), using data in the epidemic region from March 2014 to December 2015. AM is the world’s largest steel producer and is particularly important in West Africa, where the extractive industry is economically crucial.Qualitative methods, in-depth interviews (IDIs) and focus group discussions (FGDs), were used to investigate the events and channels of impact of the epidemic on the firm, as perceived by employees and contractors. Quantitative data regarding these costs were also collected. Retrospective cost analysis estimated the actual cost of preventive methods adopted.Most respondents indicated the largest cost impact was suspension of the Phase II expansion, a series of projects designed to increase iron ore production in Liberia. The next largest cost was the preventive measures adopted to counter disease spread. Total costs incurred for adopting preventive measures were USD 10.58–11.11 million. The overall direct costs of preventive measures adopted within the fence, meaning within the physical boundary of the firm’s sites, shared 30–31% of the total costs incurred. The share of external donations supporting humanitarian response was 11–12% of the total costs, followed by 7–12% of relational costs.The firm’s response during the EVD epidemic focussed on its employees and operations, which was later expanded to the wider community and then in supporting the international humanitarian response. The Ebola virus disease (EVD) epidemic of 2014 in West Africa was unprecedented, leading to a public health emergency on a new scale. The morbidity and mortality impact of the 2014 EVD epidemic was far larger than all previous EVD epidemics combined . This inExtractive projects put workers at high risk of exposure to pathogens such as the Ebola virus. These projects are frequently associated with increased contact between wildlife, humans and domestic animals, a major risk factor for the emergence of infectious disease . The extThe Ebola virus epidemic reportedly began in West Africa during late 2013. It started in Guinea and spread at an alarming rate, quickly involving Liberia, Sierra Leone, Nigeria, Senegal and Mali . This isCountries in West Africa are rich in many mineral deposits including alumina, bauxite, cement, diamonds, gold, mineral sands and iron ore , 16. TheIn the context of the 2014 EVD epidemic, it is important to document the actions of a multinational firm in their response to the epidemic, the various channels of impact of EVD epidemic, as perceived by their staff working during the period, on the operations of the firm in the extractive sector and to estimate the costs of the preventive measures that were put in place. Additionally, documenting the actions and measures taken during this period can possibly help to identify longer term benefits (or otherwise) to the firm, as well as their employees, of continuing operations during the lockdown of the country where effective travel between locations was limited. This study was therefore designed to estimate the direct and indirect costs of preventive measures of the EVD epidemic to AM and its subsidiary ArcelorMittal Liberia (AML), using data from a case study based in the outbreak region. Documenting the actions of one of the few firms that continued to operate during the EVD epidemic period may provide some benefit to other firms and business looking to build organisational resilience to epidemics in the future. Therefore, this study aims to qualitatively analyse the perception of staff and contractors of the firm to the EVD epidemic, its impact on the firm in terms of actions taken (or not taken), on supply chains, on the operational cost of the firm, and then to estimate the actual costs incurred by the firm for preventive measures adopted during the EVD epidemic period.The case study was based on AM and its subsidiary AML. The study applied a mixed methods approach to assess the firm level impact of the EVD epidemic on the case study firm with an emphasis on quantifying the total costs of the preventive measures taken by the firm during the EVD epidemic period from March 2014 till December 2015. The study used qualitative methods to investigate and map the sequence of events and the various direct and indirect impacts of the epidemic on the firm, as perceived by its employees and contractors. The quantitative data on the direct and indirect costs of the EVD epidemic to the firm was extracted from interviews, financial documents and other materials provided by AM and its employees. This data was further validated in group sessions with relevant AM personnel.Informed by the qualitative analysis and the quantitative data collected from the various departments across AML including the finance department of AML, as well as crosschecking with eternal sources, the study estimated the actual cost of the preventive measures both ‘within the fence’, which indicates expenditures made within the physical boundaries of the firm’s sites, and ‘outside the fence’, which indicates expenditures made on actions that primarily take place outside of the firm’s physical boundaries, that the firm had adopted during the epidemic period.The research study used in-depth interviews (IDIs) and focus group discussions (FGDs) to collect qualitative data as part of the case study approach. In order to facilitate and guide IDIs and FGDs, guidance notes were prepared in advance  and B. IThrough the IDIs, respondents were asked to list what they believed were the most critical systems impacted within the firm by the EVD epidemic and, if applicable to them, were asked to provide information on how the cost structure within the firm may have been affected accordingly. Questions were asked about the preventive measures taken by the firm, their knowledge and opinion about the implementation of those measures and their understanding of how it may or may not have impacted their work. If known to them, the costs of those measures were also asked about. Detailed data on the cost components identified in the qualitative study were sought from relevant departments of the firm.The FGDs were conducted at the end after all interviews were completed, where they were used to check if the key findings from the IDIs were valid and correct. The questions for the FGDs were formulated by the research team after the preliminary analysis of the first set of IDIs and were emailed to respondents in advance to ensure that they were aware of the type of questions being explored. Subsequently, the transcripts were checked by the other study team members to ensure that the questions and format of the FGDs were followed as designed.The study used interview data to identify major sources through which the epidemic impacted the different aspects of the firm’s operation and consequent operational costs. Respondents were asked to list and rank what, in their opinion, were the three major cost impacts of the EVD epidemic on the firm’s operations. It was reported that there were opportunity costs of staff time devoted to dealing with the epidemic. Relational costs of the EVD epidemic, the costs indirectly arising from the impact of the EVD on the operations of the firm, were investigated using data from qualitative interviews. The interviews were transcribed individually into Microsoft Word documents using ScribePro and analysed thematically using content analysis to derive the main concepts about the perceptions of the employees as well as key information about the decision-making within the firm.A retrospective cost analysis was performed from the firm’s perspective using the financial information extracted from the interviews, FGDs and the financial documentation provided by AM. This entailed a detailed look at the cost and expenditures data shared by the various departments of the firm – tabulating them chronologically and cross checking between sources to ensure consistency and accuracy. The policies, their costs and impacts were validated in further group sessions with participants, who had knowledge about financial expenditures, at the end of the study. Similarly, the opportunity costs of the relational items were quantified using the wage data of the firm obtained from both qualitative interviews and quantitative data.The financial information collected from the firm detailed the specific dollar costs of all items purchased during the epidemic period – both those items purchased directly for the preparedness, mitigation and response phases and, also, the items purchased in routine operations during this period to compare with data from the previous year’s purchases. This comparison helped the team develop a better understanding of dollar expenditures towards additional activities and towards routine total costs. Additionally, total production or output level data was also obtained from the relevant department for several years so that a comparison to similar periods in the previous years could be made. Finally, total cost data that covered the above two items for the firm were also obtained for the period of the epidemic.The primary source of all quantitative data was the Accounts and Payroll section of AML. The data sets used were given to the team in Excel sheets and data were shared for the 2014 epidemic period as well as data from the previous 2 years for comparison. The data shared were shared in two batches, one before the fieldwork began and one post the fieldwork, although additional clarifications and some figures were shared on direct request throughout the study period whenever gaps or missing information were perceived. Needless to say, the process was not easy and securing the data was a difficult task especially as the study took place just after the epidemic period and the firm was still operating under EVD epidemic conditions.The results from the quantitative assessment from the data provided by the firm and validated by the qualitative interviews on the perception of staff and employees of AM, along with their experience of the epidemic period are given below. The results from both these analyses were then subsequently validated by the FGDs.The quantitative data indicated firstly that the single largest cost of the EVD epidemic in 2014 to the firm was from the range of preventive measures put into place in the firm’s concession areas and raising awareness in the adjacent community. Secondly, was the in-kind donations of priority materials and direct support to national and international engagement in the health and humanitarian crisis that was verified by internal and external sources. Thirdly, Ebola-related construction costs constituted the next highest dollar value expenditure, followed finally by the additional salary paid to workers as hazard pay during the epidemic period, and evacuation of non-essential staff (NES). There were other costs, including the lost productivity from workers’ engagement with health and safety (H&S) measures during the epidemic period. The total preventive costs of the epidemic incurred by the mining firm were mainly driven by direct costs and relational or productivity costs as reported in The overall direct costs of preventive measures adopted within the fence, shared 30–31% of the total costs incurred . The shaFifty-seven percent of the preventive costs were incurred from payment to consultants  and training for putting the security and safety measures in place . The cosThe mining firm made donations to the international public health and humanitarian response communities to support the prevention and treatment of Ebola. The costs related to donations and support to the external Ebola response was approximately USD 1.27 million. The major share of the external support was for supporting the response towards the eradication of EVD, followed by building three isolation centres, donations towards the ambulance services, contract tracing, machinery and capacity to construct external ETUs, as well as other essential medical supplies as shown in The firm provided external support to many Ebola initiatives around the community. The largest share of the external support was provided to support the activities of the Red Cross (56%), followed by providing funds for the county’s regional task forces formed at the local government level to deal with the epidemic (28%), hospitals (11%), county/township services not covered under the taskforce roles (4%) and other beneficiaries including the airport, the police and other government departments (1.2%).The firm paid hazard and incentive payments to the workforce to help maintain a stable supply of workers during the epidemic period. The costs of additional salary payments during the epidemic totalled USD 2.41 million. The cost of evacuation of expats and NES was USD 1.27 million.Based on the distribution of the workforce and the hourly wage rate across employment categories , the cosThese additional construction-related costs totalled USD 1.56 million.The presentation of findings from the qualitative IDIs and FGDs is detailed next and helps provide context and background to the results in the previous section as well the general perceptions of the workforce during the epidemic period.Though not a preventive measure, the suspension of the Phase II expansion was cited the most times  by respondents. The project has been placed on hold till further notice by the firm, more due to the international market price of iron ore than the aftereffects of the Ebola epidemic. The resultant loss in overall production and revenue has significantly impacted both the firm and Liberia itself. The assessment of what the total costs of this suspension will be is beyond the scope of the study. Respondents mentioned that one reason for the suspension of Phase II in 2014 may have been due to airlines ceasing services to Monrovia, the capital city of Liberia:The [Phase II] construction was impacted because our contractors had to leave…some of them…because of Ebola fear…they did not leave because of Ebola but because of the restrictions put on travel…and they did not want to be hemmed in.The mining firm decided to delay and eventually (late 2015 – early 2016) to temporarily suspend the expansion which may have been due to the tough international market conditions for iron ore in that year (2015) in addition to the impact of the EVD epidemic.The 2014 dates [of force majeure] were more driven by the contractors themselves saying…you know we are pulling out…this [risk] is not acceptable to us – 2015 was more I suppose driven by the firm in the sense that we were actually calling the suspension to the project…but one kind of fell after the other…it was a bit of a domino…but certainly you know if Ebola had not come in theory we would have…you know those months we lost we would have had contractors on the ground and they would have been constructing and we would have been further down the road then we are now.The major cost impacts that were reported and pointed out by the respondents were part of the package of preventive measures that the firm adopted over the epidemic period. Fourteen of the 24 respondents  had the opinion that one of the key factors that separated the firm from other extractive industry members was the proactive nature of the firm in seeking information from a world-renowned health expert in the field relatively early in the process. The health expert spent three crucial weeks of April 2014 in Liberia and advised the firm on preventive measures while collaborating with International SOS, in developing their medical response to the epidemic. This intervention was important for AM both as a tool for the internal communication of risks within the organisation and for providing insight into strategies to safeguard their employees, the concession and the communities around it.Risk communication materials developed during this initial phase (in April–May 2014) helped the firm in Liberia to distribute large amounts of printed material across their concessions initially targeting employees, their families and then the wider community at large (June onwards). Some respondents  indicated that there were additional roles they had to conduct during the epidemic period. These roles included conducting a social awareness campaign on Ebola, delivering hand washing buckets and sanitizers and other activities within these communities. This was particularly for those communities inside or in close proximity to the concessions.Temperature screening and access control were some of the first steps recommended by the experts and these were quickly implemented by the firm in Liberia throughout the concessions where the firm operated. Fencing around the key operational sites in the concessions was completed by the end of June and by beginning of July temperature screening and hand washing stations were operating at all entry and exit points of the fenced zones in the firm’s concessions. These steps were quickly absorbed into the H&S culture already prevalent in the firm. Multinational extractive firms in general must follow international H&S standards and the routine training, certification and adherence to these industry standards may have contributed to the swift implementation of temperature screening and fencing protocosl throughout its operations. The strict observance of temperature screening protocols was reported positively by several respondents  as the primary reason for staying Ebola-free within the fence in the concessions throughout the epidemic period, whereas a large majority  indicated it played an important role in maintaining operations. Respondents gave several examples from memory of incidents of suspected Ebola cases being refused entry that later became confirmed EVD cases. During the epidemic period in late November 2014, AML built and maintained two separate ETUs at considerable cost. A large number of respondents , perceived the ETUs construction as the single most expensive expenditure. The two new ETU buildings were specially designed and equipped to handle three confirmed Ebola patients each  and were developed to stabilise Ebola patients until they could be evacuated to their country of origin. Several human resource policies were enacted during the epidemic that had significant cost implications for the firm. Two specific policies were indicated in the interviews, the hazard pay policy and the NES policy implemented throughout August and September 2014. The hazard pay policy was costly because of the number of employees that qualified for it. In effect, all those who were classified as essential workers would receive it. In addition to the hazard pay policy, those employees who were considered as non-essential were sent home and were also paid a salary although at a reduced rate. NES were asked to work from home where they continued to support the firm’s operations remotely. Another major cost impact was the evacuation flights arranged for ex-pats and NES. The evacuation flights were triggered after most international airlines cancelled their flights to and from Monrovia, Liberia in August 2014. A number of respondents  indicated that together the hazard pay and the NES policies had the largest cost impact of the epidemic on the firm’s operations.Some respondents  indicated that there was an administrative productivity loss  due to the preoccupation with EVD management and, also, as a direct result of some of the preventive measures taken. In early 2014, the firm began reviewing and updating emergency management plans in Liberia in case of major security incidents or natural disasters. The firm had decided at that stage to develop emergency management teams (EMTs) in Liberia as part of a crisis management infrastructure within the firm. These teams consisted of senior management and other concerned staff, as dictated by the needs of the crisis, and would be enacted on an emergency by emergency basis. These EMTs played a crucial role in responding to the EVD epidemic as there was a crisis management structure which the firm in Liberia could then build on and link to a central EMT located in London.Even before the outbreak… in Liberia…so, for example, if we had a major security incident or natural disaster…anything…we could manage it effectively from the company side. So, we put in place what was called an emergency management team… really all they consisted of was a lot of the head of departments from the CEO on to Health & Safety, Security, Communications and Medical.The EMTs connected daily for several hours a day for the duration of the outbreak and played a key role in the decision-making process, with regards to what preventive measures were to be taken and when. These meetings of senior management and staff for several hours a day during the peak months of the epidemic had significant cost implications.The qualitative interviews suggest that on average senior management spent 1.5 h per day during the peak Ebola period (August–November 2014), followed by 1 h daily during the off-peak epidemic period (December 2014–June 2015) and 0.2 h daily during the super off-peak epidemic period (July–December 2015), on these meeting. The terms peak, off-peak and super off-peak were used by the senior management team in the EMT based in London to describe the crisis period from their point of view in terms of how much time they allocated to the meetings. In the base case, only the staff time of the senior management is costed to value relational costs. The daily hours spent by senior management on Ebola-related activities during peak and off-peak period varied between the lower  and upper limit  as indicated in the qualitative interviews. We have also evaluated the time costs of all other employment categories to predict possible relational costs when an epidemic affects staff time and productivity across the board. It was inferred from statements by operational staff that all other workers spent on average 0.2 h per day on Ebola-related activities over the epidemic period (August 2014 to December 2015).During the EVD epidemic period, the firm also incurred additional construction costs for mining activities while maintaining the security and safety of its workforce when considering the risks related to Ebola transmission. These costs were incurred from constructing gates, installing washing stations and building fencing as safety measures adopted to fight Ebola.One of the crucial impacts of any disaster-related disruptions is on the supply chain of the firm. The epidemic caused considerable issues with logistics and this was confirmed in the interviews with employees across several departments within the firm. A few respondents  also indicated that some of the extra costs and bottlenecks in operations were a direct result of issues in the supply chain and logistics. This is especially true for a firm in the extractive industry working in conditions like Liberia where most supply items, if not all, are imported from other countries. However, this could not be documented through quantitative means due to the unavailability of data from the concerned department. One of the policies that could be documented from other departments was the stockpiling of several items required for the implementation of key preventative measures and steps like masks, personal protective equipment (PPE) clothing, temperature screening equipment, maintaining hand washing stations and alcohol-based hand sanitizers. The costs for those items have been included in the preventative measures’ expenditure section where appropriate rather than here.Furthermore, the interviews probed whether there were additional costs of the epidemic from the shutdown, transport costs, insurance payments and supply chain items attributable to the EVD epidemic period. The responses of key staff of the firm suggested no additional costs were incurred from any other items in addition to those already included in this analysis.The IDIs and FGDs provided the team with an understanding of the perception of employees and contractors of the firm regarding the chain of events during the epidemic. They also inform the team of the different areas of expenditure costs incurred by the firm for preventive measures adopted to stop the epidemic from entering its workforce and operational sites. The quantitative data provided by the firm helped match and compare those perceptions with the actual expenditures incurred. The qualitative results show that despite initial gaps in knowledge and awareness of emerging infectious diseases (EIDs) like Ebola, AML was able to rapidly access expertise and put into place a number of preventative measures that primarily focussed on inside the fence risk mitigation that incurred additional operational costs. The responses indicated that AML had the capacity for early detection and was flexible enough to respond quickly to the situation by changing practices and allocating the required funds for its implementation. The ability to quickly adapt infection control measures and to internalise them into existing H&S mechanisms meant AML was better prepared in June 2014 when the epidemic entered the urban areas of Liberia than it was in March 2014 at the outset of the epidemic. The level of preparedness, and to certain extent the quality of mitigation measures adopted by the firm, was documented in the study through mixed methods allowing the research team to analyse the additional cost impacts that were incurred by the firm during the epidemic period.The magnitude of the actual costs incurred by the firm largely conformed to the perceived costs identified by the respondents in the qualitative study. The actual cost of preventive measures and reduced productivity incurred by the firm was in the range of USD 10.58–11.11 million . This raAccordingly, the largest cost was generated from preventive health outlays and other containment measures implemented in the mining concession and in the community. The second largest costs were incurred from additional salary payments and the evacuation of NES, followed by Ebola-related direct construction costs, external support towards Liberia’s efforts to contain, treat and eradicate Ebola, and reduced productivity due to the EVD epidemic period.The respondents in the qualitative interviews identified the Phase II expansion as the largest cost impact of the EVD epidemic period on the firm, followed by preventive measures, external donations, consultant fees, ETU construction and hazard pay. As a consequence of the suspension of Phase II, many contractors declared force majeure, when unforeseeable circumstances prevent a contract from being fulfilled, resulting in contractors pulling out of Liberia in August and September of 2014. Although the EVD epidemic may have been responsible for the series of events that led up to this declaration, the general market prices of iron ore and other considerations also played a role. In the quantitative costing, the researchers were unable to estimate the costs associated with the Phase II expansion. However, for the other items listed, the actual costs incurred largely conformed to the perceived costs. It also needs to be highlighted that the low iron ore prices over the period likely added to the uncertainties stemming from the EVD epidemic on business continuity and expansion of iron ore mining.The study shows that there was a system in place in AML for the early monitoring of threats such as disease epidemics. Despite this, Ebola was identified as a potential EID risk only when it was confirmed in Guinea in March 2014. By this time, Ebola had already spread to Liberia. For organisations to be resilient they need as much lead time as possible before a disruption, in order to develop and implement measures that can help prevent or mitigate the impacts of a disruption on its business activities. This is especially true for EIDs that can spread unnoticed in a human or animal population for a significant period of time before being detected.Interviewees felt that one of the factors for the continued operations of the firm in Liberia during the EVD epidemic was the role attributed to communications in its corporate culture . EffectiPrivate firms in the extractive industry also typically have operations in remote locations, like the border areas of Nimba County in Liberia, and can therefore play a vital role in the early detection of EIDs if connected to local health systems. This is in the interest of both the public and private sectors to increase preparation time for mitigation strategies that can limit the extent of the impact of the disruption . The impThe timeline of AM’s response demonstrates that the firm was engaged in prevention, building and strengthening an EVD epidemic control infrastructure, for example, Ebola screening mechanisms and ETUs . These dTo put these expenditures on preventive measures and external donations in context to the epidemic timeline, Most of the respondents  indicated that by the early adoption of recommended measures, like social awareness campaigns and temperature screening, the firm was reacting proactively regardless of the mortality and morbidity rates prevalent in Liberia at the time. The timeline of the firm’s response also shows that when the number of cases per week increased in June and July 2014, the firm in Liberia already had systems in place to continue monitoring its workforce and maintain its production. This commitment to be operational allowed it to be in a position to help the communities it was based in to combat the outbreak by contributing to the building of critical EVD epidemic control infrastructures, such as isolation centers and contact tracing teams, when they were needed most at the peak of the outbreak well before the international community’s response.The timeline of response shown in There are several recommendations that can be made following this case study. Firstly, due to the importance of effective communication in the risk management process, we recommend that firms train their communications department to manage crisis coordination, both internally and externally to the communities they operate in. Risk communication vertically with government and local communities as well as horizontally with other private sector actors to form partnerships and coalitions, like EPSMG, can also contribute to operational resilience. Formulating such partnerships can allow private firms like AM to adopt wider early warning systems to monitor for critical events such as disease outbreak, with particular emphasis on being able to identify the potential impact of EIDs before it is too late. Integration of such a system into the local community and health system would also minimise the public impact of an epidemic. This requires significant collaboration and information sharing between public and private sectors in order to create such a system. Being able to act early and proactively allows firms not only to protect their own operations but also allows firms to commence and support their humanitarian response earlier and more productively.This study’s strengths are largely related to its practical applicability in the industry. The study was set in the real-world mining firm context. The views of the experienced mining staff have strengthened the study by providing a balanced and representative view of how an epidemic can affect mining operations, and how a future crisis could be handled. The study was able to report on direct costs on preventive and mitigation measures incurred by the firm over the course of the epidemic.This study also has several limitations. First, the study was conducted while the epidemic was continuing and AM was experiencing economic downturn, not only because of the epidemic but also due to falling commodity prices in the international market. Second, this study was designed to capture the effect of epidemic on both direct and indirect costs, especially the effect on supply chain items and future expansion projects, but it was not possible to obtain sufficiently detailed data to estimate such costs. Third, the qualitative and system analysis was limited by the availability of the key respondents during the study’s timeframe. These limitations stemmed largely from a high turnover of finance office and senior management staff over the course of the study, as well as a large number of redundancies, particularly in the finance division, which the company had to incur at the end of 2015.Our study found that extractive companies operating in outbreak-prone areas should introduce crisis communication training and support the creation of an early warning system for events such as EID epidemics. Importantly, such a system should be integrated into local community and health regimes in order to minimise the public and business continuity related impacts of an epidemic and limit potentially significant financial losses.The cost incurred by the mining firm for adopting preventive measures during the 2014–2015 EVD epidemic was in the range of USD 10.58 million to USD 11.11 million. The response of the mining firm during the EVD epidemic was focussed on its employees and its operations, which was then expanded to the wider community and then in supporting the international humanitarian response. This was important to building and strengthening the Ebola response infrastructure of Liberia to make a concerted effort to fight the epidemic. There are several recommendations that can be made to private firms following this case study. This includes introducing crisis communication training and the creation of an early warning system for events such as EID epidemics. Importantly, such a system should be integrated into local community and health regimes in order to minimise the public impact of an epidemic. Due to a paucity of studies examining the macroeconomic and especially microeconomic impact of Ebola, further research would help strengthen understanding of the economic impact of endemics and how firms and economies can best manage epidemics.


Coronaviruses are a family of viruses that cause disease in mammals and birds. In humans, coronaviruses cause infections on the respiratory tract that can be fatal. These viruses can cause both mild illnesses such as the common cold and lethal illnesses such as SARS, MERS, and COVID-19. Air transmission represents the principal mode by which people become infected by SARS-CoV-2. To reduce the risks of air transmission of this powerful pathogen, we devised a method of inactivation based on the propagation of electromagnetic waves in the area to be sanitized. We optimized the conditions in a controlled laboratory environment mimicking a natural airborne virus transmission and consistently achieved a 90% (tenfold) reduction of infectivity after a short treatment using a Radio Frequency (RF) wave emission with a power level that is safe for people according to most regulatory agencies, including those in Europe, USA, and Japan. To the best of our knowledge, this is the first time that SARS-CoV-2 has been shown to be inactivated through RF wave emission under conditions compatible with the presence of human beings and animals. Additional in-depth studies are warranted to extend the results to other viruses and to explore the potential implementation of this technology in different environmental conditions. Airborne pathogens are so called because they are transmitted through the air. There are several lines of evidence to support airborne transmission of SARS-CoV-2, the causative agent of COVID-19 pandemic  based onCOVID-19 is a highly contagious viral illness that had a catastrophic effect on the world population with well over 750 million cases and almost 7 million deaths worldwide as of 5 June 2023 . A totalCompared to classical pathogen inactivation, electromagnetic wave radiation can penetrate and heat rapidly and homogeneously, and resonate with microorganisms. In recent years, the applications of electromagnetism for the inactivation of pathogenic viruses have attracted increasing attention ,18,19.The viral structure is generally made up of an inside nucleic acid (RNA or DNA) and an external capsid. Nucleic acid determines the genetic and replication properties of the virus. Most emerging viruses of public health concern have an exterior envelope structure consisting of lipids and glycoproteins. Furthermore, envelope proteins determine the specificity of the receptor and act as primary antigens, which the host’s immune system can recognize. An intact structure guarantees the integrity and genetic stability of the virus.The mechanisms underlying the inactivation of viruses subjected to electromagnetic waves are closely related to the type of virus, and the frequency and power of electromagnetic waves and the viral growth medium are still largely unexplored. Recent studies have focused on the mechanisms of thermal and structural energy transfer for virus inactivation. The thermal inactivation can be attributed to the effects of electromagnetic waves on the water molecules around them. The disadvantage of such a process, compared to the SRET effect via Electromagnetic (EM) wave technique implemented in our work, is the need to generate very high power electric fields, since it has to heat the water up to 100 °C for the destruction of the virus morphology and, therefore, for the reduction of viral activity ,21. The The first series of tests was performed in collaboration with the Defense Institute for Biomedical Sciences, Rome, Italy and with the Sacco Civil Hospital, Milan, Italy. The tests were conducted between 2020 and early 2021 in the respective Biosafety Level-3 (BSL-3) laboratories by illuminating viral samples contained in small drops with microwave signals at different frequencies. To check the inactivation ratio, the illuminated viruses were analyzed by plaque assay to measure the residual infectivity of the viruses. The titer of the illuminated viruses was then compared with the titer of the non-illuminated control sets to calculate the inactivation ratio at different frequencies. The initial experiments were performed by using drops larger than those generated by human sneezing.Subsequently, an innovative method for mimicking the spread of the virus in aerosol has been implemented in cooperation with ViroStatics srl in a controlled and safe way within a BSL-3 laboratory in Alghero, Italy. Virucidal activity has been evaluated against SARS-CoV-2  in this bioaerosol experimental system according to the methods and test setup described below.v/v heat-inactivated fetal bovine serum (FBS) , 2 mM Glutamine (Life Technologies), and 1% v/v antibiotic solution (Life Technologies) at 37 °C under 5% CO2. SARS-CoV-2,  was isolated from a nasopharyngeal swab by the Department of Infectious Diseases, National Institute of Health, Rome, Italy. The virus was propagated in Vero E6 in 2% FCS until 80% of cellular lysis was observed. The supernatants containing the released viral particles were collected and centrifuged at 600× g for 5 min. The clarified supernatant was frozen and kept at −80 °C until use. Viral titer was determined by plaque assay. Viral culture of SARS-CoV-2 was conducted in the Biosafety Level-3 (BSL-3) facility of the Defense Institute for Biomedical Sciences, Rome, Italy. SARS-CoV-2 cultures and bioaerosol tests were handled in a BSL-3 laboratory at ViroStatics facilities located at the Scientific and Technological Park Porto Conte Ricerche srl . Vero E6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) , 1% antibiotic solution penicillin/streptomycin , 1% L-glutamine , i.e., complete medium, at 37 °C with 5% CO2. The human 2019-nCoV strain 2019-nCoV/Italy-INMI1, isolated in Italy (ex-China) from a sample collected on 29 January 2020, was provided by the Istituto Lazzaro Spallanzani, Rome, Italy ). The virus was propagated in Vero E6 cells as described above to obtain high titer virus (>1 × 106 TCID50/mL) and was stored at −80 °C until use. Tissue culture infectious dose (TCID50) is defined as the dilution of a virus required to infect 50% of a given cell culture.For the experiments conducted at the the Defense Institute for Biomedical Sciences, Rome, Italy (preliminary tests with large drops), a VERO E6 cell line (Cercopithecus aethiops kidney) was purchased from American Type Culture Collection (ATCC) and was cultured in a minimal essential medium (MEM) . This was complemented with 10% 2 incubator. To calculate plaque forming units (PFU), the cells were washed with sodium chloride (0.9%) physiological solution, followed by staining with a crystal violet solution.To evaluate the antiviral efficacy of electromagnetic wave radiation, a viral suspension of 300 µL SARS-CoV-2 was spotted on a cover glass and illuminated with microwave signals at different frequencies (from 6.5 to 17 GHz). Untreated SARS-CoV-2 was spotted on a cover glass and used as a positive control. The temperature and relative humidity inside the laminar flow cabinet were maintained within a narrow range for testing, specifically 20 ± 4 °C and 19 ± 5%, respectively. After the treatment, a 10-fold serial dilution of the exposed and unexposed viral suspension was inoculated (in duplicate and triplicate) in confluent monolayers of Vero E6 cells seeded in a 12-well plate and incubated for 1 h. The cells were then overlaid with MEM containing 1.5% Tragacanth and 2% FBS . The cells were incubated for 72–96 h in a COThe virucidal activity of microwaves produced by a Radio Frequency (RF) generator was evaluated against the SARS-CoV-2 Wuhan strain in a bioaerosol experimental system according to the methods described below. The replicative capacity of the virus treated with microwaves (compared to untreated virus) has been determined in an in vitro system, as follows. A virus containing aerosol was introduced in a plastic, air-proof container (0.5 L volume), and particles with a size up to 10 µm were generated with a commercially available aerosol generator 6 TCID50/mL) was obtained from infected cultures of Vero E6 cells. During the test, the chamber containing the infected aerosol was subjected to irradiation by an electromagnetic signal at the appropriate frequency (that is the speed of the wave or the distance between the start and end of each wave) and amplitude (represented by the height of the wave) to maximize the reduction of the viral load. The RF generator was controlled with a software application installed in a mobile device. The treated aerosol was then recovered by means of active impingement directly in complete medium with 2% serum contained in a special glass collector with an inlet and tangential nozzles, through which air from the plastic chamber was sucked when vacuum was applied (at 12 L/min). Viral particles were absorbed in this medium and then cultured in the laboratory for viral titer determination. The use of this system allowed us to consistently recover 5% of the aerosolized virus. The control virus was processed in the same experimental conditions with the device turned off. The replicative capacity  of the virus treated with microwaves (compared to untreated virus) was determined in an in vitro system. Vero E6 cells were seeded at a density of 20,000 cells/well into a 96-well plate in complete medium at 37 °C and 5% CO2. Twenty-four hours after seeding the cells reached ~90% confluency the fetal bovine serum concentration was decreased to 2% to avoid interference with the viral infection. The cells were then infected with 10-fold serial dilutions (8 replicates for each dilution) of the viral suspensions obtained after the impingement procedures for aerosol collection and control. The cells were then cultured for seventy-two hours, and infection was determined by observing the cytopathic effect under an inverted microscope. The viral titer of the recovered virus was determined according to the Reed and Muench method and expressed as TCID50/mL [A viral suspension of the infectious SARS-CoV-2  was used to generate the aerosol. High titer virus  is the spherical Bessel function of the first kind of the l-th order, R denotes the radius of the particle, and LV and TV are, respectively, the sound velocities of longitudinal and transverse waves.Based on Lamb’s theory , the freLV/TV ratio remains approximately 2 [LV of lipid is ~1500 m/s [LV are around 1800 m/s [LV of the uncompressed wet genomes is ~1700 m/s [LV should be in the order of or more than 1500–1800 m/s if the virus does not have a lipid envelope.Since most typical condensed matters have around 0.3 Poisson’s ratio, the mately 2 . Most vi1500 m/s  and most1800 m/s . The rep1700 m/s . Since mz and any integer m > 0:Considering the previous observation, for any variable LR/V) → 0 and (TR/V) → 0; ξ → 0, η → 0 and the eigen value Equation (1) becomes:Therefore, ) 2ξ = 1.Consequently:πν, the resonant frequency f isIn conclusion, since the angular frequency is ω = 2f, the correct estimation of LV is fundamental, while it is possible to measure the radius R of the virions with high accuracy.For the analytical estimation of Since the range of the radius of the SARS-CoV-2 particle is between 60 nm and 140 nm , the resSince the specific core-shell space charge separation for resonance dipolar interaction excitation is valid regardless of the virus shape and the confined “dipolar-like” acoustic phonon modes, we expect that a similar approach can be used for the de-activation of different types of viruses and even bacteria. For example, we believe that the charge distribution for the non-enveloped virus containing only two elements, which are the genome (RNA or DNA) and the virus-encoded protein capsid surrounding the genome , can be To perform the inactivation tests on pathogens, a demonstrator was developed whose transfer function is that of a synthesized RF generator.The unit consists of an Ultra-Wideband (UWB) frequency-tunable synthesizer operating from the C band to the Ku band, a medium power and a high-power microwave amplifier, and a digital variable attenuator to generate as output a microwave signal that is variable in frequency and amplitude .https://code.visualstudio.com/ (accessed on 20 June 2022)).To set all the possible configurations of the RF components, an embedded software written in C++ was developed on the ESP32 platform using Visual Studio Code  is the RF amplifier output power in dBm, Gant(f) is the antenna gain in dBi, and IL(f) is the cable insertion loss in dB.The system has an EIRP  (Effecti in dBmi :(6)EIRPfR is the distance in meters between the antenna and the base where the sample is placed. Moreover:E(f) is the peak value of the continuous wave (CW) sinusoidal electrical field and Z is the impedance of free space.Since the distance between the samples and the antenna is approximately 20 cm, the power density in proximity of the sample is:EIRP(f) and R, we can express the peak of the electrical field as a function of frequency.Using Equations (7) and (8), given Before starting the treatment, an appropriate calibration of the test setup, modifying the gain of the amplifier, and, consequently, the Pout to obtain the same values of the electrical field for all the frequency, was carried out to evaluate the correct inactivation of the virus for each specific value of the electric field intensity associated with each specific frequency. For this purpose, a broadband field meter was used b. The aiTo evaluate the antiviral efficacy of the resonant effect, we first measured the residual viral infectivity of SARS-CoV-2 after illumination with microwaves of different frequencies. Each identical viral sample was placed below the horn antenna and irradiated with a fixed frequency and high-level amplitude electromagnetic field . The radiated frequency is different among all the tests  with the aim to measure different levels of the inactivation ratio and, consequently, to detect the resonant frequency . The inactivation ratio (100 − (N/N0 × 100)) was calculated by comparison of the PFU count of the illuminated viruses (N) and the unilluminated control sets (N0) at different frequencies (6.5–17 GHz).As summarized in r- being ca. 80) and the absorption caused from the DMEM drops.These initial tests were performed with large size DMEM drops (300 µL), larger than those commonly spread during sneezing, coughing, or talking ,11 laid Since the viral drop size influences the inactivation capacity of microwaves, we decided to test the antiviral efficacy of the RF generator in a more physio-pathological relevant aerosol system, selecting a range of 8–10 GHz based on the previous experiments and on preliminary aerosol testing.First, we used a high electromagnetic field (400 V/m) to confirm viral inactivation in the aerosol system. Then, the emitted power was gradually reduced to 6 V/m, a level that is compatible with the presence of humans , withoutThe results presented in The viral inactivation ratio at a different value of frequency step and frequency dwell time are reported in The dwell time could be reduced to 0.05 s, but this time had to be constant. Interrupting the emission  before moving to the next frequency step resulted in a loss of activity.By applying an optimized frequency step of 20 MHz and an optimized constant dwell time of 0.05 s , the total time exposure could be reduced to one minute without a loss in antiviral activity .The inhalation of aerosol and droplets from infected individuals is recognized as the primary mode of respiratory virus transmission, with droplet dispersion influenced by size and environment.This study allowed the identification of the microwave resonant frequencies for SARS-CoV-2 and the demonstration of a reproducible virucidal effect using an RF generator. Our preliminary results, obtained in a controlled laboratory environment mimicking a natural airborne virus transmission with aerosol particles up to 10 μm in diameter, demonstrate for the first time that the viability and infectivity of SARS-CoV-2 can be reduced by 90% (tenfold) through RF wave emission. The application of this technology has the potential to aid in the reduction of virus transmission by sanitizing indoor environments where virus-laden airway fluid (greater than 5μ m in diameter) or aerosol  can remain for hours .This virucidal effect could be achieved by using an RF-wave emission system with a power level that is safe for people according to the Specific Absorption Rate (SAR) US, Canadian, European, and Japanese standards and specifications, thus offering an important method for human environment sanitization.Additional studies will be needed to confirm the results in different environmental conditions, and to demonstrate the efficacy of microwave inactivation for airborne viruses other than SARS-CoV-2, with the intent of building a library of frequencies that are effective for different viruses.


This review aims to provide an update on the current scientific understanding of various aspects of White Spot Syndrome Virus (WSSV) formation, diagnostic procedures, transmission, ecological effects, pathophysiology and management strategies. In terms of production and financial benefits, the WSSV has been the most virulent in shrimp and several other crustacean sectors around the globe. It spreads vertically from diseased broodstock to post‐larvae and horizontally by cannibalism, invertebrate vectors, freshwater and sediments. In the transfer of white spot disease (WSD) in newly stocked ponds, the survivability of WSSV in sediment is the most important variable. In typical cultural conditions, it is a highly infectious pathogen capable of inflicting total death within 3–10 days after an outbreak. Some of the current biosecurity strategies used to keep diseases out of shrimp ponds such as pond water disinfection, quarantine of new stocks before stocking and broader usage of specific pathogen‐free shrimp. The sequencing and characterisation of various WSSV strains have provided details about pathogen biology, pathogenicity and disease. To develop successful control methods, knowledge of these characteristics is essential. In several shrimp‐producing countries in Asia and the Americas, the infections produced by the WSSV have had disastrous socio‐economic consequences. As a result of international trade or migration of diseased species, the World Animal Health Organization recognised several illnesses as posing a substantial hazard to farmed shrimp. WSD is receiving much scientific research due to the potential economic effects of the virus. Research is now being done to understand better the molecular biology and pathophysiology of WSSV, as well as how to treat and prevent the virus. However, further study should be conducted in countries with more resilient host species to understand their role in mitigating disease impacts since these revelations may aid in developing a WSD treatment. The goal of this paper is to focus the molecular techniques reported till now to detect WSSV on shrimp more conveniently. Moreover, the implication of biosecurity measures to control and prevent WSSV on shrimp also mentioned along with the latest prevention application. This virus is a solitary individual from the Nimaviridae virus family, which was recently discovered  from Fujian Province, Mainland China, in 1992   have been shown to discriminate WSSV in shrimp while avoiding cross‐reactions with other viral or shrimp proteins  PCR, which relies on two pairs of primers, the reading is performed by the electrophoresis gel method. The positive results in the first episode of the standard method criteria refer to the infection of the severe white spot virus, while when the positive results are obtained in the second period, it indicates that it is a latent or carrier infection  for positive control and 10× diluted sensitivity tests to contain copies in the range of 2 × 106.4.3Another approach for quantifying DNA is quantitative fluorescence PCR (FQ‐PCR), where the result is decided by the concentration of DNA templates having fluorescent sequences Bustin . RT‐PCR 6.4.4The LAMP method is an innovative, sensitive and fast technology used in aquaculture to diagnose illness. The LAMP method developed a high sensitivity and diagnostic specificity test for identifying the WSSV. The WSSV genome DNA was used to build a set of four primers, two outside and two interior primers. The reaction time and temperatures were set at 1 h at 65°C. As contrasted to 10 fg using nested PCR, the DL of the LAMP approach was up to 1 fg (PCR). As a response, a standard LAMP technique was used to detect WSSV in the heart, stomach and lymphoid organs of diseased shrimp  and the staining and visual detection of product bands on the gel with the use of a fluorescent dye, is the most common method of product detection  appears to have improved WSSV resistance in Ecuador. Since the outbreak of the WSSV in 1999, in Central and South America, the cultural sectors for P. vannamei have been steadily rebuilding. Ecuador, for example, exported 115,000 metric tonnes in 1998, but only 38,000 metric tonnes in 2000 following the arrival of WSSV in 1999. Ecuador has since recovered, with an estimated export of 50,000 metric tons in 2003  and total hemocyte pPO , although plasma THC, total protein content and osmotic pressure were all reduced  are produced as a result of oxidative stress, and they damage DNA and proteins, impairing normal cellular activity. As a result, organisms have devised several ways to counteract such harm. To eliminate ROS and RNS, downstream enzymes such as superoxide dismutase (SOD) and glutathione reductase are often utilised, as well as upstream signalling recognition and signalling transduction molecules. Several enzymes involved in redox reactions have been researched in shrimp, with SOD being the most well researched. MnSOD and Cu/ZnSOD have been identified in shrimp. FeSOD had yet to be discovered  and supplemented mineral and vitamin diets.Consider stocking during periods of the year when you know the environment will not be subjected to extremes in temperature and salinity. It has been claimed that these pressures can trigger an epizootic in a virus‐infected population.Maintain a high‐quality diet and employ NSIS throughout your life cycle.If conceivable, stock at lower densities.Keep an eye out for WSSV‐carrying vectors in the ponds and take steps to eliminate them.Before stocking, collect phytoplankton and zooplankton from the pond and test for WSSV using PCR. Positive ponds should be avoided at all costs.Sample your ponds regularly. Ascertain that sick and dying animals are routinely collected by PCR and histology, as well as any unexpected patterns of death. If you can, harvest the shrimp at the first sign of a problem.At first, avoid purchasing nauplii or PLs from sources that may or may not be infected with the virus. When used on eggs, nauplii and PLs, iodine and water washes will likely eradicate the virus. This procedure must be followed consistently. Incubation facilities should maintain high biosecurity norms and inspect every animal batch. A hatchery should be constructed to prevent the infection from being presented in the ocean. To diagnose and screen WSSV, utilise the most sensitive and dependable demonstrative tests accessible  linked to immune function and growth, as well as potential advancements in quantitative genetics to shrimp breeding, suggest that selecting fast‐growing, disease‐resistant variants could become much more productive shortly. This could also provide insight into invertebrate antiviral immunity, which is currently unknown. In P. stylirostris, such disease‐related indicators have already been discovered (Hizer et al., SPR stands for SPR and is another frequently misunderstood acronym. It refers to a shrimp genetic feature that offers some resistance to a particular virus. SPR shrimp are usually the outcome of a specialised breeding effort to increase viral resistance. SPF and SPR are two distinct qualities. SPR shrimp may not all be SPF shrimp and vice versa. SPR shrimp strains, on the other hand, do not have to be SPF (Alday‐Sanz, 18P. vannamei (C. Lin, The movement of infected PLs mostly determines the viruses and other diseases’ mobility. As a result, if this can be stopped, the virus's transmission will be slowed. Numerous nations have found a way to accomplish this, however, the truth will surface eventually about how fruitful they will be. The virus appears to be here to stay once it takes a footing. Luckily, as with all other viral infections like BP, IHHN, TSV and MBV, the infection's effect will blur over a long period (Mani et al.,  C. Lin, . However C. Lin, . Typical C. Lin, . Because C. Lin, .Avoiding the exchange of water is another suggestion. Recent outbreaks of WSSV in South Carolina, USA, appear to be linked to the addition of water to ponds, therefore there appears to be some weight to this theory (Boyd et al., 19WSSV remains a crucial disease in shrimp production and a big danger to the global shrimp business. WSD is attracting a lot of scientific attention these days, owing to the disease's economic implications. Currently, research is being conducted to achieve a remarkable comprehension of the molecular and pathophysiology of WSSV, as well as the proper treatment and prevention of the virus. Nevertheless, additional research should be done in places with more resistant host species to comprehend their function against disease effects, as these insights could be helpful in finding a cure for WSD. SPR shrimps are preferred over SPF animals for cultivation in locations where the disease is endemic. As a result, more research should be done in the breeding program to develop SPR animals which are resistant to WSSV. Notably, various studies in the domain of WSSV immunisation have been undertaken, although their field application has not yet succeeded. As a result, intervention and development of vaccines for commercial uses can help to minimise the risks. On the other hand, advanced techniques such as shrimp farming in recirculating aquaculture systems and polyculture could be the potential strategies for WSD regulation.Conceptualisation, supervision, writing – original draft and writing – review and editing: Sk Injamamul Islam; Conceptualisation and writing – review and editing: Moslema Jahan Mou. Conceptualisation and writing – review and editing: Saloa Sanjida.The authors declare that they have no competing interests.Ethics statement is not applicable as no sample collection or questionnaires was conducted.https://publons.com/publon/10.1002/vms3.979.The peer review history for this article is available at


Viruses are simple yet highly pathogenic microorganisms that parasitize within cells and pose serious threats to the health, economic development, and social stability of both humans and animals. Therefore, it is crucial to understand the dynamic mechanism of virus infection in hosts. One effective way to achieve this is through virus tracking technology, which utilizes fluorescence imaging to track the life processes of virus particles in living cells in real-time, providing a comprehensively and detailed spatiotemporal dynamic process and mechanism of virus infection. This paper provides a broad overview of virus tracking technology, including the selection of fluorescent labels and virus labeling components, the development of imaging microscopes, and its applications in various virus studies. Additionally, we discuss the possibilities and challenges of its future development, offering theoretical guidance and technical support for effective prevention and control of the viral disease outbreaks and epidemics. These technologies have the ability to reflect the information about research subjects. When applied to the study of viruses, these technologies can be used to track their replication, transmission, and other life activities within cells or hosts. Additionally, they can aid researchers in determining the expression levels of certain genes or proteins during biological processes, making them essential for screening antiviral drugs and studying viral infections and pathogenesis.Viruses, which lack a cell structure and rely on host cells for replication, present a significant threat to the survival of humans and other animals . Their iIn this review, we provide an overview of the selection strategies for labeling and discuss in details the fluorescence labeling strategies for different viral components. We also describe the imaging instruments for virus tracking and highlighted several applications of viral labeling technologies. Additionally, we discuss the future possibilities and challenges of the viral labeling technologies.22.1Labeling viruses poses practical limitations due to their dense structure, including challenges related to labeling approaches, tag sizes, and the precise positioning of labeled viruses. Careful consideration must be given to avoid compromising the functionality and replication levels of viruses during the labeling process. To address this, appropriate labeling methods and viral components must be selected, and thorough controls should be implemented to verify that the viruses retain their functionality and infectivity. Additionally, since virus assembly typically occurs within living cells, cellular labeling techniques are necessary to visualize the early stages of assembly. These limitations, distinct from labeling nanoparticles or other objects, make virus labeling particularly demanding and complex.2.2The diameters of viruses typically range from 25 nm to 1 μm. To render viruses fluorescent, researchers have explored different techniques, one of which involves the insertion of a gene encoding fluorescent proteins (FPs) into the virus genome. By incorporating this gene, the virus becomes capable of producing the FP, resulting in the emission of fluorescent light. This method allows for direct visualization and tracking of the virus in host cells or tissues, enabling researchers to study viral replication, spread, and interactions with the host immune system or antiviral interventions. However, for larger viruses like vaccinia virus, traditional fluorescent labels such as green fluorescent protein (GFP) or red fluorescent protein (RFP) are preferred to avoid disrupting the virus’s structure and function. Conversely, influenza viruses, with an average diameter of only 100 nm, are difficult to label with traditional FPs due to their small size. For smaller viruses, chemical labels are often used to target lipids, proteins, or sugars in viruses , 8. Thes2.3The optical properties of fluorescent labels play a crucial role in visualizing viruses. Bright labels offer advantages in terms of higher temporal and spatial resolution, allowing for more detailed observations. Strong light stability is also essential as it enables longer tracking of viruses without significant signal degradation. Additionally, the spectral characteristics of fluorescent labels can be utilized to assess the quality of the labeling process. A high-quality fluorescent label should exhibit high brightness, facilitating efficient photon capture, and demonstrate excellent light stability to resist photobleaching caused by repeated excitation and de-excitation cycles. These optical properties are vital considerations for successful and reliable fluorescence labeling of viruses.33.13.1.1Specific cellular and virus envelope and capsid proteins can be labeled using FPs through genetic engineering Figure 23.1.1.1Aequorea victoria in 1962, and was first expressed in Escherichia coli and Caenorhabditis in 1994 (in vivo (Green fluorescent protein (GFP) was originally discovered by Osamu Shimomura in  in 1994 . GFP lum are capable of emitting fluorescence of different colors with the changes of excitation wavelength . These F3.1.1.3FPs have been subjected to numerous mutations, resulting in variants with various optical properties and environmental sensitivities that can change with the environmental pH , 26. pH 3.1.2Fluorescent proteins, as genetically encoded labels, have high specificity but relatively low brightness and poor light stability . Organic2+ and is often used to purify the protein. His tag has been used for live cell imaging under the development of Ni-NTA-based fluorescent probes , have proven to be essential for labeling biological systems with fluorescence. They are often excited with high energy and short wavelength due to their single-photon excitation process, while the emitted light has low energy and long wavelength characteristics . The advLipophilic fluorescent dyes possess the ability to stain cell membranes and other lipid-soluble biological structures. Enveloped viruses acquire their lipid membrane from host cell or organelle membranes. Through hydrophobic-photophilic interactions, lipophilic dyes can incorporate into the viral envelope. At high concentrations, self-quenching occurs when these dyes are integrated into virus particles. However, during virus fusion with the cell membrane under low pH conditions, the concentration decreases, leading to dequenching of the lipophilic dyes and an increase in fluorescence signals. Consequently, lipophilic dyes can be utilized to detect the release of the virus genome, with enhanced fluorescence indicating virus-plasma membrane or virus-endosome fusion , 45.For instance, influenza viruses infect cells through the endocytosis pathway, which requires the combination of the viral envelope and the endometrium to transmit the viral genetic material to the cytoplasm. The R110-labeled influenza viruses were used to study real-time hemifusion and its pore formation on the double layer of lipids. Hemifusion occurs in the rapid brightening of the individual, which is caused by the quenching of R110 . Long-ch3.23.2.1Whole virus particles package the viral genome, making it difficult for dyes to bind to the genome. However, some intercalating dyes can penetrate the external components of virus particles and label the virus genome Figure 23.2.2via the mechanism of protein-protein interaction. The third component of the system is the ATPase, which involves the final step of bacterial chromosome or plasmid separation. In its engineering form, called ANCHOR, the shortened ANCH sequence, including palindromic ParS nucleation sites, is specifically bound to ParB proteins. If ParB proteins fuse with FPs, their accumulation on the ANCH target sequence and spread to adjacent sequences will form fluorescence focuses being easy to detect, enabling the identification of ANCHOR-labeled DNA sites  in living cells .in situ hybridization (FISH) is a technology that utilizes complementary base pairing to bind a specific DNA sequence to the target sequence within cells. This allows for the direct visualization of specific nucleic acids within cells, facilitating the observation of virus-related sequences and their localization.Labeling the virus oligonucleotide enables tracking of the virus genome and the replication location. Fluorescence via a FISH probe. Then, a specific antibody is used to label the cluster of differentiation (CD)  , enablin44.1Wide-field fluorescence microscopy is a technique to excite fluorophores in samples using specific wavelength excitation and to receive fluorescence signal imaging through eyepieces or cameras. When the fluorescent molecule is irradiated by excitation light at a specific wavelength, extranuclear electrons in the ground state absorb excitation photon energy and jump to the excited state. Some extranuclear electrons of fluorescent molecules at the high energy level first jump to the metastable energy level in a non-radiation transition and then to the ground state as a radiation transition. Photons corresponding to the energy wavelength will be emitted during the radiant transition, forming fluorescent signal light, which can be captured by eyepieces or cameras for fluorescence imaging.In the wide-field fluorescence microscopy, the large field of view is entirely illuminated by excited light, and the camera can quickly and easily capture the fluorescence signals of a single sample . Objecti4.2Minsky, a researcher at Harvard University, designed the first confocal microscope in 1955 while trying to image the connections between intermediate neurons. He realized that he could significantly improve the resolution by illuminating a small part of the sample and scanning point by point, which reduced the amplitude of light scattering. The basic principle of a confocal microscope is to use point lighting and pinholes in front of detectors to eliminate out-focal signals. The laser beam emitted by the light source of the confocal system passes through the lighting pinhole and becomes a point light source P. After the point light source P enters the imaging system of the high-numeric aperture microscope through the beam splitter, an image point P is formed in some transparent samples under test. Scattering and reflected light, and other lights emitted from the image point P, pass through the objective and spectroscope in the opposite direction and are incident on the single-point detector. The single-point sensor consists of a detector and a pinhole placed in front of the detector, which can reduce the effective area of the detector. Detectors generally use photomultiplier tubes or avalanche photodiodes, and the computer finally processes the electrical signals received by the sensor.Compared with the wide-field fluorescence microscope, the confocal microscope excites only one point in the focal plane at a time. The adjacent fluorescent molecules are not excited and are unlikely to produce stray fluorescence –76. In a4.3Over the years, the resolution of wide-field and confocal microscopy has been limited by the Abbe/Rayleigh diffraction limit, which cannot distinguish structures below 200 nm. In recent years, with the emergence of new fluorescent molecular probes and the improvement of imaging methods, researchers have developed various super-resolution fluorescence microscopies. These technologies have been greatly improved the resolution of optical imaging, achieving accuracy comparable to that of electron microscopy, and enabling the visualization of nanoscale proteins (10 to 50 nm) in living cells. An essential area of super-resolution imaging applications is living cell imaging, where techniques such as saturated structured illumination microscopy (SSIM), photoactivated localization microscopy (PALM), and stimulated emission depletion (STED) microscopy –81. For 4.4Two-photon fluorescence microscopy is a novel imaging technology that combines laser scanning confocal microscopy and two-photon excitation technology. The basic principle of two-photon excitation is that, under high photon density, fluorescent molecules can absorb two long-wavelength photons simultaneously and then emit a shorter-wavelength photon after a brief excitation state. The resulting effect is that the fluorescence is excited by a photon with half the wavelength of the traditional single photon excitation method. Two-photon excitation requires a high photon density, which is achieved using a high-energy mode-locking pulse laser to avoid damaging cells. The laser used in two-photon microscope emits high peak energy and average deficient energy, with a frequency range of 80 to 100 megahertz (MHz).When using a high numerical objective lens to focus the pulsed laser photon, the photon density at the focus of the objective lens is the highest. Two-photon excitation occurs only in the focus of the objective lens, so two-photon microscope does not need to confocal pinholes, improving fluorescence detection efficiency. Traditional confocal microscopes are prone to phototoxicity due to the small size of the confocal pinholes required to obtain high-resolution images. The small aperture blocks a significant proportion of fluorescence emitted from the sample, including the fluorescence emitted from the focal plane. Consequently, the excitation light must be strong enough to obtain a sufficient signal-to-noise ratio. However, high-intensity lasers can quickly bleach the fluorescent dye during the continuous scanning, and the fluorescence signal will become weaker and weaker as the scanning process continues. Additionally, many fluorescent dye molecules produce cytotoxins such as singlet oxygen or free radicals under laser irradiation, so the scanning time and the excitation light power density should be limited in the experiments to maintain the sample’s activity.Under a two-photon fluorescence microscope, fluorescent dye molecules can absorb two low-energy photons simultaneously , and the excitation effect is equivalent to absorbing a high-energy photon with a 1/2 wavelength. For example, absorbing two red wavelength photons is equivalent to one molecule absorbing ultraviolet. Long-wavelength photons are not easily absorbed by cells, reducing the phototoxicity to living cells and light bleaching, thus serving as an effective alternative to ultraviolet excitation and avoiding damage to the sample , 86.55.1The adsorption of most viruses can be classified into two stages: non-specific adsorption and specific adsorption: Non-specific adsorption occurs when viruses come into contact with host cells through electrostatic forces. Specific adsorption, on the other hand, refers to the specific recognition and binding of viral surface proteins (antigens) to the corresponding receptors on the host cell membrane. The binding of viruses to receptors triggers downstream signals and initiates the internalization of viruses , 87. HowRecent studies have highlighted the critical role of filopodia during virus infection –92. Filo5.2via the clathrin- and caveola-independent pathways, which have a similar efficient pathway.After the virus binds to the receptor on the cell surface, there are two main strategies for virus internalization: endocytosis-independent and endocytosis-dependent internalization. The first is that the virus binds to the receptor on the cell’s surface and then fusion directly with the plasma membrane into the cell, such as the herpes simplex virus (HSV) , 94. In 5.3After the virus enters the cell, it is necessary to release its genome from the package of the capsid to a specific site or nucleus of the cytoplasm before it can start subsequent replication. As the fluorescent dyes, SYTO 82 and CypHer5 were used to label the RNA and capsid of poliovirus (PV), respectively . The gen5.4Many viruses enter the cell through the endocytosis pathway, finding that they are in intracellular vesicles and are transported to specific sites in the cell for genome release and replication. This mechanism often uses the cytoskeleton for intracellular transport, including myosin, dynamic protein, and driving protein. Therefore, virus transport is a complex and multi-step process, and viruses usually follow several pathways in the cytoplasm. Virus tracking technology can monitor the behavior of each virus-infected cell in real-time, thus providing new mechanism insights for virus infection . For exa5.5After virus genome replication and protein synthesis, the viral genome is assembled with proteins to form well-structured and infectious progeny virions. The progeny virions are released from the host cell in many ways, such as vomiting, pyrolysis, or germination. Studying the dynamic mechanism of virus assembly and release are more challenging than that of virus invasion and virus transmission, because it is difficult to label the newly synthesized viral components with fluorescent labels. For example, Liu et al. developed a recombinant pseudorabies virus (PRV) based on the Halo Tag fixed-labeled virus capsids and combined with a variety of the Halo Tag protein fluorescent ligands to study the virus entry, intracellular transportation of parental viruses, as well as the assembly of progeny virions capsids, and the diffusion movement of the nucleus and cytoplasm, to complete the tracking of the life cycle of PRV .5.6After the progeny virions are release, they continue infecting new host cells. One mechanism of this infection is the virus transmission between cells. The progeny virions invade the new host cell through direct cell-to-cell contact, which may be 100 to 1000 times more efficient than cell-free transmission . Surpris6Virus tracking technologies are crucial for studying the virus replication process and understanding the mechanisms involved in the virus life cycle, including virus adsorption and internalization, transportation, genome delivery, assembly, and egress. The development and application of virus labeling technology require interdisciplinary knowledge from biology, virology, chemistry, and physics. Despite the rapid progress made in virus labeling technology, several challenges need to be addressed in the future to improve the accuracy and efficacy of virus labeling and to expand its range of applications.Firstly, most virus particles are small, and fluorescent labels may affect the virus’s infectivity to varying degrees. To minimize this impact, a limited number of fluorescent labels should be used to attach to the virus composition. Additionally, conventional genetic manipulation methods can be used to label viral components with FPs, but the operation is complicated, the fluorescent intensity is weak, and the size of the FPs is large, which can also affect virus activity. Furthermore, continuously monitoring the virus infection process under microscope is challenging. The fluorescent labels used for virus tracing must have excellent performance, including strong fluorescence signaling, non-toxicity, anti-quenching, high stability, small size, ease of operation, and no impact on virus infection activity. For example, QDs have high fluorescence intensity and light bleach resistance, but they also have some limitations, such as large size, non-specificity, and potential impact on virus infection , 109–111Secondly, an urgent problem that needs to be addressed is continuously monitoring the entire infection process of fluorescence-labeling viruses at high resolution. While different components of the virus such as the viral envelope and capsid can be labeled with fluorescence, labeling nucleic acids remains a challenge , which lFinally, the virus infection process involves complex interaction between viral proteins and host cells. Given that some of these interactions occur between a small number of viruses and cell molecules, higher temporal and spatial resolutions are often required to efficiently monitor the relevant processes. However, fluorescence microscopy also has some limitations, such as imaging speed and resolution trade-offs, and cumbersome instrument operation. Confocal microscopy is a popular technique for living cell imaging, but it is limited by its resolution and cannot image structures smaller than 200 nm . 3-D imaDL wrote the manuscript. LP, HZ, YS, and H-JQ revised this manuscript. All authors contributed to the article and approved the submitted version.


Paris polyphylla var. yunnanensis, tentatively named Paris yunnanensis rhabdovirus 1 (PyRV1), was recently identified in Yunnan Province, China. The infected plants showed vein clearing and leaf crinkle at early stage of infection, followed by leaf yellowing and necrosis. Enveloped bacilliform particles were observed using electron microscopy. The virus was mechanically transmissible to Nicotiana bethamiana and N. glutinosa. The complete genome of PyRV1 consists of 13,509 nucleotides, the organization of which was typical of rhabdoviruses, containing six open reading frames encoding proteins N–P–P3–M–G–L on the anti-sense strand, separated by conserved intergenic regions and flanked by complementary 3′-leader and 5′-trailer sequences. The genome of PyRV1 shared highest nucleotide sequence identity (55.1%) with Sonchus yellow net virus (SYNV), and the N, P, P3, M, G, and L proteins showed 56.9%, 37.2%, 38.4%, 41.8%, 56.7%, and 49.4% amino acid sequence identities with respective proteins of SYNV, suggesting RyRV1 belongs to a new species of the genus Betanucleorhabdovirus.A novel betanucleorhabdovirus infecting  Paris polyphylla is a perennial herb belonging to the tribe Parideae of the family Melanthiaceae. Rhizome paridis  is a precious traditional Chinese medicine as the raw sources of several well-known Chinese medicines such as “Yunnan baiyao”, “Jideshengsheyaopian” and “Gongxuening Capsules”, which are used as haemostatic, antalgic and antipyretic treatment, respectively. P. polyphylla contains chemical constituents of steroidal saponins, β-ecdysone, polysaccharide, flavone glycoside and other active compounds1. Modern pharmacological studies have revealed that these chemical constituents have antimicrobial, antiviral, antihelminthic and antineoplastic activities6. P. polyphylla var. yunnanensis is distributed throughout the Yunnan-Guizhou Plateau, Sichuan province, and Chongqing city. Due to the economic and medicinal value, wild P. polyphylla var. yunnanensis was reaped excessively. As a result, wild P. polyphylla var. yunnanensis is very scarce. So, artificial cultivation of the plant is crucial to meet market demand. The large-scale cultivation of P. polyphylla var. yunnanensis may lead to the emerging of new fungal, bacterial and viral diseases that have the potential to negatively impact the yield and quality. Till to date, six viruses have been reported to infect the P. polyphylla var. yunnanensis that cause the yield losses. They include Paris polyphylla virus X (PPVX), Paris mosaic necrosis virus (PMNV), pepper mild mottle virus (PMMoV), cowpea aphid-borne mosaic virus (CABMV), Paris virus 1 (ParV1), and Yunnan paris negative-stranded virus (YPNSV)12.Rhabdoviridae family in the order Mononegavirales are important pathogens of animals and plants. The virions are typically bullet-shaped or bacilliform 100–430 nm length and 45–100 nm wide composed of a helical nucleocapsid surrounded by a matrix layer and a lipid envelope14. Their genomes consist of negative-sense single-strand RNA (− ssRNA) 10.8–16.1 kb in length with partially complementary termini15. Taxonomically, plant rhabdoviruses are assigned to the subfamily Betarhabdovirinae that includes 6 genera  based on the replication sites and morphogenesis in the cytoplasm or nucleus of infected cells, and their genome architecture16. Betanucleorhabdovirus genomes encode 6 canonical proteins in the conserved order of 3′-to-5′: nucleoprotein (N), phosphoprotein (P), movement protein (P3), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The genome lacks additional accessory open reading frames (ORFs). The genes are flanked by conserved junction regions 3′-AUUCUUUUU GG UUG-5′ separating their genes, and complementary 3′-leader and 5′-trailer sequences18. Recently, with the application of High Throughput Sequencing (HTS), more and more plant rhabdoviruses were discovered20. The previously reported betanucleorhabdoviruses infect dicot and monocot plants and several of them are transmitted by aphids in which they also replicate21.Viruses of the P. polyphylla var. yunnanensis, provisionally named Pairs yunnanensis rhabdovirus 1 (PyRV1) based on analysis of genomic organization, sequence homology and phylogeny.In this study, we used the HTS platforms and RT-PCR to determine the complete genome of a new betanucleorhabdovirus infecting Paris polyphylla var. yunnanensis plant samples were collected from the medicinal plant plantation in QuJing city of YunNan province. The diseased leaf material was homogenized at ratio 1:5 (w/v) in 0.1 mol/L sodium phosphate Buffer, pH7.0, with carborundum powder as an abrasive agent. Then it was mechanically smeared with the pestle onto the young leaves of Nicotiana benthamiana, N. glutinosa, N. tabacum var. K326 and Sonchus oleraceus and injected with a medicinal injector into the leaf vein of P. polyphylla var. yunnanensis to determine if the virus was sap-transmitted.A field survey was conducted to find out medicinal plant diseases in Yunnan province, China in 2019. During the survey, virus infected Experimental research on plant samples, including the supply of plant material, complies with institutional, national and international guidelines and legislation.P. polyphylla var. yunnanensis were directly cut in 20 µL 2.5% isoamyl alcohol until the tissues was completely homogenized. A pioloform carbon-coated copper grid was floated for 3.5 min on the crude sap to absorb the viral particles. The grids were then transferred onto one drop of 20 µL negative staining Buffer (2% ammonium molybdate at pH 6.5) and stained for 2 min. Finally, the grids were dried by absorbing moisture with filter paper. The virions were examined under a transmission electron microscope .For electron microscopy, 0.1 g symptomatic leaves from P. polyphylla var. yunnanensis leaves using TRIzol  following the manufacturer’s protocol. Ribosomal RNA was depleted using a RiboZero kit  according to the manufacturer’s protocol. Next, a cDNA library was synthesized using the TruSeq Stranded Total RNA with Ribo-Zero Gold (Illumina). The cDNA library obtained was subjected to deep sequencing using the Illumina HiSeq 2500.Total RNA was extracted from the diseased 22. Low quality reads were removed. Next, the clean reads were assembled into expressed sequence tag clusters (contigs) and de novo assembled into transcripts by using Trinity (version: 2.4) with the paired-end method23. The longest transcript was chosen as a unigene based on the similarity and length of a sequence for subsequent analysis. The assembled-contigs (above 150 nt) were subjected to local BLAST searches against the reference viral sequence (RefSeq) database of NCBI .Raw data (raw reads) were processed using Trimmomatic24. In addition, specific primers were designed based on the contig sequences obtained above to amplify and verify the genome sequence  of the rhabdovirus L gene containing a conserved polymerase motif were amplified using the previously reported degenerate primers RhabFor (GGATMTGGGGBCATCC) and RhabRev (GTCCABCCYTTTTGYC)ce Table . Total RGenomic 5′ and 3′-termini were determined using SMARTer 5′/3′ RACE kits . For the 5′-terminus, first-strand cDNA was produced with random priming according to the user manual. Rapid amplification of 5′-end was performed with the primer GPS-5′end: GATTACGCCAAGCTTGGGAAGCCCATATGTGACCCGAAGAC . A band of approximately 700 bases was obtained. For the 3′-terminus, total RNA was added with a poly(A) tail using Poly(A) Polymerase (TaKaRa), then used as a template to synthesize the first-strand cDNA of the 3′-end. Rapid amplification of the 3′-end was performed with the primer GPS-3end: GATTACGCCAAGCTTTGGAGGAGAGGAGAACACATTCCCTCC (reverse complementary with 478–504 of genomic 3′-end). A band of approximately 730 bases was obtained. The PCR products of both ends were cleaned and cloned according to the manufacturer’s instructions. Eight colonies were sequenced in both directions.https://www.ncbi.nlm.nih.gov/orffinder/). The predicted protein were verified using SMART BLAST (http://smart.embl.de/)25. Conserved and functional domains of the predicted proteins in PyRV1 were identified using the Conserved Domain Database (CDD) of the NCBI26 and the pfam database27. Transmembrane helices were predicted using the web based TMHMM Server v. 2.0 , and SignalP were used to predict signal peptide cleavage sites . The nuclear localization and export signals were predicted by cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi)28 and NetNES 1.1 29, respectively. Alignment analyses of the expected amino-acid sequences of viral proteins were performed using MEGAX software, phylogenetic trees were constructed by neighbor joining algorithm with 1000 bootstrap replicates30.Sequences were compared with those published for rhabdoviruses using DNAMAN7.0 software. Open reading frames (ORFs) of PyRV1 were predicted using the ORF Finder program of the NCBI  to detect field samples. RT-PCR was conducted using the Prime ScriptTM One Step RT-PCR Kit  according to the above-mentioned protocol. Ten plant samples with virus-like symptoms were randomly collected from plantation of P. polyphylla var. yunnanensis plants exhibited mild distortion on leaves and leaf tips rolling downward after sprouting in the spring. Vein clearing with the leaf growth and leaf distortion  confirmed the presence of this virus in N. bethamiana and N. glutinosa, but absence in N. tabacum var. K326 and S. oleraceus  identity to Sonchus yellow net virus (SYNV) isolate DSMZ PV-0052 (MT613317), 66.24–70.82% identities  to partial nucleotide sequences of other nucleorhabdovirus such as Blackcurrant-associated rhabdovirus (BCaRV) isolate Mara Eglite (OU015520), Datura yellow vein virus (DYVV) (KM823531), Zhuye pepper nucleorhabdovirus isolate ZPNu1 (ZPNRV), Cardamom vein clearing virus (CdVCV), Bacopa monnieri virus 2 (BmV2) isolate of India and Plectranthus aromaticus virus 1(PleArV1) using BLASTn. Using BLASTx, the contig shared 53.39%, 56.32% and 49.44%  identities to nucleocaspid, glycoprotein and L protein of SYNV (L32603), respectively. Five contigs of 1500–2000 bp, which also was aligned to SYNV and shared aa sequence identity from 49.0 to 53.2% with nucleocaspid, glycoprotein and L protein of SYNV, were obtained from another samples. We also detected 4 contigs of 900–3900 bp were mapped to the PMNV-cn isolate (MF509898.1) with nt sequence identity from 82.4 to 90.3%.To further investigate the virus infecting Betanucleorhabdovirus in a clade with SYNV, DYVV and BCaRV in the phylogenetic tree based on the complete genome sequences of the selected rhabdoviruses , Motif A (DExKWNxxxE), Motif B (GxEGxRQKxWT), Motif C (GxGDNQ) and Motif D (GLPxKxxExWxSx7Kx13K) were located at position 364–369, 560–568, 639–644, 711–721and 743–748, respectively. The underlined residues in motifs were specific to mononegaviruses34.The amino acid sequences of the predicted PyRV1 proteins were aligned with those of available plant rhabdoviruses. The alignment of conserved residues and motifs revealed that PyRV1 shared some conserved residues and motifs in N, G and L proteins with other nuleorhabdoviruses. No conserved residues were identified in P, P3 and M between PyRV1 and other plant rhabdoviruses. Thirteen conserved positions and one motif  were identified in the N protein, 10 conserved cysteine residues  and 2 tryptophan residues (position 231 and 405) in G protein. Sixty four conserved amino acid residues were identified in L protein. Several conserved domains involved in the RNA dependent RNA polymerase activity functionBetarhabdovirinae sub-family also indicated the closest relative of PyRV1 was SYNV. These viruses formed a cluster clearly separated from other betanucleorhabdoviruses, alphanucleorhabdoviruses, gamanucleorhabdovirus, cytorhabdoviruses, dichorhaviruses and varicosaviruses formed separate clusters well separated from each other .To understand phylogenetic relationship between PyRV1 and other plant rhabdoviruses, the maximum likelihood phylogenic trees were generated using MEGA X program. The phylogenetic tree based on complete betanucleorhabdoviral genome inferred that PyRV1 is most closely related to SYNV, with at high bootstrap value > 88 Fig. . The phyr Figure  as wouldP. polyphylla var. yunnanensis were detected by using PyRV1 specific primer derived from L conversed motifs. A 700-bp specific PCR product from one sample was amplified . The genome contained 6 genes in the order 3′–N–P–P3–M–G–L–5′, each gene being separated by a conserved IGR (UUCUUUUU GG UUG), that was common to SYNV  replicate in viroplasms in the host cell nucleus35. As has been reported for both SYNV and DYVV37, the NLS and NES were also observed in the encoded proteins of PyRV1  for establishing a new species of the genus Betanucleorhabdovirus. Therefore, PyRV1 should be considered as a new species in the genus Betanucleorhabdovirus.The presence of a novel nucleorhabdovirus in NV Table . The genVCV Fig.  and S3. Betanucleorhabdovirus has other two characteristics: should occupy different ecological niches (differences in hosts and/or arthropod vectors), can be clearly distinguished in serological tests or by nucleic acid hybridization15. Attempts were taken to isolate the virus from single local lesion through inoculation into Chenopodium quinoa leaves but could not possible. So, the virus was directly inoculated into N. benthamiana and N. glutinosa. The inoculated N. benthamiana and N. glutinosa plants exhibited vein clearing symptom that was similar to the symptoms on P. polyphylla var. yunnanensis. Inoculation followed by verification of amplicon sequencing. The virus was mechanically smeared with the pestle onto 1-year seedlings of P. polyphylla var. yunnanensis. The inoculated plants wilted after 1 week. It was not possible to detect virus from the withered leaves. When the virus was inoculated into more than 1-year aged P. polyphylla var. yunnanensis plants, did not exhibit the virial symptoms and could not detect the virus. In nature, plant rhabdoviruses are transmitted by insect vectors such aphids, leafhoppers or planthoppers21. SYNV is transmitted by aphid. So, PyRV1 may also be transmitted by aphid. However, we could not investigate the aphid in the diseased field. So, we are not sure about the vector. As a wild plant, P. polyphylla var. yunnanensis has been cultivated in a large scale for more than 20 years, several viruses have been detected in P. polyphylla var. yunnanensis12. However, back inoculation into P. polyphylla var. yunnanensis is a dilemma. The leaves of inoculated P. polyphylla var. yunnanensis exhibited clear vein and yellow symptoms similar to naturally infection  causing vein clearing and leaf crinkle disease was discovered in Betanucleorhabdovirus, based on the study of morphology and analysis of genomic organization, sequence similarity, and phylogeny. Our results also revealed the significant diversities between PyRV1 and other nucleorhabdoviruses in terms of gene sequences. Further study was needed to characterize this virus in terms of host range, morphogenesis and its insect vectors.This study identified a novel negative-sense ssRNA virus of paris yunnanensis nucleorhabdovirus (PyRV1), which we suggest belonged to a new species in the genus Supplementary Information.