Modern human activity is profoundly changing our relationship with microorganisms with the startling rise in the rate of emerging infectious diseases. Nipah virus together with Ebola virus and SARS-CoV-2 are prominent examples. Since COVID-19 and the West African Ebola virus disease outbreak, different chemical disinfectants have been developed for preventing the direct spread of viruses and their efficacy has also been evaluated. However, there are currently no published efficacy studies for the chemical disinfection of Nipah virus. In this study, the virucidal efficacy of three disinfectants  against Nipah virus was evaluated in quantitative suspension tests including. Our results showed that the > 4 log reduction achieved for all products in inactivating Nipah virus in 15 s. Even, 19% ethanol was able to inactivate Nipah virus when applied for at least 8 min contact time. Comparative analysis displayed virucidal efficacy of each of the evaluated disinfectants against SARS-CoV-2, Ebola virus and Nipah virus, with only minor differences in working concentrations and contact times required for complete inactivation. We expect that our study can assist in decontamination in healthcare settings and high level biosafety laboratories and can be beneficial to control for emerging enveloped viruses. The impact of this altered relationship can be seen by the startling rise in the rate of emergence of novel viral diseases3. Climate change, deforestation and anthropogenic factors, such as farming, industrialization and increased global travel, are all creating greater opportunity for human exposure to new pathogens, which may result in the emergence of viral disease5. Human immunodeficiency virus (HIV), avian influenza, Hendra and Nipah viruses, SARS, MERS and SARS-CoV-2, and Ebola and Marburg viruses are examples, and all of the emerging infectious diseases induced by these pathogens present a serious and increasing threat to public health and global economies. Though the lack of preparedness is multifactorial, the 2014/2015 Ebola virus disease outbreak in West Africa and the ongoing SARS-CoV-2/COVID-19 pandemic shows that the global community remains ill-prepared for these emerging infectious diseases. Viral surveillance or rapid detection, treatment, vaccination and decontamination or improved sanitation are all key component of successful infection prevention and control programs for emerging infectious viral diseases.Modern human activity is profoundly and irreversibly changing our relationship with microorganisms to far-reaching effect6. Nipah virus is a member of the Henipavirus genus of the Paramyxoviridae family, members of which include the human pathogenic viruses Hedra virus, measles virus, mumps virus and human parainfluenza virus, and is a zoonotic virus with a high case fatality rate7. It possesses a single-stranded, non-segmented, negative sense RNA genome fully encapsulated by a lipid envelop, envelope proteins, and a total of six genes  which encode 3 envelope structural proteins, nucleocapsid protein, polymerase, and phosphoprotein. The clinical and autopsy findings revealed that the clinical symptoms in an early outbreak were mostly localized to the central nervous system including drowsiness, headache, and some degree of encephalopathy7. With different clinical and epidemiological characteristics, the Bangladesh strain responsible for the more recent outbreaks is more commonly associated with respiratory symptoms, human-to-human transmission, and a higher overall case fatality rate (~ 75%), though the genomes of Nipah virus-Bangladesh and Nipah virus-Malaysia are 91.8% homologous8. Furthermore, there is growing evidence of rapid adaptation of Nipah virus to other hosts, including pigs and horses and infected humans display varying modes of transmission.Beside SARS-CoV-2, the infectious agent of the current COVID-19 pandemic as a novel coronavirus, serious diseases caused by Ebola virus and Nipah virus are listed in the World Health Organization’s List of Blueprint Priority Diseases due to their potential to cause a public health emergency and due to the absence of efficacious drugs and/or vaccines12. There are quite a few preventative measures that have been adopted to limit the spread of these virus, including those based on chemical, heat and radiation treatment17. However, the data for persistence of Nipah virus under various environmental conditions are limited18, and there are currently no published efficacy studies available for the chemical disinfection of Nipah virus, other than some methods for the safe removal and handling of virus infected samples, such as human serum or plasma and infected tissues, from BSL-4 facility21. The paucity of virucidal efficacy data for Nipah virus and certain other of the World Health Organization’s Priority List viruses has been mentioned also in a recent review by Ijaz et al.22.Since the COVID-19 and the West African Ebola virus disease outbreaks, studies have revealed that SARS-CoV-2 and Ebola virus can remain infectious for significant periods of time on multiple surface types in the environment, including plastic, glass, stainless steel, and banknotes, and even personal protective equipment (PPE)23. We summarized and compared the efficacies of the three virucidal agents for inactivation of Nipah virus, Ebola virus and SARS-CoV-2.In the present study, we evaluated the efficacy of two quaternary ammonium (QAC) biocides, including MICRO-CHEM PLUS Detergent Disinfectant Cleaner (MCP) and FWD , for inactivating Nipah virus. The virucidal efficacies determined for the dual QAC were compared with that for ethanol, one of the important active ingredients in some chemical disinfectants and used commonly as a low-level disinfectant in healthcare settings for many years. The different concentrations of MCP, FWD and Medical EtOH and contact times from 15 s to 8 min were evaluated in a quantitative suspension test according to Technical Standard for Disinfection of China2. Each treated cell monolayer was examined by light microscopy to assess cytotoxicity daily. The results showed that no obvious cytotoxicity was demonstrated in cell culture media at a 1:500 dilution of 5% MCP and FWD, when these disinfectant solutions was removed from the monolayers following 1 h incubation. Only a slight cytotoxic effects was caused by treatment with medical ethanol at 1:10 dilution, and this effect disappeared after the treatment was removed from the monolayers and cells were refed with fresh DMEM with 2% FBS medium overnight  and two dilution of Medical EtOH® were performed in cell culture medium. The MCP, FWD and Medical EtOH® dilutions were added to Vero E6 cell mono-layers in three replicates and incubated at 37 °C with 5% CODifferent disinfectant concentrations and contact periods were evaluated to observe the dose–response and time kinetics of Nipah virus inactivation. We exposed the Nipah virus for 15 s, 30 s, 1 min, 2 min, 4 min and 8 min to two QAC disinfectants at final concentrations ranging from ~ 5% to ~ 0.06% . Each disinfectant tested was effective at inactivating Nipah virus (Table 10) of FWD  was greater than that of MCP evaluated at the same concentration and contact time, again indicating superiority of FWD over MCP for inactivating Nipah virus.The results show that the dual-QAC disinfectants, MCP and FWD, are highly effective at inactivating Nipah virus within 15 s of contact time, even when they are diluted 1:9   and 19% ethanol was also able to inactivate Nipah virus but enough contact time is necessary (more than 8 min) in suspension test . The noninfectious viruses were removed after the viruses-disinfectant mixture dilutions were discarded. Next, the cells were overlaid with fresh medium. Subsequently, the cells were incubated for an optimised period to amplify the intracellular viruses. Finally, CPE was observed 4 days post infection and viral nucleic acids were extracted from the cell culture supernatant and used for one-step real-time RT-PCR with Nipah virus-specific primers to quantify the infectious virus titer after the virus inactivation treatment. The results showed that the CPE of cells infected with virus-disinfectant mixture were consistent with RF results  inactivated Nipah virus and SARS-CoV-2 quickly (less than 1 min), while MCP at the same concentration took more than 1 min to inactivate these two viruses. Medical ethanol showed similar inactivation profiles against the three emerging viruses. It was surprising that 20% medical ethanol inactivated Nipah virus completely, though more than 8 min of contact time was required in this case.These data suggest that inactivation of Ebola virus may be more difficult than inactivation of SARS-CoV-2 and Nipah virus. The FWD with the same concentration (diluted 9 × or more) needed more contact time to inactivate Ebola virus than SARV-CoV-2 and Nipah virus, and the virucidal activity of diluted MCP kept for one week at RT  showed a similar result. This indicates that these QAC disinfectants should be diluted freshly for Ebola virus inactivation. Comparatively speaking, Nipah virus may be the most susceptible virus among these emerging viruses to inactivation by the three disinfectants.6. A high rate of inter-person transmission with no intermediate hosts has been reported recently in Bangladesh and India outbreaks27. What’s more, recurring Nipah virus outbreaks have been reported almost yearly throughout South and Southeast Asia since 200128. All the information cautions us that continued researches into antiviral drug therapies and vaccines are necessary, as are more comprehensive public health measures comprising a combination of education, hygiene and necessary practices to prevent potentially future outbreaks. Use of an effective virucidal agent for disinfecting objects contaminated with Nipah virus represents an important intervention, thereby mitigating the risk of virus transmission. However, only a few related data for Nipah virus could be identified during this literature search22.As an increasingly global pandemic threat with high case fatality rate, wide host range and expanding modes of transmission coupled with the absence of effective vaccine or therapeutic agents, WHO have declared Nipah virus a priority pathogen for the research and development of diagnostic, prevention and treatment strategies. This virus, has also been included as a priority for vaccine development by the Coalition for Epidemic Preparedness Innovations (CEPI)10 inactivation of Nipah virus though the virucidal efficacy between MCP and FWD varied slightly. The slight differences in efficacy noted may be due to the different compositiosn of these QAC. As we known, QACs are cationic detergents and the cation portion consists of the central nitrogen with four attached groups, which occur in a variety of structures. These variations can affect the antimicrobial activity of the QAC in terms of dose and action against different groups of microorganisms29. Despite the inactivation effectiveness for viruses, the potential environmental impact of QACs also should be considered. As a novel disinfectant based on dual quaternary ammonium compounds, FWD lacks surfactants known as nonylphenol ethoxylates (NPE) that are considered highly toxic to the aquatic environment. Though some effective ingredients in FWD are not as stable as those in MCP, aged FWD remains effective against Nipah virus albeit with longer contact time. These results indicate that FWD may could be considered as a potential alternate for MCP.In this study, we evaluated in detail the virucidal efficacy of two QAC disinfectants (considered low-level disinfectants), against Nipah virus. Our results showed that MCP (approved by EPA) and a novel QAC disinfectant (FWD) displayed > 4 LogThe ongoing COVID-19 pandemic caused by SARS-CoV-2 has drawn broader attention and initiated widespread academic research with the goal of controlling the virus and the disease including various decontamination measures for environment and population. Disinfectants may play important roles in defense against the emerging viruses directly. We report bench-scale experiments evaluating the virucidal activity of three disinfectants against Nipah virus, compared with efficacy for inactivating SARS-CoV-2 and Ebola virus in quantitative suspension test. Two QAC disinfectants (MCP and FWD) and Medical EtOH each display rapid virucidal activity, but further inactivation experiments may have to be repeated using downstream application-specific matrices. As a continually evolving area, new disinfectant product formulations are constantly appearing in the market to meet challenges posed by emerging pathogens. Each disinfectant has its advantages and disadvantages for a particular situation. These novel disinfectants need to be evaluated before use.30. The inactivation profiles of the three evaluated disinfectants against different enveloped viruses were similar, as might be expected. Certain differences in efficacy were discovered. For example, MCP-1W (kept for one week at RT) at the concentration of about 0.56% was still able to inactivate SARS-CoV-2 and Nipah virus, but not Ebola virus after more than 8 min contact time, though all of them are enveloped virus with single-stranded RNA genome. It is important to note that the data related to the virucidal activity of some disinfectants which were determined by surrogate virus such as bacteriophages, enveloped viruses, or non-enveloped viruses should not be extrapolated for use with these emerging viruses without further proof. Owing to their high lethality, Ebola virus and Nipah virus are generally classified as biosafety level-4 (BSL-4) pathogens, and testing conducted specifically with these emerging viruses are needed to confirm expected results.Efficacy of disinfectants is dependent on the target organism(s)33. This method utilizes the host cell as an efficient tool to separate infectious and noninfectious viruses, and to enable the amplification only of viruses capable of infecting the host cell. Accompanied with real-time RT-PCR, it decreases the limit of quantitation and improves the sensitivity of detection. This method even could be used to evaluate the possibility of virus being present at levels lower than the limit of detection of a TCID50/plaque assay. And higher log inactivation values might be possible without limitations in the amount of challenge virus that can be applied. For example, when the initial titre of the virus used for the inactivation effect test is low, RF > 4 cannot be reached34. Hence our method ensured that the smallest amount of possibly non-inactivated virus could be detected, and a very long passaging time and/or large quantity of culture was not needed.In this study, we utilized integrated cell culture real-time quantitative RT-PCR method to assess the virucidal efficacy of disinfectants and addressed the advantage of this method furtherEven in the long-established fields of application, one can expect more effective, mild and eco-friendly disinfectant or virucidal agents to be employed in the various applications further. One aim of our study was to provide a summary that bridge between interested scientists from different disciplines including chemistry, biology and public health etc. By designing tailor-made disinfectants or advanced formulations, public health experts can expect more and accurate choice of disinfectants for decontamination in healthcare settings as part of infection prevention and control for emerging infectious diseases.Finally, the emerging pathogens, such as Nipah virus, Ebola virus and SARS-CoV-2, are limited to handling at high levels of biosafety containment (BSL4 and BSL-3). Use of validated methods of disinfection is an essential requirement when working with these emerging viruses in BSL-4 and BSL-3. This study may assist in disinfectant choice and in developing and improving related operating procedures for high levels of biosafety laboratories.23. Among these disinfectants, Medical EtOH and Micro-Chem Plus® are commercial, broad-spectrum disinfectants. FWD  is a dual quaternary ammonium compound (QAC) product similar to MCP, and is still in the research and development stage.In this study, the virucidal efficacies of three disinfectants against Nipah virus, Micro-Chem Plus® , FWD and Medical EtOH, were evaluated in suspension tests according to the Technical Standard for Disinfection of China2 in Dulbecco’s Modified Eagle medium (DMEM) supplemented with fetal bovine serum (FBS). Nipah virus—Bangladesh, Ebola virus-Mayinga and SARS-CoV-2 (SARS-CoV-2 China/Wuhan/WIV04/201912) were provided by National Biosafety Laboratory, Chinese Academy of Sciences, and was propagated in Vero E6 cells. All work involving infectious Nipah virus, Ebola virus and SARS-CoV-2 were performed in BSL-4 facility in National Biosafety Laboratory, Wuhan.The Vero E6 cells were obtained from the Preservation Center in Wuhan Institute of Virology, Chinese Academy of Sciences, and were cultured at 37 °C with 5% CO25. Briefly, the test products were serially diluted, and 1 ml dilution from each sample was inoculated into Vero E6cells. After 1 h of incubation at 37 °C, dilutions were discarded, and cells were overlaid with fresh medium and cultured for 4 days. The cells were observed daily for cytotoxic effects by light microscopy.The mixtures of viruses and disinfectants were diluted before testing to reduce the potential cytotoxicity. Cytotoxic effects were first assessed in Vero E6 cells using medium (DMEM with 2% FBS) and disinfectant only to ensure that diluted disinfectants was not cytotoxic, as described previously50 on Vero E6 cells using the protocol described previously25. Briefly, 96-well plates containing Vero E6 cells were incubated for 1 h at 37 °C in a 5% CO2 incubator with 0.1 ml of serial dilutions of virus stocks using 1:10 as the starting dilution. Then 0.1 ml of 2% FBS medium was added to each well. After incubation for 4 days at 37 °C in 5% CO2, CPE was observed and used to calculate the virus titer in TCID50/ml.Nipah viruses were titrated by TCID25, equal volumes of disinfectant at different concentration and Nipah virus stock were mixed for each of the inactivation experiments. Immediately after incubation for defined periods of time  at room temperature, the mixture were diluted with a large amount of medium to quench the inactivation reaction and eliminate the toxicity of disinfectant. Some samples were taken out from each dilution to infect cells. After incubating for 1 h at 37 °C in a 5% CO2 incubator, the viruses-disinfectant mixture dilutions were discarded. Next, the cells were overlaid with fresh medium. The CPE was observed after 4 days post infection and Nipah viral RNA was extracted from the supernatant. Quantitative one-step real-time RT-PCR was used to assess virus titer. For each experiment, virus control containing medium instead of disinfectant was included, and each experiment was repeated three times.As described in our previous study35. The standard curve for Nipah virus titres calculation was established using the Nipah virus stock with known titres  following the manufacturer’s instructions. The extracts were resuspended in 60 μl of Buffer AVE, aliquoted and stored at − 70 °C before RT-PCR amplification was carried out. Viral RNA was quantified by One-step real-time RT-PCR using using HiScript II One Step qRT-PCR Probe Kit (Vazyme) according to the manufacturer’s instructions and with the modified Nipah virus specific assay-NiV-NP-UD 23, the virucidal activity was determined by the difference of the logarithmic titre of the virus control minus the logarithmic titer of the test virus, reduction factor (RF). The Log10 titer and its standard deviation  were calculated as well as the variance of the RF. RF of ≥ 4 log10 was regarded as evidence of efficient virucidal activity.According to the standard requirementSupplementary Table 1.


Globally, nearly half of all deaths among children under the age of 5 years can be attributed to malaria, diarrhoea, and pneumonia. A significant proportion of these deaths occur in sub-Saharan Africa. Despite several programmes implemented in sub-Saharan Africa, the burden of these illnesses remains persistently high. To mobilise resources for such programmes it is necessary to evaluate their costs, costs-effectiveness, and affordability. This study aimed to estimate the provider costs of treating malaria, diarrhoea, and pneumonia among children under the age of 5 years in routine settings at the health facility level in rural Uganda and Mozambique.Service and cost data was collected from health facilities in midwestern Uganda and Inhambane province, Mozambique from private and public health facilities. Financial and economic costs of providing care for childhood illnesses were investigated from the provider perspective by combining a top-down and bottom-up approach to estimate unit costs and annual total costs for different types of visits for these illnesses. All costs were collected in Ugandan shillings and Mozambican meticais. Costs are presented in 2021 US dollars.In Uganda, the highest number of outpatient visits were for children with uncomplicated malaria and of inpatient admissions were for respiratory infections, including pneumonia. The highest unit cost for outpatient visits was for pneumonia (and other respiratory infections) and ranged from $0.5 to 2.3, while the highest unit cost for inpatient admissions was for malaria ($19.6). In Mozambique, the highest numbers of outpatient and inpatient admissions visits were for malaria. The highest unit costs were for malaria too, ranging from $2.5 to 4.2 for outpatient visits and $3.8 for inpatient admissions. The greatest contributors to costs in both countries were drugs and diagnostics, followed by staff.The findings highlighted the intensive resource use in the treatment of malaria and pneumonia for outpatient and inpatient cases, particularly at higher level health facilities. Timely treatment to prevent severe complications associated with these illnesses can also avoid high costs to health providers, and households.Trial registration: ClinicalTrials.gov, identifier: NCT01972321.The online version contains supplementary material available at 10.1186/s12936-022-04254-y. Globally, nearly half 45%) of all deaths among children under the age of 5 years can be attributed to malaria (8%), diarrhoea 18%), and pneumonia (19%) 8%, and p. In this5% of allThe literature identifies several challenges in the implementation and equitable delivery of programmes to combat these illnesses. These include timely care-seeking, and appropriate utilisation of services by caregivers and families at community level on the demand side ; and, inPrevious economic evaluations of childhood pneumonia, diarrhoea, and malaria treatment programmes or interventions have focussed on the costs and cost-effectiveness of vaccines and drugs –17; inseThis study aimed to estimate the provider costs of treating malaria, diarrhoea, and pneumonia (MDP), among children under the age of 5 years (outpatients and inpatients) in routine settings at the health facility level in rural Uganda and Mozambique.This study investigated the full financial and economic costs of providing care for childhood (under-5) MDP in rural settings in midwestern Uganda and in Inhambane province in Mozambique from the provider perspective in a ‘real world’ setting. Costs incurred by health facilities in the provision of outpatient and inpatient MDP services were obtained from a mix of private and public health facilities, then apportioned and analysed with a focus on the three illnesses under study. Costs were estimated by combining a top-down and bottom-up approach . Unit coThis cost estimation was conducted as part of the inSCALE  trial , which evaluated the effect of interventions to increase community health worker (CHW) supervision and performance on the coverage of appropriate treatment for children with MDP in Uganda and Mozambique. In the east and southern Africa region, these two countries have the highest burdens of the three illnesses , 26. In In Uganda, inSCALE was implemented in midwestern Uganda, which had an estimated 1.8 million people living in approximately 4000 villages, with 20% being children under 5 years of age. They are engaged in a variety of agricultural and allied occupations, including cattle herding, and fishing, and majority are able to read and write . The UgaPosto de Saúde) is the most peripheral, and provides primary care at the community level, Level II (Centro de Saúde I and II) provides services for referrals from Level I facilities for complications of childbirth, injuries, medical and surgical emergencies that cannot be responded to at a Level I facility. Levels III (Centro de Saúde III) and IV  are fundamentally oriented towards more specialized curative actions and are a reference for lower levels [In Mozambique, inSCALE was implemented in Inhambane province, with an estimated population of 1.3 million living in approximately 145 villages, with 18% being children under 5 years of age. The majority of the population are subsistence farmers with little or no literacy skills [r levels .Service and costs data were collected from health facilities in both trial sites, i.e. midwestern Uganda and Inhambane province, Mozambique. Sites were purposively selected to capture costs for different types of provider facilities and populations. Costs were collected from seven public and private health facilities in Uganda and six public health facilities in Mozambique, and covered peri-urban and rural hospitals, primary health clinics and health posts Table .Table 1SCost data at the health facility level were collected for 2013 between April 2014 and May 2014 in Uganda, and for 2014 between June 2015 and October 2015 in Mozambique. Data were collected at the facility level only. The aim was to capture all overhead costs within a facility but did not aim to capture any above facility costs . As the trial focussed on the primary care level, there was no need to exclude research costs, nor any costs of supporting changes in service delivery.Resource use was measured through a review of facility records, semi-structured interviews with key health facility personnel, and direct observation of service utilisation to ensure that all resources were included. Resources included staff salaries, equipment, supplies , utilities, furniture, and buildings. Prior to data collection, interviews, and direct observations, verbal informed consent was obtained from health facility personnel using an information sheet. All data collected were entered into structured cost data sheets created in MS Excel.Costs were classified as capital and recurrent costs . CapitalService specific costs included staff costs, equipment drugs and consumables. Staff costs included salaries and were allocated to child, adult and all-patient health using the amount of time staff spent working on those services. In the top-down approach, total staff salaries were divided based on staff time sheets and through direct observation. For the bottom-up approach, minutes used to provide a service was multiplied by the salary cost per minute. Salary costs were available from either the facility or the Ministry of Health. Equipment costs were assigned to the different services depending on what the equipment was used for. Top-down costs were allocated by the percentage of patients for each service that used the equipment. Bottom-up costs equipment costs were allocated using the minutes used as observed. In Uganda and Mozambique, equipment prices were sourced from the Ministry of Health’s procurement lists, and from the price lists of the national medical stores.International Drug Price Indicator Guide [Data on the list of drugs used by illness for the entire year were collected. In Uganda and Mozambique, data on the inventory were collected via invoice records present at each facility. In Uganda, costs of the drugs were also obtained from the facility invoice records. However, in Mozambique, drug prices were not available at the facility, as drugs are procured centrally by the Ministry of Health and distributed to health facilities. Drug prices were obtained from the 2014 or Guide . It was All costs were collected in Ugandan shillings and Mozambican meticais. Costs are presented in 2021 USD  that provided essential curative services including vaccination and prevention of local endemic diseases. Of these, one was in an urban area. The sixth health facility was a secondary level facility, a rural hospital, that provided routine surgical interventions with larger diagnostic capacity such as X-ray facilities. All health facilities provided outpatient services; additionally, Level II and III facilities provided maternity services, and the hospital provided inpatient services.In Uganda, data were collected from three government-owned and one private not-for-profit lower level (II–III) primary health facilities in rural towns or villages. Data was also collected from two rural private secondary facilities (level IV) and a rural government-owned hospital. With the exception of the Level II health facilities, all facilities provided inpatient and outpatient services. The coverage varied between facilities, with higher level health facilities providing services to larger populations. In Mozambique, five of the facilities were primary health facilities  was the highest amongst the four illnesses, irrespective of the level of facility under consideration, while the lowest was for diarrhoea. In both Mozambique and Uganda, the cost of inpatient admissions was the highest for malaria  tending to drive the majority of outpatient visits and inpatient visits in both settings. This may be because lower-level facilities, often located in rural and remote areas lack adequate health personnel, diagnostics, and drugs to accurately diagnose and provide appropriate treatment , 35. AnoIn both countries, across all facilities, the lowest outpatient and inpatient unit costs were associated with diarrhoea. In Uganda, the highest outpatient costs were associated with pneumonia and other respiratory infections, and the highest inpatient costs were associated with malaria. In Mozambique, the highest outpatient and inpatient costs were associated with malaria. In both counties, the highest costs were seen at the hospital level. This highlights the intensive resource use in the treatment of malaria and pneumonia, consistent with other studies conducted in sub-Saharan African countries –40. TimeThe cost components that had the largest contribution to the bottom-up unit costs of outpatient and inpatient visits were drugs and diagnostics, followed by staff. This is similar to what other studies have found. For example, in Ghana the largest contributors to the cost of treatment were staff, diagnostics and consumables , in ZambWhile treatment services for all three conditions were provided across all levels of health facilities, there were differences in costs between facilities, with larger facilities having higher levels of unit and total costs. This is likely because higher level facilities are more likely to receive proportionally more specialized drugs and equipment, and a larger and more varied cadre of healthcare providers. Higher costs of treatment at these facilities might be inevitable for specialised services. However, in settings where iCCM is implemented, and care and treatment for MDP may be available in a timely manner, the resource use for diagnosis and treatment at higher level facilities could be lower than observed in this study. However, in some cases, the unit costs (top-down and bottom-up estimates) were lower at higher level facilities than lower-level facilities, possible economies of scale as a larger number of patients were seen at these facilities. Improvements in efficiency and infrastructure at lower-level facilities may have a similar effect, and potentially reduce opportunity costs of higher level facilities.A bottom-up and top-down approach for estimating unit costs was used. On comparing the two, it is likely that the bottom-up cost estimates are more accurate as these were able to capture resource use for service provision in a more comprehensive way than the top-down estimates. Both sets of estimates are helpful in thinking through budget allocations at facility and national levels. The bottom-up approach can capture site level differences and differences in marginal costs, which might help the (re)allocation of resources at the level of the health facility or health centre, especially in settings where financial decision-making is decentralized. On the other hand, the top-down approach help inform the long-run national average costs of service provision, and aid the allocation of resources and priority setting .This study has a few limitations. First, in several of the facilities, cases of pneumonia were not distinguished from other respiratory infections, and in these analyses, these have been conflated into one category. As a result, there may be some variation in the unit and total costs associated with the treatment of pneumonia in these settings. However, the estimates in this study are comparable to those in previously conducted studies. Second, in Uganda, it was not possible to calculate top-down unit costs for inpatient treatment of diarrhoea as service statistics could not be obtained. However, estimates using a bottom-up approach are provided, which provide a clear understanding of resource use for this condition at the health facility level. Third, this study did not assess patient costs and, therefore, it was not possible to estimate the associated cost burden on household. For Uganda, this has been published in a separate study , which fThis study estimated the unit costs of treating MDP among children under the age of 5 years in routine settings at different types of health facilities in rural Uganda and Mozambique. The findings indicate that higher treatment costs are seen for with malaria and pneumonia, particularly if treated at higher-level facilities. Encouraging timely care-seeking at the community level and improving supply side readiness for treatment at lower-level facilities could help improve health outcomes, reduce direct and opportunity costs for the health systems and households in resource-constrained settings, and achieve universal health coverage.Additional file 1: Top down unit costs in 2013 and 2014 of visit (USD) for any illness, children under 5.


Neutralization activities against the recombinant vesicular stomatitis virus bearing SFTS virus Gn/Gc as envelope proteins were observed with three anti-Gn and six anti-Gc mAbs. Therapeutic activities were observed among anti-Gn, but not anti-Gc mAbs with neutralizing activities. These results propose an effective strategy to obtain promising therapeutic mAb candidates for SFTS treatment, and a necessity to reveal precise roles of the SFTS virus Gn/Gc proteins.Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease with a high case fatality rate caused by the SFTS virus, and currently there are no approved specific treatments. Neutralizing monoclonal antibodies (mAbs) against the virus could be a therapeutic agent in SFTS treatment, but their development has not sufficiently been carried out. In the present study, mouse and human mAbs exposed to the viral envelope proteins Gn and Gc (16 clones each) were characterized in vitro and in vivo by using recombinant proteins, cell culture with viruses, and an SFTS animal model with IFNAR Scrpatients , was lowpatients . The mecpatients ,10. Whilpatients , none ofPhenuiviridae, Gn and Gc seem to play a role in cell attachment and membrane fusion, respectively [The genome of the SFTS virus encodes four proteins, nuclear protein, glycoprotein (GP), RNA-dependent RNA polymerase, and a non-structural protein. GP is the viral envelope protein and is cleaved into Gn and Gc (Gn/Gc) during synthesis. While roles of Gn/Gc proteins have not been well explored, based on homology of amino acid sequence of the protein among viruses of ectively ,14. Actiectively ,16. TherD values 25.9 nM for MAb4-5 and 104 pM for Ab10), one of the envelope proteins of SFTS virus, and had neutralizing activities against the virus (approximately 80% inhibition at 5 μg/mL for both). The neutralizing activity of the human sera was 2000 [-/- mice [Monoclonal antibodies (mAbs) and immunoglobulins against viruses are in general expected to show therapeutic effects through inhibition of viral infection, enhancement of phagocytosis by opsonization, destruction of infected cells, and others , and havwas 2000 . Among t-/- mice . UnfortuIn the present study, to reveal the potential of mAbs as therapeutic agents in SFTS treatment, we characterized mouse and human mAbs specific to the envelope proteins of the SFTS virus, Gn and Gc, in in vitro and in vivo experiments. The mAbs characterized were prepared in the present and previous studies, obtained commercially, or made based on publicized studies. Some mAbs showing high affinities and/or neutralizing activities were examined for therapeutic effects in an SFTS animal model. Data obtained will be useful in further development of therapeutic mAbs for SFTS treatment and in profound understanding of SFTS envelope protein roles.Strep-Tag II monoclonal antibody were purchased from FUJIFILM Wako Pure Chemical Corp. , R&D Systems , and IBA-Lifesciences , respectively.Vero cells  were cultured in DMEM  supplemented with 5% heat-inactivated foetal calf serum (FCS) and antibiotics . Upon inoculation, Vero cells were maintained in DMEM with 2% FCS (DMEM-2FCS) and antibiotics. 293T cells, HuH-7 cells and NIH/3T3 cells were cultured in DMEM (Sigma) supplemented with 10% FCS and antibiotics. BTI-TN-5B1-4 (Tn5) insect cells  were cultured in EX-CELL 405 Serum-Free Medium for Insect Cells (Sigma) supplemented with antibiotics (Gibco) at 28 °C. Normal human IgG, mouse IgG1 isotype control, and anti-SFTS virus SPL030 strain was prepared as described previously . LysatesFor the preparation of a soluble form of the SFTS virus Gn, cDNA encoding amino acid position 1 to 429 of SPL030 strain GP fused with strep tag and HiBiT sequence (STH) at the C-terminus was cloned into pCAGGS plasmid . For Gc,Soluble forms of the SFTS virus Gn fused with Fc portion of mouse IgG2a at the C-terminus were prepared as follows: cDNAs encoding amino acid position 1 to 429 or 1 to 340 of SPL030 strain GP were cloned into pCAGGS plasmid containing mouse IgG2a Fc cDNA. Two resultant plasmids and a control plasmid (no insertion) for Gn429mFc, Gn340mFc, and IgkmG2aFc proteins were transfected with TransIT LT1 transfection reagent  into 293T cells cultured in Opti-MEM culture medium (Thermo Fisher Scientific). Three days post transfection, supernatants were harvested and mixed with Protein G Sepharose 4 Fast Flow  to purify Fc-containing proteins.To produce a soluble form of the SFTS virus Gn in a baculovirus expression system, cDNA encoding amino acid position 21 to 451 of SPL030 strain Gn fused with Honey Bee Mellitin secretion signal at the N-terminus and 8 × histidine at the C-terminus was cloned into pAcYM1 . RecombiMonoclonal antibodies from the SFTS patient were established as previously described . In brieA human mAb Ab10 clone specific to the SFTS virus Gn reported by Kim et al.  was prep® mice to obtain antibody-producing hybridoma and screening of hybridoma was performed with ELISA with each protein as antigens . To obtain purified mAbs, hybridoma were cultured in Hybridoma Serum-Free Medium  and HiTrap™ Protein G HP Columns (Cytiva) were used. Isotypes of mouse mAbs obtained were determined with IsoStrip™ Mouse Monoclonal Antibody Isotyping Kit . Commercially available mouse mAbs specific to SFTS virus Gn and Gc were purchased from Immune Technology Corp. . Two mAbs, C3A11 and C6C1 clones, were described previously [NIH/3T3 cells were transfected with an SFTS virus Gn/Gc expression plasmid (pC030GP)  by usingeviously .Replication-defective vesicular stomatitis virus vector (VSVΔG) encoding green fluorescent protein (GFP) and carrying SFTS virus Gn/Gc as envelope proteins  was usedTo test whether neutralizing mAbs had different inhibitory effects on VSVΔG carrying the SFTS virus Gn/Gc, mAbs, mouse IgG1 isotype control, and normal human IgG were used at 16 µg/mL before (the vector was treated with antibodies then inoculated onto Vero cells) and after (the vector was inoculated first onto Vero cells in the absence of antibodies then cultured with media containing antibodies) viral adsorption to cells. When antibodies were used before adsorption, the vector was treated with antibodies for 1 h on ice then inoculated onto Vero cells in a cold room. After 1 h incubation, inocula were removed and cells were washed with cold DMEM-2FCS three times then cultured in the absence of antibodies. When antibodies were used after adsorption, the vector was inoculated onto Vero cells without antibodies in a cold room. After 1 h incubation, inocula were removed and cells were washed with cold DMEM-2FCS three times then cultured in the presence of antibodies. Cell culture and reporter analyses were performed as described in the previous paragraph.-/-) [2 50% tissue culture infectious dose (TCID50) of SFTS virus SLP030 strain were used as a fatal animal model of SFTS. To assess the therapeutic effects of mAbs, the inoculated mice (3 to 6 mice per group) were intraperitoneally injected with indicated doses of mAbs daily between 1 day-post inoculation (dpi) and 6 dpi. Mice were observed daily up to 14 dpi.Mice lacking type I interferon receptor (IFNAR-/-)  subcutan2, DOJINDO Laboratories, Kumamoto, Japan) at optimized dilutions and incubation for 30 m. After washing, plates were incubated with peroxidase-conjugated streptavidin  at 1:2000 for 30 m. Colorization was performed with ABTS tablet (Roche) and optical densities at 405 nm were measured .SFTSVGnSTH and SFTSVGcSTH proteins (described above) were used as antigens to coat Nunc MaxiSorp™ flat-bottom (Thermo Fisher Scientific). After blocking with skim milk, antigens were first incubated with unlabeled mAbs at indicated concentrations for 30 m, followed by the addition of biotin-labeled mAbs  proteins were used as antigens to coat Nunc MaxiSorp™ flat-bottom. After blocking with skim milk, antigens were incubated with biotin-labeled mAbs followed by peroxidase-conjugated streptavidin or directly incubated with goat anti-mouse IgG (H+L) secondary antibody, HRP (Thermo Fisher Scientific). Colorization and optical density measurement were performed as described above. The ratios of densities with peroxidase-conjugated streptavidin to those with anti-mouse IgG (H+L) secondary antibody were calculated.PBMCs were obtained from SFTS-recovered patients for the preparation of human mAbs. Protocols and procedures were approved by the research ethics committee of the National Institute of Infectious Diseases for the use of human subjects (no. 539). Experiments with animals were performed in strict accordance with the Animal Experimentation Guidelines of the National Institute of Infectious Diseases. The protocol of animal experiments were approved by the Institutional Animal Care and Use Committee of the National Institute of Infectious Diseases (nos. 117143 and 118121).p-value less than 0.05 was considered significant statistically. Student’s t-test was used to determine the significance of the difference between the means of two groups.Survival curves were plotted using the Kaplan-Meier method and effects of mAb administration was evaluated using a log-rank test. A ® mice immunized with SFTS virus Gn and Gc proteins. Human mAbs MAb4-5  in ELISA and half maximal inhibitory concentrations (IC50) in a neutralization test (NT). The maximum concentration of mAbs examined were 16µg/mL. Results are shown in 50 in ELISA of the anti-Gn and the anti-Gc mAbs were between 0.016 and 2.2 μg/mL. In NT, mAb clones which had IC50 of less than 16 μg/mL were 4M5 , Ab10 , No. 22 , No. 23 , 5M5 , 5M9 , C6C1 , and Ab3 .Four human mAbs  were obtained by using PBMCs from an SFTS patient and SFTS virus Gn protein . Three murine mAbs  were established from BALB/c mice immunized with the SFTS virus Gn/Gc-expressing cells, and all the three recognized Gc protein in ELISA. Murine mAbs to Gn  and to Gc  were established from GANP4-5 , Ab10 , and Ab-/- mice. mAb doses used were 0.02 mg/day for 4M5 and 4M6 (control for 4M5) clones and 1 mg/day for the others. As shown in Based on the results obtained in ELISA, NT, and/or mAb usability, anti-Gn mAb clones  and anti-Gc mAb clones  were tested with regard to their therapeutic effects with SFTS virus-infected IFNAREpitopes recognized by anti-Gn mAbs, Ab10, 4M5, and M1-E5 were examined with SFTS virus Gn protein in competition ELISA . Among the three mAbs, as shown in Epitopes of Ab10, 4M5, and M1-E5 mAbs were further examined in ELISA with antigens including truncated Gn proteins, which were consisted with amino acid position 1 to 429 (Gn429mFc) or 1 to 340 (Gn340mFc) of the SFTS virus Gn. Both of the Ab10 and M1-E5 mAbs showed similar reactivity to Gn429mFc and Gn340mFc, however, 4M5 mAb only showed a positive signal to Gn429mFc .SFTS virus Gn and Gc proteins were used in Western blotting under reduced conditions. As shown in Epitopes recognized by anti-Gc mAbs, No. 22, C6C1, and Ab3 were examined with the SFTS virus Gc protein in competition ELISA. Among the three mAbs, the binding of biotinylated mAbs were reduced by unlabeled respective mAbs dose-dependently , indicatTo speculate which steps during viral infection were affected by neutralizing anti-Gn mAbs  and anti-Gc mAbs , mAbs were used at two different time points in NT  and results were compared between the time points. Inhibitory effects of Ab10 and M1-E5 on VSVΔG carrying the SFTS virus Gn/Gc infection were reduced when the mAbs were used after viral adsorption to cells, in comparison to those when used before . Such a 50 values or reduced viral infectivity to nearly 50% at 4 or 16 µg/mL. In contrast, any of anti-Gc mAbs examined did not show therapeutic effects at a dose of 1 mg/day x 6 days, whereas some anti-Gc mAbs showed comparable to or higher neutralizing activities than anti-Gn mAbs. From these results, we propose that to obtain promising therapeutic mAb candidates for SFTS treatment effectively, mAbs recognizing Gn protein of the SFTS virus and having neutralizing activities against the virus should be picked up during selection. Because Ab10 and 4M5 did not interfere their binding to Gn protein with each other, and the EC50 value of 4M5 was not so low (indicating low affinity), mAbs recognizing 4M5′s epitope with high affinities, if obtained, might become a promising therapeutic candidate such as Ab10. Because the number of mAbs with therapeutic effects were limited, it is unfortunately unclear whether isotypes and affinities to antigens of mAbs were important as essences to predict therapeutic effects against SFTS.Among 32 mAbs recognizing envelope proteins of SFTS virus Gn/Gc, three clones, Ab10, 4M5, and M1-E5, showed therapeutic effects in murine model of SFTS. Ab10 is a human mAb which has been reported in Kim’s study , in whicKim et al. reported that the Ab10 clone recognizes a conformational epitope existing within C-terminal half of Gn . Virus nWhereas analyses of the neutralization mechanism(s) of mAbs are not the original goal of the present study, it was observed that the inhibitory effects of two anti-Gn mAbs, Ab10 and M1-E5, on viral infection were decreased when the mAbs were used after the viral attachment to cells, suggesting that the mAbs might block the attaching step of SFTS virus infection. This is consistent with the previous work with SFTS virus-related viruses reporting a role of the Gn protein during infection . InteresResults of neutralizing activities and therapeutic effects of mAbs against SFTS virus revealed several points which should be resolved in future studies to understand SFTS pathogenesis more profoundly and enhance development of therapeutic agents. First is the role of the Gn protein of SFTS virus, especially in SFTS murine models. The anti-Gc mAb, Ab3, showed the strongest neutralizing activity among 32 mAbs examined in the present study, but did not show any therapeutic effects such as inhibition or delay of body weight loss and death upon SFTS virus infection. Although having less neutralizing activities than Ab3, some anti-Gn mAbs apparently showed therapeutic effects. Therefore, we speculate that there might be an unknown role(s) of Gn in vivo, for example, stimulation of some immune cells by binding to their surface, which eventually might lead to hyper cytokine production and could be inhibited by some anti-Gn mAbs. Second is the different involvement of Gn/Gc proteins of authentic SFTS virus particles and of VSV pseudotypes in infection to cells. VSV pseudotypes are a safe and convenient tool due to their replication incompetency and reporter expression, however there is currently no evidence which supports the identical or similar functions of Gn/Gc between authentic SFTS virus particles and VSV pseudotypes. Contribution of Gn might be greater than that of Gc in authentic viral particles and vice versa in VSV particles. Supportive of this speculation is that maximum inhibitory effects of anti-Gn mAbs were approximately 40% of control, while those of anti-Gc mAbs were 40% or less . In thisOur results in the present study propose an effective strategy to obtain promising therapeutic mAb candidates for SFTS treatment, and a necessity to reveal precise roles of the SFTS virus Gn/Gc proteins.


To retrospectively investigate the early postoperative range of motion (ROM)  after total knee arthroplasty (TKA) and to test for associations (a) with long-term outcome in terms of ROM and (b) with a disease-specific knee score.A retrospective analysis was performed in patients with previous primary TKA. Data taken from the medical records were ROM from preoperative and postoperative days 4, 7 and 10 and 1 year. As patient-reported outcome the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC Score) was taken from preoperative and one year after TKA.316 patients (330 knees) were available. Only negligible correlations were determined between ROM at twelve months postoperative and ROM in the early postoperative days . Similarly, only negligible correlations were determined between ROM in the early postoperative days  and the 1-year WOMAC.From the main findings it would seem that steepness of ROM ascent in the early postoperative days is of minor importance for (a) long-term ROM and (b) long-term knee score outcome after TKA. In daily clinical practice the question often arises as to when to discharge a patient after routine total knee arthroplasty (TKA). The fact that there is no generally accepted consensus on this question is also reflected by the length of hospital stay, which varies in general between two and 15 days  with eveRegarding associations between early postoperative ROM and long-term ROM after TKA, Bade et al. found that knee flexion at postoperative day 2 was not related to knee flexion at six months postoperative . Others Based on the above-mentioned lack of evidence it was the aim of the study to test whether early postop ROM  was associated with ROM at 12 months postoperative (Hypothesis 1) or with the WOMAC Score at 12 months postoperative (Hypothesis 2).The study design was retrospective observational. Data already available from clinical routine were analyzed after approval by the ethics committee of the Medical University. Consecutive patients who previously underwent primary TKA as part of the clinical routine were analyzed. Cases were excluded in the case of: (a) incomplete WOMAC data, (b) incomplete ROM data, (c) primary prostheses other than cruciate-retaining. Included were patients with Triathlon CR, Scorpio CR and Scorpio NRG CR implants .During the study period (January 2005 to August 2015), 2093 TKA were performed at our institution. Of those, 988 had to be excluded because either the preoperative or postoperative or both WOMAC Scores were incomplete. Of the remaining 1105 TKA 242 were excluded because other implants than above-mentioned CR implants had been used. Of the remaining 863 primary TKA only 330 cases could be included in the final analysis. The rest had to be excluded because no information on ROM was available.ROM data collected with goniometers were taken from the medical records for the following points in time: preoperative, postoperative days 4, 7 and 10 and 1 year (± 6 months) postoperative. The early postoperative measurements were taken from the obligatory daily ROM documentation of our physiotherapists. The one-year ROM was taken from the documentation of the one-year outpatient visit.For patient-reported outcome measurement the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) was applied in the German-language version . The queData were extracted from the records and stored in spreadsheet format. Further analyses were performed with SPSS. As descriptive statistics medians and inter-quartile-ranges were determined. Kolmogorov–Smirnov tests indicated that the data sets were not normally distributed. Therefore, the above-mentioned Hypotheses 1–4 were tested with Spearman correlation coefficients; alpha was defined as 0.05. With regard to Spearman's correlation coefficient, the size of the correlation was assumed as very high from 0.90 to 1.00, high from 0.70 to 0.90, moderate from 0.50 to 0.70, low from 0.30 to 0.50 and negligible from 0.00 to 0.30 .316 patients  with a median age of 70 years (IQR 12) were available for analysis.Knee ROM (Medians ± Interquartile-Ranges) changed from 110 ± 20° preoperative to 65 ± 20° on day 4, further to 85 ± 15° on day 7 and 90 ± 15° on day 10. Twelve months after surgery ROM was 110 ± 20°. The total WOMAC Score was 52 ± 28 preoperative and changed to 16 ± 29 at 12 months postoperative  Table .Table 2CSimilarly, regarding Hypothesis 2, only negligible correlations were determined between ROM in the early postoperative days  and the 12-month WOMAC outcome Table .Table 3CThe most important finding made in the study was that the ROM obtained in the early postoperative days  showed only negligible correlations with the WOMAC score at 12 months postoperative and ROM at 12 months postoperative.When attempting to compare our findings with previous research, it became obvious that evidence on this specific issue is scarce. Regarding the influence of early postoperative ROM on long-term postoperative ROM, supporting evidence comes from Bade et al., who found that knee flexion at postoperative day 2 did not influence knee flexion at six months postoperative . Our finWith regard to the influence of early postoperative ROM on long-term postoperative knee score outcome, previous researchers tested for associations between knee flexion at discharge and the Oxford knee score one year postoperative . They foOur findings are regarded as highly clinically relevant. Due to the fact that early postoperative ROM did not predict long-term knee score outcome or long-term ROM after TKA, it does not seem necessary to force ROM gain too strongly in the initial postoperative days. Probably, preoperative ROM and proper biomechanics of the TKA are predictors of much greater importance than is steepness of ROM ascent in the early postoperative period. Fast-track rehabilitation programs that aim to substantially accelerate ROM gain in the early postoperative period might also be questioned. Although the findings from the current study support the practice of not forcing ROM gain too aggressively in first postoperative weeks there should be a limit to this in patients that do not achieve a minimum of 90° knee flexion within the first postoperative weeks. Yao et al. demonstrated that these patients can successfully be treated with early manipulating under anesthesia. preoperative ROM instead early-postoperative ROM on long-term ROM outcome. The preoperative maximum knee flexion angle was found positively correlated with the flexion angles at 6 months [Other researchers investigated the effect of 6 months , 21, 22 6 months , 22–24. 6 months  and 12 m6 months , 26. Whepreoperative ROM and long-term postoperative knee scores, it appears that there are only few studies available. Naylor et al. reported that the preoperative maximum knee flexion angle was not a significant predictor of the Oxford Knee Score at 1-year postoperative [Trying to find publications on a potential relationship between perative . Similarperative . Howeverperative . In synoThe following limitations are acknowledged. This was a retrospective study with the weaknesses typically associated with such studies. We were not able to investigate for parameters other than those already available . It is regarded as significant limitation of the study that from 2093 TKA in the study period only 330 cases could be included in the analysis due to missing WOMAC or ROM data or due to the fact that other than the above-mentioned CR implants had been used. A fact which may have led to selection bias. Another weakness was that ROM was not always measured by the same investigator. It was measured by physical therapists in the early postop phase and by physicians at the 1-year outpatient visit.Its strength is that the current work generated scientific findings in a field where publications are very rare. Moreover, the findings obtained from the current work are based on case numbers far superior to those of previous research.From the main findings that early postoperative ROM did not influence long-term knee score outcome or long-term ROM after TKA, it does not seem necessary to force ROM gain too strongly in the initial postoperative days. Probably, preoperative ROM and proper biomechanics of the TKA are predictors of much greater importance than is steepness of ROM ascent in the early postoperative period.


EP153R and EP402R gene clusters were knocked out. Based on the deletion mutant, a further deletion from the MGF_360-12L, MGF_360-13L to MGF_360-14L genes was obtained. The five-genes knockout virus was designated as ASFV-ΔECM3. To investigate the efficacy and safety of the ASFV-ΔECM3 virus as a vaccine candidate, the evaluation of the virus was subsequently carried out in pigs. The results showed that the ASFV-ΔECM3 virus could induce homologous protection against the parental isolate, and no significant clinical signs or viremia were observed. These results show that the contiguous deletion mutant, ASFV-ΔECM3 encompassing the EP153R/EP402R and MGF_360-12L/13L/14L genes, could be a potential live-attenuated vaccine candidate for the prevention of ASFV infection.African swine fever virus (ASFV) represents a serious threat to the global swine industry, and there are no safe or commercially available vaccines. Previous studies have demonstrated that inactivated vaccines do not provide sufficient protection against ASFV and that attenuated vaccines are effective, but raise safety concerns. Here, we first constructed a deletion mutant in which  African swine fever (ASF), a fatal hemorrhagic and highly contagious disease in swine caused by the African swine fever virus (ASFV), has led to significant economic losses in the global swine industry  belonged to the genotype II and was isolated from a piglet with severe infection in Guangdong  were collected from specific-pathogen-free (SPF) pigs as described previously  virus, respectively. The preformed monolayers were prepared in six-well plates and infected at an MOI of 0.01. The GZ201801 virus was titrated by limiting dilution in cultures of BMDMs cells using a monoclonal antibody (mAb) against p30 protein (prepared in our lab). Briefly, 10-fold serial dilutions of viruses were added to 96-well plates. After 4 days, the cells were fixed with 4% paraformaldehyde and permeated with 0.1% Triton X-100 for 10 min, and washed with PBS. The cells were blocked with 3% BSA for 1 h and then incubated with p30 mAb for 1 h at 37°C and subsequently with Alexa488-labeled anti-mouse secondary antibody IgG (H+L) for 30 min at 37°C. Finally, the cells were observed under a fluorescence microscope . The ASFV-ΔECM3 was directly observed under a fluorescence microscope. Virus titers were calculated by the Reed and Muench method . BMDMs were transfected with 2 μg of the recombinant transfer vector p72eGFPΔEP153R/ΔEP402R using jetPEI-macrophage transfection  and infected with the GZ201801 virus at an MOI of 1 at 6 h post-transfection. After 48 h, ASFV-ΔEP153R/ΔEP402R viruses were purified in BMDMs by limiting dilution assay. In brief, the viruses were serially diluted in 96-well plates. After virus dilution, the green fluorescent cells were picked by limited dilution. BMDMs were transfected with the p72mCherryΔMGF_360-12L/13L/14L plasmid and infected with the ASFV-ΔEP153R/ΔEP402R virus at an MOI of 1 at 6 h post-transfection. After 48 h, the viruses were purified in BMDMs by limiting dilution assay. The cells exhibiting both green and red fluorescence were picked by limited dilution.The genes were knocked out and generated by homologous recombination by infection with parental virus GZ201801 and transfection with the recombination transfer vectors (p72eGFPΔEP153R/ΔEP402R and p72mCherryΔMGF_360-12L/13L/14L) described in our previous study  and freeze-thawed three times. Virus titers were calculated as described previously.The virus growth curves of the ASFV strain GZ201801 and ASFV-ΔECM3 virus were analyzed. The cells were seeded in six-well plates and infected at an MOI of 0.01. After 1 h of adsorption at 37°C under 5% COn = 5 per group) were infected with the ASFV-ΔECM3 virus or the WT virus intramuscularly (IM), both at a dose of 105 TCID50. ASFV-inoculated pigs were monitored for rectal temperature as described previously  and the immunized group (n = 5) were IM challenged with 102 HAD50 of the virulent parental GZ201801 virus and later subjected to necropsy examination at 75 days post-challenge (dpc). To evaluate pathological changes in pigs immunized with ASFV-ΔECM3, spleen, lung, liver, kidney, and mandibular lymph node tissues were collected and stained with H & E stain and TUNEL assay. The samples were collected and determined following the methods mentioned earlier.Efficacy evaluation of protection by vaccination with ASFV-ΔECM3 in pigs. To investigate the immunogenicity of the ASFV-ΔECM3 virus in pigs, 10 piglets were divided into two groups. Five pigs were immunized with 10African swine fever virus genomic DNA was extracted from tissue homogenate  and whole blood using GlinX Viral Nucleic Acid Extraction kits . The qPCR procedure was carried out on a QuantStudio 5 system  according to the protocol of ASFV dtec-qPCR test kits .® African Swine Fever Competition ELISA kits  were used to detect specific anti-ASFV antibodies in serum. For each sample, the competition percentage (S/N%) was calculated according to the manufacturer's instructions. Less than or equal to 40% was considered positive, between 40 and 50% was considered doubtful, and ≥50% was considered negative.The ID ScreenAnimal experiments were performed under Animal Biosafety Level 3 (ABSL-3) conditions in The Spirit Jinyu Biological Pharmaceutical Co., LTD and approved by the China National Accreditation Service for Conformity Assessment (CNAS) with a license of CNAS-BL0101. The protocols of ASFV infection were approved by the Ministry of Agriculture and Rural Affairs.t-test. A p-value < 0.05 was considered statistically significant.Statistical analyses were performed using unpaired, two-tailed Student's EP153R-EP402R genes from 72,837–74,465 nt in the viral genome was achieved by homologous recombination using the p72eGFPΔEP153R/ΔEP402R vector  repository, accession number: The animal study was reviewed and approved by Spirit Jinyu Company.HC and HW conceived and designed the experiments. ZX, YLiu, DD, JL, LL, LG, ZC, XL, and HC performed the experiments. YLi, WY, and QZ analyzed the data. HC and QS contributed reagents/materials/analysis tools. ZX, JL, HW, and HC wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (Grant Nos. 32170161 and U19A2039), the National Key Research and Development Program of China (Grant Nos. 2021YFD1801401 and 2021YFD1801300), and the Key-Area Research and Development Program of Guangdong Province (2019B020211003).DD and QS were employed by the Spirit Jinyu Biological Pharmaceutical Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer H-JQ declared a shared affiliation with the authors HC, ZX, YLi, JL, ZC, YLiu, WY, LL, QZ, YS, and XL at the time of the review. The handling editor XY declared a shared affiliation with the authors HC, ZX, YLi, JL, ZC, YLiu, WY, LL, QZ, YS, and XL at the time of the review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.