Culicoides species are arbovirus vectors. Effective virome profile surveillance is essential for the prevention and control of insect-borne diseases. From June to September 2021, we collected eight species of female mosquito and Culicoides on Zhoushan Island, China, and used meta-viromic sequencing to analyze their virome compositions and characteristics. The classified virus reads were distributed in 191 genera in 66 families. The virus sequences in mosquitoes with the largest proportions were Iflaviridae (30.03%), Phasmaviridae (23.09%), Xinmoviridae (21.82%), Flaviviridae (13.44%), and Rhabdoviridae (8.40%). Single-strand RNA+ viruses formed the largest proportions of viruses in all samples. Blood meals indicated that blood-sucking mosquito hosts were mainly chicken, duck, pig, and human, broadly consistent with the habitats where the mosquitoes were collected. Novel viruses of the Orthobunyavirus, Narnavirus, and Iflavirus genera were found in Culicoides by de-novo assembly. The viruses with vertebrate hosts carried by mosquitoes and Culicoides also varied widely. The analysis of unclassified viruses and deep-learning analysis of the “dark matter” in the meta-viromic sequencing data revealed the presence of a large number of unknown viruses.Mosquitoes and biting IMPORTANCE The monitoring of the viromes of mosquitoes and Culicoides, widely distributed arbovirus transmission vectors, is crucial to evaluate the risk of infectious disease transmission. In this study, the compositions of the viromes of mosquitoes and Culicoides on Zhoushan Island varied widely and were related mainly to the host species, with different host species having different core viromes. and many unknown sequences in the Culicoides viromes remain to be annotated, suggesting the presence of a large number of unknown viruses. Culicoides species are common vectors of arboviruses that cause a variety of significant diseases, including dengue virus, Zika virus, Getah virus, West Nile virus, Japanese encephalitis virus, bluetongue virus, and Banna virus  were collected at the sampling sites. The dominant mosquito species differed among sites (Table S1). About 5,000 Culicoides were collected from the sites; based on their morphological characteristics, three dominant species  were identified . The viral families commonly identified in the mosquito viromes were Iflaviridae (30.03%), Phasmaviridae (23.09%), Xinmoviridae (21.82%), Flaviviridae (13.44%), and Rhabdoviridae (8.40%), found mainly in Culex pipienspallens, Anopheles sinensis, Culex tritaeniorhynchus, and Ae. albopictus, respectively.Meta-viromic sequencing of 28 vector pools yielded an average of 9,634,984  reads per pool for virus discovery and characterization. Reads assigned to viruses represented 0.007% to 97% of all reads in each pool . The claCulicoides (Polycipiviridae), Ae. albopictus (Rhabdoviridae), and A. sinensis  contained a large number of Iflaviridae reads and was obviously an outlier, probably because the mosquitoes were full of blood DNA, and dsRNA. The major viral genome types varied among the sampled speices. For example, the major type in Culicoides and Ae. albopictus was ssRNA+, whereas that in A. sinensis was ssRNA–. Among all samples, we annotated 14 viruses with vertebrate hosts (Table S3).Among all 10 viral taxonomy, single-strand (ss)RNAAe. albopictus. Some contigs were found to have 80% to 100% aa identity with the NS5 region of dengue virus 3  and 80% identity with that of Aedes aegypti, suspected to reflect endogenous viral elements or part of the Aedes genome. No viral gene was found in the sequences annotated as dengue virus and West Nile virus, and the corresponding PCR results were negative. In Culicoides, Quang Binh virus (Flaviviridae), Wuhan mosquito virus 9 and Culex tritaeniorhynchus rhabdovirus (Rhabdoviridae), and a novel Thimiri orthobunyavirus (Bunyaviridae) were identified. A small amount of Culex flavivirus was found in Culicoides and C. tritaeniorhynchus. Wuhan mosquito virus 9 was found in midges and mosquitoes, and its sequence included open reading frames (ORFs) 1 to 3, glycoprotein, and RNA-dependent RNA polymerase (RdRp). The conserved domains include Mononeg_RNApol and paramyx_RNAcap. Phylogenetic analysis based on the RdRp showed the closest affinity to Wuhan mosquito virus 9  that were broadly consistent with the habitats where the mosquitoes were collected. Blood meals from A. subalbatus samples from the village and Ae. albopictus samples from the residential area were mainly human, and that of C. p. pallens from the residential area was mainly duck. Those of C. tritaeniorhynchus collected from the farmland and pig farm were chicken and pig, respectively. Rodent sequences were found in various samples, potentially representing a link in the mosquito-borne virus transmission chain. The viruses carried by the blood-sucking mosquitoes can be carried by rats, chickens, pigs, and humans, concordant with the blood meal calls.We analyzed blood meals in five pooled mosquito samples from four sampling sites. We compiled their corresponding Chordata contigs and chose animals that mosquitoes and Culicoides samples (Midge_3 and Midge_4), which was named Culicoides narna-like virus 1 (CNV1). The longest ORF in the 2,485-bp genome of CNV1 encodes an RdRp related closely to Sanya ochthera mantis narnavirus 2 . The narnavirus hosts phylogenetically closest to CNV1 are arthropods, including Ochthera mantis and Drosophila  sequences were found in the Midge_1 and Midge_3 samples. Parts of the L segment  and S segment  were obtained by de-novo assembly, and the L segment contained a conserved domain annotated as the Bunya RdRp. Phylogenetic analysis showed that it was closest to Thimiri orthobunyavirus , rhv_like (Picornavirus capsid protein domain_like), and RNA_helicase (Table S5). A phylogenetic tree constructed based on a polyprotein  suggested that the contig represented a novel Iflavirus, a picorna-like virus that infects arthropods (C. pipiens and showed 93.01% nucleotide identity with the reference genome (KX883461.1 3226 bp). Phylogenetic analysis showed that the RdRp gene was related most closely to Zhejiang mosquito virus 3  and Xanthi narna-like virus (QRD99904.1).De-novo read assembly revealed the presence of a novel narnavirus (Narnaviridae) in two osophila . Many Sinyavirus . We alsothropods . Anothern = 2,704,391) of the contigs were unannotated; 2,180,762 were annotated .Nearly 50%  were very abundant in Culicoides viromes, which has not been reported previously. Meta-viromic sequencing of C. impunctatus viromes revealed viruses of the Polycipiviridae family, but in only one group (Thimiri orthobunyavirus (genus Orthobunyavirus), originally isolated in birds  and infect mammals and birds  and Xincheng mosquito virus (Xinmoviridae) were found in A. sinensis and a relatively complete Xincheng mosquito virus was assembled. Similar results were obtained in previous mosquito virome studies conducted in Hubei Province, China (A. sinensis in Hubei Province (~96.7% nucleotide similarity), and other viruses have similar findings, reflecting small differences in the core viromes of mosquito species at this geographic scale.Different mosquito species may carry unique virus groups , 29, whi diverse –29. Moree, China , 51, bute, China . Speciese, China . The XinC. tritaeniorhynchus mosquitoes, for which the blood meal source was identified as chickens on a farm. This fragment accounts for 70% of the full length of the virus (GenBank no. MK951780.1). As viral proteins were found in it, we consider it to be an exogenous viral sequence. When the virus is incorporated into the mosquito's blood meal during feeding, the meta-viromic of satiated mosquitoes can reflect the viral diversity of many mammals, birds, and humans by means of xenosurveillance -treated water to remove potential surface microorganisms. RNA was dissolved in 30 μL non-RNase DEPC and frozen at −80°C.One to 10 female mosquitoes or 20 to 50 DNA library construction and sequencing were performed at the Beijing Genomics Institute. A nucleic acid microbes purification kit  was used to enrich the viruses in all samples. The remaining RNA was fragmented to about 280 bp and then reverse transcribed into cDNA, followed by second-strand synthesis. Using the synthetic double-stranded DNA, a DNA library was constructed through end repair, unique dual-index adaptor ligation, and PCR amplification. The library was qualified with a Qubit 4.0 fluorometer  and the fragment size was analyzed with a Qsep100 system . After circularization and DNA nanoball generation, the resulting libraries were sequenced with an MGI high-throughput sequencing set (PE150) on MGISEQ-2000/T7 platforms  . Clean rCulicoides, we first examined the overall proportions of nonhost reads that could be assigned to viral, bacterial, eukaryotic, and archaeal organisms. BLAST  was used to align the scaffold sequence with the NT database. Then, the sequences annotated as viruses were extracted and evaluated using CheckV software (v. 1.0.1)    and the . 1.0.1) . To idenhostdb/) . Viral phostdb/) .http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) . The threshold of identity for new sequences was set with amino acid (aa) sequences < 90% or nucleotide sequences < 85%. Conserved domains were searched for using the NCBI CD-Search tool (psb.cgi) . Maximumpsb.cgi)  with 1,0P < 0.05 were considered to be viral   was run be viral . Sequencbe viral . Proteinhttps://talk.ictvonline.org/).To verify the presence of dengue virus and West Nile virus  in the sPRJNA847438 .The raw sequencing data sets for the current study are available in the NCBI Sequence Read Archive repository, under the Bioproject with accession code


Is there any difference in health care costs for patients who undergo endoscopic vein harvesting rather than open vein harvesting during a coronary artery bypass surgical procedure?In this secondary analysis of cost outcomes from a randomized clinical trial involving 1150 participants undergoing a coronary artery bypass procedure, the use of endoscopic vein harvesting was not associated with lower costs at discharge or over time compared with open vein harvesting. None of the cost end points were significantly different.This study’s findings suggest that the choice to use endoscopic vein harvesting does not increase economic burden and may be based on surgeon and patient preferences. Value-based purchasing creates pressure to examine whether newer technologies and care processes, including new surgical techniques, yield any economic advantage.To compare health care costs and utilization between participants randomized to receive endoscopic vein harvesting (EVH) or open vein harvesting (OVH) during a coronary artery bypass grafting (CABG) procedure.This secondary economic analysis was conducted alongside the 16-site Randomized Endo-Vein Graft Prospective (REGROUP) clinical trial funded by the Department of Veterans Affairs (VA) Cooperative Studies Program. Adults scheduled for urgent or elective bypass involving a vein graft were eligible. The first participant was enrolled in September 2013, with most sites completing enrollment by March 2014. The last participant was enrolled in April 2017. A total of 1150 participants were randomized, with 574 participants receiving OVH and 576 receiving EVH. For this secondary analysis, cost and utilization data were extracted through September 30, 2020. Participants were linked to administrative data in the VA Corporate Data Warehouse and activity-based cost data starting with the index procedure.EVH vs OVH, with comparisons based on intention to treat.Discharge costs for the index procedure as well as follow-up costs  were analyzed, with results from different statistical models compared to test for robustness . All costs represented care provided or paid by the VA, standardized to 2020 US dollars.Among 1150 participants, the mean (SD) age was 66.4 (6.9) years; most participants  self-reported as American Indian or Alaska Native, 10 (0.9%) as Asian or Pacific Islander, 91 (7.9%) as Black, 62 (5.4%) as Hispanic, 974 (84.7%) as non-Hispanic White, and 6 (0.5%) as other race and/or ethnicity; data were missing for 1 participant (0.1%). The unadjusted mean (SD) costs for the index CABG procedure were $76 607 ($43 883) among patients who received EVH and $75 368 ($45 900) among those who received OVH, including facility costs, insurance costs, and physician-related costs (commonly referred to as provider costs in Centers for Medicare and Medicaid and insurance data). No significant differences were found in follow-up costs; per 90-day follow-up period, EVH was associated with a mean (SE) added cost of $302 ($225) per patient. The results were highly robust to the statistical model.In this study, EVH was not associated with a reduction in costs for the index CABG procedure or follow-up care. Therefore, the choice to provide EVH may be based on surgeon and patient preferences.NCT01850082ClinicalTrials.gov Identifier:  This secondary analysis of cost outcomes from the Randomized Endo-Vein Graft Prospective (REGROUP) clinical trial compared health care costs between participants randomized to receive endoscopic vein harvesting vs open vein harvesting during a coronary artery bypass grafting procedure. Both EVH and OVH had comparable rates of major adverse cardiac events  over a median follow-up of 2.78 years.1 Overall, leg infections were rare, but leg infections were approximately twice as common among participants receiving OVH vs EVH , although this difference was not statistically significant.Coronary artery bypass grafting (CABG) remains a treatment option for patients with ischemic heart disease. During the procedure, saphenous veins are frequently used for grafts. Harvesting the saphenous vein was traditionally performed using open vein harvesting (OVH), but endoscopic vein harvesting (EVH) has become the dominant approach since its introduction in the 1990s. The Randomized Endo-Vein Graft Prospective (REGROUP) clinical trialValue-based purchasing has encouraged more analyses of the relative costs of newer technologies, approaches, and techniques. There is a desire to examine new surgical approaches, such as EVH, to assess whether they yield any intended or unintended economic consequences. The objective of this secondary analysis of cost outcomes from the REGROUP clinical trial was to compare health care costs between participants randomized to receive EVH or OVH during a coronary artery bypass grafting (CABG) procedure. We hypothesized that EVH would be associated with lower discharge and follow-up costs (including intended and unintended events) compared with traditional OVH.CONSORT) reporting guideline for randomized clinical trials  Cooperative Studies Program, randomized 1150 participants (574 received OVH and 576 received EVH) at 16 VA medical centers. Adults scheduled for urgent or elective bypass involving a vein graft were eligible for participation. All patients provided written informed consent to participate in the clinical trial. The REGROUP clinical trial followed the Consolidated Standards of Reporting Trials  follow-up was 33.0 (19.9) months, with a maximum follow-up of 85 months.The REGROUP clinical trial was designed to assess whether OVH was superior to EVH based on a primary outcome of time to first major adverse cardiac event .2 starting with the index procedure. The analysis used a VA perspective. The cost data for VA-provided care came from the VA Managerial Cost Accounting System,3 which includes cost estimates for all VA care, including inpatient, outpatient, and pharmacy. The Managerial Cost Accounting System uses activity-based cost methods, in which the care provided to each patient is linked with unit costs. The system tracks care as measured by detailed inputs, including time in the operating room, staff time, supplies, medications, laboratory tests, and overhead. This costing method is considered among the best for economic evaluations because it reflects the resources used to provide care, and it is highly precise.4 These data have been extensively used in research, including in analyses examining the costs of other surgical approaches to CABG.6 All costs were standardized to 2020 US dollars. The VA administrative data were linked to clinical and patient data collected during the clinical trial. For VA-purchased care, we used data from the Fee Basis Claims System7 and the Program Integrity Tool.8Economic end points were not specified in the original clinical trial protocol . With su9 separating the costs of the index stay from subsequent care provided greater insights into the costs of the index stay itself, and this approach avoided the statistical challenges of combining a high-cost admission with follow-up costs that include zeros. We graphed the distribution of discharge costs using kernel densities.10Two separate cost outcomes were assessed. First, we analyzed discharge costs for the index procedure. This outcome included information costs in the surgical suite and any added costs for differences in length of stay or critical care. In previous work,12 We then examined VA-purchased care, which has expanded since the passage of the Veterans Access, Choice, and Accountability Act (commonly referred to as the Choice Act)14 and the VA Maintaining Internal Systems and Strengthening Integrated Outside Networks (MISSION) Act.15 For follow-up care, we also examined VA health care utilization; this measure can sometimes provide insights into mechanisms that may underlie any cost differences.Second, we analyzed downstream costs for follow-up care after the index stay. We combined VA costs into 90-day follow-up periods up to the last follow-up at 85 months . The first 90-day period excluded the cost of the index discharge. The 90-day periods were preferred over shorter 30-day periods, which exhibited considerably more unexplained variability (greater SEs) resulting from 3.3 times more patient observations with zero costs in the shorter period. If patients had an inpatient stay that spanned two 90-day periods, the costs were assigned to each period proportionately based on the length of stay. In the follow-up periods, we examined total VA costs along with subtotals for inpatient care and, more specifically, inpatient medical-surgical care. We also estimated total outpatient costs, outpatient medical-surgical costs, and outpatient pharmacy costs. Subtotals were based on the inpatient treating specialties and outpatient clinic.16 a modified Park test,18 and empirical goodness-of-fit tests to guide our choice. We then compared results from different statistical models to test for robustness . For the analysis of discharge costs, the Box-Cox model suggested a log transformation, the modified Park test was inconclusive, and the Hosmer-Lemeshow statistics for model fit favored an ordinary least squares approach. Thus, we chose ordinary least squares as the primary model and used a generalized linear model with a log link and gamma error distribution in the sensitivity analysis. For the follow-up costs, because the fit statistics did not favor a specific model, we used a linear model with a person random effect to account for the nonindependence of the error terms. We used generalized estimating equations with a log link, gamma error distribution, and exchangeable correlation matrix in the sensitivity analysis. For the utilization outcomes, which included inpatient stays, unique days with an outpatient visit, unique days with an outpatient visit to a medical or surgical clinic, and pharmacy prescriptions, we used a Poisson model with a person random effect. The significance threshold was P = .05, and 2-tailed tests were used for all analyses. Stata software, version 16.1 (StataCorp LLC), was used for analysis.We followed intention-to-treat principles throughout the analysis. We examined the characteristics of patients who received EVH and OVH. Next, we compared unadjusted mean costs by randomized treatment group. For the regression analysis, the protocol did not specify a preferred statistical model, so we used a Box-Cox transformation,19 As reported previously,1 the mortality rate during the active follow-up period did not exceed 10% and was not statistically different across the groups, so we made no adjustments for mortality.The Managerial Cost Accounting System data are computed using local staff time and wages. To remove variation due to wage differences across the 16 sites, we included a site fixed effect in the regression models. We controlled for the presence of diabetes, hypertension, hyperlipidemia, depression, chronic kidney disease, and chronic liver disease at baseline. These conditions have been associated with a higher prevalence of adverse events, higher expected costs, and worse patient outcomes, and any imbalance in randomization could have confounded the results. We also controlled for patient age, sex , and self-reported race and ethnicity , as collected by study coordinators on the clinical trial case report forms. We collapsed race and ethnicity categories into non-Hispanic White vs all other racial and ethnic groups given the small samples and the results of analyses revealing no differences in total costs by each racial and/or ethnic group. Costs may be associated with mortality and imbalanced survival, especially when death is common, which can add challenges to interpreting a cost analysis.P = .01 as a penalty for multiple comparisons.The clinical trial protocol did not prespecify any subgroup analyses. We explored 6 subgroups: diabetes, hypertension, hyperlipidemia, depression, chronic kidney disease, and chronic liver disease. For these exploratory post hoc analyses, we used a threshold of 1 Among 1150 participants, the mean (SD) age was 66.4 (6.9) years; 1144 participants (99.5%) were male, and 6 (0.5%) were female. With regard to race and ethnicity, 6 participants (0.5%) self-reported as American Indian or Alaska Native, 10 (0.9%) as Asian or Pacific Islander, 91 (7.9%) as Black, 62 (5.4%) as Hispanic, 974 (84.7%) as non-Hispanic White, and 6 (0.5%) as other race and/or ethnicity; data were missing for 1 participant (0.1%). At randomization, 1036 participants (90.1%) had hypertension, 993 (86.3%) had hyperlipidemia, and 577 (50.2%) had diabetes. Depression (297 participants [25.8%]), chronic kidney disease (157 participants [13.7%]), and chronic liver disease (42 participants [3.7%]) were less common. There were no significant demographic or clinical differences between patients who received EVH vs OVH.Participant characteristics are shown in The unadjusted mean (SD) discharge costs for CABG were $76 607 ($43 883) per patient among those in the EVH group and $75 368 ($45 900) per patient among those in the OVH group, including facility costs, insurance costs, and physician-related costs (commonly referred to as provider costs in Centers for Medicare and Medicaid and insurance data). Follow-up costs were highest in the year after the index procedure; mean follow-up costs then decreased to approximately $5000 per quarter, or $20 000 per year . DespiteWe found no significant differences in follow-up costs over a mean (SD) of 33.0 (19.9) months. The marginal estimate and 95% CIs are shown in Additional VA resource utilization among patients who received EVH is shown in 1 but these infections were not associated with significantly higher costs for the index stay. Although the distribution of discharge costs was skewed . The use of EVH was cost-effective at a threshold of £30 000 per quality-adjusted life-year  in 60.4% of their simulations, but the authors noted uncertainty in these estimates.22 A major factor in this uncertainty was the small difference in expected event rates. The results of our study, in combination with the clinical end points reported elsewhere,23 highlight the lack of any significant differences between EVH and OVH. In other words, neither approach was superior to the other based on clinical or economic criteria. Thus, the choice to use EVH or OVH may be best guided by surgeon and patient preferences.In 2015, Oddershede et al9 found that veterans who participate in a surgical clinical trial typically have high reliance on the VA; although it is feasible to obtain Medicare fee-for-service claims, an increasing proportion of Medicare beneficiaries enroll in Medicare Advantage, for which we do not have data. We also do not have access to private insurance claims for veterans not eligible for Medicare. Second, this study lacked patient outcome data. Although costs are part of the value equation, we would ideally have had information on patient outcomes, such as quality of life, beyond our survival data. The survival and major cardiac adverse event end points have been published elsewhere and revealed no significant differences between OVH and EVH.23 Although our study had a mean follow-up period of 33 months, most participants were still alive at follow-up and were thus censored. Future analyses, based on a pending proposal, will examine longer-term follow-up. Third, the study was conducted within the VA and included mostly men. The results may not be generalizable to women.This study has several limitations. First, we included information on VA-provided and VA-purchased care. However, we did not have cost data from other insurers. Previous analysesIn this secondary analysis of cost outcomes from the REGROUP clinical trial, the use of EVH was not associated with a reduction in costs for the index CABG procedure or follow-up care. These results suggest that the choice to use EVH vs OVH may be based on surgeon and patient preferences.


Since 1989, lumpy skin disease of cattle (LSD) has spread out of Africa via the Middle East northwards and eastwards into Russia, the Far East and South-East Asia. It is now threatening to become a worldwide pandemic, with Australia possibly next in its path. One of the research gaps on the disease concerns its main mode of transmission, most likely via flying insect vectors such as biting flies or mosquitoes. Direct or indirect contact transmission is possible, but appears to be an inefficient route, although there is evidence to support the direct contact route for the newly detected recombinant strains first isolated in Russia. In this study, we used experimental bulls and fed them via virus-inoculated feed to evaluate the indirect contact route. To provide deeper insights, we ran two parallel experiments using the same design to discover differences that involved classical field strain Dagestan/2015 LSDV and recombinant vaccine-like Saratov/2017. Following the attempted indirect contact transmission of the virus from the inoculated feed via the alimentary canal, all bulls in the Dagestan/2015 group remained healthy and did not seroconvert by the end of the experiment, whereas for those in the Saratov/2017 recombinant virus group, of the five bulls fed on virus-inoculated feed, three remained clinically healthy, while two displayed evidence of a mild infection. These results provide support for recombinant virus transmission via the alimentary canal. In addition, of particular note, the negative control in-contact bull in this group exhibited a biphasic fever at days 10 and 20, developed lesions from day 13 onwards, and seroconverted by day 31. Two explanations are feasible here: one is the in-contact animal was somehow able to feed on some of the virus-inoculated bread left over from adjacent animals, but in the case here of the individual troughs being used, that was not likely; the other is the virus was transmitted from the virus-fed animals via an airborne route. Across the infected animals, the virus was detectable in blood from days 18 to 29 and in nasal discharge from days 20 to 42. Post-mortem and histological examinations were also indicative of LSDV infection, supporting further evidence for rapid, in F transmission of this virus. This is the first report of recombinant LSDV strain transmitting via the alimentary mode. Capripoxvirinae, family Poxviridae [Lumpy skin disease (LSD) is an OIE-reportable viral disease inflecting considerable losses on farms and affecting international trade . The etixviridae . The genxviridae ,3. LSD vxviridae ,5,6,7. Txviridae . Excretexviridae . The disxviridae .Lumpy skin disease has recently received much interest after it began a rapid and aggressive spread northwards and eastwards in the Northern Hemisphere. The historical range of distribution includes most African countries and more recently, the Middle East; however, within the past decade, a dramatic expansion into the Balkans, eastern Europe, Russia, China and most of southern Asia has been experienced, with Indonesia and Singapore being some of the latest countries to experience outbreaks ,12,13,14Solid evidence pertaining to the predominant transmission route of the virus is still scarce ,20. IndiIn this study, we extend our previous findings on the non-vector-borne transmission of Saratov/2017 to demonstrate infection of cattle fed on feed inoculated with this recombinant strain versus non-transmission when using a classical field strain (Dagestan/2015).The experimental animals in this group showed no fever, or other clinical signs, and PCR and ELISA results were all negative.Bull №1. Fever was erratic up to 40 °C . An increased body temperature of up to 40 °C was observed at day 10 and day 20 , PCR-detection of viral DNA in its blood lasted 12 days, from days 18 to 29, whereas LSDV DNA was detectable in its nasal swabs for 23 days, from day 20 to 42.In Bull №3, detectable viremia via PCR lasted 3 days from day 22 to 24. No detectable DNA was identified in nasal swabs.In Bull №4, DNA was detected at days 41 and 45. Nasal swabs were positive at days 24, 29 and 45.ELISA results are provided in Only Bulls №1–3 displayed detectable seroconversion. The first detectable antibodies were observed at day 31 and increased until day 43. Bull №2, which did not receive virus via inoculated feed, displayed increasing levels of antibodies from day 31 onwards.Samples for histological analysis were derived from Bull №2 as confirmation of contact transmission. At the histological level, the skin lesion was surrounded by dense irregular connective tissue that is part of the skin derma. Under the derma are large infiltrations of lymphocytes and histiocytes, with an increasing concentration from the periphery to the center. Many cells of the lesion have undergone karyopiknosis and karyolysis A.Examination of the gastrointestinal epithelium showed focal necrotic sites accompanied by damage to subepithelial tissue B.Lungs demonstrated lobular pneumonia with necrotic sites, whereas at the histological level atelectasis and dislectases were observed C. The puDuring the subacute inflammatory stage, we observed the expansion of interalveolar septa, peribronchovascular lymphocytic-histicytic infiltration and macrofocal purulonecrotic pneumonia. The pleura was expanded and infiltrated with lymphocytes. Most cellular nuclei already attained lytic or rhexis states E.Histological examination of muscle tissue revealed thickening and lymphocytic infiltration in the intermuscular layers of connective tissues, edema and diffuse dystrophy of muscle fiber sheaves F. SerousStomoxys calcitrans) and Culicoides midges are the most likely blood-sucking vectors for natural virus transmission, but testing of field-caught specimens, including species such as horse flies, during outbreaks has yet to provide definitive confirmation [The issue of LSDV transmission has been puzzling in many respects since no conclusive proof has yet been obtained as to the dominant mechanism, applicable to real field conditions both in tropical and northern climates ,23. Earlirmation ,28,32.While attempts to achieve LSDV infection by classical field strains without using blood-sucking arthropods have been unsuccessful , field-iIn the group fed with bread soaked in culture containing strain Dagestan/2015, the experimental animals remained healthy and were PCR- and serologically-negative up to day 50 of the trial. These results agree with previous investigations, showing that in-contact transmission of classical LSDV strains is ineffective. Dagestan/2015, along with SERBIA/Bujanovac/2016, the Greek strain Evros/GR/15, the Bulgarian strain 210LSD-249/BUL/16, and the Warmbaths LW strain from South Africa are included in the group of classical field isolates, comprising cluster 1.2 ,35,36. SBy contrast, Saratov/2017 is the first recombinant strain isolated in the field, comprising a genome most likely derived from a Neethling-type virus from cluster 1.1 as the major parent and a Kenyan KSGP vaccine virus as the minor parent from cluster 1.2 ,17,36. ITwo of the five experimental animals that received virus-inoculated feed in the Saratov/2017 group exhibited very mild clinical signs, which may be overlooked by inexperienced vets, and low and erratic detectability of virus genomes in blood and nasal secretions . Three aBy contrast, Bull №2 in this group, which served as an in-contact negative control animal, became infected and displayed an even more severe disease presentation as compared to either of its flanking animals, Bull №1 or Bull №3. Infection in the in-contact animal was also evident at the histological level . Of noteAt day 18 and 20, all animals but Bull №2 tested negative in nasal swabs from each, which are normally a good source of virus. Since it takes time for virus titres to increase in the host, it is likely that at those time points virus loads in animals fed virus-soaked bread were lower than the assay detection limit, which caused the discrepancy. Importantly, the very rapid rise in body temperature may suggest Bull №2 was able to feed on some of the virus-inoculated bread left over from adjacent animals. However, this was not likely as the troughs were separated, and the animals were tethered to prevent cross-trough feeding. Another possible scenario is that some virus-soaked bread fell onto the floor and was then inadvertently kicked or pushed within reach of Bull №2. In any future trials of this nature, the investigators will endeavor to ensure that any possibility of in-contact negative control animals gaining direct access to virus-inoculated feed is excluded.It is also possible that the virus was transmitted from the virus-fed animals to Bull №2 via the airborne route. Early work on LSD raised concerns that the virus can transmit without involvement of arthropods, but at a low level of efficiency  This is As more LSDV strains emerge, including recombinants with diverse patterns of recombination and parental strain contributions, additional studies will be needed to provide clarification on all possible aspects of LSDV transmission.Despite the important knowledge gained from our study, we used only six animals per group, which may be deemed as a limitation. However, experiments published by Wolff et al. (2020 and 2022) ,44, BabiInterestingly, in-contact transmission was detected in a previous study, but the results reported here cannot be used to confirm the results of the previous study, as a different route of infection was investigated here . In thisA mild or subclinical disease presentation of animals infected with a recombinant virus in the field may enable low-level circulation of the virus between animals in a herd during a vector-free period, until favorable conditions set in for vector-assisted spread. This evidence provides a possible explanation for the LSD outbreaks in winter conditions in Russia in 2018–2019, caused by recombinant isolates. Since recombinant LSDVs are currently spreading in Eastern Asian countries, it is important to test other strains with different recombination patterns to verify the identified alternative modes of transmission. Clarifying the mechanisms by which classical and recombinant LSDVs overwinter in different climates will greatly improve control and eradication strategies.Genomic rearrangements have profound effects on phenotypic characteristics of poxviruses ,47. PastOverall, in this study we followed up on previous experiments where we had for the first time shown vector-free transmission of the recombinant strain, Saratov/2017, and here we proved that this strain, in contrast to the classical field strain, Dagestan/2015, can cause a mild disease via the alimentary route. Interestingly enough, the control animal did not receive virus-inoculated feed but contracted the virus by other means, which provides evidence that recombinant LSDV can be transmitted via alternative routes, as occurs for sheeppox and goatpox capripoxviruses. Further studies are currently underway to explore the degree to which other recombinant LSDV strains display contact infection. This information will help us better understand the relationship between phenotype and genotype of LSDV.The recombinant vaccine-derived strain, Saratov/2017 , and the3 of virus suspension of either strain containing 105 TCD50/cm3 (day 1 of experiment). The virus for the soaking bread was harvested from a fresh cell culture exhibiting at least 50% cytopathic effects. The virus-soaked bread was given to animals within an hour of harvesting. No virus recovery from the soaked bread was carried out. The feeding was repeated with the virus-inoculated bread for five consecutive days (the first five days). If an animal did not finish eating its bread on any given day, the remainder was left in the individual troughs (not shared). The animals shared the same air space but were separated by metal railings and tethered so that one animal could not reach the other animal’s trough. The freedom of movement was such that the animal could eat only from its designated trough. One animal out of the six in either group (Bull №2) was a negative control, being fed the mock-inoculated bread, infiltrated with virus-free cell culture, but shared the same airspace with the others in its group. This negative control animal was located between Bull №1 and 3. Animals were screened each day once for clinical signs of LSD such as fever, lymph node enlargement or skin nodules. Nasal swabs and blood samples were collected every other day. Nasal swabbing was carried out at least 12 h post-feeding. Serum samples were collected at days 8, 16, 22, 31, 36, and 43 of the experiment. The total duration of the experiment was 50 days, after which animals were euthanised. The euthanasia protocol  consisted of captive-bold penetration to desensitize the animals, followed by the injection of the muscle relaxant, Adilinum super, according to the manufacturer’s instructions .A total of six bulls were used in the experiment for each virus strain for assessing the potential for alimentary tract infection via ingestion of inoculated feed under biosafety level 3 and vector proof conditions. All animals in both experimental groups were 4–6 months old bulls of the Russian black pied breed, purchased from the same breeder. The bulls were tethered, but movement was not completely restricted and a degree of physical contact was possible between adjacent animals. The infection procedure was as follows: each bull was fed a piece of brown bread, weighing 100g and pre-inoculated with 5 cm® Advance Kit (LSDV116 RNA polymerase subunit) following manufacturer’s recommendations were utilized for PCR testing. Total viral DNA was extracted using a QIAamp DNA Mini Kit . All threshold values (Ct) below 38 were considered positive. Samples with low Cts (nodules) were subjected to sequencing at RPO30 and GPCR targets to confirm the identity of the strain to that used for infection as previously described [The assay targeting ORF44 of LSDV  and commescribed .® Capripox Double-Antigen Multi-species ELISA kit  was used according to the manufacturer’s instructions for detecting antibodies to LSDV in sera [N ≥ 20%: positive.The ID Screen in sera . The ELI3, followed by fixation in 10% neutral buffered formalin. The fixed specimens were processed using a TLP-720 tissue processor . Embedding was carried out via an ESD-2800 station . Sections were cut at a thickness of 0.005–0.008 cm3 using a RMD-3000 microtome station , followed by staining in hematoxilin and eosin. Light microscopy was performed using a Mikmed-6 microscope .Tissue sections for histological examination were removed in a volume of 1.0 cm


Moreover, estimates of case fatality rates were higher in Ebola  followed by Nipah , MERS  and SARS  with the overall case fatality rates of 29.86 . Bat‐originated viruses have caused several outbreaks of deadly diseases, including Nipah, Ebola, SARS and MERS in Asia and Africa in a sequential fashion. Nipah virus emerged first in Malaysia, but later, periodic outbreaks were noticed in Bangladesh and India. Similarly, the Ebola virus was detected in the African continent with neurological disorders in humans, like Nipah, seen in the Asian region. Two important coronaviruses, MERS and SARS, were introduced, both with the potential to infect respiratory passages. This paper explores the dimension of spillover events within and/or between bat–human and the epidemiological risk factors, which may lead to another pandemic occurring. Further, these processes enhance the bat‐originated virus, which utilises an intermediate host to jump into human species.Bats are the natural reservoir host for many pathogenic and non‐pathogenic viruses, potentially spilling over to humans and domestic animals directly or via an intermediate host. The ongoing COVID‐19 pandemic is the continuation of virus spillover events that have taken place over the last few decades, particularly in Asia and Africa. Therefore, these bat‐associated epidemics provide a significant number of hints, including respiratory cellular tropism, more intense susceptibility to these cell types, and overall likely to become a pandemic for the next spillover. In this systematic review, we analysed data to insight, through bat‐originated spillover in Asia and Africa. We used STATA/IC‐13 software for descriptive statistics and meta‐analysis. The random effect of meta‐analysis showed that the pooled estimates of case fatality rates of bat‐originated viral zoonotic diseases were higher in Africa (61.06%, 95%CI: 50.26 to 71.85,  Major bat‐borne zoonotic viral epidemics in Asia and Africa: A systematic review and meta‐analysis The species jump of Nipah virus from bats to humans via pigs happened in the last decade  guidelines . We identified and excluded the duplicates using EndNote and then studied the title and abstract to find the relevant articles. The relevant articles and the articles that were not clear by the title‐abstract study were subjected to full‐text collection. Only documents published in English were considered for the review Ahmed, .2.3We considered only the field reports containing bat‐originated zoonotic diseases for data abstraction. The extracted data included the country and location of the outbreak, year of sample, total sample, total death and possible associating factors such as causal agent, transmission dynamics, pathogenicity etc.2.4p value were calculated on different diseases between Asia and Africa. Variations among the studies were evaluated using the chi‐square (χ2) test on Cochran's Q statistics  followed by I2 statistics to determine the degree of heterogeneity in the study. The weights were chosen to reflect the amount of information that each study contains. A random‐effect meta‐analysis was applied using the ‘metan’ command specifying the random effects due to the high degree of heterogeneity (I2 > 75%). The outputs have been illustrated using a forest plot . Crude estimation of case fatality rate was performed by dividing the number of deaths of humans by the total number of affected and expressing the results as percentages. The crude estimate of case fatality rates, the 95% confidence interval (CI) and the 33.1We reviewed 41 published articles of the last two decades 1999–2021) that reported bat‐originated major zoonotic diseases, particularly in Asia and Africa  and 61.06% in Africa . Figure l2% = 99.0, p < 0.001).Based on the available data, the estimated pooled case fatality rates of the different types of bat‐originated zoonotic viral diseases of humans in Asia and Africa have been presented in Table l2% = 97.3, p < 0.001) followed by Nipah , MERS  and SARS . Figure l2% = 99.0, p < 0.001).The estimated pooled case fatality rates for bat originates viral zoonotic disease are presented in Table 44.1Pteropus bat species) in several countries, including Cambodia, Indonesia, Thailand, Philippines, Ghana and Madagascar  of Ebola virus disease (EVD) were similar to the CFR of the reported review (65.4%), and WHO also reported that CFR around 50% in the infected areas  and small (Pteropus hypomelanus) flying foxes were found to be the natural reservoir hosts for the Nipah virus in the post‐outbreak surveillance by the Malaysian authority , and subsequently, inter‐species transmission to humans took place via an intermediate host  and raccoon dogs (Nyctereutes procyonoides)   in a wildlife market in Shenzhen, China. The genomes of SARS‐CoV isolates from civets , including R. sinicus, R. ferrumequinum, R. macrotis, R. pearsoni and R. pusillus and have transmitted in the meat markets to amplifying hosts, such as masked palm civets, raccoon dogs and a hog badger  of the EPteropodidae family. These infected bats have been recognised in Bangladesh  and has developed its ability to infect humans in massive ways. The previous coronaviruses (MERS and SARS) have respiratory cellular tropism to allowing the ability to infect humans in continuing virus spillover. These phenomena further indicate that a more likely respiratory pathogen might evolve from wildlife, with the potentiality of causing a global pandemic. In addition, human behaviour, deforestation, wildlife trade and consumption, and the interaction with wildlife are the critical factors to transmitting viruses from bat to human. Human interactions with wildlife, especially hunting bats for meat, wildlife trade and the use of bat guano in the agricultural production system , pose a severe indication of an occurring future pandemic this part of the world. As the current pandemic has shown, an infectious disease that starts in one part of the world can spread globally in no time whatsoever. There is an urgent need for constructive conservation strategies to prevent deforestation and reduce wildlife–human interactions. A comprehensive global surveillance system to monitor the emergence of bat‐borne viruses would be an indispensable tool in helping us fight these deadly and terrifying epidemics and pandemics.The research did not receive any grant from the funding agencies.No ethical approval was required as the current work is a review.Conceived and designed the research: MMH and SAK. Data collection and verification: MMH, SAK, AZT, AI and MAI. Wrote the paper and commented: MMH, SAK, AZT, AI, MMI and MAI. All the authors read and approved the manuscript for submission to the journal for publication.https://publons.com/publon/10.1002/vms3.835.The peer review history for this article is available at


The tripartite motif protein 21 (TRIM21) belongs to the TRIM family, possessing an E3 ubiquitin ligase activity. Similar to other TRIMs, TRIM21 also contains three domains (named RBCC), including the Really Interesting New Gene (RING) domain, one or two B-Box domains (B-Box), and one PRY/SPRY domain. Notably, we found that the RING and B-Box domains are relatively more conservative than the PRY/SPRY domain, suggesting that TRIM21 of different species had similar functions. Recent results showed that TRIM21 participates in virus infection by directly interacting with viral proteins or modulating immune and inflammatory responses. TRIM21 also acts as a cytosol high-affinity antibody Fc receptor, binding to the antibody–virus complex and triggering an indirect antiviral antibody-dependent intracellular neutralization (ADIN). This paper focuses on the recent progress in the mechanism of TRIM21 during virus infection and the application prospects of TRIM21 on virus infection. The tripartite motif proteins (TRIMs) belong to the E3 ubiquitin ligase family, discovered in almost all multicellular animals ,2,3. TRI+, which confers TRIMs with E3 ubiquitin ligase activity ..33].HSV-1 lytic gene expression [After sensing the pathogen, the E3 ubiquitin ligase activity of TRIM21 promotes the degradation of IFN regulatory factor 3 (IRF3), resulting in negative regulation of the production of IFN-β ,34. TRIMpression . Highly pression . TRIM21 pression . TRIM21 pression . These rTargeting upstream molecules of the IRF pathways by TRIM21, such as DEAD-box protein 41 (DDX41), STING, and RIG-I, is also a strategy to inhibit immune responses. DDX41 belongs to DExD/H-box RNA helicases, characterized by two conserved RecA-like domains . DDX41 aTRIM protein can also target host protein, thus inhibiting immune activation and affecting virus infection. Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) is a restriction factor of retroviruses and enterovirus 71 (EV71) ,45. FurtTherefore, the ubiquitination of IRF3/7 or its upstream sensors by TRIM21, such as STING, DDX41, NMI/IFI35, and RIG-I, leads to immune suppression during virus infection. Finally, the replications of viruses, including SeV, IAV, HSV-1, ASFV, and JEV, were enhanced in TRIM21-dependent manners.Apart from negatively regulating immune responses, TRIM21 also enhances immune responses during some virus infections . For exaTRIM21 induces the destruction of the antibody-bounded virion, followed by the recognition of the exposed viral genome in the cytoplasm by DNA or RNA sensors, such as cGAS or RIG-I, and the immune responses are activated ,50. In a− monocytes was significantly lower than that of CD16+ cells and monocytes [p < 0.01) than that in monocytes [+ monocytes and macrophages during acute or chronic infections of enveloped viruses. We previously found that TRIM21 triggers immune and inflammatory responses by enhancing IFNs and proinflammatory factors, such as IL-6, IFN-β, IFN-γ, and TNF-α, in PCV2-infected cells [The level of TRIM21 in CD16onocytes . On the onocytes . These red cells .Therefore, TRIM21 has diverse activities during different virus infections, and its regulation of type I IFN and inflammatory reactions are inconsistent during various virus infections.TRIM21 also serves as a cytosolic IgG receptor, which can efficiently bind with the antibody–virus complex and induce an indirect antibody-dependent intracellular neutralization (ADIN) to degrade virion via the ubiquitin–proteasome pathway  10,14,2,55,56,57As reported, porcine TRIM21 was triggered by type I IFN during foot-and-mouth disease virus (FMDV) infection . The comMoreover, TRIM21-mediated protein degradation also activates AP-1, NF-κB, and IRF signaling pathways, thus promoting the production of inflammatory cytokines and enhancing the antiviral activities of cells ,61,67,68Antibody-coated viruses can stimulate phagocytosis, activate the formation of NLRP3-associated inflammasome, and release IL-1β . UnexpecSince TRIM21 possesses Fc receptor and E3 ubiquitin ligase activities, numerous groups have tried to develop virus infection detection and prevention methods based on TRIM21.The first application based on TRIM21 is TRIM-AWAY, designed to degrade target protein or inhibit virus propagation via a proteasomal pathway ,74. IntrTRIM21-based virus protein or antibody detection is also promising. Albecka et al. developed an electroporated antibody-dependent neutralization assay (EDNA) based on the TRIM21-mediated intracellular neutralization to detect nucleoprotein antibodies of coronaviruses, including murine hepatitis virus (MHV) and SARS-CoV-2 . The EDNIn summary, TRIM21 affects virus infection by directly regulating virus proliferation and modulating immune and inflammatory responses. However, TRIM21 affects virus infection in a virus-dependent manner. The replications of viruses, including ASFV, EV71, HPV, HSV-1, IAV, JEV, PCV2, and SeV, were enhanced by TRIM21, while TRIM21 inhibits the infections of other viruses, such as CVB3, FMDV, HBV, HCV, PEDV, and VSV, etc. The critical domains of TRIM21 are PRY/SPRY and RING, which recognize the target protein and mediate the ubiquitination of the protein, respectively. The RING domain is conserved among species and retains E3 ubiquitin ligase activity. However, the PRY/SPRY domain is not very conservative, suggesting that it can interact with various proteins and has a multitargeting characteristic. Therefore, TRIM21 is a promising target for targeted therapy or prevention strategies against virus infection.


The use of mobile devices to deliver public health interventions is rapidly increasing, particularly in low resource settings. Despite their proliferation, several mHealth interventions in developing countries fail to reach geographical scale, and long-term sustainability for most remains uncertain. There is a need to cost for such programs, to enable better planning and budgeting and tailor programs as required. Cost estimates can contribute to a more informed debate on resource allocation priorities and help make choices clearer for policymakers. This paper has two main objectives: (1) present a detailed protocol on determining the costs of a large national mHealth job aid and behavior change communication tool known as Integrated Child Development Services - Common Application Software (ICDS-CAS) in India, and (2) to present lessons for policymakers on how to ensure financial planning for scaling mHealth interventions. The study uses the Activity Based Costing—Ingredients (ABC-I) method. The major advantage of the ABC-I method is the clarity it brings to costs for each input and activity, across levels and geographies. It also accounts for indirect costs. There are five key lessons while costing for mHealth programs. First, that there are many activities and ingredients that must be budgeted for and discussed while planning and implementing mHealth programs. Second, the ABC-I method described in this paper provides great clarity on costs, yet its major limitation is the availability of data, which must be mitigated with the careful use of assumptions. Third, mHealth technology life cycles have financial implications which must be accounted for. Fourth, determining cost locations and all sources of funding including non-government sources is crucial. Fifth, since costing estimates are subject to a set of assumptions, a disaggregation of costs allows for scenario-building, which is useful while planning ahead and accounting for program changes. The evidence generated can be used for more informed debate on resource allocation priorities, given competing priorities in low- and middle-income countries. The use of mobile and wireless technologies to support the achievement of health objectives or mHealth interventions has been increasing. They have been heralded as having the ability to transform the delivery of health services . In partDespite their proliferation, a systematic review of mHealth interventions found that while the low cost of mobile technology enabled their adoption, successful expansion was often hampered by mechanisms for financial sustainability and a lack of data on the cost of programs at scale . ConsequIn the absence of quality administrative data and given financial limitations and several competing priorities in LMICs, economic evaluations are an important tool for effective prioritization of resources . Where pPoshan Tracker (Poshan means nutrition).This paper attempts to strengthen the evidence base by presenting a comprehensive, bottom-up methodology on undertaking a cost evaluation of a large-scale national mHealth program. We have applied this method to cost for such an intervention in India, known as the Integrated Child Development Services-Common Application Platform (ICDS-CAS). In September 2020, the program was replaced by another application with similar aims known as This study aims to (1) present a detailed protocol on determining the costs of a large national mHealth job aid and behavior change communication tool known as ICDS-CAS in India, and (2) to present lessons for policymakers on how to ensure financial planning for scaling mHealth interventions.There are two common starting points for any costing study. First, to develop a financial forecast for sustaining and/or expanding program activities; and second, to model the sector-wide implications of scaling up . The metLaunched in 2016, the ICDS-CAS program sought to improve service delivery of nutrition programs and had four key features. First, through a smartphone application, it digitized and automated 10 of the 11 service registers that frontline workers (FLWs) - known as Anganwadi Workers (AWWs) are expected to maintain (except the stock register). This information was then aggregated through a web-based dashboard at different sub-national levels including block, district, state, and finally at the national level. Second, since AWWs are meant to conduct regular home visits to pregnant women and lactating mothers, it had a scheduler that helped prioritize home visits. Third, the software also contained counseling videos which could be used as a job aid. Finally, with features such as a photo capture feature and GPS capability, the application helped create channels for monitoring. AWW supervisors known as Lady Supervisors (LSs) had access to real-time data . BetweenStudies have found that AWWs spend substantial amounts of time on administrative tasks . ICDS-CAYet, the program was not without its challenges. A process evaluation found that impediments to roll-out included state readiness, delays in device procurement and set-up, dashboard readiness, and low data storage space . This stThis research uses the Activity Based Costing - Ingredients (ABC-I) method which has been used in several costing studies –24. The Resources are typically scarce in LMICs, and any expenditure at scale requires careful prioritization between competing needs. Cost estimates can contribute to a more informed debate on resource allocation priorities , and helThe objective of budgeting is estimating existing revenues required and likely expenditures as well as determining future funding needs. As mHealth programs expand across LMICs, every government will have to construct a budget including start-up costs, fixed costs, and variable costs, as well as creating annual or longer-term plans. ABC-I provides a basis for that. Furthermore, since ABC-I aims to disaggregate costs, it can be helpful in creating budgets tailored to a detailed roll-out plan. For example, the program may only be launched in a limited geography or with limited features initially, and gradually expand.Additionally, as with most government interventions, it is possible that some components of mHealth programs are phased out in the future, or the component-mix and resource-mix requires change. To this end, ABC-I can provide detailed inputs on the minutiae of the program and help visualize these changes easily. For example, this can help in determining the impact of delays or changes in the implementation modalities on total program costs.Results using the ABC-I method can serve as a toolkit for governments, policymakers, donors, and practitioners. It allows for a comparison with other similar programs that could be rolled out across LMICs in the future.The following five steps were involved in application of the ABC-I approach in this cost evaluation.This process involves sequentially listing out all processes and components that go into implementing the program as desired. This includes, but is not limited to, procurement of equipment, hiring staff, training of FLWs, mid-level managers, and officials, and setting up the monitoring and upkeep procedures. This helps in creating a timeline of all activities conducted for the intervention as well. This description helps in grasping the scope of the intervention and narrowing the focus on activities that require the most time, effort, and resources.Detailed program descriptions can be created based on operational guidelines set by the implementing authority or be co-created with them. Primary documents used should ideally include the operations manual, impact pathways, training, hiring, and other process manuals, etc. Various partners need to be consulted to understand their exact role, if it is unclear from secondary sources.mutually exclusive and exhaustive AB-CCs to avoid double counting and should be sufficiently detailed, to allow for granular analysis of each element of the intervention. A descriptive report helps identify all possible activities, and to break them down by which stage they are needed – start-up, maintenance, or scale-up. Every AB-CC itself comprises various “ingredients” that come together, and the various inputs needed for each activity. For example, training activities could be described as an AB-CC, broken down into ingredients such as FLW training, training of officials and other personnel. Each ingredient requires various inputs such as space to conduct the training, trainer's remuneration, and so on.The procedure for this step is similar to the previous one and some degree of convergence between these steps is anticipated. The detailed checklist created in step one shall be used to identify Every activity must be broken down, as much as possible, into all its elements. This enables the compilation of a cost database, where all inputs and ingredients needed for a given activity are listed in detail. This database specifies the category, ingredient name and description, and a unit of measurement for each .In this step, vital categories of inputs are delineated—shared vs. non-shared costs, fixed vs. variable costs, and recurrent vs. non-recurrent costs. Capital goods represent a type of fixed and non-recurrent cost which needs to be depreciated over the assets' potential life term. These include vehicles, buildings, etc. Recurrent costs include remuneration for personnel or maintenance costs, which are purchased frequently, often in regular intervals . Sometimes for an ingredient, both recurrent and non-recurrent costs exist. For example, while the purchase of a smartphone for the mobile application is a fixed cost it is recurrent every few years because the lifespan of a phone rarely exceeds 2–3 years, whereas the phone servicing and maintenance is a recurrent cost.This step also entails determining the number of units of each input required for any given activity such as the number of hours of the training agency's time, the number of mobiles and tablets needed, the amount of office space required, and so forth.The first three steps shall occur simultaneously. Furthermore, they are iterative to an extent whereby new information should be included as and when required.For each activity and ingredient, unit costs are to be compiled from various sources including the government program budget and expenditure documents, training guidelines, procurement registers, etc. There can be certain ingredients for which exact unit costs cannot be compiled. For instance, when personnel work across multiple interventions including mHealth programs, the time they spend and therefore the remuneration paid to them solely for the mHealth program may be unclear. A mitigation strategy for such scenarios is mentioned in the subsection on limitations and mitigation.Ideally, the cost of any activity should be measured as the total sacrifice made to complete that activity. For this purpose, we may have to use market prices as well as economic costs. It is important to note, however, that market prices may not reflect true economic costs. This is particularly true for inputs that are donated, capital inputs, or have distorted or non-existent markets. For our purpose, the idea is to include time costs, personal costs, and social costs, if any.To cost for ICDS-CAS, unit costs were obtained from a central Indian state Madhya Pradesh (MP), which was one of the pilot states for the program.Private costs are those borne by individuals. Health interventions can affect the ability of people to work , or on a larger scale  without any need to re-collect prices and quantities for different scenarios.Thirdly, ABC-I accounts for indirect costs that cannot be attributed to a particular program alone. ABC allows us to apportion these costs for each input based on a direct tracing of personnel time to services . For exaThis methodology is proposed as it brings clarity to the costing exercise by using unit costs, taking into account the possibility of double counting, and by allowing to account for indirect costs .The steps listed above lead to the production of two key outputs. First, the construction of a cost profile. This profile lists out the various costs associated with each activity in a comprehensive and disaggregated manner. A cost profile can list activities based on whether they are start-up costs, maintenance costs, or costs sustained during the scale-up process. We can then visualize the shape of the intervention with greater clarity, with information on which activities and inputs cost the most, require the bulk of maintenance costs, or are the costliest aspects of scaling up. This allows comparisons across activities and can inform discussions on optimizing the intervention.Second, visualizing scale-up costs. Several mHealth interventions start as pilots but aim to scale-up. While an intervention is scaled up, input quantities, and therefore, costs may not increase in a linear fashion. This is particularly true of fixed costs, such as the physical infrastructure and overhead costs. Excluding certain inputs and costs is relatively straightforward using the ABC-I methodology proposed for this exercise. Simultaneously, it allows us to analyze the costs required to replicate the program in vastly different contexts.There were several lessons from the application of ABC-I to understand ICDS-CAS costs. They are presented below:An application of the method to costing for ICDS-CAS, found that broadly, the intervention could be categorized into 3 broad activities :Administrative costs which include all costs related to managing and organizing the implementation of ICDS-CAS. At both the central and state level, project management units were set up to manage CAS, coordinating CAS implementation across states and districts, known as the Central Program Management Unit (CPMU) and the State Program Management Unit (SPMU). These include both non-recurring costs such as facility costs (furniture and equipment) and recurring costs such as personnel costs, expenses on electricity, stationery, etc. Examples of personnel included District Coordinators (DCs), District Project Assistants (DPAs), Block Coordinators (BCs), Block Project Assistants (BPAs)Software and devices including costs for the software developer, Cloud Service Provider (CSP), and devices. Data collected through mHealth interventions requires huge cloud storage capacity. Simultaneously, device costs include not just the purchase of devices and accessories, but also costs to maintain and repair devices, and monthly usage fees.The software component includes all costs incurred on developing the CAS platform incurred primarily by the Software Development Agency (SDA). The ingredients for the software developer include program management, software development, data analytics, impact assessment and improvement, field support, and travel.Training costs include costs for the Training Agency (TA), and related training costs. The two main parts to training costs are designing and implementing the training, and providing the resources  for the same.Each of the major components  have several ingredients which need to be budgeted for. Moreover, programs must be augmented with continuous training, supervision and the provision of equipment as well as planning costs over time . An examThere are many kinds of data gaps that may exist. First, certain costs may be unavailable. With government programs, several costs might be sensitive, and access may be restricted. It is also possible that figures obtained may be estimates themselves. For instance, while applying the ABC-I method to cost for ICDS-CAS in India, detailed data on cloud storage costs, costs for maintaining the call center, and costs for the program management unit were not available and had to be inputted based on limited information. For pilot programs, it is also hard to predict how large or small certain costs would be if the program scaled up. The lack of cost data has been a challenge while evaluating other mHealth interventions as well .Second, estimating exact annual costs is difficult, given the dynamic evolution of programs. Several programs are rolled out in a phased manner, starting with a pilot. In the absence of information on how the program would evolve in the next, say 5–10 years, costing must be based on current guidelines. Therefore, changes in costing due to economies of scale, technological dividends, or a change in the implementation design if the scale-up is staggered are hard to account for.Relatedly, as programs are scaled up, costs across geographical settings might not be available in disaggregated form. In a country like India, for instance, every state may eventually have a slightly different implementation model based on their current technological and administrative capacity. This would impact how the intervention is scaled and costed. Finally, assigning costs for personnel working across interventions and programs might be challenging in absence of a detailed time-use study, as mentioned before.To mitigate some of these limitations, assumptions can play a critical role. These could be assuming proxy values for certain costs, assuming time spent by personnel on the program, more general things such as identifying different sources, and setting discount rates, inflation rates, conversion rates, etc. All these assumptions must be carefully listed out to enable replication and justified as well.The sensitivity of each assumption should also be tested i.e., how much do costs change with changing assumptions. For instance, how much do total costs vary if we assume that certain personnel spend 50% of their time on the mHealth program instead vs. if this figure were 70%. These assumptions are useful in calculating the range of possible costs under different sets of assumptions and should be used as a guide to a more comprehensive planning and budgeting exercise. An example of some assumptions, used when calculating costs for ICDS-CAS are given below.A related strategy is to leverage data collected by various studies and surveys which analyze different aspects of programming including time use of personnel. This data can be directly used while inputting unit costs, or indirectly in the form of using it in assumptions to proxy for certain costs.Overall costs of the program are the most important costs to highlight, as well as per FLW costs. Along with this, costs for each component should be noted as well. For example, for ICDS-CAS, 55% of total costs at scale would have been on buying devices. This is then followed by the cost of conducting state level training of AWWs and LSs, at 18%, and state administration at 17% of the total costs incurred.Any cost description should differentiate between recurring fixed costs, variable costs, and onetime capital costs. As an example, within the AB-CC ‘software and devices', cloud storage costs are all recurring costs, while all costs for the software developer are non-recurring.Overall, for ICDS-CAS, in year 1, non-recurring costs account for 59% of total costs with the bulk being costs for devices (75% of recurring costs) and training (15% of recurring costs) . Their pAt the same time, for financial sustainability of mHealth interventions, it is important to remember the life cycle of technology. The cyclical nature of fixed costs must therefore be accounted for over time. Over time, costs across programs can also be reduced by avoiding monolithic architecture and investing in interoperability .Certain non-recurring costs such as devices and other equipment may need to be replaced every 3–5 years. Extending this analysis over a 10-year period for ICDS-CAS and accounting for inflation and discount future spending in present time, shows a non-linear cost progression. Thus, while year 4, 7 and 10 sees significant spikes in costs due to replacement of devices and other equipment, for the remaining years costs are less than half the costs incurred in year 1 . MoreoveIn the early stage of large mHealth interventions, sources of funding may include non-government ones including donor funding, implementing organizations responsible for training etc. It is thus useful to disaggregate costs based on the source of funding for each component. Paired with plans to spend funds over time, this also ensures that the funds are sourced appropriately, and advance planning can ensure the government has funds coming through for the program. Furthermore, it should attempt to quantify any in-kind contributions they may receive from other organizations, and identify cost-share opportunities .Restrictions on the use of government money may impede implementation by impacting procurement, including the procurement of third-party software and hardware and ongoing maintenance and support from vendors with the necessary skill sets. Continued donor funding can be difficult to sustain but nevertheless provides added flexibility to be responsive to evolving needs on the ground .For ICDS-CAS, costs such as central administration and cloud storage were fully funded by the Union government. For the others, costs were shared between the Union government and states in an 80:20 ratio for large states, 95:5 for hilly states, and 100:0 for UTs, as per scheme cost-sharing norms. In India, based on this fund sharing pattern, a majority (78%) of the total scaled up costs for ICDS-CAS in year 1 was to be funded by the Union government. State governments combined would have to fund 17% of the total costs. The remaining costs for training and software development were to be borne by non-government entities.One of the major challenges of costing studies is the absence of disaggregated financial data . To addrIn the absence of information on exact time spent by the CPMU and SPMU staff on ICDS-CAS specifically, assumptions had to be made regarding personnel time, as mentioned above. Administrative costs account for only 17% of total costs and thus changing the assumption did not significantly impact the overall costs.On the other hand, software and device costs are driven primarily by device costs and the support required for configuration. The purchase price for devices can thus alter costs in a significant way. To the extent that the application doesn't change radically in the future, we can assume that device costs should not rise in the future. In fact, devices have been getting cheaper over time. However, if the application requires a better device for optimal performance, costs may rise. Therefore, total program costs are sensitive to the way the application will be structured in the future, and the way the application is used.For training, costs vary substantially based on the amount of support to be provided by the TA, whether the training is to be residential or not, and whether some groups require refresher training. For ICDS-CAS, the main assumption impacting training costs is whether training is residential or not.Mhealth interventions for health and nutrition have been studied at length. There are three primary gaps which have been highlighted which are addressed with the methods and lessons described above. First, is the reporting on program sustainability, scale-up costs, and long-term effects of the intervention . Second,Adding to the literature, the study presents the method from the first attempt at costing a large-scale national mHealth intervention in India. In India's context, applicable to other developing countries as well, there are several crucial related lessons related to scaling up. The study highlights five key lessons while costing for mHealth programs. First, that there are many activities and ingredients that must be budgeted for and discussed while planning and implementing mHealth programs. Second, the ABC-I method described in this paper provides great clarity on costs, yet its major limitation is the availability of data. Given that ABC-I aims to be as comprehensive and disaggregated as possible, a large amount of data is required. The lack of data for activities and inputs must be mitigated with the careful use of assumptions. These assumptions should be clearly listed out, and their sensitivity should be tested as well, as an indication of robustness of the costing study. Third, mHealth technology life cycles have financial implications. This includes understanding the life cycle of the technology used. For example, phones must be replaced every 2–3 years, which must be budgeted for. Fourth, in the early stages of a large mHealth intervention, sources of funding may include non-government sources. Embedding digital health solutions into health systems will require transitioning management and ownership to government partners who will have to bear the additional costs. Fifth, since costing estimates are subject to a set of assumptions, a disaggregation of costs allows for scenario-building, which is useful while planning ahead. Pilot costs are very different from costs at scale, and in fact, the component mix may be completely different, which should be kept in mind. As they say, you may pilot “apples” but have to scale “oranges” . At the As mHealth programs expand globally, governments and policymakers require a lot of information not just on their effectiveness, but on financial implications as well. For full-scale sustainability, financing for all aspects of digital health solutions needs to be integrated into routine health budgets and the budgeting processes. Gathering and estimating program costs is essential for LMICs, given that resources are typically scarce and there are several competing priorities. Project teams should also keep in mind that drafting a budget should not be a one-off event. Budgets should be revisited regularly since funding, assumptions and activities can all change. Regular reviews will assist project teams to plan appropriately for increasing their scale and managing resources efficiently . This enPoshan Tracker, and similar interventions are being launched globally. With that in mind, we hope that this analysis can help assess the resources necessary to undertake or sustain similar interventions including in low-resource settings.Exercises such as the costing method described above can be used by policymakers to plan and budget for new programs, tailor existing programs as they develop, and to determine the cost-effectiveness of any program. For India specifically, the government of India has replaced ICDS-CAS with a new mHealth intervention known as The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.AK and RS conceptualized the paper outline. RS conducted the literature review with inputs from AK. RS collected and analyzed the data, while AK reviewed the data analysis. RS wrote the first draft of the manuscript and AK reviewed it and provided feedback, suggestions, and comments. AK also edited significant sections of the manuscript. Both authors contributed to manuscript revision, read, and approved the submitted version.This study was funded by Grant No. OPP1158231 from the Bill and Melinda Gates Foundation (BMGF) to the University of California, San Francisco.The 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.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.


Continuous bioreactors for petroleum degradation and the effect factors of these bioreactors have rarely been mentioned in studies. In addition, indigenous bacteria living in seawater could influence the performance of continuous bioreactors with respect to petroleum degradation in practice. In this paper, a bioreactor fitted with immobilized petroleum–degrading bacteria beads was designed for further research. The results indicated that the diesel degradation rate of the bioreactor could remain above 50% over 27 days, while degradation performance decreased with bioremediation time. Intriguingly, the diameters of immobilized petroleum–degrading bacteria beads were reduced by 32.49% after 45 days remediation compared with the initial size of the immobilized petroleum–degrading bacteria beads. Change in immobilized petroleum–degrading bacteria beads was considered to correlate remarkably with reduced degradation efficiency. Therefore, this paper will be helpful for further study and improvement of bioreactors in the practical context of oil-spill accident recovery.  High demand for fuel, such as petroleum, has led to an increase in the number of petroleum-spill accidents. It has been estimated that more than 120 million liters of oil per year enter marine environments ,4. CrudeImmobilization technology has been at the forefront of bioremediation research and could be used to improve bacterial density and enhance the degradation of petroleum-spill pollution ,9,10,11.2 and H2O over time. Moreover, previous studies have proved that bacterial community structure is related to immobilized microbial remediating effectiveness [Researchers have elucidated the degradation mechanism of immobilized petroleum–degrading bacteria beads, which is related to bacterial degradation . The restiveness . Howevertiveness , and theIn this paper, a continual bioreactor was designed to investigate degradation performance. However, during the experiment, the degradation rate declined over time. At the same time, an interesting phenomenon attracted our attention, namely, that the volume of immobilized petroleum–degrading bacteria beads in the bioreactor reduced. In order to further enhance the effectiveness of bioreactors, this phenomenon should be investigated further, as it has only rarely been mentioned in earlier studies. A larger range of available detection methods were used to study and discuss this phenomenon in this research.2 solution dropwise to obtain immobilized petroleum–degrading bacteria beads of Shewanella algae, which were stored in CaCl2 solution for 72 h. Once they were completely formed, the immobilized petroleum–degrading bacteria beads were taken out and washed with normal saline and distilled water. All solutions mentioned in the experiment were sterilized and cooled to room temperature for use. A specific preparation procedure is shown in The composition of mineral salt medium (MSM) is displayed in With an inner diameter, length, and operating volume of 30 mm, 1000 mm, and 0.71 L, respectively, a uniform cylindrical reactor was set up for the experiment. The reactor operated continuously and steadily for 45 days. The entire experiment was run in the bioreactor, and diesel-contaminated seawater was prepared by adding diesel to seawater and stirring fully to ensure the influent diesel concentration. Synthetic diesel-contaminated seawater was pumped in from the leftmost inlet of the unit and out of the rightmost outlet. The bioreactor was equipped with sampling inlet and outlet valves, and the influents and effluents from the device were sampled once a day to measure diesel concentrations for calculation of the degradation rate. The specific design of the bioreactor is shown in R (%) is the degradation rate of diesel, C0 (mL/L) is the influent concentration of diesel, and C1 (mL/L) is the effluent concentration of diesel.The diesel in the water inlet and outlet were extracted with n-hexane in advance and then the concentrations of diesel were determined according to the ultraviolet spectrophotometry method. The degradation rates of diesel in the bioreactor were calculated using the following equation :DR = . The bacterial primers 338F (ACTCCTACGGGAGGCAGCAG) and 806R (GGACTACHVGGGTWTCTAAT) were used to amplify partial 16S rRNA genes [The immobilized petroleum–degrading bacteria beads, collected from water inlets and outlets after 45-day bioremediation , were sent to Shanghai Biotech Co., Ltd. for high-throughput sequencing, as was original seawater (OS), which was filtered through a 0.22 μm-fiber filter. In this study, DNA samples were extracted using a FastDNANA genes . Detailewww.majorbio.com, accessed on 16 March 2022) [R programming  tools were used to map community composition based on the data table in the tax_summary_a folder. Mothur  was used to cluster sequences into distance-based (97% similarity) operational taxonomic units (OTUs). Furthermore, PICRUSt was used to infer gene representation using taxonomic information from 16S rRNA genes sequencing . The datch 2022) .−1 approximately, while the effluent concentration of diesel changed on different days. As shown in In order to display the changes in degradation effects in the reactor, the influent concentrations and effluent concentrations of diesel were measured daily. During the 45-day remediation of the bioreactor, the influent concentration of diesel was kept at 10 mL LRegarding morphological changes to the immobilized petroleum–degrading bacteria beads, diameters were measured and SEM was chosen for microcosmic observation. Twenty immobilized petroleum–degrading bacteria beads for an initial sample and twenty beads for a 45-day sample were selected randomly for measurement a. Compar−1, 1400–1500 cm−1, and 900–1200 cm−1 related to cell proteins, fatty acids, glycopeptides, and phosphate groups of nucleic acid constituents, respectively, were weaker in the spectra for the immobilized degrading bacteria beads. In contrast, the peaks at 2800–3000 cm−1, representing cell membrane fatty acids, were strengthened with respect to the initial immobilized petroleum–degrading bacteria beads [−1, while the association degree was greater, with the peak being wide and the wave number being low. -OH in the sample was evidently lower than in the initial sample in To investigate the change in quantity and in the kinds of groups on the immobilized degrading bacteria beads, FTIR analysis was performed to determine the influence of the continuous process on the immobilized petroleum–degrading bacteria beads in the reactor. Such an analysis was undertaken to study the variation mechanism in the bioreactor with regard to the immobilized degrading bacteria beads. Compared with the petroleum–degrading bacteria beads initially immobilized in the bioremediation reactor, the distribution of the characteristic peaks of the beads had not changed significantly after the period of bioremediation. However, quantitative analysis of the immobilized petroleum–degrading bacteria beads in the bioreactor showed that the strength of the characteristic peaks of the immobilized degrading bacteria beads at 0 and 45 days changed considerably. According to the study of Fanesi et al., the contribution of spectral features to the prediction of the content of each macromolecule was specific, by which protein structure, phosphorylated molecules, and lipid acylchains could be characterized ,23. As sia beads . The varia beads ,25. As fShewanella algae), so that the Shannon and Chao indices were evidently both lower than the values for the immobilized petroleum–degrading bacteria beads after 45 days in the IB and EB. Change in community richness and community diversity was affected by change in environment and bacterial migration. A previous study reported that adjusting the living conditions of the immobilized petroleum–degrading bacteria beads would induce bacterial migration and community structure [Alpha diversity was used in this study to show community richness and community diversity according to the Chao index and the Shannon index, respectively. Community richness and community diversity showed similar varying trends, OS being the highest, as shown in tructure . Howevernorank_f__norank_o__SAR202_clade (14.70%) was the most abundant in the original seawater, and the family Flavobacteriaceae had the highest community structure percentages in the EB and IB . In fact, the family Shewanellaceae should be the only strain in the initial immobilized petroleum–degrading bacteria beads; however, it accounted for just 1.65% (EB) and 6.00% (IB) after 45 days in the bioreactor. Hence, the detection results indicated that the family Shewanellaceae decreased in the immobilized petroleum–degrading bacteria beads significantly, whereas the family Flavobacteriaceae increased after continuous remediation, which should be studied more in detail. The community structures in the original seawater (OS) and in the immobilized petroleum–degrading bacteria beads at the water inlet (IB) and at the outlet (EB) showed the abundances for each sample at the family level a. AdvantShewanella, many bacteria, not being abundant in seawater, could be detected in the immobilized petroleum–degrading bacteria beads, which indicated that the immobilized petroleum–degrading bacteria beads provided shelter for these bacteria and improved their growth and reproduction. The genera Alcanivorax, Shewanella, Thalassospira, Alteromonas, Tenacibaculum, and Lutibacter, were abundant in the interior of the immobilized petroleum–degrading bacteria beads, and these bacteria were all petroleum–degrading bacteria [Maribacter, belonging to the family Flavobacteriaceae, could directly be observed to be dominant in the immobilized petroleum–degrading bacteria beads in the detection results. Moreover, effluent immobilized petroleum–degrading bacteria beads showed more Maribacter than influent immobilized petroleum–degrading bacteria beads.A heatmap was constructed to analyze the change in community composition at the genus level b. Exceptbacteria ,28,29. Pbacteria ,31. HoweMaribacter was −35.65 ± 0.64%, showing a significant difference between the two samples in Maribacter in the immobilized petroleum–degrading bacteria beads at the effluent site, corresponding to the research results.Fisher’ exact test was used to compare species abundance differences between the dominant bacteria in the interiors of the immobilized petroleum–degrading bacteria beads at the inlet and outlet of the bioreactor. The confidence interval for genus Further investigation of functional abundance was carried out in order to determine the influence of continuous remediation on the immobilized petroleum–degrading bacteria. Compared with species composition, COG functional composition was similar across all samples in −1, 1500–1800 cm−1, 1400–1500 cm−1, and 900–1200 cm−1 indicated changes in microbial composition in the immobilized petroleum–degrading bacteria beads [Alcanivorax, Thalassospira, Alteromonas, Tenacibaculum, and Lutibacter, which became abundant in the interiors of the immobilized petroleum–degrading bacteria beads along with the genus Shewanella [Maribacter, was proven to decompose carbohydrates, such as alginate, which fact was linked to the FTIR detection result showing that hydrogen-bonding interactions in alginate decreased [Maribacter accounted for a higher percentage of community composition in the immobilized petroleum–degrading bacteria beads. Hence, the variation in functional genes in the immobilized petroleum–degrading bacteria beads was related to the bacteria using carbohydrates. Increasing numbers of carbohydrate-degrading bacteria would affect the physical structure of the immobilized petroleum–degrading bacteria beads; thus, when more carbohydrates were being consumed by bacteria, the degradation rate in the bioreactor would have declined over time.According to photochemical and microcosmic observations, the morphologies of the immobilized petroleum–degrading bacteria beads in the bioreactor changed obviously after 45-day continuous bioremediation. Irregular hole structure could not only induce fragility in the immobilized petroleum–degrading bacteria, it could also influence the absorption of diesel, further affecting the degrading functions of the immobilized petroleum–degrading bacteria . In the ia beads ,25,26. Cewanella ,33,34. Mecreased ,36. COG In order to investigate the decline in degradation performance in the petroleum–degrading bioreactor used for oil-spill accident recovery, microbial population dynamics were analyzed as well as the physicochemical parameters of the immobilized petroleum–degrading bacteria beads after 45 days of continuous operation. Together with the detection results for the bioreactor, the following conclusions can be drawn:(1)The bioreactor could maintain a degradation rate above 50% during earlier weeks, indicating that renewal of the immobilized petroleum bacteria beads over time would be beneficial for bioreactor performance;(2)Alcanivorax, Shewanella, and Thalassospira, and so on, becoming part of the community structure in the immobilized petroleum–degrading bacteria beads; however, the total abundance of petroleum–degrading bacteria decreased significantly;Bacterial migration led to more kinds of petroleum–degrading bacteria, such as (3)Maribacter, which consumes alginate, further changed the morphology of the immobilized petroleum–degrading bacteria beads, thus influencing the efficiency of the bioreactor [The increase in the representation of the genus oreactor ,36.Maribacter was the main reason that the immobilized petroleum–degrading bacteria beads were destroyed and that the performance of the bioreactor decreased further. Thus, it will be necessary to find a way of inhibiting the reproduction of the genus Maribacter in the bioreactor in order to improve the performance in the next study.It can be concluded that the genus